**Meet the editors**

Dr. Chandraleka Saravanan is working as an Assistant Professor at the Department of Chemistry, Urumu Dhanalakshmi College, Tiruchirappalli, Tamil Nadu, India. She has experience in the field of coordination chemistry and bioinorganic chemistry. Her current research focuses on metal complex synthesis, structural characterization and their utility in pharmacological and biological

applications. She has published 20 research papers and 1 book: *Transition Metal Complexes*: *Biological Activity*. She guided eight MPhil candidates and participated in summer school on materials science. She has presented 16 research papers in national and international conferences/seminars. She is an editorial board member of national and international journals and an examiner committee member of MSc, MPhil, and PhD in Chemistry.

Dr. Bhaskar Biswas is currently working as an Assistant Professor of Chemistry at the Surendranath College, Kolkata, India. He is actively engaged in teaching as well as in research. He is an inorganic chemist with a vast experience in synthetic coordination chemistry. He also actively engrossed in the investigation of catalytic activities of different metal complexes, supramolecular

aspects of coordination molecules and biological modeling of metelloenzymes and metalloproteins. He has published more than 50 research articles in national and international reputes. At present, Dr. Biswas is the supervisor of four research scholars on various aspects of inorganic chemistry and one research scholar (Sri Dhananjay Dey) who already received his doctoral degree under the supervision of Dr. Biswas.

Contents

**Preface VII**

Ede Bodoki

**Complexation 29**

Chapter 3 **Ligand-Protected Gold Clusters 53**

**Section 2 Ligand in Biological Chemistry 75**

Nurfadly Zain

Kurashige and Yuichi Negishi

Chapter 4 **Neuropeptides as Ligands for GPCRs 77**

**Section 1 Chemical Bonding in Ligand Chemistry 1**

Chapter 1 **Metal–Ligand Interactions in Molecular Imprinting 3**

Chapter 2 **Versatile Silylphosphine Ligands for Transition Metal**

Burcin Duan Sahbaz and Necla Birgul Iyison

Chapter 5 **Role of Soluble Fas Ligand in Severity of Dengue Disease 103**

Julio Zamora-Moreno and Virginia Montiel-Palma

Bogdan-Cezar Iacob, Andreea Elena Bodoki, Luminița Oprean and

Sakiat Hossain, Lakshmi V. Nair, Junta Inoue, Yuki Koyama, Wataru

## Contents

#### **Preface XI**

#### **Section 1 Chemical Bonding in Ligand Chemistry 1**


Preface

of chemical entities.

Ligands are a special class of species, having a significant character to control the electronic properties of metal ions through some kind of chemical attachment. They have a great abili‐ ty to enhance the reactivity and/or selectivity in controlling the physico-chemical properties

This book consists of five chapters. Chapter 1 aims to outline the beneficial role of various functional ligands in pairing of metal ions in the molecular imprinting process and to pro‐ vide an up-to-date overview of various applications in chemical sensing, separation process‐ es (stationary phases and selective sorbents), drug delivery and catalysis. Chapter 2 covers the incorporation of both Si and P functionalities in a ligand backbone (silylphosphines), along with their applicability in reactivity in catalysis. The topic of discussion in Chapter 3 focuses on preparative route for gold clusters consisting of chalcogenate (thiolate, selenolate or tellurolate) ligands. A description of their geometric/electronic structures including phys‐ ical and chemical properties is also depicted here. In Chapter 4, the concept of neuropeptide, including its intracellular maturation process and characteristics of some typical neuropep‐ tide families with common properties of their cognate GPCRs, is dealt with. Chapter 5 high‐ lights the significant contribution to soluble Fas ligand, a Type II membrane protein belonging to TNF family in the pathogenesis against dengue infection. The importance of Fas ligand and its mechanistic aspects in preventing dengue disease is also depicted here. So, the book *Ligand* describes the diversity and versatility of ligands covering structural fea‐ tures, donor-acceptor properties and secondary functions like molecular recognition. More‐ over, this book also provides a comprehensive account on the applicability: catalysis, sensors, supramolecular assembly, photochemical property, bioinorganic chemistry, and so on.

> **Dr. Chandraleka Saravanan** Department of Chemistry Urumu Dhanalakshmi College

> > Tamil Nadu, India **Dr. Bhaskar Biswas** Department of Chemistry Surendranath College West Bengal, India

## Preface

Ligands are a special class of species, having a significant character to control the electronic properties of metal ions through some kind of chemical attachment. They have a great abili‐ ty to enhance the reactivity and/or selectivity in controlling the physico-chemical properties of chemical entities.

This book consists of five chapters. Chapter 1 aims to outline the beneficial role of various functional ligands in pairing of metal ions in the molecular imprinting process and to pro‐ vide an up-to-date overview of various applications in chemical sensing, separation process‐ es (stationary phases and selective sorbents), drug delivery and catalysis. Chapter 2 covers the incorporation of both Si and P functionalities in a ligand backbone (silylphosphines), along with their applicability in reactivity in catalysis. The topic of discussion in Chapter 3 focuses on preparative route for gold clusters consisting of chalcogenate (thiolate, selenolate or tellurolate) ligands. A description of their geometric/electronic structures including phys‐ ical and chemical properties is also depicted here. In Chapter 4, the concept of neuropeptide, including its intracellular maturation process and characteristics of some typical neuropep‐ tide families with common properties of their cognate GPCRs, is dealt with. Chapter 5 high‐ lights the significant contribution to soluble Fas ligand, a Type II membrane protein belonging to TNF family in the pathogenesis against dengue infection. The importance of Fas ligand and its mechanistic aspects in preventing dengue disease is also depicted here.

So, the book *Ligand* describes the diversity and versatility of ligands covering structural fea‐ tures, donor-acceptor properties and secondary functions like molecular recognition. More‐ over, this book also provides a comprehensive account on the applicability: catalysis, sensors, supramolecular assembly, photochemical property, bioinorganic chemistry, and so on.

> **Dr. Chandraleka Saravanan** Department of Chemistry Urumu Dhanalakshmi College Tamil Nadu, India

> > **Dr. Bhaskar Biswas** Department of Chemistry Surendranath College West Bengal, India

**Section 1**

**Chemical Bonding in Ligand Chemistry**

**Chemical Bonding in Ligand Chemistry**

**Chapter 1**

**Provisional chapter**

**Metal–Ligand Interactions in Molecular Imprinting**

Molecular imprinting enables the design of highly crosslinked polymeric materials that are able to mimic natural recognition processes. Molecularly imprinted polymers exhibit binding sites with tailored selectivity toward target structures ranging from inorganic ions to biomacromolecules and even viruses or living cells. The choice of the appropriate functional monomer, crosslinker, and the nature and specificity of template–monomer interactions are critical for a successful imprinting process. The use of a metal ion mediating the interaction between the monomer and template (acting as ligands) has proven to offer a higher fidelity of imprint, which modulates the molecularly imprinted polymers (MIPs) selectivity or to endow additional features to the polymer, such as stimuli-responsiveness, catalytic activity, etc. Furthermore, limitations in using nonpolar and aprotic solvents are overcome, allowing the use of more polar solvents and even aqueous solutions as imprinting media, opening new prospects toward the imprinting of biomacromolecules (proteins, DNA, RNA, antibodies, biological receptors, etc.). This chapter aims to outline the beneficial pairing of metal ions as coordination centers and various functional ligands in the molecular imprinting process, as well as to provide an up to date overview of the various applications in chemical sensing, separation processes (stationary phases and selective sorbents), drug delivery, and catalysis.

**Keywords:** molecular imprinting, metal pivot imprinting, ion imprinting, drug delivery systems, catalysis, metal-ligand interactions, sensors,

**Metal–Ligand Interactions in Molecular Imprinting**

DOI: 10.5772/intechopen.73407

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Molecular recognition is indispensable to most of natural occurring phenomena, such as antibody–antigen immune response, ligand-receptor interactions, and enzyme catalysis. The complexity and specificity of these phenomena is the refined product of millions of years of evolution inciting scientists to search ways of mimicking these natural processes. The most

Bogdan-Cezar Iacob, Andreea Elena Bodoki,

Bogdan-Cezar Iacob, Andreea Elena Bodoki,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Luminița Oprean and Ede Bodoki

Luminița Oprean and Ede Bodoki

http://dx.doi.org/10.5772/intechopen.73407

surface imprinting, chiral analysis

**Abstract**

**1. Introduction**

**Provisional chapter**

## **Metal–Ligand Interactions in Molecular Imprinting**

**Metal–Ligand Interactions in Molecular Imprinting**

DOI: 10.5772/intechopen.73407

Bogdan-Cezar Iacob, Andreea Elena Bodoki, Luminița Oprean and Ede Bodoki Luminița Oprean and Ede Bodoki Additional information is available at the end of the chapter

Bogdan-Cezar Iacob, Andreea Elena Bodoki,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73407

#### **Abstract**

Molecular imprinting enables the design of highly crosslinked polymeric materials that are able to mimic natural recognition processes. Molecularly imprinted polymers exhibit binding sites with tailored selectivity toward target structures ranging from inorganic ions to biomacromolecules and even viruses or living cells. The choice of the appropriate functional monomer, crosslinker, and the nature and specificity of template–monomer interactions are critical for a successful imprinting process. The use of a metal ion mediating the interaction between the monomer and template (acting as ligands) has proven to offer a higher fidelity of imprint, which modulates the molecularly imprinted polymers (MIPs) selectivity or to endow additional features to the polymer, such as stimuli-responsiveness, catalytic activity, etc. Furthermore, limitations in using nonpolar and aprotic solvents are overcome, allowing the use of more polar solvents and even aqueous solutions as imprinting media, opening new prospects toward the imprinting of biomacromolecules (proteins, DNA, RNA, antibodies, biological receptors, etc.). This chapter aims to outline the beneficial pairing of metal ions as coordination centers and various functional ligands in the molecular imprinting process, as well as to provide an up to date overview of the various applications in chemical sensing, separation processes (stationary phases and selective sorbents), drug delivery, and catalysis.

**Keywords:** molecular imprinting, metal pivot imprinting, ion imprinting, drug delivery systems, catalysis, metal-ligand interactions, sensors, surface imprinting, chiral analysis

#### **1. Introduction**

Molecular recognition is indispensable to most of natural occurring phenomena, such as antibody–antigen immune response, ligand-receptor interactions, and enzyme catalysis. The complexity and specificity of these phenomena is the refined product of millions of years of evolution inciting scientists to search ways of mimicking these natural processes. The most

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

promising and advanced field of biomimetics is molecular imprinting (MI), a technique that gained popularity after the 90s, even though the first reports of imprinting date back to 1931, related to the findings of the Soviet chemist M. V. Polyakov. Compared with their natural counterparts, molecularly imprinted polymers (MIPs) possess, besides a similar selectivity, good chemical and thermal stability, ease of preparation, and low-cost production. By far, their main applications have been reported in the analytical field, and especially in separation techniques, where they have been used as stationary phases for (electro)chromatography and chiral separations and as selective sorbents in solid phase extraction [1]. They have also been applied as promising recognition elements in the development of biosensors, particularly electrochemical [2] and optical [3] ones. In the last few years, MIPs have proven to be versatile engineered materials in the construction of drug delivery systems [4] and as catalysts [5].

complex. The covalent approach or the preorganized approach employs reversible covalent bonds between the functional monomer and template, such as reversible esterification or condensation reactions (boronate ester, ketal/acetal, and Schiff's base formation) both prior to the polymerization, and also in the subsequent rebinding step of the template. This strategy leads to the generation of a higher yield of specific and more homogeneous binding sites along with reduced nonspecific adsorption. However, the applicability of the covalent imprinting approach is limited because of the small number of compounds bearing required functionalities (alcohols (diols), aldehydes, ketones, amines, and carboxylic acids). Removal of the template is generally more difficult, and the chemical cleavage must be achieved under mild conditions. A third, semi-covalent approach, (also called hybrid approach) [9], developed by Whitcombe et al. [10] combines the advantages of the previous two methods. While reversible covalent bonds are employed during the polymerization step, the re-binding is entirely

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407 5

Unfortunately, most authors neglect in their classification a different strategy, metal ion coordination, even though its first report dates back to 1985 [11]. It has been employed as an alternative to enhance template and functional monomer association in water by introducing a metal ion as mediator [12]. The use of metal ions allows the formation of a ternary complex between the functional monomer, metal ion, and template. The heteroatoms of monomer and template bind to the metal ion (generally first row transition metals) by donating electrons to the unfilled orbitals of the outer coordination sphere of the latter [13]. Coordination of metal ions to natural (e.g. structural elements of DNA, peptides, alkaloids, etc.) or synthetic ligands bearing a large variety of donor atoms has proven to be well suited for the preparation of polymers with outstanding molecular recognition properties, put into good use in a wide

Initially, the design and development of MIPs, regardless of the employed imprinting approach, aimed for the rebinding of the template with the highest selectivity. Nevertheless, instead of using the metal ion as mere mediator in the formation of the imprinted polymer, the selective rebinding of a target metal ion is often of interest. Thus, based on the principle of MI, the concept of ion imprinting has also been introduced, in which case the metal ion

In this approach, metal ions act as a bridge between the functional monomer and the template. Compared to noncovalent interactions, coordinative bonds are stronger, leading to a better stability in aqueous media. The stronger the interactions within the ternary complex, the more specific the recognition sites. Thus, the functional monomer and the template are maintained in close and fixed positions throughout the polymerization step [14]. Monomers are regularly positioned around the template via coordinate bonds, the relative motion of species is restrained, thus leading to improved imprinting factors and lower number of nonspecific binding sites. A key role in the imprinting process is represented by the nature of the

non-covalent in nature.

variety of applications fields.

assumes the role of template.

**2. Metal pivot imprinting (metal ion as mediator)**

The MI process is a relatively simple concept, enabling the synthesis of highly crosslinked polymeric materials of various formats with highly specific binding sites for target structures. The synthesis procedure is relatively easy, versatile, and straightforward, and in general, five components are required: template molecule, functional monomer(s), crosslinking monomer, solvent (porogen), and initiator. The polymers are prepared in the presence of the target molecule itself as template. The first step in MIP-preparation is the self-assembly of templatefunctional monomer into a complex to immobilize the template molecules throughout the polymerization process. After the addition of the remaining components, the polymerization is initiated and the functional monomer (linked with the template) is incorporated into the rigid 3D structure of the polymer with the functional groups locked toward the template. Upon the subsequent removal (extraction) of the template, cavities are unveiled in the structure of the rigid polymer, which are complementary in size, shape, and functionality to the template. Hence, a molecular memory is created into the polymeric matrix, which has now the ability to selectively and reversibly bind the analyte or its structural analogs. The strength of the interactions between template and monomer determines the efficiency of the imprinting process [6].

Traditionally, MI is classified according to the chemical nature of the interactions that occur during the functional monomer–template complex formation and template rebinding, into two main approaches: the noncovalent and the covalent approach [7, 8]. By far, the most frequently employed approach is the noncovalent one, based on weak, noncovalent interactions in the template-functional monomer complex formation and also in the subsequent recognition step. These interactions, such as hydrogen bonding, electrostatic interactions and van der Waals forces, are similar with those occurring in biological recognition systems. Because of the weak nature of these bonds, the formed complexes are unstable and a large excess of functional monomer, as compared to the template, is required during the polymerization step in order to favor the formation of the template-monomer assemblies. However, this excess generates a high number of heterogeneous binding sites as a result of random incorporation of the monomer's functional groups outside the imprinted cavities. Moreover, because the complex formation is governed by an equilibrium, a special attention must be paid to the employed porogenic solvent. Usually, nonpolar, aprotic solvents, such as chloroform and toluene promote the template-functional monomer association, whereas polar solvents like methanol and water tend to disrupt the noncovalent interactions in the prepolymerization complex. The covalent approach or the preorganized approach employs reversible covalent bonds between the functional monomer and template, such as reversible esterification or condensation reactions (boronate ester, ketal/acetal, and Schiff's base formation) both prior to the polymerization, and also in the subsequent rebinding step of the template. This strategy leads to the generation of a higher yield of specific and more homogeneous binding sites along with reduced nonspecific adsorption. However, the applicability of the covalent imprinting approach is limited because of the small number of compounds bearing required functionalities (alcohols (diols), aldehydes, ketones, amines, and carboxylic acids). Removal of the template is generally more difficult, and the chemical cleavage must be achieved under mild conditions. A third, semi-covalent approach, (also called hybrid approach) [9], developed by Whitcombe et al. [10] combines the advantages of the previous two methods. While reversible covalent bonds are employed during the polymerization step, the re-binding is entirely non-covalent in nature.

promising and advanced field of biomimetics is molecular imprinting (MI), a technique that gained popularity after the 90s, even though the first reports of imprinting date back to 1931, related to the findings of the Soviet chemist M. V. Polyakov. Compared with their natural counterparts, molecularly imprinted polymers (MIPs) possess, besides a similar selectivity, good chemical and thermal stability, ease of preparation, and low-cost production. By far, their main applications have been reported in the analytical field, and especially in separation techniques, where they have been used as stationary phases for (electro)chromatography and chiral separations and as selective sorbents in solid phase extraction [1]. They have also been applied as promising recognition elements in the development of biosensors, particularly electrochemical [2] and optical [3] ones. In the last few years, MIPs have proven to be versatile engineered materials in the construction of drug delivery systems [4] and as catalysts [5].

The MI process is a relatively simple concept, enabling the synthesis of highly crosslinked polymeric materials of various formats with highly specific binding sites for target structures. The synthesis procedure is relatively easy, versatile, and straightforward, and in general, five components are required: template molecule, functional monomer(s), crosslinking monomer, solvent (porogen), and initiator. The polymers are prepared in the presence of the target molecule itself as template. The first step in MIP-preparation is the self-assembly of templatefunctional monomer into a complex to immobilize the template molecules throughout the polymerization process. After the addition of the remaining components, the polymerization is initiated and the functional monomer (linked with the template) is incorporated into the rigid 3D structure of the polymer with the functional groups locked toward the template. Upon the subsequent removal (extraction) of the template, cavities are unveiled in the structure of the rigid polymer, which are complementary in size, shape, and functionality to the template. Hence, a molecular memory is created into the polymeric matrix, which has now the ability to selectively and reversibly bind the analyte or its structural analogs. The strength of the interactions between template and monomer determines the efficiency of the imprint-

Traditionally, MI is classified according to the chemical nature of the interactions that occur during the functional monomer–template complex formation and template rebinding, into two main approaches: the noncovalent and the covalent approach [7, 8]. By far, the most frequently employed approach is the noncovalent one, based on weak, noncovalent interactions in the template-functional monomer complex formation and also in the subsequent recognition step. These interactions, such as hydrogen bonding, electrostatic interactions and van der Waals forces, are similar with those occurring in biological recognition systems. Because of the weak nature of these bonds, the formed complexes are unstable and a large excess of functional monomer, as compared to the template, is required during the polymerization step in order to favor the formation of the template-monomer assemblies. However, this excess generates a high number of heterogeneous binding sites as a result of random incorporation of the monomer's functional groups outside the imprinted cavities. Moreover, because the complex formation is governed by an equilibrium, a special attention must be paid to the employed porogenic solvent. Usually, nonpolar, aprotic solvents, such as chloroform and toluene promote the template-functional monomer association, whereas polar solvents like methanol and water tend to disrupt the noncovalent interactions in the prepolymerization

ing process [6].

4 Ligand

Unfortunately, most authors neglect in their classification a different strategy, metal ion coordination, even though its first report dates back to 1985 [11]. It has been employed as an alternative to enhance template and functional monomer association in water by introducing a metal ion as mediator [12]. The use of metal ions allows the formation of a ternary complex between the functional monomer, metal ion, and template. The heteroatoms of monomer and template bind to the metal ion (generally first row transition metals) by donating electrons to the unfilled orbitals of the outer coordination sphere of the latter [13]. Coordination of metal ions to natural (e.g. structural elements of DNA, peptides, alkaloids, etc.) or synthetic ligands bearing a large variety of donor atoms has proven to be well suited for the preparation of polymers with outstanding molecular recognition properties, put into good use in a wide variety of applications fields.

Initially, the design and development of MIPs, regardless of the employed imprinting approach, aimed for the rebinding of the template with the highest selectivity. Nevertheless, instead of using the metal ion as mere mediator in the formation of the imprinted polymer, the selective rebinding of a target metal ion is often of interest. Thus, based on the principle of MI, the concept of ion imprinting has also been introduced, in which case the metal ion assumes the role of template.

## **2. Metal pivot imprinting (metal ion as mediator)**

In this approach, metal ions act as a bridge between the functional monomer and the template. Compared to noncovalent interactions, coordinative bonds are stronger, leading to a better stability in aqueous media. The stronger the interactions within the ternary complex, the more specific the recognition sites. Thus, the functional monomer and the template are maintained in close and fixed positions throughout the polymerization step [14]. Monomers are regularly positioned around the template via coordinate bonds, the relative motion of species is restrained, thus leading to improved imprinting factors and lower number of nonspecific binding sites. A key role in the imprinting process is represented by the nature of the metal ion, which needs to simultaneously meet several requirements, that is, no inhibition of the polymerization process, well-defined coordination sphere, and optimal affinity toward the template and monomer.

monomer):Fe(III):template (uric acid) was found in the coordination compound used for the surface plasmon resonance (SPR) detection of uric acid [39]. Fe(III)-MIP was developed as a drug carrier that showed larger drug loading capacity and a higher amount of drug release at its equilibrium state compared with the Fe-free MIP. Moreover, the Fe-MIP drug release rate was more controlled than that of the MIP and the non-imprinted polymer (NIP), especially at

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407 7

Metal–template binding should be stable under polymerization conditions yet labile enough to allow removal of substrates-templates. With few exceptions, [41] the design of MIPs for the selective recognition of amino acids and peptides has been limited to the traditional imprinting strategy, which employs polyacrylates with MAA as the functional monomer in organic solvents. Bulkier templates, such as macromolecules (especially proteins) are not compatible with the organic media because of their low solubility and tendency toward denaturation, thus using water as solvent is essential. However, polar solvents will interfere in templatemonomer hydrogen bonding, therefore metal-coordination interactions represent an effective alternative in imprinting biological-relevant compounds. The affinity of N-terminal histidine for Ni(II) allowed the creation of MIP receptors for peptides with exposed histidine residues [36]. It was shown that the metal ion works not just as a link between the monomer and template, it also influences the steric environment around the metal ion during polymerization step. The superior performance of metal-ion mediated imprinting compared with the metal-free approach, was demonstrated for CCK-5 [35]. MIPs produced in the presence Ni(II) showed a more than double average rebinding value and an I.F. of 1.9 with respect to the traditionally imprinted polymer. A Co(II)-mediated imprinted polymer for the bovine serum albumin (BSA) recognition (I.F. = 14.9), was synthesized and compared with the BSA ion-free MIP [17]. The Co(II)-mediated MIP presented an 8-fold increase in the I.F. and a reduced cross-selectivity by a factor of 2.5 compared with the BSA-MIP. Using Cu(II) chelation strategy, the cytochrome *c* was successfully imprinted into a supermacroporous cryogel, which was employed for template separation from a mixture of proteins (cytochrome c, lysozyme,

Protein imprinting is still a challenging task mainly because of their huge molecular size and conformational flexibility and complexity, which makes template removal and the subsequent protein rebinding onto imprinted sites very difficult. One alternative to the protein bulk imprinting is the metal-ion mediated surface imprinting in which the specific recognition sites are located at the surface of MIP. Porcine serum albumin was imprinted on the surface of silica microparticles via a metal chelating strategy in phosphate buffer [32]. The thickness of the imprinted polymer layer was about 20 nm, allowing fast binding kinetics (~1 min), the binding of protein template reaching more than 90% of the maximum capacity. Satisfactory selectivity was obtained using three competitive proteins: cytochrome c, ribonuclease B and myoglobin. Another metal-ion imprinted thin polymeric film was synthesized on the surface of cellulose nanofibers for the selective recognition and purification of hemoglobin from hemolysate [33]. The obtained MIP was able to rebind 8 times more template protein

the early stages of release [40].

**2.2. Template as ligand**

and BSA).

#### **2.1. Metal ion**

Generally, only a small number of transitional metal ions are employed as pivots in MI, such as: Co(II), Co(III), Cu(II), Ni(II), Zn(II), Cd(II), Fe(II), and Fe(III). Due to their high ability in forming coordination compounds with the majority of ligands of interest cobalt [15–26] and copper [27–33] ions are most often chosen by default. Nevertheless, in the absence of rational guidelines for matching the optimal metal pivot ion with the ligand of interest, a systematic pairing process would be required for each individual set of components.

Wu and Li [27] reported Cu(II) being complexed in the pre-polymerization step by 2 molecules of monomer (4-vinylpyridine (4-VPy)), one of template (picolinamide) and two acetates. They also have shown that the anion in the copper salt participates in the recognition process and in the complex formation [27]. Even though Cu(II) forms the most stable coordination compounds with ligands bearing N-donor atoms, the best imprinting efficiency is achieved in the case of Co(II), as evidenced in multiple studies that compared the imprinting performances of multiple metal ions [15, 18, 21]. In an attempt to separate the enantiomers of mandelic acid, the R(+) enantiomer and 4-VPy were used as template and functional monomer, respectively, alongside different metal ions as pivot (Co(II), Ni(II), Cu(II), and Zn(II)) to create imprinted monoliths [15]. The best resolutions were obtained using Co(II) and Ni(II) as mediators (RS = 1.87 and R<sup>S</sup> = 1.41, respectively), while in the case of Cu(II) and Zn(II) no separation was observed. The smallest template retention recorded in the case of Zn(II) monolith implies that no ternary complex (template:metal ion:monomer) is formed due to zinc's weak coordination capacity. In the case of Cu(II), even though it produces the most stable hexa-coordinated complexes, because of Jahn–Teller distortion, these coordination compounds are known to be susceptible to tetragonal distortion (elongation/ compression) [34]. In another study, the Co(II) mediated imprinted monolith showed the best retention (*k* = 2.75) and imprinting factors (I.F. = 3.1) for the gallic acid (template), in comparison with Ni(II) mediated polymer (*k* = 2.49) or different other ion-mediated MIP monoliths that were tested [18]. The Co(II)-mediated MIPs emerge also in other two studies [21, 22] in which ketoprofen and ketoprofen with naproxen, respectively were used as templates. In the first study, [21] the ability of molecular recognition of the ion-mediated imprinted polymers decreases in the order: Co(II) > Ni(II) > Zn(II). The binding affinity of Ni(II) to the N containing ligands (especially aminoacids containing compounds) was employed in creating imprinted polyacrylamides as artificial receptors for different peptides (cholecystokinin C-terminal pentapeptide (CCK-5) [35] and His-Alac [36]). The functional monomer (nitrilotriacetic acid) occupies four positions in the octahedral coordination sphere of Ni(II), leaving the remaining two for selective interactions with the template. Both Fe(II) and Fe(III) were successfully used in developing MIP adsorbents for tetracyclines enriching [37, 38]. It was found that Fe(II) could form a ternary complex with tetracycline (template) and methacrylic acid (MAA) (functional monomer), made of one molecule of template, one Fe(II) and four MAAs. The same mole ratio 2:1:2, methacryloyl-l-cysteine methyl ester (functional monomer):Fe(III):template (uric acid) was found in the coordination compound used for the surface plasmon resonance (SPR) detection of uric acid [39]. Fe(III)-MIP was developed as a drug carrier that showed larger drug loading capacity and a higher amount of drug release at its equilibrium state compared with the Fe-free MIP. Moreover, the Fe-MIP drug release rate was more controlled than that of the MIP and the non-imprinted polymer (NIP), especially at the early stages of release [40].

#### **2.2. Template as ligand**

metal ion, which needs to simultaneously meet several requirements, that is, no inhibition of the polymerization process, well-defined coordination sphere, and optimal affinity toward

Generally, only a small number of transitional metal ions are employed as pivots in MI, such as: Co(II), Co(III), Cu(II), Ni(II), Zn(II), Cd(II), Fe(II), and Fe(III). Due to their high ability in forming coordination compounds with the majority of ligands of interest cobalt [15–26] and copper [27–33] ions are most often chosen by default. Nevertheless, in the absence of rational guidelines for matching the optimal metal pivot ion with the ligand of interest, a systematic

Wu and Li [27] reported Cu(II) being complexed in the pre-polymerization step by 2 molecules of monomer (4-vinylpyridine (4-VPy)), one of template (picolinamide) and two acetates. They also have shown that the anion in the copper salt participates in the recognition process and in the complex formation [27]. Even though Cu(II) forms the most stable coordination compounds with ligands bearing N-donor atoms, the best imprinting efficiency is achieved in the case of Co(II), as evidenced in multiple studies that compared the imprinting performances of multiple metal ions [15, 18, 21]. In an attempt to separate the enantiomers of mandelic acid, the R(+) enantiomer and 4-VPy were used as template and functional monomer, respectively, alongside different metal ions as pivot (Co(II), Ni(II), Cu(II), and Zn(II)) to create imprinted monoliths [15]. The best resolutions were obtained using Co(II) and Ni(II) as mediators (RS = 1.87 and R<sup>S</sup> = 1.41, respectively), while in the case of Cu(II) and Zn(II) no separation was observed. The smallest template retention recorded in the case of Zn(II) monolith implies that no ternary complex (template:metal ion:monomer) is formed due to zinc's weak coordination capacity. In the case of Cu(II), even though it produces the most stable hexa-coordinated complexes, because of Jahn–Teller distortion, these coordination compounds are known to be susceptible to tetragonal distortion (elongation/ compression) [34]. In another study, the Co(II) mediated imprinted monolith showed the best retention (*k* = 2.75) and imprinting factors (I.F. = 3.1) for the gallic acid (template), in comparison with Ni(II) mediated polymer (*k* = 2.49) or different other ion-mediated MIP monoliths that were tested [18]. The Co(II)-mediated MIPs emerge also in other two studies [21, 22] in which ketoprofen and ketoprofen with naproxen, respectively were used as templates. In the first study, [21] the ability of molecular recognition of the ion-mediated imprinted polymers decreases in the order: Co(II) > Ni(II) > Zn(II). The binding affinity of Ni(II) to the N containing ligands (especially aminoacids containing compounds) was employed in creating imprinted polyacrylamides as artificial receptors for different peptides (cholecystokinin C-terminal pentapeptide (CCK-5) [35] and His-Alac [36]). The functional monomer (nitrilotriacetic acid) occupies four positions in the octahedral coordination sphere of Ni(II), leaving the remaining two for selective interactions with the template. Both Fe(II) and Fe(III) were successfully used in developing MIP adsorbents for tetracyclines enriching [37, 38]. It was found that Fe(II) could form a ternary complex with tetracycline (template) and methacrylic acid (MAA) (functional monomer), made of one molecule of template, one Fe(II) and four MAAs. The same mole ratio 2:1:2, methacryloyl-l-cysteine methyl ester (functional

pairing process would be required for each individual set of components.

the template and monomer.

**2.1. Metal ion**

6 Ligand

Metal–template binding should be stable under polymerization conditions yet labile enough to allow removal of substrates-templates. With few exceptions, [41] the design of MIPs for the selective recognition of amino acids and peptides has been limited to the traditional imprinting strategy, which employs polyacrylates with MAA as the functional monomer in organic solvents. Bulkier templates, such as macromolecules (especially proteins) are not compatible with the organic media because of their low solubility and tendency toward denaturation, thus using water as solvent is essential. However, polar solvents will interfere in templatemonomer hydrogen bonding, therefore metal-coordination interactions represent an effective alternative in imprinting biological-relevant compounds. The affinity of N-terminal histidine for Ni(II) allowed the creation of MIP receptors for peptides with exposed histidine residues [36]. It was shown that the metal ion works not just as a link between the monomer and template, it also influences the steric environment around the metal ion during polymerization step. The superior performance of metal-ion mediated imprinting compared with the metal-free approach, was demonstrated for CCK-5 [35]. MIPs produced in the presence Ni(II) showed a more than double average rebinding value and an I.F. of 1.9 with respect to the traditionally imprinted polymer. A Co(II)-mediated imprinted polymer for the bovine serum albumin (BSA) recognition (I.F. = 14.9), was synthesized and compared with the BSA ion-free MIP [17]. The Co(II)-mediated MIP presented an 8-fold increase in the I.F. and a reduced cross-selectivity by a factor of 2.5 compared with the BSA-MIP. Using Cu(II) chelation strategy, the cytochrome *c* was successfully imprinted into a supermacroporous cryogel, which was employed for template separation from a mixture of proteins (cytochrome c, lysozyme, and BSA).

Protein imprinting is still a challenging task mainly because of their huge molecular size and conformational flexibility and complexity, which makes template removal and the subsequent protein rebinding onto imprinted sites very difficult. One alternative to the protein bulk imprinting is the metal-ion mediated surface imprinting in which the specific recognition sites are located at the surface of MIP. Porcine serum albumin was imprinted on the surface of silica microparticles via a metal chelating strategy in phosphate buffer [32]. The thickness of the imprinted polymer layer was about 20 nm, allowing fast binding kinetics (~1 min), the binding of protein template reaching more than 90% of the maximum capacity. Satisfactory selectivity was obtained using three competitive proteins: cytochrome c, ribonuclease B and myoglobin. Another metal-ion imprinted thin polymeric film was synthesized on the surface of cellulose nanofibers for the selective recognition and purification of hemoglobin from hemolysate [33]. The obtained MIP was able to rebind 8 times more template protein compared to the corresponding NIP. z-Histidine was also imprinted in polar organic solvent (methanol) via the mediation of Co(II) ions [20]. However, it was found a small difference in the rebinding capacities between the polymers prepared in the presence of z-His/Co(II) and Co(II) when they were exposed to the Co(C<sup>2</sup> H3 O2 ) 2 (z-Histidine)] coordination compound.

The combination of using ionic liquid ([Bmim]BF4

by limiting the polymer swelling and shrinkage [44].

**2.3. Functional monomer**

acid [29].

the binding sites.

compound on the polymer backbone.

imprinting a polar compound, methyl gallate, exhibiting superior recognition abilities than the ion-free polymer [24]. It is assumed that the ionic liquid improves the imprinting process

A successful metal ion-imprinting process is achieved if the formation of the templatemetal ion-functional monomer ternary complex involves strong coordination interactions. Therefore, the choice of the functional monomer is very important. It must interact with the metal ion and template in a particular geometry offering the anchor point for the coordination

One approach is to synthesize the metal ion-functional monomer complex before the addition of template, complex that will be incorporated into the polymer matrix and will be preserved after template removal [16, 17, 29, 35, 36]. Thus, in the rebinding step, the MIP should be exposed only to the free-form of the template. Examples of such coordination compounds: nitrilotriacetic acid– nickel (Ni–NTA) complex [35, 36], Co-porphyrin (Co(III)tetrakis(o-aminophenyl) porphyrin [16], Co(II)-(E)-2-((2 hydrazide-(4-vinylbenzyl) hydrazono)methyl)phenol, Fe(III) chloroprotoporphyrin, vinylferrocene, Zn(II) protoporphyrin [17], and Cu(II)–N-(4-vinylbenzyl)iminodiacetic

However, in the metal-mediated imprinting, the most widely used functional monomer is by far 4-VPy, because of its ability to form strong coordination bonds with a large spectrum of divalent metals. Different metal ion-4-VPy molar ratios have been used, ranging from 1:1 [43], 1:2 [19, 20, 27, 28], 1:4 [22] up to 1:6 [15, 18, 23]. It appears that with the increasing molar ratio of functional monomer, the IFs are also increasing up to a ratio of 1:6. An excess of functional monomer is needed in order to stabilize the ternary complex and to achieve good fidelity of

Surprisingly, when 4-VPy was investigated as functional monomer versus acrylamide, and MAA, the best performances were exhibited by acrylamide-imprinted polymer, even though

However, acidic acrylates (itaconic acid [37], MAA [25, 32, 38], and acrylic acid (AA) [40]) were successfully employed in metal-ion imprinting using Fe(II) and Fe(III) as central ion.

Commonly used monomers in MI often possess the ability to universally bond a multitude of metal ions, with variable selectivity. Thus, the use of monomers and/or ligands with structural features that enable metal ion chelation has opened new perspectives in the management and analysis of metal ions. First introduced by Nishide et al. [45], the concept of metal ion imprinting has been increasingly developed during the last two decades on the principle of MI.

among the three monomers, acrylamide generates the lowest binding energy.

**3. Ion imprinting (metal ion as template)**

) and metal pivoting was employed in

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407 9

Metal chelating approach was employed for the chiral discrimination of different amino acids, like phenylalanine, tyrosine, alanine, valine, leucine, isoleucine [29], and Boc-L-Phe-OH [25] and other compounds (mandelic acid [15]). The amino acid's side group's size plays a crucial role in obtaining a good enantioselectivity. The MIPs prepared with aliphatic amino acids showed no or little enantioselectivity. Amino acids containing aromatic or heterocyclic groups yielded MIPs with good chiral discriminative properties. According to the three-point interaction model, these bulky groups are responsible for the third necessary interaction with the polymer matrix, sterically hindering the opposite enantiomer.

Regarding the smaller and simpler (no multiple functional groups) molecules, non-covalent imprinting is more difficult because of the smaller number of possible interactions between the template and functional monomer, especially in aqueous media. It is the case of formate, acetate and propionate anions, which showed no imprinting effect using 4-VPy as functional monomer. However, if these anions are part of a ternary complex with picolinamide as ligand and Cu(II) ion as mediator (during the polymerization process as well as during the rebinding step), their indirect analysis is possible [28].

Metal ion mediated approach may be an alternative for compounds with strong intramolecular hydrogen bonds that can interfere in the formation of template-monomer intermolecular hydrogen bonds, thus inhibiting the MI effect. For example, picolinamide cannot be imprinted through the noncovalent approach, but if it is included in a ternary Cu(II) complex with 4-VPy (both as ligand and monomer), the imprinted polymer showed a high molecular recognition ability [27]. Metal ion-mediated imprinting was also used to prepare different MIPs for the specific recognition of multiple drugs with high metal chelating capability: tetracyclines [37, 38], quinolones [37], ketoprofen [21], furosemide [40], and naproxen [26]. Two pharmaceutical compounds, naproxen, and ketoprofen were simultaneously imprinted using metal chelating strategy without loss of selectivity and it was found to give better results versus traditional MIPs [22]. A SPR sensor for a biological-relevant molecule (uric acid) was developed and applied for the metabolite's detection in urine [39].

Because of the stronger coordination binding compared with noncovalent imprinting, metal ion mediated imprinted polymers can be successfully used as selective sorbents for the concentration and the clean-up of different pollutants and toxic compounds (methylmercury from human hair and soil [42], organohalide pesticide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) [16], thiabendazole fungicide in citrus and soil samples [31]).

MIPs were also used as extraction media of active compounds from complicated natural products, using metal coordination interactions. Quercetin was shown to form coordination compounds with Zn(II) through 3-hydroxyl-4-ketone electron donor functionality from its structure [43]. Epigallocatechin gallate was separated from natural plant extracts employing gallic acid as a dummy template in order to reduce the MIPs manufacturing costs [18].

The combination of using ionic liquid ([Bmim]BF4 ) and metal pivoting was employed in imprinting a polar compound, methyl gallate, exhibiting superior recognition abilities than the ion-free polymer [24]. It is assumed that the ionic liquid improves the imprinting process by limiting the polymer swelling and shrinkage [44].

#### **2.3. Functional monomer**

compared to the corresponding NIP. z-Histidine was also imprinted in polar organic solvent (methanol) via the mediation of Co(II) ions [20]. However, it was found a small difference in the rebinding capacities between the polymers prepared in the presence of z-His/Co(II) and

> H3 O2 ) 2

Metal chelating approach was employed for the chiral discrimination of different amino acids, like phenylalanine, tyrosine, alanine, valine, leucine, isoleucine [29], and Boc-L-Phe-OH [25] and other compounds (mandelic acid [15]). The amino acid's side group's size plays a crucial role in obtaining a good enantioselectivity. The MIPs prepared with aliphatic amino acids showed no or little enantioselectivity. Amino acids containing aromatic or heterocyclic groups yielded MIPs with good chiral discriminative properties. According to the three-point interaction model, these bulky groups are responsible for the third necessary interaction with

Regarding the smaller and simpler (no multiple functional groups) molecules, non-covalent imprinting is more difficult because of the smaller number of possible interactions between the template and functional monomer, especially in aqueous media. It is the case of formate, acetate and propionate anions, which showed no imprinting effect using 4-VPy as functional monomer. However, if these anions are part of a ternary complex with picolinamide as ligand and Cu(II) ion as mediator (during the polymerization process as well as during the rebinding

Metal ion mediated approach may be an alternative for compounds with strong intramolecular hydrogen bonds that can interfere in the formation of template-monomer intermolecular hydrogen bonds, thus inhibiting the MI effect. For example, picolinamide cannot be imprinted through the noncovalent approach, but if it is included in a ternary Cu(II) complex with 4-VPy (both as ligand and monomer), the imprinted polymer showed a high molecular recognition ability [27]. Metal ion-mediated imprinting was also used to prepare different MIPs for the specific recognition of multiple drugs with high metal chelating capability: tetracyclines [37, 38], quinolones [37], ketoprofen [21], furosemide [40], and naproxen [26]. Two pharmaceutical compounds, naproxen, and ketoprofen were simultaneously imprinted using metal chelating strategy without loss of selectivity and it was found to give better results versus traditional MIPs [22]. A SPR sensor for a biological-relevant molecule (uric acid) was developed and

Because of the stronger coordination binding compared with noncovalent imprinting, metal ion mediated imprinted polymers can be successfully used as selective sorbents for the concentration and the clean-up of different pollutants and toxic compounds (methylmercury from human hair and soil [42], organohalide pesticide 4-(2,4-dichlorophenoxy)butyric acid

MIPs were also used as extraction media of active compounds from complicated natural products, using metal coordination interactions. Quercetin was shown to form coordination compounds with Zn(II) through 3-hydroxyl-4-ketone electron donor functionality from its structure [43]. Epigallocatechin gallate was separated from natural plant extracts employing gallic acid as a dummy template in order to reduce the MIPs manufacturing costs [18].

(z-Histidine)] coordination compound.

Co(II) when they were exposed to the Co(C<sup>2</sup>

8 Ligand

step), their indirect analysis is possible [28].

applied for the metabolite's detection in urine [39].

(2,4-DB) [16], thiabendazole fungicide in citrus and soil samples [31]).

the polymer matrix, sterically hindering the opposite enantiomer.

A successful metal ion-imprinting process is achieved if the formation of the templatemetal ion-functional monomer ternary complex involves strong coordination interactions. Therefore, the choice of the functional monomer is very important. It must interact with the metal ion and template in a particular geometry offering the anchor point for the coordination compound on the polymer backbone.

One approach is to synthesize the metal ion-functional monomer complex before the addition of template, complex that will be incorporated into the polymer matrix and will be preserved after template removal [16, 17, 29, 35, 36]. Thus, in the rebinding step, the MIP should be exposed only to the free-form of the template. Examples of such coordination compounds: nitrilotriacetic acid– nickel (Ni–NTA) complex [35, 36], Co-porphyrin (Co(III)tetrakis(o-aminophenyl) porphyrin [16], Co(II)-(E)-2-((2 hydrazide-(4-vinylbenzyl) hydrazono)methyl)phenol, Fe(III) chloroprotoporphyrin, vinylferrocene, Zn(II) protoporphyrin [17], and Cu(II)–N-(4-vinylbenzyl)iminodiacetic acid [29].

However, in the metal-mediated imprinting, the most widely used functional monomer is by far 4-VPy, because of its ability to form strong coordination bonds with a large spectrum of divalent metals. Different metal ion-4-VPy molar ratios have been used, ranging from 1:1 [43], 1:2 [19, 20, 27, 28], 1:4 [22] up to 1:6 [15, 18, 23]. It appears that with the increasing molar ratio of functional monomer, the IFs are also increasing up to a ratio of 1:6. An excess of functional monomer is needed in order to stabilize the ternary complex and to achieve good fidelity of the binding sites.

Surprisingly, when 4-VPy was investigated as functional monomer versus acrylamide, and MAA, the best performances were exhibited by acrylamide-imprinted polymer, even though among the three monomers, acrylamide generates the lowest binding energy.

However, acidic acrylates (itaconic acid [37], MAA [25, 32, 38], and acrylic acid (AA) [40]) were successfully employed in metal-ion imprinting using Fe(II) and Fe(III) as central ion.

## **3. Ion imprinting (metal ion as template)**

Commonly used monomers in MI often possess the ability to universally bond a multitude of metal ions, with variable selectivity. Thus, the use of monomers and/or ligands with structural features that enable metal ion chelation has opened new perspectives in the management and analysis of metal ions. First introduced by Nishide et al. [45], the concept of metal ion imprinting has been increasingly developed during the last two decades on the principle of MI.

The series of reviews published throughout the years offer general guidelines and concepts on the development of ion imprinted polymers (IIPs) (synthesis, characterization, types of imprinting, and assessment of analytical performance) and various applications (selective detection, sample enrichment, recovery, and decontamination of metal ions) in the biomedical and environmental fields [46–50]. Herein, several aspects on different materials, natural and synthetic, used in the design of IIPs and the particular features that allow these materials to selectively bind distinct metal ions will be pointed out.

**3.2. Surface imprinting**

acrylate (EDMA) and Cu(MAA)<sup>2</sup>

photocatalysis. Fe2

embedded in mesoporous silica.

**3.3. Chemical immobilization**

of CTS [68].

adsorbate [56].

TiO<sup>2</sup>

Chemical immobilization, trapping, and crosslinking of linear chain polymers, prepared mainly by traditional polymerization methods (bulk, precipitation, and suspension), present several drawbacks (i.e. relatively low rebinding capacity, slow mass transfer, and incomplete removal of template) that arise mostly from the restricted accessibility of the binding site, enclosed in the rigid polymeric mixture [48, 65]. Surface imprinting addresses these issues by generating binding cavities at the surface of the imprinted polymer. A thin imprinted layer is immobilized on the surface of fibers or small sized particles of organic or inorganic nature [48].

Selective sorption of Cu(II) was achieved by copolymerization of ethylene glycol dimeth-

glycidyl methacrylate on polypropylene fibers [67]. A polypropylene membrane was used as support for a Pb(II) imprinted composite material in a process that implied grafting polymerization of AA on the polypropylene membrane and subsequent covalent immobilization

appealing since the post processing of solid-phase extraction is reduced to a simple magnetic separation. Chen et al. [57] developed thiourea-modified magnetic ion imprinted CTS/TiO<sup>2</sup> for highly effective Cd(II) adsorption and simultaneous 2,4-dichlorophenol degradation via

fied CTS-Fe(III), for the effective and simultaneous removal of Cd(II) and tetracycline from water samples. The synergistic effect of tetracycline and Cd(II) adsorption was found to be due to the formation, at pH = 8, of a tetracycline-Cd(II) complex bridging the adsorbent and

Modified silica gel particles are extensively used as support for the imprinted layer because of their mechanical and chemical stability, low cost, and ease of preparation and functionalization through the silanol groups. A facile approach with good results in terms of selectivity (selectivity coefficients above 50) was reported by Zhang et al. [69] and involved the use of two commonly employed functional monomers, 4-VPy and MAA to obtain ternary Pb(II) complexes, immobilized by polymerization with EDMA and subsequently grafted on hollow mesoporous silica by co-condensation between Si-OH and EDMA. Pb(II) imprinted silica sorbents were designed using a tetradentate chelating silylating agent derived from 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane and 2-pyridinecarboxaldehyde [70] or a N,N-bidentate group in the structure of the functional monomer 4-(di(1H–pyrazol-1-yl)methyl) phenol [71]. Iminodiacetic functionalized silane ((3-glycidyloxypropyl)trimethoxysilane) [72] and the bifunctional ligand monomer [3-(γ-aminoethylamino)-propyltrimethoxysilane] [72, 73] were used for the imprinting with Ni(II) and Cd(II) ions and the imprinted sites were

The chemical immobilization technique employs bifunctional ligands that possess both polymerizable functional group (i.e. a vinyl group for free radical polymerization or a silane

Surface-imprinting modification of magnetic particles such as TiO<sup>2</sup>

O3

on the surface of a polystyrene core [66]. Li et al. grafted

magnetic particles were immobilized on carbon disulfide modi-

and Fe2

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407

O3

is particularly

11

The choice of the chelating agent, the complexation mode, the particular geometry of the coordination compound, the charge, and the size of the imprinted metal ions are key factors in determining the selectivity of the resultant imprinted polymer [34, 51, 52].

The inclusion of the metal binding entity in the polymerization matrix can be achieved through four distinct approaches: (a) crosslinking of linear chain polymers carrying metal-binding groups, (b) chemical immobilization, (c) surface imprinting, and (d) trapping of ligand in the polymeric matrix [47].

#### **3.1. Crosslinking of linear chain polymers carrying metal-binding groups**

This approach is currently used mainly with natural linear polymers, such as chitosan (CTS) and cellulose. CTS units, copolymers of glucosamine and N-glucosamine are widely used as functional monomers due to the material's abundance, lack of toxicity, biocompatibility, and biodegradability that add to its particular structure with numerous amino and hydroxyl functional groups which enable structural modifications and crosslinking [53].

The uptake of metal occurs mainly by chelation and is most likely to occur inter- or intra-CTS chains via one to four amino groups, with the nitrogen atoms in the amino and N-acetyl amino groups acting as electron donors. Upon deprotonation, hydroxyl groups may also be involved in metal ion coordination [54]. The poor selectivity, low stability in acidic solutions, and weak mechanical strength of nonimprinted raw CTS renders it inappropriate as selective metal ion sequestrant; these drawbacks, however, may be addressed by crosslinking and functionalization [53–55].

Nevertheless, crosslinking may decrease the metal uptake efficiency as, often, the functional groups of CTS involved in metal binding are also involved in the crosslinking reaction. The reactive amine and hydroxyl groups most likely to be involved in metal chelation are protected by ion imprinting prior to crosslinking [56, 57]. The commonly used crosslinkers include, but are not limited to, aldehydes (formaldehyde, glutaraldehyde, and glyoxal), heterocyclic compounds [epichlorohydrin(ECH)], and ethers [crown ethers, ethylene glycol diglycidyl ether (EGDE)]. Various modes of functionalization intended to modulate selectivity of CTS toward different metal ions have been reported: carbomethylation and thiourea/glutaraldehyde grafting for Ag(I) [58], carboxylation via ketoglutaric acid for Cu(II) [59], derivatization with aminobenzaldehyde for Ni(II), Cu(II) and Pd(II), [60, 61] dithiocarbamate for Sr(II), [62] tetraethylenpentamine for Pb(II) [63].

A different approach was employed by Hande et al. [64] for the design of a Pb(II) imprinted interpenetrating polymer by simultaneous polymerization of MAA and CTS in the presence of Pb(II) ions as template.

#### **3.2. Surface imprinting**

The series of reviews published throughout the years offer general guidelines and concepts on the development of ion imprinted polymers (IIPs) (synthesis, characterization, types of imprinting, and assessment of analytical performance) and various applications (selective detection, sample enrichment, recovery, and decontamination of metal ions) in the biomedical and environmental fields [46–50]. Herein, several aspects on different materials, natural and synthetic, used in the design of IIPs and the particular features that allow these materials

The choice of the chelating agent, the complexation mode, the particular geometry of the coordination compound, the charge, and the size of the imprinted metal ions are key factors

The inclusion of the metal binding entity in the polymerization matrix can be achieved through four distinct approaches: (a) crosslinking of linear chain polymers carrying metal-binding groups, (b) chemical immobilization, (c) surface imprinting, and (d) trapping of ligand in the

This approach is currently used mainly with natural linear polymers, such as chitosan (CTS) and cellulose. CTS units, copolymers of glucosamine and N-glucosamine are widely used as functional monomers due to the material's abundance, lack of toxicity, biocompatibility, and biodegradability that add to its particular structure with numerous amino and hydroxyl

The uptake of metal occurs mainly by chelation and is most likely to occur inter- or intra-CTS chains via one to four amino groups, with the nitrogen atoms in the amino and N-acetyl amino groups acting as electron donors. Upon deprotonation, hydroxyl groups may also be involved in metal ion coordination [54]. The poor selectivity, low stability in acidic solutions, and weak mechanical strength of nonimprinted raw CTS renders it inappropriate as selective metal ion sequestrant; these drawbacks, however, may be addressed by crosslinking and

Nevertheless, crosslinking may decrease the metal uptake efficiency as, often, the functional groups of CTS involved in metal binding are also involved in the crosslinking reaction. The reactive amine and hydroxyl groups most likely to be involved in metal chelation are protected by ion imprinting prior to crosslinking [56, 57]. The commonly used crosslinkers include, but are not limited to, aldehydes (formaldehyde, glutaraldehyde, and glyoxal), heterocyclic compounds [epichlorohydrin(ECH)], and ethers [crown ethers, ethylene glycol diglycidyl ether (EGDE)]. Various modes of functionalization intended to modulate selectivity of CTS toward different metal ions have been reported: carbomethylation and thiourea/glutaraldehyde grafting for Ag(I) [58], carboxylation via ketoglutaric acid for Cu(II) [59], derivatization with aminobenzaldehyde for Ni(II), Cu(II) and Pd(II), [60, 61] dithiocarbamate for Sr(II), [62] tetraethylenpentamine for Pb(II) [63].

A different approach was employed by Hande et al. [64] for the design of a Pb(II) imprinted interpenetrating polymer by simultaneous polymerization of MAA and CTS in the presence

in determining the selectivity of the resultant imprinted polymer [34, 51, 52].

**3.1. Crosslinking of linear chain polymers carrying metal-binding groups**

functional groups which enable structural modifications and crosslinking [53].

to selectively bind distinct metal ions will be pointed out.

polymeric matrix [47].

10 Ligand

functionalization [53–55].

of Pb(II) ions as template.

Chemical immobilization, trapping, and crosslinking of linear chain polymers, prepared mainly by traditional polymerization methods (bulk, precipitation, and suspension), present several drawbacks (i.e. relatively low rebinding capacity, slow mass transfer, and incomplete removal of template) that arise mostly from the restricted accessibility of the binding site, enclosed in the rigid polymeric mixture [48, 65]. Surface imprinting addresses these issues by generating binding cavities at the surface of the imprinted polymer. A thin imprinted layer is immobilized on the surface of fibers or small sized particles of organic or inorganic nature [48].

Selective sorption of Cu(II) was achieved by copolymerization of ethylene glycol dimethacrylate (EDMA) and Cu(MAA)<sup>2</sup> on the surface of a polystyrene core [66]. Li et al. grafted glycidyl methacrylate on polypropylene fibers [67]. A polypropylene membrane was used as support for a Pb(II) imprinted composite material in a process that implied grafting polymerization of AA on the polypropylene membrane and subsequent covalent immobilization of CTS [68].

Surface-imprinting modification of magnetic particles such as TiO<sup>2</sup> and Fe2 O3 is particularly appealing since the post processing of solid-phase extraction is reduced to a simple magnetic separation. Chen et al. [57] developed thiourea-modified magnetic ion imprinted CTS/TiO<sup>2</sup> for highly effective Cd(II) adsorption and simultaneous 2,4-dichlorophenol degradation via TiO<sup>2</sup> photocatalysis. Fe2 O3 magnetic particles were immobilized on carbon disulfide modified CTS-Fe(III), for the effective and simultaneous removal of Cd(II) and tetracycline from water samples. The synergistic effect of tetracycline and Cd(II) adsorption was found to be due to the formation, at pH = 8, of a tetracycline-Cd(II) complex bridging the adsorbent and adsorbate [56].

Modified silica gel particles are extensively used as support for the imprinted layer because of their mechanical and chemical stability, low cost, and ease of preparation and functionalization through the silanol groups. A facile approach with good results in terms of selectivity (selectivity coefficients above 50) was reported by Zhang et al. [69] and involved the use of two commonly employed functional monomers, 4-VPy and MAA to obtain ternary Pb(II) complexes, immobilized by polymerization with EDMA and subsequently grafted on hollow mesoporous silica by co-condensation between Si-OH and EDMA. Pb(II) imprinted silica sorbents were designed using a tetradentate chelating silylating agent derived from 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane and 2-pyridinecarboxaldehyde [70] or a N,N-bidentate group in the structure of the functional monomer 4-(di(1H–pyrazol-1-yl)methyl) phenol [71]. Iminodiacetic functionalized silane ((3-glycidyloxypropyl)trimethoxysilane) [72] and the bifunctional ligand monomer [3-(γ-aminoethylamino)-propyltrimethoxysilane] [72, 73] were used for the imprinting with Ni(II) and Cd(II) ions and the imprinted sites were embedded in mesoporous silica.

#### **3.3. Chemical immobilization**

The chemical immobilization technique employs bifunctional ligands that possess both polymerizable functional group (i.e. a vinyl group for free radical polymerization or a silane coupling agent for sol–gel processes), and electron donor groups for the chelation of metal ions [48, 74].

without being chemically bound to the polymeric network. The stability of the binding sites depends upon the correct immobilization of the ligand in the polymeric matrix and the pres-

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407 13

The first trapping procedure was reported by Rao et al. in 2003. The imprinted polymer was synthesized by co-polymerization of a coordination compound between Dy(III), 5,7-dichloroquino-

The entrapped species may be a metal ion:ligand binary complex, as in the case of Zn(II):8 hydroxyquinoline (1,2) and Al:8-hydroxyquinoline (1,3) coordination compounds embedded in the polymeric matrix formed by MAA and DVB [88]. In most cases, however, a ternary metal complex is formed, the metal ion being coordinated by both the ligand ensuring selectivity and the functional monomer (e.g. 4-VPy, MAA) bearing, it too, electron donating heteroatoms, and therefore coordination ability. The ternary complex can be prepared in situ, just before the polymerization step or synthesized, isolated and characterized before being introduced in the polymerization. Comparative studies on the efficiency of polymers prepared with such ternary complexes vs. binary species where the coordination environment is ensured by the presence of the ligands alone, revealed the importance of the presence of bifunctional species acting as complementary complexing agents. Alizadeh used 4-VPy as functional monomer and quinaldic acid as complexing agent to imprint Cd(II) and employed experimental design to study various binary and ternary mixtures [89]. IPs prepared from binary complexes were found to be less efficient than those prepared with ternary complexes.

Crown ethers and derivatives with cavities of appropriate size were trapped in the polymer network by using suitable functional monomers and crosslinking agents and the polymer imprinted with alkali metals ions. Dicyclohexyl-18-crown-6, [90] dibenzo-21-crown-7, [91] dibenzo-24-crown-8 ether [92] and the aza-thioether crown containing a 1,10-phennathroline subunit (5-azamethyl-2,8-dithia [9],(2,9)-1,10-phenanthrolinophane), [93] were used by Shamsipur and coll. to imprint K(I), Rb(I), Cs(I) and Ag(I) ions, respectively, in the presence

Other ligands used for ion-imprinting via trapping include isatine for Cu(II), [94] diphenylcarbazide (for Cd(II)), [95] 1,10-phenanthroline for Ag(I) [93] or neocuproine for Cd(II), [96]

As compared to chemical immobilization, the trapping approach is easier to implement. The stability of the binding sites created via the trapping approach, however, depends upon the correct immobilization of the ligand in the polymeric matrix and the presence and the integ-

Polymers have played an integral role in the advancement of drug delivery systems (DDS) through the last three decades, improving safety, efficacy, and patient compliance during

of MAA as functional monomer and EDMA as crosslinker.

rity of the ligand during and after the removal of template [48].

8-hydroxyquinoline for Ni(II), [97] etc.

**4. MIPs in drug delivery**

ence and the integrity of the ligand during and after the template removal [47, 48].

line-8-ol and 4-VPy, in the presence of divinylbenzene (DVB) as crosslinker [87].

Currently, the technique is a one-step process that implies mixing together the metal ion, the bifunctional monomer and the crosslinker prior to co-polymerization. Isolation of the binary complex prior to polymerization is a more complicated approach but it offers the advantage of the control of the amount and of the structure of the coordination compound embedded into the polymer's structure [34, 48].

Common monomers such as 4-VPy, 1-vinylimidazole, AA or acrylamide may serve for chemical immobilization, but they show low binding capacities and low selectivity. MAA was used simultaneously with 1-vinylimidazole [75] or 4-VPy [76, 77] to prepare Cd(II), Cu(II), and Zn(II) imprinted polymer particles, respectively. Considering however that the use of simple, commercially available monomers results in materials with generally low binding capacity and selectivity, new tailored bifunctional ligands, bearing both chelating functionalities and polymerizable groups, have been proposed.

Particular features that differ from those of their open-chain analogs such as controlled size and the "macrocyclic effect" that translates into high selectivity and stability, make crown ethers interesting candidates as ligands in ion-imprinting. Benzo-15-crown-5-acrylamide, 4-vinylbenzo-18-crown-6, and 2-(allyoxy)methyl-12-crown-4 have been successfully employed for the imprinting of K(I), [78] Pb(II) [79] and Li(I) ions [80, 81].

Calix[4]resorcinorene, a resorcinol-based macrocyclic compound with a bowl-shape molecular cavity formed by four resorcinol units, was used by Yusof et al. [82] to synthesize diallylaminomethyl-calix[4]-resorcinarene, as host for imprinting Pb(II) ions.

Amino acids or amino acid derivatives bearing vinylated groups, (e.g. N-methacryloyl-(L) histidine), [83, 84] vinylated SALEN, [85] [N-(4-vinybenzyl)imino]diacetic acid, [34] are other examples of bifunctional ligands that have been reported for the chelation of various metal ions and subsequent copolymerization with a suitable crosslinking agent.

Based on the ability of Hg(II) to form stable coordination compounds with thymine (T), T-Hg(II)-T interactions, Xu et al. [74] synthesized 3-isocyanatopropyltriethoxysilane, bearing thymine (T) bases as recognition elements for the imprinting of Hg(II).

Using 5-(bisulfate N,N-diallyl-N-methyl ammonium)methyl salicylaldoxime, Zhang et al. [86] anchored chelating salicylaldoxime units onto the polymer networks through quaternary ammonium cations serving as spacers.

Chemical immobilization shows the advantage of ligands not being leached out during the elution of the template. The magnitude of the imprinting effect is however rather low; this adds up to the difficulty of the vinylation procedure [47].

### **3.4. Trapping of the ligand in the polymeric matrix**

In case of trapping, ligands do not require the insertion of polymerizable functions, but instead they are used as such and are entrapped inside the network during the polymer's formation, without being chemically bound to the polymeric network. The stability of the binding sites depends upon the correct immobilization of the ligand in the polymeric matrix and the presence and the integrity of the ligand during and after the template removal [47, 48].

The first trapping procedure was reported by Rao et al. in 2003. The imprinted polymer was synthesized by co-polymerization of a coordination compound between Dy(III), 5,7-dichloroquinoline-8-ol and 4-VPy, in the presence of divinylbenzene (DVB) as crosslinker [87].

The entrapped species may be a metal ion:ligand binary complex, as in the case of Zn(II):8 hydroxyquinoline (1,2) and Al:8-hydroxyquinoline (1,3) coordination compounds embedded in the polymeric matrix formed by MAA and DVB [88]. In most cases, however, a ternary metal complex is formed, the metal ion being coordinated by both the ligand ensuring selectivity and the functional monomer (e.g. 4-VPy, MAA) bearing, it too, electron donating heteroatoms, and therefore coordination ability. The ternary complex can be prepared in situ, just before the polymerization step or synthesized, isolated and characterized before being introduced in the polymerization. Comparative studies on the efficiency of polymers prepared with such ternary complexes vs. binary species where the coordination environment is ensured by the presence of the ligands alone, revealed the importance of the presence of bifunctional species acting as complementary complexing agents. Alizadeh used 4-VPy as functional monomer and quinaldic acid as complexing agent to imprint Cd(II) and employed experimental design to study various binary and ternary mixtures [89]. IPs prepared from binary complexes were found to be less efficient than those prepared with ternary complexes.

Crown ethers and derivatives with cavities of appropriate size were trapped in the polymer network by using suitable functional monomers and crosslinking agents and the polymer imprinted with alkali metals ions. Dicyclohexyl-18-crown-6, [90] dibenzo-21-crown-7, [91] dibenzo-24-crown-8 ether [92] and the aza-thioether crown containing a 1,10-phennathroline subunit (5-azamethyl-2,8-dithia [9],(2,9)-1,10-phenanthrolinophane), [93] were used by Shamsipur and coll. to imprint K(I), Rb(I), Cs(I) and Ag(I) ions, respectively, in the presence of MAA as functional monomer and EDMA as crosslinker.

Other ligands used for ion-imprinting via trapping include isatine for Cu(II), [94] diphenylcarbazide (for Cd(II)), [95] 1,10-phenanthroline for Ag(I) [93] or neocuproine for Cd(II), [96] 8-hydroxyquinoline for Ni(II), [97] etc.

As compared to chemical immobilization, the trapping approach is easier to implement. The stability of the binding sites created via the trapping approach, however, depends upon the correct immobilization of the ligand in the polymeric matrix and the presence and the integrity of the ligand during and after the removal of template [48].

## **4. MIPs in drug delivery**

coupling agent for sol–gel processes), and electron donor groups for the chelation of metal

Currently, the technique is a one-step process that implies mixing together the metal ion, the bifunctional monomer and the crosslinker prior to co-polymerization. Isolation of the binary complex prior to polymerization is a more complicated approach but it offers the advantage of the control of the amount and of the structure of the coordination compound embedded

Common monomers such as 4-VPy, 1-vinylimidazole, AA or acrylamide may serve for chemical immobilization, but they show low binding capacities and low selectivity. MAA was used simultaneously with 1-vinylimidazole [75] or 4-VPy [76, 77] to prepare Cd(II), Cu(II), and Zn(II) imprinted polymer particles, respectively. Considering however that the use of simple, commercially available monomers results in materials with generally low binding capacity and selectivity, new tailored bifunctional ligands, bearing both chelating functionalities and

Particular features that differ from those of their open-chain analogs such as controlled size and the "macrocyclic effect" that translates into high selectivity and stability, make crown ethers interesting candidates as ligands in ion-imprinting. Benzo-15-crown-5-acrylamide, 4-vinylbenzo-18-crown-6, and 2-(allyoxy)methyl-12-crown-4 have been successfully employed

Calix[4]resorcinorene, a resorcinol-based macrocyclic compound with a bowl-shape molecular cavity formed by four resorcinol units, was used by Yusof et al. [82] to synthesize diallyl-

Amino acids or amino acid derivatives bearing vinylated groups, (e.g. N-methacryloyl-(L) histidine), [83, 84] vinylated SALEN, [85] [N-(4-vinybenzyl)imino]diacetic acid, [34] are other examples of bifunctional ligands that have been reported for the chelation of various metal

Based on the ability of Hg(II) to form stable coordination compounds with thymine (T), T-Hg(II)-T interactions, Xu et al. [74] synthesized 3-isocyanatopropyltriethoxysilane, bearing

Using 5-(bisulfate N,N-diallyl-N-methyl ammonium)methyl salicylaldoxime, Zhang et al. [86] anchored chelating salicylaldoxime units onto the polymer networks through quaternary

Chemical immobilization shows the advantage of ligands not being leached out during the elution of the template. The magnitude of the imprinting effect is however rather low; this

In case of trapping, ligands do not require the insertion of polymerizable functions, but instead they are used as such and are entrapped inside the network during the polymer's formation,

ions [48, 74].

12 Ligand

into the polymer's structure [34, 48].

polymerizable groups, have been proposed.

ammonium cations serving as spacers.

for the imprinting of K(I), [78] Pb(II) [79] and Li(I) ions [80, 81].

aminomethyl-calix[4]-resorcinarene, as host for imprinting Pb(II) ions.

ions and subsequent copolymerization with a suitable crosslinking agent.

thymine (T) bases as recognition elements for the imprinting of Hg(II).

adds up to the difficulty of the vinylation procedure [47].

**3.4. Trapping of the ligand in the polymeric matrix**

Polymers have played an integral role in the advancement of drug delivery systems (DDS) through the last three decades, improving safety, efficacy, and patient compliance during long-term medication therapy by providing sustained release of both hydrophilic and hydrophobic therapeutic agents [98]. MIPs used as excipients of solid pharmaceutical dosage forms have been tested for tuning drug release profiles and eventually protect their load from enzymatic degradation while being freight through the body, nevertheless the inherent feature of these polymers, their selectivity, has not been put to a proper use. Therefore, efforts have been made to integrate MIPs in therapeutic systems for intelligent drug release or as targeting drug vectors [99].

limitations and future prospects of such molecularly imprinted DDS may be found in different

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407 15

A wide range of biocompatible semi-synthetic and synthetic polymers have been tested as suitable imprinted frameworks for DD. PHEMA and its derivatives or nanocomposites continues to be one of the most widely used biomaterials due to their low toxicity, excellent and long-term biocompatibility (including hemocompatibility) and high resistance to degradation [111, 112]. Such molecularly imprinted biomaterials served for the fabrication of various drug-delivery systems, such as transdermal membranes, [113] ocular inserts [114, 115], and implants (sub-

Polymer biodegradability plays also an important role in the biomedical exploitation, patient compliance and safe use of such DD systems. Because PHEMA is not biodegradable, upon the release of the pharmacologically active load the implants must be removed from the body through minor surgery to avoid the formation of pseudocyst. However, in many cases, such as the localized treatment of spinal cord injuries, the use of hydrolytically degradable hydrogel implants is far more convenient. *In vivo* experiments showed that macroporous 2-ethoxyethyl methacrylate/ N-(2-hydroxypropyl) methacrylamide based hydrolysable hydrogels (adjustable degradation between 2 and 40 days) are promising candidates for

Although metal ion coordination-based imprinting has shown promise in the creation of advanced recognition and DD systems up until now, literature is rather scarce in such studies. Nevertheless, there are some noteworthy publications in this field, such as the one reporting the sustained release (5 days) of copper salicylate, a metal-based nonsteroidal anti-inflammatory drug, successfully embedded in a metal chelate imprinted polymer using 4-VPy and 2-hydroxyethyl methacrylate (HEMA) as functional monomers and EDMA as crosslinker [118]. Another interesting study is the synthesis of Co(II) mediated imprinted hydrogels containing pendent chain linked template (drug) [119]. A pH responsive drug release could be achieved in the range of pH 3–6.8 due to the presence of an imidazole group within the proximity of the polymer-drug (ester or amide) bond responsible of the catalytic hydrolysis of the hydrogel.

Design of dedicated macromolecular architectures through MI able to recognize certain target molecules as well as capable of intelligent DD and release leads to the introduction of feedback-controlled drug release systems employing stimuli-responsive gel systems. As a result of oscillatory swelling, they are able to modulate release in response to pH, temperature, ionic strength, electric fields, or specific analyte concentration differences [101, 104]. The solvation of the hydrogel's macromolecular network is rather well adjustable by the local environment,

The inherent advantages offered by metal pivot-based MI have been successfully exploited in the imprinting process of hydrogels intended for stimuli-responsive DDS. The formation and cleavage of coordination bonds between different metal ions and various drugs of interest are pH-dependent, so by a rational design they could be specifically engineered for an intended use. As such, metal-mediated imprinting of HEMA-based hydrogel backbone crosslinked with N, N-methylenebisacrylamide (MBA) has been described for the pH-responsive and

implantation into tissue defects of the central nervous system [117].

this leading to a controlled swelling and release of the payload.

reviews [99, 104, 105, 110].

cutaneous, intra-peritoneal, etc.) [116].

These tailor-made IPs would be therapeutically advantageous for several reasons as they can act as molecular trap (sequestrant) systems, [100] as reservoir for prolonged release of a particular drug, they can enable an increased loading capacity of the therapeutic formulation, facilitate environmentally or physiologically responsive intelligent release of the therapeutic agent [101] and if required, they can confer an enantioselective load or release [102, 103]. Using conventional drug formulations, repeated administration would help in building up the required therapeutic levels of the drug in various biological compartments (blood, tissues, urine, etc.); however, in case of bioactive molecules with a narrow therapeutic index (i.e. digoxin, cyclosporine, sirolimus, theophylline, warfarin, lithium, phenytoin, and flecainide) or with very short plasmatic half-life (i.e. 5-fluorouracil (5-FU), acetylcholine, GABA, catecholamines, adenosine, and NO) repeated administration could lead to elevated risks or severity of toxic side effects.

Several comprehensive reviews have been published concerning the use of MIPs in general as DDS for controlled/sustained drug release or as intelligent drug delivery (DD) platforms (responsive release systems) either for oral, ocular, transdermal, or implant-associated local delivery routes of the therapeutic agent [99–101, 104–108].

Targeted DD relies on the MIP's ability to specifically recognize certain bioreceptors, such as a cell surface epitopes, which could further convey to cellular internalization of the drug loaded carrier and subsequent release of the active pharmaceutical compound. In the initial and most simple approaches the payload of biologically active molecule was non-covalently bound (hydrogen bonding, hydrophobic interactions, charge transfer, or van der Waals forces) to the imprinted polymer network [109]. Nevertheless, the overall controllability and reliability of DDS based on noncovalent binding might not be ideal in a living organism. Therefore, as an alternative, metal ion-mediated coordinate bonds between the functional monomer and the targeted drug molecule (template) has been investigated offering higher specificity and strength, as well as spatial directionality in comparison with noncovalent bonding. Additionally, metal coordination bonds are more compatible with the polar environment of living tissues and they can be easily manipulated through changes of the local hydrogen ion concentration, a feature extremely helpful in the development of pH-responsive delivery systems. Furthermore, MIPs prepared by noncovalent imprinting methods usually require using organic solvents, which eventually leave toxic traces, incompatible with biomedical applications.

Some of the imprinted polymers employed nowadays in intelligent DD [i.e. poly(2-hydroxyethyl methacrylate, (PHEMA)] were initially employed in the early forms of the non-imprinted DDS [98]. Various aspects about the encountered recognition and drug release mechanisms, optimization of the drug loading capacity, latest trends in various routes of DD, as well as limitations and future prospects of such molecularly imprinted DDS may be found in different reviews [99, 104, 105, 110].

long-term medication therapy by providing sustained release of both hydrophilic and hydrophobic therapeutic agents [98]. MIPs used as excipients of solid pharmaceutical dosage forms have been tested for tuning drug release profiles and eventually protect their load from enzymatic degradation while being freight through the body, nevertheless the inherent feature of these polymers, their selectivity, has not been put to a proper use. Therefore, efforts have been made to integrate MIPs in therapeutic systems for intelligent drug release or as targeting

These tailor-made IPs would be therapeutically advantageous for several reasons as they can act as molecular trap (sequestrant) systems, [100] as reservoir for prolonged release of a particular drug, they can enable an increased loading capacity of the therapeutic formulation, facilitate environmentally or physiologically responsive intelligent release of the therapeutic agent [101] and if required, they can confer an enantioselective load or release [102, 103]. Using conventional drug formulations, repeated administration would help in building up the required therapeutic levels of the drug in various biological compartments (blood, tissues, urine, etc.); however, in case of bioactive molecules with a narrow therapeutic index (i.e. digoxin, cyclosporine, sirolimus, theophylline, warfarin, lithium, phenytoin, and flecainide) or with very short plasmatic half-life (i.e. 5-fluorouracil (5-FU), acetylcholine, GABA, catecholamines, adenosine, and NO) repeated administration could lead to elevated risks or

Several comprehensive reviews have been published concerning the use of MIPs in general as DDS for controlled/sustained drug release or as intelligent drug delivery (DD) platforms (responsive release systems) either for oral, ocular, transdermal, or implant-associated local

Targeted DD relies on the MIP's ability to specifically recognize certain bioreceptors, such as a cell surface epitopes, which could further convey to cellular internalization of the drug loaded carrier and subsequent release of the active pharmaceutical compound. In the initial and most simple approaches the payload of biologically active molecule was non-covalently bound (hydrogen bonding, hydrophobic interactions, charge transfer, or van der Waals forces) to the imprinted polymer network [109]. Nevertheless, the overall controllability and reliability of DDS based on noncovalent binding might not be ideal in a living organism. Therefore, as an alternative, metal ion-mediated coordinate bonds between the functional monomer and the targeted drug molecule (template) has been investigated offering higher specificity and strength, as well as spatial directionality in comparison with noncovalent bonding. Additionally, metal coordination bonds are more compatible with the polar environment of living tissues and they can be easily manipulated through changes of the local hydrogen ion concentration, a feature extremely helpful in the development of pH-responsive delivery systems. Furthermore, MIPs prepared by noncovalent imprinting methods usually require using organic solvents, which

Some of the imprinted polymers employed nowadays in intelligent DD [i.e. poly(2-hydroxyethyl methacrylate, (PHEMA)] were initially employed in the early forms of the non-imprinted DDS [98]. Various aspects about the encountered recognition and drug release mechanisms, optimization of the drug loading capacity, latest trends in various routes of DD, as well as

drug vectors [99].

14 Ligand

severity of toxic side effects.

delivery routes of the therapeutic agent [99–101, 104–108].

eventually leave toxic traces, incompatible with biomedical applications.

A wide range of biocompatible semi-synthetic and synthetic polymers have been tested as suitable imprinted frameworks for DD. PHEMA and its derivatives or nanocomposites continues to be one of the most widely used biomaterials due to their low toxicity, excellent and long-term biocompatibility (including hemocompatibility) and high resistance to degradation [111, 112].

Such molecularly imprinted biomaterials served for the fabrication of various drug-delivery systems, such as transdermal membranes, [113] ocular inserts [114, 115], and implants (subcutaneous, intra-peritoneal, etc.) [116].

Polymer biodegradability plays also an important role in the biomedical exploitation, patient compliance and safe use of such DD systems. Because PHEMA is not biodegradable, upon the release of the pharmacologically active load the implants must be removed from the body through minor surgery to avoid the formation of pseudocyst. However, in many cases, such as the localized treatment of spinal cord injuries, the use of hydrolytically degradable hydrogel implants is far more convenient. *In vivo* experiments showed that macroporous 2-ethoxyethyl methacrylate/ N-(2-hydroxypropyl) methacrylamide based hydrolysable hydrogels (adjustable degradation between 2 and 40 days) are promising candidates for implantation into tissue defects of the central nervous system [117].

Although metal ion coordination-based imprinting has shown promise in the creation of advanced recognition and DD systems up until now, literature is rather scarce in such studies. Nevertheless, there are some noteworthy publications in this field, such as the one reporting the sustained release (5 days) of copper salicylate, a metal-based nonsteroidal anti-inflammatory drug, successfully embedded in a metal chelate imprinted polymer using 4-VPy and 2-hydroxyethyl methacrylate (HEMA) as functional monomers and EDMA as crosslinker [118]. Another interesting study is the synthesis of Co(II) mediated imprinted hydrogels containing pendent chain linked template (drug) [119]. A pH responsive drug release could be achieved in the range of pH 3–6.8 due to the presence of an imidazole group within the proximity of the polymer-drug (ester or amide) bond responsible of the catalytic hydrolysis of the hydrogel.

Design of dedicated macromolecular architectures through MI able to recognize certain target molecules as well as capable of intelligent DD and release leads to the introduction of feedback-controlled drug release systems employing stimuli-responsive gel systems. As a result of oscillatory swelling, they are able to modulate release in response to pH, temperature, ionic strength, electric fields, or specific analyte concentration differences [101, 104]. The solvation of the hydrogel's macromolecular network is rather well adjustable by the local environment, this leading to a controlled swelling and release of the payload.

The inherent advantages offered by metal pivot-based MI have been successfully exploited in the imprinting process of hydrogels intended for stimuli-responsive DDS. The formation and cleavage of coordination bonds between different metal ions and various drugs of interest are pH-dependent, so by a rational design they could be specifically engineered for an intended use. As such, metal-mediated imprinting of HEMA-based hydrogel backbone crosslinked with N, N-methylenebisacrylamide (MBA) has been described for the pH-responsive and controlled release of doxorubicine (within 7 days 60% of the drug released at pH 5.0 vs. 10% at pH 7.2). The anticancer drug was loaded onto the hydrogel as a preassembled Cu(II) ion bridged complex of doxorubicin (1,2 molar ratio) as template and 4-VPy as functional monomer [120]. Although by a slightly different approach, Liang et al. reported the encapsulation in self-assembled biodegradable zein/carboxymethyl chitosan (CMCS) nanoparticles of the same anticancer drug by electrostatic interactions [121]. The nanoparticles were additionally coated by a thin layer of metal-tannic acid layer, where the metal ions (Cu(II), Ca(II)) act as stimuli-responsive crosslinking agents, controlling the release of the guest molecule.

A salicylaldiminato Co(III)-based catalyst was used for the preparation of Cibacron-reactive-reddye-imprinted MIP with tert-butyl acrylate as a functional monomer and DVB as a crosslinker. Methyl aluminoxane activated the transition-metal coordination compound, which catalyzed the polymerization of tert-butyl acrylate, and high-molar-mass polymers with very low molecular weight distributions were generated, even in the presence of the polar dye. The obtained MIP was used for the selective rebinding and preconcentration of the red dye from tap water and textiles [129]. Co(II)- and Ni(II)-imprinted hydrogel catalyst were able to significantly improve

and NH<sup>3</sup>

, Andreea Elena Bodoki2

\*Address all correspondence to: bodokie@umfcluj.ro

BH<sup>3</sup> in H2

[130]. Rare earth metal ions (Y(III), Ce(III), Nd(III), and La(III)) as doping ions were immobilized

mers (o-phenylenediamine, m-phenylenediamine), the photocatalytic activity was demonstrated on tetracycline degradation (up to 78.80%) in simulated wastewaters under visible light irradiation [131]. Last but not least, as a more stable alternative to the natural enzyme phosphotriesterase (hydrolysis of organophosphotriester pesticides), a MIP was synthesized using a paraoxon analog as template and Co(II)–imidazole coordination compound mimicking the catalytic center of the enzyme. Polymers containing the Co(II)–imidazole coordination compound showed a 20-fold higher hydrolytic activity in comparison with polymers containing only imidazole or a solution containing only Co(II) ions. Additionally, the MIP synthesized using the paraoxon analog as template showed higher paraoxon hydrolysis activity than the control NIP [132].

Work supported by the University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj-Napoca, internal grant no. 4944/11/08.03.2016 and internal grant no. 4945/19/08.03.2016.

1 Analytical Chemistry Department, "Iuliu Hațieganu" University of Medicine and

2 General and Inorganic Chemistry Department, "Iuliu Hațieganu" University of Medicine

[1] Iacob B-C, Bodoki E, Oprean R. Chiral electrochemical sensors based on molecularly imprinted polymers with pharmaceutical applications. In: Handbook of Sustainable

Polymers. Boca Raton: Pan Stanford; 2015. pp. 587-614. DOI: 10.1201/b19600-18

, Luminița Oprean<sup>2</sup>

production (total hydrolysis in 50 s at 60°C)

Metal–Ligand Interactions in Molecular Imprinting http://dx.doi.org/10.5772/intechopen.73407 17

and Ede Bodoki<sup>1</sup>

\*

Halloysite. Using two aniline derivatives as mono-

the hydrolysis kinetics of NaBH4

**Acknowledgements**

**Author details**

Bogdan-Cezar Iacob1

**References**

Pharmacy, Cluj-Napoca, Romania

and Pharmacy, Cluj-Napoca, Romania

by ion-imprinting in photocatalysts on TiO<sup>2</sup>

The intracellular conversion rate of a key anticancer agent, 5-FU, to its biologically active metabolites is very fast in the human body, however more than 80% of the administered pro-drug is inactivated by the liver (6 min plasmatic half-life) [122]. As a solution, various controlled localized DD approaches have been investigated [123, 124]. Nevertheless, the prospects of metal ion-mediated MI technology for the controlled delivery 5-FU has also been exploited by the formation of a metal-chelate complex of N-methacryloyl-L-histidine (MAH) functional comonomer and 5-FU via Cu(II) ion coordination in the prepolymerization step [125]. A free radical polymerization, crosslinking and a cryogenic processing lead to the formation of 5-FU imprinted PHEMA-N-methacryloyl-(L)-histidine methyl ester) cryogel discs, an interesting class of implantable biomaterials, particularly suitable for the controlled delivery of an antineoplastic agent directly to the site of tumor. *In vitro* studies have shown that drug release may be simply controlled by the amount of used crosslinker, whereas the delivery rate of 5-FU is further tuneable (faster at pH 4 vs. 7.4), through the influence of the coordination compound's stability, rendering the metal ion-mediated imprinted polymer pH-responsive [125].

Due to the inherent large surface area of porous metal–organic frameworks (MOFs) and to the excellent gas adsorption capacity of the active metal atoms, such structures have been described for the delivery of bioactive gas molecules, such as NO as an antithrombosis and vasodilation agent [126]. The gas can be stably stored by the covalently unsaturated metal atoms (Co or Ni) from their structure, each able to coordinate to one NO molecule (accumulating up to 7 mmol NO/g of MOF), whereas the bioactive gas is delivered through a water-triggered release. Although other porous MOFs were also described as promising DD systems, where the pharmaco-active payload is stored in a 3D network of nanoscaled cages by guest-host interactions, in the respective case the metal ion is not actively involved in the drug's (i.e. 6-FU) binding or release [127].

## **5. MIPs in catalysis**

The use of MIPs in catalysis has been gaining in interest in recent years thanks to their low cost of manufacturing, good biocompatibility, and recognition properties and excellent stability compared to their bio-analogues such as enzymes. The main objective in this field is to produce MIPs capable of showing enzyme-like activities for reactions for which no enzyme exists, or to improve the performance of the existing catalytic systems [128]. Many natural enzymes contain metal ions capable of specifically coordinate different molecules.

A salicylaldiminato Co(III)-based catalyst was used for the preparation of Cibacron-reactive-reddye-imprinted MIP with tert-butyl acrylate as a functional monomer and DVB as a crosslinker. Methyl aluminoxane activated the transition-metal coordination compound, which catalyzed the polymerization of tert-butyl acrylate, and high-molar-mass polymers with very low molecular weight distributions were generated, even in the presence of the polar dye. The obtained MIP was used for the selective rebinding and preconcentration of the red dye from tap water and textiles [129]. Co(II)- and Ni(II)-imprinted hydrogel catalyst were able to significantly improve the hydrolysis kinetics of NaBH4 and NH<sup>3</sup> BH<sup>3</sup> in H2 production (total hydrolysis in 50 s at 60°C) [130]. Rare earth metal ions (Y(III), Ce(III), Nd(III), and La(III)) as doping ions were immobilized by ion-imprinting in photocatalysts on TiO<sup>2</sup> Halloysite. Using two aniline derivatives as monomers (o-phenylenediamine, m-phenylenediamine), the photocatalytic activity was demonstrated on tetracycline degradation (up to 78.80%) in simulated wastewaters under visible light irradiation [131]. Last but not least, as a more stable alternative to the natural enzyme phosphotriesterase (hydrolysis of organophosphotriester pesticides), a MIP was synthesized using a paraoxon analog as template and Co(II)–imidazole coordination compound mimicking the catalytic center of the enzyme. Polymers containing the Co(II)–imidazole coordination compound showed a 20-fold higher hydrolytic activity in comparison with polymers containing only imidazole or a solution containing only Co(II) ions. Additionally, the MIP synthesized using the paraoxon analog as template showed higher paraoxon hydrolysis activity than the control NIP [132].

## **Acknowledgements**

controlled release of doxorubicine (within 7 days 60% of the drug released at pH 5.0 vs. 10% at pH 7.2). The anticancer drug was loaded onto the hydrogel as a preassembled Cu(II) ion bridged complex of doxorubicin (1,2 molar ratio) as template and 4-VPy as functional monomer [120]. Although by a slightly different approach, Liang et al. reported the encapsulation in self-assembled biodegradable zein/carboxymethyl chitosan (CMCS) nanoparticles of the same anticancer drug by electrostatic interactions [121]. The nanoparticles were additionally coated by a thin layer of metal-tannic acid layer, where the metal ions (Cu(II), Ca(II)) act as

The intracellular conversion rate of a key anticancer agent, 5-FU, to its biologically active metabolites is very fast in the human body, however more than 80% of the administered pro-drug is inactivated by the liver (6 min plasmatic half-life) [122]. As a solution, various controlled localized DD approaches have been investigated [123, 124]. Nevertheless, the prospects of metal ion-mediated MI technology for the controlled delivery 5-FU has also been exploited by the formation of a metal-chelate complex of N-methacryloyl-L-histidine (MAH) functional comonomer and 5-FU via Cu(II) ion coordination in the prepolymerization step [125]. A free radical polymerization, crosslinking and a cryogenic processing lead to the formation of 5-FU imprinted PHEMA-N-methacryloyl-(L)-histidine methyl ester) cryogel discs, an interesting class of implantable biomaterials, particularly suitable for the controlled delivery of an antineoplastic agent directly to the site of tumor. *In vitro* studies have shown that drug release may be simply controlled by the amount of used crosslinker, whereas the delivery rate of 5-FU is further tuneable (faster at pH 4 vs. 7.4), through the influence of the coordination compound's stability, rendering the metal ion-mediated imprinted polymer

Due to the inherent large surface area of porous metal–organic frameworks (MOFs) and to the excellent gas adsorption capacity of the active metal atoms, such structures have been described for the delivery of bioactive gas molecules, such as NO as an antithrombosis and vasodilation agent [126]. The gas can be stably stored by the covalently unsaturated metal atoms (Co or Ni) from their structure, each able to coordinate to one NO molecule (accumulating up to 7 mmol NO/g of MOF), whereas the bioactive gas is delivered through a water-triggered release. Although other porous MOFs were also described as promising DD systems, where the pharmaco-active payload is stored in a 3D network of nanoscaled cages by guest-host interactions, in the respective case the metal ion is not actively involved in the

The use of MIPs in catalysis has been gaining in interest in recent years thanks to their low cost of manufacturing, good biocompatibility, and recognition properties and excellent stability compared to their bio-analogues such as enzymes. The main objective in this field is to produce MIPs capable of showing enzyme-like activities for reactions for which no enzyme exists, or to improve the performance of the existing catalytic systems [128]. Many natural

enzymes contain metal ions capable of specifically coordinate different molecules.

stimuli-responsive crosslinking agents, controlling the release of the guest molecule.

pH-responsive [125].

16 Ligand

**5. MIPs in catalysis**

drug's (i.e. 6-FU) binding or release [127].

Work supported by the University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj-Napoca, internal grant no. 4944/11/08.03.2016 and internal grant no. 4945/19/08.03.2016.

## **Author details**

Bogdan-Cezar Iacob1 , Andreea Elena Bodoki2 , Luminița Oprean<sup>2</sup> and Ede Bodoki<sup>1</sup> \*

\*Address all correspondence to: bodokie@umfcluj.ro

1 Analytical Chemistry Department, "Iuliu Hațieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania

2 General and Inorganic Chemistry Department, "Iuliu Hațieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania

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**Chapter 2**

**Provisional chapter**

**Versatile Silylphosphine Ligands for Transition Metal**

**Versatile Silylphosphine Ligands for Transition Metal** 

In this chapter, a review throughout the literature on the chemistry of multidentate silylphosphines is presented. The incorporation of P and Si functionalities in cooperation in a single ligand backbone is exceptionally versatile, and examples of this rich chemistry stemming from the works of many research groups around the world are herein provided. The ligand systems can be flexible or rigid and incorporate varying numbers of P, Si and even other atoms. Exceptional ligand-metal systems are discussed in terms of their

**Keywords:** silicon, phosphorous, silylphosphines, transition metals, multidentate

In modern Coordination and Organometallic Chemistry, ligand design is recognised as crucial for the development of efficient and selective complexes for important transformations including medicinal chemistry, material science and catalysis. Polydentate-rigid or semi-rigid ligands constrain the geometry at the metal centre providing inherently well-defined coordination geometries for potential incoming substrates. Indeed, a good number of these metal-ligand systems are capable of performing selectively difficult activations and many research groups around the world have directed their endeavours to the study of their chemical properties.

The incorporation of dual functionalities in a single ligand backbone has also been shown to modify the properties of the compounds making them especially prone to undergo selective

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.73502

Julio Zamora-Moreno and Virginia Montiel-Palma

Julio Zamora-Moreno and Virginia Montiel-PalmaAdditional information is available at the end of the chapter

structure, reactivity and, in some cases, catalytic activity.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73502

**Complexation**

**Abstract**

ligands

**1. Introduction**

**Complexation**


#### **Versatile Silylphosphine Ligands for Transition Metal Complexation Versatile Silylphosphine Ligands for Transition Metal Complexation**

DOI: 10.5772/intechopen.73502

Julio Zamora-Moreno and Virginia Montiel-Palma Julio Zamora-Moreno and Virginia Montiel-Palma

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73502

**Abstract**

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(95)00401-7

28 Ligand

app.32072

clay.2014.06.021

209-214. DOI: 10.1016/S0003-2670(01)00933-3

In this chapter, a review throughout the literature on the chemistry of multidentate silylphosphines is presented. The incorporation of P and Si functionalities in cooperation in a single ligand backbone is exceptionally versatile, and examples of this rich chemistry stemming from the works of many research groups around the world are herein provided. The ligand systems can be flexible or rigid and incorporate varying numbers of P, Si and even other atoms. Exceptional ligand-metal systems are discussed in terms of their structure, reactivity and, in some cases, catalytic activity.

**Keywords:** silicon, phosphorous, silylphosphines, transition metals, multidentate ligands

## **1. Introduction**

In modern Coordination and Organometallic Chemistry, ligand design is recognised as crucial for the development of efficient and selective complexes for important transformations including medicinal chemistry, material science and catalysis. Polydentate-rigid or semi-rigid ligands constrain the geometry at the metal centre providing inherently well-defined coordination geometries for potential incoming substrates. Indeed, a good number of these metal-ligand systems are capable of performing selectively difficult activations and many research groups around the world have directed their endeavours to the study of their chemical properties.

The incorporation of dual functionalities in a single ligand backbone has also been shown to modify the properties of the compounds making them especially prone to undergo selective

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

transformations resulting from differing reactivity of the coordinating atoms in the ligand. A wide variety of combinations of donor atoms have been employed to date, including for example, soft and hard donor atoms in what is known as hemilabile ligands.

In this chapter, the chemistry related to silylphosphine ligands which include in their structure both a basic P as well as a Si is reviewed. Si derivatives are exceptionally good sigma donors and exert a considerably high *trans*-influence/effect, thus their coordination generates electron rich metal centres in turn capable of activating otherwise inert substrates. Phosphines have long been preferred ligands due to their ability to tune their steric and electronic properties depending on the substituents on P. The incorporation of P and Si in a ligand framework also allows for the employment of NMR spectroscopic tools deriving from 31P and 29Si nuclei.

#### **2. Silylphosphine ligands: definition, general structure and bonding**

Silylphosphines can be described as bi- or polydentate ligands bearing at least one basic phosphorous (III) atom, usually a phosphine PR3 or phosphite P(OR)3 , and at least one silicon-substituted moiety. The P (III) group is able to form a coordination bond to the transition metal, while the silyl

the P and Si atoms, there are generally a number of carbon atoms in the form of an alkyl or aryl bridges (**Scheme 1**). Else a direct P─Si bond can be established. Silylphosphines are potentially bi-, tri- or polydentate ligands, the coordination number depending on the number of P or Si

Therefore, the molecular orbitals can be described as those of the phosphine and silicon donor moieties. For example, for the non-classical bidentate coordination mode, the frontier orbitals are shown schematically in **Figure 1**. A bidentate *P*, *Si* ligand can readily coordinate to the metal centre both through the phosphorous atom via the donation of the electron lone pair on P to an empty d-orbital on the metal and through the σ-Si─H electron pair donated to a suitable empty d-orbital on the metal generating a 3c-2e non-classical bond. In both bonds, the stabilisation is given by the retro-donation of electron density of a filled d-orbital to an anti-bonding orbital. In the full oxidative addition process of the Si─H bond to the metal, due to the strong retro-donation of the d-orbital → σ\*(Si─H), the final product results in the formation of two 2c-2e bonds: M─H, M─Si. As expected, depending on the substituents on both the P and Si atoms, the molecular orbital diagrams and the energy of the HOMO and LUMO will vary. In general, it could be said according to **Figure 1**, the HOMO generally pos-

, alkane or arene molecules. Between

Versatile Silylphosphine Ligands for Transition Metal Complexation

http://dx.doi.org/10.5772/intechopen.73502

31

moiety is potentially prone to bind by means of loss of H2

**Figure 1.** Molecular orbital diagrams of the phosphine and non-classical Si─H sigma moieties.

sess a higher ligand character, while the LUMO is more metal centred.

**3. Silylphosphine ligands throughout the chemical literature: a** 

Stobart and co-workers pioneered the systematic study of transition metals bound to silylphosphine ligands. As early as in 1983, they reported the synthesis and full characteri-

moieties present in the ligand backbone.

**review**

**Scheme 1.** General structure of silylphosphine ligands and examples of main coordination modes.

Versatile Silylphosphine Ligands for Transition Metal Complexation http://dx.doi.org/10.5772/intechopen.73502 31

**Figure 1.** Molecular orbital diagrams of the phosphine and non-classical Si─H sigma moieties.

transformations resulting from differing reactivity of the coordinating atoms in the ligand. A wide variety of combinations of donor atoms have been employed to date, including for

In this chapter, the chemistry related to silylphosphine ligands which include in their structure both a basic P as well as a Si is reviewed. Si derivatives are exceptionally good sigma donors and exert a considerably high *trans*-influence/effect, thus their coordination generates electron rich metal centres in turn capable of activating otherwise inert substrates. Phosphines have long been preferred ligands due to their ability to tune their steric and electronic properties depending on the substituents on P. The incorporation of P and Si in a ligand framework also allows for the employment of NMR spectroscopic tools deriving

**2. Silylphosphine ligands: definition, general structure and bonding**

**Scheme 1.** General structure of silylphosphine ligands and examples of main coordination modes.

Silylphosphines can be described as bi- or polydentate ligands bearing at least one basic phospho-

or phosphite P(OR)3

, and at least one silicon-substituted

example, soft and hard donor atoms in what is known as hemilabile ligands.

from 31P and 29Si nuclei.

30 Ligand

rous (III) atom, usually a phosphine PR3

moiety. The P (III) group is able to form a coordination bond to the transition metal, while the silyl moiety is potentially prone to bind by means of loss of H2 , alkane or arene molecules. Between the P and Si atoms, there are generally a number of carbon atoms in the form of an alkyl or aryl bridges (**Scheme 1**). Else a direct P─Si bond can be established. Silylphosphines are potentially bi-, tri- or polydentate ligands, the coordination number depending on the number of P or Si moieties present in the ligand backbone.

Therefore, the molecular orbitals can be described as those of the phosphine and silicon donor moieties. For example, for the non-classical bidentate coordination mode, the frontier orbitals are shown schematically in **Figure 1**. A bidentate *P*, *Si* ligand can readily coordinate to the metal centre both through the phosphorous atom via the donation of the electron lone pair on P to an empty d-orbital on the metal and through the σ-Si─H electron pair donated to a suitable empty d-orbital on the metal generating a 3c-2e non-classical bond. In both bonds, the stabilisation is given by the retro-donation of electron density of a filled d-orbital to an anti-bonding orbital. In the full oxidative addition process of the Si─H bond to the metal, due to the strong retro-donation of the d-orbital → σ\*(Si─H), the final product results in the formation of two 2c-2e bonds: M─H, M─Si. As expected, depending on the substituents on both the P and Si atoms, the molecular orbital diagrams and the energy of the HOMO and LUMO will vary. In general, it could be said according to **Figure 1**, the HOMO generally possess a higher ligand character, while the LUMO is more metal centred.

## **3. Silylphosphine ligands throughout the chemical literature: a review**

Stobart and co-workers pioneered the systematic study of transition metals bound to silylphosphine ligands. As early as in 1983, they reported the synthesis and full characterisation of an extensive family of silanes modified with a phosphorous fragment connected to the silicon atom by a polymethylene chain, of general formula (XYZ)Si(CH2 )nPR2 (where X, Y, Z = Me, Ph, Cl or H; n = 1–3; R = Me or Ph;) (**Figure 2**, compounds **1–33**) [1]. The reactivity of Vaska's complex *trans*-[Ir(PPh3 )2 (CO)(Cl)] towards Ph2 P(CH2 )2 SiRR'H (compounds: **12**, **14**, **16**, **18**, **20**) was also investigated. The reaction results in the formation of air stable six-coordinated Ir(III) compounds, resulting from coordination of the ligands through the P atom and of the oxidative addition of the Si─H bond (compounds: **34**–**38**) (**Figure 3**). Furthermore, the reactions of Ph2 P(CH2 )2 SiMe2 H towards the dimers [M(μ-Cl) (COD)]2 (M = Rh, Ir; COD = 1, 5-cyclooctadiene), also afford the M(III) complexes [MCl{Ph<sup>2</sup> P(CH2 )2 SiMe2 }2 ] (M = Rh **39**; Ir **40**) which are quiral with the two phosphorous atoms in *trans* disposition while the two Si dispose in a *cis* fashion (**Figure 3**) [2]. The fact that the reactivity of complexes **39** and **40** was remarkably constrained due to the *trans*-labilising effect of the silyl groups, was exploited in their use as catalysts for transformations of organic substrates [3]. Several works reported in the literature have argued on the high extent of *trans*-influence silyl groups exercise on a transition metal centre. There are various reasons for this behaviour including an excellent sigma orbital overlap as well as a favourable electronic release of the Si [4, 5]. This is in agreement with only a few compounds exhibiting a *trans* coordination of the Si atoms in many cases as kinetic products in equilibria with their *cis* isomers [6, 7] even when employing chelating silylphosphines (*vide supra*) [8–11] (Section 8).

It was found that the ligands with two or three phosphorous atoms and a Si-H bond (compounds **41**-**52**, **Figure 4**) coordinate via oxidative addition to the metal centre (i.e. rhodium, iridium, ruthenium and platinum) and impose steric constraints on the coordination sphere in turn restraining substrate entry to sites which could suffer the strongly labilising *trans* effect of the silyl group, increasing the complexes' capabilities as catalysts [12] (**Figure 4**).

**4. Silylphosphines complexation in tetra-coordinated systems**

its reactivity with transition metal precursors. The complexes [MCl{(*ο*-C6

SiMe}] (**61**) which regenerates complex **65**, upon reaction with Ph2

phine chemistry. They introduced a *PSiP*-type ligand [(*ο*-C*<sup>6</sup>*

**Figure 4.** Design of poly(phosphino)-silane ligands reported in Ref. [12].

**Figure 3.** The reactivity of group 9 metals as reported in Ref. [2].

] provides complex [Pd{(*ο*-C6

SiMe}(SiHPh2

SiMe}] (**60)** and complex [Ni{(*ο*-C6

Turculet and co-workers have further made significant contributions in the field of silylphos-

(M = Ni or Pd) (**64**, **65**) were treated with alkyl lithium or Grignard reagents. In the case of **65** treatment with stoichiometric amounts of MeLi led to the formation of [Pd(Me){(*ο*-C6

)] (**66**) from reaction with Ph2

) bond activation. Treatment of complex **64** with MeMgBr led to [Ni(Me){(*ο*-C6

H4 -PCy2

H4 -PCy2 H*4* -PCy*<sup>2</sup>* )*2* SiMe*<sup>2</sup>*

)SiMe2

)SiMe2

SiH2

Versatile Silylphosphine Ligands for Transition Metal Complexation

http://dx.doi.org/10.5772/intechopen.73502

33

}(*ο*-C6 H4 -PCy2

}(*ο*-C6 H4 -PCy2 ] (**53**) and explored

SiClH while renders

)] (**63**) derived from

)] (**62**) resulting from

. The direct reaction of **53**

H4 -

H4 -

H4 -PCy2 )2 SiMe}]

**4.1. Square-planar geometry**

PCy2 )2

[Pd{(*ο*-C6

and [Pd2

Si-C(sp2

PCy2 ) 2 H4 -PCy2 )2

(dba)3

**Figure 2.** The silylphosphine ligands bearing alkyl bridges reported in Ref. [1].

**Figure 3.** The reactivity of group 9 metals as reported in Ref. [2].

sation of an extensive family of silanes modified with a phosphorous fragment connected

X, Y, Z = Me, Ph, Cl or H; n = 1–3; R = Me or Ph;) (**Figure 2**, compounds **1–33**) [1]. The

)2

pounds: **12**, **14**, **16**, **18**, **20**) was also investigated. The reaction results in the formation of air stable six-coordinated Ir(III) compounds, resulting from coordination of the ligands through the P atom and of the oxidative addition of the Si─H bond (compounds: **34**–**38**)

disposition while the two Si dispose in a *cis* fashion (**Figure 3**) [2]. The fact that the reactivity of complexes **39** and **40** was remarkably constrained due to the *trans*-labilising effect of the silyl groups, was exploited in their use as catalysts for transformations of organic substrates [3]. Several works reported in the literature have argued on the high extent of *trans*-influence silyl groups exercise on a transition metal centre. There are various reasons for this behaviour including an excellent sigma orbital overlap as well as a favourable electronic release of the Si [4, 5]. This is in agreement with only a few compounds exhibiting a *trans* coordination of the Si atoms in many cases as kinetic products in equilibria with their *cis* isomers [6, 7] even when employing chelating silylphosphines (*vide supra*) [8–11]

It was found that the ligands with two or three phosphorous atoms and a Si-H bond (compounds **41**-**52**, **Figure 4**) coordinate via oxidative addition to the metal centre (i.e. rhodium, iridium, ruthenium and platinum) and impose steric constraints on the coordination sphere in turn restraining substrate entry to sites which could suffer the strongly labilising *trans* effect of the silyl group, increasing the complexes' capabilities as catalysts [12]

P(CH2 )2 SiMe2

(M = Rh, Ir; COD = 1, 5-cyclooctadiene), also afford the M(III) complexes [MCl{Ph<sup>2</sup>

] (M = Rh **39**; Ir **40**) which are quiral with the two phosphorous atoms in *trans*

(CO)(Cl)] towards Ph2

)nPR2

SiRR'H (com-

P(CH2 )2

H towards the dimers [M(μ-Cl)

(where

to the silicon atom by a polymethylene chain, of general formula (XYZ)Si(CH2

reactivity of Vaska's complex *trans*-[Ir(PPh3

(**Figure 3**). Furthermore, the reactions of Ph2

**Figure 2.** The silylphosphine ligands bearing alkyl bridges reported in Ref. [1].

(COD)]2

(Section 8).

(**Figure 4**).

P(CH2 )2 SiMe2 }2

32 Ligand

**Figure 4.** Design of poly(phosphino)-silane ligands reported in Ref. [12].

## **4. Silylphosphines complexation in tetra-coordinated systems**

#### **4.1. Square-planar geometry**

Turculet and co-workers have further made significant contributions in the field of silylphosphine chemistry. They introduced a *PSiP*-type ligand [(*ο*-C*<sup>6</sup>* H*4* -PCy*<sup>2</sup>* )*2* SiMe*<sup>2</sup>* ] (**53**) and explored its reactivity with transition metal precursors. The complexes [MCl{(*ο*-C6 H4 -PCy2 )2 SiMe}] (M = Ni or Pd) (**64**, **65**) were treated with alkyl lithium or Grignard reagents. In the case of **65** treatment with stoichiometric amounts of MeLi led to the formation of [Pd(Me){(*ο*-C6 H4 - PCy2 )2 SiMe}] (**61**) which regenerates complex **65**, upon reaction with Ph2 SiClH while renders [Pd{(*ο*-C6 H4 -PCy2 )2 SiMe}(SiHPh2 )] (**66**) from reaction with Ph2 SiH2 . The direct reaction of **53** and [Pd2 (dba)3 ] provides complex [Pd{(*ο*-C6 H4 -PCy2 )SiMe2 }(*ο*-C6 H4 -PCy2 )] (**63**) derived from Si-C(sp2 ) bond activation. Treatment of complex **64** with MeMgBr led to [Ni(Me){(*ο*-C6 H4 - PCy2 ) 2 SiMe}] (**60)** and complex [Ni{(*ο*-C6 H4 -PCy2 )SiMe2 }(*ο*-C6 H4 -PCy2 )] (**62**) resulting from ligand rearrangement. Complexes **62** (Ni) and **63** (Pd) constitute rare examples of reversible Si-C(sp2 ) and Si-C(sp3 ) bond cleavage (**Figure 5**) [13, 14].

Interestingly, changes on the identity of the substituents on the P atoms in the *PSiP* ligand backbone bring about a great strategy for the coordination of ligand **55** towards group 9 metals, in particularly rendering an Ir system able to activate intermolecular arene C─H bonds. Indeed, the

(**77**); M = Ir, L = C2

) 4

] led to the formation of the Ni(0) complex [Ni{*η*<sup>2</sup>

**Figure 6.** Reactivity of **54**. Tetra-coordination is highlighted in blue and penta-coordinated in purple [21-23, 26-29].

kept in solution because the observed NMR parameters such as coupling constants *J*

*J*

) 4

)] (**81**). The Si─H, Ni─H and Ni─Si distances are 1.62(3), 1.44(2) and 2.2782(4) Å respectively, suggesting that the Si─H bond was preserved. This non-classical complexation mode is

H4 -PCy2 ) 2

able to subsequently coordinate neutral ligands, thus generating [M(L){(*ο*-C6

(M = Rh, Ir; coe = 1-cyclooctadiene) or [RhCl(PPh3

] or [CpPd(C3

] is discussed in Section 5.2 [22].

H4

)] (**82**) instead of the hypothesised hydride complex **68** which was pro-

) 3

H4 -PCy2 ) 2 SiMe}]

)] yielding complex [Pd{*η*<sup>2</sup>

SiMe}] (M = Rh, **74**; Ir, **75**), which react with

http://dx.doi.org/10.5772/intechopen.73502

(**78**); M = Ir, L = PMe3

H4 -PPh2 ) 2

> -(*ο*-C6 H4 -PPh2 ) 2 SiHMe}

to afford also **82** [21]. Likewise, the reaction of ligand

SiH ≤ 20 Hz for a complete Si─H bond cleavage) and in


Li] forming neutral three-coordinate intermediate species

Versatile Silylphosphine Ligands for Transition Metal Complexation

H5

] produced

35

(**79**); M = Rh,

SiMe}] (**71**, see

SiH = 89 Hz


reaction of **55** and [MCl(coe)2

organolithium compound [Me3

(M = Rh, L = H2

L = PMe3

C6 H4 -PPh2 ) 2

(PPh3

the monomeric complexes [MH(Cl){(*ο*-C6

(**80**)) (**Figure 7**) [20].

SiHMe}(PPh3

) 4

at 300 K and 77 Hz at 193 K are large (2

line with the conservation of the *η*<sup>2</sup>

hand, the mixture of **54** with [Pt(PPh3

**Figure 5**) with LiHBEt3

**54** with [Ni(PPh3

] 2

NPh (**76**); M = Rh, L = NH3

**4.2. Tetrahedral and trigonal pyramidal geometries**

Ligand **54** (see **Figure 5**) reacted with [Pd(PPh3

SiCH2

posed as the intermediate in the reduction of compound [PdCl{(*ο*-C6

in presence of PPh3

Moreover, the reactivity of **54** towards [PtCl2 (SEt2 )2 ] leads to the generation of [PtCl{(*ο*-C6 H4 -PPh2 )2 SiMe}] (**67**) where the ligand coordination results in adoption of a distorted square planar geometry around Pt with a persistent Cl atom bonded *trans* to the silyl group (**Figure 5**) [9].

Iwasawa and collaborators reported an interesting system for the catalytic hydrocarboxylation of allenes using the Pd(II) hydride complex [PdH{(*ο*-C6 H4 -PPh2 ) 2 SiMe}] (**68**) as the active catalyst. Their methodology also served for the synthesis of β,γ-unsaturated carboxylic acids. In general, complex [Pd(OTf){(*ο*-C6 H4 -PPh2 ) 2 SiMe}] (**69**) in catalytic ratios 1.0 mol% or 2.5 mol % was used in soft conditions of CO2 pressure (1 atm) with 150 mol% of AlEt3 or ZnEt2 for carboxylation of 1,1-disubstituted, monosubstituted or disubstituted allenes to the respective carboxylic acid or ester [15]. In addition, the chemical properties of complex **69** were described as well. To mention some, **69** undergoes transmetalation with AlEt3 followed by β-hydride elimination to generate the proposed complex **68**. Complex **69** reacted with an excess of B2 pin2 at room temperature leading to HBpin and the monoborylpalladium complex [Pd(Bpin){(*ο*-C6 H4 -PPh2 ) 2 SiMe}] (**70**) which promotes the product of borylation of styrene as well as other alkenes (**Figure 5**) [16–18].

Milstein and co-workers described the design and synthesis of the first pincer-type silanol-Pt(II) compound by using a *PSiP* ligand. The ligand {(*ο*-C6 H4 -P*i*Pr2 )2 SiH2 } (**58**) was obtained in moderate yields from the *o*-bromophosphine. It readily reacts with [(Me2 S)2 Pt(Me)Cl] at room temperature to give the bicyclic complex [PtCl{(*ο*-C6 H4 -P*i*Pr2 ) 2 SiH}] (**72**), which then undergoes autoxidation yielding the silanol complex [PtCl{(*ο*-C6 H4 -P*i*Pr2 )2 Si(OH)}] (**73**) in moderate yields (65%) (**Figure 5**) [19].

**Figure 5.** [PSiP] ligands and their square planar group 10 metal complexes [13-19, 26, 59].

Interestingly, changes on the identity of the substituents on the P atoms in the *PSiP* ligand backbone bring about a great strategy for the coordination of ligand **55** towards group 9 metals, in particularly rendering an Ir system able to activate intermolecular arene C─H bonds. Indeed, the reaction of **55** and [MCl(coe)2 ] 2 (M = Rh, Ir; coe = 1-cyclooctadiene) or [RhCl(PPh3 ) 3 ] produced the monomeric complexes [MH(Cl){(*ο*-C6 H4 -PCy2 ) 2 SiMe}] (M = Rh, **74**; Ir, **75**), which react with organolithium compound [Me3 SiCH2 Li] forming neutral three-coordinate intermediate species able to subsequently coordinate neutral ligands, thus generating [M(L){(*ο*-C6 H4 -PCy2 ) 2 SiMe}] (M = Rh, L = H2 NPh (**76**); M = Rh, L = NH3 (**77**); M = Ir, L = C2 H4 (**78**); M = Ir, L = PMe3 (**79**); M = Rh, L = PMe3 (**80**)) (**Figure 7**) [20].

#### **4.2. Tetrahedral and trigonal pyramidal geometries**

ligand rearrangement. Complexes **62** (Ni) and **63** (Pd) constitute rare examples of reversible

square planar geometry around Pt with a persistent Cl atom bonded *trans* to the silyl group

Iwasawa and collaborators reported an interesting system for the catalytic hydrocarboxylation

Their methodology also served for the synthesis of β,γ-unsaturated carboxylic acids. In general,

1,1-disubstituted, monosubstituted or disubstituted allenes to the respective carboxylic acid or ester [15]. In addition, the chemical properties of complex **69** were described as well. To mention

which promotes the product of borylation of styrene as well as other alkenes (**Figure 5**) [16–18]. Milstein and co-workers described the design and synthesis of the first pincer-type silanol-

pressure (1 atm) with 150 mol% of AlEt3

(SEt2 )2

SiMe}] (**67**) where the ligand coordination results in adoption of a distorted

H4 -PPh2 ) 2

SiMe}] (**69**) in catalytic ratios 1.0 mol% or 2.5 mol % was used

H4 -P*i*Pr2 )2 SiH2

H4 -P*i*Pr2 ) 2

H4 -P*i*Pr2 ) 2 or ZnEt2

followed by β-hydride elimination to generate

pin2

H4 -PPh2 ) 2

S)2

SiH}] (**72**), which then under-

Si(OH)}] (**73**) in moderate

] leads to the generation of [PtCl{(*ο*-

SiMe}] (**68**) as the active catalyst.

for carboxylation of

at room temperature

} (**58**) was obtained in

Pt(Me)Cl] at room

SiMe}] (**70**)

) bond cleavage (**Figure 5**) [13, 14].

Si-C(sp2

(**Figure 5**) [9].

complex [Pd(OTf){(*ο*-C6

in soft conditions of CO2

yields (65%) (**Figure 5**) [19].

C6 H4 -PPh2 )2

34 Ligand

) and Si-C(sp3

Moreover, the reactivity of **54** towards [PtCl2

of allenes using the Pd(II) hydride complex [PdH{(*ο*-C6

H4 -PPh2 ) 2

some, **69** undergoes transmetalation with AlEt3

the proposed complex **68**. Complex **69** reacted with an excess of B2

Pt(II) compound by using a *PSiP* ligand. The ligand {(*ο*-C6

goes autoxidation yielding the silanol complex [PtCl{(*ο*-C6

temperature to give the bicyclic complex [PtCl{(*ο*-C6

leading to HBpin and the monoborylpalladium complex [Pd(Bpin){(*ο*-C6

moderate yields from the *o*-bromophosphine. It readily reacts with [(Me2

**Figure 5.** [PSiP] ligands and their square planar group 10 metal complexes [13-19, 26, 59].

Ligand **54** (see **Figure 5**) reacted with [Pd(PPh3 ) 4 ] or [CpPd(C3 H5 )] yielding complex [Pd{*η*<sup>2</sup> -(*ο*-C6 H4 -PPh2 ) 2 SiHMe}(PPh3 )] (**82**) instead of the hypothesised hydride complex **68** which was proposed as the intermediate in the reduction of compound [PdCl{(*ο*-C6 H4 -PPh2 ) 2 SiMe}] (**71**, see **Figure 5**) with LiHBEt3 in presence of PPh3 to afford also **82** [21]. Likewise, the reaction of ligand **54** with [Ni(PPh3 ) 4 ] led to the formation of the Ni(0) complex [Ni{*η*<sup>2</sup> -(*ο*-C6 H4 -PPh2 ) 2 SiHMe} (PPh3 )] (**81**). The Si─H, Ni─H and Ni─Si distances are 1.62(3), 1.44(2) and 2.2782(4) Å respectively, suggesting that the Si─H bond was preserved. This non-classical complexation mode is kept in solution because the observed NMR parameters such as coupling constants *J* SiH = 89 Hz at 300 K and 77 Hz at 193 K are large (2 *J* SiH ≤ 20 Hz for a complete Si─H bond cleavage) and in line with the conservation of the *η*<sup>2</sup> -Ni(0) structure seen in solid state (**Figure 6**). On the other hand, the mixture of **54** with [Pt(PPh3 ) 4 ] is discussed in Section 5.2 [22].

**Figure 6.** Reactivity of **54**. Tetra-coordination is highlighted in blue and penta-coordinated in purple [21-23, 26-29].

Bourissou and co-workers reported the reactivity of the *PSiP* ligand **54** towards CuCl and AuCl(SMe2 ), which was subsequently followed by a stoichiometric addition of GaCl3 (complexes **84**, **85**). The addition of the gallium halide was envisioned to increase the electrophilicity of the central metal and thus to escalate the strength of non-classical σ-SiH bond interaction at the metal. In complex **84**, the coordination of the ligand occurs through the two phosphorous atoms and a weak sigma interaction Si─H⋯Cu. The spectroscopic evidence as well as computational analyses (geometry optimisations and NBO analyses) are in agreement with weak donation σ-SiH → Cu in combination with a negligible Cu → σ\*SiH backdonation in **84**. Meanwhile in the cationic gold complex **85**, the coordination of **54** took place only through the two phosphorous atoms as any non-classical Si─H bond interaction to the metal was strongly disfavoured as it was found to be by computational means 15.9 kcal/mol (**Figure 6**) [23].

**5. Silylphosphines complexation in penta-coordinated systems**

(*p*-cymene)]2

rendering a binuclear complex that preserves bridging chloride ligands [Ru(μ-Cl){(*ο*-C6

and **55**) was also observed in monomeric Ru systems. Compound **54** reacted with [RuCl2

(**91**). The X-ray diffraction study confirms the coordination of the ligand through the two phosphorous and the silicon atom adopting a facial arrangement with the silyl group occupying the basal position of a distorted square pyramidal geometry around Ru. The Cl atom disposes *trans*

BH to form octahedral-Ru hydride complex **92** in moderate yield (**Figures 6**) [26].

SiMe}(PPh3

Ni(0) **81** and Pd(0) **82** (**Figure 6**), where the Si─H bond is only slightly activated, the Pt(II) hydride complex **93** derives from the complete oxidative addition of the Si─H bond. The crystalline structure displays a trigonal bipyramidal geometry with the silyl group in the apical position in the metal centre. The opposed apical site was taken by the hydride ligand which

via oxidative addition of the Si─H bond. Two isomers: *cis* and *trans* were proposed. Depending on the phosphine choice, the isolation of one isomer was possible through a reversible σ-bond

ligand coordinates each Ru atom through two P, one Si and two Cl atoms in a distorted square pyramidal geometry, in which the silyl group occupies the apical coordination site. This Ru(II)-ligand system brings about stability and selectivity in catalytic transformations including the transfer hydrogenation of ketones. In this case, Li *et al* observed high conversion of the corresponding secondary alcohols for numerous dialkyl, diaryl, and alkyl/aryl ketones,

(**86**), which was exhaustively characterised spectroscopically (**Figure 7**). The

Bu at 80°C [25].

was carried out in the presence of PCy3

Versatile Silylphosphine Ligands for Transition Metal Complexation

http://dx.doi.org/10.5772/intechopen.73502

H4 -PR2 ) 2

] at room temperature led to the generation of five-coordi-

was made, since it could provide a means of accessing Pd(II) hydrides

)] (**94**) showed a slow reverse reaction and was obtained predomi-

derivative, the equilibrium favoured the thermodynamic isomer

and Et3

(PPh3 ) 3 ]

)]

SiMe}(PPh3

SiHMe (R = Ph or Cy; **54**

. Complex **91** reacted with

H4 -PPh2 ) 2

)] (**93**). In contrast with the derivatives of

H of 650 Hz, which is considerably small com-

ligated complex, the kinetic product *cis*-[Pd(Bpin)

)] (**95**) as a major product (**Figure 6**) [27, 28]. In relation

*J*PH = 18.9 Hz) with 195Pt satellites

*J*PtH = 890–1010 Hz); supporting


N

37

H4 -

**5.1. Square pyramidal geometry**

The reactivity of **55** with [RuCl2

employing 0.2 mol% of **86** with 5 mol% of KO*<sup>t</sup>*

**5.2. Trigonal bipyramidal geometry**

exhibiting a measured coupling constant 195Pt-1

pin2

metathesis pathway. In the case of the PMe3

H4 -PPh2 ) 2

SiMe}(PMe3

The mixture of **54** with [Pt(PPh3

nated Pt(II) complex [PtH{(*ο*-C6

)] (**83**) towards B2

nantly. In contrast, for the PPh3

*trans*-[Pd(Bpin){(*ο*-C6

The activation of Si-H bonds in ligands of general formula (*ο*-C6

in the presence of triethylamine as a base affording complex [RuCl{(*ο*-C6

to the silyl group, and the remaining site was occupied by a PPh3

)4

H4 -PPh2 )2

H NMR spectrum revealed a quartet at δ −7.92 ppm (2

the proposal that the hydride is located *trans* to Si atom (**Figure 6**) [22].

A study of the reaction of complex **82** and its related analogue [Pd{*η*<sup>2</sup>

SiMe}(PPh3

pared with some *cis*-H-Pt(II)-Si species previously reported (1

PCy2 ) 2 SiMe}]2

LiEt3

in the 1

(PMe3

{(*ο*-C6 H4 -PPh2 ) 2

Extraordinarily, **86** (described in more detail in Section 5.2) demonstrated to be a suitable precursor for the synthesis of stable 14-electron [Ru(X){(*ο*-C6 H4 -Cy2 ) 2 SiMe}] (X = O*<sup>t</sup>* Bu (**87**), N(SiMe3 ) 2 (**88**); NHPh (**89**); NH(2,6-Me2 C6 H3 ) (**90**)) complexes, donning unusual trigonal pyramidal coordination geometries explained once again by the presence of the strongly σ-donating silyl group in the apical site with the contribution of steric effects of the phosphino substituents in the equatorial plane (**Figure 7**) [24].

**Figure 7.** Reactivity of ligand **55**. Tetra-coordination is highlighted in blue and penta-coordination in purple [20, 24-25, 36].

## **5. Silylphosphines complexation in penta-coordinated systems**

#### **5.1. Square pyramidal geometry**

Bourissou and co-workers reported the reactivity of the *PSiP* ligand **54** towards CuCl and

plexes **84**, **85**). The addition of the gallium halide was envisioned to increase the electrophilicity of the central metal and thus to escalate the strength of non-classical σ-SiH bond interaction at the metal. In complex **84**, the coordination of the ligand occurs through the two phosphorous atoms and a weak sigma interaction Si─H⋯Cu. The spectroscopic evidence as well as computational analyses (geometry optimisations and NBO analyses) are in agreement with weak donation σ-SiH → Cu in combination with a negligible Cu → σ\*SiH backdonation in **84**. Meanwhile in the cationic gold complex **85**, the coordination of **54** took place only through the two phosphorous atoms as any non-classical Si─H bond interaction to the metal was strongly disfavoured as it was found to be by computational means 15.9 kcal/mol

Extraordinarily, **86** (described in more detail in Section 5.2) demonstrated to be a suitable

pyramidal coordination geometries explained once again by the presence of the strongly σ-donating silyl group in the apical site with the contribution of steric effects of the phosphino

**Figure 7.** Reactivity of ligand **55**. Tetra-coordination is highlighted in blue and penta-coordination in purple [20, 24-25, 36].

C6 H3 H4 -Cy2 ) 2

) (**90**)) complexes, donning unusual trigonal

SiMe}] (X = O*<sup>t</sup>*

precursor for the synthesis of stable 14-electron [Ru(X){(*ο*-C6

(**88**); NHPh (**89**); NH(2,6-Me2

substituents in the equatorial plane (**Figure 7**) [24].

), which was subsequently followed by a stoichiometric addition of GaCl3

(com-

Bu (**87**),

AuCl(SMe2

36 Ligand

(**Figure 6**) [23].

N(SiMe3 ) 2 The reactivity of **55** with [RuCl2 (*p*-cymene)]2 was carried out in the presence of PCy3 and Et3 N rendering a binuclear complex that preserves bridging chloride ligands [Ru(μ-Cl){(*ο*-C6 H4 - PCy2 ) 2 SiMe}]2 (**86**), which was exhaustively characterised spectroscopically (**Figure 7**). The ligand coordinates each Ru atom through two P, one Si and two Cl atoms in a distorted square pyramidal geometry, in which the silyl group occupies the apical coordination site. This Ru(II)-ligand system brings about stability and selectivity in catalytic transformations including the transfer hydrogenation of ketones. In this case, Li *et al* observed high conversion of the corresponding secondary alcohols for numerous dialkyl, diaryl, and alkyl/aryl ketones, employing 0.2 mol% of **86** with 5 mol% of KO*<sup>t</sup>* Bu at 80°C [25].

The activation of Si-H bonds in ligands of general formula (*ο*-C6 H4 -PR2 ) 2 SiHMe (R = Ph or Cy; **54** and **55**) was also observed in monomeric Ru systems. Compound **54** reacted with [RuCl2 (PPh3 ) 3 ] in the presence of triethylamine as a base affording complex [RuCl{(*ο*-C6 H4 -PPh2 ) 2 SiMe}(PPh3 )] (**91**). The X-ray diffraction study confirms the coordination of the ligand through the two phosphorous and the silicon atom adopting a facial arrangement with the silyl group occupying the basal position of a distorted square pyramidal geometry around Ru. The Cl atom disposes *trans* to the silyl group, and the remaining site was occupied by a PPh3 . Complex **91** reacted with LiEt3 BH to form octahedral-Ru hydride complex **92** in moderate yield (**Figures 6**) [26].

#### **5.2. Trigonal bipyramidal geometry**

The mixture of **54** with [Pt(PPh3 )4 ] at room temperature led to the generation of five-coordinated Pt(II) complex [PtH{(*ο*-C6 H4 -PPh2 )2 SiMe}(PPh3 )] (**93**). In contrast with the derivatives of Ni(0) **81** and Pd(0) **82** (**Figure 6**), where the Si─H bond is only slightly activated, the Pt(II) hydride complex **93** derives from the complete oxidative addition of the Si─H bond. The crystalline structure displays a trigonal bipyramidal geometry with the silyl group in the apical position in the metal centre. The opposed apical site was taken by the hydride ligand which in the 1 H NMR spectrum revealed a quartet at δ −7.92 ppm (2 *J*PH = 18.9 Hz) with 195Pt satellites exhibiting a measured coupling constant 195Pt-1 H of 650 Hz, which is considerably small compared with some *cis*-H-Pt(II)-Si species previously reported (1 *J*PtH = 890–1010 Hz); supporting the proposal that the hydride is located *trans* to Si atom (**Figure 6**) [22].

A study of the reaction of complex **82** and its related analogue [Pd{*η*<sup>2</sup> -(*ο*-C6 H4 -PPh2 ) 2 SiHMe} (PMe3 )] (**83**) towards B2 pin2 was made, since it could provide a means of accessing Pd(II) hydrides via oxidative addition of the Si─H bond. Two isomers: *cis* and *trans* were proposed. Depending on the phosphine choice, the isolation of one isomer was possible through a reversible σ-bond metathesis pathway. In the case of the PMe3 ligated complex, the kinetic product *cis*-[Pd(Bpin) {(*ο*-C6 H4 -PPh2 ) 2 SiMe}(PMe3 )] (**94**) showed a slow reverse reaction and was obtained predominantly. In contrast, for the PPh3 derivative, the equilibrium favoured the thermodynamic isomer *trans*-[Pd(Bpin){(*ο*-C6 H4 -PPh2 ) 2 SiMe}(PPh3 )] (**95**) as a major product (**Figure 6**) [27, 28]. In relation to unusual bonding modes, Sun and collaborators reported the systematic reactivity of the tridentate ligand **54** towards the low-valent nickel compound [Ni(PMe3 ) 4 ] which induced the formation of Ni(0) complexes [Ni{*η*<sup>2</sup> -(*ο*-C6 H4 -PPh2 ) 2 MeSi-H}(PMe3 )] (**96**, **Figure 6**). Complex **96** did not undergo the oxidative addition process of a Si─H bond even in the presence of independent silanes (Et3 SiH, Ph2 MeSiH). However, the reactivity with chlorosilanes Me3 SiCl or MeHSiCl2 led to the formation of Ni(II) complex [NiCl{(*ο*-C6 H4 -PPh2 ) 2 MeSi}] (**97**). The halogenated products [NiX{(*ο*-C6 H4 -PPh2 ) 2 SiMe}(PMe3 )] (X = Br (**98**); I (**99**)) were easily obtained from reaction with EtBr or MeI of complex **96** (**Figure 6**) [29].

The versatility of ligand **55** was also probed in the coordination towards Ru. With the aim of preparing highly valuable 16-electron complexes, complex **86** was reacted with monodentate phosphines. The reaction with PPh3 results in small conversion to the five-coordinated compound [Ru(Cl){(*ο*-C6 H4 -PCy2 ) 2 SiMe}(PPh3 )] (**100**) in equilibrium with **86**. Interestingly, this latter compounds are also formed from the reaction of ligand **55** and [RuCl2 (PPh3 ) 3 ] in the presence of NEt3 albeit in low yields. However, the production of the isolable penta-coordinate complex [Ru(Cl){(*ο*-C6 H4 -PCy2 ) 2 SiMe}(PMe3 )] (**101**) was possible in quantitative yields when employing **86** in solution and the smaller, more σ-electron-donating PMe3 (**Figure 7**) [30].

In complexes **74** and **75** (**Figure 7**), the ligand coordinates in a tridentate fashion through the phosphorous atoms which dispose in *trans* and the Si which sits in the equatorial plane of a trigonal bipyramidal geometry. The remaining sites were taken by the hydride derived from the ligand and a Cl atom. Remarkably, besides the intermolecular C─H activation ability imposed by the coordination of ligand **55** to Ir, complex **75** also exhibits facile N─H bond activation of ammonia and anilines while its Rh analogues undergo mainly adduct formation. Likewise, the starting complexes **74** and **75** react with lithium anilides [Li(NHR)] generating isolable anilido hydride complexes [MH(NHR){(*ο*-C6 H4 -PCy2 ) 2 SiMe}] (M = Rh, R = Ph (**102**); M = Rh, R = {2, 6-Me2 C6 H3 } (**103**); M = Ir, R = Ph (**104**); M = Ir, R = {2, 6-Me<sup>2</sup> C6 H3 } (**105**); M = Ir, R = H (**106**)) upon mixing. The new compounds were described as being very resistant to N─H bond reductive elimination even in the presence of alkyl or aryl substrates (**Figure 7**) [20].

The ligand (*ο*-C6 H4 -PPh2 ) 3 Si-H (**107**) reacts with [Ni(PPh3 ) 4 ] to yield the complex [Ni{*η*<sup>2</sup> -(*ο*-C6 H4 - PPh2 ) 2 Si-H(*ο*-C6 H4 -PPh2 )}(PMe3 )] (**109**) bearing non-classical σ-Si─H bonds. On the other hand, complex **109** undergoes thermal oxidative addition at the Ni centre and loss of PMe3 to allow the coordination of the previously uncoordinated phosphorous, thus rendering a compound of formula [NiH{(*ο*-C6 H4 -PPh2 ) 3 Si)}] (**110**). In a subsequent step, HCl was added to afford the formation of [NiCl{(*ο*-C6 H4 -PPh2 ) 3 Si)}] (**111**), which was also obtained when compound **109** was combined with one equivalent of MeHSiCl2 . Compounds [NiX{(*ο*-C6 H4 -PPh2 ) 3 Si}] (X = Br, **112**; I, **113**) were obtained from the reaction of **110** with either EtBr or MeI (**Figure 8**).

the metal. The reaction of this agostic complex with HCl leads to the formation of complex

to reduction with an additional equivalent of Na/Hg in the presence of [12]crown-4 to render

zero oxidation state and the dinitrogen ligand is less labile than in **116** because of stronger π backdonation from the more reduced metal [32]. Once again the nature of the substituents on

temperature producing a species with one uncoordinated phosphorous atom and the Si─H

H4 -P*i*Pr2

H4 -P*<sup>i</sup>* Pr2 )3

H4 -P*i*Pr2 )3

Si}] (**122**), respectively (**Figure 8**) [33].

){(*ο*-C6 H4 -PPh2 )3

> ]+ [Fe(N2

SiH(*ο*-C6

in the presence of MeMgCl at −78°C, the desired complex [FeCl{(*ο*-C6

isolated. The reactivity of **108** with metallic precursors CoCl2

*Si* systems [31-34].

Si}] (**115**). Subsequent reduction with Na/Hg amalgam under a N<sup>2</sup>

){(*ο*-C6 H4 -PPh2 )3 Si}]<sup>−</sup> atmo-

at room

was

Si}] (**119**) was

Si}] (**116**), which was again subjected

SiH (**108**) reacts with FeCl2

Versatile Silylphosphine Ligands for Transition Metal Complexation

http://dx.doi.org/10.5772/intechopen.73502

39

)}] (**118**); however, if the reaction was made

and [Ir(*μ*-Cl)(COD)]2

H4 -P*i*Pr2 )3 Si}]

H4 -P*i*Pr2 )3

Si}] (**120**), [NiCl{(*ο*-C6

, NiCl2

(**117**) in which Fe is in a

[FeCl{(*ο*-C6

bond intact [FeCl2

(**121**) and [IrHCl{(*ο*-C6

H4 -PPh2 )3

**Figure 8.** The chemistry of *P3*

sphere led to the Fe(I) complex [Fe(N2

dark purple ionic pair [Na([12]crown-4)2

{(*ο*-C6 H4 -P*i*Pr2 )2

the P atom is determinant. Indeed, ligand (*ο*-C6

found to yield the tripodal species [CoCl{(*ο*-C6

H4 -P*i*Pr2 )3

Peters and co-workers have also reported the synthesis and reactivity of silanes functionalised with phosphines and/or sulphur derivatives. In particular, the ligand **107** reacts with [Fe2 Mes4 ] leading to the formation of [Fe{(*ο*-C6 H4 -PPh2 )3 Si}Mes] (**114**), which was characterised structurally by single crystal X-ray diffraction. The analysis discloses a distorted octahedral geometry around the Fe atom in which the ligand has taken four out of the six coordination positions, a mesityl group occupies one more and the sixth site (*trans* to silyl group) is occupied by an agostic interaction (C─H⋯Fe) from a methyl group in *ortho* position of the mesityl bonded to Versatile Silylphosphine Ligands for Transition Metal Complexation http://dx.doi.org/10.5772/intechopen.73502 39

**Figure 8.** The chemistry of *P3 Si* systems [31-34].

to unusual bonding modes, Sun and collaborators reported the systematic reactivity of the tri-

not undergo the oxidative addition process of a Si─H bond even in the presence of independent

H4 -PPh2 ) 2

The versatility of ligand **55** was also probed in the coordination towards Ru. With the aim of preparing highly valuable 16-electron complexes, complex **86** was reacted with monoden-

In complexes **74** and **75** (**Figure 7**), the ligand coordinates in a tridentate fashion through the phosphorous atoms which dispose in *trans* and the Si which sits in the equatorial plane of a trigonal bipyramidal geometry. The remaining sites were taken by the hydride derived from the ligand and a Cl atom. Remarkably, besides the intermolecular C─H activation ability imposed by the coordination of ligand **55** to Ir, complex **75** also exhibits facile N─H bond activation of ammonia and anilines while its Rh analogues undergo mainly adduct formation. Likewise, the starting complexes **74** and **75** react with lithium anilides [Li(NHR)] generating isolable anilido

mixing. The new compounds were described as being very resistant to N─H bond reductive

the coordination of the previously uncoordinated phosphorous, thus rendering a compound

Peters and co-workers have also reported the synthesis and reactivity of silanes functionalised with phosphines and/or sulphur derivatives. In particular, the ligand **107** reacts with [Fe2

ally by single crystal X-ray diffraction. The analysis discloses a distorted octahedral geometry around the Fe atom in which the ligand has taken four out of the six coordination positions, a mesityl group occupies one more and the sixth site (*trans* to silyl group) is occupied by an agostic interaction (C─H⋯Fe) from a methyl group in *ortho* position of the mesityl bonded to

SiMe}(PPh3

latter compounds are also formed from the reaction of ligand **55** and [RuCl2

SiMe}(PMe3

H4 -PCy2 ) 2

Si-H (**107**) reacts with [Ni(PPh3

**113**) were obtained from the reaction of **110** with either EtBr or MeI (**Figure 8**).

H4 -PPh2 )3

complex **109** undergoes thermal oxidative addition at the Ni centre and loss of PMe3

} (**103**); M = Ir, R = Ph (**104**); M = Ir, R = {2, 6-Me<sup>2</sup>

)}(PMe3

H4 -PPh2 ) 3

combined with one equivalent of MeHSiCl2

leading to the formation of [Fe{(*ο*-C6

H4 -PPh2 ) 3

elimination even in the presence of alkyl or aryl substrates (**Figure 7**) [20].

employing **86** in solution and the smaller, more σ-electron-donating PMe3

MeSiH). However, the reactivity with chlorosilanes Me3

MeSi-H}(PMe3

albeit in low yields. However, the production of the isolable penta-coordinate

C6 H3

) 4

. Compounds [NiX{(*ο*-C6

)] (**109**) bearing non-classical σ-Si─H bonds. On the other hand,

Si)}] (**110**). In a subsequent step, HCl was added to afford the

Si)}] (**111**), which was also obtained when compound **109** was

H4 -PPh2 ) 3

Si}Mes] (**114**), which was characterised structur-

) 4

results in small conversion to the five-coordinated

)] (**100**) in equilibrium with **86**. Interestingly, this

)] (**101**) was possible in quantitative yields when

SiMe}] (M = Rh, R = Ph (**102**); M = Rh, R = {2,

] to yield the complex [Ni{*η*<sup>2</sup>

} (**105**); M = Ir, R = H (**106**)) upon

)] (X = Br (**98**); I (**99**)) were easily obtained from reaction with

] which induced the for-

SiCl or MeHSiCl2

led

] in the pres-


Si}] (X = Br, **112**; I,

to allow

Mes4 ]

)] (**96**, **Figure 6**). Complex **96** did

MeSi}] (**97**). The halogenated products

(PPh3 ) 3

(**Figure 7**) [30].

dentate ligand **54** towards the low-valent nickel compound [Ni(PMe3


mation of Ni(0) complexes [Ni{*η*<sup>2</sup>

to the formation of Ni(II) complex [NiCl{(*ο*-C6

EtBr or MeI of complex **96** (**Figure 6**) [29].

tate phosphines. The reaction with PPh3

hydride complexes [MH(NHR){(*ο*-C6

H4 -PPh2 ) 3

H4 -PPh2

SiMe}(PMe3

H4 -PCy2 ) 2

H4 -PCy2 ) 2

SiH, Ph2

H4 -PPh2 ) 2

compound [Ru(Cl){(*ο*-C6

complex [Ru(Cl){(*ο*-C6

silanes (Et3

38 Ligand

[NiX{(*ο*-C6

ence of NEt3

6-Me2 C6 H3

PPh2 ) 2

The ligand (*ο*-C6

Si-H(*ο*-C6

of formula [NiH{(*ο*-C6

formation of [NiCl{(*ο*-C6

the metal. The reaction of this agostic complex with HCl leads to the formation of complex [FeCl{(*ο*-C6 H4 -PPh2 )3 Si}] (**115**). Subsequent reduction with Na/Hg amalgam under a N<sup>2</sup> atmosphere led to the Fe(I) complex [Fe(N2 ){(*ο*-C6 H4 -PPh2 )3 Si}] (**116**), which was again subjected to reduction with an additional equivalent of Na/Hg in the presence of [12]crown-4 to render dark purple ionic pair [Na([12]crown-4)2 ]+ [Fe(N2 ){(*ο*-C6 H4 -PPh2 )3 Si}]<sup>−</sup> (**117**) in which Fe is in a zero oxidation state and the dinitrogen ligand is less labile than in **116** because of stronger π backdonation from the more reduced metal [32]. Once again the nature of the substituents on the P atom is determinant. Indeed, ligand (*ο*-C6 H4 -P*<sup>i</sup>* Pr2 )3 SiH (**108**) reacts with FeCl2 at room temperature producing a species with one uncoordinated phosphorous atom and the Si─H bond intact [FeCl2 {(*ο*-C6 H4 -P*i*Pr2 )2 SiH(*ο*-C6 H4 -P*i*Pr2 )}] (**118**); however, if the reaction was made in the presence of MeMgCl at −78°C, the desired complex [FeCl{(*ο*-C6 H4 -P*i*Pr2 )3 Si}] (**119**) was isolated. The reactivity of **108** with metallic precursors CoCl2 , NiCl2 and [Ir(*μ*-Cl)(COD)]2 was found to yield the tripodal species [CoCl{(*ο*-C6 H4 -P*i*Pr2 )3 Si}] (**120**), [NiCl{(*ο*-C6 H4 -P*i*Pr2 )3 Si}] (**121**) and [IrHCl{(*ο*-C6 H4 -P*i*Pr2 )3 Si}] (**122**), respectively (**Figure 8**) [33].

Another example of the importance of the *trans*-influence of the silyl groups on the metal coordination sphere is given by the chemical properties of [RuCl{(*ο*-C6 H4 -PPh2 ) 3 Si}] (**123**). The exchange reaction with LiPPh2 (also with LiP*i*Pr2 ) led to expected phosphide complex [Ru(PPh2 ){(*ο*-C6 H4 -PPh2 ) 3 Si}] (**124**), which decays at room temperature to the cyclometalated [Ru(PHPh2 ){Si(*ο*-C6 H4 -PPh2 ) 2 (*ο*-C6 H4 )P(*ο*-C6 H4 )Ph}] (**125**). The reaction of **123** with stoichiometric amounts of MeLi or PhCH2 MgCl leads to [Ru(Z){(*ο*-C6 H4 -PPh2 ) 3 Si}] (Z = Me (**126**); PhCH2 (**127**)). Successive loss of methane or toluene renders the formation of the unsaturated square pyramidal species [Ru{Si(*ο*-C6 H4 -PPh2 ) 2 (*ο*-C6 H4 )P(*ο*-C6 H4 )Ph}] (**128**), which in turns affords under H<sup>2</sup> or N2 atmospheres compounds [Ru(H2 )H{(*ο*-C6 H4 -PPh2 ) 3 Si}] (**129**) and [Ru(N2 )H{(*ο*-C6 H4 -PPh2 )3 Si}] (**130**) in subsequent reactions steps (**Figure 8**) [34].

signal was found at δ −17.09 (*J*

H4 -PPh2 ) 3 Si}(PMe3

PH = 79.4, 78.8 and 10.8 Hz (**Figure 9**) [31].

**7. Hybrid silylphosphines complexation**

)-NMe2

reactivity towards organolithium {(*ο*-C6

}{(*ο*-C6 H4

with group 10 precursors PdBr2

observed upon the use of PMe3

)-CHNMe-SiMe}(*η*<sup>3</sup>

ligand with one equivalent of [Ru(2-methylallyl)2

**Figure 10.** The chemistry of mixed *PSiN* ligands [36-37].

ligands is that comprising the ligands of general formula {(*ο*-C*<sup>6</sup>*

)-NMe2

preparation of *PSiN*-type ligands involved the synthesis of {(*ο*-C6

plex [FeH{(*ο*-C6

a signal in 1

with *J*

{(*ο*-C6 H4 )-P*t*Bu2

{(*ο*-C6 H4

NMe2

NMe2

{(*ο*-C6 H4

}(PMe3

PH = 71.4, 18.1 Hz) in the 1

H NMR spectrum. By comparison, com-

http://dx.doi.org/10.5772/intechopen.73502

)] (**139**) was characterised by a *ν*(Fe─H) band at 1967 cm−1 and

Versatile Silylphosphine Ligands for Transition Metal Complexation

H4

}SiHMe (**140**) was synthesised. This new-fangled ligand reacted

(cod)] to give the complexes [M(X){(*ο*-C6


H*4* )-PR*<sup>2</sup>*



which yields compounds [M(X){(*ο*-C6

nation of the P and Si atoms of the ligand as well as a C─H bond activation of the methyl group (NMe) with a hydrogenated cyclooctene, remaining on the coordination sphere of the metal. Overall, a square planar geometry around Ru centre is structurally proposed (**Figure 10**) [36].

Another example of an elegant catalytic application of systems derived of *PSiN* pincer-like

)] (M = Pd, X = Br (**148**); M = Pt, X = Cl (**149**)). The coordination of the *PSiN* ligand

)-NMe2

}. In this manner, the mixed-donor ligand

H4 )-P*t*Bu2

(cod)]. The complex resulted from the coordi-

}{(*ο*-C*<sup>6</sup>* H*4*

H4 )-P*t*Bu2

}SiHMeCl and its

H4 )-P*<sup>t</sup>* Bu2 }


H4 )-P*t*Bu2 }

)-NMe*<sup>2</sup>*

}{(*ο*-C6 H4 )-

> H4 )-

41

}SiHMe

H NMR spectrum at δ −15.00 of a triplet of doublets of doublets (tdd) multiplicity

*PSiN*-ligated complexes have also been attractive synthetic targets due to the hemilability property the presence of soft P, Si and hard N atoms could impose. The synthetic strategy for the

Li)-P*t*Bu2

H4

towards group 9 (Rh, Ir) and 8 (Ru) has also been studied. The complex [Ru{(*ο*-C6

or [PtCl2

latter complexes with OTf- led to the formation of compounds [M(OTf){(*ο*-C6

## **6. Silylphosphines complexation in hexa-coordinated systems: octahedral geometry**

Shimada and collaborators reported on the reactivity of ligands (*ο*-C6 H4 -PR2 ) 2 SiHMe (R = Cy (**55**), *i*Pr (**56**), *t*Bu (**57**); **Figure 5**) towards [Ir(μ-Cl)(COD)]2 . The complexes [IrClH{(*ο*-C6 H4 - PR2 ) 2 SiMe}] (R = Cy, **75**, *i*Pr, **131**, *t*Bu, **132**) (**Figure 7** and **9**) reacted with reducing agent Me4 N·BH4 under argon to produce the tetrahydride complexes [IrH4 {(*ο*-C6 H4 -PR2 ) 2 SiMe}] (R = Cy (**133**), *<sup>i</sup>* Pr (**134**), *<sup>t</sup>* Bu(**135**)) or under dinitrogen gas to produce rare stable Ir(III) dihydride-dinitrogen complexes of formula [IrH2 (N2 ){(*ο*-C6 H4 -PR2 ) 2 SiMe}] (R = Cy (**136**), *i*Pr (**137**)). For the last two complexes, NMR spectroscopy reveals the presence of the *fac*/*mer* isomers; the meridional and facial disposition of the *PSiP* ligand was supported by single crystal X-ray diffraction (**Figure 9**) [35].

The complex [FeH{(*ο*-C6 H4 -PPh2 ) 2 SiMe}(PMe3 ) 2 ] (**138**) was synthesised from [Fe(PMe3 ) 4 ], its *ν*(Fe─H) stretching band was found at 1870cm−1 in the IR spectrum, while a triplet of doublets (td),

**Figure 9.** (Left) Chemistry of *PSiP* complexes of group 9. (Right) Related Fe octahedral complexes [35].

signal was found at δ −17.09 (*J* PH = 71.4, 18.1 Hz) in the 1 H NMR spectrum. By comparison, complex [FeH{(*ο*-C6 H4 -PPh2 ) 3 Si}(PMe3 )] (**139**) was characterised by a *ν*(Fe─H) band at 1967 cm−1 and a signal in 1 H NMR spectrum at δ −15.00 of a triplet of doublets of doublets (tdd) multiplicity with *J* PH = 79.4, 78.8 and 10.8 Hz (**Figure 9**) [31].

## **7. Hybrid silylphosphines complexation**

Another example of the importance of the *trans*-influence of the silyl groups on the metal

Si}] (**124**), which decays at room temperature to the cyclometalated

{(*ο*-C6 H4 -PR2 ) 2

(also with LiP*i*Pr2

H4

H4 -PPh2 ) 2 (*ο*-C6 H4

atmospheres compounds [Ru(H2

SiMe}] (R = Cy, **75**, *i*Pr, **131**, *t*Bu, **132**) (**Figure 7** and **9**) reacted with reducing agent Me4

plexes, NMR spectroscopy reveals the presence of the *fac*/*mer* isomers; the meridional and facial disposition of the *PSiP* ligand was supported by single crystal X-ray diffraction (**Figure 9**) [35].

> ) 2

*ν*(Fe─H) stretching band was found at 1870cm−1 in the IR spectrum, while a triplet of doublets (td),

Bu(**135**)) or under dinitrogen gas to produce rare stable Ir(III) dihydride-dinitrogen com-

MgCl leads to [Ru(Z){(*ο*-C6

(**127**)). Successive loss of methane or toluene renders the formation of the unsatu-

Si}] (**130**) in subsequent reactions steps (**Figure 8**) [34].

H4 -PPh2 ) 3

) led to expected phosphide complex

)Ph}] (**125**). The reaction of **123** with stoichio-

H4

H4 -PPh2 ) 3

H4 -PR2 ) 2

SiMe}] (R = Cy (**136**), *i*Pr (**137**)). For the last two com-

] (**138**) was synthesised from [Fe(PMe3

. The complexes [IrClH{(*ο*-C6

SiMe}] (R = Cy (**133**), *<sup>i</sup>*

H4 -PPh2 ) 3

)P(*ο*-C6

)H{(*ο*-C6

Si}] (**123**).

Si}] (Z = Me (**126**);

)Ph}] (**128**), which in

Si}] (**129**) and

SiHMe (R = Cy

H4 -

Pr

N·BH4

) 4 ], its

coordination sphere is given by the chemical properties of [RuCl{(*ο*-C6

)P(*ο*-C6

**6. Silylphosphines complexation in hexa-coordinated systems:** 

SiMe}(PMe3

**Figure 9.** (Left) Chemistry of *PSiP* complexes of group 9. (Right) Related Fe octahedral complexes [35].

Shimada and collaborators reported on the reactivity of ligands (*ο*-C6

(**55**), *i*Pr (**56**), *t*Bu (**57**); **Figure 5**) towards [Ir(μ-Cl)(COD)]2

under argon to produce the tetrahydride complexes [IrH4

){(*ο*-C6 H4 -PR2 ) 2

(N2

H4 -PPh2 ) 2

The exchange reaction with LiPPh2

metric amounts of MeLi or PhCH2

H4 -PPh2 )3

H4 -PPh2 ) 2 (*ο*-C6 H4

rated square pyramidal species [Ru{Si(*ο*-C6

or N2

){(*ο*-C6 H4 -PPh2 ) 3

turns affords under H<sup>2</sup>

)H{(*ο*-C6

**octahedral geometry**

plexes of formula [IrH2

The complex [FeH{(*ο*-C6

){Si(*ο*-C6

[Ru(PPh2

40 Ligand

PhCH2

[Ru(N2

PR2 ) 2

(**134**), *<sup>t</sup>*

[Ru(PHPh2

*PSiN*-ligated complexes have also been attractive synthetic targets due to the hemilability property the presence of soft P, Si and hard N atoms could impose. The synthetic strategy for the preparation of *PSiN*-type ligands involved the synthesis of {(*ο*-C6 H4 )-NMe2 }SiHMeCl and its reactivity towards organolithium {(*ο*-C6 H4 Li)-P*t*Bu2 }. In this manner, the mixed-donor ligand {(*ο*-C6 H4 )-P*t*Bu2 }{(*ο*-C6 H4 )-NMe2 }SiHMe (**140**) was synthesised. This new-fangled ligand reacted with group 10 precursors PdBr2 or [PtCl2 (cod)] to give the complexes [M(X){(*ο*-C6 H4 )-P*<sup>t</sup>* Bu2 } {(*ο*-C6 H4 )-NMe2 -SiMe}] (M = Pd, X = Br (**144**); M = Pt, X = Cl (**145**)), respectively. Treatment of the latter complexes with OTf- led to the formation of compounds [M(OTf){(*ο*-C6 H4 )-P*t*Bu2 }{(*ο*-C6 H4 )- NMe2 -SiMe}] (M = Pd (**146**); Pt (**147**)) showing in the X-ray diffraction molecular structure a distance Pd-O of 2.3518(11) Å in **146**, which once again highlights the strong *trans*-influence of the silyl donor. The selective reversible de-coordination of the amine arm in **144** and **145** was observed upon the use of PMe3 which yields compounds [M(X){(*ο*-C6 H4 )-P*t*Bu2 -SiMe-(*ο*-C6 H4 )- NMe2 }(PMe3 )] (M = Pd, X = Br (**148**); M = Pt, X = Cl (**149**)). The coordination of the *PSiN* ligand towards group 9 (Rh, Ir) and 8 (Ru) has also been studied. The complex [Ru{(*ο*-C6 H4 )-P*t*Bu2 } {(*ο*-C6 H4 )-CHNMe-SiMe}(*η*<sup>3</sup> -cyclooctene)] (**150**) was achieved upon thermal reaction of the *PSiN* ligand with one equivalent of [Ru(2-methylallyl)2 (cod)]. The complex resulted from the coordination of the P and Si atoms of the ligand as well as a C─H bond activation of the methyl group (NMe) with a hydrogenated cyclooctene, remaining on the coordination sphere of the metal. Overall, a square planar geometry around Ru centre is structurally proposed (**Figure 10**) [36].

Another example of an elegant catalytic application of systems derived of *PSiN* pincer-like ligands is that comprising the ligands of general formula {(*ο*-C*<sup>6</sup>* H*4* )-PR*<sup>2</sup>* }{(*ο*-C*<sup>6</sup>* H*4* )-NMe*<sup>2</sup>* }SiHMe

**Figure 10.** The chemistry of mixed *PSiN* ligands [36-37].

(R = *t*Bu (**140**); Cy (**141**); *i*Pr (**142**); Ph (**143**)) [36]. The complexes [PtCl{(*ο*-C6 H4 )-PR2 (*ο*-C6 H4 )- NMe2 -SiMe}] (R = *t*Bu (**145**); Cy (**151**); *i*Pr (**152**); Ph (**153**)) were synthesised by the reaction with [PtCl2 *η*2 -(C2 H4 )]2 in the presence of Et3 N. In particular, the *PSiN*-platinum complex **151** successfully catalysed C─H borylation not only of highly electron deficient perfluoroarenes but also of the monofluorinated arenes, chloroarenes and benzoate (**Figure 10**) [37].

Interestingly, the synthesis of the bulkier ligand (*ο*-C6

**8. Bulky silylphosphines complexation**

**Figure 12.** Bulky carborane silyl-phosphine ligands [53].

**Figure 13.** Syntheses of bulky cage trigonal bipyramidal iron complexes [54].

compound **162** (**Figure 11**) [52].

P*i*Pr2 ) 2

C2

[Pt(*η*<sup>2</sup> -C2 H4 )(PPh3 ) 3

B10H10(SiMe2

H4 -P*i*Pr2 )2 HSi-(C6

 (**162**) was reported very recently. The lithiation of (2,6-dibromophenyl)-isopropyl thioether with *n*BuLi in one pot followed by the stoichiometric addition of **59** affords the formation of (3-bromo-2-(isopropylthio)phenyl)(bis(2-diisopropylphenylphosphino)silane, which received the same treatment that (2,6-dibromophenyl)-isopropyl, to form the desired

An example of rare kinetic stabilisation of *trans* bis(silyl) isomers was provided by the contributions of Kang, Ko and coworkers on the reaction of the bulky carborane silyl-phosphines {(R<sup>2</sup>

(R = Me (**166**); OEt (**167**)) formed by "chelate-assisted" oxidative addition. However, in the presence of dimethyl acetylenedicarboxylate, the complexes endure thermally rearrangements to the thermodynamically favoured *cis* isomers **166′** and **167′**. Besides, the reaction of

] towards **165** occurs via oxidative addition resulting in the mono(chelate)

H)} (R= Me (**163**), OEt (**164**), Ph (**165**)) towards [Pt(*η*<sup>2</sup>

which afforded extremely uncommon *trans*-bis(P,Si-chelates) [Pt{(R<sup>2</sup>

H3

http://dx.doi.org/10.5772/intechopen.73502

Versatile Silylphosphine Ligands for Transition Metal Complexation

P)C2


)S*i*Pr-SiH(*ο*-C6

H4 - 43

P)

)}2 ]

],

] or [Pt(COD)2

B10H10(SiMe2

Regarding [M(*P3 Si*)] tripodal systems, an extended series of organometallic species of general formulae [M(X){(*ο*-C6 H4 -PR2 ) 3 Si}] or [M(L){(*ο*-C6 H4 -PR2 ) 3 Si}] (R = Ph, *<sup>i</sup>* Pr; X = Me, Cl; L = H2 , N2 , CO, NH3 , N3 R', PMe3 ) has been disclosed with a variety of metals including, Fe [38–44], Co [45, 46], Ni [47], Ru [48], Os [48], Rh [45] and Ir [45], with outstanding and specific properties. Perhaps among the most novel systems, one can find the chemistry of novel hybrids (thioether/phosphine)-silane ligands (*ο*-C6 H4 -P*i*Pr2 ) 3-n(*ο*-C6 H4 -SAd)nSiH (n = 2 (**154**); 1 (**155**)) synthesised from silyl-phosphines such as (*ο*-C6 H4 -P*i*Pr2 ) 2 SiHCl, **59** (**Figure 5**, section 4.1), upon lithiation of Br(*ο*-C6 H4 )E (E = P*i*Pr2 or SAd) with varying stoichiometric amounts of Li(*ο*-C6 H4 ) E. The reactivity of these hybrids with FeCl2 afforded a new class of iron complexes featuring a S─Fe─N2 linkage (**Figure 11**) [49]. The bulky hexa-dentate ligand {(*ο*-C6 H4 -P*i*Pr2 ) 2 HSi-O-SiH(*ο*-C6 H4 -P*i*Pr2 ) 2 } (**156**) was synthesised by the controlled hydrolysis of **59** (**Figure 11**). The reactivity of **59** was also reported towards FeBr2 and NiX2 ·DME (X = Cl or Br). The complex [Fe2 (N2 ) (*μ*-H)2 {((*ο*-C6 H4 -P*i*Pr2 ) 2 Si)2 O}] (**157**) was formed in an equilibrium mixture with [Fe2 (N2 ) 2 (*μ*-H)2 {((*ο*-C6 H4 -P*i*Pr2 ) 2 Si)2 O}] (**158**), which were observed at low temperature in the IR spectrum (two *ν*(N─N) bands at 2097 and 2060 cm−1) in accordance with the determined thermodynamic parameters including a large negative entropy (−30(2) cal/mol K), consistent with the coordination of a gas molecule and a rather small enthalpy of binding (−9.0(4) kcal/mol) in line with the observation of both species at low temperature (**Figure 11**) [50]. This research is particularly relevant for the understanding of nitrogenase mimicking systems. From the reactivity of a binucleating variant of a *PSiP* ligand with NiX2 .DME (X = Cl, Br) in the presence of triethylamine, dinuclear zerovalent nickel complexes bearing both *η*<sup>2</sup> -(Si─H) and *η*<sup>2</sup> -H2 moieties were observed by the group of Peters. Theoretical studies suggest that the Ni centre facilitates H atom exchange between the *η*<sup>2</sup> -(Si─H) and *η*<sup>2</sup> -H2 ligands via interconversion with a higher valent Ni(II) isomer (compounds **159**–**161**) (**Figure 11**). This exchange has been exploited in the selective catalytic deuteration of exogenous silanes [51].

**Figure 11.** Chemistry of compound **59** and related reactions [49-52].

Interestingly, the synthesis of the bulkier ligand (*ο*-C6 H4 -P*i*Pr2 )2 HSi-(C6 H3 )S*i*Pr-SiH(*ο*-C6 H4 - P*i*Pr2 )2 (**162**) was reported very recently. The lithiation of (2,6-dibromophenyl)-isopropyl thioether with *n*BuLi in one pot followed by the stoichiometric addition of **59** affords the formation of (3-bromo-2-(isopropylthio)phenyl)(bis(2-diisopropylphenylphosphino)silane, which received the same treatment that (2,6-dibromophenyl)-isopropyl, to form the desired compound **162** (**Figure 11**) [52].

## **8. Bulky silylphosphines complexation**

(R = *t*Bu (**140**); Cy (**141**); *i*Pr (**142**); Ph (**143**)) [36]. The complexes [PtCl{(*ο*-C6

the monofluorinated arenes, chloroarenes and benzoate (**Figure 10**) [37].

Si}] or [M(L){(*ο*-C6

H4 -P*i*Pr2 ) 3-n(*ο*-C6 H4

linkage (**Figure 11**) [49]. The bulky hexa-dentate ligand {(*ο*-C6

in the presence of Et3

H4 -PR2 ) 3

thesised from silyl-phosphines such as (*ο*-C6

)E (E = P*i*Pr2

of a binucleating variant of a *PSiP* ligand with NiX2

selective catalytic deuteration of exogenous silanes [51].

**Figure 11.** Chemistry of compound **59** and related reactions [49-52].

ethylamine, dinuclear zerovalent nickel complexes bearing both *η*<sup>2</sup>

H4

ity of **59** was also reported towards FeBr2

H atom exchange between the *η*<sup>2</sup>

E. The reactivity of these hybrids with FeCl2

R', PMe3

ether/phosphine)-silane ligands (*ο*-C6


*Si*)] tripodal systems, an extended series of organometallic species of general

) has been disclosed with a variety of metals including, Fe [38–44], Co

fully catalysed C─H borylation not only of highly electron deficient perfluoroarenes but also of

H4 -PR2 ) 3

[45, 46], Ni [47], Ru [48], Os [48], Rh [45] and Ir [45], with outstanding and specific properties. Perhaps among the most novel systems, one can find the chemistry of novel hybrids (thio-

> H4 -P*i*Pr2 ) 2

and NiX2

(two *ν*(N─N) bands at 2097 and 2060 cm−1) in accordance with the determined thermodynamic parameters including a large negative entropy (−30(2) cal/mol K), consistent with the coordination of a gas molecule and a rather small enthalpy of binding (−9.0(4) kcal/mol) in line with the observation of both species at low temperature (**Figure 11**) [50]. This research is particularly relevant for the understanding of nitrogenase mimicking systems. From the reactivity

were observed by the group of Peters. Theoretical studies suggest that the Ni centre facilitates

valent Ni(II) isomer (compounds **159**–**161**) (**Figure 11**). This exchange has been exploited in the



} (**156**) was synthesised by the controlled hydrolysis of **59** (**Figure 11**). The reactiv-

O}] (**157**) was formed in an equilibrium mixture with [Fe2

O}] (**158**), which were observed at low temperature in the IR spectrum

NMe2

42 Ligand

[PtCl2 *η*2 -(C2 H4 )]2

Regarding [M(*P3*

CO, NH3

S─Fe─N2

C6 H4 -P*i*Pr2 ) 2

(*μ*-H)2

H)2

formulae [M(X){(*ο*-C6

, N3

lithiation of Br(*ο*-C6

{((*ο*-C6 H4 -P*i*Pr2 ) 2 Si)2

{((*ο*-C6 H4 -P*i*Pr2 ) 2 Si)2 H4 )-PR2

Pr; X = Me, Cl; L = H2


SiHCl, **59** (**Figure 5**, section 4.1), upon

afforded a new class of iron complexes featuring a

H4 -P*i*Pr2 ) 2

.DME (X = Cl, Br) in the presence of tri-

ligands via interconversion with a higher



moieties

·DME (X = Cl or Br). The complex [Fe2

N. In particular, the *PSiN*-platinum complex **151** success-

Si}] (R = Ph, *<sup>i</sup>*

or SAd) with varying stoichiometric amounts of Li(*ο*-C6

(*ο*-C6 H4 )-

> , N2 ,

> > H4 )

(N2 )

HSi-O-SiH(*ο*-

(N2 ) 2 (*μ*- An example of rare kinetic stabilisation of *trans* bis(silyl) isomers was provided by the contributions of Kang, Ko and coworkers on the reaction of the bulky carborane silyl-phosphines {(R<sup>2</sup> P) C2 B10H10(SiMe2 H)} (R= Me (**163**), OEt (**164**), Ph (**165**)) towards [Pt(*η*<sup>2</sup> -C2 H4 )(PPh3 ) 3 ] or [Pt(COD)2 ], which afforded extremely uncommon *trans*-bis(P,Si-chelates) [Pt{(R<sup>2</sup> P)C2 B10H10(SiMe2 )}2 ] (R = Me (**166**); OEt (**167**)) formed by "chelate-assisted" oxidative addition. However, in the presence of dimethyl acetylenedicarboxylate, the complexes endure thermally rearrangements to the thermodynamically favoured *cis* isomers **166′** and **167′**. Besides, the reaction of [Pt(*η*<sup>2</sup> -C2 H4 )(PPh3 ) 3 ] towards **165** occurs via oxidative addition resulting in the mono(chelate)

**Figure 12.** Bulky carborane silyl-phosphine ligands [53].

**Figure 13.** Syntheses of bulky cage trigonal bipyramidal iron complexes [54].

[PtH{(Ph<sup>2</sup> P)C2 B10H10(SiMe2 )}(PPh3 )] (**168**). These authors also investigated the reactivity of the same bulky carborane silyl-phosphines with the palladium precursor [Pd2 (dba)3 ] and observed that depending of the substituents over the phosphorous atoms, the *cis* isomer was exclusively formed [Pd{(R<sup>2</sup> P)C2 B10H10(SiMe2 )}2 ] (R = OEt (**169**); Ph (**170**)) or a mixture of the *trans*/*cis* isomers was detected when R = Me (**171**/**171′**) (**Figure 12**) [53].

**183** with respect to a non-benzilic phenylphosphine analogue, coupled with the presence of the non-classical Si─H bond interactions, which could undergo a low energy dissociation-coordina-

stable bis(carbometallated) complex **187**. Thus, it was reasonable to propose that the agostic interactions preceded and favoured the C─H bonds activation process [58]. Ligand *SiPSi* phos-

behaved as a pincer-like ligand capable of adopting different coordination modes at ruthenium through different degrees of Si─H bond activation. The reaction of **188** towards complex **184** yielded exclusively the formation of **189**, in which a Ru(II) centre is coordinated to one ligand

hydride ligands of the original Ru complex. The phosphorous atoms arrange in a distorted *cis* with a P-Ru-P angle 113.32(4)° in **189** which should be compared to 107.1(4)° in bis-cyclometallated **187**. This sterically encumbered arrangement of the phosphine ligands around ruthenium has been explained due to the favourable exchange of the two formally terminal and two non-

with theoretical calculations and the observed chemical behaviour of **189** in solution agree with

formulated as an 18-electron species stabilised by two unusual intramolecular ε-non-classical interactions. Complex **189** undergoes facile and reversible loss of dihydrogen to afford quantitatively 16-electron complex **190,** which is thought to preserve a single non-classical hydride as well as a terminal one. Moreover, NMR spectroscopic experiments on complex **189** show it to be very fluxional in the temperature range accessible, while hydride exchange in complex **190** takes place at the high-temperature regime but in the slow exchange indicates only one

in **185** to the final

, it

45

and two

SiH together

(**188**) was synthesised from PhP(*ο*-tolyl)2

Versatile Silylphosphine Ligands for Transition Metal Complexation

http://dx.doi.org/10.5772/intechopen.73502


tion process of the Si─H bonds, was claimed to induce the gradual loss of H2

SiMe2 H}2

**188**, through the P atom and two non-fully activated Si─H bonds preserving one PCy3

classical sigma hydrides around the metal. Certainly, the measured value of the *J*

H4 )CH2

**Figure 15.** Chemistry of silyl-benzyl phosphines bi-, tri- and tetradentate [58-60].

phinodibenzyl-silane PhP{(*ο*-C6

the presence of non-classical *η*<sup>2</sup>

Recently, the syntheses of bulky-cage trigonal bipyramidal iron complexes **174** and **175** with remote tertiary amines were reported. The synthesis of ligands **172** and **173** is shown in **Figure 13**. Once again, in this regard, the incorporation of secondary sphere interactions into iron-phosphine scaffolds is relevant to synthetic nitrogen fixation research [54].

## **9. Non-rigid and semi-rigid silylphosphines**

Sola reported tridentate systems exemplified by [IrHCl{[Ph<sup>2</sup> P(CH2 ) 3 ] 2 SiMe}] (**176**) [55] derived from the reaction of the ligand *PSiP* {[Ph<sup>2</sup> P(CH2 ) 3 ] 2 SiHMe} (**47**, **Figure 3**) with the dimeric compound [Ir(*μ*-Cl)(cod)]2 . In solution, complex **176** displays an equilibrium between the *syn* (**176**) and *anti* (**176′**) isomers in a ratio 93:7 in C6 D6 and C7 D8 , while in CDCl3 or CD2 Cl2 solutions, the ratio is *ca.* 83:17. Complex **176** (and **176′**) reacted with NaX (X = Br or I) leading to the corresponding complexes [IrHX{[Ph<sup>2</sup> P(CH2 ) 3 ] 2 SiMe}] ((X = Br (**177**); I (**178**)) also in equilibrium with their respective *syn* and *anti*-isomers (**177′**, **178′**) in similar ratios that those of **176** [56]. The mixture of isomers **176**, also reacted with Me(O3 SCF3 ) to produce the isomers *syn* **179** and *anti* **179′** with general formula [IrH(O3 SCF3 ){[Ph<sup>2</sup> P(CH2 ) 3 ] 2 SiMe}]; likewise the reactivity of **176** with AgX or HX (X = PF6 ) in the presence of a neutral ligand afforded the mixture of the respective *syn*/*anti* cationic species [IrH(L)2 {[Ph<sup>2</sup> P(CH2 ) 3 ] 2 SiMe}]+ [PF6 ] − (L = NCMe (**180***/***180′**), CO (**181***/***181′**), bipy (**182***/***182′**)) (**Figure 14**) [57].

Our research group studied the reactivity of *PSi* ligand phosphino-(benzyl)-silane Ph2 P{(*ο*-C6 H4 )CH2 SiMe2 H} (**183**) towards the complexes [RuH2 (*η*<sup>2</sup> -H2 )(PCy3 ) 2 ] (**184**) and [Ru(cod)(cot)]. Complex **185** resulted from the substitution of two molecules of dihydrogen and two of the ligands PCy3 in **184** by two ligands **183** bonded to the ruthenium atom through the phosphorous atoms and two σ-bonds of the fragments Si─H. Following loss of H2 , complex **183** slowly transformed to the cyclometalated complex **186** and subsequently into the bis(cyclometalated) **187**. When **183** was added to [Ru(cod)(cot)], the synthetic precursor of **184**, it generated directly complex **187** in very high yield. The increase on the acidity of the methylene groups of ligand

**Figure 14.** The chemistry of [IrP2 Si] [56, 57].

**183** with respect to a non-benzilic phenylphosphine analogue, coupled with the presence of the non-classical Si─H bond interactions, which could undergo a low energy dissociation-coordination process of the Si─H bonds, was claimed to induce the gradual loss of H2 in **185** to the final stable bis(carbometallated) complex **187**. Thus, it was reasonable to propose that the agostic interactions preceded and favoured the C─H bonds activation process [58]. Ligand *SiPSi* phosphinodibenzyl-silane PhP{(*ο*-C6 H4 )CH2 SiMe2 H}2 (**188**) was synthesised from PhP(*ο*-tolyl)2 , it behaved as a pincer-like ligand capable of adopting different coordination modes at ruthenium through different degrees of Si─H bond activation. The reaction of **188** towards complex **184** yielded exclusively the formation of **189**, in which a Ru(II) centre is coordinated to one ligand **188**, through the P atom and two non-fully activated Si─H bonds preserving one PCy3 and two hydride ligands of the original Ru complex. The phosphorous atoms arrange in a distorted *cis* with a P-Ru-P angle 113.32(4)° in **189** which should be compared to 107.1(4)° in bis-cyclometallated **187**. This sterically encumbered arrangement of the phosphine ligands around ruthenium has been explained due to the favourable exchange of the two formally terminal and two nonclassical sigma hydrides around the metal. Certainly, the measured value of the *J* SiH together with theoretical calculations and the observed chemical behaviour of **189** in solution agree with the presence of non-classical *η*<sup>2</sup> -Si-H character of the silyl moieties. Thus, the complex **189** was formulated as an 18-electron species stabilised by two unusual intramolecular ε-non-classical interactions. Complex **189** undergoes facile and reversible loss of dihydrogen to afford quantitatively 16-electron complex **190,** which is thought to preserve a single non-classical hydride as well as a terminal one. Moreover, NMR spectroscopic experiments on complex **189** show it to be very fluxional in the temperature range accessible, while hydride exchange in complex **190** takes place at the high-temperature regime but in the slow exchange indicates only one

[PtH{(Ph<sup>2</sup>

44 Ligand

formed [Pd{(R<sup>2</sup>

P)C2

B10H10(SiMe2

from the reaction of the ligand *PSiP* {[Ph<sup>2</sup>

*anti* **179′** with general formula [IrH(O3

respective *syn*/*anti* cationic species [IrH(L)2

CO (**181***/***181′**), bipy (**182***/***182′**)) (**Figure 14**) [57].

responding complexes [IrHX{[Ph<sup>2</sup>

**176** with AgX or HX (X = PF6

SiMe2

**Figure 14.** The chemistry of [IrP2

C6 H4 )CH2

ligands PCy3

and *anti* (**176′**) isomers in a ratio 93:7 in C6

The mixture of isomers **176**, also reacted with Me(O3

pound [Ir(*μ*-Cl)(cod)]2

P)C2

)}(PPh3

scaffolds is relevant to synthetic nitrogen fixation research [54].

**9. Non-rigid and semi-rigid silylphosphines**

Sola reported tridentate systems exemplified by [IrHCl{[Ph<sup>2</sup>

)}2

B10H10(SiMe2

was detected when R = Me (**171**/**171′**) (**Figure 12**) [53].

same bulky carborane silyl-phosphines with the palladium precursor [Pd2

that depending of the substituents over the phosphorous atoms, the *cis* isomer was exclusively

Recently, the syntheses of bulky-cage trigonal bipyramidal iron complexes **174** and **175** with remote tertiary amines were reported. The synthesis of ligands **172** and **173** is shown in **Figure 13**. Once again, in this regard, the incorporation of secondary sphere interactions into iron-phosphine

> P(CH2 ) 3 ] 2

> > D6

SCF3

P(CH2 ) 3 ] 2

H} (**183**) towards the complexes [RuH2

rous atoms and two σ-bonds of the fragments Si─H. Following loss of H2

Si] [56, 57].

the ratio is *ca.* 83:17. Complex **176** (and **176′**) reacted with NaX (X = Br or I) leading to the cor-

with their respective *syn* and *anti*-isomers (**177′**, **178′**) in similar ratios that those of **176** [56].

){[Ph<sup>2</sup>

{[Ph<sup>2</sup>

Our research group studied the reactivity of *PSi* ligand phosphino-(benzyl)-silane Ph2

Complex **185** resulted from the substitution of two molecules of dihydrogen and two of the

transformed to the cyclometalated complex **186** and subsequently into the bis(cyclometalated) **187**. When **183** was added to [Ru(cod)(cot)], the synthetic precursor of **184**, it generated directly complex **187** in very high yield. The increase on the acidity of the methylene groups of ligand

and C7

)] (**168**). These authors also investigated the reactivity of the

] (R = OEt (**169**); Ph (**170**)) or a mixture of the *trans*/*cis* isomers

P(CH2 ) 3 ] 2

, while in CDCl3

) in the presence of a neutral ligand afforded the mixture of the

. In solution, complex **176** displays an equilibrium between the *syn* (**176**)

D8

SCF3

(*η*<sup>2</sup> -H2 )(PCy3 ) 2

in **184** by two ligands **183** bonded to the ruthenium atom through the phospho-

P(CH2 ) 3 ] 2

P(CH2 ) 3 ] 2 SiMe}]+

SiHMe} (**47**, **Figure 3**) with the dimeric com-

SiMe}] ((X = Br (**177**); I (**178**)) also in equilibrium

[PF6 ] − or CD2

) to produce the isomers *syn* **179** and

SiMe}]; likewise the reactivity of

(dba)3

] and observed

SiMe}] (**176**) [55] derived

Cl2

(L = NCMe (**180***/***180′**),

] (**184**) and [Ru(cod)(cot)].

, complex **183** slowly

solutions,

P{(*ο*-

**Figure 15.** Chemistry of silyl-benzyl phosphines bi-, tri- and tetradentate [58-60].

hydride is bound to the two silicon atoms. In the solid-state X-ray diffraction analysis, a P-Ru-P angle of 154.37(3)° was determined, which is significantly more obtuse than in **189** and in **187**, as expected due to the diminished hydride exchange in **190**. The free energy Δ<sup>r</sup> *G*298 of the reaction **189** to **190** + H2 is +16.9 kJ/mol; in line with the experimentally observed conversion at 308 K [59]. The reactivity of the ligands **183**, **188** and new *PSi3* ligand P{(*ο*-C6 H4 )CH2 SiMe2 H}3 (**191**) was also investigated with compounds [M(*μ*-Cl)(cod)]2 (M = Rh, Ir) [60] and with [Pt(PPh3 ) 3 ] [8]. Compound **191** coordinates to Rh and Ir centres as a tetradentate ligand through the phosphorus and two silyl groups, while a third Si atom engages in an agostic Si─H interaction mode [60]. Complexes **192** and **193** react with adventitious water to generate dimeric siloxane compounds. Additionally, compounds **188** and **191** react with Pt as tridentate ligands leading exclusively to compounds exhibiting a very rare *trans* silyl disposition at square planar Pt (**194**, **195**). These two complexes feature ligand (**188** and **191**) in a close to meridional disposition. Complex **195** results from ligand modification at one of the benzylic positions which undergoes formation of a new C-Si bond. Furthermore, d8 Pt(II) complex **195** is the first case of a silyl-platinum complex that includes a novel C─H⋯Pt anagostic interaction (**Figure 15**).

capability of forming σ-complexes, coupled to those features of the P moieties, which can be

Versatile Silylphosphine Ligands for Transition Metal Complexation

http://dx.doi.org/10.5772/intechopen.73502

47

Throughout this chapter, it has been shown the study of transition metal systems bonded to silylphosphine ligands has thrived in the last decades, but the findings in the last years highlight the importance of their study. Numerous extraordinary systems displaying unusual bonding modes, structures or physicochemical properties have been reported to date and many more can be envisioned to be informed in the near future given the relatively accessible

However, the catalytic and other applications of these compounds have been sparingly explored; yet the potential of many of the reported systems is foreseen. We thus expect this field of chemistry to continue growing rapidly and encourage other research groups to direct

We acknowledge financial support from CONACyT-Mexico through project CB 242818 and

Center for Chemical Research, Autonomous University of the State of Morelos, Cuernavaca,

[1] Holmes-Smith RD, Osei RD, Stobart SR. Phosphinoalkylsilanes: Synthesis and spectroscopic properties of phosphino(silyl)methanes, 1-phosphino-2-silylethanes, and 1-phosphino-3-silylpropanes. Journal of the Chemical Society, Perkin Transactions. 1983;

[2] Auburn MJ, Holmes-Smith RD, Stobart SR. (Phosphinoalkyl)silyl complexes: 3. "Chelateassisted" hydrosilylation: Formation of enantiomeric and diasteroisomeric iridium(III) complexes with chelating (phosphinoethyl)silyl ligands. Journal of the American

synthesis of ligands and the seemingly unlimited structural variations.

greatly modified by the choice of substituents.

their endeavours to this fascinating area of research.

ANR-CONACyT 274001 and a PhD grant to JZ-M

Julio Zamora-Moreno and Virginia Montiel-Palma\*

Chemical Society. 1984;**106**:1314-1318

\*Address all correspondence to: vmontielp@uaem.mx

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

**Author details**

Morelos, Mexico

**References**

**I**:861-866

## **10. Applications of silylphosphines in the chemical industry**

From the examples throughout this chapter, one can safely envisage transition metal complexes of silylphosphines as active catalysts in a variety of industrial processes. The industrial application of this type of ligand systems, nevertheless, is still at its cradle with future applications expected to materialise in the mid-term.

In principle, Si and P are capable of displaying nucleophilic behaviour and both also possess the ability to displace leaving groups such as halogens, neutral/monodentate ligands, and so on, while the factors affecting their stereochemistry may also assist the complex in the attainment of specific geometries [61]. Catalysed transfer hydrogenation has been developed mainly based on complexes derived from the platinum-metals group [62], and it is applied in industrial process and organic synthesis [63]. [PSiP-Ru] species also have shown to play an excellent role in the reduction of ketones employing i PrOH as the hydrogen source. The well-known Kumada's cross-coupling reaction is an actual tool for the low-cost synthesis of styrene derivatives in the industrial scale by using Ni and Pd complexes as catalysts [64]. Some advances revealed the crucial use of phosphorous-containing compounds [65–67] and/or the very bulky donor ligands [68, 69]. Nevertheless, [PSiP-Co] systems have shown efficient conversions in relative mild reaction conditions of an aryl-Grignard reagent reaction with organic halides at 50°C for 24 h [70].

## **11. Conclusion and perspectives**

The incorporation of dual functionalities P and Si in single ligand backbones, silylphosphines, notably modifies the properties of the complexes they form, making them especially reactive and able to undergo selective transformations resulting from differing reactivity of the coordinating atoms in the ligand in conjunction with the chelate effect.

Predictably, the observed reactivity stems from the combination of the most important qualities of the Si ligands, specifically their extremely high σ-donating character and thus their capability of forming σ-complexes, coupled to those features of the P moieties, which can be greatly modified by the choice of substituents.

Throughout this chapter, it has been shown the study of transition metal systems bonded to silylphosphine ligands has thrived in the last decades, but the findings in the last years highlight the importance of their study. Numerous extraordinary systems displaying unusual bonding modes, structures or physicochemical properties have been reported to date and many more can be envisioned to be informed in the near future given the relatively accessible synthesis of ligands and the seemingly unlimited structural variations.

However, the catalytic and other applications of these compounds have been sparingly explored; yet the potential of many of the reported systems is foreseen. We thus expect this field of chemistry to continue growing rapidly and encourage other research groups to direct their endeavours to this fascinating area of research.

## **Acknowledgements**

hydride is bound to the two silicon atoms. In the solid-state X-ray diffraction analysis, a P-Ru-P angle of 154.37(3)° was determined, which is significantly more obtuse than in **189** and in **187**,

Compound **191** coordinates to Rh and Ir centres as a tetradentate ligand through the phosphorus and two silyl groups, while a third Si atom engages in an agostic Si─H interaction mode [60]. Complexes **192** and **193** react with adventitious water to generate dimeric siloxane compounds. Additionally, compounds **188** and **191** react with Pt as tridentate ligands leading exclusively to compounds exhibiting a very rare *trans* silyl disposition at square planar Pt (**194**, **195**). These two complexes feature ligand (**188** and **191**) in a close to meridional disposition. Complex **195** results from ligand modification at one of the benzylic positions which undergoes formation of a new

From the examples throughout this chapter, one can safely envisage transition metal complexes of silylphosphines as active catalysts in a variety of industrial processes. The industrial application of this type of ligand systems, nevertheless, is still at its cradle with future applica-

In principle, Si and P are capable of displaying nucleophilic behaviour and both also possess the ability to displace leaving groups such as halogens, neutral/monodentate ligands, and so on, while the factors affecting their stereochemistry may also assist the complex in the attainment of specific geometries [61]. Catalysed transfer hydrogenation has been developed mainly based on complexes derived from the platinum-metals group [62], and it is applied in industrial process and organic synthesis [63]. [PSiP-Ru] species also have shown to play an excellent role in

cross-coupling reaction is an actual tool for the low-cost synthesis of styrene derivatives in the industrial scale by using Ni and Pd complexes as catalysts [64]. Some advances revealed the crucial use of phosphorous-containing compounds [65–67] and/or the very bulky donor ligands [68, 69]. Nevertheless, [PSiP-Co] systems have shown efficient conversions in relative mild reaction conditions of an aryl-Grignard reagent reaction with organic halides at 50°C for 24 h [70].

The incorporation of dual functionalities P and Si in single ligand backbones, silylphosphines, notably modifies the properties of the complexes they form, making them especially reactive and able to undergo selective transformations resulting from differing reactivity of the coor-

Predictably, the observed reactivity stems from the combination of the most important qualities of the Si ligands, specifically their extremely high σ-donating character and thus their

dinating atoms in the ligand in conjunction with the chelate effect.

is +16.9 kJ/mol; in line with the experimentally observed conversion at 308 K

ligand P{(*ο*-C6

Pt(II) complex **195** is the first case of a silyl-platinum complex that

PrOH as the hydrogen source. The well-known Kumada's

H4 )CH2

(M = Rh, Ir) [60] and with [Pt(PPh3

*G*298 of the reac-

) 3 ] [8].

SiMe2 H}3 (**191**)

as expected due to the diminished hydride exchange in **190**. The free energy Δ<sup>r</sup>

**10. Applications of silylphosphines in the chemical industry**

[59]. The reactivity of the ligands **183**, **188** and new *PSi3*

includes a novel C─H⋯Pt anagostic interaction (**Figure 15**).

tions expected to materialise in the mid-term.

the reduction of ketones employing i

**11. Conclusion and perspectives**

was also investigated with compounds [M(*μ*-Cl)(cod)]2

tion **189** to **190** + H2

46 Ligand

C-Si bond. Furthermore, d8

We acknowledge financial support from CONACyT-Mexico through project CB 242818 and ANR-CONACyT 274001 and a PhD grant to JZ-M

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Julio Zamora-Moreno and Virginia Montiel-Palma\*

\*Address all correspondence to: vmontielp@uaem.mx

Center for Chemical Research, Autonomous University of the State of Morelos, Cuernavaca, Morelos, Mexico

## **References**


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**Chapter 3**

Provisional chapter

**Ligand-Protected Gold Clusters**

Ligand-Protected Gold Clusters

Sakiat Hossain, Lakshmi V. Nair, Junta Inoue,

Sakiat Hossain, Lakshmi V. Nair, Junta Inoue,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73441

these compounds, are also described.

and chemical properties, ligand exchange reactions

Abstract

1. Introduction

Yuki Koyama, Wataru Kurashige and Yuichi Negishi

Small gold clusters with diameters less than or equal to 2 nm (below approximately 200 atoms) possess geometric and electronic structures different from bulk gold. When these gold clusters are protected by ligands, these clusters can be treated as chemical compounds. This review focuses on gold clusters protected by chalcogenate (thiolate, selenolate, or tellurolate) ligands and describes the methods by which these clusters are synthesized as well as their geometric/electronic structures and physical and chemical properties. Recent findings regarding ligand exchange reactions, which may be used to impart functionality to

DOI: 10.5772/intechopen.73441

Keywords: gold clusters, chalcogenate, geometric and electronic structures, physical

Small gold clusters with diameters less than or equal to 2 nm (below approximately 200 atoms) possess geometric and electronic structures different from those of bulk gold [1]. The geometric structure often consists of an atomic arrangement, such as an icosahedral structure, that differs from the close-packed structure of bulk gold, as a result of reducing the surface energy. In addition, a discrete electronic structure appears rather than the continuous structure observed in the bulk element. Owing to these characteristics, small gold clusters exhibit fundamental properties and functionalities different from those of bulk gold. In addition, when these gold clusters are protected by ligands, it is possible to treat them as chemical compounds. In early

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Yuki Koyama, Wataru Kurashige and Yuichi Negishi


## **Chapter 3**

Provisional chapter

## **Ligand-Protected Gold Clusters** Ligand-Protected Gold Clusters

Sakiat Hossain, Lakshmi V. Nair, Junta Inoue, Sakiat Hossain, Lakshmi V. Nair, Junta Inoue,

Yuki Koyama, Wataru Kurashige and Yuichi Negishi Yuki Koyama, Wataru Kurashige and Yuichi Negishi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73441

#### Abstract

[58] Montiel-Palma V, Muñoz-Hernandez MA, Ayed T, Barthelat JC, Grellier M, Vendier L, Sabo-Etienne S.Agostic Si-H bond coordination assists C-H bond activation at ruthenium in bis(phosphinobenzylsilane) complexes. Chemical Communications. 2007;**0**(38):3963-3965

[59] Montiel-Palma V, Muñoz-Hernandez MA, Cuevas-Chavez CA, Vendier L, Grellier M, Sabo-

[60] Corona-Gonzalez MV, Zamora-Moreno J, Cuevas-Chavez CA, Rufino-Felipe E, Mothes-Martin E, Coppel Y, Muñoz-Hernandez MA, Vendier L, Flores-Alamo M, Grellier M, Sabo-Etienne S, Montiel-Palma V. A family of rhodium and iridium complexes with semirigid benzylsilyl phosphines: From bidentate to tetradentate coordination modes.

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[63] Ikariya T, Murata K, Noyori R. Bifunctional transition metal-based molecular catalysts for asymmetric syntheses. Organic & Biomolecular Chemistry. 2006;**4**(3):393-406 [64] Tamao K, Sumitani K, Kumada M. Selective carbon-carbon bond formation by crosscoupling of Grignard reagents with organic halides. Catalysis by nickel-phosphine com-

[65] Hua X, Masson-Makdissi J, Sullivan RJ, Newman SG. Inherent vs apparent chemoselectivity in the Kumada–Corriu cross-coupling reaction. Organic Letters. 2016;

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[68] Xi Z, Liu B, Chen W. Room-temperature Kumada cross-coupling of unactivated aryl chlorides catalyzed by N-heterocylic carbene-based nickel(II) complexes. The Journal of

[69] Iglesias MJ, Prieto A, Nicasio MC. Kumada–Tamao–Corriu coupling of heteroaromatic chlorides and aryl ethers catalyzed by (IPr)Ni(allyl)Cl. Organic Letters. 2012;

[70] Xiong Z, Li X, Zhang S, Shi Y, Sun H. Synthesis and reactivity of N-heterocyclic PSiP pincer iron and cobalt complexes and catalytic application of cobalt hydride in Kumada

chiral reactions. Platinum Metals Review. 2001, 2002;**46**(2):82-83

American Chemical Society. 2009;**131**(27):9590-9599

coupling reactions. Organometallics. 2016;**35**(3):357-363

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plexes. Journal of the American Chemical Society. 1972;**94**(12):4374-4376

pincer-type ligand at ruthenium. Inorganic Chemistry. 2013;**52**(17):9798-9806

H4 CH2

)SiMe2

H}2

: A versatile"PSi2

Hx "

Etienne S. Phosphinodi(benzylsilane) PhP{(o-C<sup>6</sup>

Dalton Transactions. 2017;**46**(27):8827-8838

**40**(12):1385-1393

52 Ligand

**18**(20):5312-5315

**76**(24):10031-10038

**14**(17):4318-4321

Small gold clusters with diameters less than or equal to 2 nm (below approximately 200 atoms) possess geometric and electronic structures different from bulk gold. When these gold clusters are protected by ligands, these clusters can be treated as chemical compounds. This review focuses on gold clusters protected by chalcogenate (thiolate, selenolate, or tellurolate) ligands and describes the methods by which these clusters are synthesized as well as their geometric/electronic structures and physical and chemical properties. Recent findings regarding ligand exchange reactions, which may be used to impart functionality to these compounds, are also described.

DOI: 10.5772/intechopen.73441

Keywords: gold clusters, chalcogenate, geometric and electronic structures, physical and chemical properties, ligand exchange reactions

#### 1. Introduction

Small gold clusters with diameters less than or equal to 2 nm (below approximately 200 atoms) possess geometric and electronic structures different from those of bulk gold [1]. The geometric structure often consists of an atomic arrangement, such as an icosahedral structure, that differs from the close-packed structure of bulk gold, as a result of reducing the surface energy. In addition, a discrete electronic structure appears rather than the continuous structure observed in the bulk element. Owing to these characteristics, small gold clusters exhibit fundamental properties and functionalities different from those of bulk gold. In addition, when these gold clusters are protected by ligands, it is possible to treat them as chemical compounds. In early

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

reaction is a very powerful means for introducing the different ligands into the pre-synthesized cluster. Although this type of reaction was discovered nearly 20 years ago [60–65], the associated mechanism was not fully understood at that time. Recently, tremendous progress has been made in terms of the precise synthesis and evaluation of metal clusters, and details of these reactions have been elucidated [66, 67]. Recent findings regarding these reactions are therefore also included

Ligand-Protected Gold Clusters

55

http://dx.doi.org/10.5772/intechopen.73441

The method used most frequently to synthesize Aun(XR)<sup>m</sup> clusters is based on the chemical reduction of gold ions in the presence of ligands in solution (Figure 2). In this approach, a gold salt and the ligand are mixed in solution to form Au-ligand complexes that are subsequently treated with a reducing agent (normally NaBH4). Aun(XR)<sup>m</sup> clusters are formed by the aggregation

Figure 2. A typical procedure for the synthesis of Aun(SR)<sup>m</sup> clusters having a well-defined chemical composition.

herein.

2. Synthesis of Aun(XR)<sup>m</sup> clusters

Figure 1. The crystal structures of (a) Aun(PR3)m, (b) Aun(SR)m, (c) Aun(SeR)m, and (d) Aun(PR3)m(SeR)l. H atoms are omitted for clarity. In [Au39(PPh3)14Cl6] 2+, C atoms are also not shown (these figures were adapted from Refs. [2, 10, 11, 29, 30, 34, 47, 48, 59]).

studies, beginning in the 1960s, phosphine was employed as a protective ligand [2–16]. Representative phosphine (PR3)-protected gold clusters (Aun(PR3)m) include [Au11(PPh3)8Cl2] + , [Au13(PMe2Ph)8Cl2] 3+, [Au39(PPh3)14Cl6] 2+, and Au55(PPh3)12Cl6 (Figure 1(a)). Unfortunately, these clusters have been found to be unstable in solution, which restricts their practical applications. In contrast, thiolate (SR)-protected gold clusters (Aun(SR)m), first synthesized by Brust et al. in 1994 (Figure 1(b)) [17], are highly stable both in solution and in the solid state, because the SR ligands form strong bonds with gold atoms. These Aun(SR)<sup>m</sup> clusters exhibit various physical and chemical properties not shown by bulk gold, such as photoluminescence and catalytic activity. For these reasons, SR ligands have become the most common choice for use with gold clusters [18–40]. Recently, the synthesis of gold clusters protected by other chalcogenates (selenolate (SeR) or tellurolate (TeR); Figure 1(c)) [41–51], by alkynes [52–54], or by two kinds of ligand (Figure 1(d)) [55–59] has also been reported. In this chapter, we focus on gold clusters protected by chalcogenates (Aun(XR)m; XR = SR, SeR, or TeR) and describe the synthetic procedures, geometric/electronic structures, and physical and chemical properties of these compounds. Moreover, the physical and chemical properties of these gold clusters are greatly affected by the type of functional group of the protecting ligand. The ligand exchange reaction is a very powerful means for introducing the different ligands into the pre-synthesized cluster. Although this type of reaction was discovered nearly 20 years ago [60–65], the associated mechanism was not fully understood at that time. Recently, tremendous progress has been made in terms of the precise synthesis and evaluation of metal clusters, and details of these reactions have been elucidated [66, 67]. Recent findings regarding these reactions are therefore also included herein.

## 2. Synthesis of Aun(XR)<sup>m</sup> clusters

studies, beginning in the 1960s, phosphine was employed as a protective ligand [2–16]. Representative phosphine (PR3)-protected gold clusters (Aun(PR3)m) include [Au11(PPh3)8Cl2]

Figure 1. The crystal structures of (a) Aun(PR3)m, (b) Aun(SR)m, (c) Aun(SeR)m, and (d) Aun(PR3)m(SeR)l. H atoms are

these clusters have been found to be unstable in solution, which restricts their practical applications. In contrast, thiolate (SR)-protected gold clusters (Aun(SR)m), first synthesized by Brust et al. in 1994 (Figure 1(b)) [17], are highly stable both in solution and in the solid state, because the SR ligands form strong bonds with gold atoms. These Aun(SR)<sup>m</sup> clusters exhibit various physical and chemical properties not shown by bulk gold, such as photoluminescence and catalytic activity. For these reasons, SR ligands have become the most common choice for use with gold clusters [18–40]. Recently, the synthesis of gold clusters protected by other chalcogenates (selenolate (SeR) or tellurolate (TeR); Figure 1(c)) [41–51], by alkynes [52–54], or by two kinds of ligand (Figure 1(d)) [55–59] has also been reported. In this chapter, we focus on gold clusters protected by chalcogenates (Aun(XR)m; XR = SR, SeR, or TeR) and describe the synthetic procedures, geometric/electronic structures, and physical and chemical properties of these compounds. Moreover, the physical and chemical properties of these gold clusters are greatly affected by the type of functional group of the protecting ligand. The ligand exchange

2+, and Au55(PPh3)12Cl6 (Figure 1(a)). Unfortunately,

2+, C atoms are also not shown (these figures were adapted from Refs. [2, 10, 11, 29,

3+, [Au39(PPh3)14Cl6]

[Au13(PMe2Ph)8Cl2]

30, 34, 47, 48, 59]).

54 Ligand

omitted for clarity. In [Au39(PPh3)14Cl6]

+ , The method used most frequently to synthesize Aun(XR)<sup>m</sup> clusters is based on the chemical reduction of gold ions in the presence of ligands in solution (Figure 2). In this approach, a gold salt and the ligand are mixed in solution to form Au-ligand complexes that are subsequently treated with a reducing agent (normally NaBH4). Aun(XR)<sup>m</sup> clusters are formed by the aggregation

Figure 2. A typical procedure for the synthesis of Aun(SR)<sup>m</sup> clusters having a well-defined chemical composition.

of the resulting gold atoms in conjunction with surface protection by the ligands [18]. In the first report published by Brust et al., dodecanethiolate was used as the ligand [17]. Because a gold salt and dodecanethiolate are soluble in different solvents, Brust transferred the gold salt from an aqueous phase to a ligand-containing toluene phase using a phase-transfer reagent (representing a two-phase system; Figure 2). In contrast, in more recent research, tetrahydrofuran (THF) has often been used as the solvent because it could dissolve both gold salt and ligand [68]. This removes the need for phase transfer of the gold salt and thereby simplifies the synthesis to a one-phase system (Figure 2). Similarly, when a hydrophilic thiol is used as the ligand, gold clusters can be synthesized in a one-phase system [69–71].

structural analysis has revealed that several Aun(SR)<sup>m</sup> clusters consist of an Au core covered with multiple dS(R)[dAudS(R)]xd staples (Figure 3(b)) [30, 85–88]. Based on the geometrical structures determined for Aun(SR)<sup>m</sup> clusters to date, it can be assumed that almost all small Aun(SR)<sup>m</sup> clusters have this type of core-shell structure. Single-crystal X-ray structural analysis has also demonstrated that small Aun(SeR)<sup>m</sup> clusters have core-shell structures similar to those of small Aun(SR)<sup>m</sup> clusters (Figure 1(c)) [47, 48]. The geometrical structure of Aun(TeR)<sup>m</sup> clusters has not yet been determined experimentally, although theoretical calculations [45, 89]

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Unlike bulk gold, small Aun(SR)<sup>m</sup> clusters have discrete electronic structures. As a result, multiple peak structures can be observed in the optical absorption spectra of these clusters. As an example, Aun(SC12H25)<sup>m</sup> clusters show multiple peak structures across the entire visible range in their optical absorption spectra up to the size of Au144(SC12H25)60 (Figure 4) [75]. Such fine peak structures are not observed in the spectra of larger clusters, although peaks that can be attributed to surface plasmon resonance absorption have been identified at approximately

Figure 4. Optical absorption spectra of films composed of Aun(SC12H25)<sup>m</sup> clusters (n = 38–520) at various temperatures

(25–290 K) (this figure was adapted from Ref. [75]).

have shown that these clusters are also likely to have a similar core-shell structure.

4. Electronic structures of Aun(XR)<sup>m</sup> clusters

The product obtained from this technique is typically a mixture of Aun(XR)<sup>m</sup> clusters having various numbers of constituent atoms. Because the physical and chemical properties of the clusters are greatly affected by the number of atoms, separation by size or conversion to stable clusters by exposure to severe conditions is required to obtain Aun(XR)<sup>m</sup> clusters with welldefined physical properties and functions (Figure 2) [18, 72]. Polyacrylamide gel electrophoresis [69–72], high-performance liquid chromatography [72–77], and solvent extraction are the most frequently applied techniques for size separation. It is also common to use an etching reaction for size convergence [72, 78–82]. In addition to these techniques, the ligand exchange method, in which the ligands of a specific Aun(XR)<sup>m</sup> cluster are replaced with other ligands, is an effective means of generating Aun(XR)<sup>m</sup> clusters with a specific chemical composition (Figure 2) [83]. Recent results associated with such ligand exchange reactions are discussed in Section 6.

## 3. Geometrical structures of Aun(XR)<sup>m</sup> clusters

Until 2007, it was believed that Aun(SR)<sup>m</sup> clusters possess a geometrical structure in which an Au core is covered with thiolate ligands (Figure 3(a)) [84]. Since then, single-crystal X-ray

Figure 3. The geometrical structures of Au38(SR)24 (a) predicted by theoretical calculations in 1999 and (b) determined by single-crystal X-ray structural analysis in 2010. The R groups have been omitted for clarity (these figures were adapted from Refs. [30, 84]).

structural analysis has revealed that several Aun(SR)<sup>m</sup> clusters consist of an Au core covered with multiple dS(R)[dAudS(R)]xd staples (Figure 3(b)) [30, 85–88]. Based on the geometrical structures determined for Aun(SR)<sup>m</sup> clusters to date, it can be assumed that almost all small Aun(SR)<sup>m</sup> clusters have this type of core-shell structure. Single-crystal X-ray structural analysis has also demonstrated that small Aun(SeR)<sup>m</sup> clusters have core-shell structures similar to those of small Aun(SR)<sup>m</sup> clusters (Figure 1(c)) [47, 48]. The geometrical structure of Aun(TeR)<sup>m</sup> clusters has not yet been determined experimentally, although theoretical calculations [45, 89] have shown that these clusters are also likely to have a similar core-shell structure.

## 4. Electronic structures of Aun(XR)<sup>m</sup> clusters

of the resulting gold atoms in conjunction with surface protection by the ligands [18]. In the first report published by Brust et al., dodecanethiolate was used as the ligand [17]. Because a gold salt and dodecanethiolate are soluble in different solvents, Brust transferred the gold salt from an aqueous phase to a ligand-containing toluene phase using a phase-transfer reagent (representing a two-phase system; Figure 2). In contrast, in more recent research, tetrahydrofuran (THF) has often been used as the solvent because it could dissolve both gold salt and ligand [68]. This removes the need for phase transfer of the gold salt and thereby simplifies the synthesis to a one-phase system (Figure 2). Similarly, when a hydrophilic thiol is used as the

The product obtained from this technique is typically a mixture of Aun(XR)<sup>m</sup> clusters having various numbers of constituent atoms. Because the physical and chemical properties of the clusters are greatly affected by the number of atoms, separation by size or conversion to stable clusters by exposure to severe conditions is required to obtain Aun(XR)<sup>m</sup> clusters with welldefined physical properties and functions (Figure 2) [18, 72]. Polyacrylamide gel electrophoresis [69–72], high-performance liquid chromatography [72–77], and solvent extraction are the most frequently applied techniques for size separation. It is also common to use an etching reaction for size convergence [72, 78–82]. In addition to these techniques, the ligand exchange method, in which the ligands of a specific Aun(XR)<sup>m</sup> cluster are replaced with other ligands, is an effective means of generating Aun(XR)<sup>m</sup> clusters with a specific chemical composition (Figure 2) [83]. Recent results associated with such ligand exchange reactions are discussed in Section 6.

Until 2007, it was believed that Aun(SR)<sup>m</sup> clusters possess a geometrical structure in which an Au core is covered with thiolate ligands (Figure 3(a)) [84]. Since then, single-crystal X-ray

Figure 3. The geometrical structures of Au38(SR)24 (a) predicted by theoretical calculations in 1999 and (b) determined by single-crystal X-ray structural analysis in 2010. The R groups have been omitted for clarity (these figures were adapted

ligand, gold clusters can be synthesized in a one-phase system [69–71].

3. Geometrical structures of Aun(XR)<sup>m</sup> clusters

from Refs. [30, 84]).

56 Ligand

Unlike bulk gold, small Aun(SR)<sup>m</sup> clusters have discrete electronic structures. As a result, multiple peak structures can be observed in the optical absorption spectra of these clusters. As an example, Aun(SC12H25)<sup>m</sup> clusters show multiple peak structures across the entire visible range in their optical absorption spectra up to the size of Au144(SC12H25)60 (Figure 4) [75]. Such fine peak structures are not observed in the spectra of larger clusters, although peaks that can be attributed to surface plasmon resonance absorption have been identified at approximately

Figure 4. Optical absorption spectra of films composed of Aun(SC12H25)<sup>m</sup> clusters (n = 38–520) at various temperatures (25–290 K) (this figure was adapted from Ref. [75]).

Figure 5. Structural changes in Aun(SC12H25)<sup>m</sup> clusters with varying numbers of gold atoms (this figure was adapted from Ref. [75]).

520 nm in their optical absorption spectra (Figure 4). Thus, the electronic structures of Au187(SC12H25)68 and larger clusters tend to resemble that of bulk gold (Figure 5) [75].

At present, the relationship between cluster size and electronic structure is not well understood for Aun(SeR)<sup>m</sup> and Aun(TeR)<sup>m</sup> clusters, because only a small number of such compounds have been studied to date. However, the researches regarding Au25(SeR)18 and Au38(SeR)24 clusters have demonstrated that changing the ligands from SR to SeR reduces the HOMO-LUMO gap of the clusters [42, 43] and that this effect becomes more pronounced in the case of clusters containing TeR in the ligand shell [45].

## 5. Physical and chemical properties of Aun(XR)<sup>m</sup> clusters

Aun(SR)<sup>m</sup> clusters exhibit size-specific electronic structures, and their physical and chemical properties also vary with size. Herein, we first discuss typical physical and chemical characteristics of such Aun(SR)<sup>m</sup> clusters.

#### 5.1. Photoluminescence

Small Aun(SR)<sup>m</sup> clusters have been shown to exhibit photoluminescence (Figure 6(a)) [18, 20, 23, 70, 71, 90]. As an example, Au25(SG)18 (SG = glutathionate) exhibits photoluminescence with an estimated quantum yield of ~1 <sup>10</sup><sup>3</sup> [71], which can be used for sensing and imaging applications [91].

redox behavior is not confined to clusters with discrete electronic structures; Aun(SR)<sup>m</sup> clusters larger than Au144(SR)60 also exhibit redox behavior as a result of quantized double-layer charging [21]. The redox properties of Aun(SR)<sup>m</sup> clusters could be applied to single-electron

Figure 6. Size-specific physical and chemical properties of Aun(SR)<sup>m</sup> clusters: (a) photoluminescence, (b) redox behavior,

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(c) optical activity, and (d) catalytic activity (these figures were adapted from Refs. [22, 90, 95]).

Several clusters, such as Au38(SR)24 and Au40(SR)24, have optical isomers with different dS(R)[dAudS(R)]xd staple (x = 1, 2) configurations [93–95] and thus are optically active [36]. Figure 6(c) presents the circular dichroism spectra of two optical isomers of Au38(SC2H4Ph)24

transistors [92].

5.3. Optical activity

#### 5.2. Redox behavior

Aun(SR)<sup>m</sup> clusters also display redox behavior [20, 21]. Figure 6(b) shows a differential pulse voltammogram obtained from Au25(SC2H4Ph)18, in which the peaks at 1.9 and 0.3 V originate from [Au25(SC2H4Ph)18] /2 and [Au25(SC2H4Ph)18] 0/ redox couples, respectively. This

Figure 6. Size-specific physical and chemical properties of Aun(SR)<sup>m</sup> clusters: (a) photoluminescence, (b) redox behavior, (c) optical activity, and (d) catalytic activity (these figures were adapted from Refs. [22, 90, 95]).

redox behavior is not confined to clusters with discrete electronic structures; Aun(SR)<sup>m</sup> clusters larger than Au144(SR)60 also exhibit redox behavior as a result of quantized double-layer charging [21]. The redox properties of Aun(SR)<sup>m</sup> clusters could be applied to single-electron transistors [92].

#### 5.3. Optical activity

520 nm in their optical absorption spectra (Figure 4). Thus, the electronic structures of

Figure 5. Structural changes in Aun(SC12H25)<sup>m</sup> clusters with varying numbers of gold atoms (this figure was adapted

At present, the relationship between cluster size and electronic structure is not well understood for Aun(SeR)<sup>m</sup> and Aun(TeR)<sup>m</sup> clusters, because only a small number of such compounds have been studied to date. However, the researches regarding Au25(SeR)18 and Au38(SeR)24 clusters have demonstrated that changing the ligands from SR to SeR reduces the HOMO-LUMO gap of the clusters [42, 43] and that this effect becomes more pronounced in the case of

Aun(SR)<sup>m</sup> clusters exhibit size-specific electronic structures, and their physical and chemical properties also vary with size. Herein, we first discuss typical physical and chemical charac-

Small Aun(SR)<sup>m</sup> clusters have been shown to exhibit photoluminescence (Figure 6(a)) [18, 20, 23, 70, 71, 90]. As an example, Au25(SG)18 (SG = glutathionate) exhibits photoluminescence with an estimated quantum yield of ~1 <sup>10</sup><sup>3</sup> [71], which can be used for sensing and imaging

Aun(SR)<sup>m</sup> clusters also display redox behavior [20, 21]. Figure 6(b) shows a differential pulse voltammogram obtained from Au25(SC2H4Ph)18, in which the peaks at 1.9 and 0.3 V orig-

0/ redox couples, respectively. This

/2 and [Au25(SC2H4Ph)18]

Au187(SC12H25)68 and larger clusters tend to resemble that of bulk gold (Figure 5) [75].

5. Physical and chemical properties of Aun(XR)<sup>m</sup> clusters

clusters containing TeR in the ligand shell [45].

teristics of such Aun(SR)<sup>m</sup> clusters.

5.1. Photoluminescence

applications [91].

from Ref. [75]).

58 Ligand

5.2. Redox behavior

inate from [Au25(SC2H4Ph)18]

Several clusters, such as Au38(SR)24 and Au40(SR)24, have optical isomers with different dS(R)[dAudS(R)]xd staple (x = 1, 2) configurations [93–95] and thus are optically active [36]. Figure 6(c) presents the circular dichroism spectra of two optical isomers of Au38(SC2H4Ph)24 [95]. The anisotropy factor associated with the optical activity of this cluster increases with wavelength up to a maximum of 4 <sup>10</sup><sup>3</sup> .

6. Ligand exchange reactions

6.1. Mechanism

reported, and these findings are discussed in this section.

As described above, Aun(SR)<sup>m</sup> clusters tend to resist degradation. However, this type of metal cluster readily exchanges its ligands with other coexisting ligands in solution (Figure 8(a)). A complete understanding of the associated mechanism would allow these reactions to be controlled, thus permitting synthesis of novel metal clusters with specific functions. Recently, more details regarding exchange reactions between metal clusters and ligands have been

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Murray et al. reported the ligand exchange reactions of this type of cluster nearly 20 years ago [60–65]. However, their research was conducted using mixtures and did not use advanced techniques such as mass spectrometry and single-crystal X-ray structural analysis to characterize the products. Therefore, a thorough understanding of the details of these reactions was not obtained. More recent research has elucidated the associated mechanism. As an example, Au25(SR)18 has a geometry in which the Au13 core is covered by six dS(R)d[AudS(R)]2d staples (Figure 9(a)). As a result, there are two types of SR units in Au25(SR)18: those in contact with the Au13 core (core-site SR; Figure 9(a)) and those at the apex of each staple (apex-site SR; Figure 9(a)) [102, 103]. Ackerson et al. performed a single-crystal X-ray structural analysis of the product obtained from the reaction of Au25(SC2H4Ph)18 (SC2H4Ph = 2-phenyl ethanethiolate)

Figure 8. A schematic diagram of ligand exchange reactions including (a) only ligand exchange, (b) induction of quasi-

isomerization, and (c) induction of size transformation.

#### 5.4. Catalytic activity

Catalytic activity is another typical size-specific property of Aun(SR)<sup>m</sup> clusters (Figure 6(d)) [22, 72]. As an example, Au25(SR)18 catalyzes the oxidation of CO, styrene, benzyl alcohol, cyclohexane, and sulfides. The same cluster also exhibits catalytic activity for the hydrogenation of nitrophenol, aldehydes, and ketones and promotes CdC coupling reactions. As noted, several Aun(SR)<sup>m</sup> clusters have optical isomers and therefore could potentially function as asymmetric catalysts [96].

#### 5.5. Effect of changing ligands

Regarding Aun(SeR)<sup>m</sup> and Aun(TeR)<sup>m</sup> clusters, it has been reported that the incorporation of SeR or TeR ligands changes the nature of the bonding between the Au atoms and the ligands [97, 98]. In the case of Aun(SeC12H25)<sup>m</sup> clusters, this effect reduces the degree of charge transfer from the Au atoms to the ligands (Figure 7(a)) such that the Audligand bond becomes much more covalent than that in Aun(SC12H25)<sup>m</sup> clusters [41]. Owing to these changes in bonding characteristics, Au25(SeR)18 (R = C12H25 or C8H17) exhibits greater resistance to degradation in solution compared with Au25(SR)18 (R = C12H25 or C8H17) (Figure 7(b)) [42, 99]. In addition to such an improved stability, the use of SeR ligands is expected to improve conductivity between the gold core and the ligands [97, 100, 101], and future work is likely to demonstrate the conductivity of Aun(SeR)<sup>m</sup> clusters. Furthermore, recent studies have found that Au25(SePh)18 exhibits catalytic activity for the reduction of 4-nitrophenol (Figure 7(c)) [48].

Figure 7. A comparison of (a) the Au L3-edge X-ray absorption near-edge structure spectra of Au25(SeC12H25)18 and Au25(SC12H25)18 and (b) the stability of Au25(SeC12H25)18 and Au25(SC12H25)18 in solution under harsh conditions. (c) Representative UV-vis optical absorption spectra acquired during the reduction of 4-nitrophenol to 4-aminophenol over Au25(SePh)18 (these figures were adapted from Refs. [42, 43, 48]).

## 6. Ligand exchange reactions

As described above, Aun(SR)<sup>m</sup> clusters tend to resist degradation. However, this type of metal cluster readily exchanges its ligands with other coexisting ligands in solution (Figure 8(a)). A complete understanding of the associated mechanism would allow these reactions to be controlled, thus permitting synthesis of novel metal clusters with specific functions. Recently, more details regarding exchange reactions between metal clusters and ligands have been reported, and these findings are discussed in this section.

#### 6.1. Mechanism

[95]. The anisotropy factor associated with the optical activity of this cluster increases with

Catalytic activity is another typical size-specific property of Aun(SR)<sup>m</sup> clusters (Figure 6(d)) [22, 72]. As an example, Au25(SR)18 catalyzes the oxidation of CO, styrene, benzyl alcohol, cyclohexane, and sulfides. The same cluster also exhibits catalytic activity for the hydrogenation of nitrophenol, aldehydes, and ketones and promotes CdC coupling reactions. As noted, several Aun(SR)<sup>m</sup> clusters have optical isomers and therefore could potentially function as

Regarding Aun(SeR)<sup>m</sup> and Aun(TeR)<sup>m</sup> clusters, it has been reported that the incorporation of SeR or TeR ligands changes the nature of the bonding between the Au atoms and the ligands [97, 98]. In the case of Aun(SeC12H25)<sup>m</sup> clusters, this effect reduces the degree of charge transfer from the Au atoms to the ligands (Figure 7(a)) such that the Audligand bond becomes much more covalent than that in Aun(SC12H25)<sup>m</sup> clusters [41]. Owing to these changes in bonding characteristics, Au25(SeR)18 (R = C12H25 or C8H17) exhibits greater resistance to degradation in solution compared with Au25(SR)18 (R = C12H25 or C8H17) (Figure 7(b)) [42, 99]. In addition to such an improved stability, the use of SeR ligands is expected to improve conductivity between the gold core and the ligands [97, 100, 101], and future work is likely to demonstrate the conductivity of Aun(SeR)<sup>m</sup> clusters. Furthermore, recent studies have found that Au25(SePh)18 exhibits catalytic

Figure 7. A comparison of (a) the Au L3-edge X-ray absorption near-edge structure spectra of Au25(SeC12H25)18 and Au25(SC12H25)18 and (b) the stability of Au25(SeC12H25)18 and Au25(SC12H25)18 in solution under harsh conditions. (c) Representative UV-vis optical absorption spectra acquired during the reduction of 4-nitrophenol to 4-aminophenol

.

wavelength up to a maximum of 4 <sup>10</sup><sup>3</sup>

5.4. Catalytic activity

60 Ligand

asymmetric catalysts [96].

5.5. Effect of changing ligands

activity for the reduction of 4-nitrophenol (Figure 7(c)) [48].

over Au25(SePh)18 (these figures were adapted from Refs. [42, 43, 48]).

Murray et al. reported the ligand exchange reactions of this type of cluster nearly 20 years ago [60–65]. However, their research was conducted using mixtures and did not use advanced techniques such as mass spectrometry and single-crystal X-ray structural analysis to characterize the products. Therefore, a thorough understanding of the details of these reactions was not obtained. More recent research has elucidated the associated mechanism. As an example, Au25(SR)18 has a geometry in which the Au13 core is covered by six dS(R)d[AudS(R)]2d staples (Figure 9(a)). As a result, there are two types of SR units in Au25(SR)18: those in contact with the Au13 core (core-site SR; Figure 9(a)) and those at the apex of each staple (apex-site SR; Figure 9(a)) [102, 103]. Ackerson et al. performed a single-crystal X-ray structural analysis of the product obtained from the reaction of Au25(SC2H4Ph)18 (SC2H4Ph = 2-phenyl ethanethiolate)

Figure 8. A schematic diagram of ligand exchange reactions including (a) only ligand exchange, (b) induction of quasiisomerization, and (c) induction of size transformation.

This same work also demonstrated that exchanging the ligands of Au28(S-c-C6H11)20 with SPh-<sup>t</sup>

Aun(SR)<sup>m</sup> cluster.

(Figure 3(b)) with <sup>t</sup>

6.3. Induction of size transformation

figures were adapted from Refs. [111, 112]).

regenerated the original geometry, meaning that the reaction was reversible (Figure 10(a)) [111]. Thus, it has recently been revealed that both ligand exchange and quasi-isomerization (as opposed to true isomerization because the ligand is different) can be induced for a particular

Researches have also shown that the introduction of a significant structural deformation via ligand exchange can result in the formation of Aun(SR)<sup>m</sup> clusters with different chemical compositions (Figure 8(c)) [102]. An example is the reaction of Au38(SC2H4Ph)24 clusters

main product (yield ~90%) (Figure 10(b)) [112]. This outcome indicates that exchange with a ligand containing a bulky functional group can affect the chemical composition of the cluster.

Figure 10. Examples of ligand exchange reactions, including (a) quasi-isomerization and (b) size transformation (these

Bu-PhSH in solution, from which Au36(SPh-<sup>t</sup>

Bu

63

Bu)24 was generated as the

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Figure 9. Preferential sites in ligand exchange reactions. (a) and (c) Geometrical structures of the products obtained from the reaction between Au25(SC2H4Ph)18 and para-bromobenzenethiol and benzeneselenol, respectively. (b) Chromatogram of the product obtained from the reaction between Au24Pd(SC2H4Ph)18 and dodecanethiol (these figures were adapted from Refs. [103, 104, 106]).

with para-bromobenzenethiol to ascertain which SR was more likely to be exchanged [103]. The results showed that Au25(SC2H4Ph)16(p-BBT)2 (p-BBT = para-bromobenzenethiolate), in which the substitution had occurred at the core-site SR ligands, was obtained (Figure 9(a)), suggesting that the ligand exchange occurred at the core-site SR locations. However, this prior work did not determine whether other structures may have been present in the reaction mixture or not. For this reason, Niihori et al. employed reversed-phase high-performance liquid chromatography to allow the high-resolution separation of the coordination isomers generated by a similar reaction and estimated the distribution of isomers in the product. It was confirmed that the product mixture mainly contained a coordination isomer in which the core-site SR ligands had been substituted (Figure 9(b)) [104]. Fernando and Aikens performed density functional theory (DFT) calculations at approximately the same time, and the results indicated that ligand exchange was likely to occur at core-site SR ligands in Au25(SR)18 [105]. These results demonstrated that ligand exchange preferentially proceeds at core-site SR ligands in Au25(SC2H4Ph)18. The research by Hossain et al. has revealed that preferential exchange at core-site SR ligands also occurs in the reaction between [Au25(SC2H4Ph)18] and other chalcogenides (Figure 9(c)) [106].

#### 6.2. Induction of quasi-isomerization

Studies have found that, in addition to ligand exchange, a change in geometry can also take place during reactions with thiol (RSH) (Figure 8(b)). This discovery originated from the prediction of the geometry of Au24(SR)20 clusters. Specifically, Jin et al. synthesized Au24(SC2H4Ph)20 in 2010 [107], after which Pei and coworkers predicted the geometry of these clusters via DFT calculations based on Au24(SCH3)20 [108]. Thereafter, Jin et al. characterized Au24(SCH2Ph-<sup>t</sup> Bu)20 (SCH2Ph-<sup>t</sup> Bu = 4-tert-butylphenylmethanethiolate) by single-crystal X-ray structural analysis but found that the resulting structure was different from that predicted by Pei's group [109]. This discrepancy prompted Jiang et al. to study the geometric structures of Au24(SR)20 clusters (R = CH3, C2H4Ph, or CH2Ph-<sup>t</sup> Bu) using DFT, leading to the conclusion that the most stable structure of a Au24(SR)20 cluster depends on the ligand [110]. At present, this theory has not been proven experimentally for Au24(SR)20. However, in 2016, Jin et al. reported that exchanging the ligands of Au28(SPh-<sup>t</sup> Bu)20 (SPh-<sup>t</sup> Bu = 4-tert-butylbenzenethiolate) with cyclohexanethiolate (S-c-C6H11) altered the skeletal structure of the cluster (Figure 10(a)) [111]. This same work also demonstrated that exchanging the ligands of Au28(S-c-C6H11)20 with SPh-<sup>t</sup> Bu regenerated the original geometry, meaning that the reaction was reversible (Figure 10(a)) [111]. Thus, it has recently been revealed that both ligand exchange and quasi-isomerization (as opposed to true isomerization because the ligand is different) can be induced for a particular Aun(SR)<sup>m</sup> cluster.

#### 6.3. Induction of size transformation

with para-bromobenzenethiol to ascertain which SR was more likely to be exchanged [103]. The results showed that Au25(SC2H4Ph)16(p-BBT)2 (p-BBT = para-bromobenzenethiolate), in which the substitution had occurred at the core-site SR ligands, was obtained (Figure 9(a)), suggesting that the ligand exchange occurred at the core-site SR locations. However, this prior work did not determine whether other structures may have been present in the reaction mixture or not. For this reason, Niihori et al. employed reversed-phase high-performance liquid chromatography to allow the high-resolution separation of the coordination isomers generated by a similar reaction and estimated the distribution of isomers in the product. It was confirmed that the product mixture mainly contained a coordination isomer in which the core-site SR ligands had been substituted (Figure 9(b)) [104]. Fernando and Aikens performed density functional theory (DFT) calculations at approximately the same time, and the results indicated that ligand exchange was likely to occur at core-site SR ligands in Au25(SR)18 [105]. These results demonstrated that ligand exchange preferentially proceeds at core-site SR ligands in Au25(SC2H4Ph)18. The research by Hossain et al. has revealed that preferential exchange at core-site SR ligands also

Figure 9. Preferential sites in ligand exchange reactions. (a) and (c) Geometrical structures of the products obtained from the reaction between Au25(SC2H4Ph)18 and para-bromobenzenethiol and benzeneselenol, respectively. (b) Chromatogram of the product obtained from the reaction between Au24Pd(SC2H4Ph)18 and dodecanethiol (these figures were adapted

Studies have found that, in addition to ligand exchange, a change in geometry can also take place during reactions with thiol (RSH) (Figure 8(b)). This discovery originated from the prediction of the geometry of Au24(SR)20 clusters. Specifically, Jin et al. synthesized Au24(SC2H4Ph)20 in 2010 [107], after which Pei and coworkers predicted the geometry of these clusters via DFT calculations based on Au24(SCH3)20 [108]. Thereafter, Jin et al. characterized

structural analysis but found that the resulting structure was different from that predicted by Pei's group [109]. This discrepancy prompted Jiang et al. to study the geometric structures of

the most stable structure of a Au24(SR)20 cluster depends on the ligand [110]. At present, this theory has not been proven experimentally for Au24(SR)20. However, in 2016, Jin et al. reported

cyclohexanethiolate (S-c-C6H11) altered the skeletal structure of the cluster (Figure 10(a)) [111].

Bu)20 (SPh-<sup>t</sup>

and other chalcogenides (Figure 9(c)) [106].

Bu) using DFT, leading to the conclusion that

Bu = 4-tert-butylbenzenethiolate) with

Bu = 4-tert-butylphenylmethanethiolate) by single-crystal X-ray

occurs in the reaction between [Au25(SC2H4Ph)18]

Bu)20 (SCH2Ph-<sup>t</sup>

Au24(SR)20 clusters (R = CH3, C2H4Ph, or CH2Ph-<sup>t</sup>

that exchanging the ligands of Au28(SPh-<sup>t</sup>

6.2. Induction of quasi-isomerization

Au24(SCH2Ph-<sup>t</sup>

from Refs. [103, 104, 106]).

62 Ligand

Researches have also shown that the introduction of a significant structural deformation via ligand exchange can result in the formation of Aun(SR)<sup>m</sup> clusters with different chemical compositions (Figure 8(c)) [102]. An example is the reaction of Au38(SC2H4Ph)24 clusters (Figure 3(b)) with <sup>t</sup> Bu-PhSH in solution, from which Au36(SPh-<sup>t</sup> Bu)24 was generated as the main product (yield ~90%) (Figure 10(b)) [112]. This outcome indicates that exchange with a ligand containing a bulky functional group can affect the chemical composition of the cluster.

Figure 10. Examples of ligand exchange reactions, including (a) quasi-isomerization and (b) size transformation (these figures were adapted from Refs. [111, 112]).

Research regarding the mechanism of such reactions has also been conducted. Jin et al. found that the following four processes occur in the reaction between Au38(SC2H4Ph)24 and <sup>t</sup> Bu-PhSH: (I) ligand exchange, (II) structural distortion, (III) disproportionation, and (IV) size focusing conversion together with further ligand exchange (Figure 10(b)) [112]. In the first process, ligand exchange occurs without size or structural transformations, while the structural distortion of the resulting Au38(SC2H4Ph)24<sup>m</sup>(SPh-<sup>t</sup> Bu)<sup>m</sup> (m > ~12) is initiated in the second process. During the third process, one Au38(SC2H4Ph)24–m(SPh-<sup>t</sup> Bu)<sup>m</sup> releases two gold atoms to form Au36 and another Au38(SC2H4Ph)24–m(SPh-<sup>t</sup> Bu)<sup>m</sup> captures these two atoms and two free ligands to form Au40(SC2H4Ph)24–m(SPh-<sup>t</sup> Bu)m+2. In the final process, the Au40(SC2H4Ph)24<sup>m</sup>(SPh-<sup>t</sup> Bu)m+2 begins to convert to Au36, such that pure Au36(SPh-<sup>t</sup> Bu)24 is eventually obtained (Figure 1(b)). Aun(SR)<sup>m</sup> clusters such as Au28(SPh-<sup>t</sup> Bu)20, Au36(SPh-<sup>t</sup> Bu)24, and Au36(S-c-C5H9)24, none of which can be generated via direct synthesis at atomic precision, have also been synthesized in a size-selective manner by inducing this kind of structural deformation [102].

Research on Innovative Areas (Coordination Asymmetry) (grant number 17H05385). Funding from the Takahashi Industrial and Economic Research Foundation, Futaba Electronics Memorial Foundation, Iwatani Naoji Foundation, Murata Science Foundation, and Ube Industries Foundation is also gratefully acknowledged. We thank Michael D. Judge, MSc, from Edanz

Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

, Junta Inoue<sup>2</sup>

1 Photocatalysis International Research Center, Tokyo University of Science, Noda, Chiba,

2 Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo,

[1] Corain B, Schmid G, Toshima N, editors. Metal Nanoclusters in Catalysis and Materials

[2] McKenzie LC, Zaikova TO, Hutchison JE. Structurally similar triphenylphosphinestabilized undecagolds, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, exhibit distinct ligand exchange pathways with glutathione. Journal of the American Chemical Society. 2014;

[3] Vollenbroek FA, Bour J, Velden JWA. Gold-phosphine cluster compounds. The reactions of

[4] Schulz-Dobrick M, Jansen M. Characterization of gold clusters by crystallization with polyoxometalates: The intercluster compounds [Au9(dpph)4][Mo8O26], [Au9(dpph)4] [PW12O40] and [Au11(PPh3)8Cl2]2[W6O19]. Zeitschrift für anorganische und allgemeine

Recueil des Travaux Chimiques des Pays-Bas. 1980;99:137-141. DOI: 10.1002/recl.

2+ (Au11L8(SCN)2]

<sup>+</sup> and [Au11L8Cl2]

+ .

Science: The Issue of Size Control. 1st ed. Amsterdam: Elsevier; 2007. 470 p

, Yuki Koyama<sup>2</sup>

, Wataru Kurashige<sup>2</sup> and

Ligand-Protected Gold Clusters

65

http://dx.doi.org/10.5772/intechopen.73441

Conflict of interest

Author details

Sakiat Hossain1

Japan

Japan

References

[Au9L8]

19800990410

Yuichi Negishi1,2\*

There are no conflicts to declare.

, Lakshmi V. Nair<sup>2</sup>

\*Address all correspondence to: negishi@rs.kagu.tus.ac.jp

136:13426-13435. DOI: 10.1021/ja5075689

3+ (L = PPh3) with L, SCN and Cl to [Au8L8]

Chemie. 2007;633:2326-2331. DOI: 10.1002/zaac.200700210

#### 6.4. Relation between ligand structure and outcome

In this way, the outcomes are significantly affected by the bulkiness of the ligand in the ligand exchange reactions. Normally, ligand exchange with alkanethiol or PhC2H4SH does not result in structural transformation, but simply leads to ligand exchange. Conversely, a bulky ligand such as <sup>t</sup> Bu-PhSH often leads to structural transformation. At present, these are no clear rules for predicting the final state of the deformed cluster (whether quasi-isomerization or size transformation). The final state seems to be related to the magnitude of the structural transformation and the possibility of isomeric structures with similar stabilities.

#### 7. Summary

This chapter summarized common methods of fabricating Aun(XR)<sup>m</sup> clusters and surveyed the various geometric and electronic structures of these compounds, as well as their physical and chemical properties. Recent discoveries regarding ligand exchange reactions capable of enhancing the functionality of these clusters were also described. Although the precise synthesis of such clusters was first reported only 13 years ago at the time of writing, many studies regarding these clusters have been conducted in the interim, all of which have significantly improved our understanding of synthetic methods as well as the structures and functions of the clusters. It is expected that more information related to Aun(XR)<sup>m</sup> clusters will be gained on the basis of continuing research, leading to the readily synthesis of metal clusters with desired functions in the near future.

### Acknowledgements

This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers JP16H04099, 16 K17480, and 17 K19040) and by the Scientific Research on Innovative Areas (Coordination Asymmetry) (grant number 17H05385). Funding from the Takahashi Industrial and Economic Research Foundation, Futaba Electronics Memorial Foundation, Iwatani Naoji Foundation, Murata Science Foundation, and Ube Industries Foundation is also gratefully acknowledged. We thank Michael D. Judge, MSc, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

## Conflict of interest

Research regarding the mechanism of such reactions has also been conducted. Jin et al. found

(I) ligand exchange, (II) structural distortion, (III) disproportionation, and (IV) size focusing conversion together with further ligand exchange (Figure 10(b)) [112]. In the first process, ligand exchange occurs without size or structural transformations, while the structural distortion of the

generated via direct synthesis at atomic precision, have also been synthesized in a size-selective

In this way, the outcomes are significantly affected by the bulkiness of the ligand in the ligand exchange reactions. Normally, ligand exchange with alkanethiol or PhC2H4SH does not result in structural transformation, but simply leads to ligand exchange. Conversely, a bulky ligand

for predicting the final state of the deformed cluster (whether quasi-isomerization or size transformation). The final state seems to be related to the magnitude of the structural transfor-

This chapter summarized common methods of fabricating Aun(XR)<sup>m</sup> clusters and surveyed the various geometric and electronic structures of these compounds, as well as their physical and chemical properties. Recent discoveries regarding ligand exchange reactions capable of enhancing the functionality of these clusters were also described. Although the precise synthesis of such clusters was first reported only 13 years ago at the time of writing, many studies regarding these clusters have been conducted in the interim, all of which have significantly improved our understanding of synthetic methods as well as the structures and functions of the clusters. It is expected that more information related to Aun(XR)<sup>m</sup> clusters will be gained on the basis of continuing research, leading to the readily synthesis of metal clusters with desired

This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers JP16H04099, 16 K17480, and 17 K19040) and by the Scientific

Bu-PhSH often leads to structural transformation. At present, these are no clear rules

Bu)m+2. In the final process, the Au40(SC2H4Ph)24<sup>m</sup>(SPh-<sup>t</sup>

Bu)<sup>m</sup> (m > ~12) is initiated in the second process. During the

Bu)<sup>m</sup> captures these two atoms and two free ligands to form

Bu)<sup>m</sup> releases two gold atoms to form Au36 and

Bu)24 is eventually obtained (Figure 1(b)). Aun(SR)<sup>m</sup>

Bu)24, and Au36(S-c-C5H9)24, none of which can be

Bu-PhSH:

Bu)m+2 begins

that the following four processes occur in the reaction between Au38(SC2H4Ph)24 and <sup>t</sup>

Bu)20, Au36(SPh-<sup>t</sup>

mation and the possibility of isomeric structures with similar stabilities.

manner by inducing this kind of structural deformation [102].

6.4. Relation between ligand structure and outcome

resulting Au38(SC2H4Ph)24<sup>m</sup>(SPh-<sup>t</sup>

another Au38(SC2H4Ph)24–m(SPh-<sup>t</sup>

Au40(SC2H4Ph)24–m(SPh-<sup>t</sup>

clusters such as Au28(SPh-<sup>t</sup>

such as <sup>t</sup>

64 Ligand

7. Summary

functions in the near future.

Acknowledgements

third process, one Au38(SC2H4Ph)24–m(SPh-<sup>t</sup>

to convert to Au36, such that pure Au36(SPh-<sup>t</sup>

There are no conflicts to declare.

## Author details

Sakiat Hossain1 , Lakshmi V. Nair<sup>2</sup> , Junta Inoue<sup>2</sup> , Yuki Koyama<sup>2</sup> , Wataru Kurashige<sup>2</sup> and Yuichi Negishi1,2\*

\*Address all correspondence to: negishi@rs.kagu.tus.ac.jp

1 Photocatalysis International Research Center, Tokyo University of Science, Noda, Chiba, Japan

2 Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan

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**Section 2**

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74 Ligand

**Chapter 4**

Provisional chapter

**Neuropeptides as Ligands for GPCRs**

Neuropeptides as Ligands for GPCRs

Burcin Duan Sahbaz and Necla Birgul Iyison

Burcin Duan Sahbaz and Necla Birgul Iyison

Additional information is available at the end of the chapter

Neuropeptides constitute an important part of the nervous system, since the simple nerve nets (i.e. of Hydra). The assigned functions of these peptides vary enormously. For instance, besides inhibiting or stimulating the release of some hormones, they can be responsible for tentacle contraction of the Hydra, dropping the tail of the lizard, postnatal care of the beetles and also aggressiveness of humans. They perform these tasks via activating their cognate GPCRs, which are hypothesized to be coevolved with their ligand neuropeptides. In this chapter, we will introduce the concept of neuropeptide, its intracellular maturation process, characteristics of some typical neuropeptide families and the common properties of their cognate GPCRs. At last, we will try to give information about

DOI: 10.5772/intechopen.73504

Neuropeptides are polypeptides expressed in and secreted from neurons. They are produced as propeptides, cleaved into smaller fragments and matured via posttranslational modifications, differing from classical neurotransmitters in size, concentration and secretion mechanisms. They are expressed everywhere in the nervous system, take role in synapsis and can

Neuropeptides constitute the most diverse class of molecules in the body. They have various roles in development, reproduction, physiology and behavior of the animals. There are at least 70 known genes coding for neuropeptide precursor proteins, called prepropeptides, in

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

the widely used methods for studying GPCR-neuropeptide interactions.

Keywords: neuropeptide, GPCR, peptide hormone, interaction

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73504

have distal target organs, as do the hormones.

Abstract

1. Introduction

## **Chapter 4**

Provisional chapter

## **Neuropeptides as Ligands for GPCRs** Neuropeptides as Ligands for GPCRs

Burcin Duan Sahbaz and Necla Birgul Iyison Burcin Duan Sahbaz and Necla Birgul Iyison

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73504

#### Abstract

Neuropeptides constitute an important part of the nervous system, since the simple nerve nets (i.e. of Hydra). The assigned functions of these peptides vary enormously. For instance, besides inhibiting or stimulating the release of some hormones, they can be responsible for tentacle contraction of the Hydra, dropping the tail of the lizard, postnatal care of the beetles and also aggressiveness of humans. They perform these tasks via activating their cognate GPCRs, which are hypothesized to be coevolved with their ligand neuropeptides. In this chapter, we will introduce the concept of neuropeptide, its intracellular maturation process, characteristics of some typical neuropeptide families and the common properties of their cognate GPCRs. At last, we will try to give information about the widely used methods for studying GPCR-neuropeptide interactions.

DOI: 10.5772/intechopen.73504

Keywords: neuropeptide, GPCR, peptide hormone, interaction

#### 1. Introduction

Neuropeptides are polypeptides expressed in and secreted from neurons. They are produced as propeptides, cleaved into smaller fragments and matured via posttranslational modifications, differing from classical neurotransmitters in size, concentration and secretion mechanisms. They are expressed everywhere in the nervous system, take role in synapsis and can have distal target organs, as do the hormones.

Neuropeptides constitute the most diverse class of molecules in the body. They have various roles in development, reproduction, physiology and behavior of the animals. There are at least 70 known genes coding for neuropeptide precursor proteins, called prepropeptides, in

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

mammals and 42 genes in Drosophila. Secreted forms are processed from these prepropeptides and can vary from 5 to 80 amino acids in length. They coexist with classical neurotransmitters; for instance, neurotensin is released with dopamine [1] or vasoactive intestinal peptide with acetylcholine [2]. They mostly act on G-protein coupled receptors (GPCRs) and transduce their signals via intracellular secondary messenger systems.

cosecreted and activating the same receptor. In a study on Drosophila genus, multiple copies of peptides from the same prepropeptide were analyzed [10], and it was found that they were highly conserved and under stabilizing selection. The numbers of peptide copies were the same within the genus (except FMRFamides). This conservation is important for receptor selectivity, affinity or the final response. Additionally, the researchers showed that the most conserved peptides were the most potent ligands for their receptors. Finally, these results on Drosophila neuropeptides supported the idea of evolutionary pressure of peptide-receptor coevolution on neuropeptide selection. This idea was proposed also for the vertebrate neuropeptides. Some regions of the vertebrate peptides are conserved, and these regions are thought to be the most important parts for functioning. For instance, the C-terminal residues of tachykinins are strictly conserved within vertebrates, and this region has roles in binding with tachykinin receptors. However, these similarities between neuropeptides of different species do not have to mean cross-reactivity with the receptors of different species [11, 12]. And this

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 79

Neuropeptides are the gene products that range from 5 to 80 amino acids in length. They born like prepropeptides, which contain an N-terminal signal sequence (between 15 and 40 residues in length). A typical signal sequence contains a positively charged region, a hydrophobic region and some polar but uncharged amino acids until the cleavage site, in the order [13]. This signal sequence is responsible for the anchorage of prepropeptide to the endoplasmic reticulum (ER) membrane via a complex called translocon, where folding and signal peptide cleavage occur. In some prepropeptides, the N-terminal region includes a signal anchor instead of a signal sequence. This signal anchor is responsible for the anchorage of precursor protein to the ER membrane but not cleaved. An example for this signal anchor can be given for the precursor of Allatostatin CC peptides of insects [14]. These signal anchors produce single-pass

Cleaved propeptides are exported to the Golgi for further processing. Mainly, two types of "trypsin-like" endopeptidases are responsible for the cleavage of propeptides. These enzymes are called proprotein convertase 1 (PC1/3) and 2 (PC2). Seven PC types are expressed in mammals, but only three PCs in fruit fly (Amontillado, Dfurin1 and Dfurin2). PCs recognize and cleave the C-terminal site of dibasic residues such as KR or RR, especially of R-X-(R/K/X)- R motif on propeptides [15]. However, cleavage preferences differ within organisms. For instance, if valine or leucine is placed in place of X, the site will become resistant to cleavage by vertebrate PC (furin) but will be efficiently cleaved by insect PCs (Dfurin) [16]. In processing of neuropeptides, mammalian PC1 and PC2 and fly Amontillado are widely expressed in

Cleaved propeptide contains a basic C-terminus, which is further cleaved by carboxypeptidase E. In order to stabilize peptide structure against degradation, C-terminal glycine of most of

fact would be a support for the discussion of peptide-receptor coevolution.

3. Processing and trafficking of neuropeptides

membrane proteins, which can act as juxtacrines in nervous system.

neurons, whereas furins have ubiquitous expression [17, 18].

## 2. Evolution of neuropeptide signaling

Simple nerve nets have evolved since the earliest animals like cnidarians (such as Hydra, sea anemones and jellyfish). Even they have no real brain, their nervous system includes secretory vesicles and produces different types of neuropeptides [3]. Neuropeptides constitute an important part of their nervous system. Therefore, neuropeptide signaling should have been evolved before the divergence of cnidarians and bilaterians, which means more than 550 million years of evolution.

Secretory vesicles of cnidarian nervous system are accumulated at the synapses [4, 5]. This may help for directed-signal transmission such as tentacle contraction of Hydra [6]. However, in vertebrates, the peptide secretory vesicles are not localized only to the synapse but distributed also along the nerve body and soma. Because neuropeptides interact mostly with GPCRs, their action mechanism is slower than classical neurotransmitters. This fact should be disadvantageous for the peptidergic nervous system of the Hydra. However, it was found that they have evolved different receptor-binding mechanisms to overcome this problem. For instance, mammalian RFamide neuropeptides activate different GPCRs and this activation leads to a slow response. On the other hand, Hydra-RFamide I and II act through a so-called peptidegated ionotropic receptor, which is a trimeric complex of ion channels [7]. This system results in an advantage like faster transmission than that of classical neuropeptide-GPCR system, in absence of classical neurotransmitters.

In evolution of neuropeptide signaling, echinoderms are the second most important because they constitute an intermediate step between Protostomia (which include fruit fly) and Deuterostomia (which include both the vertebrates and echinoderms). Echinoderm neuropeptides are suggested to be involved in unusual mechanisms such as autotomy (dropping the tail of the lizard) and regeneration, or control of stiffness of connective tissue [8].

The major assumptions behind the diversity of neuropeptide genes are tandem duplications and following substitutions. Neuropeptide sequences are conserved in most cases (such as oxytocin family). However, some neuropeptide sequences show variations, and these variations can lead to differences in half-life, receptor affinity or expression profiles. Finally, these changes can generate a pressure in the direction of neuropeptide-receptor coevolution. Additionally, the mature peptides that are processed from the same gene can have sequence variations. These variations cannot be explained by gene duplication. An example for this is 37 peptide products of metamorphosin A prepropeptide of sea anemone [9]. All of these peptides can show functional redundancy, which means that they can be coexpressed, cosecreted and activating the same receptor. In a study on Drosophila genus, multiple copies of peptides from the same prepropeptide were analyzed [10], and it was found that they were highly conserved and under stabilizing selection. The numbers of peptide copies were the same within the genus (except FMRFamides). This conservation is important for receptor selectivity, affinity or the final response. Additionally, the researchers showed that the most conserved peptides were the most potent ligands for their receptors. Finally, these results on Drosophila neuropeptides supported the idea of evolutionary pressure of peptide-receptor coevolution on neuropeptide selection. This idea was proposed also for the vertebrate neuropeptides. Some regions of the vertebrate peptides are conserved, and these regions are thought to be the most important parts for functioning. For instance, the C-terminal residues of tachykinins are strictly conserved within vertebrates, and this region has roles in binding with tachykinin receptors. However, these similarities between neuropeptides of different species do not have to mean cross-reactivity with the receptors of different species [11, 12]. And this fact would be a support for the discussion of peptide-receptor coevolution.

## 3. Processing and trafficking of neuropeptides

mammals and 42 genes in Drosophila. Secreted forms are processed from these prepropeptides and can vary from 5 to 80 amino acids in length. They coexist with classical neurotransmitters; for instance, neurotensin is released with dopamine [1] or vasoactive intestinal peptide with acetylcholine [2]. They mostly act on G-protein coupled receptors (GPCRs) and transduce their

Simple nerve nets have evolved since the earliest animals like cnidarians (such as Hydra, sea anemones and jellyfish). Even they have no real brain, their nervous system includes secretory vesicles and produces different types of neuropeptides [3]. Neuropeptides constitute an important part of their nervous system. Therefore, neuropeptide signaling should have been evolved before the divergence of cnidarians and bilaterians, which means more than 550 million years

Secretory vesicles of cnidarian nervous system are accumulated at the synapses [4, 5]. This may help for directed-signal transmission such as tentacle contraction of Hydra [6]. However, in vertebrates, the peptide secretory vesicles are not localized only to the synapse but distributed also along the nerve body and soma. Because neuropeptides interact mostly with GPCRs, their action mechanism is slower than classical neurotransmitters. This fact should be disadvantageous for the peptidergic nervous system of the Hydra. However, it was found that they have evolved different receptor-binding mechanisms to overcome this problem. For instance, mammalian RFamide neuropeptides activate different GPCRs and this activation leads to a slow response. On the other hand, Hydra-RFamide I and II act through a so-called peptidegated ionotropic receptor, which is a trimeric complex of ion channels [7]. This system results in an advantage like faster transmission than that of classical neuropeptide-GPCR system, in

In evolution of neuropeptide signaling, echinoderms are the second most important because they constitute an intermediate step between Protostomia (which include fruit fly) and Deuterostomia (which include both the vertebrates and echinoderms). Echinoderm neuropeptides are suggested to be involved in unusual mechanisms such as autotomy (dropping the tail

The major assumptions behind the diversity of neuropeptide genes are tandem duplications and following substitutions. Neuropeptide sequences are conserved in most cases (such as oxytocin family). However, some neuropeptide sequences show variations, and these variations can lead to differences in half-life, receptor affinity or expression profiles. Finally, these changes can generate a pressure in the direction of neuropeptide-receptor coevolution. Additionally, the mature peptides that are processed from the same gene can have sequence variations. These variations cannot be explained by gene duplication. An example for this is 37 peptide products of metamorphosin A prepropeptide of sea anemone [9]. All of these peptides can show functional redundancy, which means that they can be coexpressed,

of the lizard) and regeneration, or control of stiffness of connective tissue [8].

signals via intracellular secondary messenger systems.

2. Evolution of neuropeptide signaling

absence of classical neurotransmitters.

of evolution.

78 Ligand

Neuropeptides are the gene products that range from 5 to 80 amino acids in length. They born like prepropeptides, which contain an N-terminal signal sequence (between 15 and 40 residues in length). A typical signal sequence contains a positively charged region, a hydrophobic region and some polar but uncharged amino acids until the cleavage site, in the order [13]. This signal sequence is responsible for the anchorage of prepropeptide to the endoplasmic reticulum (ER) membrane via a complex called translocon, where folding and signal peptide cleavage occur. In some prepropeptides, the N-terminal region includes a signal anchor instead of a signal sequence. This signal anchor is responsible for the anchorage of precursor protein to the ER membrane but not cleaved. An example for this signal anchor can be given for the precursor of Allatostatin CC peptides of insects [14]. These signal anchors produce single-pass membrane proteins, which can act as juxtacrines in nervous system.

Cleaved propeptides are exported to the Golgi for further processing. Mainly, two types of "trypsin-like" endopeptidases are responsible for the cleavage of propeptides. These enzymes are called proprotein convertase 1 (PC1/3) and 2 (PC2). Seven PC types are expressed in mammals, but only three PCs in fruit fly (Amontillado, Dfurin1 and Dfurin2). PCs recognize and cleave the C-terminal site of dibasic residues such as KR or RR, especially of R-X-(R/K/X)- R motif on propeptides [15]. However, cleavage preferences differ within organisms. For instance, if valine or leucine is placed in place of X, the site will become resistant to cleavage by vertebrate PC (furin) but will be efficiently cleaved by insect PCs (Dfurin) [16]. In processing of neuropeptides, mammalian PC1 and PC2 and fly Amontillado are widely expressed in neurons, whereas furins have ubiquitous expression [17, 18].

Cleaved propeptide contains a basic C-terminus, which is further cleaved by carboxypeptidase E. In order to stabilize peptide structure against degradation, C-terminal glycine of most of intermediate propeptides is amidated. This amidation is a multistep process of two enzymes in invertebrates, while vertebrates have a multifunctional enzyme to perform this task, called as peptidylglycine alpha-amidating monooxygenase (PAM).

Mature peptides are transported in large dense core vesicles (DCV), which are different from small vesicles delivering classical neurotransmitters. Furthermore, posttranslational modifications occur in DCVs. These modifications may include acetylation, sulfation, glycosylation, phosphorylation and cyclization. Some peptides can be processed even after secretion to the extracellular space. For instance, it was found that CPA6 of A/B family of carboxypeptidases is secreted to the extracellular matrix, cleaves hydrophobic C-terminal residues of neuropeptides and can lead to activation of Angiotensin I while degradation of some other peptides [19].

The engagement of DCVs to the nerve terminals is a very rare event. This is because DCVs respond to the changes in Ca+2 content and hundreds of spikes are needed to stimulate a DCV to release its content [20]. Even in these rare events, very large amounts of neuropeptides are released to the synaptic cleft where they are enzymatically cleaved and degraded. On the other hand, unlike neuropeptides, classical neurotransmitters are very rapidly transported to the membrane, easily released and recycled from the synaptic cleft.

Finally, one precursor protein can generate more than one neuropeptide and these peptides can be distinct or the same. Additionally, a precursor molecule can be alternatively spliced to yield different mature neuropeptides in different cells [21].

## 4. Types, cognate GPCRs and functions

As the simplest nervous system, cnidarians express at least 17 different neuropeptides, which can be grouped in three: FMRFamide-like peptides (FLPs), GLWamides and Hym-355 [3]. The neuropeptides expressed in worm C. elegans are also classified in three major groups, depending on their structural and functional similarities [21]. These groups are called as insulin-like peptides (ILPs), FLPs and neuropeptide-like peptides (NLPs). Vertebrate neuropeptides can be clustered in a wide range of families according to sequence similarities [22]. However, in human, neuropeptides expressed from 96 different genes were clustered in 22 distinct families together with the no-family peptides and deposited in neuropeptide databases [23].

In a study of metazoan (all animals) propeptides, neuropeptides of 10 phyla were taken and clustered in about 80 families according to their similarities within propeptide sequences [22]. Twenty-two of these families showed high similarity with each other. These included FMRFamides, LWamides, myoinhibitory peptide (MIP), neuropeptide FF and gonadotropin inhibitory hormone (GnIH).

In this chapter, we will introduce some of the neuropeptides that show conservation within species (as reviewed from the study of Jékely [22]) or that are specific examples for vertebrates and exclude the ligands interacting with non-GPCR targets. Summary of all mentioned neuropeptide families is given in Table 1.

Peptide family Examples for active

peptides

FMRFamidelike peptides

FMRFamide FLP peptides CCK/gastrin-type

FMRFamide-like

 peptides

Gonadotropin-inhibitory

Vertebrates

hormone

Neuropeptide

Pyroglutamylated

RFamide peptide Prolactin-releasing

Kisspeptin

Tachykinins

 Substance P-like

Cnidaria, nematodes,

C-terminal

FXGXRamide

 Promotion of aggression,

fecundity

Stimulation

 of muscle contractions

 sexual activity and

arthropods

tachykinins

Locustatachykinins

Eledoisin Sialokinin I and II

Skin tachykinins Brain/gut tachykinins

Neurokinin

Neurokinin

 B Neuropeptide

Neuropeptide γ

Substance P

 K

 A

vertebrates

Mammals

Submammalian

 Arthropods Amphibians

C-terminal

FXGLMamide

 Ion transport Vasodilation,

Regulation of

responses

Regulation of

gonadotropin-releasing

Regulation of sensation

Regulation of

reproduction

 hormone

reproduction,

 secretion of

NK-3

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504

NK-2

NK-2

NK-1

81

inflammation

 and pain

NK-2

 smooth muscle contraction

Eledone

C-terminal

F(Y/I)GLMamide

Vasodilation,

Vasodilation

 hypotension

Invertebrates

 peptide

 FF

 Arthropods

Cnidaria

 and

C-terminal RFamide

 peptides

Mollusks Nematodes

Tetrapeptide

C-terminal FMRFamide

C-terminal QFamide

 FMRFamide

Control of feeding, Control of feeding behavior, energy

homeostasis

Cardioacceleration

Inhibition of

Nociception Control of feeding

Stimulation

 of prolactin release

Reproductive

development

gonadotropin

 release

FMRFamide

sulfakinin receptors

GPR147

NPFFR1, NPFFR2

GPR103

PrRP receptors Kisspeptin receptor

NK-1

NKRs

NK-1

NKRs

 and

reproduction,

 sensation

 Various NPRs

Cardioacceleration

nd

Expressed in

 Structural similarity

Function(s)

Cognate GPCR


intermediate propeptides is amidated. This amidation is a multistep process of two enzymes in invertebrates, while vertebrates have a multifunctional enzyme to perform this task, called as

Mature peptides are transported in large dense core vesicles (DCV), which are different from small vesicles delivering classical neurotransmitters. Furthermore, posttranslational modifications occur in DCVs. These modifications may include acetylation, sulfation, glycosylation, phosphorylation and cyclization. Some peptides can be processed even after secretion to the extracellular space. For instance, it was found that CPA6 of A/B family of carboxypeptidases is secreted to the extracellular matrix, cleaves hydrophobic C-terminal residues of neuropeptides and can lead to activation of Angiotensin I while degradation of some other peptides [19].

The engagement of DCVs to the nerve terminals is a very rare event. This is because DCVs respond to the changes in Ca+2 content and hundreds of spikes are needed to stimulate a DCV to release its content [20]. Even in these rare events, very large amounts of neuropeptides are released to the synaptic cleft where they are enzymatically cleaved and degraded. On the other hand, unlike neuropeptides, classical neurotransmitters are very rapidly transported to the

Finally, one precursor protein can generate more than one neuropeptide and these peptides can be distinct or the same. Additionally, a precursor molecule can be alternatively spliced to

As the simplest nervous system, cnidarians express at least 17 different neuropeptides, which can be grouped in three: FMRFamide-like peptides (FLPs), GLWamides and Hym-355 [3]. The neuropeptides expressed in worm C. elegans are also classified in three major groups, depending on their structural and functional similarities [21]. These groups are called as insulin-like peptides (ILPs), FLPs and neuropeptide-like peptides (NLPs). Vertebrate neuropeptides can be clustered in a wide range of families according to sequence similarities [22]. However, in human, neuropeptides expressed from 96 different genes were clustered in 22 distinct families together with the no-family peptides and deposited in

In a study of metazoan (all animals) propeptides, neuropeptides of 10 phyla were taken and clustered in about 80 families according to their similarities within propeptide sequences [22]. Twenty-two of these families showed high similarity with each other. These included FMRFamides, LWamides, myoinhibitory peptide (MIP), neuropeptide FF and gonadotropin

In this chapter, we will introduce some of the neuropeptides that show conservation within species (as reviewed from the study of Jékely [22]) or that are specific examples for vertebrates and exclude the ligands interacting with non-GPCR targets. Summary of all mentioned neuro-

peptidylglycine alpha-amidating monooxygenase (PAM).

80 Ligand

membrane, easily released and recycled from the synaptic cleft.

yield different mature neuropeptides in different cells [21].

4. Types, cognate GPCRs and functions

neuropeptide databases [23].

inhibitory hormone (GnIH).

peptide families is given in Table 1.


Peptide family Examples for active

peptides

Neuropeptide

Neuropeptide

Peptide tyrosine tyrosine

Pancreatic polypeptide

Somatostatin/

Allatostatin

 C

> allatostatin C

SST14 SST28

Galanin/

Allatostatin

 A

Arthropods

C-terminal

No

Inhibition of juvenile hormone synthesis,

structural

regulation of food intake

similarity

Nociception,

 feeding and osmotic regulation

 GalR1, GalR2 and

GalR3

PDF receptor

FGLamide

N-terminal similarity

and C-terminal

amidation

allatostatin A Galanin

> VIP/PACAP

α-Pigment dispersing

Invertebrates

 C-terminal

No

Regulation of circadian clock

structural

similarity

amidation

factor

β-Pigment dispersing

factor

VIP PHI

PHM

PHV

PACAP27 PACAP38

Table 1.

Summary of the

neuropeptide

 families and the similarities

 within these families.

Vertebrates

β-turns and

structures

α-helical

VPAC1 and VPAC2

PAC1, VPAC1 and

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504

VPAC2

83

Vertebrates

Vertebrates

 Vertebrates Arthropods

C-terminal PISCF

No

Inhibition of juvenile hormone synthesis

 AlstR-C

structural

similarity

Inhibition of growth hormone release

 SSTR1, SSTR2A, SSTR2B, SSTR3, SSTR4 and SSTR5

AST-A receptors

and a disulfide

bridge

Sequence similarity

 Vertebrates

 Y

Vertebrates

 and

C-terminal amidation and a

pancreatic polypeptide

structure

 fold

Regulation of blood pressure and feeding

behavior

Inhibition of gastric motility and electrolyte

secretion

Inhibition of pancreatic exocrine secretion

invertebrates

Y

Expressed in

 Structural similarity

Function(s)

Cognate GPCR

GnRH1 and GnRH2

receptors

Y1,2,4,5 and y6


Peptide family Examples for active

peptides

Vasopressin/

Conopressins

Invertebrates

 Sequence similarity and disulfide

bridge between the 1st and 6th

residues

oxytocin

Diuretic hormones

Mesotocin Vasopressin

Oxytocin

Myoinhibitory

Sex peptide

From cnidarians

C-terminal W(X)8 W–amide

> through annelids

> peptide/

GWamides

Myoinhibitory/allatostatin-

C-terminal W(X)6 W–amide and

disulfide bridge

B peptide Prothoracicostatic

hormone

APGWamides

Orexin/

Allatotropin

allatotropin

Orexin A Orexin B

GnRH/

Corazonin

Arthropods

N-terminal pyro-

cardioacceleration,

developmental

Phe in 8th

Mobilization

 of

carbohydrates,

 lipids and

AKH receptor

position

Following

Stimulation

 of testosterone

 and

progesterone

GnRHR

HWS

synthesis

Stimulation

 of

gonadotropin

 release

residues

proteins from the fat body

 processes

melanization

 and

Corazonin receptor

> glutamate

corazonin/

AKH

Adipokinetic

GnRH-like peptides

GnRH

 Annelids and

mollusks

Vertebrates

 hormone

 Arthropods

Mollusks Arthropods

Drosophila),

annelids

Mammals

Sequence similarity

 mollusks,

 (except

Tetrapeptide N-terminal GFK

No

Stimulation

 of juvenile hormone synthesis,

cardioacceleration,

myostimulation

structural

similarity

Sleep and wakefulness

OX1 and OX2

residues

Submammalian

vertebrates

Vertebrates

Antidiuretic

Contraction

 Increasing egg laying, reduction in the

female's receptivity, stimulation

hormone synthesis Inhibition of contractions

oviduct, ecdysteroid

hormone synthesis

Regulation of

ecdysone hormone

Sex organ growth, regulation of

reproduction,

 release of

Torso (not GPCR), also activates SPR

reproduction nd

AT receptor

 of hindgut and

MIPR

 synthesis and juvenile

 of juvenile

 of the uterus, lactation

 activity

Expressed in

 Structural similarity

Function(s) Regulation of

responses, promotion of aggression

Regulation of

Regulation of water balance

reproduction

Conopressin

Diuretic hormone

receptor

Mesotocin receptor

V1A, V1B and V2

OXTR

SPR

 receptor

inflammation

 and pain

Cognate GPCR

82 Ligand

Table 1. Summary of the neuropeptide families and the similarities within these families.

#### 4.1. FMRFamide-like peptides

Genome searches and mass spectrometry-based methods on nematode C. elegans yielded around 30 genes encoding for FLPs. These peptides share a common C-terminal motif like FMRF residues. RNAi studies on these genes showed that FLPs can have roles on different processes such as hyperactivity, timing of egg laying, number of laid eggs, fat metabolism and acetylcholine signaling [24–26]. In mollusk Macrocallista nimbosa, this neuropeptide takes role in cardioexcitatory activity [27]. FLPs are expressed in all of the animal species. However, the conserved C-terminal residues may become FMRFamide, QFamide or RFamide. In arthropods, sulfakinins, myosuppressins, RFamides and other extended FMRFamides have the common C-terminal amidated RF residues. Myosuppressins seem to be restricted to crustaceans and insects and have a role in inhibiting contractions of the hindgut, cardiac muscle and release of adipokinetic hormone [28, 29]. Extended FMRFamides of arthropods affect respiration, heart rate, gut motility and muscle contractions. Drosophila sulfakinin (drosulfakinin) was shown to regulate locomotor behavior [30], feeding behavior [31] and smooth muscle contraction [32]. FMRFamides act through two types of receptors. Most of them activate GPCRs. However, FMRFamides of snail Helix aspersa lead to an excitatory response in amiloridesensitive Na<sup>+</sup> channels [33].

expressed from different genes. Processing of propeptides of vasopressin gene produces three peptides called VP, neurophysin II and copeptin, while processing of oxytocin gene produces only OXT and neurophysin I peptides. OXT and VP bind with their corresponding neurophysins, OXT with neurophysin I and VP with neurophysin II. These neurophysins are responsible for the storage of VP and OXT inside DCVs. In physiological pH, VP and OXT do not bind with neurophysins and circulate freely in the plasma. Both mature VP and OXT are nine amino acids in length, eight of which are identical and contain a disulfide bridge between the first and sixth residues [40], while neurophysins have seven bridges. The first cysteine and the following tyrosine residues play the major role in neurophysin binding [41]. Although VP and OXT show sequence similarity, their functions differ from each other. VP has antidiuretic activity and released as a response to increased blood plasma osmolarity, while OXT has roles in contraction of the uterus and in lactation and is stimulated with suckling movement of the newborn. VP and OXT receptors constitute a big family of GPCRs. There are three types of vasopressin receptors: V1A, V1B and V2. However, only one type of oxytocin receptor was

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 85

Invertebrate homologous peptides also contain the disulfide bridge at the same position and five or six amino acids of the peptides are well conserved. The invertebrate homologs of

These peptides are expressed from Cnidaria to Annelids but not present in vertebrates. This family of peptides shares a common motif like W(X)6W and includes various similar peptides such as myoinhibitory/allatostatin-B peptide (MIP/AST-B), sex peptide (SP), prothoracicostatic

The first AST-B peptide is identified in Locusta migratoria as an MIP. It inhibits contractions of hindgut and oviduct, as well as ecdysteroid synthesis. It has a W(X)6Wamide motif on its Cterminus and is widely expressed in the central nervous system. Similar peptides are identified in Gryllus bimaculatus and found that they inhibited juvenile hormone synthesis in corpora

SP is found in Drosophila male accessory glands and regulates mating behaviors of the females. During mating, SP is released from male's ejaculatory duct and acts on the corresponding receptor on the female reproductive duct, increases egg laying and reduces the female's receptivity. This peptide is 36 amino acids in length. N-terminal eight residues are responsible for sperm binding and stimulation of juvenile hormone synthesis. Following 12 amino acids have roles in innate immune responses against bacteria. And the C-terminal 16 amino acids have role in postmating responses. A disulfide bridge is localized to the C-terminal part of the peptide. In addition, there is an internal W(X)8W motif instead of W(X)6Wamide of the others. Therefore, the tryptophan residues on both peptides seem to be important for receptor binding [44]. Sex peptide receptor (SPR) of Drosophila is CG16752, and this receptor is expressed in female reproductive organs and in the central nervous system of both genders. It is proposed to be Gαi-

coupled. MIP and SP both activate SPR, but MIP has lower affinity for this receptor [45].

vertebrate VP/OXT peptides are conopressins and diuretic hormones (DH) [43].

identified: OXTR [42].

4.4. Myoinhibitory peptide/GWamides

hormone (PTTH) and GWamides (of mollusks).

allata. Therefore, they are called as allatostatins.

#### 4.2. Tachykinins

Vertebrate tachykinins are one of the largest groups of neuropeptides expressed in both invertebrates and vertebrates. They contain conserved C-terminally amidated motifs such as FXGLM residues, while some of arthropod tachykinins show FXGXRamide conservation. These five residues are very conserved but not vital for receptor activation, instead phenylalanine at the fifth position and the C-terminal amidation are essential for their activity. They can be localized both to the brain and the gut of various organisms, as well as the skin of amphibians. They can be secreted from the enteroendocrine cells of mammals as paracrines or as true hormones.

Human tachykinin family includes neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), neuropeptide γ (NPγ) and substance P (SP), which are expressed from two genes. These peptides activate three types of GPCRs: NK-1, NK-2 and NK-3. SP interacts with NK-1, while NKA with NK-2 and NKB with NK-3. Higher concentrations of SP in patients with personality disorders were correlated with aggressive behavior [34]. It was also shown that Drosophila tachykinins have aggression-promoting functions [35] and control systemic lipid homeostasis [36]. Tachykinin-like natalisin peptide regulates sexual activity and fecundity of arthropods [37]. Another tachykinin family peptide, eledoisin, was identified from the salivary glands of mollusk Eledone in 1962 [38]. And, eledoisin and kassinin were shown to be expressed and stimulated ion transport in the frog skin [39].

#### 4.3. Vasopressin/oxytocin

Vasopressin (VP) and oxytocin (OXT) are members of the same family due to their sequence similarity. They are conserved from arthropods to mammals. Vertebrate VP/OXT peptides are expressed from different genes. Processing of propeptides of vasopressin gene produces three peptides called VP, neurophysin II and copeptin, while processing of oxytocin gene produces only OXT and neurophysin I peptides. OXT and VP bind with their corresponding neurophysins, OXT with neurophysin I and VP with neurophysin II. These neurophysins are responsible for the storage of VP and OXT inside DCVs. In physiological pH, VP and OXT do not bind with neurophysins and circulate freely in the plasma. Both mature VP and OXT are nine amino acids in length, eight of which are identical and contain a disulfide bridge between the first and sixth residues [40], while neurophysins have seven bridges. The first cysteine and the following tyrosine residues play the major role in neurophysin binding [41]. Although VP and OXT show sequence similarity, their functions differ from each other. VP has antidiuretic activity and released as a response to increased blood plasma osmolarity, while OXT has roles in contraction of the uterus and in lactation and is stimulated with suckling movement of the newborn. VP and OXT receptors constitute a big family of GPCRs. There are three types of vasopressin receptors: V1A, V1B and V2. However, only one type of oxytocin receptor was identified: OXTR [42].

Invertebrate homologous peptides also contain the disulfide bridge at the same position and five or six amino acids of the peptides are well conserved. The invertebrate homologs of vertebrate VP/OXT peptides are conopressins and diuretic hormones (DH) [43].

#### 4.4. Myoinhibitory peptide/GWamides

4.1. FMRFamide-like peptides

84 Ligand

sensitive Na<sup>+</sup> channels [33].

4.3. Vasopressin/oxytocin

4.2. Tachykinins

hormones.

Genome searches and mass spectrometry-based methods on nematode C. elegans yielded around 30 genes encoding for FLPs. These peptides share a common C-terminal motif like FMRF residues. RNAi studies on these genes showed that FLPs can have roles on different processes such as hyperactivity, timing of egg laying, number of laid eggs, fat metabolism and acetylcholine signaling [24–26]. In mollusk Macrocallista nimbosa, this neuropeptide takes role in cardioexcitatory activity [27]. FLPs are expressed in all of the animal species. However, the conserved C-terminal residues may become FMRFamide, QFamide or RFamide. In arthropods, sulfakinins, myosuppressins, RFamides and other extended FMRFamides have the common C-terminal amidated RF residues. Myosuppressins seem to be restricted to crustaceans and insects and have a role in inhibiting contractions of the hindgut, cardiac muscle and release of adipokinetic hormone [28, 29]. Extended FMRFamides of arthropods affect respiration, heart rate, gut motility and muscle contractions. Drosophila sulfakinin (drosulfakinin) was shown to regulate locomotor behavior [30], feeding behavior [31] and smooth muscle contraction [32]. FMRFamides act through two types of receptors. Most of them activate GPCRs. However, FMRFamides of snail Helix aspersa lead to an excitatory response in amiloride-

Vertebrate tachykinins are one of the largest groups of neuropeptides expressed in both invertebrates and vertebrates. They contain conserved C-terminally amidated motifs such as FXGLM residues, while some of arthropod tachykinins show FXGXRamide conservation. These five residues are very conserved but not vital for receptor activation, instead phenylalanine at the fifth position and the C-terminal amidation are essential for their activity. They can be localized both to the brain and the gut of various organisms, as well as the skin of amphibians. They can be secreted from the enteroendocrine cells of mammals as paracrines or as true

Human tachykinin family includes neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), neuropeptide γ (NPγ) and substance P (SP), which are expressed from two genes. These peptides activate three types of GPCRs: NK-1, NK-2 and NK-3. SP interacts with NK-1, while NKA with NK-2 and NKB with NK-3. Higher concentrations of SP in patients with personality disorders were correlated with aggressive behavior [34]. It was also shown that Drosophila tachykinins have aggression-promoting functions [35] and control systemic lipid homeostasis [36]. Tachykinin-like natalisin peptide regulates sexual activity and fecundity of arthropods [37]. Another tachykinin family peptide, eledoisin, was identified from the salivary glands of mollusk Eledone in 1962 [38]. And, eledoisin and kassinin were shown to be

Vasopressin (VP) and oxytocin (OXT) are members of the same family due to their sequence similarity. They are conserved from arthropods to mammals. Vertebrate VP/OXT peptides are

expressed and stimulated ion transport in the frog skin [39].

These peptides are expressed from Cnidaria to Annelids but not present in vertebrates. This family of peptides shares a common motif like W(X)6W and includes various similar peptides such as myoinhibitory/allatostatin-B peptide (MIP/AST-B), sex peptide (SP), prothoracicostatic hormone (PTTH) and GWamides (of mollusks).

The first AST-B peptide is identified in Locusta migratoria as an MIP. It inhibits contractions of hindgut and oviduct, as well as ecdysteroid synthesis. It has a W(X)6Wamide motif on its Cterminus and is widely expressed in the central nervous system. Similar peptides are identified in Gryllus bimaculatus and found that they inhibited juvenile hormone synthesis in corpora allata. Therefore, they are called as allatostatins.

SP is found in Drosophila male accessory glands and regulates mating behaviors of the females. During mating, SP is released from male's ejaculatory duct and acts on the corresponding receptor on the female reproductive duct, increases egg laying and reduces the female's receptivity. This peptide is 36 amino acids in length. N-terminal eight residues are responsible for sperm binding and stimulation of juvenile hormone synthesis. Following 12 amino acids have roles in innate immune responses against bacteria. And the C-terminal 16 amino acids have role in postmating responses. A disulfide bridge is localized to the C-terminal part of the peptide. In addition, there is an internal W(X)8W motif instead of W(X)6Wamide of the others. Therefore, the tryptophan residues on both peptides seem to be important for receptor binding [44]. Sex peptide receptor (SPR) of Drosophila is CG16752, and this receptor is expressed in female reproductive organs and in the central nervous system of both genders. It is proposed to be Gαicoupled. MIP and SP both activate SPR, but MIP has lower affinity for this receptor [45].

APGWamide is a mollusk tetrapeptide. It is mostly correlated with sex organ growth and reproduction of the animal.

4.7. Neuropeptide Y

4.8. Somatostatin/allatostatin C

4.9. Galanin/allatostatin A

GalR2 and GalR3.

Neuropeptide Y (NPY) family of vertebrate neuropeptides includes NPY, peptide tyrosine tyrosine (PYY) and pancreatic polypeptide (PP). These peptides are C-terminally amidated and show a hairpin-like structure called pancreatic polypeptide fold (PP-fold). This fold was composed of one polyproline helix and one α-helix running antiparallel to each other [56]. Five types of Y receptors (for NPY family) are expressed in mammals (Y1,2,4,5 and y6). It is proposed that hydrophobic surface of the PP-fold is responsible for receptor binding. NPY is localized to the brain, while PP and PYY are localized to the gastrointestinal tract. NPY is a highly conserved peptide from frog to

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 87

A mollusk NPY was identified in Lymnaea stagnalis via activation assays on its corresponding NPY receptor homolog [58]. This peptide was 39 amino acids in length and very similar to the vertebrate NPYs. Invertebrate NPY prepropeptides lead to two peptides, one is NPY and the other one is C-terminal peptide of NPY (CPON). The important residues that are responsible for the PP-fold of vertebrate NPYs are conserved in mollusk NPYs, but only some of them are conserved in Drosophila NPYs. Additionally, C-terminal four residues and amidation, which are essential for the activity of the peptide [59], are conserved between vertebrate and invertebrate NPYs. Lymnaea NPY has role in regulation of energy consumption processes, while the

Allatostatin C (AST-C) is the arthropod homolog of vertebrate somatostatin (SST). SST is found as the inhibitor of growth hormone release from the pituitary gland. And AST-C is the inhibitor of juvenile hormone synthesis in corpora allata. From the same SST propeptide, one peptide with 14 amino acids and another with 28 amino acids are released, which are secreted from and acting on different tissues such as central and peripheral nervous system, as well as gastrointestinal tract. Both SST and AST-C peptides exhibit a disulfide bridge, which is important for receptor affinity [11]. The pharmacophore of SST is defined with FWKT residues. And it functions for the inhibition of pituitary hormones such as growth hormone, thyroid stimulating hormone and adrenocorticotropic hormone. SST acts on six different subtypes of SST

On the other hand, AST-C has highly conserved C-terminal PISCF amino acids. In addition to

Galanin peptide is first identified in porcine intestine. Human galanin propeptide produces two peptides, galanin (30 amino acids) and galanin-message associated peptide (GMAP), after cleavage. N-terminal residues and a C-terminal amidation (except in human) of galanin are highly conserved. These peptides are expressed in both central and peripheral nervous systems and have roles in nociception, feeding and osmotic regulation, via acting on three GPCRs; GalR1,

human. Circulating NPY acts on regulation of blood pressure and eating behavior [57].

other invertebrate NPYs mostly affect food intake of the animal [60].

receptors (SSTRs), SSTR1, SSTR2A, SSTR2B, SSTR3, SSTR4 and SSTR5.

juvenile hormone inhibition, it inhibits heart muscle contraction in Drosophila.

PTTH is a homodimer of two identical peptide chains that are hold together by disulfide bridges [46]. It regulates the reproduction and release of ecdysone hormone. The target receptor of PTTH is Torso, which is a kind of receptor tyrosine kinase [47]. However, it can also activate SPR [48].

#### 4.5. Orexin/allatotropin

Allatotropin (AT) was first identified in Manduca sexta, stimulating juvenile hormone synthesis [49]. These peptides show conservation on their N- and C-termini. They include glycine, phenylalanine and a following basic residue in the order in their N-termini (for instance, GFK residues). This N-terminus is important for biological activity of the peptide. On their C-termini, aromatic amino acids are conserved, followed by an amide group (such as R-amide or Y-amide). These peptides are not identified in Drosophila, Apis mellifera and Nasonia vitripennis. However, Drosophila expresses sex peptide for the same function. Therefore, it seems that different peptides may work for the same function in different species. Additionally, AT has other roles such as cardioacceleration, stimulation of muscle contractions and myostimulation in the gut [50–52].

Orexin receptors (or hypocretin receptors) are found to be orthologs of AT receptors, via similarity on their C-terminus [22]. However, their peptide ligands are not structurally or functionally related. Orexin peptides are about 28–33 amino acids in length. They are hypothalamic neuropeptides and have roles mainly in sleep and wakefulness [53].

#### 4.6. GnRH/corazonin/AKH

Gonadotropin releasing hormone (GnRH) is the peptide-stimulating gonadotropin release in vertebrates. However, invertebrates, such as annelids and mollusks, also express GnRH-like peptides. Octopus GnRH induces synthesis of testosterone and progesterone in the ovary and testis, respectively. From tunicates to mammals, GnRH sequence shows a high conservation. It is a decapeptide that has an N-terminal pyroglutamine and following HWS residues and Cterminal PGamide residues [54].

Insects express corazonin and adipokinetic hormone (AKH), instead. Corazonin is 11 amino acids in length and has a cardioacceleratory effect in cockroaches. However, other actions are defined in other insects, such as melanization in locusts and developmental pathways in other insects such as M. sexta and Bombyx mori. AKH is generally 8–10 amino acids in length. It has an N-terminal pyroglutamate, C-terminal amidation and at least two aromatic residues in between. These aromatic residues (at positions 4 and 8) are important for receptor binding. Its structure exhibits a β-turn between these positions. It regulates mobilization of carbohydrates, lipids and proteins from the fat body. Additionally, it has roles in cardioacceleration as corazonin.

Vertebrate GnRH receptors and insect AKH receptors are closely related. Drosophila corazonin receptor is clustered in the same family of AKH and VP receptors. However, corazonin receptor is highly selective for corazonin peptide [55].

## 4.7. Neuropeptide Y

APGWamide is a mollusk tetrapeptide. It is mostly correlated with sex organ growth and

PTTH is a homodimer of two identical peptide chains that are hold together by disulfide bridges [46]. It regulates the reproduction and release of ecdysone hormone. The target receptor of PTTH is Torso, which is a kind of receptor tyrosine kinase [47]. However, it can also

Allatotropin (AT) was first identified in Manduca sexta, stimulating juvenile hormone synthesis [49]. These peptides show conservation on their N- and C-termini. They include glycine, phenylalanine and a following basic residue in the order in their N-termini (for instance, GFK residues). This N-terminus is important for biological activity of the peptide. On their C-termini, aromatic amino acids are conserved, followed by an amide group (such as R-amide or Y-amide). These peptides are not identified in Drosophila, Apis mellifera and Nasonia vitripennis. However, Drosophila expresses sex peptide for the same function. Therefore, it seems that different peptides may work for the same function in different species. Additionally, AT has other roles such as cardioacceleration, stimulation of muscle contractions and myostimulation in the gut [50–52].

Orexin receptors (or hypocretin receptors) are found to be orthologs of AT receptors, via similarity on their C-terminus [22]. However, their peptide ligands are not structurally or functionally related. Orexin peptides are about 28–33 amino acids in length. They are hypo-

Gonadotropin releasing hormone (GnRH) is the peptide-stimulating gonadotropin release in vertebrates. However, invertebrates, such as annelids and mollusks, also express GnRH-like peptides. Octopus GnRH induces synthesis of testosterone and progesterone in the ovary and testis, respectively. From tunicates to mammals, GnRH sequence shows a high conservation. It is a decapeptide that has an N-terminal pyroglutamine and following HWS residues and C-

Insects express corazonin and adipokinetic hormone (AKH), instead. Corazonin is 11 amino acids in length and has a cardioacceleratory effect in cockroaches. However, other actions are defined in other insects, such as melanization in locusts and developmental pathways in other insects such as M. sexta and Bombyx mori. AKH is generally 8–10 amino acids in length. It has an N-terminal pyroglutamate, C-terminal amidation and at least two aromatic residues in between. These aromatic residues (at positions 4 and 8) are important for receptor binding. Its structure exhibits a β-turn between these positions. It regulates mobilization of carbohydrates, lipids and

Vertebrate GnRH receptors and insect AKH receptors are closely related. Drosophila corazonin receptor is clustered in the same family of AKH and VP receptors. However, corazonin

proteins from the fat body. Additionally, it has roles in cardioacceleration as corazonin.

receptor is highly selective for corazonin peptide [55].

thalamic neuropeptides and have roles mainly in sleep and wakefulness [53].

reproduction of the animal.

activate SPR [48].

86 Ligand

4.5. Orexin/allatotropin

4.6. GnRH/corazonin/AKH

terminal PGamide residues [54].

Neuropeptide Y (NPY) family of vertebrate neuropeptides includes NPY, peptide tyrosine tyrosine (PYY) and pancreatic polypeptide (PP). These peptides are C-terminally amidated and show a hairpin-like structure called pancreatic polypeptide fold (PP-fold). This fold was composed of one polyproline helix and one α-helix running antiparallel to each other [56]. Five types of Y receptors (for NPY family) are expressed in mammals (Y1,2,4,5 and y6). It is proposed that hydrophobic surface of the PP-fold is responsible for receptor binding. NPY is localized to the brain, while PP and PYY are localized to the gastrointestinal tract. NPY is a highly conserved peptide from frog to human. Circulating NPY acts on regulation of blood pressure and eating behavior [57].

A mollusk NPY was identified in Lymnaea stagnalis via activation assays on its corresponding NPY receptor homolog [58]. This peptide was 39 amino acids in length and very similar to the vertebrate NPYs. Invertebrate NPY prepropeptides lead to two peptides, one is NPY and the other one is C-terminal peptide of NPY (CPON). The important residues that are responsible for the PP-fold of vertebrate NPYs are conserved in mollusk NPYs, but only some of them are conserved in Drosophila NPYs. Additionally, C-terminal four residues and amidation, which are essential for the activity of the peptide [59], are conserved between vertebrate and invertebrate NPYs. Lymnaea NPY has role in regulation of energy consumption processes, while the other invertebrate NPYs mostly affect food intake of the animal [60].

#### 4.8. Somatostatin/allatostatin C

Allatostatin C (AST-C) is the arthropod homolog of vertebrate somatostatin (SST). SST is found as the inhibitor of growth hormone release from the pituitary gland. And AST-C is the inhibitor of juvenile hormone synthesis in corpora allata. From the same SST propeptide, one peptide with 14 amino acids and another with 28 amino acids are released, which are secreted from and acting on different tissues such as central and peripheral nervous system, as well as gastrointestinal tract. Both SST and AST-C peptides exhibit a disulfide bridge, which is important for receptor affinity [11]. The pharmacophore of SST is defined with FWKT residues. And it functions for the inhibition of pituitary hormones such as growth hormone, thyroid stimulating hormone and adrenocorticotropic hormone. SST acts on six different subtypes of SST receptors (SSTRs), SSTR1, SSTR2A, SSTR2B, SSTR3, SSTR4 and SSTR5.

On the other hand, AST-C has highly conserved C-terminal PISCF amino acids. In addition to juvenile hormone inhibition, it inhibits heart muscle contraction in Drosophila.

#### 4.9. Galanin/allatostatin A

Galanin peptide is first identified in porcine intestine. Human galanin propeptide produces two peptides, galanin (30 amino acids) and galanin-message associated peptide (GMAP), after cleavage. N-terminal residues and a C-terminal amidation (except in human) of galanin are highly conserved. These peptides are expressed in both central and peripheral nervous systems and have roles in nociception, feeding and osmotic regulation, via acting on three GPCRs; GalR1, GalR2 and GalR3.

Allatostatin A (AST-A) exhibits a conserved C-terminal FGLamide group, which is not similar to galanin peptide. However, these two peptides activate ortholog receptors of vertebrates and arthropods. AST-A peptides are mainly expressed in brain and gut and serve for the inhibition of juvenile hormone synthesis and regulation of food intake, as similar to other AST types.

5. Common features of neuropeptide GPCRs

scaffold interfaces of peptide GPCRs are limited.

Neuropeptides activate various receptors most of which are GPCRs. Some neuropeptides as given in Section 4.4 can bind to membrane receptors that couple with receptor tyrosine kinases (i.e. insulin receptors and Torso for PTTH). Some small neuropeptides do not have defined receptors but are ligands for other peptides or enzymes (i.e. 7B2 binding to PC enzymes and neurophysins binding to VP or OXT). Most of the others interact with their cognate GPCRs from the extracellular region and activate a downstream signal transduction pathway. Peptide

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 89

Class A GPCRs exhibit two types of ligand-binding pockets. In one type, the hydrophobic ligand interacts with the transmembrane (TM) region, and the N-terminal region together with the second extracellular loop (ECL2) forms a closed lid-like structure (i.e. rhodopsin and S1P receptors that have highly hydrophobic ligands). However, in the second type, ECL2 folds over the extracellular region of the receptor and forms a pocket-like vacancy, which is exposed to the soluble environment. Peptide GPCRs show the characteristics of this latter binding pocket. Here, ECL2 comprises sheets, instead of β-hairpin loops of rhodopsin or helices of adrenergic receptors. Another feature of Class A GPCRs is the presence of a disulfide bridge between transmembrane domain 3 (TM3) and ECL2. This bridge is important for the stability of the receptor and serves as a barrier against conformational changes in this region, which is important for the ligand affinity. In a review on the defined 3D structures of Class A GPCRs, the depths of bound ligands were compared with regard to positioning of TM4 [65]. Within the Class A GPCRs that exhibit open binding pockets, amines (i.e. doxepine) were interacting deeply, while peptides and nucleoside ligands were closer to the extracellular environment. Three TM regions (TM3, TM6 and TM7) of Class A GPCRs were proposed to have consensus binding residues. These consensus amino acid positions are 3.32, 3.33, 3.36, 6.48, 6.51 and 7.39 (Ballesteros-Weinstein numbering). However, peptide receptors such as neurotensin receptor (NTSR) and allatostatin C receptor (AlstR-C) were shown to have different interactions within the TM regions. For instance, neurotensin forms salt bridges and hydrogen bonds with the Y3.29, R6.54, R6.55, F6.58 and Y7.35 residues of NTSR1 [66]. Additionally, AST-C was binding with proposed AlstR-C model from the extracellular site, except for the two amino acids of TM6 (I6.59 and F6.60 residues) [67]. In addition to these consensus residues, Venkatakrishnan et al. proposed that the positions 6.48 and 6.51, which were conserved within Class A GPCRs, might be responsible for the structural folding of the binding pocket, forming a scaffold consensus [65]. However, the evidences for these consensus residues of binding pockets and

Secretin-like neuropeptide GPCRs include the receptors for VIP/PACAP, PDF (in invertebrates), calcitonin, insect DHs, corticotropin releasing factor (CRF), GHRH and parathyroid hormone (PTH) peptides. There is less information about the structures of secretin-like neuropeptide receptors than that of rhodopsin-like receptors. Within the receptors mentioned above, the only solved full-length structures come from CRF1 receptor (PDB entry: 4Z9G) and calcitonin receptor (PDB entry: 5UZ7). Additionally, there are ligand-bound structures of glucagon

GPCRs belong to either Class A (rhodopsin-like) or Class B1 (secretin-like) receptor.

#### 4.10. Vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide

Expression of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) is restricted to vertebrates. They belong to the glucagon/secretin superfamily that also includes glucagon, secretin, growth hormone releasing hormone (GHRH) and gastric inhibitory peptide (GIH). VIP and PACAP show structural similarity on their Nterminal 27 amino acids. VIP is expressed in both central and peripheral nervous system, while PACAP in hypothalamus, central nervous system, respiratory and gastrointestinal tract. Mature VIP peptide is very well conserved in both mammals and nonmammalian vertebrates. Short PACAP (27 amino acids in length) is restricted to mammals, but the longer form (38 amino acids in length) can be found also in nonmammalian vertebrates [61]. It exhibits an α-helical structure on binding to the receptor but can fold into different secondary structures in different solutions. PACAP is responsible for the release of growth hormone, luteinizing hormone, adrenocorticotropic hormone, follicle-stimulating hormone and prolactin from the pituitary gland, acts on testis and ovary and stimulates insulin and glucagon release [62]. VIP was discovered due to its vasodilatory effects [63]. It can act as both a paracrine or a hormone. However, its half-life is very short when compared to classical hormones [64]. VIP gene produces other forms of peptides such as peptide histidine isoleucine (PHI), peptide histidine methionine (PHM) and peptide histidine valine (PHV), in different organisms. However, the information about the functions of these peptides is limited.

High similarity between PACAP and VIP peptides make them to activate the same receptors, but with different affinities. Three different PACAP receptors are identified (PAC1, VPAC1 and VPAC2). And two types of PACAP selectivity were detected in tissues. In one type, PAC1 receptor has high affinity for PACAP peptides (PACAP27 and PACAP38) and expressed in anterior pituitary and hypothalamus. For the second selectivity, VPAC1 and VPAC2 receptors showed affinity for both PACAP and VIP peptides, and this was detected in peripheral organs. All of these receptors are known to activate adenylate cyclase, leading to cAMP stimulation. In other circumstances, they can stimulate Ca+2 levels and phospholipase D.

Pigment dispersing factor (PDF) receptors are homologs of VPAC2 in invertebrates. They regulate circadian clock. In nematodes, they regulate locomotion, but in crustaceans, they regulate pigment movements in the retina.

There are other additional neuropeptide families that interact with GPCRs, such as proopiomelanocortin (POMC) family, which is typical for its precursor complexity and others. However, we will not go into details of other families in this chapter.

## 5. Common features of neuropeptide GPCRs

Allatostatin A (AST-A) exhibits a conserved C-terminal FGLamide group, which is not similar to galanin peptide. However, these two peptides activate ortholog receptors of vertebrates and arthropods. AST-A peptides are mainly expressed in brain and gut and serve for the inhibition of juvenile hormone synthesis and regulation of food intake, as similar to other

Expression of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) is restricted to vertebrates. They belong to the glucagon/secretin superfamily that also includes glucagon, secretin, growth hormone releasing hormone (GHRH) and gastric inhibitory peptide (GIH). VIP and PACAP show structural similarity on their Nterminal 27 amino acids. VIP is expressed in both central and peripheral nervous system, while PACAP in hypothalamus, central nervous system, respiratory and gastrointestinal tract. Mature VIP peptide is very well conserved in both mammals and nonmammalian vertebrates. Short PACAP (27 amino acids in length) is restricted to mammals, but the longer form (38 amino acids in length) can be found also in nonmammalian vertebrates [61]. It exhibits an α-helical structure on binding to the receptor but can fold into different secondary structures in different solutions. PACAP is responsible for the release of growth hormone, luteinizing hormone, adrenocorticotropic hormone, follicle-stimulating hormone and prolactin from the pituitary gland, acts on testis and ovary and stimulates insulin and glucagon release [62]. VIP was discovered due to its vasodilatory effects [63]. It can act as both a paracrine or a hormone. However, its half-life is very short when compared to classical hormones [64]. VIP gene produces other forms of peptides such as peptide histidine isoleucine (PHI), peptide histidine methionine (PHM) and peptide histidine valine (PHV), in different organisms. However, the information about the functions of these peptides is

High similarity between PACAP and VIP peptides make them to activate the same receptors, but with different affinities. Three different PACAP receptors are identified (PAC1, VPAC1 and VPAC2). And two types of PACAP selectivity were detected in tissues. In one type, PAC1 receptor has high affinity for PACAP peptides (PACAP27 and PACAP38) and expressed in anterior pituitary and hypothalamus. For the second selectivity, VPAC1 and VPAC2 receptors showed affinity for both PACAP and VIP peptides, and this was detected in peripheral organs. All of these receptors are known to activate adenylate cyclase, leading to cAMP stimulation. In

Pigment dispersing factor (PDF) receptors are homologs of VPAC2 in invertebrates. They regulate circadian clock. In nematodes, they regulate locomotion, but in crustaceans, they regulate

There are other additional neuropeptide families that interact with GPCRs, such as proopiomelanocortin (POMC) family, which is typical for its precursor complexity and others. However, we

other circumstances, they can stimulate Ca+2 levels and phospholipase D.

4.10. Vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide

AST types.

88 Ligand

limited.

pigment movements in the retina.

will not go into details of other families in this chapter.

Neuropeptides activate various receptors most of which are GPCRs. Some neuropeptides as given in Section 4.4 can bind to membrane receptors that couple with receptor tyrosine kinases (i.e. insulin receptors and Torso for PTTH). Some small neuropeptides do not have defined receptors but are ligands for other peptides or enzymes (i.e. 7B2 binding to PC enzymes and neurophysins binding to VP or OXT). Most of the others interact with their cognate GPCRs from the extracellular region and activate a downstream signal transduction pathway. Peptide GPCRs belong to either Class A (rhodopsin-like) or Class B1 (secretin-like) receptor.

Class A GPCRs exhibit two types of ligand-binding pockets. In one type, the hydrophobic ligand interacts with the transmembrane (TM) region, and the N-terminal region together with the second extracellular loop (ECL2) forms a closed lid-like structure (i.e. rhodopsin and S1P receptors that have highly hydrophobic ligands). However, in the second type, ECL2 folds over the extracellular region of the receptor and forms a pocket-like vacancy, which is exposed to the soluble environment. Peptide GPCRs show the characteristics of this latter binding pocket. Here, ECL2 comprises sheets, instead of β-hairpin loops of rhodopsin or helices of adrenergic receptors. Another feature of Class A GPCRs is the presence of a disulfide bridge between transmembrane domain 3 (TM3) and ECL2. This bridge is important for the stability of the receptor and serves as a barrier against conformational changes in this region, which is important for the ligand affinity. In a review on the defined 3D structures of Class A GPCRs, the depths of bound ligands were compared with regard to positioning of TM4 [65]. Within the Class A GPCRs that exhibit open binding pockets, amines (i.e. doxepine) were interacting deeply, while peptides and nucleoside ligands were closer to the extracellular environment. Three TM regions (TM3, TM6 and TM7) of Class A GPCRs were proposed to have consensus binding residues. These consensus amino acid positions are 3.32, 3.33, 3.36, 6.48, 6.51 and 7.39 (Ballesteros-Weinstein numbering). However, peptide receptors such as neurotensin receptor (NTSR) and allatostatin C receptor (AlstR-C) were shown to have different interactions within the TM regions. For instance, neurotensin forms salt bridges and hydrogen bonds with the Y3.29, R6.54, R6.55, F6.58 and Y7.35 residues of NTSR1 [66]. Additionally, AST-C was binding with proposed AlstR-C model from the extracellular site, except for the two amino acids of TM6 (I6.59 and F6.60 residues) [67]. In addition to these consensus residues, Venkatakrishnan et al. proposed that the positions 6.48 and 6.51, which were conserved within Class A GPCRs, might be responsible for the structural folding of the binding pocket, forming a scaffold consensus [65]. However, the evidences for these consensus residues of binding pockets and scaffold interfaces of peptide GPCRs are limited.

Secretin-like neuropeptide GPCRs include the receptors for VIP/PACAP, PDF (in invertebrates), calcitonin, insect DHs, corticotropin releasing factor (CRF), GHRH and parathyroid hormone (PTH) peptides. There is less information about the structures of secretin-like neuropeptide receptors than that of rhodopsin-like receptors. Within the receptors mentioned above, the only solved full-length structures come from CRF1 receptor (PDB entry: 4Z9G) and calcitonin receptor (PDB entry: 5UZ7). Additionally, there are ligand-bound structures of glucagon receptors of which the ligand is not a neuropeptide. Because glucagon receptor has the most well-known structure and secretin receptor is the most studied in this class, we will use them as examples to understand ligand binding of secretin-like neuropeptide GPCRs, even though they are not neuropeptides. Additional information comes from the N-terminal region of ligand-bound structures of PAC1 (PDB ID: 2JOD) and PTH receptor (PDB ID: 3C4M), together with the free forms of V2 receptor (PDB ID: 2X57) and GHRH receptor (PDB ID: 2XDG).

6. Methods to study neuropeptide-GPCR interactions

direct, indirect or in silico methods, or a combination of these.

challenges in working with membrane-bound proteins.

and activity-based assays.

for drug design studies.

6.1. Radioligand-binding assays

ligand interaction.

Studying the ligand interaction properties of GPCRs is an essential concept in pharmacology. Neuropeptide GPCRs contribute to the majority of drug targets in central nervous system disorders. Also, insect neuropeptide GPCRs are valuable targets for pesticide designs. Finding the binding sites, discovering agonists, antagonist and even allosteric modulators, understanding the binding affinities and thermodynamic properties and measuring retention times produce a need for case-specific types of GPCR-ligand interaction studies. These may require

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 91

Direct methods for studying GPCR-ligand interactions involve nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction and surface plasmon resonance (SPR) techniques. The information coming from these studies are deposited in Protein Data Bank (PDB) and increasing every day. However, the increase in deposition of GPCR structures is not as fast as that of soluble protein structures. For instance, most of the data coming from NMR studies include only partial GPCR structures bound with their ligands. Obtaining pure crystals of GPCRs is a challenge in X-ray analysis. And studying with hydrophobic ligands is difficult in SPR method. Therefore, we will not go in detail of these direct methods in this chapter, due to their

Indirect methods for studying GPCR-ligand interactions include fluorescent-based methods, radioligand binding, photoaffinity labeling, luminescence-based methods, force spectroscopy

In silico approaches do not yield direct or indirect evidence for GPCR-ligand interactions, but they reduce the problem space, facilitate the following assays and qualitative comparisons between molecules and can mimic the assay conditions, so that they are highly valuable tools

In this chapter, we will only focus on the indirect methods that are widely used for GPCR-

In principle of radioligand-binding assays, the ligand is previously radiolabeled and added onto the receptor, and its binding is measured quantitatively. The first study of radioligands on GPCRs is that of Lefkowitz and his collaborators where they used I125-labeled adrenocorticotropic hormone (ACTH) against ACTH receptor [81]. Since then, modifications on the method made use of membrane patches and also whole cells [82]. With the help of radioligand saturation binding, indirect binding or kinetic-binding assays can be performed and result in calculation of EC50, Kd values together with the retention time of the ligand on the receptor. Also, they show if the ligand binding is reversible or not. The major challenges of this method are the cost and half-lives of radioligands and the health issues in regard to exposure to them. Agonists cannot be distinguished from antagonists with these assays. Additionally, optimizations should be performed to minimize nonspecific binding (i.e. to the cell, to the plastic ware).

In order to understand ligand-binding features of this class, we need to look at their ligands. Neuropeptide ligands that couple with secretin-like GPCRs have a common secondary structure of at least one α-helix. As that of glucagon peptide, PACAP and CRF exhibit two αhelices. VIP, PTH and calcitonin peptides have only one helical structure. On the other hand, the common feature of these family receptors is that they have a long and complex N-terminus that may include three disulfide bridges forming an α-β-β-α fold [68]. This N-terminal region of the receptors is shown to be important in ligand binding. Provided by the experimental structure of human glucagon receptor, another region on the N-terminus was identified as "stalk" at the top of TM1. And mutagenesis studies on this stalk region proved that it was important for ligand binding, by providing a defined conformation of N-terminal loop with regard to TM1 [69, 70]. As another hypothesis, Dong et al. proposed an endogenous agonism for the N-terminal region of secretin receptor [71]. Here, binding of C-terminus of the ligand to the N-terminus of the receptor results in a conformational change that results in movement of a hidden tripeptide region and becomes an endogenous agonist for the receptor itself. This tripeptide region consists of WDN residues (inside one of the N-terminal helices) on secretin receptor, which are also conserved for calcitonin and VPAC1 neuropeptide receptors.

According to FRET study by Harikumar et al., C-terminal part of secretin peptide was in proximity to the groove above the β-hairpin of receptor N-terminus, while N-terminal part of it was in proximity to ECL3 and TM6 [72]. This model of secretin binding is proposed as a general mechanism for all secretin-like GPCRs. N-terminus of the peptide ligands was shown to be important for receptor activation (i.e. for CRF, calcitonin, glucagon and VIP) [73–76]. Deletion of this region revealed antagonism for the receptor. And C-terminus of the peptide was shown to be involved in ligand binding to the receptor (i.e. VIP, PTH and CRF) [77–79]. This binding includes hydrophobic residues of the helical structures on receptor N-terminus, as well as hydrogen bonds or salt bridges formed between the ligand and polar receptor residues. In this model, the ligand adopts an α-helical structure upon binding to the receptor. This is supported by the soluble structures of glucagon, PTH or PACAP in aqueous solution and their helical structures in organic solvents. Only calcitonin did not change in either media, due to stabilization by disulfide bridges. The salt bridges between the ligand and the receptor are thought to be responsible for the helix formation. After forming a binding helix, this structure is covered by two β-sheets of the receptor N-terminus. Exceptionally, in case of PACAP binding, the peptide wraps around the helical structures of receptor N-terminus [80].

All the details proposed for ligand binding to secretin-like GPCRs add up to a common model of "two-domain" binding. The C-terminus of the peptide is responsible for receptor binding, mostly to the N-terminus of the receptor, producing a conformational change here. And Nterminus of the peptide enters to the TM region and produces a second conformational change that will lead to signal transduction.

## 6. Methods to study neuropeptide-GPCR interactions

receptors of which the ligand is not a neuropeptide. Because glucagon receptor has the most well-known structure and secretin receptor is the most studied in this class, we will use them as examples to understand ligand binding of secretin-like neuropeptide GPCRs, even though they are not neuropeptides. Additional information comes from the N-terminal region of ligand-bound structures of PAC1 (PDB ID: 2JOD) and PTH receptor (PDB ID: 3C4M), together with the free forms of V2 receptor (PDB ID: 2X57) and GHRH receptor (PDB ID: 2XDG).

In order to understand ligand-binding features of this class, we need to look at their ligands. Neuropeptide ligands that couple with secretin-like GPCRs have a common secondary structure of at least one α-helix. As that of glucagon peptide, PACAP and CRF exhibit two αhelices. VIP, PTH and calcitonin peptides have only one helical structure. On the other hand, the common feature of these family receptors is that they have a long and complex N-terminus that may include three disulfide bridges forming an α-β-β-α fold [68]. This N-terminal region of the receptors is shown to be important in ligand binding. Provided by the experimental structure of human glucagon receptor, another region on the N-terminus was identified as "stalk" at the top of TM1. And mutagenesis studies on this stalk region proved that it was important for ligand binding, by providing a defined conformation of N-terminal loop with regard to TM1 [69, 70]. As another hypothesis, Dong et al. proposed an endogenous agonism for the N-terminal region of secretin receptor [71]. Here, binding of C-terminus of the ligand to the N-terminus of the receptor results in a conformational change that results in movement of a hidden tripeptide region and becomes an endogenous agonist for the receptor itself. This tripeptide region consists of WDN residues (inside one of the N-terminal helices) on secretin

receptor, which are also conserved for calcitonin and VPAC1 neuropeptide receptors.

that will lead to signal transduction.

90 Ligand

According to FRET study by Harikumar et al., C-terminal part of secretin peptide was in proximity to the groove above the β-hairpin of receptor N-terminus, while N-terminal part of it was in proximity to ECL3 and TM6 [72]. This model of secretin binding is proposed as a general mechanism for all secretin-like GPCRs. N-terminus of the peptide ligands was shown to be important for receptor activation (i.e. for CRF, calcitonin, glucagon and VIP) [73–76]. Deletion of this region revealed antagonism for the receptor. And C-terminus of the peptide was shown to be involved in ligand binding to the receptor (i.e. VIP, PTH and CRF) [77–79]. This binding includes hydrophobic residues of the helical structures on receptor N-terminus, as well as hydrogen bonds or salt bridges formed between the ligand and polar receptor residues. In this model, the ligand adopts an α-helical structure upon binding to the receptor. This is supported by the soluble structures of glucagon, PTH or PACAP in aqueous solution and their helical structures in organic solvents. Only calcitonin did not change in either media, due to stabilization by disulfide bridges. The salt bridges between the ligand and the receptor are thought to be responsible for the helix formation. After forming a binding helix, this structure is covered by two β-sheets of the receptor N-terminus. Exceptionally, in case of PACAP binding, the peptide wraps around the helical structures of receptor N-terminus [80]. All the details proposed for ligand binding to secretin-like GPCRs add up to a common model of "two-domain" binding. The C-terminus of the peptide is responsible for receptor binding, mostly to the N-terminus of the receptor, producing a conformational change here. And Nterminus of the peptide enters to the TM region and produces a second conformational change Studying the ligand interaction properties of GPCRs is an essential concept in pharmacology. Neuropeptide GPCRs contribute to the majority of drug targets in central nervous system disorders. Also, insect neuropeptide GPCRs are valuable targets for pesticide designs. Finding the binding sites, discovering agonists, antagonist and even allosteric modulators, understanding the binding affinities and thermodynamic properties and measuring retention times produce a need for case-specific types of GPCR-ligand interaction studies. These may require direct, indirect or in silico methods, or a combination of these.

Direct methods for studying GPCR-ligand interactions involve nuclear magnetic resonance (NMR) spectroscopy, X-ray diffraction and surface plasmon resonance (SPR) techniques. The information coming from these studies are deposited in Protein Data Bank (PDB) and increasing every day. However, the increase in deposition of GPCR structures is not as fast as that of soluble protein structures. For instance, most of the data coming from NMR studies include only partial GPCR structures bound with their ligands. Obtaining pure crystals of GPCRs is a challenge in X-ray analysis. And studying with hydrophobic ligands is difficult in SPR method. Therefore, we will not go in detail of these direct methods in this chapter, due to their challenges in working with membrane-bound proteins.

Indirect methods for studying GPCR-ligand interactions include fluorescent-based methods, radioligand binding, photoaffinity labeling, luminescence-based methods, force spectroscopy and activity-based assays.

In silico approaches do not yield direct or indirect evidence for GPCR-ligand interactions, but they reduce the problem space, facilitate the following assays and qualitative comparisons between molecules and can mimic the assay conditions, so that they are highly valuable tools for drug design studies.

In this chapter, we will only focus on the indirect methods that are widely used for GPCRligand interaction.

#### 6.1. Radioligand-binding assays

In principle of radioligand-binding assays, the ligand is previously radiolabeled and added onto the receptor, and its binding is measured quantitatively. The first study of radioligands on GPCRs is that of Lefkowitz and his collaborators where they used I125-labeled adrenocorticotropic hormone (ACTH) against ACTH receptor [81]. Since then, modifications on the method made use of membrane patches and also whole cells [82]. With the help of radioligand saturation binding, indirect binding or kinetic-binding assays can be performed and result in calculation of EC50, Kd values together with the retention time of the ligand on the receptor. Also, they show if the ligand binding is reversible or not. The major challenges of this method are the cost and half-lives of radioligands and the health issues in regard to exposure to them. Agonists cannot be distinguished from antagonists with these assays. Additionally, optimizations should be performed to minimize nonspecific binding (i.e. to the cell, to the plastic ware).

#### 6.2. Photoaffinity labeling

In the study of GPCR-ligand interactions, photoaffinity labeling (PAL) is one of the oldest methods. Here, the ligand is bound with a photoreactive group (PRG). Upon binding with the receptor, PRG is activated by UV light and forms an irreversible covalent bond with the closest residues on the receptor. This approach can be combined with immunoprecipitation and mass spectrometry to sequence the amino acids that are in proximity to the ligand-binding pocket. An example can be given as the study of Ceraudo et al. for the interaction between VIP and VPAC1 receptor. They first labeled the C-terminus of VIP with a photoreactive p-benzoyl-pphenylalanine (Bpa) group. Then they followed by cleavage and Edman sequencing. Finally, they found that the C-terminus of VIP was interacting with the N-terminus of VPAC1 [83].

FRET is based on the energy transfer between two different fluorophores when they come close to a defined distance (typically between 10 and 100 Å). In principle, emission of first fluorophore (donor) should excite the second fluorophore (acceptor). In case of GPCR-ligand interactions, different approaches can be used. First, the ligand and an extracellular domain of the receptor can be expressed in fusion with different fluorescent proteins. When the ligand is in proximity to the receptor, two fluorophores also come close to yield an energy transfer. The difference between the FRET signals of interacting and noninteracting GPCR-ligand couples gives an information about the presence of interaction. FRET can also be time-resolved so that information on kinetics of ligand binding can be achieved. This method was used for various types of receptors such as M1 muscarinic acetylcholine receptor, PTH receptors, neurokinin NK2 receptor, cholecystokinin receptor and secretin receptor [89–93]. In another approach, mapping of the ligand-binding region is possible. Here, cysteine residues can be added to different locations of the proposed binding pocket of GPCR via site-directed mutagenesis. These cysteine residues can bind with small fluorophores which would not interfere ligand binding. Additionally, the environment of the ligand-binding pocket can be assessed, via accessibility of aqueous solution and changes in quenching and polarity upon ligand binding. In a technique by Hoffman et al., tetracysteine residues were added to ICL3 and C-terminus of the GPCR. These residues can bind with FlAsH reagent, which is a small fluorophore. When used in combination with cyan fluorescent protein (CFP), conformational changes upon bind-

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 93

In principle, BRET is similar to FRET by using a bioluminescent donor on one molecule and a fluorescent acceptor on the target molecule. Generally, a luciferase (i.e. Rluc8) is used as the donor. It can be performed real time, giving quantitative information about ligand binding. It is advantageous over FRET, because it does not require an initial illumination of the donor molecule. As an example, Stoddart et al. performed BRET on beta adrenergic receptor 2 (β2AR) with an antagonist in live cells. They generated the N-terminus of the receptor with lumines-

AFM is based on the principle of single-molecule force spectroscopy, in which binding force of two single molecules is measured as a difference in laser deflection. One molecule is bound on the tip of a cantilever and the other molecule stays on a rigid surface (or on cell surface). If interaction occurs, the laser deflection from the cantilever tip differs from the state of no-interaction events. Here, the receptors can be in lipid bilayers, as performed by Pfreundschuh et al. [95] and Alsteens et al. [96], and binding the ligand to the cantilever tip. Also, the method can utilize the receptors

In AFM, direct measurements can be obtained from single molecules, and the controls can be designed to exclude nonspecific-binding events. There is no need for fluorescent, luminescent or radiolabeling of the molecules, which may interfere with the binding sites. In most of the cases, peptide ligands are much smaller than fluorescent proteins. However, in AFM, the peptides can

ing of the ligand were made possible to detect [93].

6.4. Bioluminescence resonance energy transfer (BRET)

cent donor and used a fluorescently labeled ligand [94].

on live cells directly as performed by our group for AlstR-C receptor [67].

6.5. Atomic force microscopy (AFM)

In another study, Grunbect et al. have performed site-directed mutagenesis on some proposed residues of CXCR4. These mutant residues were producing amber stop codons, which can be engineered to incorporate photocrosslinkers (i.e. BzF and azF). They have transfected HEK cells with these mutant constructs and treated the cells with the ligand. After UV activation, lysis and immunopurification of the receptor-ligand complexes, they saw that 189F residue of the receptor was in close proximity to the ligand during binding [84].

#### 6.3. Fluorescence-based methods

The use of fluorescently labeled ligands has many advantages when compared to radioactively labeled ligands. For instance, detection efficiency is higher in fluorescent ligands, and health safety issues are easier to handle for the methods utilizing fluorescent ligands. Additionally, fluorescence-based methods can generate quantitative data as given by radioligand assays (i.e. EC50, etc.). For instance, microscopy and flow cytometry can be used in real-time experiments; they can measure the amount of fluorescence that is interacting with or within the cells [85]. Dissociation rate constants (Kd) of fluorescently labeled ligands can be calculated in various approaches. First, physical separation of bound ligand from free ligand in different fractions can be measured by means of concentrations. Second, the emission intensity of the ligand changes upon binding with the receptor and this change can be measured. Third, diffusion rates of bound and free ligands differ. In an approach called fluorescence correlation spectroscopy (FCS), diffusion rate of labeled ligand can be measured on a highly sensitive confocal microscope. Another approach depends on anisotropy, which means that polarization of the molecule changes between bound and free ligands. As a fifth approach, flow cytometry can be used to detect presence of labeled ligands on receptor carrying cells or beads. At last, the most frequently used sensitive approach is called as fluorescence resonance energy transfer (FRET).

There are other methods such as fluorescence recovery after photobleaching (FRAP) that is similar to FCS in principle. However, this method is used only for GPCR oligomerization or Gprotein coupling until now [86, 87], but no study was performed on GPCR-ligand interactions yet. Another complex approach combines two-photon excitation microscopy with FCS and quantum dot technology (TPE-XCS), which seems very promising for the following days [88]. In this chapter, we will give some more detail on FRET experiments performed on GCPRligand studies that are widely preferred by the researchers.

FRET is based on the energy transfer between two different fluorophores when they come close to a defined distance (typically between 10 and 100 Å). In principle, emission of first fluorophore (donor) should excite the second fluorophore (acceptor). In case of GPCR-ligand interactions, different approaches can be used. First, the ligand and an extracellular domain of the receptor can be expressed in fusion with different fluorescent proteins. When the ligand is in proximity to the receptor, two fluorophores also come close to yield an energy transfer. The difference between the FRET signals of interacting and noninteracting GPCR-ligand couples gives an information about the presence of interaction. FRET can also be time-resolved so that information on kinetics of ligand binding can be achieved. This method was used for various types of receptors such as M1 muscarinic acetylcholine receptor, PTH receptors, neurokinin NK2 receptor, cholecystokinin receptor and secretin receptor [89–93]. In another approach, mapping of the ligand-binding region is possible. Here, cysteine residues can be added to different locations of the proposed binding pocket of GPCR via site-directed mutagenesis. These cysteine residues can bind with small fluorophores which would not interfere ligand binding. Additionally, the environment of the ligand-binding pocket can be assessed, via accessibility of aqueous solution and changes in quenching and polarity upon ligand binding. In a technique by Hoffman et al., tetracysteine residues were added to ICL3 and C-terminus of the GPCR. These residues can bind with FlAsH reagent, which is a small fluorophore. When used in combination with cyan fluorescent protein (CFP), conformational changes upon binding of the ligand were made possible to detect [93].

#### 6.4. Bioluminescence resonance energy transfer (BRET)

In principle, BRET is similar to FRET by using a bioluminescent donor on one molecule and a fluorescent acceptor on the target molecule. Generally, a luciferase (i.e. Rluc8) is used as the donor. It can be performed real time, giving quantitative information about ligand binding. It is advantageous over FRET, because it does not require an initial illumination of the donor molecule. As an example, Stoddart et al. performed BRET on beta adrenergic receptor 2 (β2AR) with an antagonist in live cells. They generated the N-terminus of the receptor with luminescent donor and used a fluorescently labeled ligand [94].

#### 6.5. Atomic force microscopy (AFM)

6.2. Photoaffinity labeling

92 Ligand

6.3. Fluorescence-based methods

In the study of GPCR-ligand interactions, photoaffinity labeling (PAL) is one of the oldest methods. Here, the ligand is bound with a photoreactive group (PRG). Upon binding with the receptor, PRG is activated by UV light and forms an irreversible covalent bond with the closest residues on the receptor. This approach can be combined with immunoprecipitation and mass spectrometry to sequence the amino acids that are in proximity to the ligand-binding pocket. An example can be given as the study of Ceraudo et al. for the interaction between VIP and VPAC1 receptor. They first labeled the C-terminus of VIP with a photoreactive p-benzoyl-pphenylalanine (Bpa) group. Then they followed by cleavage and Edman sequencing. Finally, they found that the C-terminus of VIP was interacting with the N-terminus of VPAC1 [83].

In another study, Grunbect et al. have performed site-directed mutagenesis on some proposed residues of CXCR4. These mutant residues were producing amber stop codons, which can be engineered to incorporate photocrosslinkers (i.e. BzF and azF). They have transfected HEK cells with these mutant constructs and treated the cells with the ligand. After UV activation, lysis and immunopurification of the receptor-ligand complexes, they saw that 189F residue of

The use of fluorescently labeled ligands has many advantages when compared to radioactively labeled ligands. For instance, detection efficiency is higher in fluorescent ligands, and health safety issues are easier to handle for the methods utilizing fluorescent ligands. Additionally, fluorescence-based methods can generate quantitative data as given by radioligand assays (i.e. EC50, etc.). For instance, microscopy and flow cytometry can be used in real-time experiments; they can measure the amount of fluorescence that is interacting with or within the cells [85]. Dissociation rate constants (Kd) of fluorescently labeled ligands can be calculated in various approaches. First, physical separation of bound ligand from free ligand in different fractions can be measured by means of concentrations. Second, the emission intensity of the ligand changes upon binding with the receptor and this change can be measured. Third, diffusion rates of bound and free ligands differ. In an approach called fluorescence correlation spectroscopy (FCS), diffusion rate of labeled ligand can be measured on a highly sensitive confocal microscope. Another approach depends on anisotropy, which means that polarization of the molecule changes between bound and free ligands. As a fifth approach, flow cytometry can be used to detect presence of labeled ligands on receptor carrying cells or beads. At last, the most frequently used sensitive approach is called as fluorescence resonance energy transfer (FRET). There are other methods such as fluorescence recovery after photobleaching (FRAP) that is similar to FCS in principle. However, this method is used only for GPCR oligomerization or Gprotein coupling until now [86, 87], but no study was performed on GPCR-ligand interactions yet. Another complex approach combines two-photon excitation microscopy with FCS and quantum dot technology (TPE-XCS), which seems very promising for the following days [88]. In this chapter, we will give some more detail on FRET experiments performed on GCPR-

the receptor was in close proximity to the ligand during binding [84].

ligand studies that are widely preferred by the researchers.

AFM is based on the principle of single-molecule force spectroscopy, in which binding force of two single molecules is measured as a difference in laser deflection. One molecule is bound on the tip of a cantilever and the other molecule stays on a rigid surface (or on cell surface). If interaction occurs, the laser deflection from the cantilever tip differs from the state of no-interaction events. Here, the receptors can be in lipid bilayers, as performed by Pfreundschuh et al. [95] and Alsteens et al. [96], and binding the ligand to the cantilever tip. Also, the method can utilize the receptors on live cells directly as performed by our group for AlstR-C receptor [67].

In AFM, direct measurements can be obtained from single molecules, and the controls can be designed to exclude nonspecific-binding events. There is no need for fluorescent, luminescent or radiolabeling of the molecules, which may interfere with the binding sites. In most of the cases, peptide ligands are much smaller than fluorescent proteins. However, in AFM, the peptides can

be utilized in their native forms, or they can be functionalized from defined terminal sites. The method in our study also provides the native environment of the receptor as in FRET experiments. And this makes it more advantageous than the methods analyzing purified receptors. AFM setup is suitable for working on adherent cancer cells for long hours, so that the performer can take hundreds of data points from the same cell. And ectopic expression produces enough saturation of the receptor on the surface to detect at each approaching step. Therefore, AFM seems a promising and easy way to study GPCR-ligand interactions on live cells.

caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat.

Neuropeptides as Ligands for GPCRs http://dx.doi.org/10.5772/intechopen.73504 95

[2] Dori I, Parnavelas JG. The cholinergic innervation of the rat cerebral cortex shows two distinct phases in development. Experimental Brain Research. 1989;76(2):417-423

[3] Takahashi T, Takeda N. Insight into the molecular and functional diversity of cnidarian neuropeptides. International Journal of Molecular Sciences. 2015;16:2610-2625. DOI:

[4] Westfall JA, Grimmelikhuijzen CJP. Antho-RFamide immunoreactivity in neuronal synaptic and nonsynaptic vesicles of sea anemones. The Biological Bulletin. 1993;185(1):109-114

[5] Westfall JA, Sayyar KL, Elliott CF, Grimmelikhuijzen CJP. Ultrastructural localization of antho-RWamides I and II at neuromuscular synapses in the gastrodermis and oral sphincter muscle of the sea anemone Calliactis parasitica. The Biological Bulletin. 1995;189(3):280-287

[6] Rushforth NB, Hofman F. Behavioral and electrophysiological studies of hydra. III. Com-

[7] Dürrnagel S, Kuhn A, Tsiairis CD, Williamson M, Kalbacher H. Three homologous subunits form a high affinity peptide-gated ion channel in hydra. The Journal of Biological

[8] Elphick MR. The protein precursors of peptides that affect the mechanics of connective tissue and/or muscle in the echinoderm Apostichopus japonicus. PLoS One. 2012;7(8):

[9] Leviev I, Grimmelikhuijzen CJ. Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis-inducing neuropeptide: A likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(25):11647-11651 [10] Wegener C, Gorbashov A. Molecular evolution of neuropeptides in the genus Drosophila.

[11] Kreienkamp HJ, Larusson HJ, Witte I, Roeder T, Bigul N, Hönck HH, et al. Functional annotation of two orphan G-protein coupled receptors, Drostar1 and 2, from Drosophila melanogaster and their ligands by reverse pharmacology. Journal of Biological Chemistry.

[12] Belmont M, Cazzamali G, Williamson M, Hauser F, Grimmelikhuijzen CJP. Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae. Biochemical and Biophysical Research Communications. 2006;344:160-165 [13] Nielsen H, Krogh A. Prediction of signal peptides and signal anchors by a hidden Markov model. Intelligent Systems for Molecular Biology. 1998;6:122-130. DOI: 10.1.1.47.4026 [14] Veenstra JA. Allatostatin C and its paralog allatostatin double C: The arthropod somatostatins. Insect Biochemistry and Molecular Biology. 2009;39(3):161-170. DOI: 10.1016/j.

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e44492. DOI: 10.1371/journal.pone.0044492

#### 6.6. Activity-based assays

Activity-based assays depend on the previously known downstream effects of the GPCR in cells. The advantage of these assays is that they allow discrimination of agonists from antagonists and also partial agonists. Quantification of EC50 values is possible, so that they can also be used in high-throughput pharmacological studies. Examples can be given as GTPγS (guanosine 5'-O- [gamma-thio]triphosphate) binding assays, cAMP (cyclic adenosine monophosphate) assays, IP3 (inositol triphosphate) and Ca+2 assays, TGF-α (transforming growth factor alpha) shedding assay, β-arrestin recruitment and internalization assay, dimerization assays and voltage-clamp experiments. These assays can be coupled with fluorescent techniques or site-directed mutagenesis of the receptor when required.

## 7. Conclusion

There are at least 80 genes encoding for neuropeptide precursors in human. These precursors give rise to at least 150 mature neuropeptides. And until now, at least 109 of these peptides were shown to signal via GPCRs. All these peptides and their cognate GPCRs are still being studied against the neurological disorders, which range from the simplest stress and pain relief cases to the complex schizophrenia and Alzheimer's disease. Therefore, understanding the kinetics, interactions and transduction pathways of GPCR-neuropeptide signaling systems will remain crucial for the human wealth.

## Author details

Burcin Duan Sahbaz\* and Necla Birgul Iyison

\*Address all correspondence to: burcin.duan1@boun.edu.tr

Molecular Biology and Genetics Department, Bogazici University, Istanbul, Turkey

### References

[1] Ruggeri M, Ungerstedt U, Agnati LF, Mutt V, Harfstrand A, Fuxe K. Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat. Neurochemistry International. 1987;10(4):509-520

[2] Dori I, Parnavelas JG. The cholinergic innervation of the rat cerebral cortex shows two distinct phases in development. Experimental Brain Research. 1989;76(2):417-423

be utilized in their native forms, or they can be functionalized from defined terminal sites. The method in our study also provides the native environment of the receptor as in FRET experiments. And this makes it more advantageous than the methods analyzing purified receptors. AFM setup is suitable for working on adherent cancer cells for long hours, so that the performer can take hundreds of data points from the same cell. And ectopic expression produces enough saturation of the receptor on the surface to detect at each approaching step. Therefore, AFM

Activity-based assays depend on the previously known downstream effects of the GPCR in cells. The advantage of these assays is that they allow discrimination of agonists from antagonists and also partial agonists. Quantification of EC50 values is possible, so that they can also be used in high-throughput pharmacological studies. Examples can be given as GTPγS (guanosine 5'-O- [gamma-thio]triphosphate) binding assays, cAMP (cyclic adenosine monophosphate) assays, IP3 (inositol triphosphate) and Ca+2 assays, TGF-α (transforming growth factor alpha) shedding assay, β-arrestin recruitment and internalization assay, dimerization assays and voltage-clamp experiments. These assays can be coupled with fluorescent techniques or site-directed mutagen-

There are at least 80 genes encoding for neuropeptide precursors in human. These precursors give rise to at least 150 mature neuropeptides. And until now, at least 109 of these peptides were shown to signal via GPCRs. All these peptides and their cognate GPCRs are still being studied against the neurological disorders, which range from the simplest stress and pain relief cases to the complex schizophrenia and Alzheimer's disease. Therefore, understanding the kinetics, interactions and transduction pathways of GPCR-neuropeptide signaling systems

seems a promising and easy way to study GPCR-ligand interactions on live cells.

6.6. Activity-based assays

94 Ligand

esis of the receptor when required.

will remain crucial for the human wealth.

Burcin Duan Sahbaz\* and Necla Birgul Iyison

\*Address all correspondence to: burcin.duan1@boun.edu.tr

Molecular Biology and Genetics Department, Bogazici University, Istanbul, Turkey

[1] Ruggeri M, Ungerstedt U, Agnati LF, Mutt V, Harfstrand A, Fuxe K. Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and

7. Conclusion

Author details

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**Chapter 5**

**Provisional chapter**

**Role of Soluble Fas Ligand in Severity of Dengue**

**Role of Soluble Fas Ligand in Severity of Dengue** 

DOI: 10.5772/intechopen.74617

Dengue disease, which is caused by dengue virus infection, is a major public health in the tropical and subtropical countries in the world. It has a wide spectrum of clinical manifestations ranging from an undifferentiated fever in a mild clinical form (dengue fever [DF]) to the severe clinical and potentially fatal dengue hemorrhagic fever and shock syndrome (DHF/DSS). Recently, a study has suggested that excessive inflammation and apoptosis contribute to the pathogenesis of severe dengue disease. Soluble FasL is a type II membrane protein belonging to the tumor necrosis factor (TNF) family, which induces apoptosis in Fas-bearing cells and neutrophil chemotactic functions. The apoptosis of microvascular endothelial cells may explain the plasma leakage mechanism in DHF and there was a significant increase in soluble Fas-ligand level in DHF patients compared to DF patients. It can be concluded that the soluble Fas ligand is related to the pathogenesis

**Keywords:** soluble Fas ligand, dengue fever, dengue hemorrhagic fever, apoptosis,

Dengue is a public health problem in much of the tropical and subtropical countries in the world. Two-thirds of the world's population is at risk of dengue infection; an estimated 50 million cases occur annually, and around 2.5% of those affected die [1]. Dengue has a wide spectrum of clinical presentations and often has unpredictable clinical outcomes, may be asymptomatic, or may cause undifferentiated febrile illness (viral syndrome), dengue fever (DF), or dengue hemorrhagic fever (DHF) including dengue shock syndrome (DSS), and this

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74617

**Disease**

**Disease**

Nurfadly Zain

Nurfadly Zain

**Abstract**

of dengue infection.

immune response

**1. Introduction**

#### **Role of Soluble Fas Ligand in Severity of Dengue Disease Role of Soluble Fas Ligand in Severity of Dengue Disease**

DOI: 10.5772/intechopen.74617

#### Nurfadly Zain Nurfadly Zain

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74617

#### **Abstract**

Dengue disease, which is caused by dengue virus infection, is a major public health in the tropical and subtropical countries in the world. It has a wide spectrum of clinical manifestations ranging from an undifferentiated fever in a mild clinical form (dengue fever [DF]) to the severe clinical and potentially fatal dengue hemorrhagic fever and shock syndrome (DHF/DSS). Recently, a study has suggested that excessive inflammation and apoptosis contribute to the pathogenesis of severe dengue disease. Soluble FasL is a type II membrane protein belonging to the tumor necrosis factor (TNF) family, which induces apoptosis in Fas-bearing cells and neutrophil chemotactic functions. The apoptosis of microvascular endothelial cells may explain the plasma leakage mechanism in DHF and there was a significant increase in soluble Fas-ligand level in DHF patients compared to DF patients. It can be concluded that the soluble Fas ligand is related to the pathogenesis of dengue infection.

**Keywords:** soluble Fas ligand, dengue fever, dengue hemorrhagic fever, apoptosis, immune response

#### **1. Introduction**

Dengue is a public health problem in much of the tropical and subtropical countries in the world. Two-thirds of the world's population is at risk of dengue infection; an estimated 50 million cases occur annually, and around 2.5% of those affected die [1]. Dengue has a wide spectrum of clinical presentations and often has unpredictable clinical outcomes, may be asymptomatic, or may cause undifferentiated febrile illness (viral syndrome), dengue fever (DF), or dengue hemorrhagic fever (DHF) including dengue shock syndrome (DSS), and this

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

can cause death [2]. Despite much research, pathogenesis which can explain the severity of dengue remains unclear [3, 4]. Severe dengue is characterized by plasma leakage and abnormal bleeding that can lead to shock and death [1, 2, 5]. Pathogenesis of severe dengue diseases (DHF/DSS) has been suggested to be caused by the amplified production of cytokines that ultimately targets the vascular endothelium and leads to an increase in vascular permeability [4, 6, 7]. There is currently no specific treatment for severe dengue due to gaps in understanding the underlying mechanisms.

Abnormal hemostasis and plasma leakage are the main pathophysiological hallmarks in DHF. There is no vasculitis and hence no injury to the vessel walls, and plasma leakage results from the cytokine-mediated increase in vascular permeability [14]. During inflammation, increased vascular permeability occurs primarily via changes in the integrity of inter-endothelial cell junctions. The increase vascular permeability is affected by a number of soluble factors on the endothelium and among them are thrombin, bradykinin, histamine, oxygen free radicals, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNFα) [15]. Some neutrophil products like arachidonic acid (AA) or leukotriene (LT) A4 are further processed by endothelial enzymes through transcellular metabolism before the resulting products thromboxane A2, LTB4, or LTC4 can activate their cognate receptors. Neutrophils

Role of Soluble Fas Ligand in Severity of Dengue Disease http://dx.doi.org/10.5772/intechopen.74617 105

Fas ligand is a 40-kDa type II membrane protein belonging to the tumor necrosis factor (TNF) family of proteins which induce defined cellular responses upon binding to their respective receptors (Fas receptors). The interaction of Fas ligand with its receptor induced programmed

FasL is expressed by many cell types; it is primarily recognized as associated with activated T lymphocytes and natural killer (NK) cells [17]. Fas ligand is expressed in three distinct forms:

**2.** a membranous form stored in intracellular microvesicles which are excreted into the inter-

**3.** the soluble form generated from the cleavage of the membranous molecule by matrix met-

Membrane Fas ligand can be cleaved by metalloproteinases to release soluble protein segments. The soluble and membranous forms of the Fas ligand have different functions in apoptosis. Membranous Fas ligand is the primary mediator of apoptosis through formation of trimers and higher-order structures on the cell surface, while soluble Fas ligand can have proapoptotic, antiapoptotic, and neutrophil chemotactic functions, depending on the nature of other contextual mediators in the microenvironment. Soluble Fas ligand exists as a homotrimer, which is ineffective in co-aggregating Fas receptors. Soluble Fas ligand can induce apoptosis following aggregation with fibronectin of extracellular matrix proteins to form tetramers and higher-order structures. Besides its role in apoptosis, the soluble Fas ligand is a potent inflammatory agent; it induces B cell proliferation and IgE synthesis in conjunction with IL-4, and soluble B cell activating factor (BAFF) co-stimulates B cells. Expression of soluble Fas ligand in several cell types has been shown to induce an effusive neutrophil-mediated inflammatory response, as documented in vivo by either tissue trans-

also generate reactive oxygen species that induce vascular leakage [16].

cellular milieu in response to various physiologic stimuli; and

alloproteinases within minutes of cell surface expression [9].

plant infiltration or neutrophil extravasation to the peritoneal cavity [18].

**2.2. Fas ligand**

cell death (apoptosis) [8].

**1.** a membranous form on the cell surface;

Soluble FasL is a type II membrane protein belonging to the tumor necrosis factor (TNF) family, which induces apoptosis in Fas-bearing cells [8] and neutrophil chemotactic functions [9]. A recent study showed that in addition to the immune response, apoptosis also contributes to the pathogenesis of DHF. An autopsy examination showed that dengue cases show apoptosis in liver cells, brain, intestine, and lungs. The apoptosis of microvascular endothelial cells may explain the plasma leakage mechanism in DHF [10].

## **2. Role of soluble Fas ligand in the severity of dengue disease**

#### **2.1. Dengue infection**

Dengue fever (DF) and its severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become a major international public health problem, especially in the tropical and subtropical regions around the world. An estimated 50 million infections per year occur across approximately 100 countries, with potential for further spread [1, 2]. The disease is caused by a virus belonging to the family Flaviviridae that is spread by Aedes mosquitoes. There are four distinct serotypes of dengue virus (DENV 1–4). All dengue serotypes are capable of causing diseases with a wide spectrum of clinical manifestations, ranging from an undifferentiated fever in a mild clinical form (DF) to the severe clinical and potentially fatal DHF/DSS. Infection with one serotype confers protective immunity against that serotype but not against other serotypes [11]. Dengue fever (DF) is an acute and self-limited illness manifested by fever, headache, myalgia, and arthralgia, and on physical examination there occurs rash. Laboratory tests reveal leukopenia and thrombocytopenia. The more severe dengue DHF is complicated by plasma leakage that occurs around 3–5 days after the disease. A sudden and extensive plasma leakage may result in shock or death, a phenomenon called DSS. Then, patients undergo a defervescence phase marked by an abrupt drop in body temperature, at which point the illness may either wane to recovery or proceed to serious complications [1, 5].

DENV infection in humans starts with a DENV-infected mosquito bite. DENV can replicate in a wide spectrum of cells, including liver, spleen, lymph node, kidney, and other organs, but monocytes, macrophages, and dendritic cells (DC) have been shown to be the major targets for DENV [12]. Monocytes and T lymphocytes, which are infected by DENV, produce several pro-inflammatory mediators which become sources of intense cytokine production [13].

Abnormal hemostasis and plasma leakage are the main pathophysiological hallmarks in DHF. There is no vasculitis and hence no injury to the vessel walls, and plasma leakage results from the cytokine-mediated increase in vascular permeability [14]. During inflammation, increased vascular permeability occurs primarily via changes in the integrity of inter-endothelial cell junctions. The increase vascular permeability is affected by a number of soluble factors on the endothelium and among them are thrombin, bradykinin, histamine, oxygen free radicals, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNFα) [15]. Some neutrophil products like arachidonic acid (AA) or leukotriene (LT) A4 are further processed by endothelial enzymes through transcellular metabolism before the resulting products thromboxane A2, LTB4, or LTC4 can activate their cognate receptors. Neutrophils also generate reactive oxygen species that induce vascular leakage [16].

#### **2.2. Fas ligand**

can cause death [2]. Despite much research, pathogenesis which can explain the severity of dengue remains unclear [3, 4]. Severe dengue is characterized by plasma leakage and abnormal bleeding that can lead to shock and death [1, 2, 5]. Pathogenesis of severe dengue diseases (DHF/DSS) has been suggested to be caused by the amplified production of cytokines that ultimately targets the vascular endothelium and leads to an increase in vascular permeability [4, 6, 7]. There is currently no specific treatment for severe dengue due to gaps in understanding

Soluble FasL is a type II membrane protein belonging to the tumor necrosis factor (TNF) family, which induces apoptosis in Fas-bearing cells [8] and neutrophil chemotactic functions [9]. A recent study showed that in addition to the immune response, apoptosis also contributes to the pathogenesis of DHF. An autopsy examination showed that dengue cases show apoptosis in liver cells, brain, intestine, and lungs. The apoptosis of microvascular endothelial cells may

Dengue fever (DF) and its severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become a major international public health problem, especially in the tropical and subtropical regions around the world. An estimated 50 million infections per year occur across approximately 100 countries, with potential for further spread [1, 2]. The disease is caused by a virus belonging to the family Flaviviridae that is spread by Aedes mosquitoes. There are four distinct serotypes of dengue virus (DENV 1–4). All dengue serotypes are capable of causing diseases with a wide spectrum of clinical manifestations, ranging from an undifferentiated fever in a mild clinical form (DF) to the severe clinical and potentially fatal DHF/DSS. Infection with one serotype confers protective immunity against that serotype but not against other serotypes [11]. Dengue fever (DF) is an acute and self-limited illness manifested by fever, headache, myalgia, and arthralgia, and on physical examination there occurs rash. Laboratory tests reveal leukopenia and thrombocytopenia. The more severe dengue DHF is complicated by plasma leakage that occurs around 3–5 days after the disease. A sudden and extensive plasma leakage may result in shock or death, a phenomenon called DSS. Then, patients undergo a defervescence phase marked by an abrupt drop in body temperature, at which point the illness may either wane to recovery or proceed to serious

DENV infection in humans starts with a DENV-infected mosquito bite. DENV can replicate in a wide spectrum of cells, including liver, spleen, lymph node, kidney, and other organs, but monocytes, macrophages, and dendritic cells (DC) have been shown to be the major targets for DENV [12]. Monocytes and T lymphocytes, which are infected by DENV, produce several pro-inflammatory mediators which become sources of intense cytokine production [13].

**2. Role of soluble Fas ligand in the severity of dengue disease**

the underlying mechanisms.

104 Ligand

**2.1. Dengue infection**

complications [1, 5].

explain the plasma leakage mechanism in DHF [10].

Fas ligand is a 40-kDa type II membrane protein belonging to the tumor necrosis factor (TNF) family of proteins which induce defined cellular responses upon binding to their respective receptors (Fas receptors). The interaction of Fas ligand with its receptor induced programmed cell death (apoptosis) [8].

FasL is expressed by many cell types; it is primarily recognized as associated with activated T lymphocytes and natural killer (NK) cells [17]. Fas ligand is expressed in three distinct forms:


Membrane Fas ligand can be cleaved by metalloproteinases to release soluble protein segments. The soluble and membranous forms of the Fas ligand have different functions in apoptosis. Membranous Fas ligand is the primary mediator of apoptosis through formation of trimers and higher-order structures on the cell surface, while soluble Fas ligand can have proapoptotic, antiapoptotic, and neutrophil chemotactic functions, depending on the nature of other contextual mediators in the microenvironment. Soluble Fas ligand exists as a homotrimer, which is ineffective in co-aggregating Fas receptors. Soluble Fas ligand can induce apoptosis following aggregation with fibronectin of extracellular matrix proteins to form tetramers and higher-order structures. Besides its role in apoptosis, the soluble Fas ligand is a potent inflammatory agent; it induces B cell proliferation and IgE synthesis in conjunction with IL-4, and soluble B cell activating factor (BAFF) co-stimulates B cells.

Expression of soluble Fas ligand in several cell types has been shown to induce an effusive neutrophil-mediated inflammatory response, as documented in vivo by either tissue transplant infiltration or neutrophil extravasation to the peritoneal cavity [18].

Soluble Fas ligand-binding cells express Fas receptors and lead to apoptosis whereas there are some cells that have a default death pathway that can be blocked by a survival factor such as a hormone or growth factor [19].

significantly an increase in the number of human tissues that undergo apoptosis in dengue disease [26]. Apoptosis in white blood cells, brain cells, intestine, and pulmonary endothelial cells form microvasculature in DENV cases. The apoptosis of microvascular endothelial cells

Role of Soluble Fas Ligand in Severity of Dengue Disease http://dx.doi.org/10.5772/intechopen.74617 107

The interaction between DENV and humans leads to the activation of transcription factors, cytokines, and enzymatic factors. These interactions may induce not only inflammatory responses but also apoptotic responses that influence the severity and progression of the disease. The human monocytes infected in vitro by DENV have upregulated Fas expression concomitant with the viral peak, indicating that DENV apoptosis is induced by extrinsic

The dengue patients during acute infection found that TNF-α is the first cytokine detected in patients in the peripheral blood mononuclear cell (PBMC) cultures [28]. These findings showed that TNF-α and its family members are important apoptosis mediators during DENV infection. Among the TNF-α family, the Fas ligand is related to the pathogenesis of dengue infection [29–31]. The apoptotic event is an important event in life and is involved in pathogenesis of dengue infection, and these events occur in response to the variety of signals and stimuli, both internal and external. Mitochondria play a central role in mediating intrinsic apoptotic signals. Changes in the external membrane mitochondria lead to the production of reactive oxygen species related to initial apoptotic events. The extrinsic signal that usually induced apoptosis is by a death receptor such as Fas receptor binding to Fas ligand. After signaling, an enzymatic cascade leads to the activation of a series of cysteinyl aspartate proteases known as caspases and then to cell degradation [29]. DENV can induce apoptosis in DENVinfected cells and disseminate its viral progenies to the neighbor cells. The induction of apoptosis may be an attempt by the host immune system to limit the extent of the infection [13].

Apoptotic signaling may first be triggered by the interaction of the DENV envelope protein with the endosomal membrane during the fusion process while newly synthesized viral proteins may enhance apoptosis. There was indicate the involvement of NF-kB in mediating apoptosis. DENV triggers an apoptotic pathway through phospholipase A2 (PLA2) activation

This apoptotic effect can be either directly derived from the action of arachidonic acid (AA) and superoxide anion on the mitochondria or indirectly derived from the products of apopto-

The recent study showed that soluble Fas ligand can be used as a potential marker of severity of dengue infection because the study showed that there was a significant increase in the

Soluble Fas ligand contributes to the pathogenesis of the severe dengue disease. The interactions between DENV and humans induce not only inflammatory responses but also apoptotic

to superoxide anion generation and subsequently to NF-kB activation.

soluble Fas ligand level in DHF patients compared to DF patients [33].

sis-related genes activated by NF-kB [32].

**3. Conclusions**

may be associated with plasma leakage and hemorrhage during DHF/DSS [27].

apoptotic pathway [13].

The binding of the Fas ligand to the Fas receptor results in the binding of the adapter protein Fas-associated death domain (FADD). FADD then associates with procaspase-8 via dimerization of the death effector domain forming a death-inducing signaling complex (DISC). Once caspase-8 is activated, the execution phase of apoptosis is triggered. Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases, allowing initiation of a protease cascade. One caspase activates other caspases and causes the apoptotic signaling pathway to be activated. Caspases have proteolytic activity and are able to cleave proteins at aspartic acid residues. Once caspases are initially activated, it leads an irreversible commitment towards cell death. There are 10 major caspases that have been identified and categorized into initiators (caspase-2,-8,-9,-10), effectors or executioners (caspase-3,-6,-7), and inflammatory caspases (caspase-1,-4,-5) [20].

There are morphological changes that occur during apoptosis. At the early process of apoptosis, cell shrinkage and pyknosis occur. At the cell shrinkage stage, the cells are smaller in size, the cytoplasm is dense, and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation. Furthermore, plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies. The organelle integrity is still maintained and all of this is enclosed within an intact plasma membrane. Then, apoptotic bodies are subsequently phagocytosed by macrophages degraded within phagolysosomes. There is essentially no inflammatory reaction associated with the process of apoptosis [21].

The recent studies show that soluble Fas ligand also induces cellular activation signals. Soluble Fas ligand induced monocyte responses to secrete pro-inflammatory cytokines and chemotactic factors [22, 23]. Soluble Fas ligand-induced monocyte cytokine responses were associated with rapid expression of pro-inflammatory cytokine genes, suggesting at least partial regulation at the transcriptional level and involving nuclear factor-kappa beta (NF-kB) activation. There are important maturation-dependent differences in the soluble Fas ligand that depend on the signaling pathway whether inducing apoptosis or the silent disappearance of inflammatory cells. Soluble Fas ligand may serve to activate circulating monocytes and recruited macrophages to produce pro-inflammatory mediators that can initiate acute inflammation. This is may play an important role in the regulation of innate immune responses and may contribute to the pathogenesis of a variety of clinically important inflammatory diseases [24].

#### **2.3. Role of soluble Fas ligand in pathogenesis in dengue**

Soluble Fas ligand can induce apoptosis and inflammatory responses. Recently the study has suggested that excessive inflammation and apoptosis contribute to the pathogenesis of severe dengue disease. Although elevated-level cytokines occur in DF patients, the higher level was found in severe dengue disease (DHF/DSS) [25]. The evidence has suggested that there is significantly an increase in the number of human tissues that undergo apoptosis in dengue disease [26]. Apoptosis in white blood cells, brain cells, intestine, and pulmonary endothelial cells form microvasculature in DENV cases. The apoptosis of microvascular endothelial cells may be associated with plasma leakage and hemorrhage during DHF/DSS [27].

The interaction between DENV and humans leads to the activation of transcription factors, cytokines, and enzymatic factors. These interactions may induce not only inflammatory responses but also apoptotic responses that influence the severity and progression of the disease. The human monocytes infected in vitro by DENV have upregulated Fas expression concomitant with the viral peak, indicating that DENV apoptosis is induced by extrinsic apoptotic pathway [13].

The dengue patients during acute infection found that TNF-α is the first cytokine detected in patients in the peripheral blood mononuclear cell (PBMC) cultures [28]. These findings showed that TNF-α and its family members are important apoptosis mediators during DENV infection. Among the TNF-α family, the Fas ligand is related to the pathogenesis of dengue infection [29–31]. The apoptotic event is an important event in life and is involved in pathogenesis of dengue infection, and these events occur in response to the variety of signals and stimuli, both internal and external. Mitochondria play a central role in mediating intrinsic apoptotic signals. Changes in the external membrane mitochondria lead to the production of reactive oxygen species related to initial apoptotic events. The extrinsic signal that usually induced apoptosis is by a death receptor such as Fas receptor binding to Fas ligand. After signaling, an enzymatic cascade leads to the activation of a series of cysteinyl aspartate proteases known as caspases and then to cell degradation [29]. DENV can induce apoptosis in DENVinfected cells and disseminate its viral progenies to the neighbor cells. The induction of apoptosis may be an attempt by the host immune system to limit the extent of the infection [13].

Apoptotic signaling may first be triggered by the interaction of the DENV envelope protein with the endosomal membrane during the fusion process while newly synthesized viral proteins may enhance apoptosis. There was indicate the involvement of NF-kB in mediating apoptosis. DENV triggers an apoptotic pathway through phospholipase A2 (PLA2) activation to superoxide anion generation and subsequently to NF-kB activation.

This apoptotic effect can be either directly derived from the action of arachidonic acid (AA) and superoxide anion on the mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-kB [32].

The recent study showed that soluble Fas ligand can be used as a potential marker of severity of dengue infection because the study showed that there was a significant increase in the soluble Fas ligand level in DHF patients compared to DF patients [33].

## **3. Conclusions**

Soluble Fas ligand-binding cells express Fas receptors and lead to apoptosis whereas there are some cells that have a default death pathway that can be blocked by a survival factor

The binding of the Fas ligand to the Fas receptor results in the binding of the adapter protein Fas-associated death domain (FADD). FADD then associates with procaspase-8 via dimerization of the death effector domain forming a death-inducing signaling complex (DISC). Once caspase-8 is activated, the execution phase of apoptosis is triggered. Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases, allowing initiation of a protease cascade. One caspase activates other caspases and causes the apoptotic signaling pathway to be activated. Caspases have proteolytic activity and are able to cleave proteins at aspartic acid residues. Once caspases are initially activated, it leads an irreversible commitment towards cell death. There are 10 major caspases that have been identified and categorized into initiators (caspase-2,-8,-9,-10), effectors or executioners (caspase-3,-6,-7), and inflammatory caspases

There are morphological changes that occur during apoptosis. At the early process of apoptosis, cell shrinkage and pyknosis occur. At the cell shrinkage stage, the cells are smaller in size, the cytoplasm is dense, and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation. Furthermore, plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies. The organelle integrity is still maintained and all of this is enclosed within an intact plasma membrane. Then, apoptotic bodies are subsequently phagocytosed by macrophages degraded within phagolysosomes. There is essentially no inflammatory reaction associated with the

The recent studies show that soluble Fas ligand also induces cellular activation signals. Soluble Fas ligand induced monocyte responses to secrete pro-inflammatory cytokines and chemotactic factors [22, 23]. Soluble Fas ligand-induced monocyte cytokine responses were associated with rapid expression of pro-inflammatory cytokine genes, suggesting at least partial regulation at the transcriptional level and involving nuclear factor-kappa beta (NF-kB) activation. There are important maturation-dependent differences in the soluble Fas ligand that depend on the signaling pathway whether inducing apoptosis or the silent disappearance of inflammatory cells. Soluble Fas ligand may serve to activate circulating monocytes and recruited macrophages to produce pro-inflammatory mediators that can initiate acute inflammation. This is may play an important role in the regulation of innate immune responses and may contribute to the pathogenesis of a variety of clinically important

Soluble Fas ligand can induce apoptosis and inflammatory responses. Recently the study has suggested that excessive inflammation and apoptosis contribute to the pathogenesis of severe dengue disease. Although elevated-level cytokines occur in DF patients, the higher level was found in severe dengue disease (DHF/DSS) [25]. The evidence has suggested that there is

such as a hormone or growth factor [19].

(caspase-1,-4,-5) [20].

106 Ligand

process of apoptosis [21].

inflammatory diseases [24].

**2.3. Role of soluble Fas ligand in pathogenesis in dengue**

Soluble Fas ligand contributes to the pathogenesis of the severe dengue disease. The interactions between DENV and humans induce not only inflammatory responses but also apoptotic responses that influence the severity and progression of the disease. Soluble Fas ligand can induce apoptosis and is a potent inflammatory agent.

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## **Acknowledgements**

I greatly appreciate Professor Suhartono Taat Putra (University of Airlangga) for stimulating discussions which contributed to this chapter. This work was supported by University of Muhammadiyah Sumatera Utara, Indonesia.

## **Conflict of interest**

The authors declare that they have no conflicts of interests with commercial or other affiliations.

## **Author details**

Nurfadly Zain

Address all correspondence to: drnurfadly@gmail.com

Department of Tropical Medicine, University of Muhammadiyah Sumatera Utara, Indonesia

## **References**


responses that influence the severity and progression of the disease. Soluble Fas ligand can

I greatly appreciate Professor Suhartono Taat Putra (University of Airlangga) for stimulating discussions which contributed to this chapter. This work was supported by University of

The authors declare that they have no conflicts of interests with commercial or other affiliations.

Department of Tropical Medicine, University of Muhammadiyah Sumatera Utara, Indonesia

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**Acknowledgements**

108 Ligand

**Conflict of interest**

**Author details**

Nurfadly Zain

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## *Edited by Chandraleka Saravanan and Bhaskar Biswas*

The book *Ligand* describes the diversity and versatility of ligands, covering structural features, donor-acceptor properties and secondary functions like molecular recognition. Moreover, this book also provides a comprehensive account on the applicability like catalysis, sensors, supramolecular assembly, photochemical property, bioinorganic chemistry, and so on. The advancement of fundamentals in ligand design and the control of physicochemical properties of coordination compounds has largely increased emphasis on understanding the structural and electronic features toward different perspectives in materials science. In this regard, this book has a special appeal to chemists, biologists and others. This book will be beneficial for the graduate students, teachers, researchers and other professionals who are interested to fortify and expand their knowledge in chemistry, biology, microbiology, biotechnology, materials science, environmental science and so on.

Published in London, UK © 2018 IntechOpen © eranicle / iStock

Ligand

Ligand

*Edited by Chandraleka Saravanan* 

*and Bhaskar Biswas*