**3. Proteins immobilization on cellulose**

Cotton is an excellent material for immobilized enzyme active functional textiles because, like the surface of soluble proteins, it is hydrophilic and typically non-denaturing. Many methods are now available for coupling enzymes and other biologically active compounds to solid supports [33]. Several involve the preliminary preparation of carboxymethyl or *p*amino-benzyl ether derivative of a general support such as cellulose. A simple process involves the use trichloro-1,3,5-triazine [34,35] or chlorotriazine derivatives with solubilizing groups such as methoxycarbonyl or methylcarbamoyl groups which make them very convenient reagents in the coupling with a cellulose carrier [36].

There are also known other proficient approaches to the covalent attachment of enzymes to cotton cellulose. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against *B. subtilis* was assessed. Inhibition of *B. subtilis* growth was observed to be optimal within a range of 0.3 to 0.14 mM of lysozyme. This approach has also been applied to organophopsphorous hydrolase and human neutrophil elastase. Immobilizing the chromogenic peptide substrate of human neutrophil elastase on cellulose and studying its interaction with the elastase enzyme provided colorimetric response of human neutrophil elastase [37].

Invertase was immobilized onto the cellulose membrane activated photochemicaly using 1 fluoro-2-nitro-4-azidobenzene as a photolinker and used in a flow through reactor system for conversion of sucrose to glucose and fructose [38].

Over the years, several cellulose affinity ligands have been constructed based on application of noncatalytic domain of glycosidic hydrolase (CBD). This cellulose specific anchor was originally identified in *Trichoderma reesei* and *Cellulomonas fimi*. CBDs is binding on insoluble cellulose through high-affinity noncovalent interactions [39] and it is enabling the further fusion of an antibody-binding domain (i.e., protein A, protein G, protein L). Cellulose-binding domains (CBD) are ideal immobilization domains for affinity ligands because they fold independently and do not interfere with their fusion partner. [40] Coupling to cellulose matrices orients the fusion partner away from the solid support [41] reducing steric hindrance; and their high-affinity binding to cellulose is considered nearly irreversible. [42] At present, many CBD-tagged affinity ligands are purified before attachment to their solid support matrix. [43] For large-scale applications, it could be beneficial to directly immobilize the affinity ligand at the source of production, thus avoiding the cost and time required for purification.

248 Cellulose – Medical, Pharmaceutical and Electronic Applications

followed by coupling of predefined peptides.

**3. Proteins immobilization on cellulose** 

convenient reagents in the coupling with a cellulose carrier [36].

enzyme provided colorimetric response of human neutrophil elastase [37].

for conversion of sucrose to glucose and fructose [38].

Application of cellulose as a support for synthesis of complex template-assembled synthetic proteins (TASP) by orthogonal assembly of small libraries of purified peptide building blocks has been reviewed [32]. In most cases the linear template precursor was prepared by standard solid phase peptide synthesis (SPPS) on synthetic resin with orthogonal protecting groups followed by head-to-tail cyclisation of the linear precursor peptide and anchoring the template structure on cellulose. The strategy involving cleavable linker allowed control of the progress of synthesis on polystyrene resin. Final assembly of peptides prepared under standard SPPS conditions proceeded by successive cleavage of orthogonal protecting groups

Cotton is an excellent material for immobilized enzyme active functional textiles because, like the surface of soluble proteins, it is hydrophilic and typically non-denaturing. Many methods are now available for coupling enzymes and other biologically active compounds to solid supports [33]. Several involve the preliminary preparation of carboxymethyl or *p*amino-benzyl ether derivative of a general support such as cellulose. A simple process involves the use trichloro-1,3,5-triazine [34,35] or chlorotriazine derivatives with solubilizing groups such as methoxycarbonyl or methylcarbamoyl groups which make them very

There are also known other proficient approaches to the covalent attachment of enzymes to cotton cellulose. Lysozyme was immobilized on glycine-bound cotton through a carbodiimide reaction. The attachment to cotton fibers was made through a single glycine and a glycine dipeptide esterified to cotton cellulose. Higher levels of lysozyme incorporation were evident in the diglycine-linked cotton cellulose samples. The antibacterial activity of the lysozyme-conjugated cotton cellulose against *B. subtilis* was assessed. Inhibition of *B. subtilis* growth was observed to be optimal within a range of 0.3 to 0.14 mM of lysozyme. This approach has also been applied to organophopsphorous hydrolase and human neutrophil elastase. Immobilizing the chromogenic peptide substrate of human neutrophil elastase on cellulose and studying its interaction with the elastase

Invertase was immobilized onto the cellulose membrane activated photochemicaly using 1 fluoro-2-nitro-4-azidobenzene as a photolinker and used in a flow through reactor system

Over the years, several cellulose affinity ligands have been constructed based on application of noncatalytic domain of glycosidic hydrolase (CBD). This cellulose specific anchor was originally identified in *Trichoderma reesei* and *Cellulomonas fimi*. CBDs is binding on insoluble cellulose through high-affinity noncovalent interactions [39] and it is enabling the further fusion of an antibody-binding domain (i.e., protein A, protein G, protein L). Cellulose-binding domains (CBD) are ideal immobilization domains for affinity ligands because they fold independently and do not interfere with their fusion partner. [40] Coupling to cellulose Horseradish peroxidase (HRP) was immobilized to cellulose with cellulose-binding domain (CBD) as a mediator, using a ligand selected from a phage-displayed random peptide library. A 15-mer random peptide library was panned on cellulose-coated plates covered with CBD in order to find a peptide that binds to CBD in its bound form. The sequence LHS, which was found to be an efficient binder of CBD, was fused to a synthetic gene of HRP as an affinity tag. The tagged enzyme (tHRP) was then immobilized on microcrystalline cellulose coated with CBD, thereby demonstrating the indirect immobilization of a protein to cellulose *via* three amino acids selected by phage display library and CBD [44].

As a model system, it has been developed a fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD) tethered directly onto cellulose. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, singleuse antibody purification system. This straightforward preparation of purification reagents make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals [45].

In several cases it could be beneficial to directly immobilize the affinity ligand at the source of production, thus avoiding the cost and time required for purification. A potential use of cellulose-supported affinity ligands for purification of other bioproducts from homogenates from genetically modified plants expressing recombinant proteins is under intensive studies. To examine the potential of immobilizing affinity purification ligands onto cellulose matrices in a single step, the yeast *P. pastoris* were engineered to express and secrete a chimeric protein consisting of antibody-binding proteins L and G[45] fused to a cellulosebinding domain. A similar fusion was recently reported for cell capture in hollow-fiber bioreactors. There are reports on the direct immobilization of chimeric LG-CBD proteins onto cellulosic resins for antibody purification. Both protein L and protein G domains retained dual functionality demonstrated by the specific binding and purification of scFv and IgG antibodies from complex feed stocks of yeast supernatants and tobacco plant homogenates. This is a step towards the rapid generation of inexpensive affinity purification reagents and systems, to reduce the costs associated with downstream processing of pharmaceutical products, including antibodies, from complex production systems such as genetically modified crop plants.

Copolymers having polypeptide side chains grafted on cellulose main chain were used for adhesion of fibroblasts. The factor likely to play a key role in determining the binding ability was the balance between the hydrophilicity and hydrophobicity of the main- and side-chain components [46].

## **4. Protein sensors**

Current research in the field of pathogen detection in food matrixes is aimed at creating fast and reliable detection platforms. Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications, development of diagnostic reagents and biosensors. By using engineered antibodies a pentavalent bispecific antibody were prepared by pentamerizing five single-domain antibodies and five cellulose-binding modules. This molecule was dually functional as it bound to cellulose-based filters as well as *S. aureus* cells. When impregnated in cellulose filters, the bispecific pentamer recognized *S. aureus* cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants [47]. Another approach for designing protein sensor used ultrathin films of cellulose modified on surface with small engineered peptides HWRGWV or HWRGWVA as substrate for protein detection. Primary tests run with peptide HWRGWV confirmed that there was an abundant amount of protein absorbed onto the surface, particularly with lower concentration and the sensitivity of a peptide greatly affects the ability to adsorb analytes onto the surface, demonstrating that cellulose substrates can be used to immobilize peptides which can further be used to selectively bind biomolecules [48].

Cellulose Functionalysed with Grafted Oligopeptides 251

O O

O O NH2 <sup>O</sup> <sup>O</sup> NH2

(C-terminal Aaa)

e) 20% piperidine

a') Fmoc-Gly-OH, DIC, NMI

cell-based assays huge number of

**microtiter plate**

h') gaseous NH3 i') punch press

NH2

C-terminal amino acid of peptide sequence

The method was initiated as an uncomplicated technique for the positionally addressable, parallel chemical synthesis on a membrane support. SPOT synthesis of peptides on cellulose paper is a special type of solid phase peptide synthesis (SPPS) with each spot considered as a separate reaction vessel. The general strategy for parallel peptide assembly on a cellulose

filter paper

b) 20% piperidine

c) Fmoc--Ala-OPfp c') Fmoc-Aaa-OPfp

e) 20% piperidine d) Ac2O, DIEA

g) side-chain deprotection

f) SPOT-synthesis (from C to N terminus)

membrane is shown in Figure 3.

O O

O O

peptide assay

**Figure 3.** SPOT technology procedure.

h) blocking buffer i) incubation procedure NH2

NH2

d) Ac2O, DIEA

O O

NH2

a) Fmoc--Ala-OH DIC, NMT

soluble peptydes on-supprot assay membrane-bound

Plain cellulose membranes (filter paper, chromatography paper) are commonly used as a support in the SPOT synthesis. These are porous, hydrophilic, flexible and stable in the organic solvents used for peptide synthesis. Cellulose membranes are relatively inexpensive material, which makes them very useful for biochemical and biological studies in aqueous

The sensor for human neutrophil elastase (HNE), an enzyme engaged in chronic wounds healing was prepared based on colorimetric determination of enzyme activity. For colorimetric detection of human neutrophil elastase chromogenic peptide substrate Succinyl-Ala-Ala-Pro-Ala-pNA and its analog Succinyl-Ala-Ala-Pro-Val-pNA were attached to derivatized cellulose. Cellulose was pre-treated with 3-aminopropyltriethoxysilane to form the amino-propyloxy ether of cellulose, then reacted with the HNE chromogenic paranitroanilide peptide substrates to form a covalently linked conjugate of cellulose (Cell-APsuc-Ala-Ala-Pro-Ala-pNA or Cell-AP-suc-Ala-Ala-Pro-Val-pNA) through amide bond between the Cell-AP amine and the succinyl carboxylate of the substrate. The colorimetric response of the cellulose-bound chromophore was assessed by monitoring release of pnitroaniline from the derivatized cellulose probe to determine human neutrophil elastase levels from 5.0 x 10-3 to 6.0 units per mL [49].

## **5. Epitope mapping - SPOT methodology**

The SPOT synthesis of peptides, developed by Ronald Frank [50], has become one of the most frequently used methods for synthesis and screening of peptides on arrays. The method is a very useful tool for screening solid-phase and solution-phase assays with the size of arrays changeable from a few peptides up to approximately 8000 peptides [51]. Several hundred papers regarding modification and application of the SPOT method have been published [52].

The method was initiated as an uncomplicated technique for the positionally addressable, parallel chemical synthesis on a membrane support. SPOT synthesis of peptides on cellulose paper is a special type of solid phase peptide synthesis (SPPS) with each spot considered as a separate reaction vessel. The general strategy for parallel peptide assembly on a cellulose membrane is shown in Figure 3.

**Figure 3.** SPOT technology procedure.

250 Cellulose – Medical, Pharmaceutical and Electronic Applications

Current research in the field of pathogen detection in food matrixes is aimed at creating fast and reliable detection platforms. Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications, development of diagnostic reagents and biosensors. By using engineered antibodies a pentavalent bispecific antibody were prepared by pentamerizing five single-domain antibodies and five cellulose-binding modules. This molecule was dually functional as it bound to cellulose-based filters as well as *S. aureus* cells. When impregnated in cellulose filters, the bispecific pentamer recognized *S. aureus* cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants [47]. Another approach for designing protein sensor used ultrathin films of cellulose modified on surface with small engineered peptides HWRGWV or HWRGWVA as substrate for protein detection. Primary tests run with peptide HWRGWV confirmed that there was an abundant amount of protein absorbed onto the surface, particularly with lower concentration and the sensitivity of a peptide greatly affects the ability to adsorb analytes onto the surface, demonstrating that cellulose substrates can be used to immobilize peptides which can further be used to selectively

The sensor for human neutrophil elastase (HNE), an enzyme engaged in chronic wounds healing was prepared based on colorimetric determination of enzyme activity. For colorimetric detection of human neutrophil elastase chromogenic peptide substrate Succinyl-Ala-Ala-Pro-Ala-pNA and its analog Succinyl-Ala-Ala-Pro-Val-pNA were attached to derivatized cellulose. Cellulose was pre-treated with 3-aminopropyltriethoxysilane to form the amino-propyloxy ether of cellulose, then reacted with the HNE chromogenic paranitroanilide peptide substrates to form a covalently linked conjugate of cellulose (Cell-APsuc-Ala-Ala-Pro-Ala-pNA or Cell-AP-suc-Ala-Ala-Pro-Val-pNA) through amide bond between the Cell-AP amine and the succinyl carboxylate of the substrate. The colorimetric response of the cellulose-bound chromophore was assessed by monitoring release of pnitroaniline from the derivatized cellulose probe to determine human neutrophil elastase

The SPOT synthesis of peptides, developed by Ronald Frank [50], has become one of the most frequently used methods for synthesis and screening of peptides on arrays. The method is a very useful tool for screening solid-phase and solution-phase assays with the size of arrays changeable from a few peptides up to approximately 8000 peptides [51]. Several hundred papers regarding modification and application of the SPOT method have

**4. Protein sensors** 

bind biomolecules [48].

been published [52].

levels from 5.0 x 10-3 to 6.0 units per mL [49].

**5. Epitope mapping - SPOT methodology** 

Plain cellulose membranes (filter paper, chromatography paper) are commonly used as a support in the SPOT synthesis. These are porous, hydrophilic, flexible and stable in the organic solvents used for peptide synthesis. Cellulose membranes are relatively inexpensive material, which makes them very useful for biochemical and biological studies in aqueous

and organic media. However, since cellulose is not stable against harsh chemical conditions, the SPOT synthesis was developed for the milder type of the two major SPPS strategies based on the Fmoc protection of amine function of the main peptide chain [53] and orthogonal protecting groups used for protection of side chains. [54].

Cellulose Functionalysed with Grafted Oligopeptides 253

as a linker moiety was reported by Ay and co-workers [70]. Proposed solution was applied to sorting peptides according to their *C* termini using modified membranes with the

> NH2 O <sup>R</sup> <sup>O</sup>

> > <sup>2</sup>NH NH2

N-CAPE membrane

NH2

O

CAPE-membrane

TOTD like membrane

O NH O NH2

cellulose

OH

NH2

3

H OH

TOTD

DAP

OH

ester type membrane

cellulose

O N

cellulose

O NH-Prot OH

O S O

**Scheme 4.** Different methods of amino functionalization of cellulose membranes: I) preparation of ester type membrane with amino acid; II) functionalization with epibromohydrin and subsequent reaction with TODT or DAP providing TODT or *N-*CAPE membranes; III) treatment with *N-*protected 2,3 epoxypropylamine giving CAPE membrane; IV) treatment with tosyl chloride and subsequent reaction

corresponding *C*-terminal amino acids anchored either spot- or surface-wise.

OH NH2 <sup>O</sup>

O

+

cellulose +

**I)**

**II)**

O N

OH

**III)**

**IV)**

cellulose

OH

H OH

TOTD membrane

O

S O

O Cl

with TODT giving a TOTD like membrane.

NH-Prot

cellulose <sup>+</sup> cellulose

<sup>2</sup>NH O NH2

TOTD

OH R

OH <sup>O</sup> Br

3

O NH2

3

cellulose <sup>+</sup> cellulose cellulose

Br

cellulose cellulose

## **5.1. Membrane modifications**

Cellulose membranes are still the most widely used supports for SPOT peptide synthesis. The esterification of hydroxy functions of the cellulose with an Fmoc amino acid is a convenient method to introduce a spacer molecule and, after Fmoc deprotection, a free amino function for the SPPS of peptide arrays. The stability of the cellulose to organic solvents and bases allows the synthesis of peptides by utilizing the standard Fmoc methodology. Furthermore, the hydrophilic nature of cellulose offers a high compatibility with a wide variety of biological assay systems. On the other hand, however, cellulose shows only a limited acid stability. This acid sensitivity is severely restricting side chain deprotection conditions and stimulated the search of more convenient supports. Increasing resistance of peptide-cellulose membrane linkage against various types of reagents has been achieved through the development amino-functionalized ether type membranes. Ether type membranes provide stable membrane-bonding of peptides or other compound through the chemical stability of the ether bound. The first example of this type membrane was a cellulose-aminopropyl ether membrane (CAPE membrane) prepared by the treatment of cellulose filter paper with *N-*protected 2,3-epoxypropylamine [55].

The use of epibromohydrin as an activating reagent allowed introducing reactive bromine attached to the cellulose *via* an ether bond. The bromine moiety is able to react with different diamines [56] such as DAP (1,3-diaminopropane) [57] or TOTD (4,7,10-trioxa-1,13 tridecanediamine) and aminated polyethylene-3 (PEG-3) [58]. DAP modified cellulose membranes are known as *N-*CAPE membranes [59], while membranes modified with TOTD as a trioxa or TOTD membranes [60]. An amino type linker functionalized planar cellulose support [61] has been obtained by activation of cellulose with tosyl chloride and subsequent reaction with broad variety of diamines.

An additional advantage of CAPE membranes is an excellent signal-to noise ratio during onsupport assay because of the very low background signal of this membrane [62]. Due to these properties they were applied in biological studies [63]. Table 1 has shown the characteristic of selected examples of amino-functionalized cellulose membranes.

Another approach to improve SPOT technology involves the use of linker strategies to enable cleavage of peptides from the support. An interesting linker was proposed by Frank. It is known as a Carboxy-Frank-Linker [64]. This linker allows peptide release from the solid support in aqueous solution (pH 7–8). Other linker types used in SPOT technology nowadays are the *p*-hydroxymethylbenzoic acid (HMB) linker, the Rink-amide linker [65], photolabile linker [66], the Wang linker [67], thioether moieties [68] or 4-hydroxymethylphenoxy acetic acid (HMPA) and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) linkers [69]. An attractive approach based on the use of the *C*-terminal amino acid as a linker moiety was reported by Ay and co-workers [70]. Proposed solution was applied to sorting peptides according to their *C* termini using modified membranes with the corresponding *C*-terminal amino acids anchored either spot- or surface-wise.

252 Cellulose – Medical, Pharmaceutical and Electronic Applications

**5.1. Membrane modifications** 

reaction with broad variety of diamines.

orthogonal protecting groups used for protection of side chains. [54].

cellulose filter paper with *N-*protected 2,3-epoxypropylamine [55].

and organic media. However, since cellulose is not stable against harsh chemical conditions, the SPOT synthesis was developed for the milder type of the two major SPPS strategies based on the Fmoc protection of amine function of the main peptide chain [53] and

Cellulose membranes are still the most widely used supports for SPOT peptide synthesis. The esterification of hydroxy functions of the cellulose with an Fmoc amino acid is a convenient method to introduce a spacer molecule and, after Fmoc deprotection, a free amino function for the SPPS of peptide arrays. The stability of the cellulose to organic solvents and bases allows the synthesis of peptides by utilizing the standard Fmoc methodology. Furthermore, the hydrophilic nature of cellulose offers a high compatibility with a wide variety of biological assay systems. On the other hand, however, cellulose shows only a limited acid stability. This acid sensitivity is severely restricting side chain deprotection conditions and stimulated the search of more convenient supports. Increasing resistance of peptide-cellulose membrane linkage against various types of reagents has been achieved through the development amino-functionalized ether type membranes. Ether type membranes provide stable membrane-bonding of peptides or other compound through the chemical stability of the ether bound. The first example of this type membrane was a cellulose-aminopropyl ether membrane (CAPE membrane) prepared by the treatment of

The use of epibromohydrin as an activating reagent allowed introducing reactive bromine attached to the cellulose *via* an ether bond. The bromine moiety is able to react with different diamines [56] such as DAP (1,3-diaminopropane) [57] or TOTD (4,7,10-trioxa-1,13 tridecanediamine) and aminated polyethylene-3 (PEG-3) [58]. DAP modified cellulose membranes are known as *N-*CAPE membranes [59], while membranes modified with TOTD as a trioxa or TOTD membranes [60]. An amino type linker functionalized planar cellulose support [61] has been obtained by activation of cellulose with tosyl chloride and subsequent

An additional advantage of CAPE membranes is an excellent signal-to noise ratio during onsupport assay because of the very low background signal of this membrane [62]. Due to these properties they were applied in biological studies [63]. Table 1 has shown the

Another approach to improve SPOT technology involves the use of linker strategies to enable cleavage of peptides from the support. An interesting linker was proposed by Frank. It is known as a Carboxy-Frank-Linker [64]. This linker allows peptide release from the solid support in aqueous solution (pH 7–8). Other linker types used in SPOT technology nowadays are the *p*-hydroxymethylbenzoic acid (HMB) linker, the Rink-amide linker [65], photolabile linker [66], the Wang linker [67], thioether moieties [68] or 4-hydroxymethylphenoxy acetic acid (HMPA) and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) linkers [69]. An attractive approach based on the use of the *C*-terminal amino acid

characteristic of selected examples of amino-functionalized cellulose membranes.

**Scheme 4.** Different methods of amino functionalization of cellulose membranes: I) preparation of ester type membrane with amino acid; II) functionalization with epibromohydrin and subsequent reaction with TODT or DAP providing TODT or *N-*CAPE membranes; III) treatment with *N-*protected 2,3 epoxypropylamine giving CAPE membrane; IV) treatment with tosyl chloride and subsequent reaction with TODT giving a TOTD like membrane.



Cellulose Functionalysed with Grafted Oligopeptides 255

n

O

N R1

O

N N

**c**

O

cellulose

<sup>O</sup> Me

NH

O O

n

**g**

N N <sup>O</sup> <sup>N</sup> Me <sup>O</sup>

O

cellulose

NH O

R1 O

<sup>O</sup> NH2

O <sup>n</sup>

R1

NN <sup>O</sup> <sup>N</sup> Me <sup>O</sup>

**e**

cellulose

R2 NH O

NH2

**. . .**

Different type of anchoring of the peptide chain to cellulose matrix was proposed by Kaminski [73] and co-workers. 1-Acyl-3,5-dimethyl-1,3,5-triazin-2,4,6(1H,3H,5H)-trion derivatives serve both as a spacer and linker. This isocyanuric linker has been introduced by thermal isomerization [74] of 2-acyloxy-4,6-dimethoxy-1,3,5-triazines immobilized on the cellulose support or isomerization catalysed by the presence of acids. Synthetic procedure leading to peptides anchored to cellulose by 1-acyl-3,5-dimethyl-1,3,5-triazin-2,4,6(1H,3H,5H)-trion (iso-MT) is shown in Scheme 5. In the first step chloro-triazine **a** immobilized on cellulose was treated with *N-*methylmorpholine yielding *N-*triazinylammonium chloride **b**. Then compound **b** activated carboxylic function of Fmoc-protected amino acid to superactive ester **c** [75], which

n

NH

O O

R1

O OH

N+ O

Cl-

n

NH-Fmoc

TFA/DCM (1:1) 3% TRIS + 2% H2O

standard SPOT synthesis

N H

**Scheme 5.** Synthesis of peptides with free *N-*termini anchored by iso-MT linker.

R1 <sup>O</sup>

R2

O

**f**

NN <sup>O</sup> <sup>N</sup> Me <sup>O</sup>

O

cellulose

NN <sup>O</sup> <sup>N</sup> Me

> N H

O <sup>n</sup>

R1

NN <sup>O</sup> <sup>N</sup> Me <sup>O</sup>

**d**

isomerisation piperidine

cellulose

O O O

O

finally in refluxing toluene rearranges to stable isocyanuric derivative **d**.

<sup>n</sup> NN

O

N N <sup>O</sup> <sup>N</sup> Me

DMT/NMM/BF4

N +

BF4 -

O

R2

Fmoc-NH-CH-COOH

O

Me

Cl

NMM

**a b**

cellulose cellulose

<sup>O</sup> <sup>N</sup> Me

thermal

**Table 1.** Characterization of amino-modified membranes.

In cases of classical SPOT technology in which the peptide is coupled *via* an ester bond using ß-alanine of glycine spacer, peptide can be released from the cellulose by hydrolysis at pH>9. Numerous reagents were found suitable for this goal, e.g. aqueous solutions of ammonia, sodium hydroxide, trialkylamines or lithium carbonate (see Table 2) [71]. A broadly used method for releasing of soluble peptide amides is based on the treatment of membranes with ammonia vapor [72]. When the cleavage is carried out with nucleophils in an anhydrous environment, the substitution of the ester bond leads to amides, hydrazides and other derivatives.


**Table 2.** Typical linker types bounded to cellulose supports and cleavage methods used for releasing the peptides from the membrane.

Different type of anchoring of the peptide chain to cellulose matrix was proposed by Kaminski [73] and co-workers. 1-Acyl-3,5-dimethyl-1,3,5-triazin-2,4,6(1H,3H,5H)-trion derivatives serve both as a spacer and linker. This isocyanuric linker has been introduced by thermal isomerization [74] of 2-acyloxy-4,6-dimethoxy-1,3,5-triazines immobilized on the cellulose support or isomerization catalysed by the presence of acids. Synthetic procedure leading to peptides anchored to cellulose by 1-acyl-3,5-dimethyl-1,3,5-triazin-2,4,6(1H,3H,5H)-trion (iso-MT) is shown in Scheme 5. In the first step chloro-triazine **a** immobilized on cellulose was treated with *N-*methylmorpholine yielding *N-*triazinylammonium chloride **b**. Then compound **b** activated carboxylic function of Fmoc-protected amino acid to superactive ester **c** [75], which finally in refluxing toluene rearranges to stable isocyanuric derivative **d**.

254 Cellulose – Medical, Pharmaceutical and Electronic Applications

**Table 1.** Characterization of amino-modified membranes.

and other derivatives.

the peptides from the membrane.

**Cellulose type Cellulose membrane type Capacity [μmol/cm2]** 

Amine type: TsCl, diamino-PEG-3 4.0-10.0 Amine type: TsCl, diamino-PEG-3 + linker 0.45-2.6

Ester type: glycine 0.8-1.9 Ester type: different amino acids 0.2-1.7 Ether type: CAPE (amino-epoxy) 0.05-0.20 Ether type: *N-*CAPE, trioxa 0.2-1.2

In cases of classical SPOT technology in which the peptide is coupled *via* an ester bond using ß-alanine of glycine spacer, peptide can be released from the cellulose by hydrolysis at pH>9. Numerous reagents were found suitable for this goal, e.g. aqueous solutions of ammonia, sodium hydroxide, trialkylamines or lithium carbonate (see Table 2) [71]. A broadly used method for releasing of soluble peptide amides is based on the treatment of membranes with ammonia vapor [72]. When the cleavage is carried out with nucleophils in an anhydrous environment, the substitution of the ester bond leads to amides, hydrazides

**Linker Cleavage conditions C-termini derivatives** 

Boc-Imidazol linker TFA + aq. buffer free carboxy group Allyl linker Palladium (0)-catalyst free carboxy group Boc-Lys-Pro TFA + aq. buffer diketopiperazine

(thiol + coupled by amino acid) NaOH/H2O/methanol free carboxy group haloalkyl esters NaOH/H2O/acetonitrile free carboxy group Wang linker TFA vapour free carboxy group

**Table 2.** Typical linker types bounded to cellulose supports and cleavage methods used for releasing

hydrazine hydrazide hydroxyl amine hydroxyl amide aq. NaOH free carboxylic group aq. triethylamine free carboxy group primary alkyl/aryl amine alkyl/aryl amide

Glycine (differ amino acids) gaseous ammonia amide

HMB linker gaseous ammonia amide Photo-labile linker UV irradiation at 365 nm amide Rink-amide linker TFA amide Thioether gaseous ammonia amide

(2.0-5.0)

Whatman CHr1 Ester type: β-alanine 0.4-0.6

Whatman 50 Ester type: β-alanine 0.2-0.4

Whatman 540 Ester type: β-alanine 0.2-0.6 AIMS Amine type: amino-PEG 0.4-0.6

**Scheme 5.** Synthesis of peptides with free *N-*termini anchored by iso-MT linker.

$$\underbrace{\mathbb{R}\_{\text{"{a}\text{"{e}}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblright}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedblleft}\text{\textquotedbl{\textquotedbl}}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textquotedbl}\text{\textqu$$

Cellulose Functionalysed with Grafted Oligopeptides 257

the several cases for the same antigen the strengths of reaction with antibody depends on the anchoring method. Moreover, for isocyanuric linker interactions with antibodies were

Unfortunately, cellulose is not suitable for classic SPOT peptides synthesis with free *C*-termini, due to engagement of C-terminal fragment of peptide for fixation to support. One potential solution to this problem is to synthesize peptides in a nontraditional manner (that is, from the *N-* towards the C-termini) using amino acid ethyl esters [78]. One major drawback with this approach is the increased risks of epimeriation [79] at all coupling stages due to repeated solid support-bound carboxyl activation. There have been done numerous efforts to develop effective ISPPS strategies (inverse solid-phase peptide synthesis). One of the first reports on ISPPS described the use of amino acid hydrazides [80]. More recently, amino acid 9 fluorenylmethyl (Fm) esters [81], and amino acid allyl esters [82] have been used for ISPPS. However, few if any of these amino acid derivatives are currently commercially available. The Fm ester approach looks attractive considering its similarity to standard Fmoc-based *C*towards-*N* SPPS, but Fm esters are not as stable as Fmoc amino acids, and Fm ester-based inverse peptide synthesis apparently suffers from this limitation. The Fm ester approach also suffers from significant racemization during coupling reactions. The allyl ester-based approach is practicable and appears currently to be the method most competitive with the t-butyl esterbased ISPPS method described below. However, allyl esters are also not readily available commercially, and moreover, their deprotection requires the use of 20 mol% of Pd(PPh3)4, an expensive, heavy metal-based reagent. These strategies for ISPPS, therefore, appear not to be

**5.2. Cellulose membrane-bound peptides with free C-termini** 

ideal, especially since suitable amino acid substrates are not easy available.

prepare, unstable to store, and unstable under peptide coupling conditions.

A method for solid-phase peptide synthesis on cellulose in the *N-* to *C*-direction that delivers good coupling yields and a relatively low degree of epimerization was reported by Hallberg [83] and co-workers. The optimized method involves the coupling, without preactivation, of the solid support-bound *C*-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU or TBTU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Efficiency of proposed methodology was confirmed on the synthesis of five tripeptides: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, *N, N*diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Proposed methodology can provide an attractive alternative for the solid-phase synthesis of short (six residues or less) *C*-terminally modified peptides, e.g., in library format. On the other hand amino acid silyl esters are difficult to

found more selective [77].



**Table 3.** Interaction of peptidic epitopes with free *N-*termini anchored on cellulose with antibodies.

Further stages of the synthesis included the standard SPPS conditions: deprotections of Fmoc group and subsequent condensation with Fmoc/tBu-protected amino acids by using DMT/NMM/BF4 as a coupling reagent [76]. The data summarized in Table 3 shown that in the several cases for the same antigen the strengths of reaction with antibody depends on the anchoring method. Moreover, for isocyanuric linker interactions with antibodies were found more selective [77].

## **5.2. Cellulose membrane-bound peptides with free C-termini**

256 Cellulose – Medical, Pharmaceutical and Electronic Applications

cellulose

UB-33

F-8

CSF114

cellulose

Ala-COO-cellulose

2CH O

UB-33 H2*N-*Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-

CSF114 H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asn(Glc)-Gly-His-Ser-Val-

cellulose

O

N H

Epitope Peptide sequence Reaction Specificity

Gln-Phe-Ala-Asp-Ser-Arg-Ile-COO-cellulose **-** 

Gln-Phe-Ala-Asp-Ser-Arg-Ile-β-Ala-COO-cellulose -

Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-COO-cellulose **+, m** 

Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-COO-cellulose **+, m**

O

Gln-Phe-Ala-Asp-Ser-Arg-Ile-β-Ala-iso-MT-cellulose +,**<sup>m</sup>**

cellulose +, **<sup>s</sup>** H2*N-*Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-iso-MT-cellulose +, **<sup>m</sup>**

H2*N-*Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-

H2*N-*Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-Ala-Asp-Ser-Arg-Ile-iso-MT-cellulose +,**<sup>s</sup>**

H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asn(Glc)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-iso-MT-cellulose +,**<sup>m</sup>**

H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asn(GlcAc4)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-iso-MT-

H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asp-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-iso-MT-cellulose +/-

**Table 3.** Interaction of peptidic epitopes with free *N-*termini anchored on cellulose with antibodies.

Further stages of the synthesis included the standard SPPS conditions: deprotections of Fmoc group and subsequent condensation with Fmoc/tBu-protected amino acids by using DMT/NMM/BF4 as a coupling reagent [76]. The data summarized in Table 3 shown that in

H2*N-*Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-β-Ala-iso-MT-

<sup>O</sup> <sup>N</sup> Me

N

N O

cellulose

O

(Aaa)n-NH2

H2*N-*Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe-Ala-Asp-Ser-Arg-Ile-β-Ala-β-Ala-β-Ala-COO-cellulose -

H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asn(Glc(OAac)4)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-COO-

H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asp-Gly-His-Ser-Val-Phe-

H2*N-*Thr-Pro-Arg-Val-Glu-Arg-Asn(Glc)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys- β-Ala-β-Ala-β-

O m

**0%** H2*N-*Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val-

(Aaa)n-NH2

**90%** 

**+/-**

**+, m**

**100%** 

**70%** 

+/-

Unfortunately, cellulose is not suitable for classic SPOT peptides synthesis with free *C*-termini, due to engagement of C-terminal fragment of peptide for fixation to support. One potential solution to this problem is to synthesize peptides in a nontraditional manner (that is, from the *N-* towards the C-termini) using amino acid ethyl esters [78]. One major drawback with this approach is the increased risks of epimeriation [79] at all coupling stages due to repeated solid support-bound carboxyl activation. There have been done numerous efforts to develop effective ISPPS strategies (inverse solid-phase peptide synthesis). One of the first reports on ISPPS described the use of amino acid hydrazides [80]. More recently, amino acid 9 fluorenylmethyl (Fm) esters [81], and amino acid allyl esters [82] have been used for ISPPS. However, few if any of these amino acid derivatives are currently commercially available. The Fm ester approach looks attractive considering its similarity to standard Fmoc-based *C*towards-*N* SPPS, but Fm esters are not as stable as Fmoc amino acids, and Fm ester-based inverse peptide synthesis apparently suffers from this limitation. The Fm ester approach also suffers from significant racemization during coupling reactions. The allyl ester-based approach is practicable and appears currently to be the method most competitive with the t-butyl esterbased ISPPS method described below. However, allyl esters are also not readily available commercially, and moreover, their deprotection requires the use of 20 mol% of Pd(PPh3)4, an expensive, heavy metal-based reagent. These strategies for ISPPS, therefore, appear not to be ideal, especially since suitable amino acid substrates are not easy available.

A method for solid-phase peptide synthesis on cellulose in the *N-* to *C*-direction that delivers good coupling yields and a relatively low degree of epimerization was reported by Hallberg [83] and co-workers. The optimized method involves the coupling, without preactivation, of the solid support-bound *C*-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU or TBTU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Efficiency of proposed methodology was confirmed on the synthesis of five tripeptides: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, *N, N*diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Proposed methodology can provide an attractive alternative for the solid-phase synthesis of short (six residues or less) *C*-terminally modified peptides, e.g., in library format. On the other hand amino acid silyl esters are difficult to prepare, unstable to store, and unstable under peptide coupling conditions.

The alternative strategy for ISPPS based on amino acid *t-*butyl esters was proposed by Gutheil [84] and co-workers . Favorable features of this approach are that amino acid *t*-butyl esters are stable, a large selection of them are commercially available, and the synthesis of commercially unavailable monomers is relatively straightforward. The *t*-butyl ester strategy also has the benefit that this approach is exactly the inverse of the well-developed Boc strategy for normal C-to-N peptide synthesis, and the extensive knowledge of side chain protection strategies and other chemical details can therefore be transferred from Boc chemistry to *t-*butyl ester chemistry. The effectiveness of the proposed solution has been demonstrated in the synthesis of tripeptides: Tyr-Ala-Phe, Tyr-Gly-Orn, Tyr-Ala-Val, Asn-D-Val-Leu, Asn-Leu-Glu, Gly-Ile-Thr, Phe-Ala-Gly. The consecutive incorporation of amino acids was performed in the presence of HATU as a coupling reagents using an excess of AA-OtBu\*HCl. The observed racemization of individual amino acids was <2%.

Cellulose Functionalysed with Grafted Oligopeptides 259

O

cellulose CAPE-membrane

Fmoc-Aaa0-OH, DIC, NMI

OH

O

cellulose

N H

OH

OH

O

cellulose

standard spot synthesis Aaa-1-Aaa-6, -Ala

O

N H O R

R O

0

NH2 <sup>N</sup>

**a b**

N H

> N H

> > O

N H O

O O Dmab

OH

1) Fmoc--Ala-OPfp 2) Fmoc-Glu(Dmab)-OPfp 3) HMB, TBTU, DIPEA

O

cellulose

N H

OH

O

N H

O

O


N H

peptide

**Scheme 7.** Synthesis of inverted peptides on cellulose membranes.

O

O

N H

**<sup>e</sup>** <sup>0</sup>

1) 50% TFA 2) Li2CO3 solution

R O


OH

O

N H

O

N H

O

**<sup>d</sup>** <sup>0</sup>

1) 20% piperidine 2) 1% hydrazine hydrate 3) PyBOP, NMM

O

R

N H O

O

N H N H

O

O N H

cellulose **f**

O N H

OH O

R


peptide

O

O O Dmab

H

O

O

**<sup>c</sup>** <sup>0</sup>

R

N H

O

OH

O

cellulose

O

N H

O

O

R


O

N H O

NH-Fmoc

R O

peptide

O

R N H


O

R N H


O

N H NH-Fmoc

OH

O O Dmab

**Scheme 6.** Synthesis of peptidic epitope: SIKEDVQF and CHHLDKSIKEDVQFADSRI on the cellulose plate from *N-* to *C*-terminus using DMT/NMM/BF4 as a coupling reagent.

**Scheme 7.** Synthesis of inverted peptides on cellulose membranes.

n

3) 50%TFA, 2%TRIS, 3% H2O / DCM

R1

NH2-CH-COOH

<sup>O</sup> <sup>n</sup>

<sup>O</sup> Me

O N N N

cellulose cellulose cellulose

N H <sup>O</sup> OH

cellulose

R1

<sup>n</sup> <sup>O</sup>

R1 R2

O N <sup>H</sup> <sup>O</sup> N <sup>H</sup> <sup>O</sup> N H

**Scheme 6.** Synthesis of peptidic epitope: SIKEDVQF and CHHLDKSIKEDVQFADSRI on the cellulose

R3

1) **DMT/NMM/BF4**

2) H2N-CH(R2)-OMe 3) 0.5N LiOH 4) 1N NaHSO4 1) **DMT/NMM/BF4**

2) H2N-CH(R3)-OMe 3) 0.5N LiOH 4) 1N NaHSO4

n-cycle

**-**

**-**

N N N

plate from *N-* to *C*-terminus using DMT/NMM/BF4 as a coupling reagent.

O Me <sup>N</sup> H

O N N N

N H <sup>O</sup> <sup>N</sup> <sup>H</sup> <sup>O</sup> N <sup>H</sup> <sup>O</sup> N H

R3

O O

...

R1 R2

n

<sup>O</sup> Me

O OH

...

N N N <sup>O</sup> Cl Me

> 1) 0.5N LiOH 2) 1N NaHSO4

The alternative strategy for ISPPS based on amino acid *t-*butyl esters was proposed by Gutheil [84] and co-workers . Favorable features of this approach are that amino acid *t*-butyl esters are stable, a large selection of them are commercially available, and the synthesis of commercially unavailable monomers is relatively straightforward. The *t*-butyl ester strategy also has the benefit that this approach is exactly the inverse of the well-developed Boc strategy for normal C-to-N peptide synthesis, and the extensive knowledge of side chain protection strategies and other chemical details can therefore be transferred from Boc chemistry to *t-*butyl ester chemistry. The effectiveness of the proposed solution has been demonstrated in the synthesis of tripeptides: Tyr-Ala-Phe, Tyr-Gly-Orn, Tyr-Ala-Val, Asn-D-Val-Leu, Asn-Leu-Glu, Gly-Ile-Thr, Phe-Ala-Gly. The consecutive incorporation of amino acids was performed in the presence of HATU as a coupling reagents using an excess of AA-

OtBu\*HCl. The observed racemization of individual amino acids was <2%.

Another approach to the synthesis of peptides attached to the cellulose matrix within *N*terminus and presenting free C-terminus [85] was based on the utilization of 1,3,5-triazine derivative as an anchoring group. The peptides anchored via *N-*terminal moiety to the cellulose plate, were synthesized in accord to step-by-step methodology by means of 4-(4,6 dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMT/NMM/BF4) as a coupling [86]. 2-Chloro-1,3,5-triazine fragment, used as an anchoring group, was introduced by the treatment of cellulose with 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) [87]. The first amino acid was attached to the triazine ring by the nucleophilic substitution reaction involving amine group. The oligopeptide chain was elongated in accord to step-bystep methodology in the sequence of standard reactions involving: activation of carboxylic function, coupling with the ester of appropriate amino acid, washing, capping, hydrolysis of ester moiety, and washing (Scheme 6).

Cellulose Functionalysed with Grafted Oligopeptides 261

O OH N <sup>H</sup> <sup>N</sup> H

cellulose

O OH N <sup>H</sup> <sup>N</sup> H

cellulose

O OH N

cellulose

O OH N

> O OH N

O OH N <sup>H</sup> <sup>N</sup> H

peptides (**i**) display a free *C* terminus resulting from reversal of the peptide orientation by successive thioether-cyclization/ester cleavage transformations. Key intermediates in the synthesis are the 3-brompropyl esters of Fmoc-amino acid (Fmoc-Aaa-OPBr) (**d**), the membrane-bound mercaptopropionyl cysteine adduct (**c**), the matrix-bound amino acid ester derivative (**e**), and the cyclic peptide (**h**). Critical reaction steps are the formation of

> N H S-Trt

> > **c** -Ala

Cs2CO3


O

N H S-Trt

O N H

<sup>H</sup> <sup>N</sup> <sup>O</sup> <sup>H</sup> N H S-Trt

O

**d**

S

O

O

R N H

<sup>H</sup> NH-CO-CH2-S

peptide -Ala

peptide

S OH

O

R N

O N H

> standard spot synthesis Aaa-1-Aaa-6

**e** -Ala

O

S

S

Li2CO3

**h**

H N


N <sup>H</sup> <sup>O</sup>

**Scheme 8.** Synthesis of inverted peptides on cellulose membranes allows further modification of side

<sup>H</sup> <sup>O</sup>

<sup>H</sup> <sup>N</sup> <sup>O</sup> <sup>H</sup> N H S-Trt

**f**

**g**

O N H

cellulose <sup>0</sup> 1) Fmoc--Ala-OPfp 2) 20% piperidine 3) 2,4-dinitrophenyl-bromoacetate

O

TFA, TIBS, an then Cs2CO3

<sup>H</sup> <sup>N</sup> <sup>O</sup> <sup>H</sup> N H S-Trt

S

**i** cellulose

O

O N H

cellulose 0


N H



O OH N <sup>H</sup> <sup>N</sup>

O N H

cellulose 0

2NH

O

SH

1) Fmoc--Ala-OPfp 2) 20% piperidine 3) Fmoc-Cys(Trt)-OPfp **a** 4) 20% piperidine **b**

> R <sup>O</sup> Br Fmoc-NH O

0

S

O

R N

H N

peptide

both the cleavable ester bond and the cyclic peptide.

O OH N <sup>H</sup> 2NH

cellulose

N-modified CAPE-membrane

O

R

0

H

N H

Br-CH2-CONH-B-Ala

CH2-CONH-B-Ala

OH

chains (phosphorylation).

O

R N

0

2NH

1) Mmt-S-CH2-CH2-COOH, HATU, NMI 2) Cl2CHCOOH, TFAQ, TIBS

<sup>H</sup> 2NH

peptide

An amount of natural, all-L diastereomer was sufficiently abundant for selective reaction with sera of patients with medically confirmed atherosclerosis even in the case of long epitope.

A more sophisticated approaches are based on inversion of the peptide chain following conventional (*C N*) synthesis and then modification of the *C*-terminus. Examples of inverting solid support-bound peptides [88] and methods for the generation of liberated *C*terminally modified peptides [89] *via* a cyclization/cleavage protocols are known. The first example of application of the synthesis of inverting cellulose support-bound peptides according SPOT-methodology with free *C*-termini *via* prepared by successive cyclization and re-linearization was described by Hoffmüller and Volkmer-Engert [90].

Aminopropyl ether cellulose (CAPE-membrane) (**a**) was used as the matrix. β-Alanine serves both as a spacer and to residue directly engaged in the rearrangement. Dmabglutamic acid was coupled as a bivalent linker followed by introducing hydroxymethylbenzoic acid (HMB) as a base-labile cleavable site ( **b**). The intended Cterminal amino acid was coupled through an ester bond ( **c**). The Fmoc and Dmab protecting groups on the *N-*terminus and the side chains of the glutamic acids were cleaved off and then construct cyclized ( **e**). Removal of the side chain protecting groups followed by hydrolysis of the ester bond linearizes the construct and generated free C terminus ( **f**). Even if presented above method allowed successful synthesis of inverted peptides arrays, the obtained yields were low and procedure were found troublesome and time-consuming.

Therefore Volkmer-Engert [91] and co-workers developed a more robust and efficient protocol for the preparation of cellulose membrane-bound inverted peptide arrays that could be used for widespread mapping different epitopes anchored on solid support and presenting *C*-termini peptides.

Synthesis of inverted peptides was performed on a cellulose membrane carrying a stable *N*functionalized anchor (*N-*modified cellulose-amino-hydroxypropyl ether membrane - *N-*CAPE), which retained the inverted peptides (**i**). The inverted and *N* terminally fixed peptides (**i**) display a free *C* terminus resulting from reversal of the peptide orientation by successive thioether-cyclization/ester cleavage transformations. Key intermediates in the synthesis are the 3-brompropyl esters of Fmoc-amino acid (Fmoc-Aaa-OPBr) (**d**), the membrane-bound mercaptopropionyl cysteine adduct (**c**), the matrix-bound amino acid ester derivative (**e**), and the cyclic peptide (**h**). Critical reaction steps are the formation of both the cleavable ester bond and the cyclic peptide.

260 Cellulose – Medical, Pharmaceutical and Electronic Applications

ester moiety, and washing (Scheme 6).

epitope.

time-consuming.

presenting *C*-termini peptides.

Another approach to the synthesis of peptides attached to the cellulose matrix within *N*terminus and presenting free C-terminus [85] was based on the utilization of 1,3,5-triazine derivative as an anchoring group. The peptides anchored via *N-*terminal moiety to the cellulose plate, were synthesized in accord to step-by-step methodology by means of 4-(4,6 dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMT/NMM/BF4) as a coupling [86]. 2-Chloro-1,3,5-triazine fragment, used as an anchoring group, was introduced by the treatment of cellulose with 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) [87]. The first amino acid was attached to the triazine ring by the nucleophilic substitution reaction involving amine group. The oligopeptide chain was elongated in accord to step-bystep methodology in the sequence of standard reactions involving: activation of carboxylic function, coupling with the ester of appropriate amino acid, washing, capping, hydrolysis of

An amount of natural, all-L diastereomer was sufficiently abundant for selective reaction with sera of patients with medically confirmed atherosclerosis even in the case of long

A more sophisticated approaches are based on inversion of the peptide chain following conventional (*C N*) synthesis and then modification of the *C*-terminus. Examples of inverting solid support-bound peptides [88] and methods for the generation of liberated *C*terminally modified peptides [89] *via* a cyclization/cleavage protocols are known. The first example of application of the synthesis of inverting cellulose support-bound peptides according SPOT-methodology with free *C*-termini *via* prepared by successive cyclization

Aminopropyl ether cellulose (CAPE-membrane) (**a**) was used as the matrix. β-Alanine serves both as a spacer and to residue directly engaged in the rearrangement. Dmabglutamic acid was coupled as a bivalent linker followed by introducing hydroxymethylbenzoic acid (HMB) as a base-labile cleavable site ( **b**). The intended Cterminal amino acid was coupled through an ester bond ( **c**). The Fmoc and Dmab protecting groups on the *N-*terminus and the side chains of the glutamic acids were cleaved off and then construct cyclized ( **e**). Removal of the side chain protecting groups followed by hydrolysis of the ester bond linearizes the construct and generated free C terminus ( **f**). Even if presented above method allowed successful synthesis of inverted peptides arrays, the obtained yields were low and procedure were found troublesome and

Therefore Volkmer-Engert [91] and co-workers developed a more robust and efficient protocol for the preparation of cellulose membrane-bound inverted peptide arrays that could be used for widespread mapping different epitopes anchored on solid support and

Synthesis of inverted peptides was performed on a cellulose membrane carrying a stable *N*functionalized anchor (*N-*modified cellulose-amino-hydroxypropyl ether membrane - *N-*CAPE), which retained the inverted peptides (**i**). The inverted and *N* terminally fixed

and re-linearization was described by Hoffmüller and Volkmer-Engert [90].

**Scheme 8.** Synthesis of inverted peptides on cellulose membranes allows further modification of side chains (phosphorylation).

## **5.3. Application of SPOT technology**

Cellulose was found to be the support of choice in the SPOT synthesis. The main area of application of SPOT technology is for epitope mapping:

Cellulose Functionalysed with Grafted Oligopeptides 263

acids [99]. Weiler et al. [100] described the synthesis of a PNA oligomer library, with coupling yields of >97%. The synthesis of small organic compounds is another broad field

**6. Supramolecular structures formed by self-organization of** *N-***lipidated** 

Cellulose is a polysaccharide with two different types of hydroxyl groups i.e. primary and secondary. The primary hydroxyl groups are significantly more reactive then the secondary. Since the chains of polyanhydroglucose interacts with each other in the precisely defined way these functional groups are positioned within the reasonably regular fashion on he surface of cellulose. In the crystalline region of cellulose [102] the every second primary hydroxyl groups are exposed and accessible for interaction with reagents making after the transformation relatively regular pattern of anchored molecules separated by the distance of one anhydroglucose residue. Due to this advantageous feature of the cellulose the space available in between molecules anchored on the cellulose surface is sufficient for docking another molecules. Based on this assumption Kaminski and co-workers proposed entirely new approach for designing artificial receptors. According to the proposed concept, appropriate structure of molecules anchored on cellulose creates precisely defined and functionalized space for trapping ligands as

The relatively weak bonding forces and conformational flexibility of both partners make docking of ligands to receptors difficult to study, to categorize by any kind of empirical rules, or to predict based on molecular modeling. Even in the case of interactions between relatively simple molecules, the possible bonding and repulsive forces of mutual hostguest interactions are multifaceted, very numerous, and difficult in terms of molecular modeling [103]. For the more advanced models involving flexible ligands and complex flexible receptor structures the rational construction plan of the host structure still exceeds our capabilities [104]. Thus, design of the molecular trap was done intuitively by mimicking structural features occurring in natural receptors, synthesis of the library of them by methods of combinatorial chemistry and selection of the most efficient

Strong, yet reversible binding force for the most of potential guest molecules were achieved by introducing into binding pockets most of the structural attributes responsible for weak intermolecular interactions [105]. These include hydrogen-bond donors and acceptors, lipophilic and hydrophilic fragments supplemented with π-donors and π-

All these elements were allocated inside the linear structure forming the matrix of podands in such a way as to separate the flexible *N-*lipopeptide fragment from the solid support by relatively rigid, aromatic rings. Thus, a bonding "pocket" was composed from the tethered fragments of "walls" constructed from aromatic rings, expanded with a diversity of

for the application of SPOT synthesis [101].

**peptides anchored to cellulose** 

presented on Figure 4.

representatives.

acceptors as depicted on Figure 5.


Today, experiments to identify and characterize linear antibody epitopes using peptide scans, amino acids scans, substitutional analyses, truncation libraries, deletion libraries, cyclization scans, all types of combinatorial libraries and randomly generated libraries of single peptides are standard techniques widely applied even in non-specialized laboratories [93].

The synthesis of non-peptidic compounds or peptides with non-peptidic elements has been carried out on cellulose as well as polypropylene membranes. Using the SPOT technique, one of the most frequently synthesized non-peptidic compounds are a peptoids [94]. These compounds are synthesized pure or as hybrids with peptides, so-called peptomers [95]. Zimmermann et al. [96] investigated the possibility of replacing natural amino acids by peptoidic elements. Screening of an array of 8000 hexapeptoids and peptomers was carried out by Heine et al [97]. Hoffmann et al. [98] described the transformation of a biologically active peptide into peptoid analogues while retaining biological activity. Another application of the SPOT method is the synthesis of chimeric oligomers of peptide nucleic acids [99]. Weiler et al. [100] described the synthesis of a PNA oligomer library, with coupling yields of >97%. The synthesis of small organic compounds is another broad field for the application of SPOT synthesis [101].

262 Cellulose – Medical, Pharmaceutical and Electronic Applications

application of SPOT technology is for epitope mapping:

understanding the structure-function relationships.

Cellulose was found to be the support of choice in the SPOT synthesis. The main area of

1. Physiology - Antibodies can identify the structural fragments which allow molecules to interact between themselves or with their specific receptor. They can also be useful in

2. Pathology - Understanding the mechanism by which an immune-mediated pathology develops by a precise identification of both B- and T-cell epitopes on the antigen. Antibodies can, therefore, be useful in analyzing the specificity of antibodies spontaneously formed in a number of diseases in which an immune response is an important parameter. Mapping of epitopes is also essential when one wishes to unravel

3. Preclinical evaluation of drugs or blood product derivatives - Most drugs act as haptens, that is to say that they are too small for being immunogenic. However, after

4. Vaccinations - The identification of both B- and T-cell epitopes on a micro-organism or bacterial derived products such as toxins or enzymes may have a crucial influence on the design of vaccines. This includes not only an increase efficiency of vaccines, but potentially the design of vaccines that could stimulate humoral or the cellular immune response. 5. Diagnosis and subtyping of micro-organisms - Antibodies of defined specificity are currently used to distinguish between micro-organisms that belong to the same strain or to render diagnostic test more specific. The identification of shared antigenic determinants between proteins pertaining to different families can also has an

6. Mechanism of drug action - an emerging field of interest concerning the use of

Today, experiments to identify and characterize linear antibody epitopes using peptide scans, amino acids scans, substitutional analyses, truncation libraries, deletion libraries, cyclization scans, all types of combinatorial libraries and randomly generated libraries of single peptides are standard techniques widely applied even in non-specialized laboratories

The synthesis of non-peptidic compounds or peptides with non-peptidic elements has been carried out on cellulose as well as polypropylene membranes. Using the SPOT technique, one of the most frequently synthesized non-peptidic compounds are a peptoids [94]. These compounds are synthesized pure or as hybrids with peptides, so-called peptomers [95]. Zimmermann et al. [96] investigated the possibility of replacing natural amino acids by peptoidic elements. Screening of an array of 8000 hexapeptoids and peptomers was carried out by Heine et al [97]. Hoffmann et al. [98] described the transformation of a biologically active peptide into peptoid analogues while retaining biological activity. Another application of the SPOT method is the synthesis of chimeric oligomers of peptide nucleic

the mechanisms by which immune tolerance is established and/or broken.

combination with plasma or tissue proteins, they can become immunogenic.

important impact on the understanding of cross-reactions.

antibodies to study the mechanism of action of drugs [92].

[93].

**5.3. Application of SPOT technology** 
