**3. Aldol reaction**

The aldol reaction has long been recognized as one of the most useful tools for organic chemists. The ability to form carbon-carbon bonds can generate a broad range of both natural and novel poly-hydroxylated compounds. Thus, it is the most important and valuable reaction for the preparation of pharmaceuticals, fine chemicals, and natural products. Aldolases have evolved to catalyze the metabolism and catabolism of highly oxygenated metabolites and are found in many biosynthetic pathways of

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

carbohydrates, keto acids, and some amino acids [35]. Aldolases bind their respective donor substrates with high specificity and generally will not accept any other donors, even if their structures are similar to the natural donor. The advantages of using aldolases are very high stereospecificity and environmentally benign reaction conditions [36]. However, the limited number of substrates as well as the high cost of these biocatalysts has led researchers to consider other more stable enzymes [37].

In 2003, Berglund and co-workers firstly reported the serine hydrolase *Candida antarctica* lipase B to have catalytic activity for aldol reactions [38]. Our group reported the first lipase-catalyzed asymmetric aldol reaction in 2008 [39]. However, these aldol reactions in earlier studies involving hydrolases just showed moderate activities and selectivities; some more efficient promiscuous aldol reaction have been researched and presented in the last decade.

#### **3.1 Aldol reaction**

In 2012, Guan et al. firstly demonstrated that lipase from porcine pancreas, type II (PPL II), has been observed to catalyze the direct asymmetric aldol reaction of heterocyclic ketones with aromatic aldehydes at 30°C in CH3CN/H2O (**Figure 25**) [40]. PPL II has good catalytic activity and good adaptability to different substrates. Its enantioselectivity can reach 87% ee and enantioselectivity 83:17 (anti/syn). Then PPL II has aldolase function in organic solvents.

In the same year, the same group also reported the similar asymmetric aldol reaction of aromatic and heteroaromatic aldehydes with cyclic and acyclic ketones in acetonitrile in the presence of a phosphate buffer by chymopapain, which is a cysteine proteinase isolated from the latex of the unripe fruits of *Carica papaya* [41]. Chymopapain exhibited the best catalytic activity and moderate stereoselectivity in DMSO, and the enzyme showed the best enantioselectivity of 79% ee in CH2Cl2 with low diastereoselectivity (**Figure 26**). In consideration of both diastereo- and enantioselectivities, the group chose MeCN as a suitable solvent for the asymmetric direct aldol reaction, which gave the best dr of 77:23 and a moderate ee of 76% among the tested solvents. Then, in order to further optimize the direct asymmetric aldol reaction catalyzed by papain, the effects of water content,

**Figure 25.** *Lipase-catalyzed direct asymmetric aldol reaction of heterocyclic ketones with aromatic aldehydes.*

**Figure 26.** *The asymmetric aldol reaction of 4-cyanobenzaldehyde and cyclohexanone.*

The protons were then transferred from vinyl malononitrile to His 257 to form a transition state. Subsequently, the protons were transferred from the imidazole group of His 257 to nitroolefins, and the carbon of nitroolefins were attacked by

In 2014, Ye et al. reported the preparation of 2-hydroxy-2-methyl-4-(4 nitrophenyl)-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one by Michael additioncyclization cascade reaction of p-nitrobenzalacetone with 1,3-cyclohexanedione in anhydrous media, and control experiments were conducted (**Figure 24**) [34]. The high yield was observed with *Escherichia coli* BioH esterase in DMF at 37°C. In order to preliminarily explore the mechanism of the reaction, site-directed mutagenesis was performed on the hydrolysis catalytic triad of BioH, and the results indicated "alternate-site enzyme promiscuity." Using a series of substituted phenylacetone

The aldol reaction has long been recognized as one of the most useful tools for organic chemists. The ability to form carbon-carbon bonds can generate a broad range of both natural and novel poly-hydroxylated compounds. Thus, it is the most important and valuable reaction for the preparation of pharmaceuticals, fine chemicals, and natural products. Aldolases have evolved to catalyze the metabolism and catabolism of highly oxygenated metabolites and are found in many biosynthetic pathways of

and 1,3-cyclodiketone as reactants, the yield could reach 76.3%.

nucleophilic carbon molecules to form products.

*BioH esterase-catalyzed Michael addition-cyclization cascade reaction.*

*The proposed mechanism of lipase-catalyzed vinylogous Michael addition.*

**3. Aldol reaction**

**46**

**Figure 23.**

*Molecular Biotechnology*

**Figure 24.**

reaction temperature, and the amount of buffer on the enzymatic reaction were investigated. The reaction of 4-cyanobenzaldehyde with cyclohexanone was used as a model reaction.

The authors proposed a mechanism for the chymopapain-catalyzed aldol reaction (**Figure 27**). The catalytic triad of Cys, His, and Asn formed the active site of chymopapain. Firstly, the carbonyl of the substrate ketone is coordinated in the oxygen anion pore of Asn-His binary and active center. Secondly, a proton is transferred from the ketone to the His residue to form enolate ion. Thirdly, another substrate aldehyde accepts the proton from imidazolium cation and forms a new carbon-carbon bond with ketones. Finally, the product is released from the oxyanion hole and separates from the active site.

In 2013, our group firstly reported the asymmetric aldol reaction between aromatic aldehydes and cyclic ketones by PPL (**Figure 28**) [42]. The results showed that a small amount of water could promote the catalytic activity of PPL at 37°C. A wide range of aromatic aldehydes reacted with cyclic ketones to provide the corresponding aldol products with high yields (up to 99%) and moderate to good stereoselectivity (up to 90% ee and 99:1 dr).

In 2014, Majumder and Gupta found that the properties of lipase-catalyzed reaction products of acetylacetone with 4-nitrobenzaldehyde depend on the source of lipase and reaction medium (**Figure 30**) [44]. *Mucor javanicus* lipase was found to give 70% aldol and 80% enantiomeric excess in anhydrous t-amyl alcohol. Gao and Guo et al. demonstrated the catalytic promiscuity of an acyl-peptide releasing enzyme from *Sulfolobus tokodaii* (ST0779) for aldol addition reaction for the first time, and accelerated activity was observed at elevated temperature

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

55°C is 7.78-fold higher than that of PPL at its optimum temperature of 37°C, and

The authors proposed a mechanism for the ST0779-catalyzed aldol reaction between acetone and 4-nitrobenzaldehyde (**Figure 32**). Based on the structure simulation of ST0779, the aldol reaction catalyzed by ST0779 with acetone and pnitrobenzaldehyde as model reaction was proposed. Because of its thermodynamic superiority and high affinity, acetone first enters the active site and then is accommodated by the active site residues Ser439 and His 555. Proton transfer forms a transition state of enol salts, which is stable by Ser439. Asp523 is involved in stabilizing the positive charge of His 555-protonated imidazole ring. In the next

*The lipase-catalyzed promiscuous reaction: formation of acyclic and cyclic 2:2 adducts.*

1

) of this thermostable enzyme at

) adds up to 140 times higher

(**Figure 31**) [45]. The turnover number kcat (s<sup>1</sup>

*MML-catalyzed aldol condensation using an in situ-generated acetaldehyde.*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

the molecular catalytic efficiency kcat/Km (M<sup>1</sup> s

than PPL.

**Figure 30.**

**49**

**Figure 29.**

In the same year, a simple and convenient synthesis route of series α,β-unsaturated aldehydes was formed by combining the two catalytic activities of the same enzyme with the one-pot method of aldehyde-alcohol reaction and in situ acetaldehyde formation (**Figure 29**) [43]. Lipase from *Mucor miehei* has conventional and promiscuous catalytic activities for the hydrolysis of vinyl acetate and aldol condensation with in situ-formed acetaldehyde.

**Figure 27.** *Proposed mechanism for the chymopapain-catalyzed aldol reaction.*

**Figure 28.** *The asymmetric aldol reaction between aromatic aldehydes and cyclic ketones by PPL.*

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

#### **Figure 29.**

reaction temperature, and the amount of buffer on the enzymatic reaction were investigated. The reaction of 4-cyanobenzaldehyde with cyclohexanone was used as

The authors proposed a mechanism for the chymopapain-catalyzed aldol reaction (**Figure 27**). The catalytic triad of Cys, His, and Asn formed the active site of chymopapain. Firstly, the carbonyl of the substrate ketone is coordinated in the oxygen anion pore of Asn-His binary and active center. Secondly, a proton is transferred from the ketone to the His residue to form enolate ion. Thirdly, another substrate aldehyde accepts the proton from imidazolium cation and forms a new carbon-carbon bond with ketones. Finally, the product is released from the

In 2013, our group firstly reported the asymmetric aldol reaction between aromatic aldehydes and cyclic ketones by PPL (**Figure 28**) [42]. The results showed that a small amount of water could promote the catalytic activity of PPL at 37°C. A wide range of aromatic aldehydes reacted with cyclic ketones to provide the corresponding aldol products with high yields (up to 99%) and moderate to good

In the same year, a simple and convenient synthesis route of series α,β-unsaturated aldehydes was formed by combining the two catalytic activities of the same enzyme with the one-pot method of aldehyde-alcohol reaction and in situ acetaldehyde formation (**Figure 29**) [43]. Lipase from *Mucor miehei* has conventional and promiscuous catalytic activities for the hydrolysis of vinyl acetate and aldol con-

a model reaction.

*Molecular Biotechnology*

**Figure 27.**

**Figure 28.**

**48**

oxyanion hole and separates from the active site.

stereoselectivity (up to 90% ee and 99:1 dr).

densation with in situ-formed acetaldehyde.

*Proposed mechanism for the chymopapain-catalyzed aldol reaction.*

*The asymmetric aldol reaction between aromatic aldehydes and cyclic ketones by PPL.*

*MML-catalyzed aldol condensation using an in situ-generated acetaldehyde.*

In 2014, Majumder and Gupta found that the properties of lipase-catalyzed reaction products of acetylacetone with 4-nitrobenzaldehyde depend on the source of lipase and reaction medium (**Figure 30**) [44]. *Mucor javanicus* lipase was found to give 70% aldol and 80% enantiomeric excess in anhydrous t-amyl alcohol.

Gao and Guo et al. demonstrated the catalytic promiscuity of an acyl-peptide releasing enzyme from *Sulfolobus tokodaii* (ST0779) for aldol addition reaction for the first time, and accelerated activity was observed at elevated temperature (**Figure 31**) [45]. The turnover number kcat (s<sup>1</sup> ) of this thermostable enzyme at 55°C is 7.78-fold higher than that of PPL at its optimum temperature of 37°C, and the molecular catalytic efficiency kcat/Km (M<sup>1</sup> s 1 ) adds up to 140 times higher than PPL.

The authors proposed a mechanism for the ST0779-catalyzed aldol reaction between acetone and 4-nitrobenzaldehyde (**Figure 32**). Based on the structure simulation of ST0779, the aldol reaction catalyzed by ST0779 with acetone and pnitrobenzaldehyde as model reaction was proposed. Because of its thermodynamic superiority and high affinity, acetone first enters the active site and then is accommodated by the active site residues Ser439 and His 555. Proton transfer forms a transition state of enol salts, which is stable by Ser439. Asp523 is involved in stabilizing the positive charge of His 555-protonated imidazole ring. In the next

**Figure 31.** *Aldol reactions catalyzed by ST0779 or PPL.*

enzymes formed by the combination of enzymes at different optimal temperatures and gold nanorods (GNRs) [47]. By using the photothermal effect of GRS to transfer energy quickly, coupled with the real-time and long-range regulation of enzyme activity, improving the thermal stability of the enzyme and effective catalysis of the aldol reaction can be achieved. The increase in energy inside GRS, stimulated by distant near-infrared (NIR) stimuli, leads to increased enzyme activity. The results show that the method of internal heating that transfers energy more directly to the enzyme-catalyzed site is a faster and more effective energy transfer method. The results also show that the catalytic effect of the remote-controlled nanocatalytic system at lower temperature is the same as that of the free enzyme at higher temperature, but it has the advantages of improving the stability of the enzyme and extending its service life. Specifically, PPL EGCs at room temperature exhibit the same catalytic effect as achieved by free PPL at 40°C, while ST0779EGCs and APE1547 EGCs at 33°C exhibit a higher catalytic effect than their corresponding free enzymes at 63°C. In addition, EGCs have superior catalytic efficiency and

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

*One-pot cascade for synthesis of chiral β-hydroxy ketone derivatives.*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

product yield compared with aldol addition in free enzyme systems.

The nitroaldol or Henry reaction is one of the most useful carbon-carbon bondforming reactions and has wide synthetic applications in organic chemistry. This reaction provides access to valuable racemic and optically active β-nitro alcohols, which are very useful in organic synthesis as precursors for pharmaceutical and biological purposes. In recent years, efficient nonconventional biocatalytic

Guan et al. firstly reported transglutaminase was used to catalyze Henry reactions of aliphatic, aromatic, and heteroaromatic aldehydes with nitroalkanes

(**Figure 34**) [49]. The reactions were carried out at room temperature, and the corresponding nitroalcohols were obtained in yields up to 96%.

*Enzyme-catalyzed Henry reaction of 4-nitrobenzaldehyde and nitromethane.*

**3.2 Henry (nitroaldol) reaction**

**Figure 33.**

approaches have been reported [48].

**Figure 34.**

**51**

**Figure 32.** *Proposed mechanism for ST0779-catalyzed aldol reaction between acetone and 4-nitrobenzaldehyde.*

step, the oxygen of carbonyl group in 4-nitrobenzaldehyde is protonated by protons from His 555 imidazole ring, and the carbon atoms in the same carbonyl group are neutrally attacked by oleic acid carbon to form a new C▬C bond. Finally the aldol product is released from the enzyme, and the enzyme is freed for a new reaction.

In 2016, Wu et al. demonstrated a one-pot bienzymatic cascade in organic media to synthesize chiral β-hydroxy ketones for the first time (**Figure 33**) [46]. The decarboxylative aldol reaction catalyzed by an immobilized lipase from *Mucor miehei* (MML) and the synthesis of β-hydroxy ketone catalyzed by a lipase A or B from *Candida antarctica* (CALA or CALB) were combined in this one-pot protocol, reducing the purification step between the two reactions. (S)-β-hydroxy ketones and acylated (R)-β-hydroxy ketones could be obtained under mild reaction condition. The ee values of most chiral compounds were in a range of 94–99%, while the total yields of both chiral products were all above 85%. This enzymatic one-pot chain method is still very effective, not only can it be amplified to the level of grams but also the catalyst was recovered three times.

In 2019, Gao et al. demonstrated the construction of an unencapsulated remotecontrolled nanobiocatalytic system. The system used three enzyme-conjugated gold nanorod composites (EGCs) to control reaction rates in real time by self-assembling *Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

**Figure 33.** *One-pot cascade for synthesis of chiral β-hydroxy ketone derivatives.*

enzymes formed by the combination of enzymes at different optimal temperatures and gold nanorods (GNRs) [47]. By using the photothermal effect of GRS to transfer energy quickly, coupled with the real-time and long-range regulation of enzyme activity, improving the thermal stability of the enzyme and effective catalysis of the aldol reaction can be achieved. The increase in energy inside GRS, stimulated by distant near-infrared (NIR) stimuli, leads to increased enzyme activity. The results show that the method of internal heating that transfers energy more directly to the enzyme-catalyzed site is a faster and more effective energy transfer method. The results also show that the catalytic effect of the remote-controlled nanocatalytic system at lower temperature is the same as that of the free enzyme at higher temperature, but it has the advantages of improving the stability of the enzyme and extending its service life. Specifically, PPL EGCs at room temperature exhibit the same catalytic effect as achieved by free PPL at 40°C, while ST0779EGCs and APE1547 EGCs at 33°C exhibit a higher catalytic effect than their corresponding free enzymes at 63°C. In addition, EGCs have superior catalytic efficiency and product yield compared with aldol addition in free enzyme systems.

#### **3.2 Henry (nitroaldol) reaction**

The nitroaldol or Henry reaction is one of the most useful carbon-carbon bondforming reactions and has wide synthetic applications in organic chemistry. This reaction provides access to valuable racemic and optically active β-nitro alcohols, which are very useful in organic synthesis as precursors for pharmaceutical and biological purposes. In recent years, efficient nonconventional biocatalytic approaches have been reported [48].

Guan et al. firstly reported transglutaminase was used to catalyze Henry reactions of aliphatic, aromatic, and heteroaromatic aldehydes with nitroalkanes (**Figure 34**) [49]. The reactions were carried out at room temperature, and the corresponding nitroalcohols were obtained in yields up to 96%.

**Figure 34.** *Enzyme-catalyzed Henry reaction of 4-nitrobenzaldehyde and nitromethane.*

step, the oxygen of carbonyl group in 4-nitrobenzaldehyde is protonated by protons from His 555 imidazole ring, and the carbon atoms in the same carbonyl group are neutrally attacked by oleic acid carbon to form a new C▬C bond. Finally the aldol product is released from the enzyme, and the enzyme is freed for a new reaction. In 2016, Wu et al. demonstrated a one-pot bienzymatic cascade in organic media

*Proposed mechanism for ST0779-catalyzed aldol reaction between acetone and 4-nitrobenzaldehyde.*

to synthesize chiral β-hydroxy ketones for the first time (**Figure 33**) [46]. The decarboxylative aldol reaction catalyzed by an immobilized lipase from *Mucor miehei* (MML) and the synthesis of β-hydroxy ketone catalyzed by a lipase A or B from *Candida antarctica* (CALA or CALB) were combined in this one-pot protocol, reducing the purification step between the two reactions. (S)-β-hydroxy ketones and acylated (R)-β-hydroxy ketones could be obtained under mild reaction condition. The ee values of most chiral compounds were in a range of 94–99%, while the total yields of both chiral products were all above 85%. This enzymatic one-pot chain method is still very effective, not only can it be amplified to the level of grams

In 2019, Gao et al. demonstrated the construction of an unencapsulated remotecontrolled nanobiocatalytic system. The system used three enzyme-conjugated gold nanorod composites (EGCs) to control reaction rates in real time by self-assembling

but also the catalyst was recovered three times.

**Figure 32.**

**50**

**Figure 31.**

*Molecular Biotechnology*

*Aldol reactions catalyzed by ST0779 or PPL.*

Then, the same group reported glucoamylase from *Aspergillus niger* (AnGA) catalyze Henry reactions of aromatic aldehydes and nitroalkanes in 2013 [50]. The reactions were carried out at 30°C in the mixed solvents of ethanol and water, and the corresponding β-nitro alcohols were obtained in yields of up to 99%. Experiments demonstrated that AnGA could be inhibited by the product of the Henry reaction at 80°C. This enzymatic Henry reaction has a broad substrate scope and could be facilely enlarged to gram scale. Based on the experiments with denatured and inhibited AnGA, and the comparison of natural activity and promiscuous activity, the possible mechanism was also discussed (**Figure 35**). Glu400, as a base, deprotonates the α-carbon of the nitroalkane providing intermediate I. At the same time, Glu179, as an acid, donates a proton to the carbonyl oxygen of the aldehyde generating intermediate II. Then, the a-carbon of I, as a nucleophile, attacks the carbonyl of II forming a new carbon-carbon bond. Finally, the product (β-nitro alcohol) is released from the active site.

On the other hand, Lin and co-workers demonstrated the Henry reaction can also be catalyzed in a neat organic solvent. When using the D-aminoacylase from *Escherichia coli* as the promiscuous biocatalyst, DMSO was found to be the best solvent at 50°C [51]. Interestingly, the synthesis of optically active β-nitro alcohols was achieved by a two-step strategy combining the D-aminoacylase-catalyzed nitroaldol reaction with the PSL-catalyzed resolution of the so obtained racemic βnitro alcohols (**Figure 36**) [52]. Both alcohols and acetates were isolated in good yields and high enantiomeric excess (>84% ees; >96% eep; E > 150).

In 2013, lipase A from *Aspergillus niger* was used in the Henry reaction between aromatic aldehydes and a large excess of nitroalkanes in an organic/water medium (**Figure 37**) [53]. The yield of corresponding β-nitro alcohols at 30°C reached 94%.

Gotor and co-workers reported the inexpensive carrier protein bovine serum albumin (BSA) as catalyst was firstly used in the condensation of an appropriate aldehyde with 1-nitroalkanes in aqueous media (**Figure 38**) [54]. By optimizing the reaction conditions, the yield of corresponding nitroalcohols at 30°C reached 91%.

Similarly, two other well-known lipases, *Pseudomonas cepacia* lipase and CALB, were found to catalyze the Henry reaction [55]. Nevertheless, spectroscopic experiments showed that the immobilization protocols contribute to the change in the secondary structure of the enzyme, which leads to improved conversion rates.

**3.3 Aldol (nitroaldol) reaction in untraditional solvent**

*Catalytic nitroaldol addition between different aromatic aldehydes and nitromethane.*

**Figure 37.**

**Figure 36.**

**Figure 38.**

**53**

niger*.*

Solvents for a biocatalysis reaction have experienced several generations of development. Traditional organic solvents (water miscible or water immiscible), in the form of cosolvents or second phase, can provide solutions for the abovedescribed challenges. However, organic solvents inevitably face their own

*Nitroaldol reaction between aromatic aldehydes and nitroalkanes catalyzed by lipase A from* Aspergillus

*Two-step method to obtain β-nitro alcohols and the corresponding acetates of both configurations based on a Daminoacylase-catalyzed reaction and PSL-mediated kinetic resolution using vinyl acetate as acyl donor.*

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

**Figure 35.** *Possible mechanism of the AnGA-catalyzed Henry reaction.*

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

#### **Figure 36.**

Then, the same group reported glucoamylase from *Aspergillus niger* (AnGA) catalyze Henry reactions of aromatic aldehydes and nitroalkanes in 2013 [50]. The reactions were carried out at 30°C in the mixed solvents of ethanol and water, and the corresponding β-nitro alcohols were obtained in yields of up to 99%. Experiments demonstrated that AnGA could be inhibited by the product of the Henry reaction at 80°C. This enzymatic Henry reaction has a broad substrate scope and could be facilely enlarged to gram scale. Based on the experiments with denatured and inhibited AnGA, and the comparison of natural activity and promiscuous activity, the possible mechanism was also discussed (**Figure 35**). Glu400, as a base, deprotonates the α-carbon of the nitroalkane providing intermediate I. At the same time, Glu179, as an acid, donates a proton to the carbonyl oxygen of the aldehyde generating intermediate II. Then, the a-carbon of I, as a nucleophile, attacks the carbonyl of II forming a new carbon-carbon bond. Finally, the product (β-nitro

On the other hand, Lin and co-workers demonstrated the Henry reaction can also be catalyzed in a neat organic solvent. When using the D-aminoacylase from *Escherichia coli* as the promiscuous biocatalyst, DMSO was found to be the best solvent at 50°C [51]. Interestingly, the synthesis of optically active β-nitro alcohols was achieved by a two-step strategy combining the D-aminoacylase-catalyzed nitroaldol reaction with the PSL-catalyzed resolution of the so obtained racemic βnitro alcohols (**Figure 36**) [52]. Both alcohols and acetates were isolated in good

In 2013, lipase A from *Aspergillus niger* was used in the Henry reaction between aromatic aldehydes and a large excess of nitroalkanes in an organic/water medium (**Figure 37**) [53]. The yield of corresponding β-nitro alcohols at 30°C reached 94%. Gotor and co-workers reported the inexpensive carrier protein bovine serum albumin (BSA) as catalyst was firstly used in the condensation of an appropriate aldehyde with 1-nitroalkanes in aqueous media (**Figure 38**) [54]. By optimizing the reaction conditions, the yield of corresponding nitroalcohols at 30°C reached 91%. Similarly, two other well-known lipases, *Pseudomonas cepacia* lipase and CALB, were found to catalyze the Henry reaction [55]. Nevertheless, spectroscopic experiments showed that the immobilization protocols contribute to the change in the secondary structure of the enzyme, which leads to improved conversion rates.

yields and high enantiomeric excess (>84% ees; >96% eep; E > 150).

alcohol) is released from the active site.

*Molecular Biotechnology*

**Figure 35.**

**52**

*Possible mechanism of the AnGA-catalyzed Henry reaction.*

*Two-step method to obtain β-nitro alcohols and the corresponding acetates of both configurations based on a Daminoacylase-catalyzed reaction and PSL-mediated kinetic resolution using vinyl acetate as acyl donor.*

**Figure 37.**

*Nitroaldol reaction between aromatic aldehydes and nitroalkanes catalyzed by lipase A from* Aspergillus niger*.*

**Figure 38.**

*Catalytic nitroaldol addition between different aromatic aldehydes and nitromethane.*

#### **3.3 Aldol (nitroaldol) reaction in untraditional solvent**

Solvents for a biocatalysis reaction have experienced several generations of development. Traditional organic solvents (water miscible or water immiscible), in the form of cosolvents or second phase, can provide solutions for the abovedescribed challenges. However, organic solvents inevitably face their own

disadvantages, such as high volatility, difficulty in preparation, and inhibition of the activity of biocatalysts. So the untraditional solvents such as buffer solvent, ionic liquids (ILs), and deep eutectic solvents (DESs) have attracted the interest of many groups.

excellent stereoselectivity in this efficient and recyclable room-temperature ionic liquid in the presence of moderate water. High yields of up to 99%, excellent enantioselectivities of up to 90% ee, and good diastereoselectivities of up to >99:1

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

as extraction, materials synthesis and biotransformation, and biocatalysis.

acetone, cyclopentanone, and cyclohexanone.

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

*The PPL-catalyzed asymmetric cross aldol reaction in DESs.*

**4. Multicomponent reactions (MCRs)**

*Lipase AS-catalyzed Henry reaction in DES.*

Despite the excellent performance of ILs in biocatalysis, more doubts about their ungreenness and environmental influence have been gradually presented. Deep eutectic solvents, the recognized alternative of ILs, first came to the public vision in 2001. Since then, research on DESs faced a prosperous increase in many fields, such

Gotor-Fernández and co-workers reported a promiscuous lipase-catalyzed aldol reaction has been performed for the first time in DESs in 2016. The aldol reaction between 4-nitrobenzaldehyde and acetone was examined in-depth, with excellent compatibility being found between PPL and DESs (choline chloride/glycerol mixtures) for the formation of the aldol product in high yields (**Figure 42**) [59]. The system was compatible with a series of aromatic aldehydes and ketones including

At the same year, Tian et al. explored the Henry reaction catalyzed by lipase AS using deep eutectic solvents as a reaction medium (**Figure 43**) [60]. The studies had shown that adding 30 vol% water to DES could increase the catalytic activity of enzymes. The final yield of the lipase AS-catalyzed Henry reaction was 92.2% in a DES-water mixture within only 4 h. In addition, the lipase AS activity was

improved by approximately threefold in a DES-water mixture compared with that in pure water. The methodology was also extended to the aza-Henry reaction. The

Multicomponent reactions have attracted sustained attention because they represent a powerful tool for the construction of complex molecular structures with evident advantages, such as simplified workup procedures, high overall yields, and versatile product libraries. Recently, hydrolases have allowed the development of

enantioselectivity of both Henry and aza-Henry reactions was not found.

dr were achieved.

**Figure 42.**

**Figure 43.**

**55**

In 2013, our group demonstrated bovine pancreatic lipase (BPL) was first used to catalyze the aldol reaction and acidic buffer was first used for promiscuous enzymatic aldol reaction (**Figure 39**) [56]. The highest yield (99.0%), the best dr of 96:4, and a moderate ee of 66% were observed with aromatic aldehyde and ketone by BPL in phosphate-citrate buffer (pH 5.6, 5.0 mL) at 30°C.

Porto et al. demonstrated the lipase from *Rhizopus niveus* (RNL) catalyzed by unspecific protein catalysis the aldol reactions between cyclohexanone and aromatic aldehydes in organic solvents with water or aqueous buffer solution (**Figure 40**) [57]. The reactional conditions strongly influenced the yield (0–99%) and enantioselectivities in the anti-products (6–55% ee). The aldol products with enantioselectivities in the anti-product were observed for inactive enzyme and in denaturing conditions. Therefore, the reactions in the evaluated conditions were proceeded by unspecific protein catalysis with moderate enantioselectivities and not by promiscuous activity.

Ionic liquids are the first enzyme-compatible untraditional media developed by the green and sustainable concept (given their low vapor pressure). Numerous reactions, e.g., hydrolytic and redox reactions as well as formation of C▬C bond, have been successfully performed in such ILs-containing media. We demonstrated PPL was used to catalyze asymmetric cross aldol reactions of aromatic and heteroaromatic aldehydes with various ketones in ionic liquid ([BMIM][PF6]) for the first time in 2014 (**Figure 41**) [58]. PPL exhibited high catalytic activity and

**Figure 39.** *The BPL-catalyzed asymmetric aldol reaction in buffer solution.*

#### **Figure 40.**

*Aldol reactions by lipase from* Rhizopus niveus*.*

**Figure 41.** *The PPL-catalyzed asymmetric cross aldol reaction in ionic liquid.*

### *Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

excellent stereoselectivity in this efficient and recyclable room-temperature ionic liquid in the presence of moderate water. High yields of up to 99%, excellent enantioselectivities of up to 90% ee, and good diastereoselectivities of up to >99:1 dr were achieved.

Despite the excellent performance of ILs in biocatalysis, more doubts about their ungreenness and environmental influence have been gradually presented. Deep eutectic solvents, the recognized alternative of ILs, first came to the public vision in 2001. Since then, research on DESs faced a prosperous increase in many fields, such as extraction, materials synthesis and biotransformation, and biocatalysis.

Gotor-Fernández and co-workers reported a promiscuous lipase-catalyzed aldol reaction has been performed for the first time in DESs in 2016. The aldol reaction between 4-nitrobenzaldehyde and acetone was examined in-depth, with excellent compatibility being found between PPL and DESs (choline chloride/glycerol mixtures) for the formation of the aldol product in high yields (**Figure 42**) [59]. The system was compatible with a series of aromatic aldehydes and ketones including acetone, cyclopentanone, and cyclohexanone.

At the same year, Tian et al. explored the Henry reaction catalyzed by lipase AS using deep eutectic solvents as a reaction medium (**Figure 43**) [60]. The studies had shown that adding 30 vol% water to DES could increase the catalytic activity of enzymes. The final yield of the lipase AS-catalyzed Henry reaction was 92.2% in a DES-water mixture within only 4 h. In addition, the lipase AS activity was improved by approximately threefold in a DES-water mixture compared with that in pure water. The methodology was also extended to the aza-Henry reaction. The enantioselectivity of both Henry and aza-Henry reactions was not found.

**Figure 42.** *The PPL-catalyzed asymmetric cross aldol reaction in DESs.*

**Figure 43.** *Lipase AS-catalyzed Henry reaction in DES.*

## **4. Multicomponent reactions (MCRs)**

Multicomponent reactions have attracted sustained attention because they represent a powerful tool for the construction of complex molecular structures with evident advantages, such as simplified workup procedures, high overall yields, and versatile product libraries. Recently, hydrolases have allowed the development of

disadvantages, such as high volatility, difficulty in preparation, and inhibition of the activity of biocatalysts. So the untraditional solvents such as buffer solvent, ionic liquids (ILs), and deep eutectic solvents (DESs) have attracted the interest of many

In 2013, our group demonstrated bovine pancreatic lipase (BPL) was first used

Porto et al. demonstrated the lipase from *Rhizopus niveus* (RNL) catalyzed by unspecific protein catalysis the aldol reactions between cyclohexanone and aromatic aldehydes in organic solvents with water or aqueous buffer solution (**Figure 40**) [57]. The reactional conditions strongly influenced the yield (0–99%) and enantioselectivities in the anti-products (6–55% ee). The aldol products with enantioselectivities in the anti-product were observed for inactive enzyme and in denaturing conditions. Therefore, the reactions in the evaluated conditions were proceeded by unspecific protein catalysis with moderate enantioselectivities and not by promis-

Ionic liquids are the first enzyme-compatible untraditional media developed by the green and sustainable concept (given their low vapor pressure). Numerous reactions, e.g., hydrolytic and redox reactions as well as formation of C▬C bond, have been successfully performed in such ILs-containing media. We demonstrated

PPL was used to catalyze asymmetric cross aldol reactions of aromatic and heteroaromatic aldehydes with various ketones in ionic liquid ([BMIM][PF6]) for the first time in 2014 (**Figure 41**) [58]. PPL exhibited high catalytic activity and

*The BPL-catalyzed asymmetric aldol reaction in buffer solution.*

*Aldol reactions by lipase from* Rhizopus niveus*.*

*The PPL-catalyzed asymmetric cross aldol reaction in ionic liquid.*

to catalyze the aldol reaction and acidic buffer was first used for promiscuous enzymatic aldol reaction (**Figure 39**) [56]. The highest yield (99.0%), the best dr of 96:4, and a moderate ee of 66% were observed with aromatic aldehyde and ketone

by BPL in phosphate-citrate buffer (pH 5.6, 5.0 mL) at 30°C.

groups.

*Molecular Biotechnology*

cuous activity.

**Figure 39.**

**Figure 40.**

**Figure 41.**

**54**

multiple transformations and mainly served for the synthesis of heterocyclic compounds with high complexity in high yields. In this section, we will focus on the hydrolase-catalyzed multicomponent reaction in a one-pot transformation.

complement to chemical catalysis. The control experiments with the denatured enzyme and non-enzyme proteins indicated that the specific natural fold of SGP was responsible for its stereoselectivity in the Mannich reaction. A wide range of substrates were accepted by the enzyme, and yields of up to 92%, enantioselectivities of up to 88% ee, and diastereoselectivities of up to 92:8 dr were achieved. As an example of enzyme catalytic promiscuity, this work broadens the scope of SGP-

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

In the same year, Lin et al. inspired by chemical cofactors or mediators expect some small molecules to similarly improve the enzymatic Michael addition of unactivated carbon nucleophiles [64]. They found that the CALB/acetamide cocatalyst system can effectively catalyze the Michael addition between less-activated ketones and aromatic nitroolefins. This is of particular interest because neither CALB nor acetamide can independently catalyze the reaction to any significant extent. The CALB/acetamide catalyst system is also effective for other C▬C bondforming reactions with varying degrees of success, for example, CALB-catalyzed Mannich reaction (**Figure 47**). After adding acetamide as a co-catalyst, the yield increased by 50% (from 25 to 38%). The synergistic catalytic system of the lipase and the small molecule organic catalyst will greatly expand the application prospect

After 2 years, Guan et al. reported the use of acylase I from *Aspergillus melleus* in

the asymmetric Mannich reaction (**Figure 48**) [65]. Compared to the current chemical technologies, this enzymatic reaction is more environmentally friendly and sustainable by using biocatalysts from inexpensive renewable resources. The activity and stereoselectivity of AMA can be improved by adjusting the solvent, pH, water content, temperature, substrate molar ratio, and enzyme loading. A wide range of substrates can be accepted by AMA, achieving enantioselectivities up to 89% ee, diastereoselectivities up to 90:10 dr, and yields up to 82% in the mixture of MeCN and phosphate buffer pH 8.1 (85:15 v/v 1 mL) at 30°C. This work not only provides new examples of enzyme-catalyzed reliability and potential synthetic methods of organic chemistry but may also help to better understand the metabolic

pathways of nitrogen-containing compound biosynthesis.

*Mannich reaction of (E)-N-(4-nitro-benzylidene)aniline with cyclohexanone.*

catalyzed transformations.

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

of the enzyme in organic synthesis.

**Figure 47.**

**Figure 48.**

**57**

*The AMA-catalyzed Mannich reactions.*

## **4.1 Mannich reaction**

The Mannich reaction is a typical and the first example for the hydrolasecatalyzed multicomponent reaction, which is atom-economic and a powerful synthetic method for generating carbon-carbon bonds and nitrogenous compounds. An unprecedented "one-pot," direct Mannich reaction of ketone, aldehyde, and amine catalyzed by lipase was described first in 2009 [61]. Lipase from *Mucor miehei* (MML) efficiently catalyzed the Mannich reaction (**Figure 44**).

To assess the generality of the lipase-catalyzed Mannich reaction, we extended other substrates such as cyclohexanone, butanone, and 1-hydroxy-2-propanone in more benign reaction system (ethanol/water) catalyzed by the lipase from *Candida rugosa* (CRL) [62]. It was found that a wide range of aromatic aldehydes could effectively participate in the CRL-catalyzed Mannich reaction to give the corresponding β-amino carbonyl compounds (**Figure 45**). The reaction was favored by the electron-withdrawing substituents of the aldehydes.

In 2012, Guan et al. reported the enzyme-catalyzed, direct, three-component asymmetric Mannich reaction using protease type XIV from *Streptomyces griseus* (SGP) in acetonitrile (**Figure 46**) [63]. This characteristic makes it important to develop an enzyme-catalyzed asymmetric Mannich reaction as a more sustainable

**Figure 44.** *The first lipase-catalyzed direct Mannich reaction.*

#### **Figure 45.**

*Lipase-catalyzed direct Mannich reaction of various aryl aldehydes and ketones with aniline.*

**Figure 46.** *SPG-catalyzed direct Mannich reaction.*

### *Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

complement to chemical catalysis. The control experiments with the denatured enzyme and non-enzyme proteins indicated that the specific natural fold of SGP was responsible for its stereoselectivity in the Mannich reaction. A wide range of substrates were accepted by the enzyme, and yields of up to 92%, enantioselectivities of up to 88% ee, and diastereoselectivities of up to 92:8 dr were achieved. As an example of enzyme catalytic promiscuity, this work broadens the scope of SGPcatalyzed transformations.

In the same year, Lin et al. inspired by chemical cofactors or mediators expect some small molecules to similarly improve the enzymatic Michael addition of unactivated carbon nucleophiles [64]. They found that the CALB/acetamide cocatalyst system can effectively catalyze the Michael addition between less-activated ketones and aromatic nitroolefins. This is of particular interest because neither CALB nor acetamide can independently catalyze the reaction to any significant extent. The CALB/acetamide catalyst system is also effective for other C▬C bondforming reactions with varying degrees of success, for example, CALB-catalyzed Mannich reaction (**Figure 47**). After adding acetamide as a co-catalyst, the yield increased by 50% (from 25 to 38%). The synergistic catalytic system of the lipase and the small molecule organic catalyst will greatly expand the application prospect of the enzyme in organic synthesis.

After 2 years, Guan et al. reported the use of acylase I from *Aspergillus melleus* in the asymmetric Mannich reaction (**Figure 48**) [65]. Compared to the current chemical technologies, this enzymatic reaction is more environmentally friendly and sustainable by using biocatalysts from inexpensive renewable resources. The activity and stereoselectivity of AMA can be improved by adjusting the solvent, pH, water content, temperature, substrate molar ratio, and enzyme loading. A wide range of substrates can be accepted by AMA, achieving enantioselectivities up to 89% ee, diastereoselectivities up to 90:10 dr, and yields up to 82% in the mixture of MeCN and phosphate buffer pH 8.1 (85:15 v/v 1 mL) at 30°C. This work not only provides new examples of enzyme-catalyzed reliability and potential synthetic methods of organic chemistry but may also help to better understand the metabolic pathways of nitrogen-containing compound biosynthesis.

**Figure 47.** *Mannich reaction of (E)-N-(4-nitro-benzylidene)aniline with cyclohexanone.*

**Figure 48.** *The AMA-catalyzed Mannich reactions.*

multiple transformations and mainly served for the synthesis of heterocyclic compounds with high complexity in high yields. In this section, we will focus on the hydrolase-catalyzed multicomponent reaction in a one-pot transformation.

The Mannich reaction is a typical and the first example for the hydrolasecatalyzed multicomponent reaction, which is atom-economic and a powerful synthetic method for generating carbon-carbon bonds and nitrogenous compounds. An unprecedented "one-pot," direct Mannich reaction of ketone, aldehyde, and amine catalyzed by lipase was described first in 2009 [61]. Lipase from *Mucor miehei*

To assess the generality of the lipase-catalyzed Mannich reaction, we extended other substrates such as cyclohexanone, butanone, and 1-hydroxy-2-propanone in more benign reaction system (ethanol/water) catalyzed by the lipase from *Candida rugosa* (CRL) [62]. It was found that a wide range of aromatic aldehydes could effectively participate in the CRL-catalyzed Mannich reaction to give the

corresponding β-amino carbonyl compounds (**Figure 45**). The reaction was favored

In 2012, Guan et al. reported the enzyme-catalyzed, direct, three-component asymmetric Mannich reaction using protease type XIV from *Streptomyces griseus* (SGP) in acetonitrile (**Figure 46**) [63]. This characteristic makes it important to develop an enzyme-catalyzed asymmetric Mannich reaction as a more sustainable

(MML) efficiently catalyzed the Mannich reaction (**Figure 44**).

by the electron-withdrawing substituents of the aldehydes.

*Lipase-catalyzed direct Mannich reaction of various aryl aldehydes and ketones with aniline.*

**4.1 Mannich reaction**

*Molecular Biotechnology*

**Figure 44.**

**Figure 45.**

**Figure 46.**

**56**

*SPG-catalyzed direct Mannich reaction.*

*The first lipase-catalyzed direct Mannich reaction.*

### **4.2 Biginelli reactions**

In 2013, Sinha et al. reported bovine serum albumin promoted simple and efficient one-pot procedure for synthesis of 3,4-dihydropyrimidin-2(1H)-ones including potent mitotic kinesin Eg5 inhibitor monastrol under mild reaction conditions (**Figure 49**) [66]. After the reaction conditions are optimized, the yields reached up to 82% in EtOH at 60°C. The catalyst recyclability and gram-scale synthesis have also been demonstrated to enhance the practical utility of process.

Followed by our group, we reported trypsin as a multifunctional catalyst for synthesis of 3,4-dihydropyrimidin-2(1H)-ones by the Biginelli reaction of urea, βdicarbonyl compounds, and in situ-formed acetaldehyde (**Figure 50**) [67]. This one-pot multistep reaction consists of two relatively independent reactions, both of which are catalyzed by trypsin. First is the transesterification of ethyl acetate and isobutanol at 60°C to produce in situ acetaldehyde, followed by in situ-generated acetaldehyde, urea, and β-dicarbonyl compounds for Biginelli reaction. The first reaction continuously provides a substance for the second reaction, effectively reducing the volatilization loss, oxidation, and polymerization of acetaldehyde and avoiding the negative influence of excess acetaldehyde on the enzyme. Under optimal conditions, a wide range of substrates participate in the reaction and provide the target product in high yield.

In 2017, CALB-catalyzed for synthesis of 3,4-dihydropyrimidin-2(1H)-ones by a tandem multicomponent reaction in one pot (**Figure 51**) has been reported [68]. Several control experiments were performed using acetaldehydes directly to explore the possible mechanism of this procedure. Moreover, owing to the distinct modularity and highly efficient features of the MCR, it assembles libraries of structurally diverse products and provides an exceptional synthesis tool for the discovery of the minimal deep-blue luminogen in the solid state, namely, a single ring. A few of the compounds show deep-blue emissions which only contain a single ring. This is an important application of green biocatalytic promiscuity for constructing a wide variety of new materials.

was very low (only 25%). The yield of the product was slightly improved using a mixed solvent (the ratio of MTBE to acetylacetone was 6:4), and the molar ratio of 4-nitrobenzaldehyde acetamide was 1:4 at 50 mg/ml lipase. When the lipase con-

They proposed a reasonable mechanism of the reaction, wherein Asp-His dyad and oxyanion hole in the active site stabilized acetamide (**Figure 53**). This activated acetamide reacted with 1,3-dicarbonyl compounds to form an intermediate, which upon subsequent hydrolysis by CALB formed an enamine intermediate. During this period, a CALB-catalyzed Knoevenagel condensation reaction of the 1,3-dicarbonyl compound with aldehyde formed a separate intermediate (α,β-unsaturated carbonyl compound). Subsequently, the intermediate that is stabilized by the catalytic center of the lipase forms the final product (1,4-dihydropyridine) by Michael addi-

In 2017, our group reported a series of 1,4-dihydropyridines was produced via facile enzymatic Hantzsch reactions in one pot, using acetaldehydes/aromatic aldehydes prepared in situ (**Figure 54**) [70]. After screening several parameters on a model reaction, the tandem process afforded 1a in 80% yield. This approach provided an opportunity to discover novel libraries of AIEEgens that contain the minimum requirement necessary for AIEE behavior, namely, a single ring.

Meanwhile, we found that certain 1,4-DHPs could stain the mitochondria in live

cells with high selectivity but without obvious guiding units (such as cationic groups). Taking one of the 1,4-DHPs as an example, we found that it exhibited excellent photostability and storage stability and that it could be utilized in applications such as real-time imaging, long-term tracking of mitochondrial morphological changes, and viscosity mapping (**Figure 55**). We believe that the use of biocatalysis

centration (100 mg/mL) was increased, the yield increased sharply.

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

tion and intramolecular condensation.

**Figure 52.**

**59**

**Figure 51.**

*Lipase-catalyzed Hantzch-type reaction.*

*CALB-initiated tandem Biginelli reaction.*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

#### **4.3 Hantzsch reaction**

Lin et al. reported a three-component (aldehyde, 1,3-dicarbonyl compound, and acetamide) Hantzsch-type reaction in anhydrous solvent, which gave 1,4 dihydropyridines in moderate to good yields (**Figure 52**) [69]. The group used acetamide as a new source of ammonia. Initially the yield of the reaction with CALB

**Figure 50.**

*Trypsin-catalyzed Biginelli reaction using an in situ-generated acetaldehyde.*

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

**Figure 51.** *CALB-initiated tandem Biginelli reaction.*

**4.2 Biginelli reactions**

*Molecular Biotechnology*

vide the target product in high yield.

wide variety of new materials.

**4.3 Hantzsch reaction**

*BSA-catalyzed Biginelli reaction.*

**Figure 49.**

**Figure 50.**

**58**

In 2013, Sinha et al. reported bovine serum albumin promoted simple and efficient one-pot procedure for synthesis of 3,4-dihydropyrimidin-2(1H)-ones including potent mitotic kinesin Eg5 inhibitor monastrol under mild reaction conditions (**Figure 49**) [66]. After the reaction conditions are optimized, the yields reached up to 82% in EtOH at 60°C. The catalyst recyclability and gram-scale synthesis have also been demonstrated to enhance the practical utility of process. Followed by our group, we reported trypsin as a multifunctional catalyst for synthesis of 3,4-dihydropyrimidin-2(1H)-ones by the Biginelli reaction of urea, βdicarbonyl compounds, and in situ-formed acetaldehyde (**Figure 50**) [67]. This one-pot multistep reaction consists of two relatively independent reactions, both of which are catalyzed by trypsin. First is the transesterification of ethyl acetate and isobutanol at 60°C to produce in situ acetaldehyde, followed by in situ-generated acetaldehyde, urea, and β-dicarbonyl compounds for Biginelli reaction. The first reaction continuously provides a substance for the second reaction, effectively reducing the volatilization loss, oxidation, and polymerization of acetaldehyde and avoiding the negative influence of excess acetaldehyde on the enzyme. Under optimal conditions, a wide range of substrates participate in the reaction and pro-

In 2017, CALB-catalyzed for synthesis of 3,4-dihydropyrimidin-2(1H)-ones by a tandem multicomponent reaction in one pot (**Figure 51**) has been reported [68]. Several control experiments were performed using acetaldehydes directly to explore the possible mechanism of this procedure. Moreover, owing to the distinct modularity and highly efficient features of the MCR, it assembles libraries of structurally diverse products and provides an exceptional synthesis tool for the discovery of the minimal deep-blue luminogen in the solid state, namely, a single ring. A few of the compounds show deep-blue emissions which only contain a single ring. This is an important application of green biocatalytic promiscuity for constructing a

Lin et al. reported a three-component (aldehyde, 1,3-dicarbonyl compound, and

acetamide) Hantzsch-type reaction in anhydrous solvent, which gave 1,4 dihydropyridines in moderate to good yields (**Figure 52**) [69]. The group used acetamide as a new source of ammonia. Initially the yield of the reaction with CALB

*Trypsin-catalyzed Biginelli reaction using an in situ-generated acetaldehyde.*

**Figure 52.** *Lipase-catalyzed Hantzch-type reaction.*

was very low (only 25%). The yield of the product was slightly improved using a mixed solvent (the ratio of MTBE to acetylacetone was 6:4), and the molar ratio of 4-nitrobenzaldehyde acetamide was 1:4 at 50 mg/ml lipase. When the lipase concentration (100 mg/mL) was increased, the yield increased sharply.

They proposed a reasonable mechanism of the reaction, wherein Asp-His dyad and oxyanion hole in the active site stabilized acetamide (**Figure 53**). This activated acetamide reacted with 1,3-dicarbonyl compounds to form an intermediate, which upon subsequent hydrolysis by CALB formed an enamine intermediate. During this period, a CALB-catalyzed Knoevenagel condensation reaction of the 1,3-dicarbonyl compound with aldehyde formed a separate intermediate (α,β-unsaturated carbonyl compound). Subsequently, the intermediate that is stabilized by the catalytic center of the lipase forms the final product (1,4-dihydropyridine) by Michael addition and intramolecular condensation.

In 2017, our group reported a series of 1,4-dihydropyridines was produced via facile enzymatic Hantzsch reactions in one pot, using acetaldehydes/aromatic aldehydes prepared in situ (**Figure 54**) [70]. After screening several parameters on a model reaction, the tandem process afforded 1a in 80% yield. This approach provided an opportunity to discover novel libraries of AIEEgens that contain the minimum requirement necessary for AIEE behavior, namely, a single ring.

Meanwhile, we found that certain 1,4-DHPs could stain the mitochondria in live cells with high selectivity but without obvious guiding units (such as cationic groups). Taking one of the 1,4-DHPs as an example, we found that it exhibited excellent photostability and storage stability and that it could be utilized in applications such as real-time imaging, long-term tracking of mitochondrial morphological changes, and viscosity mapping (**Figure 55**). We believe that the use of biocatalysis

**Figure 54.**

**Figure 55.**

**Figure 56.**

**61**

*milling conditions.*

*the HeLa cells. Scale bar: 7.5 μm.*

*Synthesis of 1 and 2 through one-pot multicomponent reactions.*

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

*Confocal fluorescence images of HeLa cells stained with 2 h and MitoTracker® Deep Red FM (MTDR). (A) Fluorescent image of 2h (10.0 μM) in HeLa cells, collected at 410-460 nm and λex = 405 nm. (B) Fluorescent image of MTDR (1.0 μM), collected at 660-740 nm and λex = 633 nm. (C) Merged image of A and B. (D) Overlay of fluorescence and bright-field image. (E) Intensity profiles of linear regions of interest (ROI) across*

*Lipozyme® RM IM-catalyzed rapid synthesis of 1,4-DHP calcium antagonists and derivatives under ball*

**Figure 53.**

*Proposed mechanism of lipase-catalyzed Hantzsch-type reaction of an aldehyde with acetamide and 1,3 dicarbonyl compounds.*

could be simplified with workup procedures and could provide avenues for discovering a wide variety of new materials.

In recently, Ye et al. reported solvent-free quick synthesis of 1,4-DHP calcium antagonists felodipine, nitrendipine, nifedipine, and nemadipine B and their derivatives by Lipozyme® RM IM-catalyzed multicomponent reactions of aromatic aldehyde, alkyl acetoacetate, and alkyl 3-aminocrotonate under ball milling conditions (**Figure 56**) [71]. The product was obtained in moderate yield (up to 86.8%), and the effects of the reaction conditions were investigated, including catalyst loading,

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

**Figure 54.** *Synthesis of 1 and 2 through one-pot multicomponent reactions.*

#### **Figure 55.**

*Confocal fluorescence images of HeLa cells stained with 2 h and MitoTracker® Deep Red FM (MTDR). (A) Fluorescent image of 2h (10.0 μM) in HeLa cells, collected at 410-460 nm and λex = 405 nm. (B) Fluorescent image of MTDR (1.0 μM), collected at 660-740 nm and λex = 633 nm. (C) Merged image of A and B. (D) Overlay of fluorescence and bright-field image. (E) Intensity profiles of linear regions of interest (ROI) across the HeLa cells. Scale bar: 7.5 μm.*

**Figure 56.**

*Lipozyme® RM IM-catalyzed rapid synthesis of 1,4-DHP calcium antagonists and derivatives under ball milling conditions.*

could be simplified with workup procedures and could provide avenues for discov-

*Proposed mechanism of lipase-catalyzed Hantzsch-type reaction of an aldehyde with acetamide and 1,3-*

In recently, Ye et al. reported solvent-free quick synthesis of 1,4-DHP calcium antagonists felodipine, nitrendipine, nifedipine, and nemadipine B and their derivatives by Lipozyme® RM IM-catalyzed multicomponent reactions of aromatic aldehyde, alkyl acetoacetate, and alkyl 3-aminocrotonate under ball milling conditions (**Figure 56**) [71]. The product was obtained in moderate yield (up to 86.8%), and the effects of the reaction conditions were investigated, including catalyst loading,

ering a wide variety of new materials.

**Figure 53.**

**60**

*dicarbonyl compounds.*

*Molecular Biotechnology*

grinding aid, and milling frequency. The reaction features environmentally friendly, simple, and efficient operation. A major feature distinguishing this enzyme promiscuity from previously reported work is the use of mechanochemical ball milling techniques that overcome disadvantages such as long reaction times and the use of hazardous organic solvents. This work demonstrates the potential application of mixed enzyme-catalyzed reactions for drug synthesis under ball milling conditions.

processes are particularly favorable for creating compound libraries for medicinal chemistry lead finding and for functional chromophores in materials sciences.

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

This chapter has reviewed some examples of various types of hydrolase catalytic promiscuous reactions and their applications in the past decade. Several different types of hydrolases catalyzed carbon-carbon or carbon-heteroatom formation reactions have been discussed: aldol reactions, Michael reactions, and multicomponent

From these examples, it is clear that enzymes that display catalytic promiscuity can provide new opportunities for organic synthesis. Exploiting enzyme catalytic promiscuous reactions might lead to new, efficient, and stable catalysts with alternative activity and could provide more promising and green synthetic methods for organic chemistry. The development of protein engineering and enzyme engineering can extend the application of metagenome libraries and find enzymes with specific promiscuous behavior. We believe the progress in the area of biocatalytic

The authors gratefully acknowledge funding of our research in this area by grants from the Natural Science Foundation of Guangdong Province (Grant No. 2018A030307022) and the Special Innovation Projects of Common Universities in

1 School of Chemistry and Chemical Engineering, Lingnan Normal University,

2 Key Laboratory of Green Chemistry and Technology, Ministry of Education,

© 2019 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,

promiscuity will greatly extend the useful applications of enzymes.

Guangdong Province (Grant No.2018KTSCX126).

\*

\*Address all correspondence to: wnchem@scu.edu.cn

provided the original work is properly cited.

College of Chemistry, Sichuan University, Chengdu, P.R. China

**5. Summary**

reactions.

**Acknowledgements**

**Author details**

Zhanjiang, China

**63**

Yun Wang<sup>1</sup> and Na Wang<sup>2</sup>

## **4.4 Ugi reaction**

In 2013, Berlozecki et al. reported for the first time an enzyme-catalyzed Ugi reaction that has many advantages over previous reactions, such as good reaction at room temperature and extensive solvent selection (**Figure 57**) [72]. In this threecomponent reaction, the aldehyde, amine, and isocyanide are condensed to form a dipeptide. Of all the selected lipases, Novozym 435 had the highest yield of 75%.

Recently, Thomas et al. reported the concatenation of the Ugi four-component synthesis, and the CALB-catalyzed aminolysis of the intermediary formed Ugi methyl ester products furnishes a novel consecutive five-component reaction for the formation of triamides (**Figure 58**) [73]. This one-pot method is compatible with metal catalysis methods such as copper-catalyzed alkyl azide ring addition and Suzuki cross-coupling or both in a one-pot process.

The mild reaction conditions make this sequence superior to the stepwise process with isolation of the Ugi product and even more favorable than other basecatalyzed or microwave-assisted aminolyses. This efficient scaffold forming

**Figure 57.** *CALB-catalyzed Ugi reaction.*

**Figure 58.** *Consecutive six-component U-4CR-CALB-catalyzed aminolysis-Suzuki cross-coupling sequence of biaryls.*

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

processes are particularly favorable for creating compound libraries for medicinal chemistry lead finding and for functional chromophores in materials sciences.
