**2. Michael addition**

As a result, reactions that generally tend to be "cleaner" and laborious can largely be omitted; and (5) enzymes can catalyze a broad spectrum of reactions. There is an enzyme-catalyzed process equivalent to almost every type of organic reaction, such as oxidation, hydrolysis, addition, halogenation, alkylation, and isomerization. In addition, many enzymes accept unnatural substrates, and genetic engineering can further alter their stability, broaden their substrate specificity, and increase their specific activity. Thus, the application of enzymes in synthesis thus represents a remarkable opportunity for the development of industrial chemical and pharma-

Although it is well known that a given enzyme is able to catalyze a specific reaction efficiently, some unexpected experimental results have indicated that many enzymes have catalytic promiscuity [8–12]. Enzyme promiscuity is classified into three categories: (a) condition promiscuity, which is an enzyme's ability to work under unexpected condition; (b) substrate promiscuity, which is an enzyme's ability to work with unexpected substrates; and (c) catalytic promiscuity, which is an enzyme's ability to catalyze unexpected reactions. Among them, catalytic promiscuity has gained much attention as it opens a wide scope for the industrial

During the past decade, biocatalytic promiscuity, as a new frontier extending the

Hydrolases (such as lipase, protease, acylase) have received extensive attention as biocatalysts for a long time due to their many attractive properties like stability in

use of enzymes in organic synthesis, has received considerable attention and expanded rapidly. A classic example of promiscuous enzymatic behavior is pyruvate decarboxylase, which not only decarboxylates pyruvate but also links acetaldehyde and benzaldehyde to form R-phenylacetylcarbinol. The use of pyruvate decarboxylase to form carbon–carbon bonds, which does not occur in the natural reaction, was first studied in 1921 and was applied in industry today [13]. As one of the most rapidly growing areas in enzymology, multifunctional biocatalytic reactions not only highlights the existing catalysts but may provide novel and practical synthetic pathways which are not currently available. Miao et al. reviewed enzyme promiscuity for carbon-carbon bond-forming reactions like aldol couplings, Michael(-type) additions, Mannich reactions, Henry reactions, and Knoevenagel condensations [14]. Gotor-Fernández et al. also highlighted the hydrolase-catalyzed

reactions for nonconventional transformations in the same year [15].

ceutical processes [4–7].

*Molecular Biotechnology*

application of enzymes.

**Figure 1.**

**34**

*Hydrolase-catalyzed promiscuous reactions.*

Michael addition is a 1,4-addition of a nucleophile to α,β-unsaturated compounds, and it is one of the most fundamental and important reactions for the formation of carbon-carbon bonds and carbon-heteroatom bonds in organic synthesis. Michael addition reactions are traditionally catalyzed under strong basic or acidic conditions, which can cause unwanted side reactions such as further condensation or polymerization of α,β-unsaturated compounds. Thus, biocatalysis can afford a green and facile method for organic synthesis. Among different biocatalysts, hydrolases such as protease and lipase have been widely used as a green and efficient catalyst for Michael addition.

#### **2.1 Carbon-heteroatom bond formation Michael addition**

Michael addition is the early promiscuous reaction catalyzed by hydrolase. In 1986, Kitazume et al. reported the hydrolytic enzyme-catalyzed stereospecific Michael addition reactions in buffer solution (pH = 8.0) at 40–41°C (**Figure 2**) [16]. This discovery overthrows the long erroneous concept of enzymology that "biocatalysis must be carried out in aqueous solution," making many organic reactions that cannot be carried out in water be completed in organic solvents and greatly expanding the application scope of enzymes as catalysts. Moreover, enzymes are frequently more stable in organic solvents than in water. Thus, some research groups began to focus on enzyme-catalyzed Michael addition reactions in organic solvents.

Lin and Gotor et al. firstly reported the hydrolase-catalyzed Michael addition of imidazole with acrylates catalyzed by alkaline protease from *Bacillus subtilis* in organic solvent in 2004 [17, 18]. Subsequently, other hydrolase-catalyzed Michael addition reactions were reported. In 2010, Bhanage et al. developed an efficient protocol for the regioselective aza-Michael addition of amines with acrylates using

**Figure 2.** *The first hydrolase-catalyzed Michael addition in buffer.* *Candida antarctica* lipase B (CALB) as a biocatalyst at 60°C (**Figure 3**) [19]. The universality of the reaction, including the reactions of various primary and secondary amines with different acrylates, was also studied. Higher yields were obtained.

Gotor et al. have explored new synthetic possibilities of commercially available protease from *Bacillus licheniformis* immobilized as cross-linked enzyme aggregates (Alcalase-CLEA) in 2011, since the CLEA immobilization improves the stability of the protein toward denaturalization by heating, organic solvents, and autoproteolysis [20]. Alcalase-CLEA has achieved the best results in the aza-Michael addition of secondary amines to acrylonitrile (**Figure 4**). In all cases the formations of the corresponding Michael adduct were faster than in the absence of biocatalyst, but also in comparison with the inhibited enzyme, the reaction rates being highly dependent of the amine structure.

beneficial to the chemoselective 1,2-addition, using TBME, dioxane, or toluene under overnight (15 h) gentle magnetic stirring at 50°C (**Figure 6**) [22]. Under these conditions, the yield of acrylamide was good at the Gram scale. S-trans-z and e-diphenylamine were formed as by-products. The reactions worked well with other primary amines but not with secondary amines that only gave the corresponding aminoacrylates. The chemoselectivity of CALB with N- and Snucleophiles was also checked. The transesterification also worked in good yields in TBME, toluene, or dioxane. The best yields (near quantitative) were observed when the reactions were carried out in open vessels under gentle magnetic stirring at 50°

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

*Lipase-catalyzed aza-Michael addition of amines to α,β-unsaturated esters.*

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

In the same year, Franssen and co-workers demonstrated lipases from *Pseudomonas stutzeri* (PSL) and *Chromobacterium viscosum* (CVL) are excellent catalysts for the aza-Michael addition of amines to substituted or unsubstituted acrylates with high product selectivity and good yields (**Figure 7**) [23]. Comparative studies of other lipases, including Novoxin 435, have proven ineffective. The selective Michael addition of diamines to these substituted acrylates was also realized. In this paper, the catalytic effects of various lipases on aza-Michael addition reaction, especially on the lipase catalysis of *Pseudomonas aeruginosa* OM2 and PSL, are introduced. The 1,4-adducts of acrylate and benzylamine have high yield and

Chemoselective synthesis of N-protected β-amino esters involving lipasecatalyzed aza-Michael additions is mainly hampered by the two electrophilic sites present on these compounds. In order to control the chemoselectivity, a solvent engineering strategy based on the thermodynamic behavior of products in media of different polarity was designed by Castillo et al. (**Figure 8**) [24]. This strategy can

for 6 h.

**Figure 5.**

selectivity.

**Figure 6.**

**37**

*Reactivity of benzylamine with ethyl propiolate.*

C

In 2012, Baldessari et al. firstly reported the synthesis of N-substituted β-amino esters by application of *Rhizomucor miehei* lipase in aza-Michael addition of monoand bifunctional amines to α,β-unsaturated esters [21]. The authors selected ethyl acrylate and propyl acrylate as the Michael acceptors and different alkylamines, alkanolamines, and diamines as the Michael donors (**Figure 5**). The reactions were carried out in low-polarity solvents (hexane, toluene, and diisopropyl ether (DIPE)) at 30°C for 16 h with yields from 12 to 100%. Subsequently, the authors investigated the effect of the reaction conditions on the Michael addition systematically, such as commercially available enzyme sources, organic solvents, and the structure of the Michael acceptor and donor. The results showed that the alkanolamines in n-hexane were not selective and double Michael adducts could be obtained. Substrate concentration also plays an important role in enhancing the catalytic effect of enzymes on spontaneous reactions. High substrate concentration limits the efficiency of biocatalysts.

In 2013, Demeunynck et al. have optimized the lipase-biocatalyzed addition of benzylamine to ethyl propiolate. Immobilized *Candida antarctica* lipase B was

#### **Figure 3.**

*CALB-catalyzed aza-Michael addition of amine to acrylate.*

**Figure 4.** *Hydrolase-catalyzed Michael-type additions between secondary amines and acrylonitrile.* *Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

#### **Figure 5.**

*Candida antarctica* lipase B (CALB) as a biocatalyst at 60°C (**Figure 3**) [19]. The universality of the reaction, including the reactions of various primary and secondary amines with different acrylates, was also studied. Higher yields were obtained. Gotor et al. have explored new synthetic possibilities of commercially available protease from *Bacillus licheniformis* immobilized as cross-linked enzyme aggregates (Alcalase-CLEA) in 2011, since the CLEA immobilization improves the stability of

In 2012, Baldessari et al. firstly reported the synthesis of N-substituted β-amino esters by application of *Rhizomucor miehei* lipase in aza-Michael addition of monoand bifunctional amines to α,β-unsaturated esters [21]. The authors selected ethyl acrylate and propyl acrylate as the Michael acceptors and different alkylamines, alkanolamines, and diamines as the Michael donors (**Figure 5**). The reactions were carried out in low-polarity solvents (hexane, toluene, and diisopropyl ether (DIPE)) at 30°C for 16 h with yields from 12 to 100%. Subsequently, the authors investigated the effect of the reaction conditions on the Michael addition systematically, such as commercially available enzyme sources, organic solvents, and the structure of the Michael acceptor and donor. The results showed that the alkanolamines in n-hexane were not selective and double Michael adducts could be obtained. Substrate concentration also plays an important role in enhancing the catalytic effect of enzymes on spontaneous reactions. High substrate concentration limits the efficiency of

In 2013, Demeunynck et al. have optimized the lipase-biocatalyzed addition of benzylamine to ethyl propiolate. Immobilized *Candida antarctica* lipase B was

the protein toward denaturalization by heating, organic solvents, and autoproteolysis [20]. Alcalase-CLEA has achieved the best results in the aza-Michael addition of secondary amines to acrylonitrile (**Figure 4**). In all cases the formations of the corresponding Michael adduct were faster than in the absence of biocatalyst, but also in comparison with the inhibited enzyme, the reaction rates

being highly dependent of the amine structure.

*CALB-catalyzed aza-Michael addition of amine to acrylate.*

*Hydrolase-catalyzed Michael-type additions between secondary amines and acrylonitrile.*

biocatalysts.

*Molecular Biotechnology*

**Figure 3.**

**Figure 4.**

**36**

*Lipase-catalyzed aza-Michael addition of amines to α,β-unsaturated esters.*

beneficial to the chemoselective 1,2-addition, using TBME, dioxane, or toluene under overnight (15 h) gentle magnetic stirring at 50°C (**Figure 6**) [22]. Under these conditions, the yield of acrylamide was good at the Gram scale. S-trans-z and e-diphenylamine were formed as by-products. The reactions worked well with other primary amines but not with secondary amines that only gave the corresponding aminoacrylates. The chemoselectivity of CALB with N- and Snucleophiles was also checked. The transesterification also worked in good yields in TBME, toluene, or dioxane. The best yields (near quantitative) were observed when the reactions were carried out in open vessels under gentle magnetic stirring at 50° C for 6 h.

In the same year, Franssen and co-workers demonstrated lipases from *Pseudomonas stutzeri* (PSL) and *Chromobacterium viscosum* (CVL) are excellent catalysts for the aza-Michael addition of amines to substituted or unsubstituted acrylates with high product selectivity and good yields (**Figure 7**) [23]. Comparative studies of other lipases, including Novoxin 435, have proven ineffective. The selective Michael addition of diamines to these substituted acrylates was also realized. In this paper, the catalytic effects of various lipases on aza-Michael addition reaction, especially on the lipase catalysis of *Pseudomonas aeruginosa* OM2 and PSL, are introduced. The 1,4-adducts of acrylate and benzylamine have high yield and selectivity.

Chemoselective synthesis of N-protected β-amino esters involving lipasecatalyzed aza-Michael additions is mainly hampered by the two electrophilic sites present on these compounds. In order to control the chemoselectivity, a solvent engineering strategy based on the thermodynamic behavior of products in media of different polarity was designed by Castillo et al. (**Figure 8**) [24]. This strategy can

**Figure 6.** *Reactivity of benzylamine with ethyl propiolate.*

addition product, obtained in 2M2B, by a resolution process with CALB on the

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

Our group demonstrated that 3-substituted 2H-chromene derivatives were synthesized via biocatalytic domino oxa-Michael/aldol condensations (**Figure 10**) [26]. α-Amylase from *Bacillus subtilis* shows excellent catalytic activity and exerts good adaptability to different substrates in the reaction. The reaction conditions including organic solvents, water content, temperature, molar ratio of substrates, and

It is generally believed that the hydrolysis site of hydrolase is also the active site of its miscible catalysis. On this basis, the possible mechanism of domino reaction catalyzed by hydrolase was proposed. First, salicylaldehyde was activated by amino residues and oxygen anions of amylase. Then methyl vinyl ketone is attacked by activated salicylaldehyde, and a new C▬O bond is formed by oxa-Michael addition reaction. Next, an intramolecular aldol reaction begins to form carbon–carbon bonds. Finally, the adduct was dehydrated, and the required product was released

Very recently, our group conducted an aza-Michael addition of aniline compounds and acrylate derivatives catalyzed by CALB and several mutants in order to investigate reaction mechanistic (**Figure 12**) [27]. The influence factors of the reaction were discussed systematically, including solvent, enzyme loading, temperature, and time of reaction. On this basis, dozens of substrates with different structures were conducted to occur aza-Michael addition on the optimized conditions. The results demonstrated that the structures of substrates had a great influ-

Four different reaction intermediates (Intermediate 1, 2, 3, and 4) were matched with the catalytic activity site of CALB to perform molecular docking simulation (**Figure 13A**). We can see that the binding mode of all the four intermediates with the active site is basically the same. The binding modes of four intermediates with CALB catalytic active sites were analyzed, in order to further study the blinding modes of aza-Michael addition intermediates and CALB and the driving forces of their mutual recognition. As shown in **Figure 13b**, it can be seen from the figure

enantiomers of aza-Michael addition product.

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

enzyme loading were optimized.

(**Figure 11**).

ence on the activity.

**Figure 10.**

**39**

*α-Amylase-catalyzed synthesis of 3-substituted 2H-chromene derivatives.*

**Figure 7.**

*Lipase-catalyzed aza-Michael addition of amines with different substituted acrylates.*

**Figure 8.** *Chemoselectivity of the addition of benzylamine to α,β-unsaturated esters.*

obtain highly selective aza-Michael adducts from benzylamine and different acrylates. Ammonia hydrolysis is avoided in almost all reactions with n-hexane (a nonpolar solvent) as solvent, while the corresponding Michael adduct is synthesized in 53–78% yield. On the contrary, if the reaction is carried out in polar solvents (e.g., 2-methyl-2-butanol (2M2B)), the product of ammonia hydrolysis will be advantageous. Thermodynamic analysis of these processes using the actual solvation conductor-like screening model (COSMO-RS) helps to understand some key factors affecting chemical selectivity and confirms that reliable estimates of the thermodynamic interactions between solutes and solvents allow adequate selection of reaction media that may lead to chemical selectivity.

Reaction media has an important effect on the yield and chemo- and enantioselectivity of biocatalytic reaction. Solvent engineering is an effective tool to direct chemo- and enantioselectivity of the aza-Michael addition and the subsequent kinetic resolution of the Michael adduct [25]. In the reaction of benzylamine and methyl crotonate catalyzed by CALB, three possible adducts can be isolated: aminolysis product, aza-Michael addition product, and double addition product (**Figure 9**). The authors selected n-hexane and 2-methyl-2-butanol (two solvents of opposite polarity) as solvents in the experiments. The Michael adduct is favored in more hydrophobic media, while the amide product in more polar solvents, and the best values of ee for aza-Michael adduct were obtained in almost 100% 2M2B. The experiment results clarify the origin of the enantiomeric excess of the aza-Michael

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

addition product, obtained in 2M2B, by a resolution process with CALB on the enantiomers of aza-Michael addition product.

Our group demonstrated that 3-substituted 2H-chromene derivatives were synthesized via biocatalytic domino oxa-Michael/aldol condensations (**Figure 10**) [26]. α-Amylase from *Bacillus subtilis* shows excellent catalytic activity and exerts good adaptability to different substrates in the reaction. The reaction conditions including organic solvents, water content, temperature, molar ratio of substrates, and enzyme loading were optimized.

It is generally believed that the hydrolysis site of hydrolase is also the active site of its miscible catalysis. On this basis, the possible mechanism of domino reaction catalyzed by hydrolase was proposed. First, salicylaldehyde was activated by amino residues and oxygen anions of amylase. Then methyl vinyl ketone is attacked by activated salicylaldehyde, and a new C▬O bond is formed by oxa-Michael addition reaction. Next, an intramolecular aldol reaction begins to form carbon–carbon bonds. Finally, the adduct was dehydrated, and the required product was released (**Figure 11**).

Very recently, our group conducted an aza-Michael addition of aniline compounds and acrylate derivatives catalyzed by CALB and several mutants in order to investigate reaction mechanistic (**Figure 12**) [27]. The influence factors of the reaction were discussed systematically, including solvent, enzyme loading, temperature, and time of reaction. On this basis, dozens of substrates with different structures were conducted to occur aza-Michael addition on the optimized conditions. The results demonstrated that the structures of substrates had a great influence on the activity.

Four different reaction intermediates (Intermediate 1, 2, 3, and 4) were matched with the catalytic activity site of CALB to perform molecular docking simulation (**Figure 13A**). We can see that the binding mode of all the four intermediates with the active site is basically the same. The binding modes of four intermediates with CALB catalytic active sites were analyzed, in order to further study the blinding modes of aza-Michael addition intermediates and CALB and the driving forces of their mutual recognition. As shown in **Figure 13b**, it can be seen from the figure

**Figure 10.** *α-Amylase-catalyzed synthesis of 3-substituted 2H-chromene derivatives.*

obtain highly selective aza-Michael adducts from benzylamine and different acrylates. Ammonia hydrolysis is avoided in almost all reactions with n-hexane (a nonpolar solvent) as solvent, while the corresponding Michael adduct is synthesized in 53–78% yield. On the contrary, if the reaction is carried out in polar solvents (e.g., 2-methyl-2-butanol (2M2B)), the product of ammonia hydrolysis will be advantageous. Thermodynamic analysis of these processes using the actual solvation conductor-like screening model (COSMO-RS) helps to understand some key factors affecting chemical selectivity and confirms that reliable estimates of the thermodynamic interactions between solutes and solvents allow adequate selection of reac-

*Lipase-catalyzed aza-Michael addition of amines with different substituted acrylates.*

Reaction media has an important effect on the yield and chemo- and enantioselectivity of biocatalytic reaction. Solvent engineering is an effective tool to direct chemo- and enantioselectivity of the aza-Michael addition and the subsequent kinetic resolution of the Michael adduct [25]. In the reaction of benzylamine and methyl crotonate catalyzed by CALB, three possible adducts can be isolated: aminolysis product, aza-Michael addition product, and double addition product (**Figure 9**). The authors selected n-hexane and 2-methyl-2-butanol (two solvents of opposite polarity) as solvents in the experiments. The Michael adduct is favored in more hydrophobic media, while the amide product in more polar solvents, and the best values of ee for aza-Michael adduct were obtained in almost 100% 2M2B. The experiment results clarify the origin of the enantiomeric excess of the aza-Michael

tion media that may lead to chemical selectivity.

*Lipase-catalyzed addition and aminolysis reaction of benzylamine to methyl crotonate.*

*Chemoselectivity of the addition of benzylamine to α,β-unsaturated esters.*

**Figure 7.**

*Molecular Biotechnology*

**Figure 8.**

**Figure 9.**

**38**

that the binding modes of the four intermediates with the CALB catalytic chamber

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

In order to determine the catalytic activity of CALB, three mutants and wildtype CALB were expressed in *E. coli* and were purified to catalyze the aza-Michael addition reaction. The results showed that aza-Michael activity could be dramatically decreased by the mutation of active sites: neither mutant S105 A nor mutant

However, the mutant I189 A still had a weak catalytic effect on this reaction. Based on these experimental results, the molecular docking was carried out, and the mechanism of aza-Michael addition catalyzed by CALB was studied, and a reasonable reaction mechanism was proposed (**Figure 14**). This helped to explain the effect of substrate structure on the reaction. The substituents of substrates affect the interaction with CALB active sites. Some substituents enhance the binding of substrates and facilitate the reaction. In the whole process, the Ser105 and His224 residues played an important role in proton transfer. Without these two residues, the proton transfer would be blocked, and the aza-Michael addition could not be possible. Besides, the Ile189 residue forms hydrophobic interaction with the benzene ring of the substrate, which makes the substrate more stable in the active cavity. The biocatalytic thia-Michael reaction is an attractive strategy to develop C▬S bond-forming reactions. In 2012, Kiełbasinski and co-workers have reported the use of a number of lipases including PPL, MJL, CALB, and PSL in the addition

of benzenethiol to racemic phenyl vinyl sulfoxide or 2-phosphono-2,3-

*Proposed reaction mechanism of aza-Michael addition catalyzed by CALB.*

didehydrothiolane S-oxide in organic solvents at room temperature (**Figure 15**) [28]. The addition of piperidine to phenyl vinyl sulfoxide in chloroform is carried out in both enzymatic and non-catalytic processes, while in the former, the reaction rate is 2.5 times faster. Conversely, the conjugate addition of phenylmercaptan with phenyl vinyl sulfoxide is only carried out in the presence of enzymes and ethanol as solvent. In any case, the product is not enantiomerically enriched. However, in the

are basically the same.

**Figure 14.**

**41**

H224 could catalyze the reaction.

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

**Figure 11.**

*The proposed mechanism for the α-amylase-catalyzed synthesis of 3-substituted 2H-chromene derivative.*

#### **Figure 12.**

*CALB and mutants catalyzed aza-Michael addition.*

#### **Figure 13.**

*Molecular docking simulation of CALB with four different reaction intermediates. (A)Hydrophobic matching of the four reaction intermediates with CAL B. The cavity represents the CAL B catalytic pocket which is able to bind and orient the substrates. The blue surface represents hydrophilic while the orange surface represents hydrophobic. The substrates are shown in the pocket in ball-and-stick representation with the atom of substrate coloured according to their atom types (carbon, grey; nitrogen, blue; oxygen, red; chlorine, green). (B)Three-dimensional model of the binding between four aza-Michael addition intermediates (1, 2, 3 and 4) and the CAL B active site. The protein is shown in grey with interacting residues shown as a sky blue stick model. The intermediate is shown as a yellow stick model, and the blue dotted lines indicate the hydrogen bonds between the intermediate and the active site of CAL B.*

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

that the binding modes of the four intermediates with the CALB catalytic chamber are basically the same.

In order to determine the catalytic activity of CALB, three mutants and wildtype CALB were expressed in *E. coli* and were purified to catalyze the aza-Michael addition reaction. The results showed that aza-Michael activity could be dramatically decreased by the mutation of active sites: neither mutant S105 A nor mutant H224 could catalyze the reaction.

However, the mutant I189 A still had a weak catalytic effect on this reaction. Based on these experimental results, the molecular docking was carried out, and the mechanism of aza-Michael addition catalyzed by CALB was studied, and a reasonable reaction mechanism was proposed (**Figure 14**). This helped to explain the effect of substrate structure on the reaction. The substituents of substrates affect the interaction with CALB active sites. Some substituents enhance the binding of substrates and facilitate the reaction. In the whole process, the Ser105 and His224 residues played an important role in proton transfer. Without these two residues, the proton transfer would be blocked, and the aza-Michael addition could not be possible. Besides, the Ile189 residue forms hydrophobic interaction with the benzene ring of the substrate, which makes the substrate more stable in the active cavity.

The biocatalytic thia-Michael reaction is an attractive strategy to develop C▬S bond-forming reactions. In 2012, Kiełbasinski and co-workers have reported the use of a number of lipases including PPL, MJL, CALB, and PSL in the addition of benzenethiol to racemic phenyl vinyl sulfoxide or 2-phosphono-2,3 didehydrothiolane S-oxide in organic solvents at room temperature (**Figure 15**) [28]. The addition of piperidine to phenyl vinyl sulfoxide in chloroform is carried out in both enzymatic and non-catalytic processes, while in the former, the reaction rate is 2.5 times faster. Conversely, the conjugate addition of phenylmercaptan with phenyl vinyl sulfoxide is only carried out in the presence of enzymes and ethanol as solvent. In any case, the product is not enantiomerically enriched. However, in the

**Figure 14.** *Proposed reaction mechanism of aza-Michael addition catalyzed by CALB.*

**Figure 11.**

*Molecular Biotechnology*

**Figure 12.**

**Figure 13.**

**40**

*CALB and mutants catalyzed aza-Michael addition.*

*between the intermediate and the active site of CAL B.*

*The proposed mechanism for the α-amylase-catalyzed synthesis of 3-substituted 2H-chromene derivative.*

*Molecular docking simulation of CALB with four different reaction intermediates. (A)Hydrophobic matching of the four reaction intermediates with CAL B. The cavity represents the CAL B catalytic pocket which is able to bind and orient the substrates. The blue surface represents hydrophilic while the orange surface represents hydrophobic. The substrates are shown in the pocket in ball-and-stick representation with the atom of substrate coloured according to their atom types (carbon, grey; nitrogen, blue; oxygen, red; chlorine, green). (B)Three-dimensional model of the binding between four aza-Michael addition intermediates (1, 2, 3 and 4) and the CAL B active site. The protein is shown in grey with interacting residues shown as a sky blue stick model. The intermediate is shown as a yellow stick model, and the blue dotted lines indicate the hydrogen bonds*

**Figure 15.** *The Michael addition of benzenethiol to racemic phenyl vinyl sulfoxide or 2-phosphono-2,3-didehydrothiolane S-oxide.*

presence of various lipases, the addition of phenylmercaptan to a better Michael receptor, cyclic sulfonyl alkylphosphonate, in some cases resulted in up to 25% optical purity of the product and the recovered substrate.

Then, some mechanistic considerations are presented in the studies. The authors proposed sulfoxide oxygen atoms are bound to the "oxygen anion pore" of the enzyme activity site by hydrogen bond. Conversely, histidine catalyzed by binary enhances the nucleophilicity of sulfur centers in phenylmercaptan molecules. Although the interaction of the latter is the same as Michael's addition of mercaptan to enols, the H-binding of sulfoxide oxygen atom must be different from that of carbonyl oxygen atom, which results in the lower catalytic efficiency of the enzyme for the reaction. It is well known that oxygen anion holes bind to the transition state better than the ground state. When lipase catalyzes ester hydrolysis, the intermediate oxygen anion is tetrahedral. Although the sulfoxide group is tetrahedral, which indicates that the bonding of sulfoxide group should be uniform, compared with the oxygen anion, the sulfoxide group has no negative charge on the intermediate oxygen atom, which significantly reduces the strength of hydrogen bond. In addition, for the Michael addition of nucleophilic reagents, the intermediate oxygen anion is planar, which reduces the space requirement and makes it more suitable for the oxygen anion pore than the tetrahedral sulfoxide intermediate (**Figure 16**).

is suitable for adding large amounts of 1,3-dicarbonyl compounds and cyclohexanone to aromatic and heteroaromatic nitroolefins and cyclohexenone in DMSO in the presence of water under mild reaction conditions (**Figure 18**) [30]. The enantioselectivities up to 83% ee and yields up to 90% were achieved.

*Assumed mechanism of the conjugate addition of benzenethiol to racemic phenyl vinyl sulfoxide.*

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

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

*PPL-catalyzed C▬S bond-forming reaction between cinnamaldehyde and thiophenol.*

Then, the same research group explored porcine pancreatic lipase (PPL) which was used as a biocatalyst to catalyze the Michael addition of 4-hydroxycoumarin to α,β-unsaturated enones in organic medium in the presence of water to synthesize warfarin and derivatives in 2012 (**Figure 19**) [31]. The products were obtained in moderate to high yields (up to 95%) with none or low enantioselectivities (up to 28% ee). The influence of reaction conditions including solvents, temperature, and molar ratio of substrates was systematically investigated. It was the first time

Sometimes Michael adducts are not the final targeted compounds. We studied lipase-catalyzed Michael addition between nitrostyrene and acetylacetone in DMSO in the presence of water under mild reaction conditions in 2011 [32]. Two possible adducts can be isolated: the routine Michael addition product and cyclic product

*Michael reaction of cyclohexenone and acetylacetone catalyzed by lipozyme TLIM in different solvents.*

warfarin and derivatives were prepared using a biocatalyst.

**Figure 16.**

**Figure 17.**

**Figure 18.**

**43**

In 2014, Domingues and co-workers firstly reported the reaction between cinnamaldehyde and thiophenol. Several hydrolases such as PPL, lipozyme, chymosin, and papain have demonstrated different levels of activities, and PPL has found application on the multigram scale (**Figure 17**) [29]. These reactions were carried out at room temperature, and good or excellent sulfur Michael adducts were obtained. The scheme describes the use of EtOH as a solvent and fewer enzymes. The chymosin and papain were used as biocatalysts for organic reactions for the first time.

#### **2.2 Carbon-carbon bond formation Michael addition**

C▬C bond-forming reactions are one of the mainstays of organic chemistry. In this field the hydrolase-catalyzed Michael reaction also has numerous applications in synthetic chemistry.

In 2011, the asymmetric C▬C Michael addition catalyzed by lipozyme TLIM (immobilized lipase from *Thermomyces lanuginosus*) in organic medium in the presence of water was reported for the first time by Guan et al. The biocatalytic reaction *Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

**Figure 16.** *Assumed mechanism of the conjugate addition of benzenethiol to racemic phenyl vinyl sulfoxide.*

**Figure 17.** *PPL-catalyzed C▬S bond-forming reaction between cinnamaldehyde and thiophenol.*

is suitable for adding large amounts of 1,3-dicarbonyl compounds and cyclohexanone to aromatic and heteroaromatic nitroolefins and cyclohexenone in DMSO in the presence of water under mild reaction conditions (**Figure 18**) [30]. The enantioselectivities up to 83% ee and yields up to 90% were achieved.

Then, the same research group explored porcine pancreatic lipase (PPL) which was used as a biocatalyst to catalyze the Michael addition of 4-hydroxycoumarin to α,β-unsaturated enones in organic medium in the presence of water to synthesize warfarin and derivatives in 2012 (**Figure 19**) [31]. The products were obtained in moderate to high yields (up to 95%) with none or low enantioselectivities (up to 28% ee). The influence of reaction conditions including solvents, temperature, and molar ratio of substrates was systematically investigated. It was the first time warfarin and derivatives were prepared using a biocatalyst.

Sometimes Michael adducts are not the final targeted compounds. We studied lipase-catalyzed Michael addition between nitrostyrene and acetylacetone in DMSO in the presence of water under mild reaction conditions in 2011 [32]. Two possible adducts can be isolated: the routine Michael addition product and cyclic product

**Figure 18.** *Michael reaction of cyclohexenone and acetylacetone catalyzed by lipozyme TLIM in different solvents.*

presence of various lipases, the addition of phenylmercaptan to a better Michael receptor, cyclic sulfonyl alkylphosphonate, in some cases resulted in up to 25%

*The Michael addition of benzenethiol to racemic phenyl vinyl sulfoxide or 2-phosphono-2,3-didehydrothiolane*

proposed sulfoxide oxygen atoms are bound to the "oxygen anion pore" of the enzyme activity site by hydrogen bond. Conversely, histidine catalyzed by binary enhances the nucleophilicity of sulfur centers in phenylmercaptan molecules. Although the interaction of the latter is the same as Michael's addition of mercaptan to enols, the H-binding of sulfoxide oxygen atom must be different from that of carbonyl oxygen atom, which results in the lower catalytic efficiency of the enzyme for the reaction. It is well known that oxygen anion holes bind to the transition state better than the ground state. When lipase catalyzes ester hydrolysis, the intermediate oxygen anion is tetrahedral. Although the sulfoxide group is tetrahedral, which indicates that the bonding of sulfoxide group should be uniform, compared with the oxygen anion, the sulfoxide group has no negative charge on the intermediate oxygen atom, which significantly reduces the strength of hydrogen bond. In addition, for the Michael addition of nucleophilic reagents, the intermediate oxygen anion is planar, which reduces the space requirement and makes it more suitable for the oxygen anion pore than the tetrahedral sulfoxide intermediate (**Figure 16**). In 2014, Domingues and co-workers firstly reported the reaction between cinnamaldehyde and thiophenol. Several hydrolases such as PPL, lipozyme,

Then, some mechanistic considerations are presented in the studies. The authors

chymosin, and papain have demonstrated different levels of activities, and PPL has found application on the multigram scale (**Figure 17**) [29]. These reactions were carried out at room temperature, and good or excellent sulfur Michael adducts were obtained. The scheme describes the use of EtOH as a solvent and fewer enzymes. The chymosin and papain were used as biocatalysts for organic reactions for the

C▬C bond-forming reactions are one of the mainstays of organic chemistry. In this field the hydrolase-catalyzed Michael reaction also has numerous applications

In 2011, the asymmetric C▬C Michael addition catalyzed by lipozyme TLIM (immobilized lipase from *Thermomyces lanuginosus*) in organic medium in the presence of water was reported for the first time by Guan et al. The biocatalytic reaction

optical purity of the product and the recovered substrate.

**2.2 Carbon-carbon bond formation Michael addition**

first time.

**42**

**Figure 15.**

*Molecular Biotechnology*

*S-oxide.*

in synthetic chemistry.

**Figure 19.**

*The Michael addition of 4-hydroxycoumarin 1 to α,β-unsaturated enones for the formation of warfarin and derivatives.*

**Figure 20.**

*Lipase-catalyzed reaction of nitrostyrene and acetylacetone.*

(**Figure 20**). With the aim to get cyclic product in more efficient manner, the catalytic activities of several lipases were firstly tested in mixed ethanol/water solvents. Among the tested lipases, PPL showed the best activity. And according to the single-crystal X-ray diffraction analysis of cyclic products, the reaction was confirmed to give the product oximes with Z-stereoselectivity.

Then, our group reported for the first time lipase-catalyzed direct vinylogous Michael addition reactions of vinyl malononitriles to nitroalkenes (**Figure 21**) [33]. A series of nitroalkenes reacted with vinyl malononitriles to produce the corresponding products with moderate to high yields in the presence of Lipozyme® (immobilized lipase from *Mucor miehei*). The excellent diastereoselective products were produced in all reactions in acetonitrile at 30°C for 48 h. The enzyme has only a very slight loss of catalyst efficiency after being reused for seven consecutive cycles of the reaction in the previously determined optimized conditions.

The reaction mechanism was studied by computational simulation approach using dock. Based on the proposed catalytic mechanism of Michael reaction, two docking process of the substrates with the amino acids of the active site were performed. The calculation results explained the experimental results that the lipase possessed specific substrate selectivity. To further elucidate the different catalytic effects of RML, CALB, and CRL, structural characteristics of their active site were analyzed, respectively (**Figure 22**). The docking results showed that once vinyl malononitrile was occupied by nitrostyrene, it could not be docked with the active site of CRL. It indicates that the active site is too narrow to bind both two substrates at the same time, so it could not catalyze the direct Michael addition reaction. As for CALB, the active site seems big enough for both nitrostyrene and vinyl malononitrile, but the docking results showed that nitrostyrene blocked proton

transferring from vinyl malononitrile to histidine, which may make it unable to

*The comparison of RML, CRL, and CALB: (A) the docking result of RML and substrates, (B) the docking*

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

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

Reaction mechanism of the lipase-catalyzed direct vinylogous Michael addition reaction has been proposed (**Figure 23**). First, nitroalkenes bind to oxygen anion pores and were stabilized by three hydrogen bonds with Leu145 and Ser82.

catalyze the direct vinylogous Michael addition.

*result of CRL and substrates and (C) the docking result of CALB and substrates.*

**Figure 22.**

**45**

**Figure 21.** *Lipase-catalyzed direct vinylogous Michael addition reaction.*

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

#### **Figure 22.**

(**Figure 20**). With the aim to get cyclic product in more efficient manner, the catalytic activities of several lipases were firstly tested in mixed ethanol/water solvents. Among the tested lipases, PPL showed the best activity. And according to the single-crystal X-ray diffraction analysis of cyclic products, the reaction was

*The Michael addition of 4-hydroxycoumarin 1 to α,β-unsaturated enones for the formation of warfarin and*

Then, our group reported for the first time lipase-catalyzed direct vinylogous Michael addition reactions of vinyl malononitriles to nitroalkenes (**Figure 21**) [33].

corresponding products with moderate to high yields in the presence of Lipozyme® (immobilized lipase from *Mucor miehei*). The excellent diastereoselective products were produced in all reactions in acetonitrile at 30°C for 48 h. The enzyme has only a very slight loss of catalyst efficiency after being reused for seven consecutive cycles of the reaction in the previously determined optimized conditions.

The reaction mechanism was studied by computational simulation approach using dock. Based on the proposed catalytic mechanism of Michael reaction, two docking process of the substrates with the amino acids of the active site were performed. The calculation results explained the experimental results that the lipase possessed specific substrate selectivity. To further elucidate the different catalytic effects of RML, CALB, and CRL, structural characteristics of their active site were analyzed, respectively (**Figure 22**). The docking results showed that once vinyl malononitrile was occupied by nitrostyrene, it could not be docked with the active site of CRL. It indicates that the active site is too narrow to bind both two substrates at the same time, so it could not catalyze the direct Michael addition reaction. As for CALB, the active site seems big enough for both nitrostyrene and vinyl malononitrile, but the docking results showed that nitrostyrene blocked proton

confirmed to give the product oximes with Z-stereoselectivity.

*Lipase-catalyzed reaction of nitrostyrene and acetylacetone.*

**Figure 19.**

*Molecular Biotechnology*

*derivatives.*

**Figure 20.**

**Figure 21.**

**44**

*Lipase-catalyzed direct vinylogous Michael addition reaction.*

A series of nitroalkenes reacted with vinyl malononitriles to produce the

*The comparison of RML, CRL, and CALB: (A) the docking result of RML and substrates, (B) the docking result of CRL and substrates and (C) the docking result of CALB and substrates.*

transferring from vinyl malononitrile to histidine, which may make it unable to catalyze the direct vinylogous Michael addition.

Reaction mechanism of the lipase-catalyzed direct vinylogous Michael addition reaction has been proposed (**Figure 23**). First, nitroalkenes bind to oxygen anion pores and were stabilized by three hydrogen bonds with Leu145 and Ser82.

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

In 2003, Berglund and co-workers firstly reported the serine hydrolase *Candida*

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

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

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,

CH2Cl2 with low diastereoselectivity (**Figure 26**). In consideration of both

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

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

biocatalysts has led researchers to consider other more stable enzymes [37].

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

been researched and presented in the last decade.

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

PPL II has aldolase function in organic solvents.

**3.1 Aldol reaction**

**Figure 25.**

**Figure 26.**

**47**

*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

**Figure 23.** *The proposed mechanism of lipase-catalyzed vinylogous Michael addition.*

**Figure 24.** *BioH esterase-catalyzed Michael addition-cyclization cascade reaction.*

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 nucleophilic carbon molecules to form products.

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