Michael lipase catalyzed

Figure 3.5 Michael addition catalyzed by the SerlOSAla C. antarctica lipase B mutant.

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

Scheme 5.20 Lipase-catalyzed Michael addition of amines to acrylonitrile.

In a similar way, lipases catalyze Michael addition of amines, thiols [110], and even 1,3-dicarbonyl derivatives [111, 112] to a,/ -unsaturated carbonyl compounds (Scheme 5.21). 

Scheme 5.21 Lipase-catalyzed Michael additions to a./J-unsaturated carbonyl compounds.

Scheme 5.22 Quantum-chemical model system of the lipase-catalyzed Michael addition of methanethiol to acrolein [110],...

Other authors have described the lipase-catalyzed chemoselective acylation of alcohols in the presence of phenolic moities [14], the protease-catalyzed acylation of the 17-amino moiety of an estradiol derivative [15], the chemoselectivity in the aminolysis reaction of methyl acrylate (amide formation vs the favored Michael addition) catalyzed by Candida antarctica lipase (Novozym 435) [16], and the lipase preference for the O-esterification in the presence of thiol moieties, as, for instance, in 2-mercaptoethanol and dithiotreitol [17]. This last finding was recently exploited for the synthesis of thiol end-functionalized polyesters by enzymatic polymerization of e-caprolactone initiated by 2-mercaptoethanol (Figure 6.2)... 

Recently, even examples of lipase-catalyzed Michael additions and aldol condensations have appeared [7]. These are dramatic examples of catalytic promiscuity, that is, the ability of an enzyme to catalyze more distinctly different chemical transformations [8], Often such activities are explained in terms of the active site offering a scaffold in which substrates adopt favorable conformations and/ or reactants are brought together in a desired geometry. Accordingly, after being observed in wild-type enzymes, these side activities can often be enhanced in site-directed variants, in which residues in or close to the active site are mutated. 

In 1992, Desmaele utiUzed enamine chemistry in a Michael addition to obtain chiral precursor 193 that gave vertinolide s tetronic ring (194) afler ketahzation, oxidation to lactone, and elimination (Scheme 1.31) [108]. After 4years, Matsuo and Sakaguchi [109] employed their chiral l,3-dioxolan-4-one 195 to obtain the central unsaturated lactone 196 after side-chain functionalization and condensation with triethyl-2-phosphonopropionate derived anion. Finally, in 2006, Takabe and coworkers [110], based on their previous work on the synthesis of racemic vertinolide, applied a lipase-catalyzed resolution on a 5-hydroxymethylene derivative of 197 to reach the natural enantiomer after five more synthetic steps (Scheme 1.31). 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

Lipase from C.antarctica also catalyzes carbon-carbon bond formation through aldol condensation of hexanal. The reaction is believed to proceed according to the same mechanism as the Michael additions [113]. Lipase from Pseudomonas sp. 

Even an entirely different enzyme can be changed to the one that has enolase activity. One representative example is the changing of a lipase to an aldolase utilizing the basicity of the catalytic triad via a simple mutation. The resulting promiscuous lipase has been demonstrated to catalyze the aldol reaction and Michael addition as shown in Fig. 23. 

The potential of enzymes as practical catalysts is well described, and their activity and selectivity (stereo-, chemo-, and regioselectivity) for catalyzed reactions cover a broad range. Enzymes clearly constitute very powerful green tools for catalyzing synthetic chemical processes. In this context, the continuous increase of the market for enantiopure fine chemicals places enzymes as suitable catalysts for green synthetic processes. Catalytic promiscuity of enzymes in nonaqueous environments has been widely described and is related to the ability of a single active site to catalyze more than one chemical transformation for example, lipase B from Candida antarctica (CALB) is able to catalyze aldol additions, Michael-type additions, and so on [4]. 

Stereospecific Michael addition reactions also may be catalyzed by hydrolytic enzymes (Scheme 2.205). When ot-trifluoromethyl propenoic acid was subjected to the action of various proteases, lipases and esterases in the presence of a nucleophile (NuH), such as water, amines, and thiols, chiral propanoic acids were obtained in moderate optical purity [1513]. The reaction mechanism probably involves the formation of an acyl enzyme intermediate (Sect. 2.1.1, Scheme 2.1). Being an activated derivative, the latter is more electrophilic than the free carboxylate and undergoes an asymmetric Michael addition by the nucleophile, directed by the chiral environment of the enzyme. In contrast to these observations made with crude hydrolase preparations, the rational design of a Michaelase from a lipase-scaffold gave disappointingly low stereoselectivities [1514-1517]. 

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