Enzymatic deacylation

The transformations described thus far were catalyzed by enzymes in their traditional hydrolytic mode. More recent developments in the area of enzymatic catalysis in nonaqueous media (11,16,33—35) have significantly broadened the repertoire of hydrolytic enzymes. The acyl—enzyme intermediate formed in the first step of the reaction via acylation of the enzyme s active site nucleophile can be deacylated in the absence of water by a number of... 

Adipoyl moiety is first attached ia order to provide an appropriately spaced proximal carbonyl group. The esters are oxidized enzymatically and then deacylated. The procedure results ia the synthesis of diols (119) with excellent enantiomeric purity (ee 96—98%) ia 72—92% yield. 

Another important enzymatic process in the production of 7-ADCA, for use in the production of semi-synthetic cephalosporins, is the hydrolysis of 7-aminocephalosporanic add (7-ACA) by the enzyme acetyl esterase. This process, again using immobilisation techniques, is illustrated in Figure 6.16. Hie deacylated product can be used, for example, as an intermediate in the production of the important oral cephalosporin cefuroxime. We will return to cephalosporin antibiotics later in this chapter. 

Enzymes are the catalyst per excellence for reactions in water, which is their natural habitat. Moreover, the use of enzymes often circumvents the need for functional group protection and deprotection steps. For example, enzymatic hydrolysis of penicillin G to 6-APA (Fig. 2.30) proceeds in one step at ambient temperature while chemical deacylation requires three steps, a temperature of - 40 C and various stoichiometric reagents, leading to a high E factor. 

However, the main stimulus for switching from chemical to enzymatic deacylation was to avoid the use of dichloromethane as solvent (Brugging et al, 1998). The enzymatic process acounts for most of the several thousands of tons of 6-APA produced annually on a worldwide basis. 

Figure 2.30. Enzymatic versus chemical deacylation of pencillin G.

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

DKR of secondary alcohol is achieved by coupling enzyme-catalyzed resolution with metal-catalyzed racemization. For efficient DKR, these catalyhc reactions must be compatible with each other. In the case of DKR of secondary alcohol with the lipase-ruthenium combinahon, the use of a proper acyl donor (required for enzymatic reaction) is parhcularly crucial because metal catalyst can react with the acyl donor or its deacylated form. Popular vinyl acetate is incompatible with all the ruthenium complexes, while isopropenyl acetate can be used with most monomeric ruthenium complexes. p-Chlorophenyl acetate (PCPA) is the best acyl donor for use with dimeric ruthenium complex 1. On the other hand, reaction temperature is another crucial factor. Many enzymes lose their activities at elevated temperatures. Thus, the racemizahon catalyst should show good catalytic efficiency at room temperature to be combined with these enzymes. One representative example is subtilisin. This enzyme rapidly loses catalytic activities at elevated temperatures and gradually even at ambient temperature. It therefore is compatible with the racemization catalysts 6-9, showing good activities at ambient temperature. In case the racemization catalyst requires an elevated temperature, CALB is the best counterpart. 

In order to recover both amines in ophcaUy achve form the amide is hydrolyzed chemically by reachon with NaOH in aqueous ethylene glycol at 150 °C. This brute force method would certainly lead to problems with amines containing other functional groups and is in stark contrast to the elegant enzymatic procedure used for the first step. Hence, an overall greener process can be obtained by employing an enzymatic deacylation step in what we have called an easy-on/easy-off process... 

This process has many benefits in the context of green chemistry it involves two enzymatic steps, in a one-pot procedure, in water as solvent at ambient temperature. It has one shortcoming, however-penicillin acylase generally works well only with amines containing an aromatic moiety and poor enantioselectivities are often observed with simple aliphatic amines. Hence, for the easy-on/easy-off resolution of aliphatic amines a hybrid form was developed in which a hpase [Candida antarctica hpase B (CALB)] was used for the acylation step and peniciUin acylase for the deacylahon step [22]. The structure of the acyl donor was also optimized to combine a high enanhoselectivity in the first step with facile deacylation in the second step. It was found that pyridyl-3-acetic acid esters gave optimum results (see Scheme 6.8). 

The first enzymatic polymerizations of substituted lactones were performed by Kobayashi and coworkers using Pseudomonas fluorescens lipase or CALB as the biocatalyst [90-92]. A clear enantiopreference was observed for different lactone monomers, resulting in the formation of optically active polymers. More recently, a systematic study was performed by Al-Azemi et al. [93] and Peelers et al. [83] on the ROP of 4-alkyl-substituted CLs using Novozym 435. Peelers et al. studied the selectivity and the rates as a function of the substituent size with the aim of elucidating the mechanism and the rate-determining step in these polymerizations. Enantio-enriched polymers were obtained, but the selectivity decreased drastically with the increase in substituent size [83]. Remarkably for 4-propyl-e-caprolactone, the selectivity was for the (R)-enantiomer in a polymerization, whereas it was S)-selective in the hydrolysis reaction. Comparison of the selectivity in the hydrolysis reaction (Fig. 10b) with that of the polymerization reaction (Scheme 8a) revealed that the more bulky the alkyl substituent, the more important the deacylation step becomes as the rate-determining step. 

For resolution of the racemate 12 two different procedures can be applied 124 the en-antioselective enzymatic deacylation of chloroacetyl-DL-a-aminosuberic acid at pH 7.2 with Taka-acylase or the enantioselective salt precipitation of Z-dl-Asu-OH with D-tyrosine hydrazide according to the method of Vogler et alJ25 Complete enzymatic digestion is achieved in about ten days at 37 °C, and the optically pure L-enantiomer is obtained in 80% yield but the overall efficiency is lower than that of the chemical method. Fractional crystallization affords in good yields the Z-l-Asu-OH derivative 13 which is then used directly as a suitably protected intermediate in subsequent derivatization steps (see Scheme 6). Moreover, the recovery of the D-enantiomer from the mother liquors is also easy in this case. 

In favorable cases, enzymatic acylation and deacylation are stereochemically complementary and provide access to both enantiomers. 

Early in vitro data indicated that ring expansion of adipyl-6-APA or penicillin G was either nonexistent or barely detectable [45,70], Therefore, an alternative route to 7-ADCA through the enzymatic deacylation of DAOC (Fig. 3) requiring the fermentative production of DAOC at economically feasible levels was pursued. Elimination of DACS activity would allow C. acremonium to produce DAOC as its end product (Fig. 1). Before it was known that DAOCS and DACS activities in C. acremonium were catalyzed by a single bifunctional enzyme,... 

Enzymatic Deacylation of Echinocandins and Related Antifungal Agents... 

Enzymatic deacylation of acyl side-chain and peptide nucleus analogs of ECB, containing a cyclic hexapeptide, was examined with the purified native deacylase under the reaction conditions optimized with ECB, as described previously [25], Unless specified, the deacylation reaction was conducted in the presence of... 

Figure 4 Enzymatic deacylation of immobilized ECB using filtrate versus concentrate.

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