Acylation and Deacylation

The hydroxy group undergoes 0-acylation and deacylation (79JHC689). These reactions of functionalized hydroxyfurazans are valuable methods for modification of these compounds. Thus, hydroxybifurazan 248 was aroylated with benzoyl chloride in the presence of pyridine with concomitant cleavage of the unsubstituted furazan ring to give nitrile 262 (Scheme 170) (75LA1029). 

Table 12. Enantioselectivities in the acylation and deacylation steps in the burst kinetics of the reaction of (Z)-Phe-PNP(52)...

The ratios of these slopes for L- and D-esters are shown in Table 12. The kL/kD values of the acylation step in the CTAB micelle are very close to those in Table 9, as they should be. It is interesting to note that the second deacylation step also occurs enantioselectively. Presumably it is due to the deacylation ocurring by the attack of a zinc ion-coordinated hydroxide ion which, in principle, should be enantioselective as in the hydroxyl group of the ligand. Alternatively, the enantioselectivity is also expected when the free hydroxide ion attack the coordinated carbonyl groups of the acyl-intermediate with the zinc ion. At any rate, the rates of both steps of acylation and deacylation for the L-esters are larger than those for the D-esters in the CTAB micelle. However, in the Triton X-100 micelle, the deacylation step for the D-esters become faster than for the L-esters. 

Fig. 21 Rate-pH dependence of the acylation and deacylation steps in the chymotrypsin-catalysed hydrolysis of 4-nitrophenyl trimethylacetate...

The major steps in the catalytic process, namely reversible binding, acylation, and deacylation, are schematically represented in Fig. 3.3 with chy-motrypsin as an example. 

The solvent present in biphasic reactions can still have an effect on the enzyme even though the enzyme functions primarily in an aqueous microenvironment. A particularly dramatic example is the lipase AH (lipase from Burkholderia cepac/fl)-catalysed desym-metrization of prochiral 1,4-dihydropyridine dicarboxylic esters, where either enantiomer can be accessed in high enantioselectivity by using either water-saturated cyclohexane or diisopropyl ether (DIPE) respectively (Scheme 1.60). The acyl group used in acylation and deacylation can also have a dramatic effect on enantioselectivity. " ... 

There is now convincing evidence that an acyl chymotrypsin intermediate is formed from both specific and non-specific substrates (Bender and Kezdy, 1964 Bender et al., 1964). This intermediate is undoubtedly an acylserine. Acyl- and phosphorylserine derivatives have been isolated and identified. In view of evidence such as a D2 O solvent isotope effect ( h2oAd2o) 2-3 for both acylation and deacylation (Bender and Hamilton, 1962), alcohol and amine nucleophiles showing little dependence on the p/iTa-value of the nucleophile in reaction with furoyl enzyme (Inward and Jencks, 1965), and the influence of increasing steric bulk in the acyl group (Fife and Milstien, 1967 Milstien and Fife, 1968,.1969), consistent... 

Acylation and deacylation in equation (13) proceed through similar transition states. If deacylation occurs through attack of an alcohol molecule R OH rather than water on the carbonyl carbon atom, then deacylation is the microscopic reverse of acylation. Bender and coworkers (Bender and Kezdy, 1965) have demonstrated the symmetry of the reaction about the acyl enzyme in reactions in which reversibility can be observed. 

Both acylation and deacylation can be performed with a lipase as shown in Scheme 6.9 in which a CALB CLEA was used in the deacylation step that, not surprisingly, was rather slow compared to deacylation with pen acylase. 

Reaction Mechanism of Lipases and Implications for Monomer Acceptance in the Acylation and Deacylation Step... 

Fig. 1 Catalytic mechanism of CALB showing an acylation and deacylation step and the formation of a covalently bound acyl-enzyme intermediate bottom right) [16]...

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

In the catalytically active complex 4-Ba the negative poles and the polyether bridge act as working units that perform cooperatively in providing the driving force for the formation of the complex itself, whereas the metal ion serves as an electrophilic catalyst both in the acylation and deacylation steps. The crucial importance of the polyether bridge is demonstrated by the disappearance of any catalytic activity upon replacement by two methoxy groups. 

The Chymotrypsin Mechanism Involves Acylation and Deacylation of a Ser Residue... 

Chromophoric acyl group,4,5 The spectrum of the furylacryloyl group depends on the polarity of the surrounding medium, and also on the nature of the moiety to which it is attached. The spectrum of furylacryloyl-L-tyrosine ethyl ester changes slightly when it is bound to chymotrypsin. There are also further changes on formation of the acylenzyme and on the subsequent hydrolysis. The rate constants for acylation and deacylation and the dissociation constant of the Michaelis complex may be measured by the appropriate experiments. 

The strategy is to measure the rate constants k2 and k3 of the acylenzyme mechanism (equation 7.1) and to show that each of these is either greater than or equal to the value of kCM for the overall reaction in the steady state (i.e., apply rules 2 and 3 of section Al). This requires (1) choosing a substrate (e.g., an ester of phenylalanine, tyrosine, or tryptophan) that leads to accumulation of the acylenzyme, (2) choosing reaction conditions under which the acylation and deacylation steps may be studied separately, and (3) finding an assay that is convenient for use in pre-steady state kinetics. The experiments chosen here illustrate stopped-flow spectrophotometry and chromopboric procedures. 

The /3-amino add (S)-ethyl-3-amino-4-pentynoate is a chiral synthon used in the synthesis of xemilofiban hydrochloride, an anti-platelet agent. (R)- and (S)-enanti-omers of ethyl 3-amino-4-pentynoate were obtained in enantiomerically pure form by employing penicillin acylase (Pen G acylase, PGA, Chapter 7, Section 7.5.1) (Figure 13.32) (Topgi, 1999). Both the acylation and deacylation modes were ap-... 

Baldessari, A. Mangone, C. P. Gros, E. G. Lipase-catalyzed acylation and deacylation reactions of pyridoxine, a member of the vitamin-B6 group. Helv. Chim. Acta 1998, 82, 2407-2413. 

Studies on acylation and deacylation of trypsinlike enzymes by nophenyl eaters of substituted benzoic acids have shown (hat the rate of descylstion is influenced by the benzoic acid substituent. There is a linear correlation between the rate of deacylation and the inductive effect of the substituent (Fig. 3). This is also true of all trypsinlike serine proteases tested. However, the... 

Fig. 9.22 CaLB for acylation and deacylation steps in amine resolution.

While there is no doubt that bound NAD enhances acylation (117) as well as deacylation (70), it is difficult to reconcile the above-mentioned cooperative effects with the observation (165) that NAD occupation of one site in the rabbit tetramer did not affect the catalytic activity of the other three sites toward aldehyde substrates. Evidence against a slow isomerization of the T->R variety, which might be expected to affect thiol reactivity in other subunits, is the fact that the sturgeon apoenzyme is almost instantaneously active in acylation, following the addition of NAD (116). The nature of the NAD enhancement of acylation and deacylation is of considerable interest since it has a bearing on the mechanism of enzyme catalysis in general, and also because it explains the NAD requirement of a number of the minor activities of GPD (see Section... 

The accumulation of the acyl intermediate (PVIm-Ac ) was established unequivocally by studying the acylation and deacylation behaviors of the polymer separately. Overberger and Glowaky (60) allowed polyvinylimidazole 1 to react with loi -chain acyl substrates SJJ" 15 and separated the partially acylated polymer by utilizing gel permeation chromatography (Sephadex LH-20). Table 4—3 gives first-order rate coie stants for acylation (kobs) and deacylation (ka) reactions, is larger than kobs fot... 

The largest turnover rate is realized by the proper balance of acylation and deacylation, and found for imidazole-containing miceUes (SA -InvCTAB), bifunctional micelles (LImHA-CTAB), a polyethyleneimine derivative(D(10%)-PEI-Im(15%) (Section 6—2), and bifimctional polymers(EIm -HA and PVP -HA) (Section S—3). It is remarkable that the turnover rate of synthetic catalysts can amount to 10 to 20% of that of a-chjonotrypsin, although it is to be noted that PNPA is never a good substrate for o-chymotrypsin. Comparable rates are observed for acylation and deacylation, when they are compared separately. 

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