Acetylation, enzymatic

Depending on the probe drug used and on the experimental method, 2 or 3 acetylator types can be described slow, intermediate and rapid, the intermediate one being not always distinguished from the rapid one. Phenotype distribution has been considered as a continuous variable (Meisel 2002). Due to slow post-natal maturation of the acetylation enzymatic systems, the acetylation status is evolving in newborns and infants, and depends on the probe drug used (Rane 1999). 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

ACOCH2CF3, porcine pancreatic lipase, THF, 60 h, 77% yield. This enzymatic method was used to acetylate selectively the primary hydroxyl group of a variety of carbohydrates. 

Citrate synthase catalyzes the metabolically important formation of citrate from ace-tyl-CoA and oxaloacetate [68]. Asp-375 (numbering for pig CS) has been shown to be the base for the rate-limiting deprotonation of acetyl-CoA (Fig. 5) [69]. An intennediate (which subsequently attacks the second substrate, oxaloacetate) is believed to be formed in this step the intermediate is thought to be stabilized by a hydrogen bond with His-274. It is uncertain from the experimental data whether this intermediate is the enolate or enol of acetyl-CoA related questions arise in several similar enzymatic reactions such as that catalyzed by triosephosphate isomerase. From the relative pK values of Asp-375... 

CS indicated that the enolate of acetyl-CoA is significantly more stable than the enol or a proton-sharing enolic form and thus do not support the proposal that a low barrier hydrogen bond is involved in catalysis in CS. This study demonstrates the practial application of high level QM-MM studies to the elucidation of mechanistic details of an enzymatic reaction that are otherwise unclear. 

Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis. It allosterically inhibits pyruvate kinase (as noted in Chapter 19) and activates pyruvate carboxylase. Because it also allosterically inhibits pyruvate dehydrogenase (the enzymatic link between glycolysis and the TCA cycle), the cellular fate of pyruvate is strongly dependent on acetyl-CoA levels. A rise in... 

Preparation of PhAcOZ amino acids proceeds from the chloroformate, and cleavage is accomplished enzymatically with penicillin G acylase (pH 7 phosphate buffer, 25°, NaHS03, 40-88% yield). In a related approach, the 4-ace-toxy derivative is used, but in this case deprotection is achieved using the lipase, acetyl esterase, from oranges (pH 7, NaCl buffer, 45°, 57-70% 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. 

Figure 6.16 Production of 7-aminodeacetyicephalosporanic acid from 7-ACA using an immobilised acetyl esterase. Following enzymatic removal of the acetyl group from 7-ACA, a 3-hydroxymethyl cephalosporin is obtained that can serve as intermediate in the production of cefuroxime.

Several L-amino acids are produced on a large scale by enzymatic resolution of N-acetyl-D,L-amino adds (Figure A8.4). Acylase immobilised on DEAE-Sephadex is for example employed in a continuous process while Degussa uses the free acylase retained in a membrane reactor. In the latter process the advantage of reuse of the enzyme and homogeneous catalysis are combined. 

Figure A8.4 Commercial process for the enzymatic production of L- and D-amino acids from N-acetyl-D,L-amino acids.

Figure A8.11 Enzymatic resolution of N-acetyl-D,L-amino add esters.

The protein was oxidized and reduced as described in Table 3. This table and the assays for binding of L12 to depleted ribosomes, poly(U>directed polyphenylalanine synthesis, and enzymatic acetylation of L12 to form L7 are from Caldwell and coworkers37. 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

Conduritols and inositols are cyclic polyalcohols with significant biological activity. The presence of four stereogenic centers in the stmcture of conduritols allows the existence of 10 stereoisomers. Enzymatic methods have been reported for the resolution of racemic mixtures or the desymmetrization of meso-conduritols. For example, Mucor miehei lipase (MML) showed enantiomeric discrimination between all-(R) and all-(S) stereoisomers ofconduritol E tetraacetate (Figure 6.52). Alcoholysis resulted in the removal of the four acetyl groups ofthe all-(R) enantiomer whereas the all-(S) enantiomer was recovered [141]. 

Acetyl-CoA carboxylase is required to convert acetyl-CoA to malonyl-CoA. In turn, fatty acid synthase, a multienzyme complex of one polypeptide chain with seven separate enzymatic activities, catalyzes the assembly of palmitate from one acetyl-CoA and seven malonyl-CoA molecules. 

The exact role of these coactivators is presendy under intensive investigation. Many of these proteins have intrinsic enzymatic activities. This is particularly interesting in view of the fact that acetylation, phosphorylation, methylation, and ubiquitination—as well as proteolysis and cellular translocation—have been proposed to alter the activity of some of these coregulators and their targets. 

The solubility of iridoids depends on their state (free, glycosylated, acetylated), but usually they are extracted with polar solvents methanol, ethanol, aqueous alcohols, and rarely acetone. Iridoid glycosides are more or less stable some of them are very sensitive to acids and alkalis. Some iridoid glycosides such as aucubin suffer color modification after chemical or enzymatic hydrolysis they give first a blue to green... 

When a reverse procedure was applied, i.e. enzymatic acetylation of racemic 3, formed in situ from the appropriate aldehydes and thiols, the reaction proceeded under the conditions of dynamic kinetic resolution and gave enantiomerically enriched acetates 2 with 65-90% yields and with ees up to 95% (Equation 2). It must be mentioned that the addition of silica proved crucial, as in its absence no racemization of the initially formed substrates 3 occurred and the reaction stopped at the 50% conversion. 

The results presented in Tables 3 and 4 deserve some comments. First, a variety of enzymes, including whole-cell preparations, proved suitable for the resolution of different hydroxyalkanephosphorus compounds, giving both unreacted substrates and the products of the enzymatic transformation in good yields and, in some cases, even with full stereoselectivity. Application of both methodologies, acylation of hydroxy substrates rac-41 and rac-43 or the reverse (hydrolysis of the acylated substrates rac-42 and rac-44), enables one to obtain each desired enantiomer of the product. This turned out to be particularly important in those cases when a chemical transformation OH OAc or reverse was difficult to perform. As an example, our work is shown in Scheme 3. In this case, chemical hydrolysis of the acetyl derivative 46 proved difficult due to some side reactions and therefore an enzymatic hydrolysis, using the same enzyme as that in the acylation reaction, was applied. Not only did this provide access to the desired hydroxy derivative 45 but it also allowed to improve its enantiomeric excess. In this way. 

Interestingly, for the transformation of both the racemic 1-hydroxyalkanephosphonates 41 and 2-hydroxyalkanephosphonates 43 into almost enantiopure acetyl derivatives 42 and 44, respectively, a dynamic kinetic resolution procedure was applied. Pamies and BackvalP used the enzymatic kinetic resolution in combination with a ruthenium-catalysed alcohol racemization and obtained the appropriate O-acetyl derivatives in high yields and with almost full stereoselectivity (Equation 25, Table 5). It should be mentioned that lowering... 

There are several other examples of C-chiral hydroxy phosphorus compounds which were obtained in enantiomerically enriched forms using enzymatic methodology. Thus, a 5-l-diethylphosphonomethyl-2-hydroxycyclohexane 48 was resolved into enantiomers by enzymatic acetylation the highest enantioselectivity was achieved using lipase PS in THF or lipase AK without solvent and vinyl acetate as the acetylating agent (Equation 26). ... 

The aminoalkanephosphonic acids which bear an additional hydroxy group in the molecule were usually resolved via enzymatic acylation of this hydroxy group. For example, resolution of //-Cbz-phosphoserine dimethyl ester 60 using various lipases gave poor results. However, lipase PS-promoted acetylation of //-Chz-phosphoisoserine diethyl ester 61 gave both the unreacted substrate 61 and the 0-acetylated product 62 with almost 100% enantiomeric excess (E = 1000). ... 

The first P-chiral hydroxy phosphoryl compounds that were enzymatically resolved into enantiomers were o-hydroxyaryl phosphines and their oxides 75. The resolution was achieved via enzyme-assisted hydrolysis of their O-acetyl derivatives 74, the most effective enzymes being CE and Upase from C. rugosa (CRL) (Equation 35). The highest enanfioselectivity was observed in the case of naphthyl derivatives (Equation 36), having a P=0 moiety. ... 

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