Methyl esters enzyme-catalyzed formation

The complex thioamide lolrestat (8) is an inhibitor of aldose reductase. This enzyme catalyzes the reduction of glucose to sorbitol. The enzyme is not very active, but in diabetic individuals where blood glucose levels can. spike to quite high levels in tissues where insulin is not required for glucose uptake (nerve, kidney, retina and lens) sorbitol is formed by the action of aldose reductase and contributes to diabetic complications very prominent among which are eye problems (diabetic retinopathy). Tolrestat is intended for oral administration to prevent this. One of its syntheses proceeds by conversion of 6-methoxy-5-(trifluoroniethyl)naphthalene-l-carboxyl-ic acid (6) to its acid chloride followed by carboxamide formation (7) with methyl N-methyl sarcosinate. Reaction of amide 7 with phosphorous pentasulfide produces the methyl ester thioamide which, on treatment with KOH, hydrolyzes to tolrestat (8) 2[. 

When 10 (R configuration about phosphorus) was treated with ribo-nuclease in aqueous methanol, nucleophilic attack by methanol in the enzyme-catalyzed process led to formation of a methyl ester, 11, which has been shown by X-ray analysis to be the isomer with the R configuration about the phosphorus (60) ... 

Capillary gas chromatographic determination of optical purities, investigation of the conversion of potential precursors, and characterization of enzymes catalyzing these reactions were applied to study the biogenesis of chiral volatiles in plants and microorganisms. Major pineapple constituents are present as mixtures of enantiomers. Reductions, chain elongation, and hydration were shown to be involved in the biosynthesis of hydroxy acid esters and lactones. Reduction of methyl ketones and subsequent enantioselective metabolization by Penicillium citrinum were studied as model reactions to rationalize ratios of enantiomers of secondary alcohols in natural systems. The formation of optically pure enantiomers of aliphatic secondary alcohols and hydroxy acid esters using oxidoreductases from baker s yeast was demonstrated. 

In addition to the hydrolysis of cocaine, the purified human liver cocaine methyl ester hydrolase also catalyzed the ethyl transesterification of cocaine with ethanol to form cocaethylene and methanol as shown in figure 1 (Dean et al. 1991 Brzezinski et al. 1994). Both the hydrolytic and the ethyl transesterification reactions increased as the two activities were analyzed in protein fractions obtained during the enzyme purifi-cation by column chromatography. This suggests that the separate activities are catalyzed by the same enzyme. The Km values for cocaine and ethanol of the purified enzyme at pH 7.3 were 116 M and 43 mM, respectively. The carboxylesterase also catalyzes the formation of ethyloleate from oleic acid and ethanol (Tsujita and Okuda 1992 Brzezinski et al. 1994). Other hydrolases or ester transferases have been reported to catalyze similar substrate "ethylation" reactions. 

Methionine-enriched protein was produced also from an enzymatically prehydrolyzed milk protein using an enzymatic peptide modification method with a-chymotrypsin as catalyst. Amino acid incorporation leading to methionine enrichment of the product proceeded via formation of covalent bonds. The concentration of the substrate was 25% (w/v). Methionine was added to the reaction mixture in the form of methionine methyl ester hydrochloride. An ester/substrate ratio of 1 5 was used in the enzymatic peptide modification reaction. The methionine content of the product was twice as high as that of the substrate. The slight change in the degree of hydrolysis revealed that part of the amino acids were bound to the peptide chains and that transpeptidation was the main force during this enzyme-catalyzed reaction. The newly incorporated Met was located in C- and N-termi-nals in a ratio of 3 1 [82],... 

Enzyme-Catalyzed Methyl or Polyol Ester Formation... 

Tyrosine hydroxylase Inhibitors - Perhaps the greatest effort is being directed toward studies of the enzyme tyrosine hydroxylase which represents the rate limiting step in catecholamine synthesis. This enzyme catalyzes the conversion of tyrosine to DOPA and Is localized In the particulate fraction of the cell sedimenting at 16,000 x g. Inhibition of this enzyme has been found to be the most effective means of blocking the formation of norepinephrine. Much of the biochemistry of this and other enzymes associated with catecholamines was discussed at the Second Symposium on Catecholamines and published in 1966. Hundreds of compoumds have been examined for anti-tyrosine hydroxylase activity, but only a few exhibited Inhibitor activity vitro, and these were mainly analogues of tyrosine or its catechol metabolites. It has become apparent, however, that there is not always a relationship between vitro and to vivo activity. Of the many compounds thus far tested, two which have shown In vivo activity are a-methyl-1-tyroslne (a-MT) and H44/68 (the methyl ester-HCl of c MT), both acting as competitive antimetabolites of tyrosine. 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

A two-electron oxidation of N-acetyltyrosine ethyl ester with mushroom tyrosinase, or with periodate, afforded the N-acetyIdopa ester 142, together with the (Z)-enamide 145 and the 6-acetoxydopa amide 146 (Fig. 40) (284). It is assumed that 145 originates from dopaquinone 143 via 144 by tautomerization. Michael addition of acetate to quinone 143 is believed to be the origin of 146. The formation of quinone methide 144 from dopa ester 142 by tyrosinase is reminiscent of the formation of iminochromes and quinone methides catalyzed by this enzyme in their formation from a-methyl dopa ester (285), and such reactions may well occur in mammalian systems. 

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