Esterase ester hydrolysis

Esterase Ester hydrolysis, -formation Pig liver esterase proteases Few esterases organic solvents State of the art, novel esterases ... 

Fig. 30. Pig liver esterase-catalyzed hydrolysis of anionic DMS-ester complexes...

Figure 8-1. Lipase/esterase-catalyzed ester hydrolysis.

Estane, commercial block copolymer, 7 648t Ester—acid interchange, 10 492-493 Ester—alcohol interchange, 10 491—492 Esterases, 3 675-676 Ester bonds, in the wood cell wall, 21 15 Ester carboxylates, 24 144, 145 Ester-ester interchange, 10 493 Ester hydrolysis, 10 497, 502—503 Esterifiable acids, 10 490 Esterification, 9 283, 320 10 471-496. 

FIGURE 6.3 Mechanism for esterase-catalyzed hydrolysis of esters and amides. 

Metabolism Glucuronidation N-demethylation Ester hydrolysis to morphine Glucuronidation demethylation (CYP2D6) Ester hydrolysis N-demethylation N-Dealkylation, then hydroxylation N-Demethylation Plasma and tissue esterases... 

L. D. Sutton, J. S. Stout, D. M. Quinn, Dependence of Transition-State Structure on Acyl Chain Length for Cholesterol Esterase Catalyzed Hydrolysis of Lipid p-Nitrophen-yl Esters ,./. Am. Chem. Soc. 1990, 112, 8398-8403. 

Figure 2 Ester hydrolysis. Design of a stable analogue of the tetrahedral transition state to be used as a hapten to generate catalytic antibodies with an esterase activity.

The above-mentioned facts have important consequences on the stereochemical outcome of the kinetic resolution of asymmetrically substituted epoxides. In the majority of kinetic resolutions of esters (e.g. by ester hydrolysis and synthesis using lipases, esterases and proteases) the absolute configuration at the stereogenic centre(s) always remains the same throughout the reaction. In contrast, the enzymatic hydrolysis of epoxides may take place via attack on either carbon of the oxirane ring (Scheme 7) and it is the structure of the substrate and of the enzyme involved which determine the regioselec-tivity of the attack [53, 58-611. As a consequence, the absolute configuration of both the product and substrate from a kinetic resolution of a racemic... 

The special case of the endogenous transmitter acetylcholine illustrates well the high velocity of ester hydrolysis. Acetylcholine is broken down at its sites of release and action by acetylcholinesterase (pp. 100,102) so rapidly as to negate its therapeutic use. Hydrolysis of other esters catalyzed by various esterases is slower, though relatively fast in comparison with other biotransformations. The local anesthetic, procaine, is a case in point it exerts its action at the site of application while being largely devoid of undesirable effects at other locations because it is inactivated by hydrolysis during absorption from its site of application. 

A new class of lipophilic ferrichrome analogs carrying acetoxymethyl ester moieties 203 has been synthesized and shown to penetrate rapidly through cell membranes. Intracellular esterase mediated hydrolysis transformed the lipophilic termini from hydrophobic to hydrophilic. The intracellular retention was visualized in hepatoma cells by labeling these analogs with a fluorescent naphthy-imide probe . Their fluorescence analogs retained their fluorescent properties for extended periods in comparison to hydrophobic derivatives 204 lacking the cleavable substituents. 

Another colorimetric assay for testing the enantioselectivity of lipases or esterases in ester hydrolysis reactions is based on a different principle (75). To simulate the state of competitive conditions of an enzymatic process, the so-called Quick-ii-Test... 

In what appears to be a particularly irmovative development in the area of UV/ Vis-based ee screening systems, the determination of the enantiomeric purity of chiral alcohols 9 is based on a new concept of using two enantioselective enzymes to modify the product (84). The method allows the determination of ee values independent of the concentration, which may be of significant advantage in directed evolution projects. It can be used in three different biocatalytic processes, namely biohydroxylation of alkanes, reductase-catalyzed reduction of ketones, and lipase-or esterase-catalyzed ester hydrolysis. 

Later, oxyanion holes were also discovered in other proteases, such as the cysteine protease papain, and in esterases and lipases, enzymes capable of esterification or ester hydrolysis. Interestingly, in these esterases, sometimes up to three hydrogen bond donors can be located within 3 A of the carbonyl oxygen atom, whereas such triple hydrogen bonding motifs have not yet been found in the proteases. 

Hydrolyses Esters and Amides. The plasma, liver, kidney, and intestines contain a wide variety of nonspecific amidases and esterases. These catalyze the metabolism of esters and amides, ultimately leading to the formation of amines, alcohols, and carboxylic acids. Kinetically, amide hydrolysis is much slower than ester hydrolysis. These hydrolyses may exhibit stereoselectivity. 

In standard aqueous media, hydrolases are enzymes which are able to hydrolyse covalent bonds (Fig. 1). Three classes of hydrolases are used industrially osidases (glycosidic linkage hydrolysis), proteinoses (peptidic linkage hydrolysis) and esterases (ester linkage hydrolysis). 

Because the ester hydrolysis leads to a change in acidity, as in hydrolytic lipase-or esterase-catalyzed kinetic resolution, an appropriate pH indicator can be used for quantification [14,15]). In an optimized version (Kazlauskas test) [15], a linear correlation between the amount of acid generated and the degree of protonation of the indicator was ensured by using a buffer (e. g., iV,iV-bis(2-hydroxyethyl)-2-(aminoethanesulfonic acid) (= BES), and a pH indicator (e. g., / -nitrophenol) having the same pKa value. The advantage of this system relates to the fact that p-nitrophenol esters are not necessary, i. e., normal substrates such as methyl esters 10 can be used. 

The evolved enzymes were further characterized for their ability to carry out the desired pNB ester hydrolysis. Figure 4 shows the specific reaction rates for enzymes from the first four generations in 1% and 15% DMF. Each successive generation catalyst is more effective than its parent, and the best, pNB esterase 4-54B9, is 15 times more productive than wild type in 1% DMF. In 15% DMF, this enzyme makes product at 4 times the rate of the wild type enzyme in 1 % organic solvent. The impact of this improvement is not only the increased productivity of the evolved enzyme, but also in the 4-fold increase in solubility of the substrate in 15% DMF. The increased solubility reduces the size of the reactor and the downstream processes required to produce and purify a given amount of product. The 2-fold increase in enzyme expression level further reduces process costs. 

Flydrolysis is an important metabolic reaction for drugs whose structures contain ester and amide groups. All types of ester and amide can be metabolized by this route. Ester hydrolysis is often catalysed by specific esterases in the liver, kidney and other tissues as well as non-specific esterases such as acetylcholinesterases and pseudocholinesterases in the plasma. Amide hydrolysis is also catalysed by non-specific esterases in the plasma as well as amidases in the liver. More specific enzyme systems are able to hydrolyse sulphate and glucur-onate conjugates as well as hydrate epoxides, glycosides and other moieties. 

Oral bioavailability of mibefradil is dose dependent and ranges from 37% to over 90% with doses of 10 mg or 160 mg, respectively. The plasma half-life is 17 to 25 hours after multiple doses, and it is more than 99% protein bound (15). The metabolism of mibefradil is mediated by two pathways esterase-catalyzed hydrolysis of the ester side chain to yield an alcohol metabolite and CYP3A4-catalyzed oxidation. After chronic dosing, the oxidative pathway becomes less important and the plasma level of the alcohol metabolite of mibefradil increases. In animal models, the pharmacological effect of the alcohol metabolite is about 10% compared to that of the parent compound. After metabolic inactivation, mibefradil is excreted into the bile (75%) and urine (25%), with less than 3% excreted unchanged in the urine. 

---