Subtilisin ester hydrolysis

In addition to chemical composition, as discussed in Sect. 3, the route of self-assembly also significantly affects the resulting structure because different kineti-cally folded structures may be formed. For example, subtilisin-triggered formation of Fmoc-Ls via ester hydrolysis gives rise to hollow nanotubular structures [22], whereas Fmoc-Ls gel formed by the thermolysin-catalysed reversed hydrolysis of the Fmoc-L/L2 system gives rise to nanofibrillar morphology [21]. 

Figure 10.16 Subtilisin-catalyzed hydrolysis of N-acylamino acid esters.

Researchers at the biotech company EntreMed, Inc., have recently prepared and tested 2-phthalimidino-glutaric acid analogs of thalidomide and found them to be potent inhibitors of tumor metastasis [28], The key to the success of their synthesis was a resolution via enantioselective ester hydrolysis catalyzed by ChiroCLEC -BL, the CLC form of the protease subtilisin. The authors were able to isolate both enantiomers of the desired product with good optical purity (95% ee) (see Fig. 7). 

Proteases such as a-chymotrypsin, papain, and subtilisin are also useful biocatalysts for regio-selective or stereoselective hydrolytic biotransformations. For example, dibenzyl esters of aspartic and glutamic acid can be selectively deprotected at the 1-position by subtilisin-catalyzed hydrolysis (Fig. 6). In addition, a-chymotrypsin is used in the kinetic resolution of a-nitro-a-methyl carboxylates, which results in l-configured enantiomers of the unhydrolyzed esters with high optical purity (>95% e.e.). ... 

Fig. 6 Regio-selective ester-hydrolysis catalyzed by subtilisin.

Table 11.1-9. Subtilisin-catalyzed hydrolysis of racemic and prochiral esters.

Horseradish peroxidase Trypsin (protease) Subtilisin (protease) Phenol polymerization Transpeptidation Ester hydrolysis Ethylacetate Butan-l,4-diol Dioxane, chloroform, etc. 

Scheme 2.50 Mirror-image oiicaitation of the eatalytie machinery of Candida rugosa lipase and the protease subtilisin and enantiocomplcanentary ester hydrolysis using Mucor sp. lipase and a-chymotrypsin...

Ricks, E.E., Estrada-Vades, M.C., McLean, T.L. and Iacobucci, G.A. (1992) Highly enantioselective hydrolysis of (/ ,Sl-phenylalanine isopropyl ester by subtilisin Carlsberg. Continuous synthesis of (Sl-phenylalanine in a hollow fibre/liquid membrane reactor. Biotechnology Progress, 8, 197-203. 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

Other biocatalytic methods of producing D-p-hydroxyphenylglycine have not proved competitive, for instance transaminase based processes require glutamate to be supplied. Others include the hydrolysis of N-acyl derivatives by acylase and amides by aminopeptidase (DSM), the use of L-specrfic hydantoinases and immobilised subtilisin for the resolution of D,L-2-acetamido-/>-hydroxyphenylacetic acid methyl ester (Bayer). 

Subtilisin is an endoprotease that has been used in the enantioselective hydrolysis of N-acylamino acid esters (Figure 10.16) into the corresponding (S)-amino acid derivatives. An organic solvent, such as acetonitrile, is often added to improve the solubility of the amino acid derivative, and this function can also be performed by an ionic liquid mixture [133, 134, 135]. 

Alternative methods for the synthesis of peptide aldehydes include reduction of acid halides, phenyl esters, thioesters, and anhydrides prepared from corresponding acids, isoxazolidides, and the hydrolysis of thiazolidine peptides 17,54-56 Enzymes such as thermolysin, subtilisin, and pronase E have proven valuable as effective semisynthetic alternatives 40,57 5 62 ... 

A considerably simpler approach in the context of a biocatalytic pathway was reported by Sidler et al. (Scheme 4.16). Here, the methyl ester 45 could be hydrolyzed selectively by the protease subtilisin (lipases and esterases were unreactive), allowing hydrolysis of the unwanted (R)-enantiomer. The desired (S)-45 was recovered from the solution in 80-90% chemical yield (98% ee) and was further manipulated into (S) L-771,668 [191]. 

A similar resolution has also been achieved on large scale <20040PD22>. The KR of racemic isoxazoline 312 catalyzed by enzymes was studied. The best result was obtained with lipase B from Candida antarctica (CALB), which hydrolyzed the ethyl ester of (—)-312 to the corresponding monoacid (—)-313. The reaction, which was run in 0.1 M phosphate buffer/acetone at room temperature, spontaneously stopped at 50% conversion to yield monoacid (—)-313 and the residual ester (- -)-312 with ees higher than 99% <2004TA3079>. The C-5 epimer of 312 underwent enantioselective hydrolysis (>99% ee) of the methyl ester linked to C-5 in the presence of the protease proleather (subtilisin Carlsberg), whereas CALB and other lipases were not able to resolve it (Equation 53). 

The first protease-catalyzed reaction in ILs was the Z-aspartame synthesis (Scheme 10.7) from carbobenzoxy-L-aspartate and L-phenylalanine methyl ester catalyzed by thermolysin in [BMIM] [PF ] [ 14]. Subtilisin is a serine protease responsible for the conversion of A -acyl amino acid ester to the corresponding amino acid derivatives. Zhao et al. [90] have used subtilisin in water with 15% [EtPy][CF3COO] as cosolvent to hydrolytically convert a series of A -acyl amino acid esters often with higher enantioselectivity than with organic cosolvent like acetonitrile (Scheme 10.8, Table 10.2). They specifically achieved l-serine and L-4-chlorophenylalanine with an enantiomeric access (ee) of-90% and -35% product yield which was not possible with acetonitrile as a cosolvent [90]. Another example is hydrolysis of A-unprotected amino acid ester in the presence of a cysteine protease known as papain. Liu et al. 

Several types of enzymes have found uses in LADD compositions [4,48], Most common are proteases, amylases, and lipases, which attack proteinaceous, starchy, and fatty soils, respectively. Proteases work by hydrolyzing peptide bonds in proteins. Proteases differ in their specificity toward peptide bonds. The typical protease used in LADD formulations, bacterial alkaline protease (subtilisin), is very nonspecific. That is, it will attack all types of peptide bonds in proteins. In contrast to proteases, amylases catalyze the hydrolysis of starch. They attack the internal ether bonds between glucose units, yielding shorter, water-soluble chains called dextrins. Lipases work by hydrolyzing the ester bonds in fats and oils. Often, combinations are used because of the specificity of each kind to one type of soil. The commercially available enzymes are listed in Table 9.6. 

Immobilized enzymes are currently the object of considerable interest. This is due to the expected benefits over soluble enzymes or alternative technologies. The number of applications of immobilized enzymes is increasing steadily [5]. Occasionally, however, experimental investigations have produced unexpected results such as a significant reduction or even an increase in activity compared with soluble enzymes. Thus, cross-linked crystals of subtilisin showed 27 times less activity in the aqueous hydrolysis of an amino acid ester compared to equal amounts of soluble enzyme [6]. On the other hand, in the application of lipoprotein lipase in the solvent-mediated synthesis of esters there was a 40-fold increase in activity using immobilized or otherwise modified enzyme preparations as compared to enzyme powder [7]. 

Cross-linked crystals from subtilisin exhibited 27 times less activity than soluble subtilisin in the hydrolysis of benzoyl-L-phenylalanine ethyl ester. Denaturation of the enzyme and restrictions from substrate-dependent internal diffusion were ruled out. A shift in the pH-dependence of the maximum activity to higher pH-values was observed which was explained by inter molecular electrostatic... 

A lipase has been used to convert solid triolein (glycerol trioleate) to the monooleate by treatment with glycerol at 8°C.75 Other lipases have been used in the hydrolysis and transesterification of oils, as well as in the esterification of fatty acids without solvents.76 Peptides can be produced from eutectic mixtures of amino acid derivatives with the addition of a small amount of solvent.77 Immobilized sub-tilisin and thermolysin were used with 19-24% water or an alcohol to produce polypeptides. Subtilisin on celite (a di-atomaceous earth) was used to convert a mixture of L-phenylalanine ethyl ester and L-leucinamide containing 10% triethyleneglycol dimethyl ether to L-phenylala-nineleucinamide in 83% yield. Addition of 30% 2 1 ethanol/water reduced the time needed from 40 to 4 h. The enzyme could be used three more times. These reaction mixtures contained 0.13-0.75 g peptide per g reaction mixture compared with 0.015-0.035 when the reaction was... 

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