Racemic amides, enantioselective hydrolysis

DSM has developed a widely applicable industrial process for production of en-antiomerically pure amino acids by enantioselective hydrolysis of racemic amino acid amides. These precursor compounds can easily be obtained by alkaline... 

Scientists at Shell began work onthe enantioselective hydrolysis of racemic amides in the early 1990s 5]. Enzyme mediated hydrolysis of racemic N 1 phenylethylace tamide 2 using whole cells of Arthrobacter sp. enabled the production of enantio merically pure (S) 1 and (f ) 2 (Figure 14.2). The use of whole cells was not optimal and long reaction times were required to obtain pure (R) 2, as the selectivity of the catalyst was not very high. 

Typical commercial enzymes reported for resolution of amino acids were tested. Whole cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids" the acylase-catalyzed resolution of Xacyl amino acids had extremely low rates toward a-dialkylated amino acids and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-ethylhexanoic amide. Finally, a large-scale screening of hydrolytic enzymes for enantioselective hydrolysis of racemic amino esters was carried out. Of all the enzymes and microorganisms screened, pig hver esterase (PLE) and Humicola langinosa lipase (Lipase CE, Amano) were the only ones found to catalyze the hydrolysis of the substrate (Scheme 9.6). 

DSM has developed an industrial process for the preparation of (D)- and (L)-amino acids, which is based on the enantioselective hydrolysis of racemic amino acid amides using amidases, for example from Pseudomonasputida. It is often not necessary to isolate the pure enzyme standardised whole-cell or crude enzyme preparations can be used instead. It is noteworthy that in some cases the enzyme activity can be increased up to ten-fold by the addition of magnesium salts. The enzymes accommodate a broad spectrmn of substrates with considerable selectivity. Typical products are (L)-phenylalanine and (L)-homophenyl-alanine. 

Snell, D. and Colby, J., Enantioselective hydrolysis of racemic ibuprofen amide to S-(+)-t txpiofeiihy Shodococcus Ai210,EnzyrrL Microb. Tech., 24,160,1999. 

Enzymatic hydrolysis of the racemic amides by the amino amidase from M, neoaurum affords the (5)-a,a-disubstituted amino acids and the (i )-a,a-disubstituted amino acid amides (15) in almost 100% e.e. at 50% conversion for most a-methyl-substituted compounds E > 200) [43] (see Scheme 7 and Table 7). Only for glycine amides wititi two small substituents the enantioselectivity is decreased for example, for isovaline amide die enantiomeric ratio E = 9 and for (a-Me)allylglycine amide = 40 [44]. Also a-H-amino acid amides are substrates and are hydrolyzed enantioselectively in contrast, however, dipeptides are not hydrolyzed [45]. For all a-methyl-substituted substrates the activity is high. Reactions performed at 5-10 w/w% substrate solutions in water (pH 8, 37 C) with 0.3-1.0 w/w% of freeze-dried biocatalyst are in general completed (i.e., 50% conversion) after 5-48 h. Increasing the size of the small substituent to ethyl, propyl, or allyl dramatically reduces the activity, especially if the large substituent contains no —CHj— spacer at the chiral center. Due to the longer reaction times the enantioselectivity is also reduced [44]. 

Naproxen, (S)-2-(6-methoxy-2-naphthyl)propanoic acid 126 is a nonsteroidal anti-inflammatory and analgesic agent first developed by Syntex [220,221]. Biologically active desired S-naproxen has been prepared by enantioselective hydrolysis of the methyl ester of naproxen by esterase derived from Bacillus subtilis Thai 1-8 [222]. The esterase was subsequently clone in Escherichia coli with over 800-fold ipcrease in activity of enzyme. The resolution of racemic naproxen amide and ketoprofen amides has been demonstrated by amidases from Rhodococcus erythropolis MP50 and Rhodococcus sp. C311 (223-226). 5-Naproxen 126 and 5-ketoprofen 127 (Fig. 44) were obtained in 40% yields (theoretical maximum yield is 50%) and 97% e.e. Recently, the enantioselective esterification of naproxen has been demonstrated using lipase from Candida cylindraceae in isooctane as solvent and trimethylsilyl as alcohol. The undesired isomer of naproxen was esterified leaving desired S isomer unreacted [227]. 

Compared to a maximal bioconversion yield of 50% in racemic resolutions, asymmetric syntheses have a theoretical yield of 100%. Such an ideal yield was nearly reached with another R. rhodochrous ATCC 21197, which transformed disubstituted malononitriles such as butylmethylmalononitrile to the corresponding (7 )-amide carboxylic acid with high enantiomeric excesses and yields [80]. The reaction proceeded via a fast, nonstereospecific hydration of the starting dinitrile followed by a slow, enantioselective hydrolysis of the diamide intermediate by the amidase (Fig. 27). 

Enantioselective hydrolysis of their racemic esters or amides, or... 

Racemic a-amino amides and a-hydroxy amides have been hydrolyzed enantio-selectively by amidases. Both L-selective and o-selective amidases are known. For example, a purified L-selective amidase from Ochrobactrum anthropi combines a very broad substrate specificity with a high enantioselectivity on a-hydrogen and a,a-disubstituted a-amino acid amides, a-hydroxyacid amides, and a-N-hydroxya-mino acid amides [102]. A racemase (a-amino-e-caprolactam racemase, EC 5.1.1.15) converts the o-aminopeptidase-catalyzed hydrolysis of a-amino acid amides into a DKR (Figure 6.38) [103]. 

Kanegafuchi Chemical Industries produce D-p-hydroxyphenyl glycine, which is a key raw material for the semisynthetic penicillins ampicillin and amoxycillin. Here, an enantioselective hydantoinase is applied to convert the hydantoin to the D-p-hydroxyphenyl glycine. The quantitative conversion of the amide hydrolysis is achieved because of the in situ racemization of the unreacted hydantoins. Under the conditions of enzymatic hydrolysis, the starting material readily racemizes. Therefore, this process enables the stereospecific preparation of various amino acids at a conversion of 100% [38]. 

Enantioselective enzymatic amide hydrolyses can also be applied for the preparation of optically active organosilicon compounds. The first example of this is the kinetic resolution of the racemic [l-(phenylacetamido)ethyl] silane rac-84 using immobilized penicillin G acylase (PGA E.C. 3.5.1.11) from Escherichia coli as the biocatalyst (Scheme 18)69. (R)-selective hydrolysis of rac-84 yielded the corresponding (l-aminoethyl)silane (R)-85 which was obtained on a preparative scale in 40% yield (relative to rac-84). The enantiomeric purity of the biotransformation product was 92% ee. This method has not yet been used for the synthesis of optically active silicon compounds with the silicon atom as the center of chirality. 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

Most of the product, i.e. iV-diphenylphosphoramides, are crystalline and their ees can often be improved to > 98% ee by the recrystallization.213 TV-diphenyl-phosphinyl group can be easily removed by acid hydrolysis (3 M HC1-THF,23 p-Ts0H-Me0H-H2021c) and the corresponding chiral amines are obtained without racemization. In addition, V-acylimines are utilized in the enantioselective addition of diethylzinc promoted by DBNE 1 affording chiral amides with up to 76% ee.24... 

Enantioselective cleavage of non-peptide amide bonds is also important in the production of optically active amino acids (Scheme 3.12). Carboxy-peptidases often are the enzymes of choice in this area of work these enzymes catalyse the hydrolysis of an amide function which is close to a carboxylic acid group. The rate of hydrolysis is usually increased if R (Scheme 3.12) is an aromatic unit or a large aliphatic moiety. For example, thrco-jS-phenylserine R = PhCH(OH) has been resolved by incubation of the racemic JV-trifluoroacetate with carboxypeptidase-A, with the optically pure (L)-enantiomer being obtained in a good yield. 

Stereospecific nitrilases were used for the conversion of a-arninonitiiles to optically active L-amino acids (Fig. 4). In an early investigation, L-alanine was formed by an l-specific nitrilase from alginate-immobilized cells of Acinetobacter sp. APN [25]. A decrease of the enantioselectivity with the time was supposed to be caused by a racemase forming d- from L-alanine. The stereoinversion of racemic a-aminopropionitrile led to a conversion yield above 50%. Similar L-a-amino acid preparations showed no stereoinversion and additionally accumulated the D-amide due to the presence of a nitrile hydratase/ amidase system [26,27]. Additionally, a number of L-a-amino acids were synthesized by a 45-kDa monomeric nitrilase from R. rhodochrous PA-34 [28]. Remarkable in this case was the preferential hydrolysis of a-aminopropionitrile to D-alanine in contrast to the l-alanine formation by the Acinetobacter nitrilase (Fig. 4). 

Evidence for the enantioselectivity of the nitrile hydratase was given by the purified enzyme catalyzing the hydration of the (5)-nitrile at least 50 times faster than the hydrolysis of the (i )-nitrile [51]. The strain was also capable of a two-step hydrolysis of racemic ibuprofen and naproxen nitriles to the corresponding (S)-acids in enantiomeric purities above 90% e.e., however, with the stereoselectivity residing primarily in the amidase. In this case, the analysis of enantioselectivity was complicated due to the product inhibition of (i )-nitrile hydration by enzymatically formed (5)-amide. On the basis of the initial rate of appearance, the nitrile hydratase showed a slight preference for the (R) enantiomers of... 

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