Acylated amino acids hydrolysis

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

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. 

Fig. 8.26. The two-step activation of oxazolidin-5-one derivatives of peptides and N-acy/ amino acids (8.190). Hydrolysis (Reaction a) yields an A-(l-hydroxyalkyl) derivative that breaks down to liberate the peptide or A-acyl amino acid (Reaction b) [247] [248],...

G. M. Whitesides, Kinetic resolution of unnatural and rarely occurring amino adds enantioselective hydrolysis of N-acyl amino acids catalyzed by acylase I,... 

The hydrolysis of an IV-acylated amino acid by an enzyme provides a resolution method to amino acids. Because the starting materials are readily available in the racemic series by the Schotten-Baumann reaction, the method can be cost effective (Scheme 2.21).68-71 The L-amino acid product can be separated by crystallization, whereas the D-amino acid, which is still /V-acylated, can be recycled by being resubjected to the Schotten-Baumann conditions used for the next batch. Tanabe has developed a process with an immobilized enzyme,72 73 whereas Degussa uses the method in a membrane reactor.69 74 The process is used to make L-methionine. 

Aminoacylase. Production of L-amino acids by Tanabe Seiyaku Company using aminoacylase adsorbed on DEAE-Sephadex represents the first industrial use of an immobilized enzyme (3,29). The process uses the enzyme to resolve the racemic mixture of an amino acid, derived by chemical synthesis, by biospecific hydrolysis of the acyl-amino acid followed by separation of the L-amino acid from the acyl-D-amino acid by crystallization. The D-forms are then racemized and passed back through the reactor thus improving the yield. The entire process, including a fixed-bed bioreactor, is a continuous, automated operation. 

Several hydrolytic enzymes other than esterases have been applied for synthetic purposes. One important subject is the chemoenzymatic preparation of amino acids. An industrial method for the synthesis of unnatural d- or L-amino acids employs the enzymatic hydrolysis of hydantoins, prepared by Bucherer-Bergs condensation using either D- or L-hydantoinase (cf Section 3.2.1.4) [33]. Another efficient method of preparing natural and unnatural amino acids is the two-step synthesis which features a Pd-catalyzed amidocarbonylation (eq. (2) cf Section 2.1.2.4) to afford racemic A-acyl amino acids followed by enantioselective hydrolysis using various acylases [34]. 

Chenault, H. K. Dahmer, J. Whitesides, G. M., Kinetic Resolution of Unnatural and Rarely Occurring Amino Acids Enantioselective Hydrolysis of N-Acyl Amino Acids Catalyzed by... 

When the enzyme is used to catalyse the synthesis of a peptide bond, the solvent is either non-aqueous or contains only a low concentration of water. In addition, of course, an amino component such as an amino acid or peptide ester replaces the water in the second step. Obviously, the amino component must be unprotonated for reaction to succeed. Synthesis is favoured over hydrolysis of the resultant peptide because an amide is kinetically a much worse substrate for a proteinase than is an ester. The rapid acylation of a proteinase by an TV-protected amino acid or peptide aryl ester can be demonstrated experimentally using a stopped-flow apparatus with spectrophotometric facilities. A rapid burst of phenol is followed by steady-state release, showing that acylation of the enzyme is faster than hydrolysis of the acy-lated enzyme. No such burst is detectable if, for example, an TV-acylated amino acid anilide is used as substrate. In fact, acylation is the rate-determining step with amide substrates. 

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. 

The properties of the N-acyl amino acid derivative and the amino acid itself can be important if crystallization is employed to ensure high optical purity. However, in the experience of the authors, it is seldom a problem. Only 3-pyridylalanine could not be purified to high optical purity by recrystallization. Use of enzymes to enhance enantiomeric excess as well as allowing the use of mild hydrolysis conditions has been advocated by others [12]. Amino acids are surprisingly stable to acid-induced racemization. 

The enzymatic hydrolysis of N-acylamino acids has been known for a century and was first detected in aqueous kidney preparations 3. Based on the finding that this enzymatic hydrolysis proceeds enantiospecifically 2, Greenstein and coworkers developed a general and very attractive procedure for the resolution of a vast number of racemic N-acylated amino acids to the corresponding L-amino acids catalyzed by aminoacylase (E.C. 3.5.1.14) whereas the N-acetyl-D-amino acid does not react13 (Fig. 12.3-1). 

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 Af-acyl 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 hpase (Lipase CE, Amano) were the only ones found to catalyze the hydrolysis of the substrate (Scheme 9.6). 

The bislactim heterocycle behaves like a masked amino acid moiety and, after (9-acylation, acidic hydrolysis furnishes the A-acyl amino acid ester 644 as a single diastereomer (Scheme 88). The acetyl group migrates from oxygen to nitrogen on distillation of the product. 

Process and reaction monitoring. We have used TLC to monitor the time required for completion of the methylation reaction (Shantha, Decker and Hennig, 1993) and to study the formation of artifacts due to methylation of phthalates present in lipid samples (Shantha and Ackman, 1991b). TLC has been used to study the extent of hydrolysis of lipase-catalysed hydrolysis of triglycerides, phospholipase-catalysed hydrolysis of phospholipids and to study the activity of the enzymes. We have used TLC to study the extent of synthesis of acylated amino acids, and TLC has also been used to study the success of radiosynthesis. 

Aminoacylases catalyze the hydrolysis of A-acyl amino acid derivatives, with the acyl groups preferably being acetyl, chloroacetyl, or propionyl. Alternatively, the corresponding A-carbamoyl- and A-formyl derivatives can be used [132], Enzymes of the amino acylase type have been isolated from hog kidney, and from Aspergillus or Penicillium spp. [133-135]. The versatility of this type of enzyme has been demonstrated by the resolution of racemic iV-acetyl tryptophan, -phenylalanine, and -methionine on an industrial scale using colunm reactors (Scheme 2.15) [136, 137]. 

Amino acylases catalyse the hydrolysis of N-acyl-L-aminoacids (Figure 6.28) but not of the corresponding D-isomers. This reaction is comparable to that catalysed by penicillin acylase which in fact shows a similar selectivity when hydrolysing amides other than penicillin G. If the racemic mixture of an amino acid is first acylated and is then treated with the acylase, the product will be a mixture of optically pure L-amino acid and predominantly D-acyl amino acid. These are easily separated on the basis of their solubilities at the isoelectric point of the amino acid. The pure L-amino acid can be crystallized from the mixture leaving the residual acyl amino acid to be racemized and recycled through the process. 

Description. Sarcosinates (or salts of acyl amino acids) are the condensation products of fatty acids with iV-methylglycine (CH3-NH-CH2-COOH) (or sarcosine). The properties of sarcosinates are similar to those of isethionates. The TEA salts are more soluble than sodium salts around neutral pH. Because of the internal amide chemical function, sarcosinates undergo hydrolysis at extreme pH ranges. 

Alpha-chymotrypsin (Fig. 2) catalyzes the facile hydrolysis of peptide bonds, in particular those adjacent to the carboxyl group of aromatic amino acids (tryptophan, tyrosine, phenylalanine) as well as a variety of esters derived from similar N-acylated amino acids. The enzyme... 

Enzymatic hydrolysis is also used for the preparation of L-amino acids. Racemic D- and L-amino acids and their acyl-derivatives obtained chemically can be resolved enzymatically to yield their natural L-forms. Aminoacylases such as that from Pispergillus OTj e specifically hydrolyze L-enantiomers of acyl-DL-amino acids. The resulting L-amino acid can be separated readily from the unchanged acyl-D form which is racemized and subjected to further hydrolysis. Several L-amino acids, eg, methionine [63-68-3], phenylalanine [63-91-2], tryptophan [73-22-3], and valine [72-18-4] have been manufactured by this process in Japan and production costs have been reduced by 40% through the appHcation of immobilized cell technology (75). Cyclohexane chloride, which is a by-product in nylon manufacture, is chemically converted to DL-amino-S-caprolactam [105-60-2] (23) which is resolved and/or racemized to (24)... 

Enzymatic Method. L-Amino acids can be produced by the enzymatic hydrolysis of chemically synthesized DL-amino acids or derivatives such as esters, hydantoins, carbamates, amides, and acylates (24). The enzyme which hydrolyzes the L-isomer specifically has been found in microbial sources. The resulting L-amino acid is isolated through routine chemical or physical processes. The D-isomer which remains unchanged is racemized chemically or enzymatically and the process is recycled. Conversely, enzymes which act specifically on D-isomers have been found. Thus various D-amino acids have been... 

Oxazol-5(2H)-one, 2-benzylidene-4-methyl-tautomerism, 6, 186 Oxazol-5(2ff)-one, 2-methylene-isomerization, 6, 226 Oxazol-5(2H)-one, 2-trifluoromethyl-acylation, 6, 201 Oxazol-5(4ff)-one, 4-allyl-thermal rearrangements, 6, 199 Oxazol-5(4H)-one, 4(arylmethylene)-Friedel-Crafts reactions, 6, 205 geometrical isomerism, 6, 185 Oxazol-5(4ff)-one, 4-benzylidene-2-phenyl-configuration, 6, 185 photorearrangement, 6, 201 Oxazol-5(4ff)-one, 4-benzyl-2-methyl-Friedel-Crafts reactions, 6, 205 Oxazol-5(4ff)-one, 4-methylene-in amino acid synthesis, 6, 203 Oxazol-5(4ff) -one. 2-trifluoromethyl-hydrolysis, 6, 206 Oxazolones... 

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