Amides enzymatic hydrolysis

The basis of the process leading to the enantiomerically pure acid is essentially the same as that for a-H-a-amino acids. However, in this case, a ketone is used as the starting material which undergoes a Strecker reaction, followed by hydrolysis of the resulting aminonitrile to form the racemic a-alkyl-a-amino acid amide. Enzymatic hydrolysis results in the formation of the L-a-alkyl-a-amino acid (Fig. 12.2-2). 

Whereas acid catalyzed hydrolysis of peptides cleaves amide bonds indiscriminately and eventually breaks all of them enzymatic hydrolysis is much more selective and is the method used to convert a peptide into smaller fragments... 

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... 

Hydrolysis of Nitriles. The chemical hydrolysis of nitriles to acids takes place only under strong acidic or basic conditions and may be accompanied by formation of unwanted and sometimes toxic by-products. Enzymatic hydrolysis of nitriles by nitrile hydratases, nittilases, and amidases is often advantageous since amides or acids can be produced under very mild conditions and in a stereo- or regioselective manner (114,115). 

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. 

Some companies are successfully integrating chemo- and biocatalytic transformations in multi-step syntheses. An elegant example is the Lonza nicotinamide process mentioned earlier (.see Fig. 2.34). The raw material, 2-methylpentane-1,5-diamine, is produced by hydrogenation of 2-methylglutaronitrile, a byproduct of the manufacture of nylon-6,6 intermediates by hydrocyanation of butadiene. The process involves a zeolite-catalysed cyciization in the vapour phase, followed by palladium-catalysed dehydrogenation, vapour-pha.se ammoxidation with NH3/O2 over an oxide catalyst, and, finally, enzymatic hydrolysis of a nitrile to an amide. 

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]. 

The amidocarbonylation of aldehydes provides highly efficient access to N-acyl a-amino acid derivatives by the reaction of the ubiquitous and cheap starting materials aldehyde, amide, and carbon monoxide under transition metal-catalysis [1,2]. Wakamatsu serendipitously discovered this reaction when observing the formation of amino acid derivatives as by-products in the cobalt-catalyzed oxo reaction of acrylonitrile [3-5]. The reaction was further elaborated to an efficient cobalt- or palladium-catalyzed one-step synthesis of racemic N-acyl a-amino acids [6-8] (Scheme 1). Besides the range of direct applications, such as pharmaceuticals and detergents, racemic N-acetyl a-amino acids are important intermediates in the synthesis of enantiomeri-cally pure a-amino acids via enzymatic hydrolysis [9]. 

A simple example in this class with which to begin is A,A-diethyl-m-to-luamide 0V,/V-dicthyl-3-mcthylbenzamidc, DEET, 4.82), an extensively used topical insect repellant. The hydrolysis product 3-methylbenzoic acid was detected in the urine of rats dosed intraperitoneally or topically with DEET. However, amide hydrolysis represented only a minor pathway, the major metabolites resulting from methyl oxidation and A-dealkylation [52], Treatment of rats with /V,/V-dicthylbcnzamidc (4.83), a contaminant in DEET, produced the same urinary metabolites as its secondary analogue, A-ethylbenzamide (see Sect. 4.3.1.2). This observation can be explained by invoking a metabolic pathway that involves initial oxidative mono-A-deethylation followed by enzymatic hydrolysis of the secondary amide to form ethylamine and benzoic acid [47], Since diethylamide was not detected in these experiments, it appears that A,A-diethylbenzamide cannot be hydrolyzed by amidases, perhaps due to the increased steric bulk of the tertiary amido group. 

Niclosamide (4.158), which carries the three substituents, was hydrolyzed by neither mammalian liver nor helminth tissue preparations. The resistance of niclosamide to enzymatic hydrolysis was explained by steric hindrance caused by the two substituents adjacent to the amide bond [102], But, since monosubstitution also considerably reduced hydrolysis, one can postulate that H-bonding and electronic effects also contributed to the hydrolytic stability of niclosamide. 

In neutral or alkaline buffer solution at 37°, the hydrolytic breakdown of A-formylbenzamide (4.166, R=H) produced only benzamide (4.167). In contrast, the higher homologues were hydrolyzed at the two amide bonds, with benzamide (4.167) and benzoic acid (4.168) formed in a 3 2 ratio. Plasma-catalyzed hydrolysis occurred predominantly at the distal amide bond to produce benzamide. Under these conditions, hydrolysis was very rapid for N-formylbenzamide (80% hydrolysis in 15 min, i. e., ca. 500-fold faster than under abiotic conditions). The rate of enzymatic hydrolysis was also markedly influenced by the length of the A-acyl group, and decreased in the order H>Me>Bu>Pr>Et. 

A. H. Kahns, H. Bundgaard, A-Acyl Derivatives as Prodrug Forms of Amides Chemical Stability and Enzymatic Hydrolysis of Various A-Acyl and A-Alkoxylcarbonyl Amide Derivatives , Int. J. Pharmaceut. 1991, 71, 31-43. 

The 3-(2-hydroxy-4,6-dimethylphenyl)-3-methylbutanoic acid shown in Fig. 8.23, as well as another phenylpropanoic derivative presented below, have been used as pro-moieties to prepare a number of prodrugs of therapeutic peptides [169] [238], Of interest here is that the pro-moiety is linked to the peptide by both amide and ester bonds to form a cyclic, double prodrug, the two-step activation of which is schematized in Fig. 8.24. Briefly, enzymatic hydrolysis of the ester bond unmasks a nucleophile (in this case, a phenol) that carries out an intramolecular attack on the amide bond, resulting in cy-clization of the pro-moiety and elimination of the peptide. [Leu5]enkephalin was one of the therapeutic peptides used to validate the concept, as illustrated in Fig. 8.25 [241],... 

Both anandamide and 2-AG are inactivated by enzymatic hydrolysis (Goparaju et al. 1998). Fatty acid amide hydrolase (FAAH) is an enzyme that catalyses their hydrolysis. High concentrations of FAAH were found in the cerebellum, hippocampus and neocortex of rat brain, which are also rich in cannabinoid receptors. Further, there is a complementary pattern of distribution of FAAH and the CBl receptor. For example, in the cerebellum, FAAH is found in the cell bodies of Purkinje cells and the CBl receptor is found in the axons of granule cells and basket cells, which are presynaptic to Purkinje cells. 2-AG may also be inactivated by direct esterification into membrane phospholipids. Cannabinoid Receptors... 

Transition metal catalysts and biocatalysts can be combined in tandem in very effective ways as shown by the following example (Scheme 2.21). An immobilized rhodium complex-catalyzed hydrogenahon of 46 was followed by enzymatic hydrolysis of the amide and ester groups of 47 to afford alanine (S)-9 in high conversion and enanhomeric excess. Removal of the hydrogenation catalyst by filtration prior to addition of enzyme led to improved yields when porcine kidney acylase 1 was used, although the acylase from Aspergillus melleus was unaffected by residual catalyst [23]. 

Analogous to the reactions of chiral alcohols, enantiomerically pure amines can be prepared by (D)KR of the racemate via enzymatic acylation. In the case of alcohols the subsequent hydrolysis of the ester product to the enantiomerically pure alcohol is trivial and is generally not even mentioned. In contrast, the product of enzymatic acylation of an amine is an amide and hydrolysis of an amide is by no means trivial, often requiring forcing conditions. 

A free carboxylic acid group also enhances the antibacterial spectmm in the penicillin series. Acylation of 6-APA (2-4) with the half-acid chloride (6-1) from benzyl phenylmalonate leads to the amide (6-2). Removal of the benzyl protecting group by catalytic hydrogenation [8] or by enzymatic hydrolysis [9] affords carben-cillin (6-3). A similar sequence starting with 3-thiophenylmalonic acid leads to the considerably more potent analogue ticarcillin (6-4) [10]. 

A particularly interesting case is the partial hydrolysis of the racemic N-tri-fluoroacetyl derivative 59 with hog kidney aminoacylase (HKA) (EC 3.5.1.14) to prepare chiral isomers of 2-trifluoromethylalanine 60 [53]. The stereochemical preference of hog kidney aminoacylase is to hydrolyze amino acid amides bearing the larger C-2 substituent in the pro-S position [54]. The enzymatic hydrolysis of compound 60 follows this trend. 

Groups susceptible to enzymatic hydrolysis chiefly esters (including phosphate esters), and also amides but this is more equivocal. 

Enzymatic Hydrolysis Reactions of Esters. Xenobiotic compounds containing esters or other acid derivatives in their structures (e.g., amides, carbamates, ureas, etc., see Table 17.3) are often readily hydrolyzed by microorganisms. To understand how enzymatic steps can be used to transform these substances, it is instructive to consider the hydrolases (i.e., enzymes that catalyze hydrolysis reactions) used by organisms to split naturally occurring analogs (e.g., fatty acid esters in lipids or amides in proteins). The same chemical processes, and possibly even some of the same enzymes themselves, are involved in the hydrolysis of xenobiotic substrates. 

Furthermore, the sulfonamide bond is expected to possess enhanced metabolic stability with structural similarities to the tetrahedral transition state involved in amide bond enzymatic hydrolysis, thus making sulfonamide peptides interesting candidates in the development of protease inhibitors and new drugs. The oligomers and polymers should also be interesting molecular scaffolds, with specific secondary structures enforced by hydrogen bonding)100,101 ... 

Peptide thioesters (Section 15.1.10) are generally prepared by coupling protected amino acids or peptides with thiols and are used for enzymatic hydrolysis. Peptide dithioesters, used to study the structures of endothiopeptides (Section 15.1.11), may be prepared by the reaction of peptide nitriles with thiols followed by thiolysis (Pinner reaction). Peptide vinyl sulfones (Section 15.1.12), inhibitors of various cysteine proteases, are prepared from N-protected C-terminal aldehydes with sulfonylphosphonates. Peptide nitriles (Section 15.1.13) prepared by dehydration of peptide amides, acylation of a-amino nitriles, or the reaction of Mannich adducts with alkali cyanides, are relatively weak inhibitors of serine proteases. 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

Figure 3.6 Typical path of enzymatic hydrolysis of esters and amides. ...

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