Amino amidase

III. AMINO AMIDASE FROM MYCOBACTERIUM NEOAURUM ATCC 25795... 

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]. 

The amino amidase from M. neoaurum is active over a broad pH range (vide infra) in which no natural hydrolysis of the amides is observed. 

Figure 9 Effect of the pH (a) and temperature (b) on the initial activity of the purified L-amino amidase from M. neoaurum ATCC 25795.

Thus, a versatile toolbox of (amino)amidases is now available for the resolution of a very broad range of both a-H- and a,a-disubstituted amino acid amides. So far the applicability of this toolbox has been demonstrated for more than 100 natural and synthetic anoino acids. The amino acids can be obtained in enantiomerically pure form for both the D and the l configuration. 

Several classes of enzymes have been used to separate stereoisomers of a-H-and a-disubstituted amino acids, eg amidases, nitrilases, hydantoinases, acylases and esterases. 

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

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]. 

The hydrolysis with a (R)-specific amidase from Klebsiella oxytoca resolved 2-hydroxy- and 2-amino-3,3,3-trifluoro-2-methylpropionamide, giving the (R)-acid and the remaining (S)-amide (Figure 6.39) [104,105]. 

Figure 6.39 Resolution of 2-hydroxy- and 2-amino-3,3,3-trifluoro-2-methylpropionamide by an amidase.

An amidase from Ochrobactrum anthropi strain NCIMB 40321 has a wide snbstrate versatility for L-amides, primarily those with an a-amino gronp (Sonke et al. 2005), while the condensation prodnct of nrea and formaldehyde H2N-[CONH-CH2NH] -CONH2 is hydrolyzed by another strain of 0. anthropi (Jahns et al. 1997). 

An alternative two-step biocatalytic route, first developed at Glaxo in the 1970s, utilized a D-amino acid oxidase and an amidase to provide 7-ACA under physiological conditions (Scheme 1.12). This process has since been established in several companies, with minor modifications. In fact, 7-ACA was manufactured by GSK at Ulverston (Cumbria, UK) using both the chemical and biocatalytic processes in parallel for a period of 2 years during which time the environmental benefits of the biocatalytic process were assessed (see Section 1.6). 

A potential versatile route into a-amino acids and their derivatives is via a combination of (i) nitrile hydratase/amidase-mediated conversion of substituted malo-nonitriles to the corresponding amide/acid followed by (ii) stereospecific Hofmann rearrangement of the amide group to the corresponding amine. Using a series of a,a-disubstituted malononitriles 14, cyanocarboxamides 15 and bis-carboxamides 16, the substrate specificity of the nitrile hydratase and amidase from Rhodococcus rhodochrous IF015564 was initially examined (Scheme 2.7). The amidase hydrolyzed the diamide 16 to produce (R)-17 with 95% conversion and 98%e.e. Amide 17 was then chemically converted to a precursor of (S)-a-methyldopa. It was found... 

Hensel et al. have also reported the use of a-amino nitriles as substrates (Scheme 2.10). They discovered a number of new bacterial isolates with stereose-lechve nitrile hydratase achvity. A combination of stereoselechve nitrile hydratases and amidases was shown to be responsible for the production of phenylglycine 24 from nitrile 22 via amide 23. By investigating five isolates both (R)- and (S)-phenylglycine were produced in greater than 99% e.e. [12]. 

Scheme 2.11 Synthesis of amino acids from amino nitriles using a nitrile hydratase and amidase.

Fig. 1 Phylogenetic tree based on amino acid sequences of the polyamidase from Nocardia farcinica and other highly homologous bacterial amidases as well as amidases with substrate specificity for 6-aminohexanoate oligomers [20]...

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

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