Amine dehydrogenases reaction scheme

A variation on the transamination approach that also starts with an a-keto acid substrate is to perform a reductive amination catalyzed by amino acid dehydrogenases (dHs) (Scheme 9.31) in combination with the formate dH cofactor recycling system, although other reducing systems can be used. " The generation of carbon dioxide from formate drives the coupled reactions to completion. 

Scheme 12 The reaction mechanism of CTQ-dependent quinohemoprotein amine dehydrogenase.

A new NAD -dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique with a synthetic substrate N-[1-DL-(carboxyl)ethyl]-L-phenylalanine [15]. We purified and characterized an NAD+-dependent secondary amine dicarboxylic acid dehydrogenase, and named it opine dehydrogenase (ODH). The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids (Scheme 1). 

The (reversible) transformation of an a-ketocarboxyhc acid in presence of ammonia and one equivalent of NAD(P)H furnishes the corresponding a-amino acid and is catalyzed by amino acid dehydrogenases [EC 1.4.1.X] [962]. Despite major differences in its mechanism, this reaction bears a strong resemblance to carbonyl group reduction and it formally respresents a reductive amination (Scheme 2.133). As deduced for L-Leu-dehydrogenase [963], the a-ketoacid substrate is positioned in the active site between two Lys-residues. Nucleophihc attack by NH3 leads to a hemiaminal intermediate, which eliminates H2O to form an iminium species. The latter is reduced by a hydride from nicotinamide forming the L-amino acid. Since this mechanism is highly tuned for a-keto/a-amino acids, it is clear that a neutral Schiff base cannot be accepted as substrate. 

In 2012, Kroutil and co-workers reported the first amination reaction of primary alcohols with ammonium chloride by an artificial multi-enzyme-catalyzed cascade method (Scheme 29) [173]. The authors assumed that the reaction might proceed by two steps. Initially, the alcohol was oxidized by an alcohol dehydrogenase (ADH), consuming NAD" " and leading to the formation of the aldehyde and NADH. Then, the aldehyde intermediate was aminated with an amine donor L-alanine by a w-transaminase (w-TA). Finally, by combining ADH-hT (ADH from Bacillus stearothermophilus) with CV-w-TA (w-TA from Chromobacterium violaceuni), the amination of various primary alcohols successfully afforded the corresponding primary amines in 2-99 % yields. 

When the lithium enolate 84 is added to propenal (tvithout transmetala-tion), the diastereomeric esters 87a and 87b are formed in the ratio 92 8. In this reaction the crude mixture 87a/87b tvas hydrolyzed to give the carboxylic acid (R)-88 in 83.5% ee. To obtain the enantiomerically pure 3-hydroxy-4-pentenoic acid, enrichment tvas performed by single recrystallization of the ammonium salt, formed from (S)-l-phenylethylamine. When the amine has been liberated from the salt the carboxylic acid (R)-88 is obtained in >99.8% ee and 41% overall yield (Scheme 1.18) [132]. The (S) enantiomer, but not the ( R) enantiomer, of 3-hydroxy-4-pentenoic acid 88 (both prepared according to this procedure) has been sho vn to be a substrate for the enzyme 3-hydroxybutanoate dehydrogenase - another example of the different biological activity of enantiomeric compounds [133]. 

Very recently, Kroutil and coworkers extended the substrate scope also to primary alcohols in a sequence of ADH-catalyzed oxidation and co-transaminase-catalyzed reductive amination of the intermediate aldehyde. Both reactions were connected via alanine dehydrogenase, which mediated the regeneration of both NAD" and alanine (as amine donor for the reductive amination, Scheme 8.17) [82]. 

Scheme 11.13 Deracemization of racemic amino acids by cascade reactions, including DAAOs-catalyzed reactions and reductive amination reactions catalyzed either by amino acid dehydrogenases (a) or transaminases (b).

A drawback of using lactate dehydrogenase as a biocatalyst to remove pyruvate from the reaction equilibrium is the need for the NADH cofactor. Another possibility to eliminate the coproduct is the application of a p5uruvate decarboxylase (Scheme 29.6b). A cofactor is not required, and the resulting products of pyruvate decarboxylation, acetaldehyde, and CO are highly volatile, shifting the equilibrium toward the product [68]. Several pyruvate decarboxylases from yeast and bacteria are commercially available and are active at the same pH value as the transaminase required for the asymmetric synthesis of chiral amines. 

Given that transaminases are not able to aminate alcohols, one possibility for the synthesis of chiral amino alcohols is an enzyme cascade reaction carried out with whole cells [36]. Three enz3nnes cascaded in series were expressed in E. colt first the alcohol dehydrogenase oxidized tiie alcohol to the corresponding aldehyde, which is converted into the amine by the transaminase as shown in Scheme 29.15. The recycling of pyruvate and cofactor regeneration were achieved by the alanine dehydrogenase. 

Under the optimized reaction conditions, co-transami-nase was used as the enzyme (ATA-113 for preparing the (5)-enantiomers and ATA-117 for synthesizing the (R)-enantiomers of the product amines), sodium phosphate buffer for maintaining the pH at 7, and ammonium formate and formate dehydrogenase to recycle the alanine. The variety of synthesized of chiral amines are shown in the following scheme (Scheme 39.42). 

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