L- hydantoins

Chemically synthesised D,L-hydantoins prepared from the corresponding aldehydes via die Bucherer Berg reaction are converted by the bacterial cells (Bacillus brevis), containing a D-spedfic hydantoinase, to a mixture of D-N-carbamoyl amino acid and L-hydantoin. The latter compound undergoes rapid and spontaneous racemisation under the conditions of the reaction, therefore, in principle 100% of the hydantoin is converted into the D-N-carbamoyl compound. The D-amino add is obtained after treatment of the D-N-carbamoyl compound with nitrous add. This process is operated on an industrial scale by the Japanese firm Kanegafuchi. 

Siemann, M., Syldatk, C. and Wagner, F. (1993) Detection and comparison of strains with selective L-hydantoin cleaving activity using polyclonal antibodies. Biotechnol. 

D,L-hydantoin W-carbamoyl-D-amino acid D-amino acid... 

The racemic 2-azabicyclo[2.2.1]heptane-3-carboxylic acid was obtained by alkaline hydrolysis (24 h reflux in 4 N NaOH/MeOH 3/1 yield 78%) of the 4-phenyl-2,4-diazatricyclo[5.2.1.02-5]decane-3,5-dione-(l)- hydantoin obtained... 

Dihydiopyiiinidinase- (hydantoinase-) based processes have been successfully employed for the production of D-amino adds, particularly D-p-hydroxyphenylglydne (7,25,77,78). D,L-hydantoins are chemically synthesized from the corresponding aldehydes using the Bucherer-Berg... 

The reaction concept with this new hydantoinase-based biocatalyst is economically highly attractive since it represents a dynamic kinetic resolution process converting a racemic hydantoin (theoretically) quantitatively into the enantiomerically pure L-enantiomer [19]. The L-hydantoinase and subsequently the L-carbamoylase hydrolyze the L-hydantoin, l-11, enantioselectively forming the desired L-amino acid, l-2. In addition, the presence of a racemase guarantees a sufficient racemiza-tion of the remaining D-hydantoin, d-11. Thus, a quantitative one-pot conversion of a racemic hydantoin into the desired optically active a-amino acid is achieved. The basic principles of this biocatalytic process in which three enzymes (hydan-toinase, carbamoylase, and racemase) are integrated is shown schematically in Fig. 9. 

All microorganisms producing D-aminoacylases commonly produce L-aminoacy-lases as well. Therefore, to reach high optical purity of the D-amino acids produced from the respective N-acetyl-D,L-amino acids, the D-aminoacylases have to be separated from the L-aminoacylases (Table 12.3-13). However, this is a disadvantage in view of an industrial application since additional purification steps lead to more expensive enzymes and thus add costs to the whole production process. This is one of several reasons why it is widely accepted today that the production of D-amino acids by enzyme-catalyzed hydrolysis of D,L-hydantoins seems to be more promising than the D-aminoacylase route via N-acetyl-D,L-amino acids. The enzyme-catalyzed synthesis of D-amino acids from the respective D,L-hydantoins is described in Chapter 12.4. 

Figure 17-15. Enzymatic synthesis of d- or L-amino acids from 5-substituted D,L-hydantoins through N-carbamoyl-D- or L-amino acids.

D-p-Hydroxyphenyl glycine is a key raw material for the semisynthetic penicillins such as ampidllin and amoxycillin. It is also used in photographic developers. Racemic hydantoins are synthesized starting from phenol derivatives, glyoxylic acid and urea via the Mannich condensation (Fig. 19-28). The D-specific hydantoinase is applied as immobilized whole cells in a batch reactor. The unreacted L-hydantoins are readily racemized under the alkaline conditions (pH 8) of enzymatic hydrolysis, yielding quantitative conversion. This process enables the stereospecific preparation of various amino acids, such as L-tryptophane, L-phenylalanine, D-valine, D-alanine... 

Basic conditions have also been exploited to effect equilibration between D- and L-hydantoins. Building on these known protocols for hydantoin racemisation, Garcia and Azerad developed an efficient route to a number of D-phenylglycine derivatives, which were mono- or disubstituted in the aromatic ring (Scheme 3.40). ... 

Very recently in the course of the study of the urinary products of histidine dissimilation Brown and Kies discovered L-hydantoin-5-propionic acid 255). This compound was observed in the urine of the rat and the... 

More recently Brown and Kies (S65) reported the formation from histidine and the excretion in the urine of hydantoin- propionic acid, and also its formation by liver extracts of guinea pig and the rat. The substrate for the oxidation was shown very probably to be imidazolone-propionic acid. No oxidation or formation of hydantoinpropionic acid could be demonstrated if the urocanase activity was first destroyed. The L-hydantoin-5-propionic acid was isolated by chromati raphy, and crystallized. Its identity was unequivocally established. 

Although many enzymological and genetic studies have been conducted, the physiological explanation for L-specific transformation of hydantoin has not yet been established. They are specific only for L-hydantoins and iV-carbamoyl-L-amino acids whose existence in nature is still unclear, with the exception of L-5-caiboxymethylhydantoin and A-carbamoyl-L-aspartate. 

The hydantoinases hydrolyzing both d- and L-hydantoin were also found in several bacteria. They could be divided into two groups one needs ATP for its activity and the other does not. The ATP-requiring enzyme was purified and cloned from Pseudomonas sp. strain NS 671 [37,38]. The enzyme consists of two subunits with differing molecular mass of 76 and 65 kDa, and preferably hydrolyzes L-hydantoin. The enzyme that does not require ATP was purified and cloned from a moderate thermophilic bacterium B. stearothermophilus NS 1122A [39,40] and Arthrobacter sp. DSM 3745 [41]. The Bacillus enzyme is homotetrameric with molecular mass of 200 kDa. Although the monomer had no activity. 

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