5-monosubstituted hydantoins

On the other hand, when a similar photoreaction is carried out on hydantoin or its 5-monosubstituted derivatives in the presence of ben2ophenone, the hydrogen atom at C-5—H is abstracted and the resulting radical couples with that of ben2ophenone (56) ... 

The ureidocarbonylation reaction provides access to hydantoins containing diverse substituents in the 1-, 3-, and 5-positions with good selectivities (Table 2) [37]. With monosubstituted ureas, 3-substituted hydantoins are obtained. 

It has been shown recently that papain exhibits hydantoinase activity. This enzyme of plant origin hydrolyzes not only 5-monosubstituted but also 5,5-disubstituted hydantoins to the corresponding N-carbamoylamino acids. Since chemical hydrolysis of the latter yields the corresponding amino acids, this approach may be of interest in amino acid synthesis [145],... 

D-p-Hydroxyphenylglycine is an important component of certain semi-synthetic antibiotics such as the semi-synthetic cephalosporins cefadroxil and cefatrizine and the semi-synthetic penicillin amoxicillin, with a combined world market in excess of 3 x 10 /a. Synthesis was possible from DL-5-monosubstituted hydantoins (cyclic ureides of amino acids) provided that a selective D-hydantoinase could be found, which would be competitive with chemical methods. 

Gross, C., Syldatk, C. and Wagner, F. (1987) Screening method for micro-organisms producing L-amino acids from D,L-5-monosubstituted hydantoins. Biotechn. Techniques, I, 85-90. 

In contrast to acyl amino acids (pKa > 30) or amides, most 5-monosubstituted hydantoins racemize comparatively easily phenyl-substituted ones even racemize spontaneously at slightly alkaline conditions as their pK.d is around 8 (Kato, 1987). Under spontaneous or enzymatic racemization (Pietzsch, 1990), racemic hydantoins with the help of enantioselective d- or L-hydantoinases and the respective carb-... 

D. Cotoras and F. Wagner, Stereospecific hydrolysis of 5-monosubstituted hydantoins, Eur. Congr. Biotechnol., 3rd 1984, 1, 351-6. 

C. Gross, C. Syldatk, and F. Wagner, Screening method for microorganisms produdng L-amino adds from DL-5-monosubstituted hydantoins, Biotechnol. Technol. 1987, 1, 85-90. 

D-p-Hydroxyphenylglycine and its derivatives are important as side-chain precursors for semisynthetic penicillins and cepharosporines. Yamada and coworkers of our laboratory found that these amino acids can be efficiently prepared from the corresponding 5-monosubstituted hydantoins using the microbial enzyme D-hydantoinase [4]. 

Many kinds of enzymes with different substrate specificities are involved in hydantoin hydrolysis. Ogawa et al. [10] found two hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4. These enzymes were purified to homogeneity and characterized (Table 1). One hydrolyzed dihydropyrimidines and 5-monosubstituted hydantoins to the corresponding AT-carbamoyl amino acids. Since the hydrolysis of 5-substituted hydantoins by this enzyme was D-stereo-specific, this enzyme was identified as D-hydantoinase, which is identical with dihydropyrimidinase. The other one preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin. Because there have been no reports on enzymes which show same substrate specificity as this enzyme, it is considered to be a novel enzyme, which should be called imidase [10]. 

Since D-hydantoinase was identified as dihydropyrimidinase, it is proposed that D-amino acid production from DL-5-monosubstituted hydantoins involves the action of the series of enzymes involved in the pyrimidine degradation pathway. Based on this proposal, D-decarbamoylase was thought to be identical with P-ureidopropionase (EC 3.5.1.6) which functions in pyrimidine metabolism. 

Hydantoinases and decarbamoylases have been applied for the production of optically active amino acids from DL-5-monosubstituted hydantoins. A variety of enzymes have been reported elsewhere. Runser et al. [33] reported the occurrence of D-hydantoinase without dihydropyrimidinase activity. Watabe et al. [34] reported that an ATP-dependent hydantoin-hydrolyzing enzyme is involved in the L-amino acid production from DL-5-monosubstituted hydantoin by Pseudomonas sp. NS671. This enzyme shows no stereospecificity. Hydan-toinase showing no stereospecificity and not requiring ATP was also reported [35]. Recently, hydantoin-racemizing enzymes were found [36,37], These enzymes make it possible to totally convert racemic substrates, which only slowly racemize under reaction conditions, to a single stereoisomer. The combinations of these hydantoin-transforming enzymes provide a variety of processes for optically active amino acid production (Fig. 4). 

Syldatk, C., Muller, R., Siemann, M., Krohn, K., Wagner, F. Microbial and Enzymatic Production of D-Amino Acids from DL-5-Monosubstituted Hydantoins. In Biocatalytic Production of Amino Acids and Derivatives, Rozzell, J. D., Wagner, F. Eds., Carl Hanser Verlag Munich, 1992, p. 75. 

One of the most widely used enzymatic methods for D-amino acid production is the hydantoinase process [4]. The great advantage of this process is that, potentially, any optically pure D-amino acid can be obtained using the corresponding substrate from a wide spectrum of D,L-5-monosubstituted hydantoins, which are readily accessible by chemical synthesis [5]. In this cascade of reachons the chemically synthesized D,L-5-monosubstituted hydantoin ring is first hydrolyzed by a stereoselective hydantoinase enzyme (D-hydantoinase). Further hydrolysis of the resulting N-carbamoyl D-amino acid to the free D-amino acid is catalyzed... 

Figure 12.1 Reaction mechanism of the 5-monosubstituted hydantoins keto-enol tautomerism under alkaline conditions.

Chemical racemization of the 5-monosubstituted hydantoins proceeds via keto-enol tautomerism under alkaline conditions, as shown in Figure 12.1 [6]. The racemization velocity is highly dependent on the buUdness and electronic factors of the substituent in 5-position (see Table 12.1) and is usually a very slow process... 

Figure 12.2 Reaction scheme for the hydrolysis of d,l-5-monosubstituted hydantoin derivatives to the corresponding D-amino acids.

Given the important role that hydantoin racemase plays in the production of optically pure D-amino acids, allowing the racemization of 5-monosubstituted... 

Although on the whole the hydantoin racemases have shown high thermal stability, with optimal activity at 55 °C (Table 12.2), the optimal temperatures for the ones from Pseudomonas and Sinorhizobium decrease to 45 and 40 °C, respectively. However, the optimal pH is higher than 8, except for both hydantoin racemases from Agrobacterium. This low alkaline pH avoids chemical racemization. Consequently, the racemization of the d- or L-5-monosubstituted hydantoins in an industrial process will only occur enzymatically. 

Figure 12.7 Reaction profile of the enzymatic racemization of D- and L-5-monosubstituted hydantoin to the racemic mixture by hydantoin racemase enzyme monitored by chiral HPLC (see the methodology in [28]).

Additional binding experiments conducted by fluorescence measurements with C76A mutant and D- and L-isopropylhydantoin and L-ethylhydantoin (D-ethylhydantoin is an inhibitor) showed that this mutant is unable to bind the D-isomers of the substrates. The same experiments carried out with C181A mutant proved that this mutant was not able to bind the L-isomers. These results indicate that cysteine 76 is responsible for the recognition of D-isomers of the 5-monosubstituted hydantoins, whereas cysteine 181 recognizes the L-isomers. 

From these results, the deduced model is that of a two-base mechanism (Figure 12.12). In this model, when a D-isomer of a 5-monosubstituted hydantoin is... 

Figure 12.14 Initial reaction rate for the production of different optically pure D-amino acids from 5-monosubstituted hydantoins using systems 1 and 2. TRP, D-tryptophan TYR, D-tyrosine pH PC, D-p-hydroxyphenyl glycine VAL, D-valine ...

System 1 was able to hydrolyze the 5-monosubstituted hydantoins faster than system 2 for the production of almost all the D-amino acids studied. System 1 was slightly slower than system 2 only for the production of the aromatic amino acids D-tyrosine and D-phenylglycine. This agrees with previously described results, finding that AtHyuAl enzyme (included in system 1) was more viable for industrial application than AtHyuA2 (included in system 2) due to its higher substrate affinity and racemization velocity [25]. 

The development of this multienzymatic system for the production of D-amino acids from any D,L-5-monosubstituted hydantoin allows the hydantoinase process to produce not only two amino acids, such as D-phenylglycine and D-p-hydroxy-phenylglycine (as explained at the beginning of the chapter), but also many non-natural D-amino acids that could be components of potential pharmaceuticals. 

As has been pointed out by Habermeier ( ), the non-equivalence of the 1- and 3- positions of the hydantoin ring readily permitted monosubstitution. Subsequent glycidylation provided the diepoxide JV. 

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