Hydantoin racemase racemization

Racemic hydantoins result from the reaction of carbonyl compounds with potassium cyanide and ammonium carbonate or the reaction of the corresponding cyanohydrins with ammonium carbonate (Bucherer-Bergs reaction). Hydantoins racemize readily under basic conditions or in the presence of hydantoin racemase, thus allowing DKR (Figure 6.43). Hydantoinases (EC 3.5.2.2), either isolated enzymes or whole microorganisms, catalyze the hydrolysis of five-substituted... 

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]).

Figure 12.8 Enzymatic racemization of the D-isomer ( ) and L-isomer (O) of (a) 5-benzylhydantoin and (b) 5-ethylhydantoin by hydantoin racemase 1 from A. tumefaciens C58 (AtHyuAl).

The amino acid sequences of known hydantoin racemases present two highly conserved cysteines around positions 75 and 180 (see the asterisks in Figure 12.4). The enzymes involved in the racemization/epimerization of different substrates such as glutamate racemase and diaminopimelate epimerase present two cyste-... 

Figure 12.9 Enzymatic racemization by hydantoin racemase from S. meiiioti CECT 4114 of the D-isomer (O) and L-isomer ( ) of 5-methylthioethylhydantoin. Chemical racemization of the D-isomer (V) and L-isomer (T) of each substrate was also measured at the same intervals.

Figure 12.12 Proposed racemization mechanism of hydantoin racemase using a two-base mechanism. The hypothetical character of the intermediate structure is indicated by the dashed lines surrounding the figure.

From reports in the early literature resting cell bioconversions of hydantoin derivatives, which do not racemize with high velocities, indicated an enzymatic racemization and the presence of a hydantoin racemase. In addition, the chemical and the enzymatic racemization proceed via the keto-enol tautomerism, which is shown in Fig. 12.4-20. Stabilizing effects on the enolate structure such as electronegative substituents are responsible for the velocity of the racemization12, 71. Increased racemization rates can be also seen at more alkaline pH-values and with increased temperatures[71. 

The first hydantoin racemase acting on a cyclic amide substrate reported in the literature was the allantoin racemase (E.C. 5.1.99.3) (Fig. 12.4-4). This enzyme enables several bacteria to use both allantoin enantiomers as substrates120-221. Racemic mixtures of allantoin, e. g. from plant materials, can be completely metabo-... 

The fast and total conversion of i-5-isopropylhydantoin to D-valine by resting microbial cells led Battilotti et al. 0 to the suggestion that a hydantoin racemase might be responsible for the racemization of the L-enantiomer. The first hydantoin racemase to be described in detail was a 5-arylalkylhydantoin racemase, which was isolated and purified from Arthrobacter sp. DSM 3747155, 144> 145l. Its substrate specificity is shown in Fig. 12.4-21. 

For example, n-p-hydroxyphenylglydne, a key intermediate in the synthesis of semisynthetic cephalosporins and penicillins, is currently manufactured on a multi-thousand ton scale. The hydantoinase-catalysed reaction is also suitable for the production of unnatural D-amino acids, although the in situ racemization of the remaining substrate via keto-enol tautomerization is generally slow. To facilitate the stereoinversion, base or hydantoin racemase of Pseudomonas and Arthrobacter strains is often used. 

The only stable product of the industrial method is para-nitrophenyl hydantoine the other proposed but not isolated intermediates are in parentheses. The hydantoine derivative possesses a stereogenic center on the C-5 atom and is racemic. Separation of enantiomers on an industrial scale is completed by selective biocat-alytic hydrolysis of / -enantiomer by hydantoinases, a group of hydrolytic enzymes. The wrong S-enantiomer can be easily racemized by heating in weak basic medium or by the enzyme hydantoin racemase and racemate recycled to separation. 

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]. 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

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. 

When one of the substituents is hydrogen some of these enzymes function as both aminohydralases and as racemases, leading to 100% conversion of racemic hydantoin to just one amino acid enantiomer others selectively hydrolyse one hydantoin enantiomer leaving the other unchanged. Whether the pure DorL amino acid is produced depends on the particular enzyme system involved. The second step, cleavage of the intermediate N-carbamoyl aminoacid to the free aminoacid can be enzymatic or chemical, but in either case is achieved without racemization under relatively mild conditions. 

Recombinant whole cells in particular turned out to be very attractive for bio-transformations in which more than one recombinant enzyme is needed such as redox reactions with in situ cofactor regeneration or hydrolysis with mrdtiple enzymes. With respect to the latter one, the dynamic kinetic resolution of hy-dantoins by a whole-cell catalyst that simultaneously overexpresses a racemase, a hydantoinase and a carbamoylase is a popular and industrially relevant example (Scheme 2.8) [23,24]. These cells convert a racemic hydantoin (an easily accessible substrate) to the corresponding enantiomerically pure d- or L-amino add with both high conversion and enantioselectivity. 

---