Dynamic resolution hydantoin

The other major class of cyclic amino acid derivative used in dynamic resolution reactions is the hydantoin group. Like oxazolinones, hydantoins readily undergo racemisation under mild conditions. Systems involving a two step procedure using D-hydantoinase and a carbamoylase were reported to provide a route to D-amino acids[S2, 53). Dynamic resolution of a p-hydroxyphenyl substituted hydantoin was reported in 1987[541. Using the intact cells of Pseudomonas sp. AJ-11220, the amino acid was prepared in over 90% yield, as shown in Fig. 9-24. This hydrolytic procedure leads directly to the amino acid, and the same enantiomer of product, the D-amino acid, was obtained independently of the stereochemistry of the substrate. 

Here the biotransformation competes with the classical chemical route (Fig. 19-29), which employs bromocamphorsulfonic acid (Br-CAS) as the resolving agent. In both routes phenol is used as raw material since p-hydroxybenzaldehyde is too expensive. The hydantoinase process for phenylglycines does not necessarily need an extra racemization step since the hydantoin is racemized in situ at an alkaline pH. Because of the dynamic resolution in the case of this biotransformation, higher yields are reached. 

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]. 

Figure 6.43 Dynamic kinetic resolution of (rac)-hydantoins by a D-hydantoinase.

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

In elegant work the enantioselectivity of a hydantoinase from Arthrobader species for the production of l-methionine in Escherichia coli has been inverted [87]. The approach is similar to the one used in the evolution of (S)- and ( -selective lipases (see above). All known hydantoinases are selective for D-5-(2-methylthioethyl)hydantoin (d-18) which leads to the accumulation of N-carbamoyl-D-methionine (d-19), conversion being complete if the conditions of dynamic kinetic resolution are upheld [88], in this case by the use of a racemase or pH >8 (Fig. 11.22). 

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. 

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. 

Basic principle for the synthesis of optically pure l- and o-amino acids via Strecker synthesis of racemic hydantoins and their (dynamic) kinetic resolution and stepwise hydrolysis toward the amino acids. KOCN, potassium cyanate HYD, hydantoin 28 CARB-AA,... 

Synthesis of t-a-amino acids via dynamic kinetic resolution of hydantoins with a recombinant whole-cell catalyst containing a racemase, i-hydantoinase, and L-carbamolyase. 

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