Hydantoinases resolution

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.

Other biocatalytic methods of producing D-p-hydroxyphenylglycine have not proved competitive, for instance transaminase based processes require glutamate to be supplied. Others include the hydrolysis of N-acyl derivatives by acylase and amides by aminopeptidase (DSM), the use of L-specrfic hydantoinases and immobilised subtilisin for the resolution of D,L-2-acetamido-/>-hydroxyphenylacetic acid methyl ester (Bayer). 

Facing difficulties with chemical methods, development work was aimed at finding a biocata-lytic process to resolve the amino acids. Typical commercial enzymes reported for resolution of amino acids were tested. Whole-cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids 106 112 the acylase catalyzed resolution of Af-acyl amino acids had extremely low rates (often zero) of catalysis toward a-dialkylated amino acids 113114 and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-... 

A method that has been used to approach 100% theoretical yield in asymmetric syntheses is dynamic kinetic resolution, or DKR. Although this method has been practiced based on strictly chemical reactions, only those chemoenzymatic DKR reactions will be discussed here. Most often, the enzyme used by this method is a hydrolase (lipase, esterase, protease), but other enzymes such as hydantoinases, /V-acylamino acid racemases, and dehydrogenases have also been exploited to effectively carry out DKR reactions.196 For additional details the reader is directed to the many review articles written on DKR.197 206... 

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). 

In the case of o-pheny Igly cine and D-/ -hydroxphcnylglycinc manufacture, several producers are believed to have started the hydantoinase route (Scheme 17). Both enzymatic and classic resolution use, as the starting material, benzalde-... 

A classic example of a typical enzymatic resolution on an industrial scale is the acylase-mediated production of L-methionine. This method has also been applied for the production of L-phenylalanine and L-valine. In addition to acylases, amidases, hydantoinases, and /i-lactam hydrolases represent versatile biocatalysts for the production of optically active L-amino acids. A schematic overview of the different type of enzymatic resolutions for the synthesis of L-amino acids is given in Fig. 2. 

Resolution processes based on the use of hydantoinases have been well-known for a long time, in particular for the production of D-amino acids [16], By the 1970s... 

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. 

In summary, a broad range of large-scale applicable biocatalytic methodologies have been developed for the production of L-amino acids in technical quantities. Among these industrially feasible routes, enzymatic resolutions play an important role. In particular, L-aminoacylases, L-amidases, L-hydantoinases in combination with L-carbamoylases, and /l-lactam hydrolases are efficient and technically suitable biocatalysts. In addition, attractive manufacturing processes for L-amino acids by means of asymmetric (bio-)catalytic routes has been realized. Successful examples are reductive amination, transamination, and addition of ammonia to rx,/fun-saturated carbonyl compounds, respectively. 

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. 

Table 9-3. Dynamic resolution using hydantoinase enzymes.

Production of D-p-Hydroxyphenyl Glycine by Dynamic Resolution with Hydantoinase from Bacillus brevis (E.C. 3.5.2.2) 81831... 

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. 

There are stUl other opportunities in using elegant enzymatic synthesis methods, for instance, the hydrolysis ofN-acetyl-(D)-amino acids from racemic mixtures by (L)-acylase cleavage, by dynamic kinetic resolution, or by employing a race-mase and a hydantoinase. 

Scheme 2.18 Enzymatic resolution of hydantoins via the hydantoinase method...

In contrast to the above-mentioned amino acid resolution methods involving amino acid esters, -amides, or Af-acylamino acids where the natural L-enantiomer is preferably hydrolyzed from a racemic mixture, hydantoinases usually convert the opposite D-enantiomer [153-155], and L-hydantoinases are known to a lesser extent... 

Although at present this chemoenzymatic method might not seem as versatile as the original hydantoinase process, the results on dihydropyrimidinases indicate a clear opportunity for the production of enantioenriched or enantiopure and P -amino acids by kinetic resolution ivhen the enantioselectivity of the enzyme is very high. Thus, further research is needed to find (or create) new enzymes with higher enantioselectivity. 

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. 

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. 

---