Hydantoin racemization

Both pure L- and D-amino acids can be made using hydantoinase enzymes. These enzymes catalyze the stereoselective hydrolysis of racemic hydantoins such as (50) which is used for the production of D-alanine (15) (58). 

Figure 2.13 Reactions and enzymes involved in the production of L-amino acids from racemic hydantoins by the three-enzyme hydantoinase process [55],...

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... 

A novel reaction of pyroglutamate (6) and an isocyanate promoted by NaH in THF leads to functionalized hydantoins (7) in good yields. The reaction involves the ring closure of intermediate (8) by a nucleophilic attack on the carbonyl of the ester function followed by expulsion of an alkoxide anion resulting in the formation of the bicyclic intermediate (9). The alkoxide anion in turn can open this bicyclic intermediate with formation of anions (10) and (11) leading to the final racemic hydantoins (7) (Scheme 3).8... 

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-... 

The bacterial D-hydantoinase has been isolated as crystals from cells of Pseudomonas putida (= P. striata) (Table 1) [5]. Because the purified enzyme showed the highest activity and affinity toward dihydrouracil, the enzyme was identified as dihydropyrimidinase (EC. 3.5.2.2). Interestingly, the enzyme also attacked a variety of aliphatic and aromatic D-5-mono-substituted hydantoins, yielding the corresponding D-form of N-carbamoyl-a-amino acids. Thus, the enzyme can be used for the preparation of various D-amino acids. Under the conditions used for the enzymatic hydrolysis of hydantoin at pH 8 to 10, the L-isomers of the remaining hydantoins are racemized through base catalysis. Therefore, the racemic hydantoins can be converted quantitatively into N-carbamoyl-D-amino acids through this step. 

R. Olivieri, E. Fascetti, L. Angelini, and L. Degen, Microbial transformation of racemic hydantoins to D-amino adds. Biotech. Bioeng., 23 2173 (1981). 

Rgure A8.8 Enzymatic processes for the production of opticaiiy active a-amino adds via resolution of the racemic hydantoins. 

De Julian-Ortiz et al. calculated connectivity indices to the fourth order and proposed charge indices (G, / ) for racemic hydantoins, racemic a-amino acids, and racemic arylamides. The best correlation equation for racemic hydantoins separated on p-cyclodextrin column using 10% methanol as a mobile phase is ... 

D-p-Hydroxyphenylglycine by hydrolysis of the racemic hydantoin D-specific hydantoinase... 

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. 

The hydantoinase method has become of significant interest for preparative organic chemistry total conversion of racemic hydantoins, synthesized by well-established chemical methods to nearly 100% optically pure products is possible using free or immobilized microbial cells or enzymes. Further, it is possible to prepare a wide range of optically pure d- as well as i-amino acids by this method. 

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... 

The enzyme, Af-carbamoyl o-amino acid amidohydrolase can be incorporated with hydantoinase to produce o-amino acids in one step from the corresponding racemic hydantoins. Because many of the five-substituted hydantoins undergo spontaneous racemization under the enzymatic reaction conditions (pH > 8), complete conversion from racemic hydantoin to the o-amino acid can be achieved. The starting hydantoins are prepared by the Bucherer-Bergs reaction (Scheme 9.34), the simple condensation of potassium cyanide and ammonium carbonate with the... 

Lactam antibiotics (penicillins and cephalosporins) Hydantoinase The enzyme is used for preparation of D-phenylglycine and D-p-hydroxyphenyl-glycine, which are chiral intermediates for racemic hydantoins [82]... 

A process for the production of D-a-amino acids has been developed by Roche Diagnostics based on the enzyme D-hydantoinase [115]. The recombinant protein was covalently fixed onto a carrier and used for the synthesis of a broad range of natural and artificial D-amino acids (132, Scheme 41). Starting from racemic hydantoins d/l-130, the enzyme exclusively hydrolyzed d-130 to 131, and new d-130 was internally produced by continuous in situ racemization of l-130. The process worked especially well with 5-(p-hydroxyphenyl)- and 6-phenylhydantoin, affording the corresponding amino acids 132 in high yield and optical purity. The number of reuse cycles until 50% of the initial enzyme activity was reached was calculated to be as high as - 200. Unfortunately, this process has never been used in the production of D-amino acids, as diazotation was found to be too noxious and complicated. 

Sorbinil 282 is a compound of potential therapeutic interest, because it prevents or alleviates the chronic complications of diabetes mellitus, due to its ability to inhibit the enzyme aldose reductase. Sarges et al. prepared 282 and its enantiomer by the reaction sequence shown in Scheme 12.71, involving a brucine resolution of the racemic hydantoin precursor 284. " The free base of brucine forms a crystalline complex with 282, whereas the other enantiomer of 282 only forms a crystalline complex with brucine hydrochloride. Since this resolution technique does not work with certain congeners of sorbinil, a synthesis via an asymmetric induction sequence (Scheme 12.72) has also been developed that seems generically applicable to optically active spiro hydantoins. Both methodsrequired 2,3-dihydro-6-fluoro-47/-l-benzopyran-4-one 283 and the introduction of the amino acid functionality... 

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. 

An interesting and useful example of synthesis of a-amino acids has been recently reported by Wuts and coworkers, describing a scalable process to prepare the INOS inhibitor PHA-399733 42 using the Bucherer-Bergs hydantoin synthesis as key step to introduce the amino acid function present in the final skeleton (Scheme 10.11) [41]. The methodology was applicable to the isolation of 39.9 kg (75% yield) of the racemic hydantoin 41, and the desired enantiomer was then opened in basic conditions giving the corresponding amino acid moiety. 

The authors developed a scalable route for the synthesis of the intermediate core 45 in 12 steps and 0.4% yield and successfully implemented at a pilot plant scale. In this multistep synthesis, racemic hydantoin 43 was obtained after crystallization in 4 1 mixture of diastereomers. Separation of enantiomers by diastereomeric salt formation with (/ )-2-phenylglycerol and further crystallization followed by salt break gave single isomer (-)-44, with the targeted l-glutamic acid configuration. 

The oxazole analogue (90) of the antitumour agent tiazofurin has been prepared from 2,S-anhydro-D-allonic acid by a fairly conventional annulation.1 1 An interesting photochemical ring expansion was used (Scheme 12) in a synthesis of the racemic hydantoin C-riboside (91), the starting material illustrated being made by a Diels-Alder-hydroxylation sequence. 1 2 Further extensions of the application of Wittig reactions for the... 

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,... 

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