PROCESS FOR THE PREPARATION OF ALLYSINE ACETAL

Abstract: The present invention relates to the preparation of compounds of formula (I) from the corresponding hydantoins by means of an enzymatic process. The L-compound is preferably formed.

Description: Process for the preparation of allysine acetal The present invention is directed towards the preparation of compounds of the general formula (I) Such compounds are prepared in particular by means of an enzymatic process from hydantoins of the general formula (II) Compounds of formula (I) are suitable intermediates for the preparation of pharmaceuticals described in US 5552397, WO 9738705 and in J. ed. Chem. 42, 305 (1999) . In J. Med. Chem. 42, 305 (1999) there is mentioned a synthesis route for the preparation of a structural unit - an α-amino-ε-caprolactam derivative - of the pharmaceutically active compounds. That structural unit is obtained with the aid of expensive reagents in a process that is rather disadvantageous for a robust commercial process . The preparation of compounds such as (I) from hydantoins such as (II) by means of Arthrobacter sp. is already known from JP 99206397. However, that document does not describe the advantageous racemisation. The conversion of hydantoins by means of hydantoinases and specific carba oylases is already known from DE19529211.1. The spontaneous chemical racemisation of hydantoins for the preparation of enantiomerically enriched a ino acids is to be found in DE-P4137581.5-44. Accordingly, the object of the present invention was to provide a further enzymatic process for the preparation of the desired compounds. In particular, that process was to be more simple to carry out and hence more suitable for use in a large-scale process. That and further objects, which are not referred to in detail but which can be inferred in an obvious manner from the prior art, are achieved by the provision of a process having the features of the present claim 1. Advantageous embodiments of the process according to the invention are protected in the sub-claims that are dependent on claim 1. Claim 6 protects an advantageous use. Owing to the fact that, in a process for the preparation of allysine acetal of the general formula (I) there are used as starting material hydantoins of the general formula (II) wherein in formulae (I) and (II) R represents (Cι-C 8 )- alkyl, (C 2 -C 4 ) -alkylenyl, preferably ethylenyl, (C 6 -Cι 8 )- aryl, (C 7 -Cι 9 ) -aralkyl, (Cι-C 8 ) -acyl, wherein the latter are subjected to a reaction with hydantoinases and D- or L- specific carbamoylases, as well as to a spontaneous and/or enzyme-catalysed in situ racemisation, and wherein the enzymes involved may be used in free form, in immobilised form or in a form enclosed in cells, the desired compounds such as (I) are obtained in a manner that is surprising for a large-scale process but advantageous. The reaction sequence according to the invention has hitherto not been applied in the prior art to the present compounds. It is therefore to be regarded as surprising that the labile acetal protecting group is stable under the reaction conditions and allysine acetal having an optical purity >99%ee can be generated in a very high yield from 100% of the hydantoin in an overall yield >85%. As has been mentioned, the process according to the invention may be carried out partially enzymatically or completely enzymatically. In addition to the use of the free enzymes in a reaction batch, special preference is given, however, to a process in which there is used a so- called whole cell catalyst which has a cloned gene coding for a hydantoin racemase, a hydantoinase and an L- or D- specific carbamoylase. Such organisms are known in principle from US 60/157427 (seq. 4/hydantoinase, 5/hydantoin racemase, 6/carbamoylase) or US 09/407062 (seq. 1/hydantoinase, 2/hydantoin racemase, 3/carbamoylase) . Accordingly, the disclosure of those specifications is regarded as being included herein, especially the disclosure of the relevant amino acid sequences in the sequence listings. That is the case especially of US 60/157427. The use of a total cell catalyst having an L-specific carbamoylase is most particularly advantageous. It is thus possible to obtain the desired optical antipode of allysine acetal for the preparation of the pharmaceutically active substances. In principle, the total cell catalyst may be any suitable expression system that comes into consideration for that purpose to those skilled in the art. Special preference is given, however, to the use of a recombinant bacterium, preferably E. coli, for the purpose. Advantageous E. coli strains are: JM109, NM 522, JM105, RR1, DH5α, TOP 10 " or HB101. In order to carry out the invention, the following procedure is generally followed: the substrate (II) is brought into contact with the enzymes in a suitable solvent, preferably water, at an optimum pH value for hydantoinase and carbamoylase of approximately from 5.5 to 8.5, preferably from 6.5 to 8, and at an optimum temperature for the enzyme activity of approximately from 20°C to 40°C, preferably from 25°C to 35°C. It may be advantageous to add metal salts that have a positive effect on the enzyme activities, such as CoCl 2 or MgCl 2 , MnCl 2 , etc.. During cleavage of the hydantoins into the optically enriched amino acids, the hydantoins racemise spontaneously. However, in order to accelerate that reaction, the hydantoin that remains can be racemised enzymatically in situ and is thus available for cleavage into the amino acid again. For reasons of time, therefore, 5 enzymatic racemisation that proceeds simultaneously with the conversion of the hydantoin to the amino acid is preferably strived for. All the hydantoin can thus be converted into the amino acid in one step. As has been stated, that may be effected using enzymes that are 10. available separately, which may be in free or immobilised form, or using enzymes that are enclosed in a microorganism (US 60/157427) . The process according to the invention may be carried out in sequential reaction batches or continuously in a so- 15 called enzyme-membrane reactor (Wandrey et al . in Jahrbuch 1998, Verfahrenstechnik und Chemieingenieurwesen, VDI p. 151 ff; Kragl et al . Angew. Chem. 1996, 6, 684 f) . In a further embodiment, the invention relates to the use of the acetals prepared according to the invention in a 20 synthesis for the preparation of active ingredients, especially pharmaceuticals, having biological activity. (Cι~C 8 ) -Alkyl is to be regarded as being methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl or octyl, including all 25 their isomers formed due to different positions of the double bond. (Cι-C 8 ) -Alkylenyl is to be understood as meaning alkyl bridges having from 2 to 8 carbon atoms, the substituents being in the 1- and n-position, such as, for example, 30 ethylenyl, propylenyl, etc.. A (C 6 -Ci 8 ) -aryl radical is to be understood as being an aromatic radical having from 6 to 18 carbon atoms. It includes in particular compounds such as phenyl, naphthyl, anthryl, phenanthryl, biphenyl radicals. A (C7-C19) -aralkyl radical is a (C 6 -C 18 ) -aryl radical bonded to the molecule via a (Cι~C 8 ) -alkyl radical. A (C 1 -Cs)-acyl radical denotes a (Cι-C 8 ) -alkyl radical that is bonded to the molecule via a C=0 function. The structures of the compounds shown relate to both optical isomers. Example : 30 g (wet weight) of E. coli cells JM109 (pOM22, pOM21) (US 60/157427) were mixed together with 100 mM DL-allysine hydantoin and ImM CoCl2 at pH 7.8 in 1 litre of water. The reaction mixture was then left for 4 hours at 37°C. The cells were then centrifuged off (45 min, 8000 rpm, 4°C, Beckman Coulter JA-10 rotor) and the supernatant was analysed by means of HPLC. After 4 hours, a yield of L- allysine acetal of >85% having an optical purity of >99%ee was obtained.

Claims: Patent claims: 1. Process for the preparation of allysine acetal of the general formula (I) starting from hydantoins of the general formula (II) wherein in formulae' (I) and (II) R represents (Cj-Cs) - alkyl, (C 2 -C 4 ) -alkylene, (C 6 -Cι 8 ) -aryl, (C-C 19 )- aralkyl, (Cχ-C 8 ) -acyl, by reaction with hydantoinases and D- or L-specific carbamoylases, as well as a spontaneous and/or enzyme- catalysed in situ racemisation, wherein the enzymes involved may be used in free form, in immobilised form or in a form enclosed in cells. 2. Process according to claim 1, characterised in that there is used a total cell catalyst which has a cloned gene coding for a hydantoin racemase, a hydantoinase and an L- or D-specific carbamoylase. 3. Process according to claim 2, characterised in that the total cell catalyst has an L-specific carbamoylase. 4. Process according to claim 1, 2 and/or 3, characterised in that the total cell catalyst is a recombinant bacterium, preferably E. coli. 5. Process according to one or more of the preceding claims, characterised in that the operation is carried out in an enzyme-membrane reactor. 6. Use of the acetals prepared according to any one of the preceding claims in a synthesis for the preparation of active ingredients, especially pharmaceuticals, having biological activity.

Inventors: DRAUZ KARLHEINZ, KLEMENT INGO, KRIMMER HANS-PETER, MAY OLIVER, REICHERT DIETMAR, DRAUZ, KARLHEINZ, KLEMENT, INGO, KRIMMER, HANS-PETER, MAY, OLIVER, REICHERT, DIETMAR
Applicants: DEGUSSA, DEGUSSA AG
Application year: 2001
Main classification: C12P13
IPC codes: C12P13