NL1019416C2 - Process for preparing an enantiomerically enriched α-amino acid. - Google Patents

Description

- 1 -

METHOD FOR PREPARING A

ENANTIOOMER ENRICHED A-AMINO ACID

The invention relates to a process for preparing an enantiomerically enriched α-amino acid in a one-pot reaction in the presence of a hydantoinase and an enantioselective carbamoylase.

Enantiomerically enriched α-amino acids can be prepared enzymatically from a mixture of the corresponding D and L-5 substituted hydantoin. Such a method is known from EP 0 775 748. A mixture of D-10 and L-5-substituted hydantoin is hereby enantioselectively converted in a one-pot reaction to the corresponding D-α-amino acid in the presence of a hydantoinase and a D-carbamoylase. Enantiomerically enriched α-amino acids are important building blocks for biologically active preparations such as β-lactam antibiotics, peptide hormones and pesticides.

A drawback of this method is that the starting material for the preparation of the enantiomerically enriched α-amino acid, the corresponding 5-substituted hydantoin, is generally difficult to handle due to the poor water solubility of most 5-substituted hydantoins.

The invention has for its object to provide a simple method for the preparation of an enantiomerically enriched α-amino acid, wherein a more manageable starting material than a 5-substituted hydantoin can be used. This is achieved according to the invention by converting an N-carbamoyl-L-α-amino acid or N-carbamoyl-D-α-amino acid to the corresponding D-α-amino acid or L-α-amino acid in the presence of a D-carbamoylase or L-carbamoylase, respectively.

Surprisingly, it has been found that the conversion of N-carbamoyl-L-α-amino acids or N-carbamoyl-D-α-amino acids to D-and-L-α-amino acids proceeds quickly and with a high yield. Since N-carbamoyl-Da-amino acid or N-carbamoyl-La-amino acid can be easily prepared from the corresponding Da-amino acid or La-amino acid, a method is also provided with the invention in which Da-amino acids are produced quickly and with a high yields can be prepared from the corresponding La amino acids (and vice versa). L-a-amino acids are often inexpensive and available on a large scale. Moreover, the process according to the invention is particularly suitable for the preparation of enantiomerically enriched α-amino acids, which when they are chemically converted to 1. ·; The corresponding hydantoins provide less stable hydantoins (so that during the chemical conversion of this enantiomerically enriched α-amino acids into the corresponding hydantoins there is much by-product formation). Examples of such α-amino acids are serine, asparagine, glutamine, lysine, threonine and cysteine.

The term carbamoylase is known from the literature. To those skilled in the art, carbamoylase is understood to be an enzyme with carbamoylase activity, i.e., the ability to catalyze the conversion of N-carbamoyl-α-amino acid to the corresponding α-amino acid. The person skilled in the art understands D-carbamoylase to be an enzyme which preferably catalyzes the conversion of N-carbamoyl-D-10 α-amino acid to the corresponding D-α-amino acid. The D-carbamoylase involved in the process for the preparation of a Da-amino acid according to the invention is preferably at least 90% selective, more in particular at least 95% selective, even more particularly at least 98% selective and the most particularly at least 99% selective. In the context of the invention, 90% selectivity of the D-carbamoylase means that the D-carbamoylase converts the N-carbamoyl (D, L) -α-amino acid to 90% Da-amino acid and 10% La- amino acid at a total conversion of 50%, which corresponds to an enantiomeric excess (ee) of 80% of the Da-amino acid at a conversion of 50%. Naturally, the same applies to L-carbamoylases.

The term hydantoinase is known from the literature. To those skilled in the art, hydantoinase is understood to be an enzyme with hydantoinase activity, i.e., the ability to catalyze the reaction of 5-substituted hydantoin to the corresponding N-carbamoyl-α-amino acid and vice versa. The hydantoinase activity required for the method of the invention may be from one or more hydantoinase. The enantioselectivity of the hydantoinase (s) involved in the conversion of N-carbamoyl-L-α-amino acid or N-carbamoyl-D-α-amino acid to the corresponding D-α-amino acid or L-α-amino acid is not particularly critical. Both hydantoinases known as D-selective and L-selectively can be used in a process for the preparation of a Da-amino acid or La-amino acid from the corresponding N-carbamoyl-La-amino acid or N-carbamoyl-Da-amino acid respectively according to the present invention.

Preferably, the conversion of N-carbamoyl-L-α-amino acid or N-carbamoyl-D-α-amino acid to the corresponding D-α-amino acid or L-α-amino acid according to the invention is carried out in the presence of -3-a hydantoin racemase. Hydantoin racemases are known from the literature (e.g. from WO 01/23582 A1). The person skilled in the art defines a hydantoin racemase as an enzyme with hydantoin racemase activity, i.e. the ability to catalyze the racemization of the L-5-substituted hydantoin (or D-5-substituted hydantoin).

In the process according to the invention, it is of course also possible to use any mixture of N-carbamoyl-Da-amino acid and N-carbamoyl-La-amino acid instead of N-carbamoyl-La-amino acid (or N-carbamoyl-Da-amino acid, respectively) in the preparation of the corresponding Da amino acid 10 (respectively La amino acid).

The temperature and the pH are not particularly critical in the conversion of N-carbamoyl-L-α-amino acid or N-carbamoyl-D-α-amino acid to the corresponding D-α-amino acid or L-α-amino acid according to the invention. Preferably, however, the conversion is carried out at a pH between 5 and 10. In particular, the conversion is carried out at a pH between 6.0 and 7.5. The temperature of the conversion of N-carbamoyl-La-amino acid or N-carbamoyl-Da-amino acid to the corresponding Da-amino acid or La-amino acid is preferably chosen between 0 and 80 ° C, in particular between 10 and 50 ° C, more in particular between 35 and 45 ° C. The reaction conditions in the process according to the invention are preferably chosen such that the enantiomerically enriched α-amino acid formed crystallizes out, so that the recovery of the enantiomerically enriched α-amino acid can be done more easily.

Preferably, the method for converting N-carbamoyl-La-amino acid to the corresponding Da-amino acid or converting N-carbamoyl-Da-amino acid to the corresponding La-amino acid is used in the preparation of a Da-amino acid from the corresponding La amino acid or the preparation of a La amino acid from the corresponding Da amino acid. Use is made here of the same method as described above, to which an additional step is added, namely the preparation of N-30 carbamoyl-La-amino acid from La-amino acid, respectively the preparation of N-carbamoyl-Da-amino acid from Da-amino acid . The preparation of N-carbamoyl-α-amino acid from the corresponding α-amino acid can, for example, be done chemically by contacting the α-amino acid with, for example, MOCN, with M being alkali metal, preferably potassium or sodium. The temperature used is not critical here: the reaction preferably proceeds at temperatures between approximately -5 and 100 ° C, preferably at temperatures between 0 and 80 ° C; the pH of the chemical reaction is preferably chosen between 8 and 11, more particularly between 9 and 10.

Preferably, the preparation of D-α-amino acid from the corresponding L-α-amino acid (or vice versa) is carried out via the corresponding N-carbamoyl-L-α-amino acid (or the corresponding N-carbamoyl-D-α-amino acid) in one pot. This has the advantage that no intermediate recovery of the N-carbamoyl-α-amino acid has to take place.

The invention is in no way limited by the form in which the enzymes are used for the present invention, provided that the enzymes have at least one of the following activities: hydantoinase activity, enantioselective carbamoylase activity or hydantoin racemase activity. The different enzymes can each be present in a specific form independently of each other. The enzymes can be present, for example, both as crude enzyme solution and as purified enzyme. The enzymes can also be present, for example, in (permeabilized and / or immobilized) cells, which naturally or through genetic modification possess the desired enzymes / enzyme activity, or from a lysate of cells with such activity. The enzymes can for example also be used in immobilized form or in chemically modified form. It will be clear to the person skilled in the art that in the method according to the invention mutants of naturally occurring (wild-type) enzymes with hydantoinase and / or enantioselective carbamoylase and / or hydantoin racemase activity can also be used. Mutants of wild-type enzymes can be made, for example, by mutating the DNA encoding the wild-type enzymes by mutagenesis techniques known to those skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) which encodes the DNA for an enzyme that differs at least one amino acid from the wild-type enzyme and by expressing the mutated DNA in a suitable (host) cell.

Suitable hydantoinases and / or enantioselective carbamoylases and / or hydantoin racemases can be located in or come from, for example, the microorganisms, which usually also provide the enzymes for the hydantoinase carbamoylase processes, e.g. organisms of the following genera: Pseudomonas, preferably Pseudomonas sp, more particularly Pseudomonas sp. FERM BP 1900, 1 0 1 ·> '^ 1 - ν' -5-

Hansenula, Aarobacterium, preferably Aqrobacterium so. or Aqrobacterium radiobacter more in particular Aqrobacterium sp. IP 1-671 or Aqrobacterium radiobacter NRRLB 11291, Aerobacter, preferably Aerobacter cloacae, more in particular Aerobacter cloacae IAM 1221, Aeromonas. Bacillus, preferably Bacillus macroides. more in particular Bacillus macroides ATCC 12905, Brevibacterium. Flavobacterium. Serratia, Micrococcus. Arthrobacter. preferably Arthrobacter aurescens. more in particular Arthrobacter aurescens DSM 3747 or Paracoccus.

Most preferably, one or more of the enzymes used in the conversion of N-carbamoyl-L-α-amino acid or N-carbamoyl-D-α-amino acid to the corresponding D-α-amino acid or L-α-amino acid according to the invention are derived from Aqrobacterium radiobacter.

The invention can be used in the preparation of both proteinogenic and non-proteinogenic D and L enantiomers of α-amino acids of formula R-CH (NH 2) -COOH, wherein R represents an amino acid residue group, for example an unsubstituted or substituted alkyl group with for example 1-20 C atoms or substituted or unsubstituted (hetero) aryl group with for example 1-20 C atoms. Examples of α-amino acids of formula R-CH (NH 2) -COOH are: alanine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, asparagine, glutamine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine , arginine, citrulline, phenylalanine, 3-fluorophenylalanine. The invention is preferably used in the preparation of the D or L enantiomers of methionine or leucine.

The suspected effect of the preparation of a Da-amino acid (La-amino acid respectively) in a single-pot reaction in the presence of a hydantoinase and a D-carbamoylase (L-carbamoylase, respectively) starting from N-carbamoyl-La-amino acid / La-amino acid (N-carbamoyl-Da-amino acid / Da-amino acid, respectively) is illustrated in Figure 1. Figure 1 shows the preparation from La-amino acid to Da-amino acid (from Da-amino acid to the corresponding La-amino acid, respectively); the compounds 1, 2 and 3 then represent connections with the L configuration (D configuration respectively) and the compounds 4, 5, and 6 connections with the D configuration (L configuration respectively). R stands for an amino acid residue group as defined above.

For the sake of clarity, the figure is illustrated by the preparation of D-α-amino acid from the corresponding L-α-amino acid / N-35 carbamoyl-L-α-amino acid in a one-pot reaction in the presence of a -6-hydantoinase and a D-carbamoylase. The operation of the invention for the preparation of Da-amino acid from the corresponding La-amino acid / N-carbamoyl-La-amino acid is analogous to that of the preparation of La-amino acid from the corresponding Da-amino acid / N-carbamoyl-Da- amino acid. For the preparation of La-amino acid from the corresponding Da-amino acid / N-carbamoyl-Da-amino acid, the explanation can be read by replacing the D for all configurations by the L and vice versa and by instead of D-carbamoylase, L-carbamoylase to read.

The preparation of D-α-amino acid from the corresponding L-α-amino acid / N-carbamoyl-L-α-amino acid is shown in the reaction from 1 to 2.

The conversion of N-carbamoyl-La-amino acid to the corresponding Da-amino acid is shown in the reactions from 2 to 6. The N-carbamoyl-La-amino acid (2) can be used by the hydantoinase present in the one-pot reaction (b ) are converted to the corresponding L-5 substituted hydantoin (3). The L-5 substituted hydantoin is then racemized in situ (reaction a) to D, L-5 substituted hydantoin (3 and 4). Racemization of L-5 substituted hydantoin can occur spontaneously, but can also be effected, for example, with the aid of heating or a hydantoin racemase. The resulting racemic mixture of D, La amino acid hydantoin (3 and 4) is then (according to the method described in EP 0 775 748 A2) converted into the corresponding Da amino acid (6), making use of the presence of a hydantoinase (b) and a D-carbamoylase (c).

The invention is illustrated below with reference to the following examples without, however, being limited thereto.

Examples

EXAMPLE 1

To 100 ml of water was added 17.4 g (0.10 mol) of N-carbamoyl-L-leucine, after which the pH was adjusted to 7.2 with the aid of 10 N NaOH. After this the volume of water was replenished to 150 ml. This mixture was heated to 40 ° C. Subsequently, under a nitrogen atmosphere, the enzymatic reaction was started by adding 18 g of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by titration with 3 mol / l H3 PO4. The composition of the reaction mixture was monitored by HPLC-35 analysis. After a reaction time of 43 hours, a D-leucine concentration of 67 g / l -7- was measured, which corresponds to a conversion of> 95% based on the N-carbamoyl-L-leucine. The enantiomeric excess of the D-leucine formed was> 98%.

Example II

A slurry of 17.9 g (0.093 mol) of N-carbamoyl-D, L-methionine in 115 ml of water was transferred to a double-walled glass reactor. The mixture was heated to 40 ° C and adjusted to pH 7.2 with 10 N NaOH, after which the mixture was made up to a volume of 170 ml. After this, 12 g of an Agrobacterium radiobacter cell suspension was added, after which the pH of the reaction mixture was kept constant at pH 7.2 by the addition of 3 mol / l of H3 PO4. The reaction was conducted under a nitrogen atmosphere. The composition of the reaction mixture was analyzed by HPLC analysis. After a reaction time of approximately 29 hours, a conversion of> 88% based on N-carbamoyl-D, L-15 methionine was achieved. The enantiomeric excess of the D-methionine formed was> 96%.

EXAMPLE III

To 100 ml of water was added 24.0 g (0.12 mol) of N-carbamoyl-L-methionine, after which the pH was adjusted to 7.2 with the aid of 10 N NaOH. After this the volume of replenished water to 170 ml. This mixture was heated to 40 ° C. Subsequently, under a nitrogen atmosphere, the enzymatic reaction was started by adding 14.2 g of an Agrobacterium radiobacter cell suspension. The pH of the reaction mixture was kept constant at pH 7.2 by titration with 3 mol / l H3 PO4. The composition of the reaction mixture was monitored by HPLC analysis. After a reaction time of 44 hours, the reaction was stopped. The results of this experiment are shown in Figure 2, with the conversion (c) in percent as a function of time in hours (t (h)). FIG. 2 shows that after more than 40 hours of reaction a conversion of approximately 65% based on N-carbamoyl-L-methionine (S) has been achieved. The enantiomeric excess of the D-methionine (P) formed was 97.8%.

EXAMPLE IV

To 300 ml of water was added 66 µg (0.50 mol) of L-leucine and 44.0 g (0.525 mol) of KOCN. The mixture was stirred at room temperature under a nitrogen atmosphere overnight. It was determined by HPLC that the -8 conversion was> 99%. The volume of this solution was 350 ml. After cooling the reaction mixture, 70 ml of this mixture was brought to pH 7.2 by using. concentrated phosphoric acid and heated to 40 ° C under a nitrogen atmosphere. The reaction mixture was made up with water to a volume of 150 ml. After this, 19 g of an Agrobacterium radiobacter cell suspension was added, after which the pH of the reaction mixture was kept constant at pH 7.2 by the addition of 3 mol / l of H3 PO4.

The composition of the reaction mixture was analyzed by HPLC analysis. After a reaction time of approximately 42 hours, a D-leucine concentration of 67 g / l was measured, which corresponds to a conversion of> 95% on the basis of the N-carbamoyl-L-leucine. The enantiomeric excess of the D-leucine formed was> 98%.

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Claims (10)

Method for preparing an enantiomerically enriched α-amino acid in a one-pot reaction in the presence of a hydantoinase and an enantioselective carbamoylase, characterized in that an N-carbamoyl-La-amino acid or N-carbamoyl-Da-amino acid is converted to the corresponding Da-amino acid or La-amino acid in the presence of a D-carbamoylase or L-carbamoylase, respectively. 2. A method according to claim 1, characterized in that any mixture of N-carbamoyl-D-a-amino acid and N-carbamoyl-L-a-amino acid is converted into the corresponding D-a amino acid or into the corresponding L-a amino acid. 3. A method according to any one of claims 1-2, characterized in that the N-carbamoyl-La-amino acid or N-carbamoyl-Da-amino acid is prepared from the corresponding La-amino acid or the corresponding Da-amino acid (or the corresponding random mixture of La amino acid and Da amino acid). 4. Method according to claim 3, characterized in that the preparation of D-a-amino acid from the corresponding L-a-amino acid or the preparation of L-a-amino acid from the corresponding D-a-amino acid is carried out in one pot. Method according to one of claims 1 to 4, characterized in that the D-carbamoylase or the L-carbamoylase, respectively, is at least 99% selective. 6. A method according to any one of claims 1-5, characterized in that the conversion takes place in the presence of a hydantoin racemase. Method according to any of claims 1-6, characterized in that the conversion is carried out at a pH between 6.0 and 7.5. A method according to any one of claims 1-7, characterized in that the conversion is carried out at a temperature between 35 and 45 ° C. A method according to any one of claims 1-7, characterized in that one or more enzymes derived from Aqrobacterium radiobacter are used for the conversion. The method according to any of claims 1-8, characterized in that the enantiomerically enriched α-amino acid is enantiomerically enriched leucine or methionine.