Process for preparing non-proteinogenic L-amino acids

Details Process for preparing non-proteinogenic L-amino acids Process for preparing non-proteinogenic L-amino acids

2002-03-22

2003-06-17

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S232000

Reexamination Certificate

active

06579705

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a process for preparing non-proteinogenic L-amino acids by means of enzymic biotransformation.
2. The Prior Art
Non-proteinogenic amino acids are amino acids which are not used in nature as building blocks for protein biosynthesis and are thereby to be clearly demarcated from the 20 proteinogenic amino acids. Within the meaning of the present invention, the very rare amino acid L-selenocysteine, which does indeed occur in proteins, is classified among the non-proteinogenic amino acids.
Non-proteinogenic amino acids constitute interesting compounds, for example for producing pharmaceutical and agricultural active compounds. They are able, as an active compound or as part of an active compound, to imitate the structure of natural amino acids in a type of molecular mimicry and thereby modulate the natural reaction, for example in the case of receptor interactions. Furthermore, as chiral compounds, they can, quite generally, serve as synthetic building blocks within the context of the “chiral pool”.
Present methods for preparing non-proteinogenic amino acids in enantiomerically pure form are for the most part based on syntheses which are elaborate and which, furthermore, usually only permit access to one particular compound. Only a few methods enable different compounds to be prepared by simply replacing a starting compound. In most cases, the syntheses are chemical syntheses which, for their part, usually already proceed from chiral building blocks. Other methods combine the chemical synthesis of racemates with a racemate resolution which is frequently carried out enzymically.
In addition, some enzymic methods which make use of prochiral compounds and which enable non-proteinogenic amino acids to be synthesized stereoselectively have also been described. Thus, it is possible to employ transaminases to prepare various non-proteinogenic amino acids from &agr;-keto acids using L-glutamic acid as the amino donor (Taylor et al. 1998,
TIBTECH
16: 412-418). Another example is the synthesis of L-tert-leucine using leucine dehydrogenase (Drauz 1997,
Chimia
51: 310-314).
The patent application DE 10046934 (registered on 21.09.2000 by the same applicant) describes a particularly simple method for preparing non-proteinogenic amino acids by means of direct fermentation of microorganisms. This method uses organisms whose cysteine metabolism is deregulated and which therefore supply a high level of O-acetyl-L-serine. In cysteine metabolism, this compound serves as a biosynthetic precursor of L-cysteine. The latter is formed by substituting the acetate group at the &bgr; position with a thiol radical. This reaction, which is termed &bgr; substitution, is catalyzed by enzymes of the O-acetyl-L-serine sulfhydrylase class [EC 4.2.99.8]. When nucleophilic substances belonging to particular compound classes (thiols, azoles and/or isoxazolinones) are fed in during the fermentation, these compounds enter into &bgr; substitution, thereby achieving the production of non-proteinogenic L-amino acids. The structure of the respective radicals of the amino acids which are prepared are thus dictated by the nucleophilic compound which is supplied.
A problem with this method is that the nucleophilic compounds which are fed in should not be metered in at too high a rate since, otherwise, the compound itself, or the resulting amino acid, can elicit toxic effects on the metabolism of the microorganisms. This applies, in particular, to many thiol compounds since, as redox-active substances, they possess toxicity at higher concentrations. Furthermore, it is very problematical to use thiol compounds in fermentative methods because, when the fermenter is intensively aerated, they tend toward oxidation and, without mechanical provisions, cause a significant degree of obnoxious odor. Because of their high toxicity, it is not possible, either, to feed in azide or cyanide, which are known to enter into &bgr; substitution when O-acetyl-L-serine sulfhydrylases are used (Flint et al., 1996,
J. Biol. Chem.
271: 16053-16067).
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a process for preparing non-proteinogenic L-amino acids, which process makes it possible to use nucleophilic compounds, in particular toxic compounds as well, which are metered in at a high rate.
This object is achieved according to the present invention by means of an enzymic biotransformation method in which O-acetyl-L-serine is reacted with a nucleophilic compound, while using an O-acetyl-L-serine sulfhydrylase as catalyst, to give a non-proteinogenic L-amino acid, which comprises the process being carried out at a pH in the range between pH 5.0 and 7.4.
This process makes it possible to synthesize a large number of non-proteinogenic, enantiomerically pure L-amino acids, some of which are novel, on an industrial scale.
O-Acetyl-L-serine sulfhydrylases are known. They have so far been isolated from a very wide variety of plants and microorganisms. Those which have been investigated to the greatest extent are the corresponding bacterial enzymes isolated from
Salmonella typhimurium.
In this organism, there are two O-acetyl-L-serine sulfhydrylase enzymes, which are designated OASS-A and OASS-B, respectively. The pertinent genes are likewise known and are termed cysK and cysM, respectively. Although the two enzymes possess very similar reaction mechanisms, they only exhibit 45% identity on the basis of their amino acid sequences.
OASS-B (CysM) has been reported to be able, in contrast to OASS-A (CysK), to catalyze a reaction of O-acetyl-L-serine with thiosulfate to give S-sulfocysteine. This reaction plays an important role in the growth of the bacteria when thiosulfate is the sole sulfur source.
Furthermore, comparisons of the sequences of O-acetyl-L-serine genes from various organisms indicate that there are two phylogenetic groups (Kitabatake et al., 2000,
J. Bacteriol.
182: 143-145).
Salmonella typhimurium
CysK and
Escherichia coli
CysK form a large group together with the O-acetyl-L-serine sulfhydrylases from other Eubacteria, from methanogenic Archaea and from plants. By contrast,
Salmonella typhimurium
CysM and
Escherichia coli
CysM are located in a very small family together with the O-acetyl-L-serine sulfhydrylases from hyperthermophilic Archaea (e.g. Pyrococcus, Sulfolobus and Thermoplasma).
Within the meaning of the present invention, O-acetyl-L-serine sulfhydrylases are distinguished by the fact that they are able to catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. Both CysM-related and CysK-related enzymes are therefore O-acetyl-L-serine sulfhydrylases within the meaning of the present invention.
Although a few publications show that O-acetyl-L-serine sulfhydrylases from the CysK group exhibit a relatively broad substrate spectrum (Ikegami & Murakoshi, 1994,
Phytochemistry
35: 1089-1104; Flint et al., 1996,
J. Biol. Chem.
271:16053-16067), the possibility of using O-acetyl-L-serine sulfhydrylases industrially for producing non-proteinogenic amino acids has not previously been considered.
The crucial reason which has previously stood in the way of using O-acetyl-L-serine sulfhydrylases to implement the enzymic preparation of non-proteinogenic amino acids industrially lies in the fact that O-acetyl-L-serine is unstable precisely in the pH range which corresponds to the activity range of the O-acetyl-L-serine sulfhydrylases.
O-Acetyl-L-serine isomerizes to N-acetyl-L-serine in dependence on the pH. The reaction is irreversible and, for example, at a pH of 7.6 extremely rapid, with rates of 1%×min
−1
. The rate of reaction falls as the pH is lowered such that the compound is stable at pH 4.0, for example. The mechanism of the reaction is based on an intramolecular, nucleophilic attack of the deprotonated amino group on the carbonyl carbon of the acyl radical (Tai et al. 1995,
Biochemistry
34: 12311-12322).
By contrast, the pH optimum of O-acetyl-L-serine sulfhydrylases lies in

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