Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Patent
1995-08-25
1999-02-09
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
536 232, 536 237, 540362, 564226, 564230, 435228, C12N 980, C07D20508
Patent
active
058692991
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the preparation of novel compounds by enzymic reactions, specifically the preparation of b- lactams particularly clavams such as clavulanic acid by the action of enzymes on appropriate precursors.
Clavulanic acid is heptane-2-carboxylic acid.
Clavulanic acid is a potent inhibitor of b-lactamase enzymes and is a compound of great clinical value since it protects b-lactamase-labile b-lactam antibiotics from degradation.
British Patent Specification No. 1 508 977 describes a clavam derivative known as clavulanic acid, which is a compound produced by Streptomyces clavulilerus ATCC 27064 or a high yielding mutant thereof.
The biosynthetic route to the important product clavulanic acid a key ingredient of the antibiotic AUGMENTIN (Trade Mark ) has been the subject of considerable study. It has been reported in EP A 0 213 914 that clavulanic acid is formed by the action of a series of enzymes on a precursor proclavaminic acid (F) ##STR1##
The earlier steps in the biosynthetic pathway have been more difficult to elucidate however the precise sequence of events has not hitherto been elucidated. Tests for assessing the utilisation of arginine and ornithine as specific precursors for clavulanic acid are described in Appl. Environ Microbiol. 1986, 52(4), 892-7.
We have now identified new intermediates and processes for their conversion into clavulanic acid. Accordingly the present invention provides a process for preparing clavulanic acid and other clavam derivatives from any one of compounds of formula (I) ##STR2## wherein R.sup.1 is ##STR3## wherein R.sup.2 =H or C.sub.1-6 alkyl or compounds of formula (II) ##STR4## wherein R.sup.1 is ##STR5## where R.sup.2 .dbd.H or C.sub.1-6 alkyl or compounds of formula (III) ##STR6## wherein R is H or OH and wherein R.sup.1 is ##STR7## where R.sup.2 .dbd.H or C.sub.1-6 allyl, using an enzyme system from Streptomyces, preferably S. clavuligerus.
By `other clavams` we mean compounds containing a (1979), 282!.
Accordingly the present invention provides new compounds useful in the preparation of clavams in particular clavulanic acid. According to the present invention there is provided a compound of formula (I): ##STR8## wherein R.sup.1 is ##STR9## wherein R.sup.2 .dbd.H or C.sub.1-6 alkyl.
We have now found that a precursor of compound (F) is a compound of formula (C): ##STR10##
Accordingly in a preferred aspect there is provided a compound (C):which is threo-3-hydroxy-5-guanidino-2-(2-oxo-azetidin-1-yl) pentanoic acid preferably (2S-3R) or derivatives thereof. ##STR11##
according to the method of J. E. Miller and J. J. Bischoff, Synthesis 1986, 777, or related methods known in the art.
According to the present invention there is also provided an enzyme having amidinohydrolase activity which is capable of removing the elements of urea from compounds bearing a guanidino group and in particular for converting (C) into (F) under suitable conditions.
Suitably the enzyme system is derived from a microorganism, in particular a species of Streptomyces. Preferably the enzyme is prepared from Streptomyces according to Example 1 described hereinafter. Alternatively, the enzyme may be produced from a recombinant source.
The enzyme may additionally be useful in converting a guanidino group into an amino group when caused to react with other substrates, for example (2S)-5-guanidino-2-(2-oxo-azetidin-1-yl)pentanoic acid (Compound B as defined below).
The enzyme having amidinohydrolase activity (hereinafter amidino hydrolyase) is obtainable from Streptomyces species, preferably Streptomyces clavuligerus. The amidinohydrolase enzyme is preferably in purified form, advantageously substantially pure form.
In a further aspect of the invention there is provided a method of preparing the amidinohydrolase by treating S. clavuligerus mycelium by centrifugation and ultrasonification followed by fractionation by ion-exchange chromatography.
In yet a further aspect of the invention there is provided a protein having amidinohydrolase activity having substantially the sequence
REFERENCES:
patent: 5118617 (1992-06-01), Ortega et al.
Brown, et al., Structures of Three Novel .beta.-Lactams isolated from Streptomyces clavuligerus, J.C.S. Chem. Comm, pp. 282-283 (1979).
Baggaley, et al., "Studies on the Biosynthesis of Clavulanic Acid, Part 4..sup.1 Synthetic Routes to the Monocyclic .beta.-Lactam Precursor, Proclavaminic Acid", J. Chem Soc. Perkin Trans. 1, pp. 1513-1520 (1990).
Baggaley, et al., "Studies on the Biosynthesis of Clavulanic Acid. Part 5..sup.1 Absolute Stereochemistry of Proclavaminic Acid, the Monocyclic Biosynthetic Precursor of Clavulanic Acid", J. Chem Soc. Perkin Trans. 1, pp. 1521-1533 (1990).
Chemical Abstracts, vol. 113, No. 3, 16 Jul. 1990, Abstract No. 18542q.
Chemical Abstracts, vol. 105, No. 21, 24 Nov. 1986, Abstract No. 187381b.
Baggaley Keith Howard
Baldwin Jack Edward
Earl Alison Jane
Edwards Jeffrey
Elson Stephen William
Kerekes Zoltan
Ketter James
King William T.
Lentz Edward T.
SmithKline Beecham p.l.c.
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