Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1997-05-29
2000-03-07
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435190, C12D 762
Patent
active
060338822
DESCRIPTION:
BRIEF SUMMARY
This invention relates to chiral synthesis; more particularly, it relates to a broad specificity 2-hydroxy carboxylic acid dehydrogenase initially obtained from Lactobacillus delbrueckii ssp. Bulgaricus, but subsequently cloned, to sequence variants thereof and to the use of such enzymes as catalysts capable of preparation of the (R)-isomer at the 2-hydroxy group.
As will be appreciated, there are several aspects to the present invention. Firstly, the present enzyme was isolated from the above source by conventional means, but it is conveniently over-produced by cloning in an E. coli system, for example. Secondly, although the present enzyme, however obtained, is useful in the production of (R)-2-hydroxy acids, sequence variants thereof modified to supress inhibition of the enzyme activity by substrate offer further advantages. Such may be obtained by site directed mutagenesis. Thirdly, the present enzyme and sequence variants thereof may be used to catalyse the production of (R)-hydroxy derivatives of 2-keto acids, and hence esters, for example, which are unsubstituted in the 3-position, but may be substituted in the 4-position or beyond by a wide range of substituents including aliphatics, aromatics, heterocyclics and nitrogen-containing moieties.
In one embodiment, the present invention provides an enzyme, a 2-hydroxy carboxylic acid dehydrogenase, characterised in that it is obtainable from Lactobacillus delbrueckii ssp. Bulgaricus and in that it catalyses the production of (R)-hydroxy derivatives of 2-keto acids, which are unsubstituted in the 3-position, but which may be substituted in the 4-position or beyond.
In another embodiment, the present invention provides a process for the production of such an enzyme characterised in that it comprises isolation from Lactobacillus delbrueckii ssp. Bulgaricus or cloning.
In a further embodiment, the present invention provides a sequence variant of such an enzyme, which may be produced by such a process, characterised in that it has been modified so as to supress inhibition of the enzyme activity by substrate. For example, in one preferred illustration, Histidine 206 may be replaced by glutamine.
In yet another embodiment, the present invention provides a process for the production of such a variant characterised in that it comprises site directed mutagenesis.
In still another embodiment, the present invention provides a process for the production of a (R)-hydroxy derivative of a 2-keto acid characterised in that it comprises contacting the acid with such an enzyme or a sequence variant thereof, which may be produced by such processes. Preferably, the conditions, such as pH, are modified so as to provide maximal rate.
By way of illustration, the present invention, or even LDH obtained from Staphylococcus epidermidis and other sources, may be applied to the preparation of 3-cyclopentyl-2(R)-hydroxypropanoic acid.
Having indicated the nature and scope of the present invention, it will now be described in more detail.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 illustrates what is believed to be the active site of the 2-hydroxy carboxylic acid of the present invention. FIG. 2 graphically illustrates a comparison of H206Q and variants of H206, showing that the Vmax for H206Q increased 8-fold.
The synthesis of chiral 2-hydroxy acids is of considerable importance. These compounds are versatile synthetic intermediaries that may be converted to a variety of compounds with retention of chirality at C-2. These include epoxides, alkyl esters, hydrazinyl esters, .alpha.-N-alkoxyamino esters and .alpha.-amino esters. Reactions involving nucleophilic substitution at the 2-position are optimally effected by way of the corresponding 2-triflate esters that are generated in situ and reacted directly with the chosen nucleophile.
The availability of chiral 2-hydroxy acids and esters possessing an additional prochiral functional group in the side-chain offers potential for the synthesis of compounds containing two or more chiral centres. The hydroxyl group at C-2 may be expected to pr
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Hateley Martin John
Holbrook Joseph John
Johnsen Keyji
Willis Christine Louise
Genzyme Corporation
Patterson Jr. Charles L.
Tegfeldt Jennifer
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