Bioprocess Technology for Synthesis of Chiral Compounds

Summary

Commercial routes to industrially-important pharmaceutical and agrichemical compounds can often be developed more economically when separation of enantiomeric intermediates/products is possible. Vanderbilt University seeks to license technologies, originally invented at DuPont, that allow such separations to be performed via novel biocatalysts. Vanderbilt's technology can be used for the production of chiral tertiary esters and/ or enantiomeric amides. In the case of the former class of compounds, our technology is somewhat unique in its ability to operate on carbonyl groups alpha to a tertiary center. See the following description for more information about the current status of this technology and the associated patent estate.

Licensee(s) with an interest in expanding their enzyme biocatalyst 'toolbox' are sought. A potential commercialization scenario could include licensee development of the technology from its current state through the production of testing/marketing quantities of cloned, expressed protein. Vanderbilt's laboratories and expertise could then be used to screen for separation efficacy against a variety of agreed-upon compounds or classes of compounds.

The final step in this scenario would, of course, include licensee sales and marketing of the new biocatalyst(s) as a complement to current product offerings.

The patents available for license teach a methodology for the selection and enrichment of R or S enantiomers from a racemic mixture. Over four hundred potential sources of biocatalysts were evaluated to perform the enantiospecific reactions.

The enrichment of enantiomers of α tertiary carboxylic acid esters (US Patent Number 5580783) makes use of enzymes obtained from microorganisms originally found in soil samples. More than 400 sources of enzymes were evaluated for stereospecificity and stereoselectivity, those that yielded an enantiomeric excess (E value) for the desired R or S form of greater than 50%. The enzymes were classified as most preferred (E>50%), more preferred (E>90%), even more preferred (E>95%) and most preferred (E>99%). The most preferred enzymes came from the following microorganisms: Rhodococcus rhodochrous, Tricophyton concetricum, Beauveria bassiana and Beauveria nivea. Biocatalysis is achieved using whole cell preparations or enzyme preparations (partially or highly purified). Selective addition of one or more of these enzymes to the racemic mixture results in enrichment of the remaining R (or S) carboxylic acid ester. This enriched product is separated from the unwanted products (R (or S) carboxylic acid or the R or S hydroxyl acid ester) by chiral HPLC, using glycoprotein columns. Thismethod can also be used to enrich for R (or S) α tertiary carboxylic acids or R (or S) hydroxy acid esters by selective biocatalysis of the corresponding mixture of enantiomers of α tertiary carboxylic acid esters.

The enrichment of 2-Alkanoic Acid Amides from nitriles (US Patent Number 5593871) makes use of nitrile hydratase enzymes that preferentially hydrolyze only one enantiomer of a racemic mixture of nitriles. Microorganisms representing three different genera (Pseudomonas, Moraxella and Serratia) were isolated from a soil sample. The strains were selected for their ability to grow on a nitrile enriched growth media. The strains were then grown and harvested such that subsequent biocatalytic experiments were performed with a cell paste. The cell pastes were then assessed in terms of stereospecificity and stereoselectivity. Stereospecific enzymes were those that yielded an enantiomeric ratio (E) for the desired enantiomer of greater than 7, while stereoselective enzymes had ratios of E less than 7. Enzymes were classified as preferred (E value greater than 8.5) or most preferred (E greater than 10).

These nitrile hydratase enzymes convert the racemic nitrile mixture, in one step, into one made of the R (or S) form of the nitrile and the R (or S) form of the amide -- two compounds that are easily separated due to their distinct chemical and physical properties.

Estimates for the chiral intermediates market (FROST) is $875.1 million, with an anticipated CAGR of 7.5%.

Patents are held in Germany, Japan, United States, Israel, Great Britain, France and Switzerland for carboxylic acid ester technology. Nitrile compound technology is protected in Austria, Belgium, Denmark, France, Germany, Great Britain, Italy, Japan, Netherlands, Spain, Switzerland and United States.

U.S. Patent # 5,580,783: Enzymatic process for the preparation of chiral alpha-tertiary carboxylic acid esters

U.S. Patent # 5,593,871: Process for the preparation of enantiometric 2-alkanoic acid amides from nitriles