A precursor of Lotrafiban ( a potent non-peptidic antagonist that inhibits platelet aggregation). The S enantiomer is necessary for activity.Hydrolysis of racemic W22-Acetate by Lipase Novozyme 435 results in only the S isomer product
Title: Lipase catalysed resolution of the Lotrafiban intermediate 2,3,4,5-tetrahydro-4-methyl-3-oxo-1 H-1,4-benzodiazepine-2-acetic acid methyl ester in ionic liquids: comparison to the industrial t-butanol process Roberts NJ, Seago A, Carey JS, Freer R, Preston C, Lye GJ Ref: Green Chem, 6:475 , 2004 : PubMed
The Candida antarctica lipase B (Novozyme 435) catalysed resolution of2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid methyl ester (SB-235349), a key Lotrafiban intermediate, has been investigated in six ionic liquids including [BMIM][PF6] and [BMIM][N(SO2CF3)2]. The initial rate and final yield of the reaction have subsequently been determined in [BMIM][PF6] as a function of initial substrateconcentration (5-40 g L-1), temperature (25-100 oC) and initial water content (3-15% H2O w/w). In each case the results have been compared to those obtained for the optimised industrial process operated in t-butanol (88% v/v). Simply replacing the organic solvent with an ionic liquid under otherwise identical reaction conditions reduced the rate of conversion. However, exploiting the increased solubility of the substrate in ionic liquids and the ability to operate at higher temperatures increased the overall rate of reaction by a factor of four while maintaining the same overall yield of 47%. In each case the ee of the product was 99%. Further experiments demonstrated the ability to re-use the enzyme over 10 reaction cycles and suggested that solute mass transfer in ionic liquids might be an issue for reactions carried out at larger scale. Overall the results suggest that ionic liquids can be very favourable reaction media for industrial bioconversion processes, which also overcome many of the safety and environmental concerns of conventional organic solvents.
BACKGROUND:
A novel bacterial esterase that cleaves esters on halogenated cyclic compounds has been isolated from an Alcaligenes species. This esterase 713 is encoded by a 1062 base pair gene. The presence of a leader sequence of 27 amino acids suggests that this enzyme is exported from the cytosol. Esterase 713 has been over-expressed in Agrobacterium without this leader sequence. Its amino acid sequence shows no significant homology to any known protein sequence.
RESULTS:
The crystal structure of esterase 713 has been determined by multiple isomorphous replacement and refined to 1. 1 A resolution. The subunits of this dimeric enzyme comprise a single domain with an alpha/beta hydrolase fold. The catalytic triad has been identified as Ser206-His298-Glu230. The acidic residue of the catalytic triad (Glu230) is located on the beta6 strand of the alpha/beta hydrolase fold, whereas most other alpha/beta hydrolase enzymes have the acidic residue located on the beta7 strand. The oxyanion hole is formed by the mainchain nitrogens of Cys71 and Gln207 as identified by the binding of a substrate analogue, (S)-7-iodo-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1, 4-benzodiazepine-2-acetic acid. Cys71 forms a disulphide bond with the neighbouring Cys72.
CONCLUSIONS:
Despite negligible sequence homology, esterase 713 has structural similarities to a number of other esterases and lipases. Residues of the oxyanion hole were confirmed by structural comparison with Rhizomucor miehei lipase. It is proposed that completion of a functional active site requires the formation of the disulphide bond between adjacent residues Cys71 and Cys72 on export of the esterase into the oxidising environment of the periplasmic space.
