Efficient Reaction Conditions for Dynamic Enzymatic Resolution

Identification of Efficient Reaction Conditions for Dynamic Enzymatic Resolution 

Our screening technique consisted of preparing lmL mixtures of the thioester 10, enzyme and Triton X-100 in phosphate buffer at 40 °C. Any reaction that might occur was monitored by chiral HPLC [39] which indicated the presence of hydrolysis, and if so, whether it was stereoselective. While the medium was basic, we were not overly concerned whether racemization occurred at this point our goal was to find an enzyme/substrate match that resolved in high yield and eep. 

Thioester Yield1,1 Thioester Yield 1 Thioester Yield 1 Thioester Yield 1 

This study revealed that only the linear (non-branched) alkyl thioesters 10 a, b, and c (ethyl, 1-propyl, and 1-butyl) were hydrolyzed efficiently ( 24 hours) with appreciable enantioselectivity ( 85%eep) for some enzymes [41]. The presence of any a or / branching led to, at best, low conversion and poor eep. This is in marked contrast to the ester 9 [13], which of course possesses a / methyl group and indicated that the demands of the enzyme s active site are stricter for thioesters as compared with esters. The reaction rate and eep for all three linear alkyl thioesters were so good that we decided to forego further optimization of the choice of substrate at that time. We felt that the choice of enzyme and thioester class was sufficient for the first pilot-plant run. 

Concurrent with the above research, we were considering reaction conditions for racemization. An initial test experiment based on Prof. Drueckhammer s conditions [26] convinced us that the search for acceptable racemizing conditions would not be arduous. A chiral thioester was prepared from (R)-l with thionyl chloride followed by 2-butyl mercaptan (94% yield). When this was mixed with toluene, l,8-diazabicyclo[5.4.0]undec-l-ene and Triton X-100 in phosphate buffer at pH 9.2, the heterogeneous mixture racemized completely in only 25 min. While the substrate had also degraded, such rapid racemization convinced us that we would be able to discover optimized conditions in a reasonable time period. 

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