Candida antarctica lipase B (CAL-B) exhibits remarkable enantioselectivity for various chiral sec-alcohols, and the enantioselectivity is structurally well-understood. Two substituents at the chiral center of a sec-alcohol separately bind two pockets, namely, large and medium binding pockets. It has been believed that the medium pocket is too small to accommodate a large substituent (larger than an ethyl group), and thus, bulky secalcohols bearing two large substituents have been regarded as a poor substrate for CAL-B. However, we found that CAL-B can catalyze the transesterification of N-Boc-protected rac-2-amino-1-phenylethanol (1a) enantioselectively with a moderate reaction rate. X-ray crystallography and computer modeling revealed that the rotation of the Leu278 side chain creates a space to accept the N-Boc-aminomethylene group of 1a. Moreover, a sec-alcohol substrate with less than one hydrogen atom at the γ-position from the hydroxyl group is required to achieve a moderate reaction rate. On the basis of this observation, we diversified bulky N-Boc-protected rac-2-amino-1-arylethanols for the transesterifications with high enantioselectivities (E > 200).
Most lipases resolve secondary alcohols in accordance with the "Kazlauskas rule" to give the R enantiomers. In a similar manner to other lipases, Candida rugosa lipase (CRL) exhibits R enantioselectivity towards heptan-2-ol, although the enantiomeric ratio (E) is low (E=1.6). However, unexpected enantioselectivity (i.e., S enantioselectivity, E=58) of CRL towards 4-(tert-butoxycarbonylamino)butan-2-ol, which has a similar chain length to heptan-2-ol, has been observed. To develop a deeper understanding of the molecular basis for this unusual enantioselectivity, we have conducted a series of molecular modeling and substrate engineering experiments. The results of these computational and experimental analyses indicated that a hydrogen bond between the Ser450 residue and the nitrogen atom of the carbamate group is critical to stabilize the transition state of the S enantiomer.
Both enantiomers of optically pure 4‐bromo‐3‐hydroxybutanoate, which is an important chiral building block in the syntheses of various biologically active compounds including statins, were synthesized from rac‐4‐bromomethyl‐β‐lactone through kinetic resolution. Candida antarctica lipase B (CAL‐B) enantioselectively catalyzes the ring opening of the β‐lactone with ethanol to yield ethyl (R)‐4‐bromo‐3‐hydroxybutanoate with high enantioselectivity (E>200). The unreacted (S)‐4‐bromomethyl‐β‐lactone was converted to ethyl (S)‐4‐bromo‐3‐hydroxybutanoate (>99% ee), which can be further transformed to ethyl (R)‐4‐cyano‐3‐hydroxybutanoate, through an acid‐catalyzed ring opening in ethanol. Molecular modeling revealed that the stereocenter of the fast‐reacting enantiomer, (R)‐bromomethyl‐β‐lactone, is ∼2 Å from the reacting carbonyl carbon. In addition, the slow‐reacting enantiomer, (S)‐4‐bromomethyl‐β‐lactone, encounters steric hindrance between the bromo substituent and the side chain of the Leu278 residue, while the fast‐reacting enantiomer does not have any steric clash.
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