Mutant Lipase‐Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec‐Alcohols: A Thermodynamic Analysis of Enantioselectivity

Abstract

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1-phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild-type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.

Highly Focused Library‐Based Engineering of Candida antarctica Lipase B with (S)‐Selectivity Towards sec‐Alcohols

“…To obtain ( S )‐selective mutants for rac ‐ 1 a , which meant inverting the Kazlauskas rule, the stereoselective pocket of alcohols should be changed to accommodate the phenylalkanol substituents, which are larger than the ethyl group. An early study has identified W104 as the most important hot spot in the alcohol‐binding pocket of CALB for the reversed ( S )‐selectivity of sec ‐alcohols, and W104 should be replaced by smaller amino acids to enlarge the volume of the stereoselectivity pocket. According to the X‐ray structure of CALB (PDB code 1TCA, Figure A), we selected four important hydrophobic residues W104, L278, A281 and A282 (sites A, C, and B as shown in Figure B) surrounding the alcohol‐binding pocket as mutagenesis targets.…”

“…Next, the substrate range of the best ( S )‐selective mutant WB15 evolved for the model substrate rac ‐ 1 a was determined. Moreover, considering that all previous studies on ( S )‐selectivity of W104A (WB2) were carried out for the acylation reaction of racemic phenylalkanols in nonaqueous media, it is valuable to determine the stereoselectivity of W104A in the hydrolytic kinetic resolution of phenylalkanol esters. The comparison results of WT, WB2 and the best WB15 variants as the catalysts for hydrolytic kinetic resolution of a series of phenylalkanol esters are listed in Table .…”

“…Concerning the inversion of the stereoselectivity of CALB towards certain sec ‐alcohols, Hult and coworkers reported an important CALB mutation, W104A, which was obtained by a rational design and being situated in the alcohol‐binding pocket, was able to accommodate much larger groups than the wild‐type (WT) CALB, and this change transformed the highly ( R )‐selective WT into an ( S )‐selective mutant . However, the enantioselectivity of W104A was unsatisfactory, the E values were low (2< E <12) for some typical sec ‐alcohols, such as phenylethanol, phenylpropanol and 2‐hexanol, in their acylation reactions, although these E values could be increased by an appropriate choice of solvents in combination with a higher temperature and longer alkyl chains of sec ‐alcohols . Thus, it is highly desirable to explore other mutations at the W104 hot spot or supplementary mutations at other important sites surrounding the alcohol‐binding pocket to obtain CALB mutants with higher ( S )‐selectivity than W104A.…”

Customizing the Enantioselectivity of a Cyclohexanone Monooxygenase by a Strategy Combining “Size‐Probes” with in silico Study

“…Some examples revealed that there was an interesting relationship between the size of substrates and the reshaped active pockets of enzymes . Hult and coworkers reported that the mutant W104A of Candida antarctica lipase B had increased enantioselectivity toward various secondary alcohols with increasing chain length of the alkyl moiety on the chiral central atom . It would be expected that in the restricted space of the reshaped pocket, an array of compounds with the increasing chain length would face the decreasing degrees of freedom.…”

Preparation of pharmaceutically important enantiomer components through lipase catalysed acylation and hydrolysis

“…It is known, that the temperature can influence both the E and the reaction rate of a lipase-catalysed reaction. 136,137 Therefore, reactions were performed at 2, 25 and 30 °C. The E and the reaction rate increased slightly with increasing temperature.…”