A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantioselectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an E value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently.kinetic resolution | library design | protein design | enzyme catalysis

PostprintThis is the accepted version of a paper published in Chemical Communications. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.

Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site. The small but smart libraries provide highly enantioselective epoxide hydrolase or lipase mutants, respectively.

An efficient one-pot synthesis of N-heteroaryl iodonium triflates from the corresponding N-heteroaryl iodide and arene has been developed. The reaction conditions resemble our previous one-pot syntheses, with suitable modifications to allow N-heteroaryl groups. The reaction time is only 30 min, and no anion exchange is required. The obtained iodonium salts were isolated in a protonated form, these salts can either be employed directly in applications or be deprotonated prior to use. The aryl groups were chosen to induce chemoselective transfer of the heteroaryl moiety to various nucleophiles. The reactivity and chemoselectivity of these iodonium salts were demonstrated by selectively introducing a pyridyl moiety onto both oxygen and carbon nucleophiles in good yields.

A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His6-tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild-type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested.