Candida antarctica Lipase A-Based Enantiorecognition of a Highly Strained 4-Dibenzocyclooctynol (DIBO) Used for PET Imaging

Abstract

The enantiomers of aromatic 4-dibenzocyclooctynol (DIBO), used for radiolabeling and subsequent conjugation of biomolecules to form radioligands for positron emission tomography (PET), were separated by kinetic resolution using lipase A from Candida antarctica (CAL-A). In optimized conditions, (R)-DIBO [(R)-1, ee 95%] and its acetylated (S)-ester [(S)-2, ee 96%] were isolated. In silico docking results explained the ability of CAL-A to differentiate the enantiomers of DIBO and to accommodate various acyl donors. Anhydrous MgCl2 was used for binding water from the reaction medium and, thus, for obtaining higher conversion by preventing hydrolysis of the product (S)-2 into the starting material. Since the presence of hydrated MgCl2·6H2O also allowed high conversion or effect on enantioselectivity, Mg2+ ion was suspected to interact with the enzyme. Binding site predictions indicated at least two sites of interest; one in the lid domain at the bottom of the acyl binding pocket and another at the interface of the hydrolase and flap domains, just above the active site.

Tuning Selectivity in CalA Lipase: Beyond Tunnel Engineering

“…Interestingly, they are in the neighboring region of two previously reported substitutions at residues V290 and V286 at the bottom of the tunnel (Figure S10), which also decrease hydrolysis of fatty acids longer than C18 . This reinforces the impact of that region in modifying fatty ester binding, It should be noted that Mg 2+ has been proposed to bind near residue N294, which could have indirect effects on the reaction rate when mutating this residue …”

“… 13 This reinforces the impact of that region in modifying fatty ester binding, It should be noted that Mg 2+ has been proposed to bind near residue N294, which could have indirect effects on the reaction rate when mutating this residue. 76 …”

“…13 This reinforces the impact of that region in modifying fatty ester binding, It should be noted that Mg 2+ has been proposed to bind near residue N294, which could have indirect effects on the reaction rate when mutating this residue. 76 Long-Chain Selective Variant LS_66: Epistasis between Two Gatekeeper Residues Disfavors the Hydrolysis of Short-Chain Substrates. Selectivity for long-chain over short-chain substrates has been more difficult to engineer in CalA than the inverse.…”