Pig liver esterases PLE1 and PLE6: heterologous expression, hydrolysis of common antibiotics and pharmacological consequences

Carboxylesterases, historically referred as non-specific esterases, are ubiquitous hydrolases with high catalytic efficiency. Without exceptions, all mammalian species studied contain multiple forms of carboxylesterases. While having been widely studied in humans and experimental animals, these enzymes remain to be characterized in farm animals. In this study, we showed that pig liver esterase 1 (PLE1) and pig liver esterase 6 (PLE6) were highly active toward amoxicillin (AMO) and ampicillin (AMP), two major antibiotics that are widely used in food-supplements. Mass-spectrometric analysis established that the hydrolysis occurred at the β-lactam amide bond and the hydrolysis drastically decreased or completely eliminated the antibacterial activity. Furthermore, hydrolytic activity and proteomic analysis suggested that trace PLEs existed in pig plasma and contributed little to the hydrolysis of AMO and AMP. These results suggested that carboxylesterases-based hydrolysis determines the therapeutic intensity of these and related antibiotics and the magnitude of the determination occurs in a species-dependent manner.

Developing Multicompartment Biopolymer Hydrogel Beads for Tandem Chemoenzymatic One-Pot Process

Chemoenzymatic processes have been gaining interest to implement sustainable reaction steps or even create new synthetic routes. In this study, we combined Grubbs’ second-generation catalyst with pig liver esterase and conducted a chemoenzymatic one-pot process in a tandem mode. To address sustainability, we encapsulated the catalysts in biopolymer hydrogel beads and conducted the reaction cascade in an aqueous medium. Unfortunately, conducting the process in tandem led to increased side product formation. We then created core-shell beads with catalysts located in different compartments, which notably enhanced the selectivity towards the desired product compared to homogeneously distributing both catalysts within the matrix. Finally, we designed a specific large-sized bead with a diameter of 13.5 mm to increase the diffusion route of the Grubbs’ catalyst-containing shell. This design forced the ring-closing metathesis to occur first before the substrate could diffuse into the pig liver esterase-containing core, thus enhancing the selectivity to 75%. This study contributes to addressing reaction-related issues by designing specific immobilisates for chemoenzymatic processes.

Preparation of Sterically Demanding 2,2-Disubstituted-2-Hydroxy Acids by Enzymatic Hydrolysis

Preparation of optically-pure derivatives of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid of general structure 2 was accomplished by enzymatic hydrolysis of the correspondent esters. A screening with commercial hydrolases using the methyl ester of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid (1a) showed that crude pig liver esterase (PLE) was the only preparation with catalytic activity. Low enantioselectivity was observed with substrates 1a–d, whereas PLE-catalysed hydrolysis of 1e proceeded with good enantioselectivity (E = 28), after optimization. Enhancement of the enantioselectivity was obtained by chemical re-esterification of enantiomerically enriched 2e, followed by sequential enzymatic hydrolysis with PLE. The preparation of optically-pure (S)-2e was validated on multi-milligram scale.

The Li Synthesis of Daphenylline

The genus Daphniphyllum consists of 25–30 species of evergreen trees and shrubs of south Asia. The leaves and roots are widely used in Chinese herbal medicine. About 250 alkaloids, many with complex polycyclic structures, have been isolated from these species. Of these, daphenylline 3 is unique in incorporating a benzene ring. Ang Li of the Shanghai Institute of Organic Chemistry envisioned (Nature Chem. 2013, 5, 679) a route to 3 based on the diastereoselective intramolecular Michael cyclization of 1 to 2. Following the work of Piers (J. Org. Chem. 1996, 61, 8439), the preparation of 1 began with the Birch reduction of 4, followed by hydrolysis. Epoxidation followed by elimination and acetylation led to the racemic acetate 5. Hydrolysis with pig liver esterase left one enantiomer of the acetate, that was transesterified with methoxide to give 6 in high ee. Mitsunobu coupling of 6 with the o-nitrobenzenesulfonamide 7 gave 8. After some experimentation, selective α¢-silylation was effected with TBDPSOTf, setting the stage for gold-catalyzed Conia cyclization to 9. Deprotection of the amine fol­lowed by acylation with 10 gave 1, that cyclized smoothly to 2 as a 10:1 ratio of diastereomers. The arene of 3 was constructed by converting 2 into the corresponding vinyl tri­flate. Pd-mediated coupling with 11 gave 12. Under irradiation with strict exclusion of oxygen, 12 cyclized to the dihydro aromatic, that on warming with DBU in the pres­ence of air was oxidized to 13. To close the last ring of 3, the ketone 13 was further oxidized to the enone 14. Desilylation of 14 followed by exposure to Ph3P/I2 gave the iodide 15, that was cyclized under reductive free radical conditions to 16. The hydrogenation of 16 under Pd catalysis delivered the incorrect diastereomer, perhaps because migration to the endocyclic alkene preceded reduction. This problem was solved by using the Crabtree Ir catalyst. Modified Krapcho decarbomethoxylation then gave 17, that was reduced to daphenylline 3.