Biocatalytic Syntheses of Antiplatelet Metabolites of the Thienopyridines Clopidogrel and Prasugrel Using Fungal Peroxygenases

Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y12 receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL−1 of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h−1 H2O2 and 20% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS2 spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays.

Tricyclic Derivative of Acyclovir and Its Esters in Relation to the Esters of Acyclovir Enzymatic Stability: Enzymatic Stability Study

The 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-6-(4-methoxyphenyl)-9-oxo-5H-imidazo[1,2-a]–purine (6-(4-MeOPh)-TACV) was selected to assess the enzymatic stability of the tricyclic acyclovir derivatives from the imidazo[1,2-a]-purine group. The parent compound and its esters (acetyl, isobutyryl, pivaloyl, nicotinic, ethoxycarbonyl) were subjected to kinetic studies and compared with the stability of analogous acyclovir (ACV) esters. The enzymatic hydrolysis was observed in vitro in a medium of 80% human plasma in the absence and presence of porcine liver esterase (PLE). The tests were carried out at 37 °C. To determine the kinetic parameters (kobs., t0.5) of the observed reaction, the validated HPLC-UV method in the reversed phase was used. The HPLC-MS/MS method was used to identify the degradation products under the tested conditions. In summary, it was found that 6-(4-MeOPh)-TACV esters are more susceptible to esterase metabolism than ACV esters. It was confirmed by HPLC-MS/MS that in the plasma, the main product of their hydrolysis is 6-(4-MeOPh)-TACV and not ACV, which confirms that their antiviral activity observed in vitro does not result from ring degradation.

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.

Review: Peran Enzim dalam Meningkatkan Kualitas Kopi

Indonesia is the third largest coffee producing nation in the world after Brazil and Vietnam. The types and the characteristics of Indonesian coffee are different in each area but the main important factor of consumer acceptance depends on its bitterness level. Chlorogenic acid lactone is a compounds that play an important role as contributor to the coffee bitterness that are formed during the roasting process of coffee bean because of its precursor chlorogenic acid. Chlorogenic acid is commonly found in many plants. One of them can be found in coffee with high concentration. Chlorogenic acid lactone (bitter compound) can be hydrolyzed to chlorogenic acid (non-bitter compound) using hog liver esterase and chlorogenate esterase. This study aimed to analyze the use of these enzymes to decrease the level of bitterness in coffee. The results indicated that HLE and chlorogenate esterase effectively hydrolyzed chlorogenic acid lactones in coffee. Based on the sensory test, coffee extracts treated with enzymes were less bitter than the untreated coffee extracts. If it was associated with Indonesian local coffee then the method can be done with chlorogenate esterase that was in accordance with the legal guarantee of halal product.