Biocatalytic synthesis of oxadiazole thioethers and evaluation of their antitumor activity.

A multicomponent enzyme-catalyzed process is suggested for the synthesis of a novel series of 1,3,4-oxadiazole thioether derivatives with yields ranging from 65 to 94%. Novozym 435, the immobilized form of Candida antarctica lipase B (CALB), was found to efficiently catalyze the reaction. The products were evaluated for antitumor activities against two cancer cell lines, HT-29 (human colorectal cancer cell) and HepG2 (human liver cancer cell), by MTT assays. Among them, two compounds exhibited higher antitumor activities, for both cell lines, compared to doxorubicin. In silico molecular docking and computational ADME analysis were performed to propose a mode of action for the anti-cancer activities and to predict drug-likeness, respectively.

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Biocatalytic stereoselective synthesis of pyrrolidine-2,3-diones containing all-carbon quaternary stereocenters.

Highly functionalized pyrrolidine-2,3-diones can be synthesized efficiently and stereoselectively under mild conditions using a biocatalytic approach. The reaction led to the formation of new all-carbon quaternary stereocenters from Myceliophthora thermophila laccase (Novozym 51003) catalyzed oxidation of catechols to ortho-quinones and subsequent 1,4-addition with 3-hydroxy-1,5-dihydro-2H-pyrrol-2-ones. The reaction was conducted with various substituents on both reactants, resulting in 13 products in moderate to good yields (42-91%). The same 15 reactions were also tested with K3Fe(CN)6 as a catalyst, but here only one reaction resulted in a product (60% yield).

Diarylation of thiazolopyrimidines by laccase and their in vitro evaluation as antitumor agents.

A mild and efficient method was developed for the synthesis of new derivatives of thiazolo[3,2-a] pyrimidin-3(2H)-ones from available starting materials based on the oxidation of catechols to ortho-quinone by Myceliophthora thermophila laccase (Novozym 51,003) and 1,4-addition of active methylene carbon to these in situ generated intermediates in moderate to good yields (35-93%). The structure of the products was confirmed through 1H NMR, 13C NMR, HMBC, HSQC, DEPT-135, and mass spectroscopy techniques. These novel compounds were evaluated as active antitumor agents against human colorectal adenocarcinoma and liver adenocarcinoma cell lines. All compounds displayed potent inhibition activities against the HT-29 cell line with IC50 values of 9.8-35.9 µM, superior to the positive control doxorubicin, and most showed potent anticancer activities against the HepG2 cell line.

A multi-component approach for co-immobilization of lipases on silica-coated magnetic nanoparticles: improving biodiesel production from waste cooking oil.

The capability of multi-component reactions in rapid immobilization of enzymes was considered for co-immobilization of Thermomyces lanuginous lipase (TLL) and Candida antarctica lipase B (CALB) [TLL: CALB]; Rhizomucor miehei lipase (RML) and CALB [RML: CALB] on amine-functionalized silica-coated magnetic nanoparticles (Fe3O4@SiO2-NH2). Immobilization of different ratios of lipases was performed within 3 h under mild conditions; producing specific activity ranging from 29 to 35 U/mg for TLL:CALB and 21-34 U/mg for RML:CALB. The co-immobilized derivatives showed improved co-solvent and thermal stability compared to the corresponding free enzymes. All the derivatives were also used to catalyze the transesterification of waste cooking oil with methanol to produce biodiesel (fatty acid methyl esters). Response surface method (RSM) and a central composite rotatable design (CCRD) were used to study the effects of different factors on the FAME yield. Fe3O4@SiO2-NH2-RML-CALB and Fe3O4@SiO2-NH2-TLL-CALB had maximum FAME yields of 99-80%, respectively.

Immobilization of Thermomyces lanuginosus lipase through isocyanide-based multi component reaction on multi-walled carbon nanotube: application for kinetic resolution of rac-ibuprofen.

Thermomyces lanuginosus lipase (TLL) was immobilized on epoxy functionalized hydroxyl multi-walled carbon nanotube (MWCNT-OH) via an isocyanide-based multicomponent reaction. The immobilization process was carried out in deionized water (pH 7.0) at room temperature resulting in loading of 600 mg enzyme/g of support with specific activity 16.9 U/mg. An immobilization yield of 100% was obtained with the expressed activity 60%. The immobilized preparation exhibited an increased thermal stability with 49% residual activity at 75 °C compared with 19% for the free enzyme at the same temperature. Solvent stability in a high ratio of DMSO was improved from 52% in free TLL to 75% in immobilized TLL. The immobilized preparation was used for kinetic resolution of rac-ibuprofen through esterification of ibuprofen in isooctane as solvent. The best result was obtained with ethanol at 45 °C and molar ratio of 2.5:1 ethanol:ibuprofen in 1 ml isooctane with 99% eep and E-value 300.

Biocatalytic decarboxylative Michael addition for synthesis of 1,4-benzoxazinone derivatives.

The Candida antarctica lipase B (Novozym 435) is found to catalyze a novel decarboxylative Michael addition in vinylogous carbamate systems for the synthesis of 1,4-benzoxazinone derivatives. The reaction goes through Michael addition, ester hydrolysis and decarboxylation. A possible mechanism is suggested, with simultaneous lipase-catalyzed Michael addition and ester hydrolysis. The present methodology offers formation of complex products through multi-step reactions in a one pot process under mild and facile reaction conditions with moderate to high yields (51-90%) and no side product formation. The reaction seems to be is a great example of enzymatic promiscuity.

A multi-component reaction for covalent immobilization of lipases on amine-functionalized magnetic nanoparticles: production of biodiesel from waste cooking oil.

A new approach was used for the immobilization of Thermomyces lanuginosus lipase (TLL), Candida antarctica lipase B (CALB), and Rhizomucor miehei lipase (RML) on amine-functionalized magnetic nanoparticles (Fe3O4@SiO2-NH2) via a multi-component reaction route (using cyclohexyl isocyanide). The used method offered a single-step and very fast process for covalent attachment of the lipases under extremely mild reaction conditions (25 °C, water, and pH 7.0). Rapid and simple immobilization of 20 mg of RML, TLL, and CALB on 1 g of the support produced 100%, 98.5%, and 99.2% immobilization yields, respectively, after 2 h of incubation. The immobilized derivatives were then used for biodiesel production from waste cooking oil. Response surface methodology (RSM) in combination with central composite rotatable design (CCRD) was employed to evaluate and optimize the biodiesel production. The effect of some parameters such as catalyst amount, reaction temperature, methanol concentration, water content for TLL or water-adsorbent for RML and CALB, and ratio of t-butanol (wt%) were investigated on the fatty acid methyl esters (FAME) yield.

Improvement of biodiesel production from palm oil by co-immobilization of Thermomyces lanuginosa lipase and Candida antarctica lipase B: Optimization using response surface methodology.

Candida antarctica lipase B (CALB) and Thermomyces lanuginose lipase (TLL) were co-immobilized on epoxy functionalized silica gel via an isocyanide-based multicomponent reaction. The immobilization process was carried out in water (pH 7) at 25 °C, rapidly (3 h) resulting in high immobilization yields (100%) with a loading of 10 mg enzyme/g support. The immobilized preparations were used to produce biodiesel by transesterification of palm oil. In an optimization study, response surface methodology (RSM) and central composite rotatable design (CCRD) methods were used to study the effect of five independent factors including temperature, methanol to oil ratio, t-butanol concentration and CALB:TLL ratio on the yield of biodiesel production. The optimum combinations for the reaction were CALB:TLL ratio (2.1:1), t-butanol (45 wt%), temperature (47 °C), methanol: oil ratio (2.3). This resulted in a FAME yield of 94%, very close to the predicted value of 98%.