RESUMEN
(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6â Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3â mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.
Asunto(s)
Oxigenasas , Podofilotoxina , Oxigenasas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , Especificidad por SustratoRESUMEN
In this study, we report the identification of a new shikonin-phenoxyacetic acid derivative, as an inhibitor of tubulin. A series of compounds were prepared; among them, compound 16 [(R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-(4- phenoxyphenyl) acetate] potently inhibited the function of microtubules, inducing cell growth inhibition, apoptosis of cancer cell lines in a concentration and time-dependent manner. Molecular docking involving 16 at the vinblastine binding site of tubulin indicated that a phenoxy moiety interacted with tubulin via hydrogen bonding with asparaginate (Asn) and tyrosine (Tyr). Analysis of microtubules with confocal microscopy demonstrated that 16 altered the microtubule architecture and exhibited a significant reduction in microtubule density. Cell cycle assay further proved that HepG2 cells were blocked in G2/M phase. Our study provides a new, promising compound for the development of tubulin inhibitors by proposing a new target for the anticancer activity of shikonin.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Naftoquinonas/química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Sitios de Unión , Relación Dosis-Respuesta a Droga , Células Hep G2/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Microtúbulos/efectos de los fármacos , Simulación del Acoplamiento Molecular , Terapia Molecular Dirigida , Vinblastina/metabolismoRESUMEN
A series of shikonin derivatives (1-13) that were acylated selectively by various thiophene or indol carboxylic acids at the side chain of shikonin were synthesized, and their biological activities were also evaluated as potential tubulin inhibitors. Among them, compound 3 ((R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-(1H-indol-3-yl)propanoate) and compound 8 ((R)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 2-(thiophen-3-yl)acetate) exhibited good antiproliferative activity of A875 (IC50 = 0.005 ± 0.001 µm, 0.009 ± 0.002 µm) and HeLa (IC50 = 11.84 ± 0.64 µm, 4.62 ± 0.31 µm) cancer cell lines in vitro, respectively. Shikonin (IC50 = 0.46 ± 0.002 µm, 4.80 ± 0.48 µm) and colchicine (IC50 = 0.75 ± 0.05 µm, 17.79 ± 0.76 µm) were used as references. Meanwhile, they also showed the most potent growth inhibitory activity against tubulin (IC50 of 3.96 ± 0.13 µm and 3.05 ± 0.30 µm, respectively), which were compared with shikonin (IC50 = 15.20 ± 0.25 µm) and colchicine (IC50 = 3.50 ± 0.35 µm). Furthermore, from the results of flow cytometer, we found compound 3 can really inhibit HeLa cell proliferation and has low cell toxicity. Based on the preliminary results, compound 3 with potent inhibitory activity in tumor growth may be a potential anticancer agent.