Regio- and enantioselective microbial hydroxylation and evaluation of cytotoxic activity of β-cyclocitral-derived halolactones

RESEARCH ARTICLE Regio- and enantioselective microbial hydroxylation and evaluation of cytotoxic activity of β-cyclocitral-derived halolactones Marcelina Mazur1*, Witold Gładkowski1, Višnja Gaurina Srček2, Kristina Radošević2, Gabriela Maciejewska3, Czesław Wawrzeńczyk1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Mazur M, Gładkowski W, Srček VG, Radošević K, Maciejewska G, Wawrzeńczyk C (2017) Regio- and enantioselective microbial hydroxylation and evaluation of cytotoxic activity of β-cyclocitral-derived halolactones. PLoS ONE 12 (8): e0183429. https://doi.org/10.1371/journal. pone.0183429 Editor: Pankaj Kumar Arora, MJP Rohilkhand University, INDIA Received: May 18, 2017 Accepted: August 2, 2017 1 Department of Chemistry, Wrocław University of Environmental and Life Sciences, Wrocław, Poland, 2 Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia, 3 Central Laboratory of the Instrumental Analysis, Wrocław University of Technology, Wrocław, Poland * Abstract Three β-cyclocitral-derived halolactones, which exhibit antifeedant activity towards storage product pests, were subjected to microbial transformation processes. Among the thirty tested strains of filamentous fungi and yeast, the most effective biocatalysts were Absidia cylindrospora AM336, Mortierella isabellina AM212 and Mortierella vinaceae AM149. As a result of regio- and enantioselective hydroxylation four new oxygenated derivatives were obtained. Regardless of the biocatalyst applied, the δ-iodo- and δ-bromo-γ-lactones were hydroxylated in an inactivated position C-5 of cyclohexane ring. The analogous transformation of chlorolactone was observed in Mortierella isabellina AM212 culture but in the case of two other biocatalysts the hydroxy group was introduced at C-3 position. All obtained hydroxylactones were enantiomerically pure (ee = 100%) or enriched (ee = 50%). The highest enantioselectivity of hydroxylation was observed for M. isabellina AM212. The cytotoxic activity of halolactones was also examined by WST-1 assay wherein tested compounds did not exhibit significant effect on the viability of tumor HeLa and normal CHO-K1 cells. Published: August 24, 2017 Copyright: © 2017 Mazur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was financially supported by Ministry of Science and Higher Education, Poland (grant number B010/00031/16). Publication supported by Wrocław Centre of Biotechnology, programme the Leading National Research Centre (KNOW) for years 2014-2018. The funders had no role in study design, data collection and analysis, Introduction The hydroxylation of “inactivated” position in substrate structure leading to enantiomerically pure or enriched products arouse great interest but it is difficult to carry out by traditional chemical synthesis. One of the most efficient methods of incorporation the hydroxy group into organic molecule is the application of microorganisms [1,2,3]. It is also worth to notice that processes catalyzed by the microorganisms have less harmful impact on natural environment than methods of organic synthesis [4]. The biotransformations catalyzed by whole cells are convenient and useful biotechnological tools, especially taking into consideration that majority of hydroxylation processes are mediated by membrane enzymes hence they cannot be easily purified. Different microorganisms can be applied to the enantioselective hydroxylation processes [5, 6]. In our present work we tested thirty strains of filamentous fungi and yeast from seventeen different genus. Three strains of filamentous fungi from Absidia and PLOS ONE | https://doi.org/10.1371/journal.pone.0183429 August 24, 2017 1 / 12 Regio- and enantioselective microbial hydroxylation and evaluation of cytotoxic activity of halolactones decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Mortierella genus were found to be efficient catalysts. The different Mortierella species are investigated as a potent producer of arachidonic acid but only few examples of their use for biohydroxylation are described in literature [7–9]. More examples of hydroxylation can be found for Absidia genus [10–12]. Different Absidia strains are able to transform biologically active compounds [13,14], including those with steroid skeleton [15,16] or lactone ring [17–21]. In this work we present the hydroxylation of β-cyclocitral-derived halolactones by three fungal strains (Absidia cylindrospora AM336, Mortierella isabellina AM212 and Mortierella vinaceae AM149). Parent compounds exhibit antifeedant activity [22] hence it is important to establish the possible metabolic pathway of those compounds in natural environment. Biotransformations of halolactones involving the hydroxylation reaction which entails the increase of their polarity is essential during their further biodegradation. Additionally the previous reports revealed the anticancer potential of some δ-halo-γ-lactones [23, 24] which prompted us to check also the cytotoxic activity of halolactones derived from β-cyclocitral. Material and methods Analysis The progress of transformations as well as the purity of isolated products were checked by Thin Layer Chromatography (TLC, silicagel couted aluminium plates, DC-Alufolien Kieselgel 60 F254, Merck) and gas chromatography (GC). GC analysis was performed on a Agilent Technologies 6890N instrument using Agilent DB-17 capillary column ((50%-phenyl)-methylpolysiloxane 30 m × 0.25 mm × 0.25 μm) and the hydrogen as the carrier gas. The temperature programme was as follows: injector 250˚C, detector (FID) 300˚C, column temperature: 80˚C (1min), 80–200˚C (rate 20˚C min−1), 200–300˚C (rate 30˚C min−1), 300˚C (2 min). The enantiomeric excesses of biotransformation products were calculated on the basis of GC analysis using CP Chirasil-Dex CB column (25 m x 0.25 mm x 0.25 μm) at the following conditions: injector 200˚C, detector (FID) 250˚C, column temperature: 75˚C (hold 1 min), 75–190˚C (rate 0.42˚C min-1), 190˚C (hold 1 min), 190–200 (rate 0.5˚C min-1), 200˚C (hold 10 min). The biotransformation products were purified by column chromatography on silica gel (Kieselgel 60, 230–400 mesh, Merck). The NMR spectra (1H, 13C NMR and correlation spectra: 1H-1H COSY, 1H-13C HMQC, 1 H-13C HMBC) were recorded in a CDCl3 solution on a Brüker Avance II 600 MHz spectrometer. Residual solvent signals (δH = 7.26, δC = 77.0) were used as references for chemical shifts. IR spectra were determined using Mattson IR 300 Thermo Nicolet spectrophotometer. The melting points (uncorrected) wer (...truncated)