Abstract
The diterpene ent-18,19-dihydroxytrachylobane was biotransformed for the first time by Rhizopus stolonifer, and yielded the new ent-11β,18,19-trihydroxytrachylobane derivative besides the new ent-kaur-11-ene diterpenes ent-16α,18,19-trihydroxykaur-11-ene and ent-18,19-dihydroxy-16α-methoxykaur-11-ene. Their structures were determined by spectrometric methods.
ent-trachylobane diterpene; ent-kaur-11-ene diterpenes; biotransformation; Rhizopus stolonifer ; fungus
Introduction
Secondary metabolites transformation by fungi is considered a useful tool for the production of new compounds under environmentally friendly conditions. Most biotransformation of natural products involves chemo-, regio-, and/or stereoselective reactions and yield products which are difficult to be obtained by chemical methods.11 Liu, J-H.; Yu, B-Y.; Curr. Org. Chem. 2010, 14, 1400.
Thus, as part of our research program on the production of novel compounds by microbial transformation of secondary metabolites,22 Nunes, F. M.; Santos, G. F.; Saraiva, N. N.; Trapp, M. A.; Mattos, M. C.; Oliveira, M. C. F.; Rodrigues-Filho, E.; Appl. Catal., A 2013, 468, 88. the biotransformation of the ent-trachyloban diterpene 1(Figure 1) by the fungus R. stolonifer was investigated. To the best of our knowledge this constitutes the first report of microbial transformation of 1.
Chemical structures of ent-18,19-dihydroxytrachylobane (1) and its biotransformation products 2-4.
Studies on fungal biotransformation of ent-trachylobane diterpenes are reported in the literature.33 Silva, E. A.; Takahashi, J. A.; Oliveira, A. B.; J. Braz. Chem. Soc. 2002, 13, 101.
4 Leverrier, A.; Martin, M-T.; Servy, C.; Ouazzani, J.; Retailleau, P.; Awang, K.; Mukhtar, M. R.; Guéritte, F.; Litaudon, M.; J. Nat. Prod. 2010, 73, 1121.
5 Fraga, B. M.; Gonzalez-Vallejo, V.; Guillermo, R.; J. Nat. Prod. 2011, 74, 1985.
6 Fraga, B. M.; Bressa, C.; Gonzalez-Vallejo, V.; González, P.; Guillermo, R.; Phytochemistry 2012, 81, 60.-77 Diaz, C. E.; Fraga, B. M.; Gonzalez, A. G.; Gonzalez, P.; Hanson, J. R.; Hernandez, M. G.; Phytochemistry 1984, 23, 2813. Most of the products are hydroxylated ent-trachylobane derivatives formed by Csp33 Silva, E. A.; Takahashi, J. A.; Oliveira, A. B.; J. Braz. Chem. Soc. 2002, 13, 101.oxidation of carbons 1, 2, 3, 7, 17 or 19, or ent-kaur- 11-ene diterpenes (with or without C-9 hydroxylation) formed by backbone rearrangement of the ent-trachylobane. In fact, the isolation of ent-kaur-11-ene diterpenes only from plants that also produce ent-trachylobane diterpenes strongly suggests that these latter compounds are probable precursors of ent-kaur-11-ene diterpenes.33 Silva, E. A.; Takahashi, J. A.; Oliveira, A. B.; J. Braz. Chem. Soc. 2002, 13, 101.,55 Fraga, B. M.; Gonzalez-Vallejo, V.; Guillermo, R.; J. Nat. Prod. 2011, 74, 1985.
Herein, we report the production of three new compounds identified as ent-11β,18,19-trihydroxytrachylobane (2), ent-16α,18,19-trihydroxykaur-11-ene (3) and ent-18,19-dihydroxy-16α-methoxykaur-11-ene (4) by fungal biotransformation of ent-18,19-dihydroxytrachylobane (1). The structures of these compounds were established mainly on the basis of their 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data.
Experimental
General procedure
Melting points were determined on a Micro-Química MQAPF-302 and Mettler Toledo FP62 apparatus, and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Optical rotations were determined on a Perkin-Elmer P-2000 and Perkin-Elmer 341 polarimeters. NMR spectra (1H, 13C, DEPT, COSY, HSQC and HMBC) were recorded in CD3OD (Tedia®, with tetramethylsilane (TMS) as internal standard) on Bruker Avance DPX 300 (300 MHz) and Avance DPX 500 (500 MHz) spectrometers. High-resolution mass spectra (MS) were obtained on a Shimadzu LC-MS IT-TOF spectrometer equipped with an electrospray ionisation (ESI) source in positive and negative modes.
Analytical thin-layer chromatography (TLC) was performed on pre-coated 0.25 mm thick plates of silica gel 60 F254, and the spots were visualized under a UV lamp (254 nm) and by spraying with a solution of perchloric acidvanilin in EtOH, followed by heating. High performance liquid chromatography (HPLC) analyses were done on a Shimadzu instrument equipped with a LC-20AT high-pressure pump, a SPD-M20A photodiode array detector, and a normal phase Phenomenex® (10 × 150 mm, 5 µm) column. Hexane and isopropanol from Tedia®, previously filtered on nylon membrane (0.22 µm, Phenomenex®), were used as solvents. A wavelength of 254 nm was used in the isolation of the compounds by high performance liquid chromatography with photodiode array detection (HPLC-DAD).
Potato-dextrose-broth was purchased from HIMEDIA® , and all other chemical compounds were from Vetec® and Synth®.
Fungal material
R. stolonifer (strain BRF-130) was isolated from wheat seed and identified by observation of key morphological characters at the Mycology Laboratory of the Universidade Federal do Ceará (Fortaleza-CE, Brazil). The key morphological characters observed were irregular in shape often polygonal, globose, ovoid or elliptical unicellular striate sporangiospores produced on globose sporangiophores forming a cluster with branched rhizoids at the base and chlamydospores absent in the stolons.
Biotransformation of compound 1
Mycelial plugs (5 mm diameter) of the fungal colony previously grown for 7 days in potato-dextrose-agar were transferred to seven 250 mL Erlenmeyer flasks each containing 100 mL of potato-dextrose (24 g L-1) broth. After 7 days under static condition at room temperature (ca. 28 °C), the mycelium in each flask was separated from the liquid medium by vacuum filtration (aseptic conditions), and was transferred to a 250 mL Erlenmeyer flask containing 100 mL of phosphate buffer solution (pH 7) providing a total of seven flasks. Compound 1 (30 mg per flask) was added to six of these flasks, and one flask (no compound added) was used as the control. After 21 days in a shaker (150 rpm and 28 °C), the mycelium was separated by vacuum filtration. The liquid portion from each flask was extracted with EtOAc (3 × 50 mL) after saturation with NaCl, and the organic layer was dried with anhydrous Na2SO4, filtered and concentrated. After TLC analysis (EtOAc:hexane, 8:2) all extracts (except from the control experiment) were combined to afford 88.7 mg of the total extract. Purification of this extract by HPLC (wavelength: 254 nm; flow rate: 3 mL min-1; injection volume: 200 µL; sample concentration: 20 mg mL-1; isocratic mixture: hexane:iPrOH 28%) afforded compounds 2 (RT: 6.19 min, 10.0 mg, 5.3%), 3(RT: 4.82 min, 9.8 mg, 5.3%) and 4 (RT: 5.43 min, 6.0 mg, 3.1%).
Ent-11β,18,19-trihydroxytrachylobane (2)
White solid; m.p. 158.3-160.7 °C; [α]20D -26.73 (c 0.1, MeOH); IR (film on ZnSe) νmax/ cm-1 3348, 2923, 2852, 1031; HRMS m/z calcd.: C20H32O3Na+: 343.2244; found: 343.2215 [M + Na]+; 1H NMR (300 and 500 MHz, CD3OD) and 13C NMR (75 and 125 MHz, CD3OD) see Tables 1 and 2.
Ent-16α,18,19-trihydroxykaur-11-ene (3)
Amorphous solid; [α]20D -71.66 (c 0.1, MeOH); IR (film on ZnSe) νmax/ cm-1 3284, 3020, 2922, 2842, 1663, 1026; HRMS m/z calcd.: C20H32O3Na+: 343.2244; found: 343.2251 [M + Na]+; 1H NMR (300 and 500 MHz, CD3OD) and 13C NMR (75 and 125 MHz, CD3OD) see Tables 1 and 2.
Ent-18,19-dihydroxy-16α-methoxykaur-11-ene (4)
White solid; m.p. 123.6-124.5 °C; [α]20D -7.96 (c 1.82, MeOH); IR (film on ZnSe) νmax/ cm-1 3373, 2920, 2851, 1440, 1019; HRMS m/z calcd.: C21H34O3Na+: 357.2400; found: 357.2403 [M + Na]+; 1H NMR (300 and 500 MHz, CD3OD) and 13C NMR (75 and 125 MHz, CD3OD) see Tables 1 and 2.
Results and Discussion
The ent-18,19-dihydroxytrachylobane (1) was recently described as the major compound in the hexane extract of roots of Croton floribundus.88 Uchôa, P. K. S.; Silva-Jr, J. N.; Silveira, E. R.; Lima, M. A. S.; Braz-Filho, R.; Costa-Lotufo, L. V.; Araújo, A. J.; Moraes, M. O.; Pessoa, C.; Quim. Nova 2013, 36, 778. This secondary metabolite was submitted to biotransformation by R. stolonifer, and yielded the new compounds 2 (5.3%), 3 (5.3%) and 4 (3.1%) as products.
1H NMR spectrum of compound 2 showed very similar signals to those described for the ent-18,19-dihydroxytrachylobane (1). This spectrum showed the shielded signals at δ 0.83 (m, H-13) and 1.02 (t, J 4.0 Hz, H-12) of the tetrasubstituted cyclopropane ring, the two angular methyl groups at δ 1.15 (s, 3H-17) and 0.90 (s, 3H-20), and the deshielding signals at δ 3.76 (d, 1H, J 11.2 Hz, H-19α), 3.50 (d, 1H, J 11.2 Hz, H-19β), 3.52 (d, 1H, J 11.0 Hz, H-18α) and 3.46 (d, 1H, J 11.0 Hz, H-18β), relative to two oxymethylene groups attached to the quaternary carbon at C-4. The only light difference was the presence of the additional signal at δ 4.11 (s, 1H, H-11) that was attributed to one oxymethine group.
The 13C NMR spectrum of 2 also resembled that of compound 1, except that the chemical shift of the C-11 (δ 73.17) was deshielded compared to the same carbon in 1 (δ 21.1), Table 2. These data led to the deduction that the methylene carbon C-11 in compound 1 was hydroxylated in 2.
On the basis of the long-range correlations observed in the HMBC spectrum (Figure 2), the assignment of the relative position of the hydroxyl group at C-11 was determined by correlations of the hydrogen at δ 4.11 (H-11) from oxymethine group with the carbons at δ 25.65 (C-13), 48.12 (C-8) and 25.10 (C-16), and between the hydrogen at δ 0.83 (H-13) with the carbon at δ 73.17 (C-11). The relative stereochemistry of the hydroxyl group was established by the NOESY experiment. In particular, the β-orientation of hydroxyl group was determined by the diagnostic NOE cross-peaks observed between the carbinol methine at δ 4.11 (H-11) and the methyl group at δ 0.90 (CH3-20), Figure 2. From the foregoing evidence, compound 2 was identified as the new ent-11β,18,19-trihydroxytrachylobane, and its molecular formula C20H32O3 was confirmed by HRMS.
Comparison of the 1H NMR data of compound 3 with those of 1 revealed some major differences between them, particularly in relation to the absence of the shielded cyclopropane protons, and the presence of two additional olefinic protons at δ 5.92 (t, 1H, J 8.2 Hz, H-12) and 5.55 (dd, 1H, J 8.2 and 3.6 Hz, H-11). These foregoing evidences suggested the opening of the cyclopropane ring on 3 and the possible formation of an ent-kaur-11-ene-derivative, as already reported during the biotransformation reactions of other trachyloban diterpenes.33 Silva, E. A.; Takahashi, J. A.; Oliveira, A. B.; J. Braz. Chem. Soc. 2002, 13, 101.
4 Leverrier, A.; Martin, M-T.; Servy, C.; Ouazzani, J.; Retailleau, P.; Awang, K.; Mukhtar, M. R.; Guéritte, F.; Litaudon, M.; J. Nat. Prod. 2010, 73, 1121.-55 Fraga, B. M.; Gonzalez-Vallejo, V.; Guillermo, R.; J. Nat. Prod. 2011, 74, 1985.
Further evidences for this suggestion were possible by analysis of 13C NMR (broad band and DEPT) spectra that showed signals relative to a disubstituted double bond at δ 133.95 (C-12) and 128.28 (C-11), and the additional signal of one oxymethine group at δ 84.42 (C-16). The deshielded values of the carbon resonances at δ 59.73 (C-15), 51.18 (C-13) and 25.92 (C-17), compared to those data of compound 1, indicated these carbons in a geminal position to the oxygenated function.
The HMBC correlations (Figure 2) observed between the olefinic hydrogens at δ 5.92 (H-12) with the carbons at δ 84.42 (C-16), 63.97 (C-9) and 35.18 (C-14), as well as the correlation of the other olefinic at δ 5.55 (H-11) and with the carbons at δ 51.18 (C-13), 44.51 (C-8) and 39.31 (C-10) undoubtedly determined the location of the double bond position at C11-C12 and the hydroxyl group at C-16, respectively. The relative stereochemistry of the hydroxyl group at C-16 was established by the NOESY experiment, which showed cross-peaks between the hydrogens CH3-17, H-9 and H-5 and indicated that they were β-oriented (Figure 2). The above data established the structure of compound 3 as a rearranged diterpene skeleton named ent-16α,18,19-trihydroxykaur-11-ene. This structure is in agreement with the molecular formula C20H32O3, which was determined by HREIMS analysis. Additionally, all 13C NMR data of compound 3, especially from the rearranged moiety (carbons 11-17), are in accordance with those reported for ent-kaur-11-ene-derivatives.33 Silva, E. A.; Takahashi, J. A.; Oliveira, A. B.; J. Braz. Chem. Soc. 2002, 13, 101.,44 Leverrier, A.; Martin, M-T.; Servy, C.; Ouazzani, J.; Retailleau, P.; Awang, K.; Mukhtar, M. R.; Guéritte, F.; Litaudon, M.; J. Nat. Prod. 2010, 73, 1121.
The 1H NMR spectrum of compound 4 indicated a close relationship with those observed for 3, through the signals relative to double bond at δ 5.59 (dd, J 8.5 and 3.7 Hz, H-11) and 5.89 (t, J 8.5 Hz, H-12), and the two oxymethylenes at δ 3.56 (d, J 11.3 Hz, H-18α) and 3.47 (d, J 11.3 Hz, H-18β), and 3.54 (d, J 12.1 Hz, H-19α) and 3.49 (d, J 12.1 Hz, 19β). A slight difference was found by the presence of the extra singlet at δ 3.13 (s, OCH3) relative to one methoxyl group.
The proposition that hydroxyl group of compound 3 was methoxylated on 4 was suggested by comparison of their 13C NMR data, that δisplayed one additional signal at δ 49.82 relative to the methoxyl group. The long-range connectivities in the HMBC spectrum between the methoxyl hydrogens at δ 3.13 (OCH3) with the carbon at δ 90.10 (C-16) confirmed this proposition. As observed on the 3, the relative stereochemistry of the methoxyl group at C-16 was defined as α-oriented by the same correlations observed on compound 3, in the NOESY experiment.
In addition to the NMR data, analysis of 4 by HRMS allowed the establishment of its molecular formula as C21H32O3. Thus, the structure of compound 4 was determined to be the new ent-18,19-dihydroxy-16αmethoxykaur-11-ene. Methylation of the hydroxyl group at C-16 of an ent-kaur-11-ene diterpene by Rhizopus arrhizus has been reported previously.44 Leverrier, A.; Martin, M-T.; Servy, C.; Ouazzani, J.; Retailleau, P.; Awang, K.; Mukhtar, M. R.; Guéritte, F.; Litaudon, M.; J. Nat. Prod. 2010, 73, 1121.
Conclusion
In summary, the ent-trachyloban diterpene ent-18,19-dihydroxytrachylobane (1) was biotransformed by R. stolonifer, and produced the new ent-11β,18,19-trihydroxytrachylobane (2), and the new ent-kaurene diterpenes ent-16α,18,19-trihydroxykaur-11-ene (3) and ent-18,19-dihydroxy-16α-methoxykaur-11-ene (4). The formation of derivative 2 involved the first hydroxylation of C-11 of ent-trachyloban diterpene skeleton by fungus, and compounds 3 and 4 were probably produced by backbone rearrangement of 2. These results corroborate the potential application of microbial transformation of natural products for the formation of new compounds.
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Supplementary InformationThe 1D and 2D NMR spectra data associated with this article are available free of charge at http://jbcs.sbq.org.br as a PDF file.
Acknowledgments
The authors wish to acknowledge Fundação Cearense de Apoio a Pesquisa (FUNCAP), Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support. We also thank CENAUREMN and LEMANOR for NMR and HRMS spectra, respectively, and Prof A. A. Leslie Gunatilaka (The University of Arizona, USA) for discussion and comments on the manuscript.
References
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1Liu, J-H.; Yu, B-Y.; Curr. Org. Chem. 2010, 14, 1400.
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2Nunes, F. M.; Santos, G. F.; Saraiva, N. N.; Trapp, M. A.; Mattos, M. C.; Oliveira, M. C. F.; Rodrigues-Filho, E.; Appl. Catal., A 2013, 468, 88.
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3Silva, E. A.; Takahashi, J. A.; Oliveira, A. B.; J. Braz. Chem. Soc 2002, 13, 101.
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4Leverrier, A.; Martin, M-T.; Servy, C.; Ouazzani, J.; Retailleau, P.; Awang, K.; Mukhtar, M. R.; Guéritte, F.; Litaudon, M.; J. Nat. Prod. 2010, 73, 1121.
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5Fraga, B. M.; Gonzalez-Vallejo, V.; Guillermo, R.; J. Nat. Prod. 2011, 74, 1985.
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6Fraga, B. M.; Bressa, C.; Gonzalez-Vallejo, V.; González, P.; Guillermo, R.; Phytochemistry 2012, 81, 60.
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7Diaz, C. E.; Fraga, B. M.; Gonzalez, A. G.; Gonzalez, P.; Hanson, J. R.; Hernandez, M. G.; Phytochemistry 1984, 23, 2813.
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8Uchôa, P. K. S.; Silva-Jr, J. N.; Silveira, E. R.; Lima, M. A. S.; Braz-Filho, R.; Costa-Lotufo, L. V.; Araújo, A. J.; Moraes, M. O.; Pessoa, C.; Quim. Nova 2013, 36, 778.
Publication Dates
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Publication in this collection
May 2015
History
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Received
10 Dec 2014 -
Accepted
20 Mar 2015
