Hydroxylation of the Labdane Diterpene Cupressic Acid by Fusarium graminearum

Abstracts

Cupressic acid [13-hydroxy-8(17),14-labdadien-19-oic acid], a diterpene obtained from Araucarea angustifolia elegans was biotransformed by Fusarium graminearum producing four hydroxylated diterpene derivatives, which were identified by chemical and physical methods as 3beta,13-dihydroxy-8(17),14-labdadien-19-oic acid, 7alpha,13-drihydroxy-8(17),14-labdadien-19-oic acid, 8,13,17-trihydroxy-14-labden-19-oic acid and 13,14,15-trihydroxy-8(17)-labden-19-oic acid.

Fusarium; biotransformation; labdane diterpene; 8(17); 14-labdadien-19-oic acid

Culturas do fungo Fusarium graminearum sob agitação na presença do diterpeno ácido cupréssico [ácido 13-hidróxi-(17),14-labdadien-19-óico], obtido de Araucarea angustifolia elegans, produziram quatro derivados diterpênicos hidroxilados os quais foram identificados por métodos químicos e físicos como sendo os ácidos 3beta,13-diidróxi-8(17),14-labdadien-19-óico, 7alfa,13-diidróxi-8(17),14-labdadien-19-óico, 8,13,17-triidróxi-14-labdaden-19-óico e 13,14,17-triidróxi-14-labdaden-19-óico.

Article

Hydroxylation of the Labdane Diterpene Cupressic Acid by Fusarium graminearum

Edson Rodrigues-Filho ^{* a},Rodrigo F. Magnani^a, Weiping Xie^b, Chester J. Mirocha^b and Sadanan V. Pathre^b

^aDepartamento de Química, Universidade Federal de São Carlos, CP 676, 13565-905, São Carlos - SP, Brazil

^bDepartment of Plant Pathology, University of Minnesota, St. Paul, MN - USA, 55108-6030

Culturas do fungo Fusarium graminearum sob agitação na presença do diterpeno ácido cupréssico [ácido 13-hidróxi-(17),14-labdadien-19-óico], obtido de Araucarea angustifolia elegans, produziram quatro derivados diterpênicos hidroxilados os quais foram identificados por métodos químicos e físicos como sendo os ácidos 3b,13-diidróxi-8(17),14-labdadien-19-óico, 7a,13-diidróxi-8(17),14-labdadien-19-óico, 8,13,17-triidróxi-14-labdaden-19-óico e 13,14,17-triidróxi-14-labdaden-19-óico.

Cupressic acid [13-hydroxy-8(17),14-labdadien-19-oic acid], a diterpene obtained from Araucarea angustifolia elegans was biotransformed by Fusarium graminearum producing four hydroxylated diterpene derivatives, which were identified by chemical and physical methods as 3b,13-dihydroxy-8(17),14-labdadien-19-oic acid, 7a,13-drihydroxy-8(17),14-labdadien-19-oic acid, 8,13,17-trihydroxy-14-labden-19-oic acid and 13,14,15-trihydroxy-8(17)-labden-19-oic acid.

Keywords: Fusarium, biotransformation, labdane diterpene, 8(17),14-labdadien-19-oic acid

Introduction

Biotransformation of natural products by fungi is an old tool, now becoming fashionable again to selectively introduce functional groups in different positions of carbon skeletons.^1-3 Diterpenes have received special attention as a substrate^4,5 for biotransformation, since some of these substances are endogenously produced by fungal species; gibberellic acid, for instance, is an endogenous metabolite of the Fusarium species in some sexual stage.^6-8 Besides gibberellic acid derivatives, labdane diterpenes have also been used as substrates for biotransformation by Fusarium^5,8,9 in an attempt to convert them into analogous forskolins, which are important bioactive compounds.^5,10,11 We recently discovered that the plant Araucaria angustifolia elegans produces high yields of cupressic acid and abietane diterpenes.¹² An antifungal bioassay to test these diterpenes against Leucoagaricus gongylophorus, a fungus living symbiotically with the leaf-cutting ant Atta sexdens rubropilosa, revealed them to be inactive compounds.¹² We therefore decided to study the ability of fungi from Fusaria group to metabolize these diterpene acids. This paper reports the results we obtained from a fermentation experiment using Fusarium graminearum.

Results and Discussion

Isolation procedures, structural identification and antifungal bioassay of cupressic acid and other diterpenes will be published elsewhere. The biotransformation products 2-6 were esterified with diazomethane prior to isolation and structural identification. The ¹³C NMR data (Table 1) obtained for the compounds 2a-5b, compared with the extensive NMR study of the cupressic acid methyl ester¹² and other related labdane diterpenes,^13-16 were the most important elucidative elements, and were supported by other 2D spectra used for confirmation. The most important features observed in the NMR data for 1a, which were used for purposes of comparison for the identification of the hydroxylated bioproducts, were the presence of the two double bonds in C-8(17) (d 148.1 and 106.4) and C-14 (d 145.1 and 111.5) and a shielded methylene carbon signal at d 19.9, which were ascribed to C-2.

A broad band at 3398 cm^-1, originated from a hydroxyl group, was detected in the IR absortion spectrum of 2a. In conjunction with this IR information, the peak at m/z 332 (M^+• - H₂O) indicated the molecular formula C₂₁H₃₄O₄. In the ¹H NMR spectrum, compound 2a showed the presence of hydrogen from two double bonds, as in the precursor 1, and a pair of doublets for a carbynolic hydrogen (d 3.17). Only six methylene carbons were detected in the ¹³C NMR spectrum (DEPT135° experiment) of 2a. In addition, the shielded methylene signal at c.a. d 20 was not present in its ¹³C NMR spectrum. These NMR data indicate that hydroxylation occurred in ring A. The split pattern shown by the carbynolic hydrogen (dd) may be attributed to a hydrogen in C-1 or C-3. In comparison with the signals for C-4 and C-10 for compound 1a, C-4 is 5.1 ppm deshielded and C-10 showed the same chemical shift, indicating the positioning of the hydroxyl group at C-3 in 2a. The b orientation for this hydroxyl was deduced from the split observed for H-3 (dd, J 3 and 8 Hz), which was corroborated by TOCSY NMR experiments. Moreover, the expected g-gauche shielding effect of an a-hydroxyl group at C-3 over C-5 and C-1,^13,14 which was detected almost in the same d as the one in 1a, was not observed. Thus, the new diterpene produced by fermentation is 3b-hydroxy-cupressic acid or 3b,13-dihydroxy-8(17),14-labdadien-19-oic acid (2), which appears to be a new substance.

Based on the IR (n 3399 cm^-1) and MS (m/z 332, M^+. - H₂O) data, we concluded that compound 3a is a diol isomer of 2a. Furthermore, in comparison with 1a, the chemical shifts of the carbons involved in ring A and the side chain at C-9 were almost the same. The ¹³C and ¹H NMR spectra also showed that 2a contained both double bonds present in precursor 1a. The main differences observed were in the carbons in ring B. The positioning of the hydroxyl group at C-7 was based on the deshielding influence of the hydroxyl on the double bond carbons at C-8(17) (Dd 1.0 and 3.4, respectively) compared to 1a, and on the HMBC correlation of H-7 (d 4.40) and C-17 (d 109.8). This positioning, as well as the a configuration for the hydroxyl group, were confirmed by the g-gauche shielding effect of 7.8 and 6.3 ppm over carbons C-5 and C-9, respectively.^14,16 Also, the NMR signal of the H-7 nuclei is a triplet due to an equatorial-equatorial and equatorial-axial coupling with 2H-6, and shows nOe with ¹H-17a. These data indicated a structure of 7-hydroxy-cupressic acid or 7a,13-dihydroxy-8(17),14-labdadien-19-oic acid (3) for this new diterpene produced in the biotransformation experiment.

The last two hydroxylated products were also a pair of isomers. The IR (n 3425-3280 cm^-1) and ¹³C NMR [d 75.8 (C), 60.1 (CH₂) and 74.3 (C) for 4a and 74.8 (C), 74.1 (CH) and 63.3 (CH₂) for 5a]¹⁷ data, together with the absence of signals in the ¹³C spectra that could be ascribed to the double bonds at C-8(17) for 4a and C-14 for 5a, indicated that these two isomers were the triol derivatives of 1a. The assignments of the ¹³C chemical shifts shown in Table 1 were obtained from an analysis of HMQC and HMBC experiments of compounds 4a and 5a and from the acetylated derivative 5b. The configurations of the stereogenic carbons in triols 4a and 5a are not easily established,¹⁷ but our NMR data indicated that only one major triol isomer was produced in each case.

Triols 4a and 5a may have been produced via epoxidation of the double bonds, followed by hydrolization of the epoxide. The epoxide would have been a possible intermediate to the partial 8,13-oximonoil labdane structure present in the forskolin derivatives.¹⁰ In our experiment, we obtained a fraction from the culture extract whose NMR and GCMS data indicated a mixture of the many isomers represented by structure 6. We are now studying the most suitable conditions to optimize the production of these tetrahydropiran ethers.

The carboxyl group at C-19 in precursor 1 apparently was not responsible for the induction of hydroxylation at C-3, since sclareol ([8,13-dihydroxy-14-labdene]), a biosynthetic parent compound of 1, produced hydroxylated compounds at C-3 under microbial fermentation using other filamentous fungi.^18,19

Experimental

General experimental procedures. The ¹H and ¹³C NMR experiments were recorded using a VARIAN GEMNI or a BRUKER DRX spectrometer, which were operated, respectively, at 500 and 400 MHz for ¹H and 125 and 100 MHz for ¹³C, using deutero chloroform (CDCl₃) as solvent, with TMS as the internal standard. MS data were collected using a Micromass Platform II instrument (Micromass, Wythenshawe, Manchester, UK) equipped with an EI/CI ion source and coupled to a Carlo Erba GC 8000 series. The column used was a DB-1 30 m x 0.5 mm x 0.25 mm from J&W.

Isolation of the starting material 1. Cupressic acid (1) was isolated from the resin exudated from Araucarea angustifolia. The crystalline resin was extracted with chloroform and the diterpenes were isolated by extensive silica gel column chromatographic procedures (n-heptane:AcOEt + 0.5 % acetic acid in a gradient elution). Compound 1 was characterized by NMR spectral data.

Microorganism, media and culture conditions. Fusarium graminearum was isolated from the soil in St. Paul, Minnesota, USA in February of 1982 and stored in sterilized soil. Samples of this isolate were stored under 4 °C in Dr. Mirocha's collection (Mycotoxin Laboratory, University of Minnesota, Department of Plant Pathology, College of Agriculture, St. Paul Campus) under number 38. The fungus was seeded in a Petri dish containing PDA (potato-dextrose-agar) and allowed to grow for 4 days. Six 5-liter Erlenmeyer flasks, each containing 600 mL of liquid medium (80g glucose, 0.48g NH₄NO₃, 5.0g KH₂PO₄, 1.0g MgSO₄, 0.1g FeSO₄, 0.015g Cu SO₄, 0.161g ZnSO₄, 0.01g MnSO₄, and 0.1g (NH₄)₂MoO₄ dissolved in 1.5L of distilled water) were inoculated with pieces of the PDA culture containing micelium and were allowed to grow in a shaker (110 rpm) at 25 °C. Substrate 1 (600mg dissolved in acetone, 100mg per flask) was added after 6 days of growth. The growth was interrupted after an additional 14-day period.

Extraction, purification and analysis of microbial transformation products. The fermentation was harvested by filtering the mycelium and the culture filtrate was acidified to pH 3 and extracted with ethyl acetate. The solvent was evaporated under vacuum in a rotary distiller and the residue thus obtained was methylated with diazomethane. The resulting methyl esters were chromatographed on a silica gel column and eluted with gradient mixtures of n-hexane and ethyl acetate. Final purification of products 2a and 3a was achieved on a silica gel column isocratically eluted with petrol ether and ethyl acetate [4:1] with 1% of acetic acid. Triols 4a and 5a were eluted from the column with petrol ether and ethyl acetate [2:1] with 1% of acetic acid. The methyl ester 5a was acetylated with acetic anhydride in pyridine and the acetate derivative purified by silica gel CC. The following physical data were used to identify the hydroxylated diterpenes:

2a:White oil; IR n/cm^-1: 3398, 2944, 2869, 1718, 1643, 1446, 1377, 1228, 1156, 914 and 733 (KBr); EIMS: m/z(%) 350(M^+• not detected, C₂₁H₃₄O₄), 332(3, M^+•- H₂O), 314(4, M^+•- 2xH₂O), 299(4, M^+•- 2xH₂O - ^•CH₃), 282(2), 255(8), 239(8), 201(10), 187(12%), 173(13), 159(22), 145(20), 133(32), 121(35), 119(78), 107(76), 105(67), 93(78), 81(70), 79(66), 71(62), 67(53) and 55(100); ¹H-NMR (CDCl₃, 500 MHz) d 3.17 (dd, J 9 and 3 Hz, H-3), 5.85 (dd, J 17 and 11 Hz, H-14), 5.13 (dd, J 17 and 1 Hz, H-15a), 5.20 (dd, J 11 and 1 Hz, H-15b), 1.21 (s, H-16), 4.47 (brs, H-17a), 4.83(brs, H-17b), 1.19(s, H-18), 0.52 (s, H-20), 3.61 (s,OCH₃); ¹³C-NMR (CDCl₃, 100 MHz): Table 1.

3a: White oil; IR n/cm^-1: 3399, 2947, 2873, 1720, 1645, 1441, 1379, 1230, 1153, 913 and 739 (KBr); EIMS: m/z(%) 350(M^+• not detected, C₂₁H₃₄O₄)_, 332(2, M^+•- H₂O), 314(3, M^+•- 2xH₂O), 299(5, M^+•- 2xH₂O - ^•CH₃), 282(3), 255(10), 239(6), 145(21), 133(27), 131(32), 123(93), 121(52), 109(68), 107(64), 105(47), 93(68), 91(69), 81(100), 79(88), 71(57) and 67(63); ¹H-NMR (CDCl₃, 400 MHz) d 4.40(t, J 1 Hz, H-7), 5.90(dd, J 17 and 11 Hz, H-14), 5.06(dd, J 17 and 1 Hz, H-15a), 5.21(dd, J 17 and 1 Hz, H-15b), 1.23(s, H-16), 4.67(brs, H-17a), 5.07(brs, H-17b), 1.18(s, H-18), 0.49(s, H-20), 3.62(s, OCH₃); ¹³C-NMR (CDCl₃, 100 MHz): Table 1.

4a: White oil; IR n/cm^-1: 3410, 2941, 2870, 1720, 1622, 1435, 1365, 1200, 1130 and 740 (KBr); EIMS: m/z(%) 368(M^+• not detected, C₂₁H₃₆O₅), 350(1, M^+•- H₂O), 332(2, M^+•- 2xH₂O) and 299 (4, M^+•- 2xH₂O - ^•CH₃); ¹H-NMR (CDCl₃, 500 MHz) d 5.86(dd, J 11 and 17 Hz, H-14), 5.03(dd, J 17 and 1 Hz, H-15a), 5.21(dd, J 11 and 1 Hz, H-15b), 1.24(s, H-16), 3.41(d, J 10 Hz, H-17a), 3.62(d, J 10 Hz, H-17b), 1.18(s, H-18), 0.51(s, H-20), 3.60(s, OCH₃); ¹³C-NMR (CDCl₃, 100 MHz): Table 1.

5b: White oil; IR n/cm^-1: 3371, 2950, 2889, 1740, 1716, 1632, 1417, 1388, 1210, 1125 and 801 (KBr); EIMS: m/z (%) 452(C₂₅H₄₀O₇, M^+•), 434(1, M^+•- H₂O), 392(3, M^+•- AcOH), 374(5, M^+•- AcOH - H₂O ), 314(7, M^+•- 2xAcOH - H₂O ), 289(4), 262(5), 229(9), 188(8), 181(12), 161(11), 147(13), 133(15), 121(100), 107(32), 93(35), 81(42) and 79(23); ¹H-NMR (CDCl₃, 400 MHz) d 5.80(dd, J 9 and 3 Hz, H-14), 4.48(dd, J 13 and 3 Hz, H-15a), 4.11(dd, J 13 and 9 Hz, H-15b), 1.12(s, H-16), 4.85(brs, H-17a), 4.50(brs, H-17b), 1.09(s, H-18), 0.52(s, H-20), 3.52(s, OCH₃); ¹³C-NMR (CDCl₃, 100 MHz): Table 1.

Acknowledgement

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for financial support and research fellowships.

References

12. Unpublished results.

Received: October 26, 2000

Published on the web: March 18, 2002

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  25 June 2002
• Date of issue
  2002

History

• Accepted
  18 Mar 2002
• Received
  26 Oct 2000