Hydroxylation of the Labdane Diterpene Cupressic Acid by Fusarium graminearum

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 3 ,13-diidróxi-8(17),14-labdadien-19-óico, 7 ,13-diidróxi-8(17),14labdadien-19-óico, 8,13,17-triidróxi-14-labdaden-19-óico e 13,14,17-triidróxi-14-labdaden-19-óico.


Introduction
7][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. 5,10,11e recently discovered that the plant Araucaria angustifolia elegans produces high yields of cupressic acid and abietane diterpenes. 12An 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. 12We 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 13 C NMR data (Table 1) obtained for the compounds 2a-5b, compared with the extensive NMR study of the cupressic acid methyl ester 12 and other related labdane diterpenes, [13][14][15][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 +• -H 2 O) indicated the molecular formula C 21 H 34 O 4 .In the 1 H 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 13 C NMR spectrum (DEPT135° experiment) of 2a.In addition, the shielded methylene signal at c.a. d 20 was not present in its 13 C 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 ggauche 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 Table 1. 13  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 +. -H 2 O) 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 13 C and 1 H 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. 14,16lso, 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 1 H-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 13 C NMR [d 75.8 (C), 60.1 (CH 2 ) and 74.3 (C) for 4a and 74.8 (C), 74.1 (CH) and 63.3 (CH 2 ) for 5a] 17 data, together with the absence of signals in the 13 C 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 13 C 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. 10In 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. 18,19perimental General experimental procedures.The 1 H and 13 C NMR experiments were recorded using a VARIAN GEMNI or a BRUKER DRX spectrometer, which were operated, respectively, at 500 and 400 MHz for 1 H and 125 and 100 MHz for 13 C, using deutero chloroform (CDCl 3 ) 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 (nheptane: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 NH 4 NO 3 , 5.0g KH 2 PO 4 , 1.0g MgSO 4 , 0.1g FeSO 4 , 0.015g Cu SO 4 , 0.161g ZnSO 4 , 0.01g MnSO 4 , and 0.1g (NH 4 ) 2 MoO 4 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