The Incubation of 13α,17-Dihydroxystemodane with Cephalosporium aphidicola

The biotransformation of 13α,17-dihydroxystemodane (3) with the fungus Cephalosporium aphidicola afforded 13α,17,18-trihydroxystemodane (4), 3β,13α,17-tri-hydroxystemodane (5), 13α,17-dihydroxy-stemodan-18-oic acid (6), 3β,11β,13α,17-tetra-hydroxystemodane (7), 11β,13α,17,18-tetrahydroxystemodane (8) and 3β,13α,17,18-tetra-hydroxystemodane (9). The hydroxylation at C-18 of the substrate points to a biosynthetically-directed transformation, because aphidicolin (2) is hydroxylated at this carbon. However, the C-3(β) and C-11(β) hydroxylations seem to indicate a xenobiotic biotransformation.


Introduction
Microbiological transformations can be divided into two groups: xenobiotic biotransformations, in which the substrate is strange to the transforming organism, and biosynthetically-directed OPEN ACCESS transformations, also known as "analogue biosynthesis", in which the substrate possesses a structure analogous to a natural biosynthetic intermediate found in the microorganism [1,2]. We have carried out both types of biotransformations using the fungi Mucor plumbeus [3] and Gibberella fujikuroi [4] respectively. Now, in this work we have used another fungus, Cephalosporium aphidicola, which occupies the borderline between the xenobiotic and biosynthetically-directed biotransformations, because it achieves both [5][6][7][8].
Diterpenes with a stemodane skeleton (i.e., 1) have a structural similarity with aphidicolin (2), an antiviral substance and a inhibitor of DNA polymerase, which was isolated from C. aphidicola [9], although the C/D ring junctions and the configuration at C-13 in stemodanes and aphidicolanes are different (Scheme 1). Thus, biotransformations of stemodane diterpenes, stemodin and stemodinone, with this fungus, have been carried out [10,11]. Some of us had isolated 13,17-dihydroxystemodane (3), and analogous compounds of this type, from Stemonia chilensis, a plant that grows in the littoral zone of central Chile [12]. This compound had been incubated with M. plumbeus [13], a fungus used in xenobiotic biotransformations. Scheme 1. Stemodane (1) and aphidicolin (2).
The metabolite 4 showed in the HRMS spectrum the ion of higher mass at m/z 304.2407, formed from the molecular ion by loss of water, which indicated its molecular formula, C 20 H 34 O 3 . Thus, a new oxygen had been introduced in the molecule during the incubation. In the 1 H-NMR spectrum the signal of a new AB system appears at  3.11 and 3.35 (1H each, d, J = 11 Hz). 13 C-NMR spectrum showed a new signal at  72.6 (t). This last value is characteristic of an equatorial -CH 2 OH group at C-4 [14,15], confirmed in the HMBC experiment with correlations of H-18 with C-3, and H-19 with C-3, C-5 and C-18. Therefore, the structure of this compound was determined as 13,17,18-trihydroxystemodane (4).
Compound 6 was obtained as its diacetate 6a by acetylation of the fractions containing it. The molecular formula of 6a was determined as C 24 H 38 O 6 considering HRMS data. Therefore, the substrate had gained two oxygens and lost two hydrogens during the fermentation. The two oxygens must form a part of an acid, because in the 13 C-NMR spectrum a new signal was detected at  182.0, typical of this group, while the disappearance of a methyl signal was noted. The presence of this new group was confirmed because 6a formed a methyl ester (compound 6am) by treatment with diazomethane. We considered that the C-18 acid must be formed by oxidation of the corresponding alcohol in 4 (Scheme 2), and consequently assigned the structure of 13,17-dihydroxystemodan-18-oic acid (6) to the original metabolite formed in the biotransformation. Compound 8 was obtained as its triacetate 8a, the mass spectrum of which showed a peak at m/z 446.2687 formed from the molecular ion by loss of water. Thus, its molecular formula was determined as C 26 H 40 O 7 . Its NMR spectra showed two -CH 2 OAc groups, one corresponding to the acetylated C-17 alcohol of the substrate, and the other, formed by acetylation of an hydroxyl group introduced in the incubation, resonates at  H 3.61 and 3.98 (each 1H, d, J = 10.8 Hz) and at  C 73.3 (t). These signals are characteristic of an equatorial acetoxymethylene group at C-4 [14,15]. Other signals observed in the spectra of 8a were those of an oxymethine group at  H 5.36 (t, J = 7.6 Hz) and  C 71.7(d). These chemical shifts and couplings were analogous to those of an 11-acetoxy derivative described in the biotransformation of the substrate 3 by M. plumbeus [13]. The HMBC experiment of 8a confirmed these assignments with the following crosspeaks: H-11 with C-8; H-18 with C-3, C-4 and C-5; H-19 with C-3, C-4, C-5 and C-18; H-20 with C-1, C-5, C-9 and C-10. Thus, the structure 11β,13,17,18tetrahydroxystemodane was assigned to the metabolite 8 (Scheme 2) obtained in this fermentation.
Acetylation and chromatography of the fractions containing 9 led to the triacetate 9a, which is an isomer of 8a. In addition to the signals of the 17-CH 2 OAc, in the 1 H-NMR spectrum of the triacetate 8a another acetoxymethylene group was detected at  H 3.67 and 3.88 (each 1H, d, J = 11.6 Hz) and  C 65.9(t), which was assigned to C-4 with an -equatorial configuration. Thus, in the HMBC experiment the main observed correlations were: H-3 with C-1, C-2, C-4, C-18 and C-19; H-18 with C-3, C-5 and C-19; H-19 with C-3, C-4, C-5 and C-18. These crosspeaks also showed that another acetoxy group was located at C-3, with resonances of this oxymethine at  C 74.6 and  H 4.78 (dd, J = 11.7 and 4.1 Hz). The coupling constant of this geminal proton to this acetoxy group indicated a -equatorial configuration for this oxygenated function. Consequently, the structure of the original alcohol formed in the feeding was determined as 3,13,17,18-tetrahydroxystemodane (9).

General Procedures
1 H-and 13 C-NMR spectra were recorded at 500.13 and 125.03 MHz, respectively, in a Bruker AMX-500 spectrometer. Mass spectra were taken at 70 eV (probe) in a Micromass Autospec spectrometer. HPLC was performed using a Beckman System Gold 125P. Purification by HPLC was achieved using a silica gel column (Ultrasphere Si 5 m, 10 × 250 mm). Dry column chromatography was carried out on silica gel Merck 0.040-0.063 mm.

Microorganism
The fungus strain Cephalosporium aphidicola IMI 68689 was a gift from Prof. J. R. Hanson, School of Chemistry, University of Sussex, UK.