Pergamon PII: S0031-9422(96)00845-X Phytochemistry, Vol. 45, No. 2, pp. 283-291, 1997 © 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0031-9422/97 $17.00+0.00 BIOTRANSFORMATION OF ENT-6o~-ACETOXY- AND ENT-6- KETOMANOYL OXIDES WITH RHIZOPUS NIGRICANS AND CUR VULARIA L UNA TA CULTURES ANDI~S GARCiA-GRANADOS,* JUAN J. GUTII~RREZ,ANTONIOMARTiNEZ, FRANCISCO RIVASand JOSI~M. ARIAS~ Instituto de Biotecnologia, Grupo de Investigaci6n en Biotecnologia y Quimica de Productos Naturales. Departamento de Quimica Org~nica, Facultad de Ciencias, Universidad de Granada, Granada, Spain; l"Departamento de Microbiologia, Facultad de Ciencias, Universidad de Granada, Granada, Spain (Received in revised form 10 October 1996) Key Word Index--Rhizopus niyricans; Curvularia lunata; biotransformation; diterpenes; ent- manoyl oxides. Abstract--Microbial transformation of ent-16,18-dihydroxy-6-oxo-13-epi-manoyl oxide by Rhizopus niyricans produced ent-1 lfl-hydroxy, ent-20-hydroxy and ent-3fl-hydroxy derivatives. Biotransformation of ent-16,18- dihydroxy-6-oxomanoyl oxide gave ent-I lfl-hydroxy and ent-3fl-hydroxy derivatives. However, biotransfor- mations of these substrates with Curvularia lunata produced only ent-1 lfl-hydroxy derivatives. Incubation of ent-18-hydroxy-6-oxo-13-epi-manoyl oxide with R. nigricans produced 14,15-epoxy derivatives with and with- out hydroxylation at C-3, whereas microbial transformation of its epimer at C-13 gave ent-11 fl-hydroxylated derivatives. © 1997 Elsevier Science Ltd. All rights reserved INTRODUCTION In recent years, microbial biotransformation pro- cesses have been used to introduce hydroxyl groups at difficult positions on diterpenoid compounds [1]. Chemical-microbiological methods constitute an alternative way to obtain new polyoxygenated com- pounds from abundant natural products. Highly func- tionalized manoyl oxides, mainly forskolin (1), have shown interesting biological properties [2, 3]. We are currently assaying an extensive series of chemical- microbiological routes of semisynthesis of differently functionalized manoyl oxides of the enantio series with both configurations at C-13. In previous papers we reported the biotransformations of ent-manoyl oxides functionalized at C-16 and C-18 with Rhizopus niy- ricans [4-6], Curvularia lunata [7, 8], Fusarium mon- iliforme [9] and Cunninyhamella elegans [9]. Some of the manoyl oxides obtained show interesting bio- logical activities [6, 8]. To complete these studies, we now describe the biotransformation with Rhizopus nigricans and Curvularia lunata of other ent-manoyl oxides epimers at C-13 functionalized at C-6, C-16 and C-18 or C-6 and C-18. The starting material was andalusol (2) [I0]. Acetylation of this product produced 6,18-diacetyl- *Author to whom correspondence should be addressed. andalusol (3) [11]. Treatment of product 3 with MCPBA yielded ent-manoyl oxide epimers at C-13 with a hydroxyl group at C-16 (4 and 5) [5]. On the other hand, treatment of product 3 with TiCL pro- duced ent-manoyl oxides without functionalization at C-16 (6 and 7) [12]. We have studied the influence of these structural differences on the action of the microorganisms. RESULTS AND DISCUSSION The biotransformation of diacetate 6 by Rhizopus niyricans over a period of 3 days produced only 6% of one metabolite, 8. Comparison of its IH NMR spectrum with that of the substrate (6) showed that the typical ABX system had disappeared from the vinyl protons; three signals were seen to be due to protons geminal to an oxygen atom: t5 3.06 (1H, dd, Jl = 4.5, J2 = 3.0 Hz), 2.78 (1H, dd, Jl = J2 = 4.5 Hz) and 2.48 (IH, dd, Jl = 4.5, J2 = 3.0 Hz). These data indicated that the microorganism had accomplished the epoxidation of the double bond of the substrate (6) to give 14,15-epoxy derivative; this process was confirmed by the 13C NMR spectrum. The configuration at C-14 could not be determined from the available data. Metabolite 8 is, therefore, a (13 R) - en t - 6~, 18 - diacetoxy - 8~, 13; 14~, 15 - diepoxy - labdane. 283