The biotransformation of ent-kaur-16-en-19-oic acid by Rhizopus stolonifer E.A. Silva a , J.A. Takahashi a, *, M.A.D. Boaventura a , A.B. Oliveira b a Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Minas Gerais, Av. Presidente Antoˆnio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil b Departamento de Produtos Farmaceˆuticos, Faculdade de Farma´cia, Universidade Federal de Minas Gerais, Av. Olega´rio Maciel, 2360, 30180-112 Belo Horizonte, MG, Brazil Received 28 August 1998; received in revised form 21 February 1999 Abstract Microbial transformation of ent-kaur-16-en-19-oic acid was carried out with R. stolonifer. After seven days of incubation, two metabolites, ent-7a-hydroxy-kaur-16-en-19-oic acid and ent-12b-hydroxy-kaur-9(11),16-dien-19-oic acid, were isolated as a result of hydroxylation and hydroxylation/dehydrogenation, respectively. Incubation for 15 days also aorded ent-16b,17-dihydroxy- kauran-19-oic acid. The metabolites were identified by spectroscopic methods. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Rhizopus stolonifer; Microbial transformation; Ent-kaur-16-en-19-oic acid 1. Introduction Microorganisms are able to transform a huge variety of organic compounds, such as hydrocarbons, terpe- noids, steroids, alkaloids, antibiotics and amino-acids (Kieslich, 1976). Many compounds of therapeutic and/ or industrial interest are obtained by microbial trans- formation (Turner, 1994). Ent-kaur-16-en-19-oic acid (1), a diterpene that occurs in some species of the Brazilian flora of Xylopia (Annonaceae) (Takahashi, Boaventura, Bayma & Oliveira, 1995) and Wedelia (Asteraceae) (Yang et al., 1986), is known to have plant growth stimulating activity (Eall, Yeung & Pharis, 1996). Derivatives of 1 possess other biological activities, such as antimicrobial (Davino, Giesbrecht & Roque, 1989), antitumoral (Kupchan, Court, Dailey, Gilmore & Bryan, 1972), trypanosomicidal (Costa, Albuquerque & Vichnewski, 1996) and anti-HIV (Wu et al., 1996). As part of a program of diterpenoid transformation by microorganisms, we carried out the transformation of ent-kaur-16-en-19-oic acid (1), using R. stolonifer. 2. Results and discussion Incubation of 1 with R. stolonifer for seven days yielded compounds 2 and 3, the former being the major product (4.7%). The IR spectrum of 2 showed typical bands for carbon–carbon double bond stretch- ing (1650 cm 1 ), carboxyl (1700 cm 1 ) and hydroxyl (3480 cm 1 ) groups. 1 H NMR and 13 C NMR spectra of 2 showed signals corresponding to the same func- tional group at C-19 and to the C-16 double bond in comparison with starting material. New signals at d H 3.63 and d C 77.12 indicated that a hydroxylation had occurred. Assignments of the 13 C NMR signals for compound 2 indicated that the hydroxyl group was introduced at C-7. This was confirmed by the down- field shifts of C-6 and C-8 signals and the upfield shifts of C-5, C-9 and C-15 due to the g-gauche eect. The axial hydroxylation at C-7 was identified by the g- gauche eect (Hasan, Healey & Waterman, 1982; Hutchison, Lewer & Macmillan, 1984). Thus, g-gauche interactions observed between the hydroxy substituent Phytochemistry 52 (1999) 397–400 0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(99)00219-8 * Corresponding author.