The possibility of transforming betulone formed by bio-oxidation of betulin by intact R. rhodochrous IEGM 66 cells was studied. The corresponding mono- and diglutaryl derivatives were prepared from betulone and its oxime. Beckmann fragmentation of 28-benzoyloxy-3-hydroxyiminolup-20(29)-ene synthesized 3,4-seco- lupane derivatives. Polycyclic triterpenoids with an oxidized 3-hydroxyl are convenient starting platforms for preparing semi-synthetic A-seco-triterpenoids and derivatives with an annelated heterocyclic moiety, many of which exhibit antiviral or cytotoxic activity. The structure of the pentacyclic triterpenoid betulin, which is commonly used to develop new pharmacologically active derivatives, includes two hydroxyls, a primary one on C-28 and a secondary one on C-3 (1, 2). Chemical approaches enabling oxidation of the secondary 3-hydroxyl of betulin to form betulone were reported. However, as a rule, traditional oxidants formed mixtures of products (3) whereas a more selective three-step synthesis of betulone included additional introduction/removal steps for protecting the primary hydroxyl of betulin (4). Biocatalytic technology for one-step oxidation of structurally complicated terpene substrates is being actively pursued as an alternative to chemical methods (5). Thus, a one-step method for bioconversion of betulin into betulone using intact mycelial fungal cells was reported. Application of them for preparative purposes was limited by the low betulin concentration and its low conversion yields (6, 7). Recently, we demonstrated that actinobacteria of the genus Rhodococcus could be used as effective biocatalysts for biotransformation of betulin (1) into betulone (2) and proposed a biocatalytic method for preparing betulone using dormant IEGM 66 R. rhodochrous cells (8, 9). Herein the synthesis of new derivatives from betulone, obtained by a one-step regioselective biotransformation of betulin, is reported. A suspension of intact IEGM 66 R. rhodochrous cells in phosphate buffer was used for preparative biocatalytic oxidation of betulin over 24 h (9). The target compound 2 (36.8% yield) was isolated by flash chromatography from the obtained extract of the biotransformation products. Reaction of 2 with 3,3-dimethylglutaric acid anhydride produced ester 3 (58% yield). The PMR spectrum of 3 showed a weak-field shift of the doublets for the C-28 methylene protons (3.84 and 4.27 ppm) compared with those of starting 2 and characteristic resonances for the 3,3-dimethylglutaric acid. The 13 C NMR spectrum of 3 exhibited resonances at 173.76 and 183.25 ppm for the ester and carboxyl C atoms. Treatment of ester 3 or the mixture of biotransformation products with hydroxylamine hydrochloride in EtOH:Py produced oximes 4 or 5 in yields of 69 and 24%, respectively. IR spectra of oximes 4 and 5 showed strong bands at 1640-1642 cm -1 for hydroxylamine C=N stretching vibrations. The C-3 resonance in 13 C NMR spectra was observed at 167.13-168.61 ppm. Reaction of oxime 5 with 3,3-dimethylglutaric acid anhydride afforded diester 6. The doubled integrated intensity of the corresponding proton resonances at 2.42-2.46 ppm confirmed that 6 contained two 3,3-dimethylglutaric acids.