Backbone Rearrangement and Novel Derivatives from the Biotransformation of Trachyloban-19-oic Acid by Rhizopus stolonifer

O ácido traquilobânico (2) foi incubado com Rhizopus stolonifer, com o objetivo de se sintetizar novos derivados diterpênicos. Após vinte dias, quatro metabólitos foram extraídos, isolados e caracterizados, dos quais dois apresentaram-se hidroxilados nas posições 7β (3) e 17 (5). Dois derivados rearranjados também foram isolados, os ácidos ent-16β-hidroxi-caur-11-en-19-óico (4) e 9α,16β-diidroxi-caur-11-en-19-óico (6). O rearranjo do esqueleto traquilobânico levando a derivados do tipo ent-16β-hidroxi-caur-11-eno é condizente com a proposta biogenética que limita a ocorrência deste grupo raro de diterpenos caurânicos somente em plantas contendo representantes com esqueleto traquilobânico. Nenhum relato anterior sobre o isolamento dos diterpenos 5 e 6 foi encontrado na literatura.


Introduction
Microbial transformation of organic compounds is a valuable synthetic biocatalytic methodology to carry on regio-and stereoselective reactions.It is one of the few methodologies available for the functionalization of inactivated carbon atoms making possible to carry on reactions that have no equivalent in conventional chemistry.Steroid and terpenoid bioconversions, particularly hydroxylation, is an area in which biocatalysis is very useful.We have been exploring this methodology for structural modification of abundant kaurane and trachylobane diterpenoids, such as ent-kaur-16-en-19-oic 1 (1) and trachyloban-19-oic acids (2).These compounds and several relatives disclose a wide spectrum of biological activities such as anti-microbial, anti-tumor, trypanocidal, antifeeding and anti-HIV. 2,3,4Trachyloban-19-oic acid (2) is a tetracyclic diterpenoid, closely related to the ent-kaur-16-ene series.Its natural occurrence in plants of the genus Xylopia (Annonaceae) 5 and Helianthus (Compositae) 4 is well reported.In this paper we report the hydroxylation of 2 by the biological reagent Rhizopus stolonifer aiming at the preparation of novel compounds for biological screening.Four biotransformated products were formed (3, 4, 5 and 6), the two later being novel diterpene derivatives (5 and 6).Compounds 4 and 5 were isolated and characterized as their methyl esters (4a and 5a) after esterification with diazomethane of a mixture eluted from the column chromatography of crude biotransformation extract.

Results and Discussion
Trachylobanic acid (2) was isolated from seeds of Xylopia sericea and characterized by m.p., IR, 1 H NMR and 13 C NMR (Table 1) spectra. 5Its methyl ester (2a) was prepared, for comparison purposes, from ethereal diazomethane. 13C NMR data for compound 2a are presented in Table 1.After twenty days of incubation of compound 2 with R. stolonifer, the aqueous extract was filtered, extracted and submitted to fractionation over silica gel column.Compounds 3 and 6 were isolated as pure compounds.Compounds 4 and 5 were recovered as a mixture and their separation was carried out by silica gel column chromatography after esterification of the mixture.
Mass spectrum of product 3 showed the M + peak at m/z 318 in accordance with the molecular formula C 20 H 30 O 3 .A peak observed at m/z 300 corresponding to M + -H 2 O was an indication that a hydroxylation of 2 had occurred.The 1 H NMR spectrum of 3 showed typical signals of cyclopropane hydrogens, H-12 and H-13 signals appearing as a double triplet at δ 0.65 (J 7.8 Hz) and a double doublet at δ 0.92 (J 7.8 and 3.1 Hz), respectively.Two singlets were also observed at δ 1.21 and 1.41 corresponding to six and three hydrogens, respectively, which were assigned to the hydrogens of C-17, C-20 and C-18.The triplet at δ 3.86 (J 5.5 Hz) is characteristic of hydrogen in a hydroxylated carbon and it is consistent with the signal observed in the 13 C NMR spectrum at δ74.5.The assignments of the remaining signals were made by analysis of DEPT experiment and comparison with the 13 C NMR spectrum of trachyloban-19-oic acid (2).Disappearance of the signal corresponding to C-7 in the 13 C NMR spectrum, the downfield shift of C-6 and C-8 signals and the γ-gauche effect observed for C-5, C-9 and C-15 indicated that the hydroxylation had occurred at C-7.This was confirmed by a 1 H-1 H COSY experiment that showed correlations of H-7 (δ 3.86) with the methylene hydrogens at C-6 (δ 2.43 and δ 2.45).HMQC experiment permitted the assignments of the most important chemical shifts for identification of 3 as, for example, the correlation of the signals of H-6, H-14 and H-15.NOESY experiment allowed the definition of the β-configuration for the hydroxyl group at C-7 due to the correlations of H-7 (δ 3.86) with H-6 (δ 2.43 and δ 2.45), H-15α (δ 1.74) and H-14β (δ 1.30).The spectral data for 3 are in accordance with the literature. 6ompounds 4 and 5 were isolated as their methyl esters (4a and 5a) after esterification from diazomethane of a fraction obtained from a column chromatography of the crude biotransformated product.Comparison of the spectral data for 4a with those of methyl trachyloban-19oate (2a) showed only few similarities and, for explaining this difference, a rearrangement, leading to a molecule with a kaurene skeleton was proposed.The molecular formula C 21 H 32 O 3 was deduced from the MS molecular ion [M + 332] of 4a.Its 1 H NMR spectrum showed three methyl singlets at δ 0.90, δ 1.19 and δ 1.58 as well as a double doublet at δ 5.58 (J 9.8 and 3.7Hz) and a triplet at δ 6.05 (J 8.0Hz) corresponding to two olefin methinic hydrogens.The multiplicities observed for the olefinic signals indicate the occurrence of a dehydrogenation between C-11 and C-12.A singlet at δ 3.61 was assigned to the hydrogens of the methoxyl group and a double doublet at δ 2.52 (J12.4 and 3.0 Hz) corresponds to the signal of H-13.The 13 C NMR spectrum for 4a showed signals of two olefinic carbons (δ 127.0 and δ 133.7) confirming that a dehydrogenation had occurred in the molecule of 2. No signal that could be assigned to hydrogen bounded to a hydroxylated carbon was found in the 1 H NMR spectrum, but the presence of a novel signal at δ 82.9 was observed in the 13 C NMR spectrum and indicated that a quaternary carbon was linked to a hydroxyl group. 7,8The assignments of the remaining carbons resulted from the analysis of a DEPT experiment (Table 1).MS showed a peak at m/z 314 [M + -H 2 O] confirming the presence of a hydroxyl group.A typical peak of 16-hydroxy-kaurenes is the m/z 274 [M + -58] that is formed from the molecular ion by cleavage between C-13 and C-16 with the migration of the H NMR spectrum of the third metabolite, the methyl ester 5a showed signals at δ 0.98 and δ 1.19, characteristic of cyclopropane hydrogens in a trachylobane skeleton.Two singlets at δ 0.86 and δ 1.16 (H-18 and H-20, respectively), and a singlet at δ 3.62 (OMe) were also observed.A double doublet centered at δ 3.94 (J 11.0 and 8.0 Hz) suggested the presence of a hydroxyl group in this molecule.This proposal was supported by the 13 C NMR spectrum and DEPT experiment that exhibited a signal at δ 66.2 characteristic of a carbon of a primary alcohol.HMQC experiment showed correlation of the hydrogen signal at δ 3.62 with the carbon signal at δ 50.8, as observed for methyl trachyloban-19-oate (2a) and a downfield shift of the C-16 signal (from δ 22.4 to δ 30.6) (Table 1).NOESY experiment confirmed the localization of the hydroxyl group at C-17 by showing correlation of H-17 (δ 3.94) with H-12 (δ 0.98), H-13 (δ 1.19), H-15α (δ 1.58) and H-15β (δ 1.86).Structure 5a was then proposed and it is in accordance with the peaks observed in MS.The peak at m/ z 313 was formed by loss of one molecule of water and a hydrogen atom.Peaks at m/z 298 [313 -CH 3 ] and m/z 254 [313 -CO 2 CH 3 ] and m/z 238 [298 -HCO 2 CH 3 ] were also observed.Loss of HCO 2 CH 3 gave a peak at m/z 272 that, after a rearrangement with loss of one molecule of water and a hydrogen atom, gave a peak at m/z 253.The later, by loss of a CH 3 group led to the peak at m/z 238.
Analysis of the fourth metabolite ( 6) was based on IR, MS, 1 H NMR, 13 C NMR data, DEPT, COSY and HMQC experiments.They indicated that 6 was structurally related to compound 4. Its IR spectrum showed typical absorptions for hydroxyl (3400 cm -1 ) and carboxyl (1690 cm -1 ) groups.The 1 H NMR spectrum presented singlets for three methyl groups at δ 1.39, δ 1.48 and δ 1.55 that are characteristic of H-20, H-18 and H-17, respectively.The broad doublet at δ 5.72 (J 9.9 Hz) and the broadened double doublet at δ 6.04 (J 9.7 and 6.7 Hz) are due to the olefinic hydrogens, H-11 and H-12, respectively, as confirmed by the correlations (COSY) observed among H-12 (δ 6.04), H-11 (δ 5.72) and H-13 (δ 2.50).The 13 C NMR spectrum and DEPT experiment indicated that modifications had occurred on B, C and D rings (Table 1) and that both hydroxylation and rearrangement had occurred in rings C and D. The signals at δ 81.0 and δ 79.6 of the 13 C NMR spectrum are characteristic of sp 3 -oxygenated carbons and were not observed in the DEPT experiment, indicating that the hydroxyl groups were bound to quaternary carbons.The signal at δ 81.0 was assigned to C-16, as for compound 4a, while the signal at δ 79.6 was attributed to C-9 since this signal has appeared in substitution to the corresponding one (δ 61.6) in the spectrum of compound 4a.The deshielding effect of the hydroxyl group reflected at C-8 and C-10 that, as expected, presented higher δ values in comparison with 4a.The stereochemistry of the hydroxyl group at C-9 was defined as β due to the γ-gauche effect observed for C-1, C-5 and C-7.MS supports the structure proposed for 6.Peak at m/z 334 (M + ) was compatible with the molecular formula C 20 H 30 O 4 .The formation of peaks at m/z 316 and 298, by successive loss of two molecules of water, confirmed the presence of two hydroxyl groups in the molecule.The peaks at m/z 288 and 270 were consecutively formed by loss of HCO 2 H and water.The peaks at m/z 276 and 230 were due to a rearrangement, loss of a 2-propenol molecule, followed by loss of HCO 2 H. Methyl ester 6a has been reported by the literature, 10 but no mention to the isolation of 6 was found.
From the biosynthetic point of view, it is interesting to note that ent-16β-hydroxy-kaur-11-ene derivatives are rare in nature and have only been found in plants from which diterpenes with a trachylobane skeleton have also been isolated. 7,8Thus, compounds 4 and 6 have probably been formed by enzymatic abstraction of hydrogen at C-11, assisting the cleavage of the cyclopropane ring and forming a carbenium ion that, after neutralization with water, gave products 4 and 6.

Conclusion
Hydroxylation of inactivated carbons remains the most explored area in microbial transformation of organic compounds.This reaction makes possible the preparation of countless novel diterpenoids derivatives inaccessible by chemical means.Novel successful biotransformations, such as the trachylobanic acid (2) rearrangement we are describing bring about novel reactions that can be largely explored for a shorter synthesis of new chemical compounds of biological interest.

Experimental Procedures
Melting points were determined on a Metler FPS apparatus and are uncorrected.IR spectra were recorded on a Shimadzu/IR-408 spectrophotometer.Crystalline samples were measured in KBr discs (1.0 mg%).IR absorption bands were expressed in cm -1 . 1 H NMR spectra were recorded in CDCl 3 and C 6 D 6 at 400MHz (Bruker Avance DPX 400), at room temperature, with TMS as internal standard. 13C NMR spectra were determined at 100 MHz, in CDCl 3 and C 6 D 6 soln.. Assignments of chemical shifts were made with the aid of distortionless enhancement by polarization transfer (DEPT) using a flip angle of 135° and bidimensional experiments (NOESY, COSY and HMQC).MS were determined on an Autospec model VG spectrometer.Silica used for flash chromatography was Merck 230-400 mesh.

Isolation of 2
Trachyloban-19-oic acid (2) was isolated from a hexane extract of Xylopia sericea seeds by silica gel column chromatography. 5
Media and culture conditions.PDA medium containing potato infusion (200 g), dextrose (20 g), agar (15 g) at pH 6.5 was used for storage of R. stolonifer.For the biotransformation experiments, a medium composed of dextrose (20 g), peptone (5 g), yeast extract (3 g), KH 2 PO 4 (5 g) at pH 5.6 in H 2 O (1 L) was used.The microorganism was grown by a two-stage fermentation procedure.Erlenmeyer flasks (500 mL) containing 100 mL of medium were inoculated with a suspension of R. stolonifer.Incubations were maintained at 28° in a rotary shaker, operating at 150 rpm for 48 h.An aliquot (3.0 mL) was used to inoculate similar flasks that were incubated under the same conditions.After 24 h of incubation, a solution of substrate 2 (0.8g) was added in dimethylformamide (DMF) to a final concentration of 0.1 g L -1 and incubation was continued for twenty days.
Extraction, purification and analysis of microbial transformation products.After twenty days of incubation, the cultures were filtered off and the aqueous layer was extracted with EtOAc (3 x 200 mL).The extracts were combined, dried over anhydrous sodium sulphate, filtered and the solvent was removed under reduced pressure, in a rotary evaporator, to give 1.2 g of residue.Flash column chromatography afforded 3 (19.8mg, 2.4%) and 5 (34.0 mg, 4.2%) by elution with n-hexane/EtOAc (8:2).
The residue obtained from some fractions (15.0 mg) was esterified with diazomethane in ethyl ether and the reaction product was submitted to silica gel column chromatography leading to the separation of 4a (6.0 mg, 0.74%) and 5a (5.0 mg, 0.62%).