Biotransformation of Digitoxigenin by Fusarium Ciliatum

A biotransformação da digitoxigenina (1) por Fusarium ciliatum foi investigada. O cardenolídeo 1 foi obtido a partir da hidrólise ácida da digitoxina e a reação também forneceu os produtos secundários -digitoxigenina (2) e -digitoxigenina (3). A reação de biotransformação foi realizada em um período de 5 dias e três produtos foram isolados e identificados a partir de análises espectrométricas: digoxigenina (4), digoxigenona (5) e digitoxigenona (6). A obtenção dos dois últimos cardenolídeos nas condições empregadas é inédita. A investigação da cinética da reação de biotransformação indicou 6 como o primeiro produto a ser formado, seguido dos derivados hidroxilados 4 e 5, sugerindo o envolvimento de enzima distintas na hiroxilação em 12 e na oxidação em C-3 da digitoxigenina (1). A reação de biotransformação também foi realizada com a digitoxina, mas F. ciliatum se mostrou incapaz de promover sua hidroxilação.


Introduction
Digoxin (Figure 1), a Digitalis cardenolide, is still the drug of choice for the treatment of congestive heart failure, acting as a selective inhibitor of the Na + ,K + ATPase enzyme.The Digitalis cardenolides are divided into 6 series, based on their genin part, termed A through F. Glycosides of the A series, like lanatoside A, are the most abundant ones, whereas C-type compounds, such as lanatoside C and digoxin, are those clinically used. 1 The steroid nucleus of the C glycosides differ from the A glycosides only at the C-12 position, the former bearing a hydroxyl group.Lanatoside C and its derivative digoxin are industrially obtained from the leaves of Digitalis lanata and lanatoside A constitutes a side product.Therefore, it is of interest to find out a process to convert the A-type cardenolides into the corresponding C-type compounds.Several plant cell cultures have been investigated as a synthetic tool to obtain derivatives of Digitalis cardenolides and the biotransformation of digitoxin into digoxin was successfully accomplished with D. lanata cells in a semi-continuous process. 2 Filamentous fungi usually present rates of biomass growth higher than those of plant cell cultures, what turns out fungal transformations more feasible for large scale applications. 35][6][7] The second species also promoted oxidation of the C-3 OH and digoxigenone was obtained. 6F. lini was also investigated for the biotransformation of digitoxin, without success, 5 whereas no report was found on the transformation of this substrate by F. ciliatum.Since digitoxin bears a sugar chain at C-3, impairing the oxidation of this position, F. ciliatum seems to be a suitable candidate to transform digitoxin into digoxin.Therefore, the main goal of the present work was to study the biotransformation of digitoxigenin (1) by F. ciliatum and to carry out the reaction with digitoxin aiming at the production of digoxin.

Materials and Methods
General 1 H NMR, 13 C NMR, 1 H 1 H COSY and HMQC spectra were recorded on a Bruker DRX-400 spectrometer ( 1 H 400 MHz and 13 C 100 MHz) using TMS as internal standard for both nuclei.Chemical shifts ( ) are given in ppm and J couplings in Hertz (Hz).

Chemicals
Acetonitrile chromatographic grade LiChrosolv and digitoxin were obtained from Merck (Germany).Water was purified using a Milli-Q 50 purification system (Millipore, USA).
The biotransformation of digitoxin (10 mg/flask) was carried out in the conditions described for digitoxigenin (1).The reaction was stopped after 11 days and the products were immediately extracted as previously described.The biotransformation of digitoxin in the presence of digitoxigenin (8 mg and 2 mg/flask, respectively) was also performed.In one of the experiments, the flasks were submitted to sonication for 30 min, prior to incubation, whereas in the other, 0.15% m/m Tween 80 was added to the reaction medium.

Kinetics of digitoxigenin biotransformation
The biotransformation conditions were those previously described.The reaction was monitored by removing portions (2.7 mL) of the culture medium with a syringe, at regular intervals (36, 60, 84, 108, 132 and 156 h) and the cardenolides were analyzed by RP-HPLC, after appropriate extraction.HPLC analysis were carried out in a Merck-Hitachi apparatus (Germany) composed of intelligent pump L-6200A, autosampler AS-2000A, UV-VIS detector L-4250 and integrator D-2500.An ODS column (250 × 4.0 mm I.D., 5 m) was employed (Merck, Germany) with temperature of 40 °C, flow rate of 1.0 mL min -1 and wave length of 220 nm.A gradient elution of H 2 O (A) and 84% aqueous CH 3 CN (B) was performed: 0 min 76% A, 24% B; 16.5 min 58% A, 42% B; 22.5 min 52% A, 48% B; 30 min 28% A, 72% B; 35 min 11% A, 89% B. The identification of the components was accomplished by comparing the retention time of the products with those of the isolated cardenolides, injected in the same conditions.

Results and Discussion
Digitoxigenin (1), employed as substrate for biotransformation in the present work, was obtained by the acid hydrolysis of digitoxin under previously described conditions, 8 with some modifications (Figure 2).The temperature of the hydrolysis reaction was held at 55 °C, for 35 min, to minimize the formation of the side product digitoxigenin-monodigitoxoside.After isolation and purification of the obtained digitoxigenin (1) by preparative RP-HPLC, its purity and authenticity was attested by NMR analysis and by data comparison with authentic samples (Table 1).
Besides digitoxigenin (1) (80%), two other products were obtained from the hydrolysis of digitoxin, resulting from dehydration of the hydroxyl group at C-14 (Figure 2).Proton chemical shifts of compound 2 were generally consistent with those of digitoxigenin (1), the major In a similar way, 13 C NMR spectrum of product 3 revealed the signals of two olefinic non-hydrogenated carbons at 129.7 and 138.3, respectively assigned to C-8 and C-14, which allowed identifying the other dehydration derivative as 8-14 -digitoxigenin (3).
Excepting the diamagnetic shifting observed for H-7 ( 1.90), H-7 ( 2.18) and H-9 ( 2.38), credited to the vicinity of the double bond, all the other chemical shifts were in agreement with those observed for digitoxigenin (1).Hence, these data allowed the unequivocal identification of compound 3 as 8-14 -digitoxigenin.
Three compounds were isolated from the biotransformation of digitoxigenin by Fusarium ciliatum (Figure 3).Structure elucidation was accomplished by 1D and 2D NMR techniques, using digitoxigenin (1) as model compound and also by comparison with spectral data reported for other cardenolides.
The 1 H NMR spectrum of compound 4 showed a complex profile and the only signals easily assigned were those of H-3 ( 4.14), H-21( 4.90 and 4.81) and H-22 ( 5.94), which confirmed the presence of the hydroxyl group at C-3 and the , unsaturated lactone ring.Besides, the singlets at 0.81 and 0.97 were readily assigned to the methyl groups at C-18 and C-19.The signal at 3.4 (J 11.0 Hz and 6.4 Hz, 1H, dd) was assigned to a methine hydrogen bearing a hydroxyl group, thus indicating a hydroxylated derivative.
The hydroxylation position was defined on the basis of the chemical shift changes observed for compound 4 in comparison to digitoxigenin (1).Hence, the resonances of C-18 ( 8.9), H-18 ( 0.81) and H-11 ( 1.27) exhibited diamagnetic shifts in comparison to those registered for digitoxigenin (1) ( 15.7, 0.88 and 1.46, respectively), allowing to locate the hydroxyl group at the 12 position.These shifts were rationalized as resulting from anisotropic effects of the C-23 carbonyl group and the C-20/22 double bond which, in turn, are the outcome of conformational changes of the steroidal frame or the , -unsaturated lactone ring.These data led to the unambiguous identification of 4 as digoxigenin.
Similarly to digoxigenin (4), the 1 H NMR spectrum of 5 showed a signal at 3.47 (J 11.8 Hz and 4.2 Hz, 1H, dd) attributed to a hydroxylated methine group.Likewise 4, the hydroxylation site of compound 5 was deduced from the paramagnetic shifting of the signals from H-18, C-18    process and afforded 100% yield of digoxigenone (5). 6,7  the present work, shortening of the biotransformation reaction time to 5 days afforded the products digoxigenin (4), digoxigenone (5) and digitoxigenone (6) (Figure 3).In order to explain these results, we decided to investigate the biotransformation kinetics of the reaction.Monitoring of the incubation medium by HPLC analysis disclosed digitoxigenone (6) as the first product to be formed (Figure 4), followed by the more polar hydroxylated compounds digoxigenin (4) and digoxigenone (5), that were detected after 24 hours (Figure 5).These data suggest the involvement of different enzymes in the 12 hydroxylation and C-3 oxydation of digitoxigenin (1).After 156 hours, the consumption of the substrate digitoxigenin (1) was almost complete, resulting in the formation of digoxigenin (4).In addition, hydroxylation of the first formed product digitoxigenone (6) occurred in a great extension, leading to the production of digoxigenone ( 5).Although we did not detect the transformation of compound 4 into 5, in our 5 days process, digoxigenone (5) seems to be the final transformation product of digitoxigenin (1) by F. ciliatum, once it has been previously obtained in a 100% yield, employing a 11 days process. 6,7 triangular hydroxylation model has been proposed for the biotransformation of 5-androstanes by F. ciliatum (syn.1][12][13] The 5-androstanes evaluated in those studies possess a hydroxyl or a carbonyl group at C-3 and as a result C-12 and C-15 hydroxylated derivatives were obtained.According to the model,

and H- 11 ,
which indicated a 12 -hydroxylated derivative.Another feature of the 1 H NMR spectrum of 5 was the absence of the characteristic H-3 signal at ca. 4.10 in the spectra of 1 and 4, which pointed out the oxidation of the C-3 OH.This was confirmed by the chemical shifts observed for H-2 ( 2.53), H-2 ( 2.11), H-4 ( 2.78) and H-4 ( 1.95), within the range expected for -carbonyl protons,9 as well as by the carbonyl resonance at 215.7, revealed by the 13 C NMR spectrum.Excepting these signals, the other resonances registered for 5 were in agreement with those obtained for digitoxigenin(1) and confirmed the integrity of the cardenolide skeleton.Based on these findings the structure of 5 was assured as digoxigenone.The carbon-13 and proton chemical shifts observed for compound 6 were generally consistent with those of digitoxigenin (1), excepting the absence of the H-3 resonance signal, likewise 5, which indicated the oxidation of the C-3 OH.This was confirmed by the paramagnetic shifts of the resonances from H-2 ( 2.34), H-2 ( 2.21), H-4 ( 2.62) and H-4 ( 2.13), as a consequence of the vicinity of the C-3 carbonyl.The assignment of C-1 ( 39.8) was accomplished by a HMQC experiment, which disclosed cross peaks with H-1 ( 1.59) and H-1 ( 1.46), whereas the correlation of these hydrogens with H-2 ( 2.34) was revealed by COSY spectroscopy.The oxidation of the C-3 OH was also evidenced by the carbonyl signal at 212.3 in the13 C NMR spectrum, allowing the unambiguous identification of 6 as digitoxigenone.The biotransformation of digitoxigenin (1) by F. ciliatum has been previously carried out on a 11 days