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Eclética Química

Print version ISSN 0100-4670On-line version ISSN 1678-4618

Eclet. Quím. vol.34 no.1 São Paulo  2009 

Di-And Tri-Hydroxylated Kaurane Derivatives From Microbial Transformation Of Ent-Kaur-16-En-19-Ol By Cephalosporium Aphidicola And Their Allelopathic Activity On Lactuca Sativa (Lettuce)



D. Rocha, J. A. Takahashi; M. A. D. Boaventura*

Departamento de Química, ICEx, Universidade Federal de Minas Gerais, Belo Horizonte, MG, CEP 31270-901 , Brazil




The use of microorganisms to induce chemical modifications in organic molecules is a very useful tool in organic synthesis, to obtain biologically active substances. The fungus Cephalosporium aphidicola is known by its ability to hydroxylate several skeleton positions of many classes of organic compounds. In this work, the microbial transformation of ent-kaur-16-en-19-ol (1) by C. aphidicola, afforded two hydroxylated compounds, ent-kauran-16β,19-diol (2) and ent-kauran-16β,17,19-triol (3). Their structures were established by 1D and 2D-NMR studies. Both compounds were tested for their action on the growth of radical and shoot of Lactuca sativa.

Key words: Biotransformations, Cephalosporium aphidicola, ent-kaur-16-en-19-ol, ent-kauran-16β, 19-diol, ent-kauran-16β,17,19-triol.




Biotransformations use living organisms or isolated enzimes to modify chemical structures and have some advantages over conventional chemical reactions [1]. This methodology have been showing a quick development along the time and represents a potential alternative for obtaining biologically active compounds of difficult access by classic synthetic methods [2,3].

Kaurane diterpenes are widely distributed in nature [4-6] and present very important biological activities, such as antitumor [7] anti-HIV [8], trypanocidal [9-10] antimicrobial [11] and allelopathic [12-13], among others. Ent-kaur-16-en-19-oic and ent-kaur-9(11),16-dien-19-oic acids and their chemical transformations derivatives are very used substrates for biotransformations [14-17]. Kaurane diterpenes are also intermediates in the biosynthesis of a number of plant and fungal metabolites, including gibberellins, a group of diterpene lactones, as GA3, used in agriculture to stimulate plant growth. Allelopathic activity of ent-kaur-16-en-19-oic and ent-kaur-9(11),16-dien-19-oic acids and derivatives obtained by chemical transformations of them were previously evaluated by our research group [12-13].

The fungus C. aphidicola promotes hydroxylations in different classes of organic compounds and has been widely used in biotransformations of kaurane diterpenes [18-20].

In this work, we describe the biotransformation of ent-kaur-16-en-19-ol (1) by C. aphidicola. Two hydroxylated compounds, ent-kauran-16β, 19-diol (2) and ent-kauran-16β,17,19-triol (3), were isolated (Scheme 1) and tested over their action on the growth of radical and shoot of Lactuca sativa (lettuce). This is the first time these compounds were obtained from this substrate using C. aphidicola. This is also the first report on their allelopathic activity by the best of our knowledge.


Materials And Methods


Solvents (hexane, dichloromethane, ethyl acetate, methanol) from VETEC (Brazil), and were of PA grade. Silica gel Merck (Darmstadt, Germany) 70-240 and 230-400 mesh were used for chromatography column and silica gel Merck 60G was used for thin layer chromatography. Reagents to prepare the culture medium were purchased from Difco (Sparks, MD, USA). Melting points were determined with a Kofler hot plate apparatus and are uncorrected. Nuclear Magnetic Resonance (NMR) spectra (1D and 2D) were recorded in CDCl3, at room temperature, on a Bruker Avance DRX 400 MHz, from Bruker Analytic, Ettlingen, Germany. Ent-kaur-16-en-19-ol (1) was furnished by Dr. Henriete S. Vieira, Departamento de Química, ICEx, UFMG, Brazil.


Stock culture of C. aphidicola (CCT 2163) was maintained on PDA under refrigeration and small sections of this agar were transferred to Erlenmeyer flasks containing a liquid medium (200 mL/flask) comprised of: glucose (50.0 g/L), NaH2PO4 (5.0 g/L), MnSO4.6H2O (2.0 g/L), KCl (1.0 g/L), glicine (2.0 g/L) and 2.0 mL of trace elements solution [CO(NO3)2, 0.10 g/L, FeSO4, 1.0 g/L, CuSO4, 0.15 g/L, ZnSO4, 1.61 g/L, MnSO4, 0.10 g/L, (NH4)6Mo7O24 .4H2O, 1.0 g/L] of distilled water; pH was adjusted to 5 with 10% HCl solution and the culture was incubated over magnetic stirring for 3 days. Cells in suspension (15 mL portions) were transferred to new Erlenmeyer flasks and reached suitable growth after 24 h. Then, the substrate 1 (0.69 g) was added (0.5 mL/flask, 53 mg/mL). After 13 days, the flasks contents were extracted with ethyl acetate and the solvent was taken out under vacuum. Residue (1.37 g) was chromatographed over silica gel (21.5 g), using hexane, CH2Cl2, AcOEt and CH3OH, either neat or in mixtures of increasing polarity. A group of fractions (G-7, 81.5 mg), eluted from CH2Cl2/AcOEt 1:1, was rechromatographed over silica gel (3.2. g), using the same solvent system. G-7-2 (25.0 mg) gave an amorphous white solid [3.7 mg, ent-kauran-16β,19-diol, (2)], corresponding to a single spot by TLC (hexane/AcOEt 75:25), by another silica gel column chromatography. G-7-3 (17.0 mg), after new purification by silica gel column chromatography, gave a crystalline white solid [12.8 mg, ent-kauran-16β,17,19-triol (3)], also pure by TLC (eluents: hexane/AcOEt 70:30).


Lactuca sativa (cv Grand Rapids) seeds were purchased from Isla Pak, RS, Brazil. All undersized and damaged seeds were discarded. Germination and growth were conducted in 100 mm Petri dishes containing a 9.0 cm sheet of Whatman no. 1 filter paper as suport. Then, 25 lettuce seeds were placed per dish with 10 mL of a test (10-4, 10-6 and 10-8 M) or a control solution. All solutions were prepared with deionized water and their pH values [buffered with 10 mM 2-(N-morpholino) ethanesulfonic acid, MES] were adjusted to 6.0 - 6.5 with NaOH solution. Concentrations lower than 10-4 M were obtained by dilution series. All tests were triplicated. Dishes were covered with Parafilm to reduce evaporation and incubated in the dark at 25 ºC, in a controlled-environment growth chamber, for 5 days. After this time, number of germinated seeds were counted (a seed was considered to be germinated when the radicle was at least 0.2 mm long), the lengths of radical and shoots were measured (using a pachymeter). During the measurement process, the dishes were kept at 4 ºC to avoid subsequent growth. The osmotic pressure values were measured on a microsmometer and ranged between 30 and 38 mOsmolar [15].

Data Analysis

The effects on germination and growth are given as percent differences from control, and consist of the differences (in cm) between mean values of seeds with tested compounds and mean values for control (seeds grown without addition of tested compounds)/ mean values for control x 100. Thus, zero represents the control, positive values represent stimulation of the studied parameter and negative values represent inhibition.

The data were evaluated using Student's t-tests and the differences between the experiment and control were significant at a value of P<0.05.

The results obtained for compounds 2 and 3 are shown in Figure 2.



Results and Discussion

Compounds 2 and 3 were identified by 1D and 2D 1H and 13C- NMR as products of mono and di-hydroxylation, respectively, of the original C16-C17 double bond of substrate 1, since in the 1H- NMR spectrum of both products, the unsaturated exocyclic methylene signals, present on starting material at δH 4.73 and 4.79 were not observed . Also, this spectrum showed the signals for C-19 hydroxymethyl AB system for both products, those of 2 at δH 3.44 (1H, d, J=11.2 Hz) and 3.72 (1H, d, J=11.2 Hz). The 1H NMR of 2 exhibited signals for two methyl groups at δH 0.95 (3H, s, H-18) and 1.01 (3H, s, H-20) and for a new methyl group at δH 1.35. 13C-NMR spectrum and DEPT experiment showed a chemical shift at δC 65.6, of the C-19 oxygenated methylene carbon, from the starting material. Additionally, signals for nine methylene carbons, three methine carbons, and four quaternary carbons (including a new oxygenated carbon at δC 79.3) were observed. In order to carry on a more detailed spectroscopic analysis of the products, bidimensional spectra (HSQC, HMBC and ROESY) were obtained for 2, since they are of special help for complete attribution of terpene compounds [21]. HSQC spectrum showed the correlations between the signal at δH 1.82 with δC 49.0, attributed to C-13, by comparison to the spectroscopic data of the starting material; the signals at δH 1.43 and 1.48 were correlated to a signal at δC 18.0 (C-11); the signals at δC 1.57 and 1.88 to a signal at δC 37.5 (C-14). A correlation of the signal at δH 0.99 with the signal at δC 57.0 (C-9) was also observed. The signals corresponding to methine positions 5, 9 and 13 appeared at δH 0.91 and δC 56.8; at δH 0.99 and δC 57.0; and at δH 1.82 and δC 49.0, respectively. Also, the signal at δH 1.35 (3H) was correlated, in the HSQC spectrum, to a signal at δC 24.4, and was associated with C-17, since this was the only option for the introduction of an additional methyl group in the molecule, and consequently the new oxygenated quaternary carbon signal observed at δC 79.3 can be associated to C-16.

In the HMBC spectrum, 3J correlations between H19β (δH 3.72) with C-18 (δC 27.0) and between H19β (δH 3.44) with C-3 (δC 35.7) were observed. Besides, correlations between hydrogens of the methyl group at C17 (δH 1.35) and C13 (δC 49.0), C15 (δC 57.9) and C16 (δC 78.3) were further evidence of the presence of a methyl group at C-17. Therefore, 2 was identified as the product of monohydroxylation of the original exocyclic double bond from starting material (Table 1).



The configuration of C-16 was established by analysis of ROESY spectrum, where the correlation of CH3-20 (δH 1.01) with H-14α, at δH 1.57, was observed. Thus, by using HSQC spectrum, the signal of H-14β was located at δH 1.88. No ROESY correlation was observed between H-14β and H-17 (δH 1.35). Also by ROESY spectrum, the signal related to H-11α was found to be δH 1.43, due to its correlation with H-9 (δH 0.99) therefore by HSQC spectrum H-11β (δH 1.48) was located and, as this signal correlates with CH3-17 (δH 1.35) in the ROESY spectrum, the later is unequivocally at the α-position (Figure 1). Thus, 2 was proposed to be ent-kauran-16β,19-diol. This compound was already isolated as natural product from stems of Aristolochia rodriguesii [22] and leaf and stems of Ozothamnus hookeri (Asteraceae) [23]; 2 was also obtained by chemical modification of ent-kaur-16-en-19-oic acid [2].

NMR spectra of product 3 were not consistent with the presence of a methyl group at C-17, since only a new signal in 1H- NMR spectrum at δH 4.03 (2H) was observed. Two new oxygenated carbon signals were detected in 13C NMR spectrum, at δC 78.7 and 71.8 (quaternary and methylene, respectively, according with DEPT experiment). HSQC spectrum presented correlations between the signal at δH 0.95 with δC 57.1, associated with C-9; at δH 1.36 and 1.48 with δC 18.1 (C-11); at δH 1.57 and 1.84 with δC 37.5 (C-14). This spectrum also showed the correlation between the signal at δH 4.03 with the signal at δC 71.8. Since, the signals related to the exocyclic double bond of starting material are not present in the NMR spectra of 3, these signals can be only associated to a hydoxymethyl group at 17 position. Therefore, the signal related to a quaternary carbon, at δC 78.7, was attributed to C-16. The β-hydroxy stereochemistry of C-16 was defined, as for 2, based on a ROESY experiment (Figure 1), mainly due to the correlation observed between H-11β (δH 1.48) and 17-CH2OH (δH 4.03). Compound 3 was, then, proposed to be the ent-kauran-16β,17,19-triol. This compound was already isolated as natural product from leaf and stems of Ozothamnus hookeri (Asteraceae) [23], from the fern Lindsaea javanensis, as 19-O-β-D-glucopyranoside [24] and from aerial parts of Bahia glandulosa, as the 19-acyl derivative [25].

The effect of compounds 2 and 3 on radical and shoot growth of L. sativa, in three different concentrations, was tested and the results are shown in Figure 3. Both diol and triol inhibited radical growth in all three concentrations, and the best result was observed for compound 2 at the concentration of 10-4 M. On shoot growth, the effect of the tested compounds was stimulatory at the concentrations of 10-4 and 10-6 M. At 10-8 M, both biotransformation products 2 and 3 inhibited shoot growth. The best growth stimulatory concentration was 10-6 M. With kaurenol (1), the compound used to provide derivatives 2 and 3, radical and shoot growth were also inhibited at the higher concentration, while stimulation of both occurred at middle concentration [14].



The authors thank CNPq for ADR, JAT and MADB grants, to FAPEMIG and to IFS for financial help.


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Received November 15 2008
Accepted December 20 2008




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