C25 steroid epimers produced by Penicillium janthinellum, a fungus isolated from fruits Melia azedarach

Abstracts

A plant-derived fungus, Penicillium janthinellum, obtained from Melia azedarach, produced ergosterol and ergosterol 5alpha,8alpha-peroxide along with a mixture of rare C25 steroid epimers. The C25 steroids, named neocyclocitrinols, shows exactly the same tetracyclic ring system present in cyclocitrinol, which was isolated from a sponge-derived Penicillium citrinum, with the same bicyclo [4:4:1] skeleton at A/B rings, but showing different side chains. The P. janthinellum was cultivated over white corn and the three steroids were isolated by several silica gel based chromatographic procedures and identified by extensive NMR methods, mainly ¹H - 13C correlations and ¹H - ¹H COSY and TOCSY. The biosynthetic origin of the cyclocitrinols is also discussed.

endophytic fungi; Melia azedarach; Penicillium janthinellum; neocyclocitrinols

O fungo Penicillium janthinellum, obtido de Melia azedarach, produziu ergosterol, ergosterol 5alfa,8alfa-peróxido e uma mistura de esteróides C25 epiméricos de ocorrência restrita na natureza. Os esteróides C25, denominados neociclocitrinóis, possuem o mesmo sistema tetracíclico de anéis presente no ciclocitrinol, o qual foi isolado do fungo Penicillium citrinum, associado a uma esponja, com o mesmo esqueleto biciclo[4:4:1] nos anéis A/B, mas com cadeias laterais diferentes. O P. janthinellum isolado de M. azedarach, foi cultivado sobre milho branco e os três esteróides foram isolados por vários procedimentos cromatográficos e identificados por cuidadosa análise dos dados de RMN, principalmente correlações ¹H - 13C em duas dimensões bem como COSY e TOCSY ¹H - ¹H. A origem biossintética dos ciclocitrinóis é discutida.

ARTICLE

C25 steroid epimers produced by Penicillium janthinellum, a fungus isolated from fruits Melia azedarach

Andrey M. do Rosário Marinho^I; Edson Rodrigues-Filho^{*, I}; Antônio Gilberto Ferreira^I; Lourivaldo S. Santos^II

^IDepartamento de Química, Universidade Federal de São Carlos, CP 676, 13.565-905 São Carlos - SP, Brazil

^IIDepartamento de Química, Universidade Federal do Pará, Av. Augusto Corrêa, 1 - 66075-970 Belém - PA, Brazil

ABSTRACT

A plant-derived fungus, Penicillium janthinellum, obtained from Melia azedarach, produced ergosterol and ergosterol 5a,8a-peroxide along with a mixture of rare C25 steroid epimers. The C25 steroids, named neocyclocitrinols, shows exactly the same tetracyclic ring system present in cyclocitrinol, which was isolated from a sponge-derived Penicillium citrinum, with the same bicyclo [4:4:1] skeleton at A/B rings, but showing different side chains. The P. janthinellum was cultivated over white corn and the three steroids were isolated by several silica gel based chromatographic procedures and identified by extensive NMR methods, mainly ¹H – ¹³C correlations and ¹H – ¹H COSY and TOCSY. The biosynthetic origin of the cyclocitrinols is also discussed.

Keywords: endophytic fungi, Melia azedarach, Penicillium janthinellum, neocyclocitrinols

RESUMO

O fungo Penicillium janthinellum, obtido de Melia azedarach, produziu ergosterol, ergosterol 5a,8a-peróxido e uma mistura de esteróides C25 epiméricos de ocorrência restrita na natureza. Os esteróides C25, denominados neociclocitrinóis, possuem o mesmo sistema tetracíclico de anéis presente no ciclocitrinol, o qual foi isolado do fungo Penicillium citrinum, associado a uma esponja, com o mesmo esqueleto biciclo[4:4:1] nos anéis A/B, mas com cadeias laterais diferentes. O P. janthinellum isolado de M. azedarach, foi cultivado sobre milho branco e os três esteróides foram isolados por vários procedimentos cromatográficos e identificados por cuidadosa análise dos dados de RMN, principalmente correlações ¹H – ¹³C em duas dimensões bem como COSY e TOCSY ¹H – ¹H. A origem biossintética dos ciclocitrinóis é discutida.

Introduction

Steroid and steroid derived secondary metabolites are frequently found in filamentous fungi.¹ Ergosterol (1) is the precursor of many other microbial steroids.² Most of the ergosterol metabolism begins with an oxidative process in double-bonds at C-5(6) and C-7(8) letting to ergosterol 5,8-peroxide,³ being the endo regioisomer (5a,8a-peroxide) (2) the most frequent, and then hydroxylated products at A/B/C rings are formed.⁴ In the present work we have found these sorts of compounds in our chemical studies of endophytic filamentous fungi associated with Meliaceae plants. We previously described the production of polyketides by a Penicillium janthinellum, isolated from fruits of Melia azedarach.⁵ Now we report the production of a pair of steroid epimers with a very unusual C25 skeleton, in addition to the regular steroids (1 and 2), when the fungus was cultivated over white corn. These epimers, which are represented here by the structural formula 3, contains a rare bicyclo [4:4:1] system at rings A/B, as in cyclocitrinol (4) and isocyclocitrinol (5) obtained by Amagata et al.⁶ from P. citrinum, isolated from a marine sponge.⁶ Herein we present spectroscopic evidences, which further support the bicycle system of these compounds and firmly establish the side chain of the new steroid neocyclocitrinol (3). Biosynthetic considerations regard the origin of these steroids are also discussed.

Results and Discussion

The methanol extract obtained from a twenty-days old culture of P. janthinellum, was partitioned between methanol and hexane. The steroids 1 and 2 accumulated in the hexane phase and were isolated by silica gel open column chromatography and identified as ergosterol and ergosterol 5a,8a-peroxide respectively, by comparison of their ¹H and ¹³C NMR spectra with those reported in the literature.^7,8 The neocyclocitrinols (3) were obtained from the methanol phase. The hydroxyanthraquinones we reported before in another cultivation of the fungus,⁵ were also obtained from this methanol phase.

The compound 3 was analyzed by atmosphere pressure chemical ionization mass spectrometry (APCIMS) in positive ion mode. The full scan mass spectrum obtained showed a base peak at m/z 401, which was later identified as [M+H]⁺, and minor ions at m/z 423 ([M+Na]⁺) and 442 ([M+H+AcN]⁺). The daughter ion scan, using m/z 401 as the precursor ion, which was accelerated at 30 eV to collide with argon (Ar), produced an spectrum showing steroid profile, with clusters of intense peaks at lighter ions region (m/z 100-200) and low abundant ions at high masses. The later region shows ion fragments corresponding to three losses of water (m/z 383, 365 and 347), suggesting a trihydroxylated steroid. These MS spectra, in conjunction with the interpretation of the NMR data discussed below, indicated the molecular formula C₂₅H₃₆O₄ (400 Da) for compound 3.

The signals in ¹H and ¹³C NMR spectra of compound 3 did not show pattern for common steroids, as did the MS data. A careful inspection on these NMR spectra indicated that most of the signals were duplicated in an intensity ratio of 1:1. This was suggestive for the presence of two epimeric compounds. The ¹H NMR spectrum of 3 showed only one signal for methyl group (d 0.55, s, CH₃-18) attached to a quaternary sp³ hybrized carbon. Following the ¹H – ¹³C HMBC correlations of this methyl group, it was possible to find the signals of C-17 (d 61.04), C-14 (d 56.53), C-13 (d 48.90) and C-12 (d 39.04) in the ¹³C spectrum. These HMBC correlations (see Table 1) along with careful analysis of the ¹H – ¹H COSY spectrum (Figure 1a), established the bicyclo [4:3:0], frequently found in rings C/D of steroids.⁹ The H-9 (d 2.80, dd J 5.7 and 12 Hz) and H-14 (d 2.21, ddd J 1.5, 7.8 and 9.2 Hz) are correlated (HMBC) with two and one carbon-carbon double bond, respectively. These two double-bonds are therefore part of rings A/B. The absence of an additional methyl group (CH₃-19) in this part of the molecule indicated that compound 3 is not a regular steroid.

A literature search covering the recent publications on fungi steroid resulted in one hit showing good agreement with the spectroscopic data of compound 3. The steroid cyclocitrinol (4) was obtained from a sponge-derived P. citrinum and had its structure elegantly elucidated by NMR spectroscopic data, mainly ¹H – ¹H COSY and ¹H – ¹³C HMBC, and single crystal X-ray diffraction analysis.⁶ The NMR data of 4, measured in chloroform-d solution, are almost identical with those we obtained for 3 (Table 1), mainly with regard the tetracyclic ring system. Slight differences in chemical shift of ¹H and ¹³C were detected, since our data were obtained in methanol-d₄, a polar-protic solvent. Besides the correlations of H-9 and H-14 with the double-bond carbons at C-1(10) (d 123.30/123.28, C-1 and 147.70, C-10) and C-7(8) (d 125.53/125.50, C-7 and 160.39/160.30, C-8) respectively, the correlation of the carbonyl carbon at d 207.84 with two pair of methylenic hydrogens (d 1.62-1.50, H-4a and 2.74, H-4b; d 2.57-2.47, H-19) were important guide to establish the bicyclo system at A/B rings. This partial structure was also confirmed by analysis of the ¹H-¹H COSY and TOCSY experiments (Figures 1a and 1b, respectively). Thus, the vinillyc hydrogen H-1 is strongly coupled with the methylenic hydrogens H-2a (d 2.11, ddd J 2.8, 7.2 and 13.8 Hz) and H-2b (d 2.38, ddd J 6.8, 11.2 and 13.8 Hz) (Figure 1a). The TOCSY spectra shows correlations between the hydrogens forming the spin-spin net H-1, H-2a, H-2b, H-3, H-4a, H-4b, H-5 and H-19 (see Figure 1b). The presence of a double-bound in such a position in the bicycle system, which means a bridge-head position, could lead to an unstable molecule. But, in this case, geometry optimization by computational software (HyperChem)¹⁰ showed that the four atoms directly attached to the sp² carbons C-1 (H-1, C-2) and C-10 (C-9 and C-19) are located in the same plane. An approximated shape, resulted from these calculations, is given in Figure 2.

The NMR data indicated that the constitutional steroids isomers 3 and 4 differ only by their side chain at C-17. The signal for H-17 (d 2.29) in the HMBC spectrum of 3 is correlated with two sp² carbons, suggesting the presence of a carbon-carbon double bound at C-20(22) (d 139.97/139.43, C-20; 128.02/127.58, C-22). This is in agreement with the deshielding effect (Dd +6.34) over C-17 (d 61.04) in 3 when compared with 4. The HMBC correlations of CH₃-21 (d 1.67/1.65, d J 1.0Hz) with C-17, C-20 and C-22, along with the other correlations reported in Table 1, firmly indicated that steroid 3 contains a different side chain. The COSY spectrum (Figure 1a) shows correlations for H-22 (d 5.26/5.17) with a carbinolic hydrogen (d 4.18/4.04, H-23), which is also coupled with another carbinolic hydrogen (d 3.60/3.47, H-24). A methyl group (d 1.02/1.00, CH₃-28) is coupled with H-24. These spin-spin connections were confirmed by the TOCSY experiment (Figure 1b). The duplication of the signals in the NMR spectra of 3 was more drastic for the side chain atoms, indicating that the epimerized center is located at C-23 or C-24, the only stereocenters at this part of the molecule. It was not found any HPLC condition to separate these epimers in preparative scale. Therefore the physical data of isomers 23,24-erythro and 23,24-threo were not individually ascribed.

The biosynthetic origin of these compounds (3-5) is not clear. Cyclocitrinol (4) was first isolated from a terrestrial P. citrinum and reported as a new sesterterpene.¹¹ When it was re-isolated, along with isocyclocitrinol (5), from a Penicillium living in a marine substrate, its structure was revised on the basis of X-ray crystallography studies and an steroidal skeleton was suggested.⁶ Based on the co-occurrence of steroids 1 and 2 with 3, we suggested a chemically plausible route to the biclyclic system in the cyclocitrinols (3-5) (Scheme 1). Assuming the participation of enzymes able to make C-19 in ergosterol (1) a good electrophilic center, the electron in orbital p at C-5 can act as a nucleophile, forming a C-5 – C-19 bond. This process can be favored by a nucleophilic oxidation at C-6 in 1a to produce the intermediate 1b. The cyclopropane ring in 1b can be opened by deprotonation at C-1, with the help of the carbonyl group at C-6, producing the cyclocitrinols 3-5.

The side chain of these steroids can also originate from ergosterol (1) (Scheme 1). Thus, oxidations at C-22, C-23 and C-25 of 1 produce the intermediate 1c, which is very suitable to eliminate acetone and produce 1d. The intermediate 1d has the carbon skeleton of the steroids 3-5. Further oxidations and rearrangement would lead to the cyclocitrinols.

If ergosterol (1) is confirmed as the precursor of these steroids, the enzymes involved in those process (Scheme 1) must be investigated, since conversion of steroids by living microbes or pure enzymes is nowadays a good tool for pharmaceutical industries.¹² Isocyclocitrinol (5) and its 22-acetyl derivative were inactive against human tumor cell lines and some bacterias.⁶ None of these cyclocitrinols 3-5 were tested in any other bioassay. These compounds deserve careful attention of researches, since they are very unusual steroidal compounds.

Experimental

General procedures

Optical rotations were measured on a Perkin Elmer 241 polarimeter. UV spectra were obtained in methanol solution on a HEWLETT PACKARD 8452-A spectrophotometer, and IR spectra were measured with a BOMEN MB-102 spectrophotometer in KBr pellets. Low-resolution APCIMS data were acquired in positive ion mode, using a MICROMASS QUATTRO-LC instrument equipped with an API "Z-spray" ion source. ¹H and ¹³C NMR experiments were recorded on a BRUKER DRX-400 spectrometer with CD₃OD as the solvent and TMS as the internal standard.

Plant material and microorganism

Details of plant material collection and fungus isolation were given in previous publications. ^5,13

White corn culture of P. janthinellum and isolation of the steroids

Large scale cultivation of the fungus on white corn was conducted exactly the same way described before.⁵ But, this time the MeOH added to stop the fungus growth was filtered under gravity, concentrated to 2.0 L and partitioned with n-C₆H₁₄ (3 x 500 mL). After evaporated under reduced pressure each phase were subjected to a low-pressure silica gel CC eluted with n-C₆H₁₄, EtOAc and MeOH gradient. The n-C₆H₁₄ extract produced the steroids 1 (85 mg) and 2 (29 mg) after repeatedly chromatography on silica gel CC [n-C₆H₁₄:EtOAc:MeOH (80:18:02), isocratic] and crystallization from MeOH. The MeOH phase of the initial partitioning was concentrated to dryness and subjected to a low pressure fast chromatography eluted with CH₂Cl₂ (100%), CH₂Cl₂: MeOH (9:1) and MeOH (100%). The steroid 3 (8.7 mg) was purified from the second fraction by flash chromatography on silica gel CC [n-C₆H₁₄:CHCl₃ :EtOH (60:35:05), isocratic].

Neocyclocitrinols (3)

White amorphous powder; [a]_D²⁵ + 70.1° (MeOH, c 0.001); UV l_max /nm (CH₂Cl₂): 265; IR (KBr) n_max /cm^-1: 3414, 2921, 1669, 1639, 1635; ¹H NMR (400 MHz, CD₃OD): see Table 1; ¹³C NMR (100 MHz, CD₃OD): see Table 1; APCIMS (full scan, pos. ion mode): m/z (rel. int.): 442 [M+H+AcN]⁺ (29), 423 [M+Na]⁺ (6), 401 [M+H]⁺ (100), 383 [M+H-H₂O]⁺ (29); APCIMS (daughter ion scan, 30 eV): 401 [M+H]⁺ (61), 383 (10), 365 (7), 355 (8), 347 (7), 339 (12), 329 (13), 279 (21), 267 (22), 237 (24), 223 (14), 209 (23), 187 (64), 173 (83), 159 (53), 147 (62), 145 (100), 133 (58), 121 (48), 119 (49), 107 (33).

Acknowledgements

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for financial support and research fellowships

Received: August 2, 2004

Published on the web: August 30, 2005

FAPESP helped in meeting the publication costs of this article.

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Publication Dates

• Publication in this collection
  20 Jan 2006
• Date of issue
  Nov 2005

History

• Received
  02 Aug 2004