Callyaerins A–F and H, new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa

Dedicated to the memory of Cho Cho Min
https://doi.org/10.1016/j.bmc.2010.06.012Get rights and content

Abstract

Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new cytotoxic cyclic peptides callyaerins A–F (16) and H (8). Their structures were determined using extensive 1D (1H, 13C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5–9 amino acids and side chains of 2–5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the template for ring closure and afforded the linkage to the peptidic side chain which was always initiated with a proline moiety. All peptides contained three or more proline residues and the remaining residues were predominantly hydrophobic residues with all amino acids present in the l form. Callyaerins A–F (16) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited strong activity against the L5178Y cell line with ED50 values of 0.39 and 0.48 μM, respectively. On the other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans.

Introduction

Marine sponges are a rich source of bioactive cyclic peptides and depsipeptides with unique structures involving a wide variety of unusual amino acids and other building blocks.1, 2, 3, 4 Structure elucidation of such compounds can be quite challenging insofar as they defy traditional amino acid sequencing strategies. Many cyclic peptides isolated from marine sponges exhibit interesting biological properties including a reverse of multi-drug resistance in tumour cells,5 cytotoxicity,6 HIV inhibition,7, 8 and nematocidal activity.9 The genus Callyspongia includes 182 species, of which around 15 species only have been investigated chemically. Sponges belonging to the genus have been known to yield biologically active polyacetylenic10 and nitrotetradecenyl pyridine11 compounds with different chain lengths and unsaturation positions. Nematocidal depsipeptides, phoriospongin A and B were isolated from the Australian marine sponge Callyspongia bilamellata.9 To date only our previous work describing a preliminary investigation of the metabolic diversity of the Indonesian sponge Callyspongia aerizusa has provided an insight into the chemical potential of this sponge species. We were able to isolate a new cytotoxic cyclic peptide callyaerin G (7)12 and found indications of a considerable number of other cyclic peptides present in the extract. These peptides were related analogues of callynormine A, a cyclic peptide isolated from the southern Kenyan sponge Callyspongia abnormis.13 As a continuation of this work we have now completed a detailed investigation of the bioactive ethyl acetate fraction of this sponge. In addition to the callyaerin G (7) previously described by us,12 here we describe the isolation, structure elucidation, and biological activities of a further seven callyaerin congeners of variable ring size and side-chain length. Most of the isolated compounds displayed strong activity against the tumour cell line L5178Y, and varying degrees of antibacterial and antifungal properties.

Section snippets

Results and discussion

The EtOAc soluble extract of the sponge C. aerizusa was fractionated using silica gel column chromatography and the resulting fractions were subjected to MTT assays toward the tumour cell lines L5178Y, HeLa, and PC12. The active fractions were subjected to semi-preparative HPLC to afford fractions containing seven novel cyclic peptides that were sufficiently pure to allow structural studies. In all cases we have used a combination of homo- and heteronuclear NMR techniques, in combination with

Conclusion

Investigation of the Indonesian Callyspongia aerisuza sponge yielded novel proline-rich peptides, the callyaerins. The analogues showed biological activity in a number of assays and indicated their cytotoxicity increased with the number of proline residues present in the molecule. This study adds considerably to our knowledge of the secondary metabolites produced by the genus Callyspongia which has been predominated until now by polyacetylenic10 and nitrotetradecenyl pyridine11 metabolites.

General experimental procedures

The UV spectra were determined using a Perkin–Elmer double beam spectrophotometer Model 550 S, attached to a Hitachi recorder Model 561, using 1 cm quartz cell. Optical rotations were measured on a Perkin–Elmer Model 341 LC polarimeter. ESI- and LC–MS spectra were obtained with a Thermofinnigan LCQ DECA mass spectrometer coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. HRESIMS were determined on a Micromass Q-Tof 2 mass spectrometer. Column chromatography was

Acknowledgement

S. R. M. Ibrahim wishes to thank the Egyptian Government for a scholarship. We are indebted to Professor Dr. W. E. G. Müller (Institute für Physiologische Chemie, Duesbergweg 6, D-55099 Mainz, Germany) for cytotoxicity testing. This project was supported by grants of the BMBF and MOST awarded to Professor Peter Proksch and Professor Wenhan Lin.

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Permanent address: Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.

Present address: Malaria Drug Resistance and Chemotherapy Laboratory, The Queensland Institute of Medical Research, 300 Herston Rd., Herston, Brisbane, Queensland 4006, Australia.

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