FULL PAPER * E-mail: drrajivdahiya@rediffmail.com; Tel.: 0091-96302-29885, 0091-755-4285308 Received October 22, 2010; revised April 28, 2011; accepted May 5, 2011. Chin. J. Chem. 2011, 29, 1911—1916 © 2011 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1911 Synthesis and Pharmacological Studies on a Cyclooligopeptide from Marine Bacteria Rajiv, Dahiya* ,a Hemendra, Gautam b a Department of Pharmaceutical Chemistry, Globus College of Pharmacy, Bhojpur Road, Bhopal-462 045, Madhya Pradesh, India b Department of Pharmacology, Institute of Pharmacy, Invertis University, Bareilly-243 123, Uttar Pradesh India A natural proline-rich tetrapeptide cyclo-prolyl-leucyl-prolyl-phenylalanyl was prepared employing solu- tion-phase method of peptide synthesis through coupling of dipeptide fragments Boc-l-Pro-l-Leu-OH and l-Pro-l-Phe-OMe which utilizes diisopropylcarbodiimide (DIPC) as coupling agent and N-methylmorpholine (NMM) as the base. Deprotection of linear tetrapeptide unit followed by its cyclization provided a cyclopeptide, identical in all aspects to the natural molecule. Pharmacological evaluation showed cytotoxic, antifungal and antihelmintic po- tential of synthesized peptide against Dalton’s Lymphoma Ascites (DLA) and Ehrlich’s Ascites Carcinoma (EAC) cell lines, pathogenic dermatophytes and earthworms. Keywords marine bacteria, natural product, solution phase synthesis, peptides, biological activity, cytotoxicity Introduction From decades, cyclic peptides continue to be chal- lenging targets for chemical synthesis. 1 The review of literature indicates that natural cyclooligopeptides from marine bacteria, fungi and actinomycete 2-4 possess di- verse pharmacological activities, including cytotoxicity, anti-dinoflagellate activity, antimicrobial activity and inhibitory activity against enzyme sortase B and prote- ase. 5-9 A natural cyclotetrapeptide, cyclo-prolyl-leucyl- prolyl-phenylalanyl, has been isolated from the marine bacteria Pseudomonas sp. and Pseudoalteromonas sp., associated with the seaweed Diginea sp. and the sponge Halisarca ectofibrosa and its structure was elucidated by LC-MS and 2D NMR data. 10 Only minute quantities of this bioactive cyclopeptide obtained from natural re- sources (1 mg from chloroform-methanol soluble frac- tions of the bacterial culture) restricted scientists to in- vestigate its biological profile in detail. Further, the wide spread increase of resistance towards conventional drugs encourages the development of novel moieties with unexploited mechanisms of action. Hence, keeping in view the biological potential of natural cyclooli- gopeptides 11,12 and in continuation of our efforts on synthesis of natural peptides, 13-16 present investigation was directed toward the synthesis of a naturally occur- ring cyclotetrapeptide (4) employing solution-phase synthesis method. Furthermore, synthesized peptide was further evaluated for its cytotoxic, antimicrobial and antihelmintic potential. Experimental Materials and methods Melting point was determined in open capillaries and is uncorrected. IR spectra were recorded on a Shimadzu 8700 FTIR spectrophotometer and 1 H/ 13 C NMR spectra were taken on a Bruker AC NMR spectrometer (300 MHz) using deuterated methanol as solvent and TMS as internal standard. The mass spectrum was recorded on a JMS-DX 303 mass spectrometer operating at 70 eV by ESIMS/MS. Optical rotation of synthesized compounds was measured on automatic polarimeter at 25 ℃ using sodium lamp. Elemental analyses of all compounds were performed on Vario EL III elemental analyzer. Purity of all synthesized peptide derivatives was checked by TLC on precoated silica gel G plates. Preparation of linear dipeptide fragments 1, 2 l-Amino acid methyl ester hydrochloride (0.01 mol) was dissolved in tetrahydrofuran (THF, 25 mL). To this solution, N-methylmorpholin (NMM, 2.21 mL, 0.021 mol) was added at 0 ℃ and the reaction mixture was stirred for 10 min. Boc-l-amino acid (0.01 mol) was dissolved in THF (25 mL) followed by addition of DIPC (1.26 g, 0.01 mol) or EDC•HCl (1.92 g, 0.01 mol) and HOBt (1.34 g, 0.01 mol). Resulting mixture was added to above solution with constant shaking and the stirring was continued for 24 h. The reaction mixture was filtered and the residue was washed with THF (25 mL) and added to the filtrate. The filtrate was washed with 5% NaHCO 3 and saturated NaCl solutions. The organic layer was dried over anhydrous Na 2 SO 4 , filtered