Journal of Peptide Science J. Peptide Sci. 6: 19–25 (2000) Chemical Synthesis, Characterization and Activity of RK-1, a Novel  -Defensin-related Peptide NICOLA F. DAWSON a , DAVID J. CRAIK b , AILSA M. MCMANUS b , STUART G. DASHPER c , ERIC C. REYNOLDS c , GEOFFREY W. TREGEAR a , LASZLO OTVOS JR d and JOHN D. WADE a, * a Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia b Centre for Drug Design and Development, University of Queensland, Brisbane, Queensland 4072, Australia c The School of Dental Science, University of Melbourne, Melbourne, Victoria 3000, Australia d The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA Received 2 August 1999 Accepted 5 August 1999 Abstract: The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial  -defensin family, was synthesized by the continuous flow Fmoc-solid phase method. The crude, cleaved and S -reduced linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by 1 H-NMR spectroscopy and confirmed to be a Cys 1 -Cys 6 , Cys 2 -Cys 4 , Cys 3 -Cys 5 arrangement similar to other mammalian  -defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd. Keywords: antibacterial assay;  -defensin; disulfide bond assignment; Fmoc-solid phase peptide synthesis; 1 H-NMR spectroscopy; mass spectroscopy; RK-1 INTRODUCTION Defensins are small (ca 30 amino acids) cystine- rich, polycationic antimicrobial peptides that are involved in eukaryotic host defences against gram- positive bacteria, fungi and enveloped viruses [1,2]. They are also believed to contribute to the regula- tion of the inflammatory response. There are two classes of mammalian defensins known as the  - and  -defensins, each of which possesses a distinct cystine motif [2]. A recently isolated rabbit kidney- derived peptide, termed RK-1, was shown to have an identical cysteine residue number and spatial distribution to human defensin suggesting it may be a bona fide member of the corticostatin/ -de- fensin family of peptides [3] (Figure 1). The peptide was demonstrated to have significant growth in- hibitory activity against Escherichia coli, the patho- gen most commonly associated with kidney and urinary tract infections, and is postulated to play an important role in renal pathophysiology. A second  -defensin-like peptide, RK-2, has also been re- cently identified in the rabbit kidney establishing the existence of a new subfamily of  -defensins in this organ [4]. Abbreviations: CD spectroscopy, circular dichroism spectroscopy; CSI, chemical shift indice; DMF, dimethylformamide; DMSO, dimethylsulfoxide; DQF-COSY, double-quantum filtered correla- tion spectroscopy; MALDITOF-MS, matrix-assisted laser desorp- tion time of flight mass spectrometry; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; RP-HPLC, reversed-phase high performance liquid chromatogra- phy; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TFA, tri- fluoroacetic acid; TFE, trifluoroethanol; TOCSY, total correlation spectroscopy. * Correspondence to: Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victo- ria 3052, Australia. E-mail: j.wade@hfi.unimelb.edu.au Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd. CCC 1075–2617/2000/0100019-07$17.50