Conformations of peptides containing Z-0 ,fl-dehydroleucine (AZLeu). A comparison of Boc-Pro-AZ Leu-GIy-NHEt and Boc-Pro-AZPhe-Gly-NHEt K. Uma and P. Balaram Molecular Biophysics Unit, Indian Institute of Science, Bangalore-12, India Paramjeet Kant, Ashwani Kumar Sharma and V. S. Chauhan*¢ Department of Chemistry, University of Delhi, Delhi-7, India (Received 7 May 1988; revised 7 September 1988) Two tripeptides of the type Boc-Pro-AZX-Gly-NHEt (where X = Leu, Phe) have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r, and i.r. spectroscopy. These conformational studies indicated that AZLeu, similar to AZPhe, has a strong tendency to stabilize folded Type II r-turn conformations when present at i + 2 position. Keywords: Conformation;dehydroleucine; dehydrophenylalanine; r-turn; nuclear Overhausereffects Introduction The conformational characteristics of peptides containing ct,fl-dehydroamino acid residues have attracted recent interest, in view of their potential use in the design of structurally constrained analogues of biologically active peptides 1-6. These studies have been largely focused on peptides containing Z-dehydrophenylalanine (AZPhe) (Figure I). Limited theoretical analysis of the model dipeptide of AAla 1 and a crystal structure determination of Boc-AZLeu-OH 7 are representative of the few conforma- tional studies on other ct,fl-dehydroamino acids. We describe in this report synthesis and n.m.r, studies in solution of the protected tripeptide Boc-Pro-AZLeu-Gly - NHEt 1 and provide a comparison with the corresponding AZPhe analogue, Boc-Pro-AZPhe-Gly-NHEt 2. Peptides 1 and 2 were chosen in view of the occurrence of the Pro-Leu-Gly-NH2 sequence as the C-terminal tripeptide fragment in oxytocin and also the MSH release activity noted for the free tripeptide amide s'9. Experimental Melting points are uncorrected. Thin layer chromato- graphy (t.l.c.) was performed on silica gel plates using the following solvent systems: (1) CHCI3-MeOH (9:1), (2) n-BuOH-AcOH-H20 (4:1:1), (3) n-BuOH-AcOH-H20 (3:1:1). The compounds were located with a ninhydrin spray. * To whom correspondence should be addressed ¢ Presentaddress: I.C.G.E.B.,NII Campus, ShaheedJeet Singh Marg, New Delhi-110067, India The amino acid residues are in the e-configuration. Standard abbreviations for amino acid derivatives and peptides are used according to the IUPAC-IUB Commission on Biochemical Nomenclature, (Eur. J. Biochemistry 1984, 138, 9-37). Optical rotations were determined with a Jasco DIP 360 polarimeter. I.r. spectra in solution were recorded on a Perkin-Elmer 257 spectrometer. Both the peptides were shown to yield a single peak by h.p.l.c. (Dupont 8800) on a Zorbax RP-18 column (4 mm × 250 mm, particle size 10 ltm), using a methanol/water gradient: 50-70% MeOH in 20 min; flow rate: 0.8 ml/min; detection: u.v. (226 nm). N.m.r. studies were carried out on a WH-270 MHz FT n.m.r, spectrometer equipped with an Aspect 2000 computer at the Sophisticated Instruments Facility, Indian Institute of Science. Difference nuclear Overhauser effect (n.O.e.) studies were carried out as described earlier 1°. The peptide concentration used for all studies is 10 mg/ml. Synthesis Boc-Pro-AZLeu-Gly-NHEt (tert-butyloxycarbonyl- prolyl-~,fl-(Z) dehydroleucylglycine ethylamide) 1 Z-GIy- NHEt 11 (2,0 g, 8.5 mmol) was dissolved in a 30% solution of HBr in AcOH (11.3 ml) and the mixture was stirred at room temperature for 30 min. Excess reagent was removed under reduced pressure. The resulting hydrobromide salt of glycine ethylamide (HBr.H-GIy-NHEt) was precipi- Figure 1 ? o ? o II AZLeu /xzPhe Structure of AZLeu and AZPhe residues 0141-8130/89/030169-03 $03.00 rt3 1989 Butterworth & Co. (Publishers) Ltd Int. J. Biol. Macromol., 1989, Vol. 11, June 169