Communication Copper(II) complexes of tetrapeptides containing cysteinyl and histidinyl residues Vassiliki Magafa, Spyros P. Perlepes and George Stavropoulos* Department of Chemistry, University of Patras, 265 00 Patras, Greece It is becoming increasingly apparent that the coordina- tion chemistry of oligopeptides with Cys-X-Y-Cys and His-X-Y-Cys sequences (X, Y  variable amino acid residues) is an important theme in transition metal and bioinorganic chemistry (1,2) . The Cys-X-Y-Cys sequence is often encountered in the metal binding site of several metalloproteins, such as iron±sulfur proteins, e.g. rub- redoxins (3) and ferrodoxins (4) , high potential iron±sulfur proteins (5) , metallothioneins (6) , zinc proteins (7) etc. The amino acid sequence His-X-Y-Cys is also of bioinor- ganic importance since it has been found in the active site of copper (8) , iron±sulfur (9) and zinc (10) proteins. We are currently seeking to develop the area of transition metal/Cys-X-Y-Cys and His-X-Y-Cys peptide interac- tions (11,12) ; our goals are: (i) to elucidate the role of these sequences in the biological activity and mechanism of action of the above metallobiomolecules, and (ii) to ®nd possible new and more eective agents for the elimina- tion of heavy metals from the human organism or from contaminated waste waters. Here we report the prepa- ration and preliminary characterization of copper(II) complexes of tetrapeptide ligands containing the Cys-X-Y-Cys and His-X-Y-Cys sequences. Experimental Most manipulations were carried out under an atmo- sphere of dry N 2 using standard Schlenk techniques. Solvents were purchased from commercial sources and puri®ed by standard techniques (13) . All amino acids employed were of the L con®guration and were pur- chased from Peptide Institute Inc. and Bachem. The amino acid derivatives used in the peptide synthesis were prepared by literature methods (12) and puri®ed by ¯ash column chromatography on silica gel 60, 230±400 mesh (ASTM) purchased from Merck. Their purity was con- trolled by t.l.c. Copper and chloride analyses were car- ried out using standard gravimetric and volumetric methods. Microanalyses for C, H and N were performed by the University of Ioannina (Greece) Microanalytical Laboratory using an EA 1108 Carlo Erba analyser. Sodium analysis was performed using a Perkin Elmer 2380 atomic absorption spectrophotometer. Physico- chemical measurements and spectroscopic techniques were carried out by published methods (12) . Peptide synthesis The precursor tetrapeptides Boc-Cys(Bzl)-Ser(Bzl)-Ala- Cys(Bzl)-NH 2 (a), Boc-Cys(Bzl)-Gly-Ala-Cys(Bzl)-NH 2 (b), Boc-His(N im -Boc)-Ser(Bzl)-Ala-Cys(Bzl)-NH 2 (c) and Boc-Cys(Acm)-Gly-Ala-Cys(Acm)-OMe (d), where Boc  t-butyloxycarbonyl, Bzl  benzyl and Acm  acetamidomethyl, were synthesized by standard liquid phase methods (active esters (14) ) using dicyclohexylcarbodi- imide (DCC, Merck)/1-hydroxybenzotriazole (Aldrich) and benzotriazolyl N-oxytridimethylaminophosphonium hexa- ¯uorophosphate (BOP, Nova). Preparation of the complexes The complexes Na[CuClL 1 (H 2 O) 2 ] á 2H 2 O (1), Na- Cu 2 ClL 2 2 H 2 O 4  H 2 O (2), [CuClL 3 (H 2 O) 2 ] (3) and [Cu 2 Cl 4 L 4 (H 2 O) 2 ] (4), where L 1  the doubly depro- tonated tetrapeptide Boc-Cys-Ser-Ala-Cys-NH 2 , L 2  doubly deprotonated tetrapeptide Boc-Cys-Gly-Ala-Cys- NH 2 ,L 3  the monoanion of Boc-His-Ser-Ala-Cys-NH 2 and L 4  the neutral peptide (d), were prepared by the reactions of CuCl 2 á 2H 2 O and the precursor peptides (a), (b), (c) and (d), respectively, as follows. For the preparation of (1), (2) and (3), 0.2 mmol of peptides (a), (b) or (c) were dissolved in liquid NH 3 (50 cm 3 ), distilled over metallic Na, under stirring. Small pieces of Na were added to the solution until the appearance of a deep blue colour, lasting for 4±5 min. The excess of Na was destroyed with the addition of few drops of AcOH. Ammonia was then evaporated to dryness under stirring and bubbling of N 2 . The solid residue was dissolved in air-free MeOH and evaporated in vacuo to dryness. The process was repeated and the residue obtained was dis- solved in the minimum vol of air-free H 2 O under stir- ring. The pH of the solution was adjusted to 7.5 using a 2 M NaOH solution and to this was added 0.2 mmol of CuCl 2 á 2H 2 O. The green precipitates formed were stir- red at room temperature for 18 h and left undisturbed at 5 °C for another 24 h. The solids were collected by ®l- tration, washed with EtOH and Et 2 O, and dried in vacuo over P 4 O 10 . Yields were typically 60±70%. For the preparation of complex (4), 0.2 mmol of the peptide (d) was dissolved in the minimum amount of distilled and air-free H 2 O or a mixture of MeOH:H 2 O (50:50, v/ v). To this solution, a solution of 0.4 mmol of Cu- Cl 2 á 2H 2 O in the same solvent was added. The resulting green solid was isolated as described above for (1), (2) and (3). Yield: 70%. Results and discussion Peptides (a), (b), (c) and (d) were characterized by elemental analyses, i.r., u.v., 1 H-n.m.r. (both 1D and 2D) and 13 C-n.m.r. spectroscopies. The Bzl and N im -Boc groups of (a), (b) and (c) were cleaved upon treatment with liquid NH 3 /Na (15) prior to complex preparation. 0340±4285 Ó 1998 Chapman & Hall Transition Met. Chem., 23, 105±107 (1998) Tetrapeptide Cu II complexes 105 * Author to whom all correspondence should be directed.