George Stavropoulos

Substance P C-terminal octapeptide analogues augment tumor necrosis factor-α release by human blood monocytes and macrophages

We have investigated the effects of the substance P C-terminal octapeptide analogues [Pro4, Glu (OBzl)11] SP4-11, [Hyp4, Glu(OBzl)11] SP4-11, [cHyp4, Glu(OBzl)11] SP4-11 and [kPro4, Glu(OBzl)11] SP4-11 on the constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF-alpha) in both freshly isolated human blood monocytes (FIBM) and monocyte-derived macrophages (MDM). The cells were treated with substance P and the substance P analogues at various concentrations (10-14 to 10-6 M) in the presence or absence of LPS and culture supernatants were analyzed for TNF-alpha as measured by an enzyme immunosorbent assay (ELISA). Monocytes and macrophages treated with the substance P analogues alone increased TNF-alpha secretion at a magnitude similar to the effect of entire undecapeptide substance P. The stimulatory effects of the substance P analogues on TNF-alpha secretion are inhibited by substance P antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the substance P receptor), indicating that these effects are specific and substance P receptor-mediated. Treatment of monocytes and macrophages with the substance P analogues in combination with LPS, however, showed no synergistic interaction in upregulation of TNF-alpha. These data indicate that the biological effect of substance P on TNF-alpha production by human monocytes and macrophages depends mainly on the sequence of the C-terminal region of the molecule.

NMR study of the interaction of platinum(II) and palladium(II) complex ions with His–Ala and His–Gly–Ala

Abstract 1 H, 13 C and 195 Pt NMR spectroscopies are used to identify the products and the binding sites of the complex salts [Pd(dien)(D 2 O)](NO 3 ) 2 , [Pt(dien)(D 2 O)](NO 3 ) 2 , [Pd(en)(D 2 O) 2 ](NO 3 ) 2 , cis -[(NH 3 ) 2 Pt(D 2 O) 2 ](NO 3 ) 2 , K 2 PdCl 4 and K 2 PtCl 4 towards the peptides His–Ala and His–Gly–Ala, in aqueous solutions as a function of pD. The 1 H, and 13 C NMR assignments were made by two-dimensional homo- and heteronuclear experiments for the two ligands His–Ala and His–Gly–Ala. The results show that all Pd(II) salts can substitute protons from all protonation sites of histidine (–NH 2 , N3, N1 of imidazole) at very acidic media (pH 2 ,N3 chelates. The complex cation [Pd(dien)(D 2 O)] 2+ forms N3,N1 imidazole bridges. The Pt(II) salts react with the carboxylate terminal groups of the peptides at pH 3 ( 195 Pt NMR range −2000 to −2500 ppm) and form also –NH 2 ,N3 chelates in weakly alkaline media ( 195 Pt NMR range −2200 to −2900 ppm). [Pt(dien)(D 2 O)] 2+ does not form bridges but coordinates separately to N1 and N3, with the N3 bound isomer predominating and more so in the case of His–Gly–Ala. K 2 PtCl 4 forms 1:2 chelates in alkaline solutions, in contrast to K 2 PdCl 4 that forms 1:1 chelates at pH 1. At higher pH values, precipitates are formed.

Darstellung und einsatz von N-Fmoc-O-Trt-hydroxyaminosäuren zur “solid phase” synthese von peptiden

Zusammenfassung The preparation of the N-Fmoc-O-Trt derivatives of serine, threonine and tyrosine is described. Their usefulness in peptide synthesis has been determined in the successful solid phase preparation of the partially protected ACTH (fragment 1–10) and peptide T 12 . The latter, having six hydroxy amino acid side chains protected with Trt groups, can be quantitatively cleaved from the applied 2-chlorotrityl resin with simultaneous side chain deprotection.

Fmoc-His(Mmt)-OH und Fmoc-His(Mtt)-OH. Zwei nue histidin-derivative Nim-geschützt mit säure-hochempfindlichen gruppen. Darstellung, eigenschaften und einsatz in der peptidsynthese

Zusammenfassung The use of 4-methoxytrityl (monomethoxytrityl, Mmt) and 4-methyl-trityl (Mtt) groups of N1m-protection of Fmoc-His is described. Both groups being unaffected by base, e.g. piperidine and diethylamine, can be quantitatively removed under very mild conditions, in comparison with trityl groups. The solid phase peptide synthesis of megainin I (fragment 1–10) on 2-chlorotrityl resin showed the excellent suitability of the above derivatives of histidine for peptide synthesis.

Preparation of polymer-bound trityl-hydrazines and their application in the solid phase synthesis of partially protected peptide hydrazides

Polymer-bound N-tritylhydrazines 4 were easily prepared by reacting polymeric tritylchlorides 3 with hydrazine. Subsequently, compounds 4 have been successfully applied to the solid phase synthesis of partially protected peptide hydrazides using 1-hydroxybenzotriazolyl esters of Fmoc- or Trt-amino acids. The synthesized peptide hydrazides can be quantitatively split off from the resins by mild acidic treatment, while the benzyl- and tert-butyl protecting groups remain unaffected.

Interaction of Hg(II) with tetrapeptides containing cysteinyl and histidinyl residues

Abstract The mercury(II) complexes [(Boc-Cys-Ser-Ala-Cys-NH 2 ) 2 Hg 2 Cl]Na·4H 2 O ( 1 ), (Boc-Cys-Ser(Bzl)-Ala-Cys-NH 2 ) 2 Hg 3 Cl 2 ( 2 ), (Boc-Cys-Ser(Bzl)-Ala-Cys-NH 2 )Hg 2 Cl 2 ( 3 ), [(Boc-Cys-Gly-Ala-Cys-NH 2 ) 2 Hg 2 Cl]Na·3H 2 O ( 4 ), (Boc-Cys-Gly-Ala-Cys-OCH 3 ) 2 Hg 4 Cl 2 ·2H 2 O ( 5 ), (Boc-Cys-Gly-Ala-Cys-OCH 3 )Hg 2 Cl 2 ·2H 2 O ( 6 ), (Boc-His-Ser(Bzl)-Ala-Cys-NH 2 )Hg 2 Cl 2 ( 7 ) and [(Boc-His-Ser(Bzl)-Ala-Cys-NH 2 )Hg 2 Cl 2 ]Cl ( 8 ) were synthesized from HgCl 2 and the corresponding free or S -acetamidomethyl (Acm) and N un -Boc protected peptides, by different synthetic routes, Acm and N un -Boc protecting groups are removed upon mercury(II) coordination, while the Bzl group was removed selectively prior to reactions of the peptides with mercury(II). The peptides and the complexes formed were characterized by elemental analysis, conductivity measurements, IR, UV, and 1D and 2D 1 H and 13 C NMR spectroscopies. In addition, 199 Hg NMR experiments suggest that mercury(II) ions exchange rapidly between chloride ions and imidazole nitrogens. Conformational studies of the peptides in the complexes formed, by ROESY experiments, suggested macromolecular chelate structures with mercury(II).

Kainic acid as conformationally constrained glutamic acid analog in peptide synthesis

Abstract A synthetic protocol is described which allows the incorporation of the γ-amide (Kan) and the γ-benzyl ester [Kai(Bzl)] and ether [Kol(Bzl)] of kainic acid (Kai) into peptide chains as exemplified with the synthesis of the substance P (SP) analogs [Kan5]-SP5−11, [Glp5. Kan6]-SP5−11. [Kan6]-SP6 11.[Glp5. Kai(Bzl)11]-SP5−11 and [Glp5. Kol(Bzl)11]-SP5−11.

Copper(II) complexes of tetrapeptides containing cysteinyl and histidinyl residues

It is becoming increasingly apparent that the coordination 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. rubredoxins(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 bioinorganic 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 interactions(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 find possible new and more effective agents for the elimination of heavy metals from the human organism or from contaminated waste waters. Here we report the preparation and preliminary characterization of copper(II) complexes of tetrapeptide ligands containing the Cys-X-Y-Cys and His-X-Y-Cys sequences.

Synthesis and proposed conformations of l-prolyl-l-leucyl-β-alanine alkylamides

Abstract The synthesis of H-Pro-Leu-β-Ala-NH 2 , H-Pro-Leu-β-Ala-NHCH 3 and H-Pro-Leu-β-Ala-N(CH 3 ) 2 is described. On the basis of IR and 1 H NMR spectral data, a 7-membered ring including the NH of β-alanine with the CO of proline should be assigned for the H-Pro-Leu-β-Ala-N(CH 3 ) 2 . Consequently, the plausible conformations for H-Pro-Leu-β-Ala-NH 2 and H-Pro-Leu-β-Ala-NHCH 3 derive from the formation of an 11-membered ring, between the trans amide proton and the CO of Pro, or from the formation of an 8-membered ring, between this carboxamide proton and the CO of Leu, plus the aforementioned 7-membered ring.

Cyclic peptide analogs of 558-565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants.

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.