Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed.

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers.

Tuftsin-AZT conjugate: potential macrophage targeting for AIDS therapy.

The IgG-derived immunomodulating peptide tuftsin, Thr-Lys-Pro-Arg, is recognized by specific receptors on phagocytic cells, notably macrophages, and is capable of targeting proteins and peptides to these sites. Aiming to target 3'-azido-3'-deoxythymidine (AZT) to HIV-infected macrophages, a conjugate of AZT with tuftsin was synthesized. The AZT-tuftsin chimera possesses the characteristic capacities of its two components.

[D-Ala2]-Deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and conformational investigations.

The synthesis is described of a [D-Ala2]-deltorphin I peptoid analogue in which all amino acid residues have been substituted by the corresponding N-alkylglycine residues. The [D-Ala2]-deltorphin I retropeptoid was also prepared as well as [Ala1 ,D-Ala2]-deltorphin 1 and the corresponding peptoid. Structural investigations by FT-IR and fluorescence measurements were carried out on the synthetic analogues and on some [D-Ala2]-deltorphin 1 peptide-peptoid hybrids previously prepared.

Synthesis, conformation and biological activity of dermorphin and deltorphin I analogues containing N-alkylglycine in place of residues in position 1, 3, 5 and 6.

Syntheses are described of new dermorphin and [D-Ala2]deltorphin I analogues in which the phenylalanine, the tyrosine or the valine residues have been substituted by the corresponding N-alkylglycine residues. Structural investigations by CD measurements in different solvents and preliminary pharmacological experiments were carried out on the resulting peptide-peptoid hybrids. The contribution from aromatic side chain residues is prominent in the CD spectra of dermorphin analogues and the assignment of a prevailing secondary structure could be questionable.

Opioid peptides: synthesis and biological properties of [(N gamma-glucosyl,N gamma-methoxy)-alpha, gamma-diamino-(S)-butanoyl]4-deltorphin-1-neoglycopeptide and related analogues.

The [D-Ala2]deltorphin 1 sequence in which the aspartic acid residue is replaced by the N gamma-OCH3-alpha, gamma-diamino (S) butanoyl residue was synthesized using the Fmoc-chemistry-based solid phase procedure. The resulting deltorphin analogue was chemoselectively glucosylated by reaction with unprotected D-glucose (Glc). The Asn4-, (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-galactopyranosyl)-Asn4- and the (2-acetamido-2-deoxy-D-galactopyranosyl)-Asn4-deltorphin I were also prepared for comparison.

Antimicrobial peptides: synthesis and antibacterial activity of linear and cyclic drosocin and apidaecin 1b analogues.

Drosocin and apidaecin Ib are two insect antimicrobial peptides showing a significant sequence homology and a common mechanism of action, which includes stereoselective elements but is devoid of any pore-forming activity. A substantial difference between the two peptides is the presence in the drosocin sequence of an O-glycosylated threonine residue, which is important for its antimicrobial activity. Through the synthesis of a series of differently glycosylated drosocin analogues, we have shown that the antimicrobial activity against several Gram-negative bacteria appears to be modulated by the sugar moiety (Gal vs GalNAc) and the type of glycosidic linkage (alpha-O-, beta-O-, or alpha-C-).

Conformational investigations on glycosylated asparagine-oligopeptides of increasing chain length.

Stepwise solution syntheses of the homo-oligomers Boc-(Asn)n-NHCH3 (n = 1-5; I1-5), Boc-[[GlcNAc(Ac)3beta]Asn]n-NHCH3 (n = 1-8; II1-8), and Boc-[(GlcNAcbeta)Asn]n-NHCH3 (n = 1-8; III1-8) are described. Members of the series III were obtained by deacetylation of the corresponding members of the series II. The conformational preferences of the N-protected homo-peptides of the three series were investigated by spectroscopic techniques.

Synthesis, conformation and biological activity of linear and cyclic Thr6-bradykinin analogues containing N-benzylglycine in place of phenylalanine.

Three linear Thr6-bradykinin analogues in which either one or both the two phenylalanine residues in the peptide sequence have been substituted by N-benzylglycine (BzlGly) and their head-to-tail cyclic analogues were synthesized and tested on an isolated rat duodenum preparation. The linear (BzlGly5,Thr6-BK, BzlGly8,Thr6-BK and BzlGly(5,8),Thr6-BK) and the cyclic (cyclo BzlGly5,Thr6-BK, cyclo BzlGly8,Thr6-BK and cyclo BzlGly(5,8),Thr6-BK) peptoid-like analogues were characterized by amino acid analysis, optical rotation, analytical HPLC and MALDI-TOF mass spectroscopy. The conformational features of both the linear and cyclic derivatives were investigated by FT-IR and CD measurements.

Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine, thiol and aspartyl proteases.

We synthesized short chromogenic peptidyl-Arg-p-nitroanilides containing either (Galbeta)Ser or (Glcalpha,beta)Tyr at P2 or P3 sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl-X-Arg-pNa and Acetyl-X-Phe-Arg-pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe8-His-Leu-Val-Ile-His-Asn14 of human angiotensinogen, in which [GlcNAcbeta]Asn was introduced before Phe8 and/or after His13 and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]-ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair.

Conformational investigations on glycosylated threonine-oligopeptides of increasing chain length.

Stepwise solution syntheses are described of the homo-oligomers Z-(Thr)n-NHCH3 (n=1-4, I1-4), Z-¿[Gal(Ac)4beta]Thr¿n-NHCH3(n=1-5, II1-5) and Z-[(Galbeta)Thr]n-NHCH3 (n=1-5, III1-5). Members of the III1-5 series were obtained by de-acetylation of the corresponding oligomers of the II1-5 series. The conformational preferences of the terminally protected homo-peptides of the three series were investigated by FT-IR absorption spectroscopy both in the solid state and in CDCl3 solution, at various concentrations.

Synthesis and biological activities of head-to-tail cyclic bradykinin analogues of varying ring size.

Syntheses of cyclic kinin analogues with different backbone atom numbers are described. Cyclization, by either the O-benzotriazolyl-N,N,N',N'-tetramethyluronium tetrafluoroborate/1-hydroxybenzotriazole/diisopropylethyl amine (TBTU-HOBt-DIPEA) or the diphenylphosphoryl azide (DPPA) procedure of linear peptides prepared by the solid-phase method based on the g-fluorenyl methyloxycarbonyl chemistry, was used for preparing cyclo-Gly-Ile-Ile-Gly-bradykinin, cyclo-Lys-kallidin (cyclo-Lys-Lys-bradykinin) and cyclo-des Arg-bradykinin. Peptides were characterized by amino acid analysis, optical rotation, analytical high-performance liquid chromatography and matrix-assisted laser desorption ionization-time flight mass spectrometry.

Cyclic analogues of Thr6-bradykinin, N epsilon-Lys-bradykinin and endo-Lys8a-vespulakinin 1.

Syntheses are described of the endo-Lys8a-vespulakinin 1 and of cyclo-Thr6- and cyclo-N epsilon-Lys-bradykinin. The linear peptides covering the entire sequences of endo-Lys8a-VSK-1 and Thr6-BK, and the decapeptide containing all residues constituting Lys-BK, with a Arg-Lys peptide bond involving the epsilon-amino function of lysine, were prepared by the solid-phase procedure based on Fmoc chemistry. Cyclization was carried out by the diphenylphosphorazide method.

Cyclic analogues of wasp kinins from Vespa analis and Vespa tropica.

Syntheses are described of two bradykinin-like kinins isolated from Vespa analis (G-R-P-P-G-F-S-P-F-R-V-I, VSK-A) and Vespa tropica (G-R-P-Hyp-G-F-S-P-F-R-V-V, VSK-T) and of their cyclic analogues. Linear dodecapeptides were prepared by the solid-phase procedure based on Fmoc-chemistry, and cyclization was carried out by the diphenyl-phosphorylazide method. Peptide were characterized by amino acid analysis, optical rotation, analytical HPLC and FAB-MS.

Synthesis and biological activity of some linear and cyclic kinin analogues.

Syntheses are described of some linear and cyclic kinin analogues. Cyclization, by the diphenyl-phosphorazide method, of linear peptides prepared by the solid-phase procedure based on Fmoc chemistry, was used for preparing cyclo-bradykinin and cyclo-kallidin (cyclo-Lys-bradykinin). Removal of the protecting group from the lysine side chain of cyclo-kallidin followed by acylation with the N-terminal sequence of vespulakinin 1 (VSK 1), Fmoc-Thr(tBu)-Ala-Thr(tBu)-Thr(tBu)-Arg(Pmc)-Arg(Pmc)-Gly-OH, by the Bop-HOBt procedure, yielded the protected N epsilon-(1-8 VSK 1)-cyclo-N alpha-kallidin, which was deblocked by acid treatment and purified by semi-preparative HPLC.

Synthetic immunochemistry of glycohexapeptide analogues characteristic of oncofetal fibronectin. Solid-phase synthesis and antigenic activity.

Monoclonal antibody FDC-6, and its second-generation antibodies FDB-1 and FDB-4, are able to distinguish between fibronectin (FN) from fetal or cancer tissue (onco-FN) vs. FN from normal adult tissue and plasma (nor-FN). The epitope structure recognized by the above antibodies is the glycohexapeptide H-Val-(GalNAc-alpha)Thr-His-Pro-Gly-Tyr-OH (P2).

Synthesis and biological activity of [L-hydroxyproline]3-tuftsin analogue and its alpha- or beta-O-D-glucosylated derivatives.

Syntheses are described of the Hyp3-tuftsin analogue and of its derivatives alpha- or beta-O-glycosylated at the side chain function of the hydroxyproline residue. The carbohydrate-free tetrapeptide was prepared by reacting Z-Thr-Lys(Z)-OH with H-Hyp-Arg(NO2)-OBzl by the mixed anhydride procedure. In the synthesis of the alpha-glycosylated analogue the O-glycosyl amino acid was incorporated by reacting Boc-(Glc alpha+beta)Hyp-OH with H-Arg(NO2)-OBzl through the same procedure.

Synthesis and biological activity of the mono- and di-galactosyl-vespulakinin 1 analogues.

Syntheses are described of some mono- and di-glycosylated analogues of vespulakinin 1. The solid phase procedure, based on the Fmoc chemistry, was used to prepare (Gal alpha)Thr3-vespulakinin 1, (Gal beta)Thr3-vespulakinin 1 and the di-glycosylated analogue ((Gal alpha)Thr3, (Gal alpha)Thr4-vespulakinin 1. The beta-glycosylated derivative was also prepared by the continuous flow variant of the Fmoc polyamide method.

Solution synthesis of the glyco-hexapeptide sequence of the human oncofetal fibronectin defined by monoclonal antibody FDC-6.

The glyco-hexapeptide sequence H-Val-(GalNAc-alpha)Thr-His-Pro-Gly-Tyr-OH, was synthesized in solution by the segment condensation procedure and the stepwise procedure. A peracetylated, O-galactosaminyl threonine derivative was used for incorporating the glycosylated amino acid residue into the peptide chain. A consistent racemization occurred during the acylation of H-His-Pro-Gly-Tyr(Bzl)-OBzl with Z-Val-[GalNAc(Ac)3-alpha]Thr-OH by the BOP-HOBt procedure and the D-allothreonine containing glyco-hexapeptide was isolated in about 20% yield.

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production.

Six Thr1 (O-glyco)-derivatives of the "phagocytosis stimulating peptide" tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(beta-D-GlcNAc)-Ser-Thr-OH, which correspond to the "tuftsin-region" at the Fc-domain of immunoglobulin G (amino acid residues 289-299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.

Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide.

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galactopyranosyl unit alpha- or beta-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289-299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)3 alpha]Thr-OH and Fmoc-[GalNAc(Ac)3 beta]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO2)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy.

Synthesis of O-glycosylated tuftsins by utilizing threonine derivatives containing an unprotected monosaccharide moiety.

Synthesis is described of four tuftsin derivatives containing a D-glucopyranosyl or a D-galactopyranosyl unit covalently linked to the hydroxy side chain function of the threonine residue through either an alpha or beta O-glycosidic linkage. Fmoc-threonine derivatives containing the suitable unprotected sugar were used for incorporating the O-glycosylated amino acid residue. Z-Thr[alpha-Glc(OBzl)4]-OBzl and Z-Thr[alpha-Gal(OBzl)4]-OBzl were prepared from the tetra-O-benzylated sugar and Z-Thr-OBzl by the trichloroacetimidate method in the presence of trimethylsilyl trifluoromethane sulfonate.

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins.

Sequential glycopeptides [Thr(beta-D-galactose)-Ala-Ala]n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d.

Synthesis, conformation, and biological activity of the carbohydrate-free vespulakinin 1.

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.

Synthesis and biological activity of tuftsin and rigin derivatives containing monosaccharides or monosaccharide derivatives.

Synthesis of some modified rigins is described in which either D-gluconic acid or 2-amino-2-deoxy-beta-D-glucopyranose have been linked to the parent molecule through amide bonds involving the alpha-amino function, alpha-carboxyl function or the gamma-amide function of glutamine in position 2. Glu2-rigin and D-gluconyl-Glu2-rigin have also been synthesized. Binding and phagocytosis assays have been carried out on the rigin derivatives and on some glycosylated tuftsin derivatives as well.