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Peptide nucleic acid (PNA) analogs based on Nα-(thymin-1-ylacetyl)ornithine were previously shown to form triplexes with complementary RNA. In order to obtain optically pure compounds for hybridization experiments, chiral monomers based on D- or L-ornithine, Nδ-Fmoc-Nα-(thymin-1-ylacetyl)ornithine 2 and Nδ-Fmoc-Nα-(uracil-1-ylacetyl)ornithine 3 were synthesized either by a one-step or by a simple three-step procedure starting from Nδ-protected ornithine; the latter procedure led to enantiomerically pure products. Oligomerization of 2 and 3 was carried out either in solution, or by solid-phase peptide synthesis (SPPS) on an MBHA-Rink amide resin. The oligomers turned out to contain large amounts of epimerization products, especially those obtained by SPPS. Therefore, we examined carefully the parameters which may be involved in epimerization: the nature of the coupling reagent, of the base, and the addition mode. Coupling of the monomer L-3 was performed under various conditions. Lower racemization was found to occur when using (7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as coupling agent and 2,4,6-trimethylpyridine (sym-collidine, TMP) as base, without preactivation, leading to a residual 4% of the D-enantiomer. By applying a procedure based on the stepwise addition of the base the D-enantiomer content was reduced to less than 1%. Using this procedure, a decamer of L-3 was synthesized, which was shown to contain less than 2% of the D-ornithine derivative.

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.

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. Stepwise elongation of the C-terminal tetrapeptide with Fmoc-[GalNAc(Ac)3-alpha]Thr-OH and Z-Val-OH, in the presence of the same coupling reagents, yielded the L-threonine containing diastereoisomer without detectable racemization. A side product, the Nim-ethoxycarbonylated hexapeptide derivative, formed during the EEDQ-mediated condensation of Fmoc-[GalNAc(Ac)3-alpha]Thr-OH with the C-terminal tetrapeptide, was isolated and characterized. Preliminary studies showed that the synthetic glycohexapeptide is a good competitive inhibitor of the binding of the FDC-6 monoclonal antibody to the oncofetal fibronectin, supporting the idea that it should represent the minimum essential structure required for the FDC-6 activity.

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. The alpha glycosylated threonine derivatives were converted into Fmoc-Thr(alpha-Glc)-OH and Fmoc-Thr(alpha-Gal)-OH by catalytic hydrogenation followed by acylation with Fmoc-OSu. beta-Glucosylation and beta-galactosylation of threonine were carried out by reacting the proper per-O-acetylated sugar with Z-Thr-OBzl and boron trifluoride ethyl etherate in dichloromethane. Catalytic hydrogenation of the beta-O-glycosylated threonine derivatives followed by acylation with Fmoc-OSu and deacetylation with methanolic hydrazine yielded Fmoc-Thr(beta-Glc)-OH and Fmoc-Thr(beta-Gal)-OH, respectively. The O-glycosylated threonine derivatives were then reacted with H-Lys(Z)-Pro-Arg(NO2)-OBzl in the presence of DCC and HOBt and the resulting glycosylated tuftsin derivatives were fully deblocked by catalytic hydrogenation, purified by HPLC, and characterized by optical rotation, amino acid analysis, and 1H NMR. The beta-galactosylated tuftsin was also prepared by the continuous flow solid phase procedure.

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). In order to define further the specificity of the reactive site, we synthesized various glycopeptides based on the unglycosylated hexapeptide sequence (P1) and compared their reactivities with these antibodies. In continuation of our structure-activity relationship studies the (Asn3,Ala5)-glycohexapeptide analogue (P3) was synthesized by a solid-phase procedure. The [Ala(CN)3,Ala5]-glycopeptide (P4), owing to dehydration of the asparagine side chain amide during carboxyl activation of Fmoc-Asn-OH, was also isolated. Fmoc-[GalNAc(Ac)3-alpha]Thr-OH was used for incorporating the glycosylated amino acid residue. For the sake of comparison the epitope P2 and the hexapeptide sequence P1 were also synthesized. The final products were characterized by elemental and amino acid analyses, optical rotation, analytical HPLC, proton NMR and fast-atom bombardment mass spectroscopy. Synthetic analogues were applied to inhibit onco-FN specific MAbs FDB-1, FDB-4 and FDC-6 binding to immobilized onco-FN, and their activities were compared with onco-FN and nor-FN. P2 exhibited an activity similar to that of an intact molecule of onco-FN. Deglycosylation (P1) or replacement of amino acid (P3, P4) greatly reduced activity. Data clearly showed that P2 was the minimal essential structure of the epitope in onco-FN defined by MAbs FDB-1, FDB-4 and FDC-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Derivatives of the tetraguanidinated bovine trypsin-kallikrein inhibitor lacking up to four amino-acid residues have been prepared by Edman degradation of the amino-terminal Arg-Pro-Asp-Phe sequence. Comparative studies on the native inhibitor, tetraguanidinated inhibitor, and truncated sequences demonstrated that none of the four N-terminal amino-acid residues are essential for the regeneration of native structure and biological activity by air oxidation of the reduced molecule. However residues 2, 3, and 4, although to a different extent, are important for the protein stability and significantly affect the conformation of the tetraguanidinated inhibitor and the rate of the reactivation process. Removal of the terminal positive charge from Arg 1 by diazotization gave a fully active deaminated, tetraguanidinated inhibitor but the lack of the salt bridge interaction between the amino and the carboxyl terminus of the protein prevented the correct folding of the reduced molecule by air oxidation as shown by activity measurements and conformational studies. The stability constants and the standard free energies of binding of the complexes between trypsin and the different inhibitor derivatives have been determined.

The synthesis, by fragment condensation, of protected peptides related to the N-terminal (positions 1-14) and C-terminal (positions 36-52) portions of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The alpha-carboxyl function of the lysyl residue in position 14 was used as the free acid, or protected by the tert-butyloxycarbonylhydrazide. Two different synthetic pathways were used in the preparation of the 36-52 sequence of the inhibitor. The identity and purity of the synthesized peptide derivatives were established by amino acid analysis of acid hydrolysates, optical rotation and this layer chromatography in two solvent sytems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values and enzymic digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.

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. The amino-terminal octapeptide sequence of vespulakinin 1, Fmoc-Thr(tBu)-Ala-Thr(tBu)-Thr(tBu)-Arg(Pmc)-Arg(Pmc)-Arg(Pmc)-Gly-OH, and its N alpha-Boc-[(Gal beta)Thr3, (Gal beta)Thr4]-analogue, were used to prepare N alpha-(1-8 VSK 1)-cyclo-N epsilon-kallidin and N alpha-[(Gal beta)Thr3, (Gal beta)Thr4, 1-8 VSK 1]-cyclo-N epsilon-kallidin. Peptides and glycopeptides were characterized by amino-acid analysis, optical rotation, analytical HPLC and FAB-MS. Consistent with previous findings, preliminary pharmacological experiments on smooth muscle preparations showed that the cyclic, or partially cyclic, analogues were significatively less potent than the linear ones.

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. The diglycosylated 1-8 VSK 1 sequence Boc-Thr(tBu)-Ala-(Gal beta)Thr-(Gal beta)Thr-Arg(Pmc)-Arg(Pmc)-Gly-OH was also synthesized by the solid-phase procedure and used to prepare the N epsilon-[(Gal beta)Thr3, (Gal beta)Thr4, 1-8 VSK 1]-cyclo-N-alpha- kallidin. Peptides and glycopeptides were characterized by amino acid analysis, optical rotation, analytical HPLC and FAB-MS. Preliminary pharmacological experiments showed that the cyclic kinin analogues are much less potent then bradykinin but still show specific bradykinin-like actions that support the hypothesis of the presence of a pharmacophore in the centre of the (brady)kinin molecule.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys0]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr6]-KL (YKL), [Trp6]-KL (WKL), cyclo-([Tyr6]-KL) (YCKL) and cyclo-([Trp6]-KL) (WCKL). The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL. The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds. The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.

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. The affinity of the new compounds for the delta-opioid receptor was expressed by the inhibition constant (Ki) of the binding of the delta-receptor selective ligand [3H]naltrindole (NTI) to rat brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the mu-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the delta-opioid receptor agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect drastically the Ki and IC50 values for delta-sites, suggesting that an electrostatic interaction does not play an essential role in the binding to delta-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to delta-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the gamma-nitrogen of the Dab residue in comparison with the Asn amide side chain.

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. Preliminary molecular mechanics calculations were also performed on some synthetic peptides. Pharmacological screening using the relaxation of the isolated rat duodenum preparation showed that incorporation of N-benzylglycine at positions 5 and/or 8 in the linear Thr6-BK causes a substantial decrease in potency. Comparable incorporation in cyclo Thr6-BK, at position 8, or 5 and 8, resulted in nearly inactive analogues. However, cyclo BzlGly5,Thr6-BK showed a potency which is of the same order of magnitude as for cyclo-BK and cyclo Thr6-BK.

Three linear bradykinin (BK) analogues, Lys-Lys-BK, Nle-Lys-BK and Lys-Nle-BK and their head-to-tail cyclic analogues, along with cyclo-Nle-Nle-BK and cyclo-Lys-Lys-[Trp5]BK, were synthesized and tested on an isolated rat duodenum preparation. All kinins, except the [Trp5]-analogue, cause relaxation with EC50 values in the picomolar range. The most potent linear analogue (Lys-Nle-BK) is about 40 times more active than BK and the most potent cyclic kinin (cyclo-Nle-Lys-BK) is about 6 times more active. Present results suggest that the significant potency of cyclo-Lys-Lys-BK, the earlier most potent cyclic kinin which is only a little less potent than linear BK, depends on the ring size rather than on the presence of the extra basic residues.

The crystal structure of the fully protected glycotripeptide N -benzyloxycarbonyl- O -(2,3,4,6-tetra- O -acetyl-β- d -galactopyranosyl)- l -threonyl-α-aminoisobutyryl-α-aminoisobutyric acid tert -butyl ester [Z-(β- d -GalAc 4 )- l -Thr-Aib-Aib-O t Bu] has been determined by X-ray diffraction. The peptide backbone is fully extended at Thr(1), left-handed helical at Aib(2), while it is right-handed helical at Aib(3). The fully extended conformation at the N-terminal Thr residue is stabilized by an intramolecular H-bond involving the N-1–H and O-1C-1 groups (intramolecularly H-bonded C 5 conformation). Two additional intramolecular H-bonds are observed, involving the peptide N–H groups of Aib(2) and Aib(3) as the donors, and the pyranosidic O-5 oxygen atom and the carbonyl oxygen atom of the acetoxy group on C-6 of the sugar, respectively, as the acceptors. Owing to the peptide–sugar H-bonds, the peptide backbone is forced to adopt a conformation dramatically different from the β-bend/3 10 -helical conformation usually observed for Aib-rich peptides. The implications and limitations of these findings on the effect of O -glycosylation on the conformation of natural peptides are briefly outlined.

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

This study was undertaken to determine the importance of the central aromatic moiety in the kallidin and cyclokallidin molecules, using the relaxation of the isolated duodenum of the rat. Replacement in kallidin of the central phenylalanine by tryptophan increased the potency from an EC50 of 3 x 10(-10)M to 2 x 10(-12)M. Replacement by tyrosine decreased the potency to an EC50 of 8 x 10(-8)M. In cyclo-kallidin (EC50: 10(-8)M) the potencies were decreased: cyclo-Trp6-kallidin showed an EC50 of 10(-6)M and cyclo-Tyr6-kallidin of 3 x 10(-7)M. The relaxation of the rat duodenum by linear and cyclic kinins was potentiated by the bradykinin potentiating peptide BPP5a and antagonized by the B2 antagonist HOE-140. At a concentration of 10(-9)M, HOE-140 significantly decreased the potencies of bradykinin and cyclo-kallidin, but not of the B1 agonist desArg9-bradykinin.

The conversion of a nitrile to an oxazoline group in an amino acid side chain promoted by platinum(II) was investigated. While benzyloxycarbonyl-β-cyano-alanine (Z-Ala(CN)OH) did not give any evidence of coordination of the nitrile group to Pt(II) in different complexes, the corresponding methyl ester Z-Ala(CN)OCH 3 readily afforded the Pt(II)-nitrile complex trans -[Pt(CF 3 ){Z-Ala(CN)OCH 3 }(PPh 3 ) 2 ][BF 4 ] in good yield by reaction with the cationic derivative trans -[Pt(CF 3 ) 2 (solv)][BF 4 ] (solv = CH 2 Cl 2 ). The reactivity of the CN group in the complex trans -[Pt(CF 3 ){Z-Ala(CN)OCH 3 }(PPh 3 ) 2 ][BF 4 ] with ClCH 2 CH 2 O − to the oxazoline ligand 2 was examined. The presence of the oxazoline ring in the final products was evidenced by IR, 1 H and 13 C NMR data and confirmed by FAB and MALDI mass spectra, but its isolation was hampered by the cleavage of the oxazoline ring under the chromatographic conditions applied.

The conformation of the natural peptide threonine6 (Thr6)-bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Phe8-Arg9, was investigated in DMSO by nmr spectroscopy and computer simulations. The structural analysis of the Thr6-peptide is made particularly interesting by the fact that despite the high sequence homology with native bradykinin (only one conservative substitution: Ser6/Thr6) there is a marked and significant difference in the biological profiles of the two peptides. The nmr spectra indicate a relatively flexible structure with the presence of an N-terminal turn. Standard distance geometry calculations failed to produce structures in accord with the experimental observations; the resulting structures are indeed too rigid and conformationally restricted for the nmr data. The results of ensemble calculations reveal conformational changes occurring rapidly on the nmr time scale and allow for the establishment of a series of disordered conformations, prevalently extended with a partially populated turn in residues 2–5, which when considered together, as an average, fulfill the experimental restraints. The structural characterization of (Thr6)-bradykinin supports the hypothesis of the significant role of the residue at position 6 on both conformation as well as biological activities and suggests a N-terminal turn as a possible bioactive conformation. © 1997 John Wiley & Sons, Inc. Biopoly 40: 561–569, 1996

The conformation of the head-to-tail cyclic analogue of bradykinin in DMSO was investigated by nmr. Three sets of resonances were detected and fully assigned. These were attributed to the presence of three stable conformers, two of which were exchanging on the nmr time scale. A fourth, incomplete set of resonances was detected but not assigned. The three major conformers differ in the conformation at the three X-Pro bonds present. From nuclear Overhauser effect spectroscopy (NOESY) spectra, three sets of interproton distances were derived and used in NOE-restrained distance geometry calculations. The resulting structures were refined by energy minimization to yield families of structures. Conformer I is characterized by the presence of two type VIb β-turns between Arg1 and Gly4 and between Phe5 and Phe8. The first β-turn is present also in conformer II, while an inverse γ-turn bridging Pro3 is the most pronounced structural feature of conformer III. © 1995 John Wiley & Sons, Inc.

Glycopeptide analogues of ribonuclease S-peptide, des 16–20 [(Gal β)Thr3]-S-peptide and des 16–20 [(Gal β)Thr6]-S-peptide, were synthesized by the solid-phase procedure based on the Fmoc chemistry. Reassociation of the synthetic glycopeptides with S-protein yielded enzymatically active, glycosylated RNase S′ variants. The conformational properties of the S-peptide analogues in aqueous buffer and in the presence of trifluoroethanol were investigated by circular dichroism spectroscopy. The activation curves of the S-protein with varying amounts of the synthetic analogues, using C > p as the substrate, were determined and the dissociation constants (Kd), the free energies of binding (−ΔG), and the kinetic parameters (Km and k2) for the RNase S′ adducts were calculated and compared with the corresponding values obtained for the S-peptide. The properties of the semisynthetic, glycosylated enzymes agree with the results of previous investigations on the conformational and catalytic features of S-peptide analogues and related RNase S′.

Several aspects of the mechanism of action of Pro-rich antimicrobial peptides, together with their low toxicity ill mammalian cells, make them good candidates for the development of new antibiotic agents. We investigated the effect induced in the insect antimicrobial peptide apidaecin Ib by the replacement of a single arginine/leucine residue witha N-substituted glycine. The resulting peptoid-peptide hybrids are more resistant to proteolysis and devoid of any significant cytotoxic activity, but moving the [NArg]residue from the N- to the C-terminal end of the molecule progressively reduces the antibacterial activity. Cell uptake experiments in E. coli cells suggest that the loss of antibacterial activity of [NArg(17)]apidaecin is a consequence of its inability to translocate into bacterial cells. Conversely, apidaecin and its peptoid-peptide hybrids are able to cross the plasma membrane ill eukaryotic cells and to diffuse in the cytosol, although their translocating ability is far less effective than that of other known cell permeant peptides.

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide, exhibiting a wide spectrum of biological activities in mammals. However, the clinical applications of VIP are mainly hampered because of its rapid degradation in vivo. Peptide glycosylation, a procedure frequently used to increase peptide resistance to proteolytic degradation and consequently increase peptide metabolic stability, has not been performed yet on VIP. The presence of three N-glycosylation sites on VIP receptor type 1 (VPAC1) was previously demonstrated. Therefore, glycosylation of the VIP ligand could potentially increase its receptor affinity because of glyco-glyco interactions between the ligand and the receptor. In order to enhance VIP's metabolic stability and to increase its ligand-receptor binding/activation, eight glycosylated VIP derivatives were successfully synthesized by the solid-phase procedure. Each VIP analog was monoglycosylated by a monosaccharide addition to one amino-acid residue along the sequence. Glycosylation did not affect the alpha-helical structure shown by the native VIP in organic environment. Few glycosylated VIP analogs displayed highly potent VPAC1 receptor binding and cAMP-induced activation; only 4-6 fold lower in comparison to the native VIP. Furthermore, the peptide analog glycosylated on Thr11 ([11Glyc]VIP) showed a significantly enhanced stability toward trypsin enzymatic degradation in comparison to VIP. Analysis of the degradation products of [11Glyc]VIP showed that differently from VIP, incubation of the peptide [11Glyc]VIP with trypsin resulted in no cleavage at the Arg12-Leu13 peptide bond, suggesting that VIP glycosylation may lead to enhanced metabolic stability.

Syntheses of the [Lys7]- and [Hyp6,Lys7]-dermorphin analogues in which either Tyr5 or Hyp6 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 biological potency of the glucosylated analogues was compared with that of the µ-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr5 or Hyp6 reduces the potency of both [Lys7]-dermorphin and [Hyp6,Lys7]-dermorphin. The effect induced by the Tyr5 glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp6 residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp6,Lys7]-, [Hyp(βGlc)6,Lys7]- and [Tyr(βGlc)5,Lys7]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8–10 times less active than the parent peptide, but its analgesic effect lasts significantly longer. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.

Syntheses are described of new endomorphin 1 and 2 peptoid-peptide hybrids in which Tyr(1), and either one or both Phe(3) and Phe(4) have been replaced by N-substituted-glycine. The preparation is also described of two glycosylated Hyp(2)-endomorphin 2 analogues in which either 2,3,4,6-tetra-O-acetyl glucose or glucose are beta-O-glycosidically linked to the hydroxyproline residue. The Hyp(2)-endomorphin sequences have also been elongate by adding a C-terminal P-alanine residue and several linear dimers have been prepared by coupling either the native peptides or the modified analogues. The cyclo endomorphin 2 has also been synthesized. Preliminary pharmacological experiments on isolated organ preparations showed that the agonist activities of both endomorphin 1 and 2 are not significantly affected by the Pro/Hyp substitution. Phe(4)/Nphe substitution in the endomorphin 1 reduced the potency on guinea pig ileum (GPI) by about 100 times and abolished the agonist activity on mouse vas deferens (MVD) preparation. The decrease of the agonist activity induced by modification of one phenylalanine residue only, either Phe(3) or Phe(4), is lower on endomorphin 2. Either modification of both Phe(3) and Phe(4) or glycosylation of the Hyp(2)-endomorphin 2 cancelled any agonist activity on both preparations. The linear peptide dimers [endomorphin 112, [endomorphin 2](2), [Hyp(2)-endomorphin 1](2), [Hyp(2)-endomorphin 2](2), [Hyp(2)-endomorphin 1-Hyp(2)-endomorphin 2](2) or [Hyp(2)-endomorphin 2-Hyp(2)-endomorphin 1](2), are 7-19 times less potent than endomorphin 1 on GPI and significantly less active than endomorphins 1 and 2 on MVD. The other afforded modifications significantly affected or abolished the agonist activity of the resulting endomorphin analogues on both GPI and MVD preparations.

Syntheses are described of the nociceptin (1-13) amide [NC(1-13)-NH2] and of several analogues in which either one or both the phenylalanine residues (positions I and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and beta-alanine, respectively. The preparation is also described of NC(1-13)-NH2 analogues in which either galactose or N-acetyl-galactosamine are beta-O-glycosidically linked to Thr(5) and/or to Ser(10). Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe(4), Thr(5), Ala(7) and Arg(8) are crucial residues for OP4 receptor activation. Manipulation of Phe(1) yielded peptides endowed with antagonist activity but [Nphe(1)] NC(1-13)-NH2 acted as an antagonist still possessing weak agonist activity. Introduction of the beta A1a residue either in position 7 or 11 of the [Nphe(1)] NC(1-13)-NH2 sequence, abolished any residual agonist activity and [Nphe1, beta Ala(7)] NC(1-13)-NH2 and [Nphe(1), beta Ala11] NC(1-13)-NH2 acted as competitive antagonists only. Modification of both Ala(7) and Ala(11) abolished the antagonist activity of [Nphe(1)]NC(1-13)-NH2 probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(beta Gal)(10)] NC(1-13)-NH2 displayed an activity comparable with that of NC(1-13)-NH2, [Ser(beta GalNAc)(10)] NC(1-13)-NH2 and [beta Ala(11)] NC(1-13)-NH2 were five and 10 times less active, respectively.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-α-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-α-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 α-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 α-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

The conformational properties of the homo oligomers of increasing chain length Boc-(Asn)n-NHMe (n = 2, 4, 5), (GlcNAc-β-Asn)n-NHMe (n = 2, 4, 5, 8) and Boc-[GlcNAc(Ac)3-β-Asn]n-NHMe (n = 2, 4, 5) were studied by using NOE experiments and molecular dynamic calculations (MD). Sequential NOEs and medium range NOEs, including (i,i+2) interactions, were detected by ROESY experiments and quantified. The calculated inter-proton distances are longer than those characteristic of β-turn secondary structures. Owing to the large conformational motions expected for linear peptides, MD simulations were performed without NMR constraints, with explicit water and by applying different treatments of the electrostatic interactions. In agreement with the NOE results, the simulations showed, for all peptides, the presence of both folded and unfolded structures. The existence of significant populations of β-turn structures can be excluded for all the examined compounds, but two families of structures were more often recognized. The first one with sinusoidal or S-shaped forms, and another family of large turns together with some more extended conformations. Only the glycosylated pentapeptide shows in vacuo a large amount of structures with helical shaped form. The results achieved in water and in DMSO are compared and discussed, together with the effect of the glycosylation. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

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. Thus, like AZT, it inhibits reverse transcriptase activity and HIV-antigen expression, and similarly to tuftsin, it stimulates IL-1 release from mouse macrophages and augments the immunogenic function of the cells. Importantly, the conjugate is not cytotoxic to T-cells. The results suggest that the AZT-tuftsin conjugate might have potential use in AIDS therapy.

Glycoproteins consist of carbohydrates covalently linked with proteins. They are widely distributed in nature both in animals and plants, and have also been discovered in some bacteria and viruses. Practically all cells produce glycoproteins and the ubiquitous occurrence of these glycoconjugates reflect their vital function in many physiological and pathological processes. Glycopeptides, which retain the critical carbohydrate-peptide linkage region of a glycoprotein but lack its size and complexity, are much more amenable to study and are important models for glycoproteins. The selective chemical synthesis of glycopeptides, either in solution or by solid phase procedure, and more recently enzymatic synthesis, has made remarkable progress over the last years. Strategies for generating glycopeptide mimics with non-native sugar-peptide linkages have been recently described which use chemoselective ligation, the selective covalent coupling of mutually and uniquely reactive functional groups under mild, aqueous conditions, without protecting groups or activation procedures.

Enzyme activities along the kynurenine pathway, liver tryptophan 2,3-dioxygenase, small intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase, involved in the catabolism of tryptophan, were studied in male adult Wistar albino rats. Intestine superoxide dismutase and serum tryptophan were also determined. Hepatic tryptophan 2,3-dioxygenase is present both as holoenzyme and apoenzyme, but the total activity is inferior to that of intestine indole 2,3-dioxygenase which, therefore, actively oxidizes tryptophan in rats. However, this activity is inhibited by scavengers for the superoxide anion, such as superoxide dismutase, which also shows to be active in small intestine of rat. However, the more active enzymes appeared to be kynurenine 3-monooxygenase and 3-hydroxyanthranilate 3,4-dioxygenase. The former is equally active in both liver and kidney, the latter is more active in liver. Kynurenine-oxoglutarate transaminase is much more active in kidney than in liver, and much more active than kynureninase, which shows similar activities in both tissues. In contrast to the high activity of 3-hydroxyanthranilate 3,4-dioxygenase, aminocarboxymuconate-semialdehyde decarboxylase is 30-35 times less active, showing the efficiency of conversion of tryptophan to NAD. These data demonstrate that rat is a useful animal model for studying tryptophan metabolism along the kynurenine pathway. Serum tryptophan appeared to be 90% bound to proteins. Results demonstrate that, in rat, tryptophan is mainly metabolised along the kynurenine pathway. Therefore, rat is a suitable animal model for studying tryptophan metabolism in the pathological field.

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. In the CD spectra of deltorphin analogues the aromatic contribution is lower and the dichroic curves indicate the predominance of random conformer populations. The disappearance of the aromatic contribution in the [Ntyr1,D-Ala2]-deltorphin spectrum could be explained in terms of high conformational freedom of the N-terminal residue. The kinetics of degradation of the synthetic peptoids digestion by rat and human plasma enzymes were compared with that of [Leu5]-enkephalin. The binding to opioid receptors was tested on crude membrane preparations from CHO cells stably transfected with the mu- and delta-opioid receptors. The biological potency of peptoids was compared with that of dermorphin in GPI preparations and with that of deltorphin I in MVD preparations. All the substitutions produced a dramatic decrease in the affinity of the peptide-peptoid hybrids for both the mu- and delta-opioid receptors. Nval5 and/or Nval6 containing hybrids behaved as mu-opioid receptor agonists and elicit a dose-dependent analgesia (tail-flick test) when injected i.c.v. in rats.

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. The affinity of the new compounds for the delta-opioid receptor was expressed by the inhibition constant (Ki) of the binding of the delta-receptor selective ligand [3H]naltrindole (NTI) to rat brain membrane preparations. The in vitro biological activity of the synthetic peptides was compared with that of the mu-opioid receptor agonist dermorphin in guinea pig ileum (GPI) preparations and with that of the delta-opioid receptor agonist deltorphin I in mouse vas deferens (MVD) preparations. The substitution of Asp4 with Asn failed to affect drastically the Ki and IC50 values for delta-sites, suggesting that an electrostatic interaction does not play an essential role in the binding to delta-opioid sites. The steric hindrance of the side chain of the residue in position 4 affects binding to delta-sites. The increase of the Ki value is smaller when the sugar-peptide linkage involves the gamma-nitrogen of the Dab residue in comparison with the Asn amide side chain.

Bioactive peptides, exhibit a remarkable range of biological properties, but their utilization for clinical purposes such as diagnostic or therapeutics has some serious drawbacks and disadvantages. The main obstacles are their susceptibility to attack by proteolytic enzymes, their poor bioavailability, and for some of them, the difficulty of exerting central nervous effects qfter peripheral administration. Many efforts will be made in the near future by peptide chemists, in diverse areas of therapeutics such as the search of new drugs, active on the nervous system and capable of producing those typical, analgesic effects which led to the clinical use of opiates, but devoid of undesirable properties. Another object deals with anti-microbial peptides. Under the selective pressure exerted by the use and misuse of conventional antibiotics, bacteria are rapidly gaining resistance to these drugs and the search for novel antibacterial agents is in high demand.

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-). The insertion of a glycosylated threonine residue in the apidaecin Ib sequence improves the sequence homology with drosocin but reduces the antimicrobial activity. To gain information on the possible bioactive conformation of these peptides, we synthesized an unglycosylated cyclic analogue of drosocin, containing an intrachain disulfide bond, and the head-to-tail cyclic analogues of drosocin and apidaecin, as well as their corresponding cyclic dimers. Only the large cyclic dimer of apidaecin partially retained the antimicrobial activity, suggesting that a bending of the peptide chain, in particular in the middle of the molecule, is not a structural element characteristic of the bioactive conformation of drosocin and apidaecin. Experiments aimed at testing the effect of selected drosocin and apidaecin peptides on biological membranes showed that some peptides display a moderate hemolytic activity and that a dissociation between antibacterial activity and cytotoxicity to eukaryotic cells can be achieved in differently glycosylated peptide analogues.

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. Proton NMR measurements in CDCl3 and in DMSO-d6 were also carried out and the effect of temperature variation on the chemical shifts of amide protons was determined in DMSO-d6 (range 298-335 K) and in CDCl3 (range 298-320K). CD spectra were recorded in water and in TFE. Solubility problems prevented measurements in CDCl3 solution for Z-(Thr)4-NHCH3 and for the entire III1-5 series. The existence of unordered structures in the carbohydrate-free oligomers and of more or less extended, organized structures in the glycosylated derivatives is indicated by the NMR and IR measurements. The sugar moieties apparently show a structure-inducing effect on the peptide chain.

Synthesis, characterization, and conformational analysis by FT-IR absorption, 1H NMR and X-ray diffraction techniques are described for a series of side-chain O-glycosylated Thr peptides of different main-chain length rich in the helicogenic Aib residue. The results obtained, compared with those of related peptides containing side-chain protected Thr and Ser residues and host Aib homo-oligomers, also reported in this work, provided new information on the preferred conformation of the naturally occurring antifreeze glycopeptides.

Most antimicrobial peptides kill bacteria by acting on the cell membrane. A direct correlation between the antibiotic effect and the ability to permeabilize has been found, for example, for magainins, cecropins and dermaseptins [1], Several alternative pathways have, however, been proposed as the killing mechanism for other antimicrobial peptides [2], sometimes requiring the recognition of a specific bacterial target. Recent results upheld the model of permease/transporter-mediated uptake in bacterial cells [3] for three small proline-rich peptides: apidaecin, drosocin and pyrrhocoricin, originally isolated from insects [4]. Information about the bioactive conformation of these peptides are thus fundamental to elucidate their mechanism of action. A popular approach to better define the structural features required for an effective binding of the peptide to the receptor/docking molecule, is to restrict its conformational freedom by introducing some conformational constrains, e.g. cyclization.

N-Benzylglycine (Nphe) is an achiral structural isomer of phenylalanine that may be useful as an amino acid replacement in SAR studies. The first incorporation of Nphe into a biologically active peptide was reported by using bradykinin (BK) [1]. In continuation of our investigations on the structure-activity relationship of BK and BK analogues [2] we focused our attention on Thr6-BK (H-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH), a native kinin discovered in the venom of a solitary wasp and present also in ant venom and in frog skin. Thr6-BK proved to be about 10 times more potent than BK in the insect central nervous system [3] as well as in stimulating smooth muscle contraction [4]. The structural analysis of Thr6-BK and its analogues is made particularly interesting by the fact that despite the high sequence homology with native BK (only one conservative substitution: Ser6/Thr6) there is a marked and significant difference in the biological profile of the two peptides. The conformation of the native Thr6-BK has been already investigated in DMSO by NMR spectroscopy and computer simulations [5]. We report in this communication the synthesis and some preliminary pharmacological experiments and structural investigations of new linear and cyclic Thr6-BK analogues in which either one or both the Phe residues in the peptide sequence have been substituted by N-benzylglycine.

In the present paper we describe the synthesis of some dermorphin and deltorphin analogues beta-O- and alpha-C-glycosylated on the C-terminal amino acid residue and report their opioid receptor affinity and selectivity as well as their analgesic potency after subcutaneous injection in mice.

Three synthetic vespulakinin analogues either with or without carbohydrate moieties and mastoparan B isolated from Vespa basalis venom were investigated for their immunogenic activity and solution conformation. Mice immunized with these wasp venom peptides, with the exception of (Gal alpha)Thr3, (Gal alpha)Thr4-vespulakinin 1, showed positive antibody responses. However, the response elicited by mastoparan B was much higher than those induced by vespulakinin analogues. The class of antibody induced by these peptides was identified as an IgG1 isotype with kappa-light chain, suggesting stimulation of a T-cell-dependent immune response by these peptides. According to the circular dichroism spectra of these peptides, the structures of the vespulakinin analogues in solution were largely unordered, while mastoparan B exhibited a conformation rich in alpha-helices. The presence of carbohydrate moieties and the rather random structure in vespulakinins may interfere with T-cell recognition of the peptides, leading to lower immune responses.

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. The conformational features of the cyclic and linear kinins were determined by circular dichroism measurements in water, 95% trifluoroethanol and 8 M guanidinium chloride. Consistent with previous findings, preliminary pharmacological experiments on smooth muscle preparations showed that cyclic wasp kinins were 50-100 times less potent than their linear analogues. Moreover, cyclo-VSK-A and cyclo-VSK-T behave like kininase inhibitors by preventing the degradation of straight kinins.

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. The alpha-glucosylated dipeptide was isolated from the diastereomeric mixture, selectively deblocked, and acylated with Z-Thr-Lys(Z)-OH by the mixed anhydride procedure. In the preparation of the beta-glucosylated analogue the BOP procedure was used for reacting Boc-[Glc(Ac)4 beta]Hyp-OH with H-Arg(NO)2-OBzl was well as for the final coupling to tetrapeptide. Removal of protecting groups from crude tetrapeptides was achieved by catalytic hydrogenation. Deacetylation of the sugar moiety of the beta-glucosylated tetrapeptide was achieved by treatment with sodium methoxide in methanol. The synthetic compounds were isolated by ion exchange chromatography, and characterized by elemental analysis, amino acid analysis, optical rotation and proton NMR. Their capacity to evoke the release of interleukin 1 from mouse peritoneal macrophages and to modulate immunogenic activity of antigen-fed cells was evaluated, in comparison with tuftsin and rigin. All of the analogues were found to possess tuftsin-like activity.

1. The pharmacological properties of four synthetic analogues of the wasp neurotoxin, Vespulakinin 1, were studied using a cascade of mammalian smooth muscle preparations and the synaptic transmission from the cockroach cereal nerves to a giant interneuron. 2. All analogues have an extremely slow bradykinin-like effect on the smooth muscles. The carbohydrate-free and the two mono-glycosylated analogues are about equally active with bradykinin. 3. The double glycosylated derivative is about 5 times more potent than bradykinin. 4. All analogues have two different effects on synaptic transmission in the insect CNS—at first a direct and reversible block of excitatory nicotinic transmission with a concurrent activation of the inhibitory GABA-ergic system and, secondly, a delayed irreversible block of the transmission, comparable to the block described earlier for bradykinin and Thr 6 -bradykinin. 5. For the synaptic transmission in the insect CNS the double glycosylated kinin is about 5 times more potent than bradykinin.

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. The synthesized glycopeptides were purified and characterized by amino acid analysis, optical rotation, analytical HPLC, 1H- and 13C-NMR and FAB-MS. Preliminary pharmacological experiments showed that the carbohydrate-free vespulakinin 1 is less active than bradykinin (about 0.3 times on a molar basis) when tested by guinea pig rectum contraction, and the two monoglycosylated analogues are equiactive (about 0.9 times the bradykinin activity). The most active derivative, the (Gal alpha)Thr3, (Gal alpha)Thr4-vespulakinin 1 analogue, was about 2.5 times as active as bradykinin.

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.