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. 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.

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. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P3 it is unfavorable, in contrast to the P2 position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac-(Gal3)Ser-Arg-pNa in comparison to Ac-Ser-Arg-pNa, and by the hydrolysis of Ac-(Glcalpha,beta)Tyr-Arg-pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P'4 glycosylated peptide [Abz-F-H-L-V-I-H-(GIcNAcbeta)N-E-EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K(m) and higher k(cat) values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P'4 and P4 glycosylated substrates.

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. 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 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. Pharmacological experiments showed that cyclo-Gly-Ile-Ile-Gly-bradykinin (39 backbone atoms) and cyclo-Lys-bradykinin (30 backbone atoms) are about equipotent, when tested on the relaxation of the isolated rat duodenum preparation. The potency of cyclo-des Arg-bradykinin is at least three orders of magnitude lower. The potency of cyclo-Lys-Lys-bradykinin (33 backbone atoms) is one tenth the activity of bradykinin but about 10 times higher than the potency of the above-mentioned cyclokinins and makes the latter analogue the most potent end-to-end cyclic analogue known currently. The present results, in agreement with data from earlier reports, seem to indicate that the enhancement of the number of backbone atoms in the cyclic kinins first increases and subsequently decreases the potency, whereas a reduction in the atom number from 27 to 24 causes a dramatic decrease in potency.

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. 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.

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. 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.

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. 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.

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). 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)

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. 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.

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. 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.

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. 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.

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 the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetamido-2-deoxy-beta-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N2-fluorenylmethyloxycarbonyl-N4-(2-acetamido-3,4,6-tri-O-acetyl-2 -deoxy-beta-D - glucopyranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotected, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.

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. 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.

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. and 1H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze glycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r. data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50 degrees C are compatible with the presence of a small amount of alpha-helix or 3(10) helix. Since the glyco-hexapeptide is too short to form an alpha-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 3(10) helix is formed. The same is observed for the 21-residue glycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of beta-structure.

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.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(But)-Ala-Thr(But)-Thr(But)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO2)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO2)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.

Synthesis of modified tuftsins containing monosaccharides or monosaccharide derivatives.

Synthesis of some modified tuftsins is described in which a monosaccharide or a monosaccharide derivative was incorporated in the molecule. Acylation of H-Thr-Lys(Z)-Pro-Arg(NO2)-OBzl with D(+)-gluco-1,5-lactone followed by catalytic hydrogenation gave N alpha-gluconyl-tuftsin. Glycosylation of the carboxyl function of the C-terminal arginine has been achieved by reacting, through the mixed anhydride procedure, Boc-Thr-Lys(Z)-Pro-OH with 2-deoxy-2-(NG-nitroargininamido)-D-glucopyranose followed by catalytic hydrogenation and trifluoroacetic acid treatment. O-Glucosyl-tuftsin has been prepared by reacting o-nitrophenyl N-benzyloxycarbonyl-O-[(alpha + beta) 2,3,4,6-tetra-O-benzyl-D-glucopyranosyl]-threoninate with H-Lys(Z)-Pro-Arg(NO2)-OBzl in the presence of 1-hydroxybenzotriazole. Flash chromatography on silica gel allowed a partial separation of the diastereoisomers, one of which has been isolated in a reasonable yield. The single diastereoisomer and the alpha + beta anomeric mixture were separately deblocked by catalytic hydrogenation and purified by RP-HPLC.

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. Of all the tested peptides only rigin enhanced the phagocytic capacity of mouse peritoneal macrophages to the same extent as tuftsin. The peptides H-Thr-Lys-Pro-Arg-NH-Glc and N alpha-gluconyl-Gly-Glu-Pro-Arg-OH slightly enhanced phagocytosis. H-Thr[(alpha + beta)-O-glucosyl]-Lys-Pro-Arg-OH was found to displace 3H-tuftsin even better than tuftsin but lacked the ability to stimulate phagocytosis.

Synthesis of N alpha-, and C alpha-derivatives of (D)Ala2, Leu5-enkephalin.

Some (D)Ala2, Leu5-Enkephalin derivatives containing hydrophilic or hydrophobic moieties have been synthesized by the solution method. Improvement in the analgesic effect was achieved by increasing the hydrophilicity of the parent compound through covalent linkage of the 2-deoxy-D-glucopyranosyl residue to the carboxyl terminal amide nitrogen of leucine 5. Reductive glucosamination of (D)Ala2, Leu5-Enkephalin practically abolishes activity and increase in hydrophobicity lowers the potency of the parent compound in both the in vitro and in vivo tests.