Laura Biondi

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.

Opioid peptides: synthesis and biological properties of [(Nγ-glucosyl,Nγ-methoxy)-α,γ-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. 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.

N-alkylation of amino acids during hydrogenolytic deprotection

Abstract Partial N-alkylation of amino acids was observed during hydrogenolytic deprotection.

Helix induction potential of N-terminal α-methyl, α-amino acids

A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.

Synthesis of the protected glycotetrapeptide corresponding to the N-terminal amino acid sequence of [Thr3-0-α-D-galactopyranosyl]-vespulakinin 1

Abstract The synthesis of the protected glycopeptide Z-Thr(tBu)-Ala-Thr-[α-D-Gal(Bzl) 4 ]-Thr(tBu)-NHNH-Boc was performed in solution by a stepwise coupling strategy. Fmoc-Thr[(α+β)-D- Gal(Bzl) 4 ]-0Su, obtained from the reducing sugar and the protected amino acid by the trifluoromethanesulfonic anhydride procedure, was reacted with H-Thr(tBu)-NHNH-Boc. The two diastereoisomers of the resulting glycosylated dipeptide derivative were purified and separated by column chromatography. The anomeric configuration of the 0-glycosidic linkage was assigned by 1 H-NMR-spectroscopy. The α-anomeric glycodipeptide, obtained in a larger amount, was selectively deblocked at the amino function and acylated with Z-Thr(tBu)-Ala-OH in the presence of either IIDQ or EEDQ, to afford the desired protected glycotetrapeptide.

Detection and quantitative micro-determination of nitro- 2-pyridyl amino acids by thin-layer chromatography on precoated sheets

Abstract The separation of 3-nitro-2-pyridyl- and of 5-nitro-2-pyridyl-amino acids on Eastman Kodak Chromagram sheets is described, together with their detection and their quantitative spetrophotometric microanalysis. The advantages of this method over the conventional procedure are discussed.

On the Role of Basic Residues of Head-to-Tail Cyclic Bradykinin Analogues on Rat Duodenum Relaxation

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.

Helix induction potential of N-terminal alpha-methyl, alpha-amino acids

A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.

Semisynthetic glycoproteins: preparation of glycosylated ribonuclease S′ analogues

Abstract 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′.

Linear and cyclic Thr6-Bradykinin analogues containing N-benzylglycine as a replacement for residues Phe5 and Phe8

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.