Mauro Ginanneschi

Modeling of copper(II) sites in proteins based on histidyl and glycyl residues

The complexes between copper(II) and four synthetic tetrapeptides bearing a single histidine residue within the sequence (AcHGGG, AcGHGG, AcGGHG and AcGGGH, respectively), have been investigated by potentiometric and spectroscopic methods (UV-Vis, circular dichroism and electron paramagnetic resonance). Potentiometric studies in the pH range 4-12 allowed identification and quantitative determination of the species present in solution for each copper-peptide complex. In all cases, upon raising pH, copper(II) coordination starts from the imidazole nitrogen of the His; afterwards three deprotonated amide nitrogens are progressively involved in copper coordination, except in the case of AcGHGG. Based on the potentiometric and spectroscopic results, detailed molecular structures are proposed for the dominant copper(II) tetrapeptide species existing in solution, either at neutral or alkaline pH. The structural consequences of the presence and of the location of a unique histidine residue within the tetrameric sequence are specifically analyzed. Results are discussed in relation to the modeling of copper(II) binding sites in proteins, particular emphasis being devoted to the copper complexes of the prion protein.

Spectroscopic and potentiometric study of the SOD mimic system copper(II)/acetyl-L-histidylglycyl-L-histidylglycine.

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the N-acetylated tetrapeptide HisGlyHisGly were determined in aqueous solution in the pH range 2-11. The potentiometric and spectroscopic data (UV-Vis, CD, EPR and Raman scattering) show that acetylation of the amino terminal group induces drastic changes in the coordination properties of AcHGHG compared to HGHG. The N3 atoms of the histidine side chains are the first anchoring sites of the copper(II) ion. At pH 4.7 and 5.6 both the imidazole rings cooperate in the formation of a 2N equatorial set, while, at higher pH values, 3N and 4N complexes are formed through the coordination of peptide N- atoms. The logbeta values of the copper complexes of AcHGHG are by far lower than those of the corresponding species in the parent CuII-HGHG system.

Novel sst5-Selective Somatostatin Dicarba-Analogues: Synthesis and Conformation−Affinity Relationships

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

Novel Octreotide Dicarba-analogues with High Affinity and Different Selectivity for Somatostatin Receptors

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

A synthetic glycopeptide of human myelin oligodendrocyte glycoprotein to detect antibody responses in multiple sclerosis and other neurological diseases

Glycopeptides of hMOG(30-50) containing a glucosyl moiety on the side-chains of Asn, Ser or Hyp at position 31 were synthesised. Antibody titres to hMOG(30-50) and to its glucoderivatives were measured by ELISA in sera of patients affected by different neurological diseases. Anti-hMOG(30-50) antibodies were detected only using the glycopeptide [Asn31(N-Glc)]hMOG(30-50).

Reactions of medicinally relevant gold compounds with the C-terminal motif of thioredoxin reductase elucidated by MS analysis.

The tetrapeptide Ac-Gly-[Cys-Sec]-Gly-NH(2), reproducing the C-terminal motif of the selenoenzyme thioredoxin reductase, was designed and synthesized, and its reactions with a few medicinally relevant gold(i,iii) compounds investigated by ESI-MS. Remarkably, the main reaction products could be unambiguously identified providing valuable insight into the likely mechanisms of enzyme inhibition by gold compounds.

Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds.

Cyclisation of histidine containing peptides in the solid-phase by anchoring the imidazole ring to trityl resins

Head-to-tail histidine containing cyclopeptides can be efficiently synthesised by a three-dimensional orthogonal solid-phase strategy (Fmoc/tBu/allyl) via anchoring the imidazole ring to trityl-resins. Furthermore, Fmoc-His(Trt-®-OA1 can be a useful starting support for the preparation of diketopiperazine combinatorial libraries.

Impact of ring size on the copper(II) coordination abilities of cyclic tetrapeptides

A new, 14-membered, tetraza cyclic tetrapeptide containing histidine and lysine side-chains, c(beta(3)homoLysdHisbeta-AlaHis), was designed, synthesized and characterized; its copper(II) binding properties were investigated in dependence of pH by potentiometric and spectroscopic methods. In line with previous studies of similar systems, the progressive involvement of amide nitrogens in copper(II) coordination was evidenced for pH values greater than 6. At physiological pH the dominant species consists of a copper(II) center coordinated by two amide nitrogens, an imidazole nitrogen and a water molecule. In contrast, at pH values higher than 8.7, a copper(II) coordination environment consisting of four amide nitrogens in the equatorial plane and the axial imidazole ligands is formed as clearly indicated by spectroscopic data and theoretical calculations. The behavior of this 14-membered cyclic tetrapeptide is compared to that of its 12-membered cyclic analog, particular attention being paid to the effects of ring size on the respective copper(II) binding abilities.

Spectroscopic and potentiometric study of copper(II) complexes with l-histidyl-glycyl-l-histidyl-glycine in aqueous solution

Abstract The complex formation between copper(II) and the tetrapeptide l -histidyl-glycyl- l -histidyl-glycine (HL) has been studied in aqueous solution in the pH range 2–10.5, by potentiometric and spectroscopic methods (visible, CD, EPR, 1 H NMR and Raman scattering). Between pH 3 and 6 the species [CuHL] 2+ and [CuH −1 L] have been detected. The former complex co-ordinates through two nitrogen and two oxygen atoms in the equatorial plane while, for the latter, the spectroscopic data (particularly Raman spectra) suggests an unusual structure with the two imidazole rings and two peptide NH in the plane. The species at pH 7, [CuH −2 L] − , is involved in the co-ordination by the amino group, the His 1 imidazole and two peptide nitrogens, while, for the complex [CuH −3 L] 2− , which seems to be predominant in alkaline medium, a third deprotonated backbone NH, replaces the imidazole nucleus.