Anna Maria Papini

Synthesis and Conformational Analysis of a Cyclic Peptide Obtained via i to i+4 Intramolecular Side-Chain to Side-Chain Azide-Alkyne 1,3-Dipolar Cycloaddition

Intramolecular side-chain to side-chain cyclization is an established approach to achieve stabilization of specific conformations and a recognized strategy to improve resistance toward proteolytic degradation. To this end, cyclizations, which are bioisosteric to the lactam-type side-chain to side-chain modification and do not require orthogonal protection schemes, are of great interest. Herein, we report the employment of Cu(I)-catalyzed 1,3-dipolar cycloaddition of side chains modified with azido and alkynyl functions and explore alternative synthetic routes to efficiently generate 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptides. The solid-phase assembly of the linear precursor including epsilon-azido norleucine and the propargylglycine (Pra) in positions i and i+4, respectively, was accomplished by either subjecting the resin-bound peptide to selective on-resin diazo transformation of a Lys into the Nle(epsilon-N3) or the incorporation of Fmoc-Nle(epsilon-N3)-OH during the stepwise build-up of the resin-bound peptide 1b. Solution-phase Cu(I)-catalyzed 1,3-dipolar cycloaddition converts the linear precursor Ac-Lys-Gly-Nle(epsilon-N3)-Ser-Ile-Gln-Pra-Leu-Arg-NH2 (2) into the 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptide [Ac-Lys-Gly-Xaa(&(1))-Ser-Ile-Gln-Yaa(&(2))-Leu-Arg-NH2][(&(1)(CH2)4-1,4-[1,2,3]triazolyl-CH2&(2))] (3). The conformational preferences of the model cyclopeptide 3 (III), which is derived from the sequence of a highly helical and potent i to i+4 side-chain to side-chain lactam-containing antagonist of parathyroid hormone-related peptide (PTHrP), are compared to the corresponding lactam analogue Ac[Lys(13)(&(1)),Asp(17)(&(2))]hPTHrP(11-19)NH2 (II). CD and NMR studies of 3 and II in water/hexafluoroacetone (HFA) (50:50, v/v) revealed a high prevalence of turn-helical structures involving in particular the cyclic regions of the molecule. Despite a slight difference of the backbone arrangement, the side-chains of Ser, Gln, and Ile located at the i+1 to i+3 of the ring-forming sequences share the same spatial orientation. Both cyclopeptides differ regarding the location of the turn-helical segment, which in II involves noncyclized residues while in 3 it overlaps with residues involved in the cyclic structure. Therefore, the synthetic accessibility and conformational similarity of i to i+4 side-chain to side-chain cyclopeptide containing the 1,4-disubstituted [1,2,3]triazolyl moiety to the lactam-type one may result in similar bioactivities.

Di-(2-Ethylhexyl) Phthalate and Autism Spectrum Disorders:

ASDs (autism spectrum disorders) are a complex group of neurodevelopment disorders, still poorly understood, steadily rising in frequency and treatment refractory. Extensive research has been so far unable to explain the aetiology of this condition, whereas a growing body of evidence suggests the involvement of environmental factors. Phthalates, given their extensive use and their persistence, are ubiquitous environmental contaminants. They are EDs (endocrine disruptors) suspected to interfere with neurodevelopment. Therefore they represent interesting candidate risk factors for ASD pathogenesis. The aim of this study was to evaluate the levels of the primary and secondary metabolites of DEHP [di-(2-ethylhexyl) phthalate] in children with ASD. A total of 48 children with ASD (male: 36, female: 12; mean age: 11±5 years) and age- and sex-comparable 45 HCs (healthy controls; male: 25, female: 20; mean age: 12±5 years) were enrolled. A diagnostic methodology, based on the determination of urinary concentrations of DEHP metabolites by HPLC-ESI-MS (HPLC electrospray ionization MS), was applied to urine spot samples. MEHP [mono-(2-ethylhexenyl) 1,2-benzenedicarboxylate], 6-OH-MEHP [mono-(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate], 5-OH-MEHP [mono-(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate] and 5-oxo-MEHP [mono-(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate] were measured and compared with unequivocally characterized, pure synthetic compounds (>98%) taken as standard. In ASD patients, significant increase in 5-OH-MEHP (52.1%, median 0.18) and 5-oxo-MEHP (46.0%, median 0.096) urinary concentrations were detected, with a significant positive correlation between 5-OH-MEHP and 5-oxo-MEHP (rs = 0.668, P<0.0001). The fully oxidized form 5-oxo-MEHP showed 91.1% specificity in identifying patients with ASDs. Our findings demonstrate for the first time an association between phthalates exposure and ASDs, thus suggesting a previously unrecognized role for these ubiquitous environmental contaminants in the pathogenesis of autism.

Modulatory action of galanin on responses due to antidromic activation of peripheral terminals of capsaicin-sensitive sensory nerves

Galanin inhibited, in a concentration-dependent manner (EC50 7.2 nM), the positive inotropic response produced by field stimulation of isolated left atria from reserpine-pretreated guinea-pigs (in the presence of atropine). These responses were shown to involve antidromic activation of capsaicin-sensitive primary afferents. On the other hand, galanin did not affect the inotropic response to capsaicin or calcitonin gene-related peptide, the putative endogenous mediator released from sensory nerves. Galanin-(1-10) was at least 10,000 times less potent than the parent peptide, while galanin-(25-29) was ineffective. Likewise, galanin inhibited the non-cholinergic contraction produced by field stimulation of the isolated guinea-pig bronchus but not the contraction produced by exogenous neurokinin A. These findings indicate a prejunctional neuromodulatory action of galanin on the excitability of peripheral terminals of capsaicin-sensitive sensory nerves.

The glycopeptide CSF114(Glc) detects serum antibodies in multiple sclerosis

Synthetic glycopeptides have the potential to detect antibodies in multiple sclerosis (MS). In the present study, we analyzed the antibodies (IgM class, IgG class and IgG subclasses) to the synthetic glycopeptide CSF114(Glc) in the serum of 186 MS patients, 166 blood donors (BDs), 25 patients affected by meningitis/encephalitis, 41 affected by systemic lupus erythematosus (SLE) and 49 affected by rheumatoid arthritis (RA). The IgM antibody level to CSF114(Glc) was significantly increased in MS patients versus BDs (p<0.001) or versus other autoimmune diseases (SLE or RA, p<0.001). The IgG response was restricted to the subclass IgG2. IgM antibodies to CSF114(Glc) were found in 30% of relapsing/remitting MS patients and, at lower levels, in subjects affected by meningitis/encephalitis. The study of antibodies to CSF114(Glc) is a new, potential immunological marker of MS.

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.

Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli

Background: Actinobacillus pleuropneumoniae N-glycosyltransferase is a cytoplasmic glycosyltransferase catalyzing N-glycosylation of polypeptides. Results: In depth analysis of a reconstituted A. pleuropneumoniae glycosylation system in Escherichia coli showed a surprisingly relaxed peptide substrate specificity of N-glycosyltransferase. Conclusion: N-Glycosyltransferase constitutes a general glycosylation system with a preference for autotransporters. Significance: Our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria. N-Linked protein glycosylation is a frequent post-translational modification that can be found in all three domains of life. In a canonical, highly conserved pathway, an oligosaccharide is transferred by a membrane-bound oligosaccharyltransferase from a lipid donor to asparagines in the sequon NX(S/T) of secreted polypeptides. The δ-proteobacterium Actinobacillus pleuropneumoniae encodes an unusual pathway for N-linked protein glycosylation. This pathway takes place in the cytoplasm and is mediated by a soluble N-glycosyltransferase (NGT) that uses nucleotide-activated monosaccharides to glycosylate asparagine residues. To characterize the process of cytoplasmic N-glycosylation in more detail, we studied the glycosylation in A. pleuropneumoniae and functionally transferred the glycosylation system to Escherichia coli. N-Linked glucose specific human sera were used for the analysis of the glycosylation process. We identified autotransporter adhesins as the preferred protein substrate of NGT in vivo, and in depth analysis of the modified sites in E. coli revealed a surprisingly relaxed peptide substrate specificity. Although NX(S/T) is the preferred acceptor sequon, we detected glycosylation of alternative sequons, including modification of glutamine and serine residues. We also demonstrate the use of NGT to glycosylate heterologous proteins. Therefore, our study could provide the basis for a novel route for the engineering of N-glycoproteins in bacteria.

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).

Side chain-to-side chain cyclization by click reaction.

CuI‐catalyzed azide‐alkyne 1,3‐dipolar Huisgens cycloaddition (CuAAC) is a click reaction that has drawn a lot of attention, in general, and in the field of peptide and protein sciences, in particular. Among several reported applications, the preparation of novel heterodetic cyclopeptides by an intramolecular side chain‐to‐side chain CuAAC, forming a 1,4‐disubstituted[1,2,3]triazolyl‐containing bridge, is of great interest. Herein, we provide a detailed protocol for the syntheses of model heterodetic cyclopeptides as a prototypical intramolecular CuAAC, using as a model a sequence derived from parathyroid hormone‐related protein. Copyright