Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83-99).

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83-99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.

Glycopeptide-based antibody detection in multiple sclerosis by surface plasmon resonance.

In multiple sclerosis (MS) the gold standard for the diagnosis and prognosis is, up to now, the use of magnetic resonance imaging markers. No alternative simpler assays proven of use, except for cerebrospinal fluid analysis, have been provided in MS diagnosis. Therefore, there is a need to develop non-invasive, sensitive, simple new techniques for the clinical routine. Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera using the surface plasmon resonance technology. The previously described glycopeptide antigen CSF114(Glc) has been immobilized on a gold sensor chip and the method has been optimized for real-time specific autoantibody detection directly in sera. A population of 60 healthy blood donors and 61 multiple sclerosis patients has been screened. The receiver operating characteristic (ROC)-based analysis has established the optimal diagnostic cut-off value for the method obtaining a sensitivity of 36% and a specificity of 95%. Sample sera have been also screened with a previously validated ELISA.

Conventional and microwave-assisted SPPS approach: a comparative synthesis of PTHrP(1-34)NH(2).

Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we compared Fmoc/tBu MW-assisted SPPS of 1-34 N-terminal fragment of parathyroid hormone-related peptide (PTHrP) with its conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the stepwise elongation of the resin-bound peptide, monitoring was conducted by performing MW-assisted mini-cleavages and analyzing them by UPLC-ESI-MS. Identification of some deletion sequences was helpful to recognize critical couplings and as such helped to guide the introduction of MW irradiations to these stages.

Microwave-assisted reaction of glycosylamine with aspartic acid.

The synthesis of N-protected glycosyl amino acids from amines has been investigated and it was found that, under microwave conditions, glycosylamines could be hydrolyzed leading to new products containing a glycosyl ester linkage. The efficiency of the microwave-induced glycosylation of aspartic acid was studied comparing the microwave activity between amide and ester bond formation. Different sugar moieties have been employed to demonstrate the simple and reproducible coupling methodology. New glycosyl ester compounds were further characterized by NMR spectroscopy.

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.

Synthetic peptides reproducing tissue transglutaminase-gliadin complex neo-epitopes as probes for antibody detection in celiac disease patients' sera.

Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients' sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients' sera before gluten-free diet. The peptide II, containing Ac-tTG(553-564)-NH2 sequence cross-linked with deamidated Ac-α2-Glia(63-71)-NH2, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553-564)-NH2 and Ac-α2-Glia(63-71)-NH2 and the corresponding cross-linked peptide II were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments.

A new biotin derivative-DOTA conjugate as a candidate for pretargeted diagnosis and therapy of tumors.

The synthesis of a new biotin derivative, the (CO) reduced N-aminohexyl biotinamido derivative, designed to be serum biotinidase resistant, and its conjugation to the chelator DOTA through an amide bond at one of the four carboxymethyl chains are described. The (90)Y-labeled conjugate was able to bind avidin at different Av/conjugate molar ratios with good results. The preclinical results indicate that this new biotin-DOTA conjugate is a good candidate for pretargeted diagnosis and therapy of tumors.

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.

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.

IgG and IgM antibodies to the refolded MOG(1-125) extracellular domain in humans.

Antibodies to MOG in serum have a dubious prognostic value in multiple sclerosis. The MOG recombinant protein conformational properties relevant to the antigenic activity are unknown. We employed a solid-phase ELISA based on a product (rMOG(ED)(His)(6)) expressed in E. coli after subcloning the cDNA of the extracellular domain of rat MOG, performing a refolding procedure on column and affinity purification. The far-UV Circular Dichroism (CD) spectra of rMOG(ED)(His)(6) showed a ß-sheet, a characteristic feature of the Ig-fold. However, in MS sera and controls we failed to detected IgM or IgG antibodies.

Posttranslationally modified peptides efficiently mimicking neoantigens: a challenge for theragnostics of autoimmune diseases.

We report the design, synthesis, and immunological evaluation of a series of glycopeptide analogues of the previously described antigenic probe CSF114(Glc), with the aim of understanding the importance of N-glycosylation on Asn residue in multiple sclerosis antibody recognition. The glucopeptide, characterized by a ß-turn conformation which is fundamental for a correct presentation of the epitope, has been modified by introducing various natural glycoamino acids in position 7. The new glycopeptides were evaluated by measuring the IgG and IgM antibody titer in multiple sclerosis patients' and normal blood donors' sera. Moreover, we achieved the efficient synthetic strategy of new Asn derivative bearing N-acetylneuraminic acid (Neu5Ac), linked by an N-glycosidic bond, on the side chain of the Asn residue orthogonally protected for Fmoc/tBu SPPS.

Ferrocenyl glycopeptides as electrochemical probes to detect autoantibodies in multiple sclerosis patients' sera.

Glycopeptide analogues of CSF114(Glc), modified at N-terminus with new ferrocenyl carboxylic acid and a new ferrocenyl-thiphosphino amino acid, were used to implement a new electrochemical biosensor for autoantibody detection in multiple sclerosis. The ferrocenyl moiety of these "electrochemical probes" did not affect autoantibody recognition both in SP-ELISA and in inhibition experiments. By electrochemical monitoring the interactions of the modified peptides Fc-CSF114(Glc) and 4-FcPhP(S)Abu-CSF114(Glc) with the autoantibodies, we demonstrated that autoantibodies could be detected with a sensitivity comparable to ELISA method. The new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology.

Fmoc-protected iminosugar modified asparagine derivatives as building blocks for glycomimetics-containing peptides.

CSF114(Glc) is the first synthetic Multiple Sclerosis Antigenic Probe able to identify autoantibodies in a statistically significant number of Multiple Sclerosis patients. The beta-turn conformation of this glucopeptide is fundamental for a correct presentation of the epitope Asn(Glc). To verify the influence of sugar mimics in antibody recognition in Multiple Sclerosis, we synthesized Fmoc-protected Asn derivatives containing alkaloid-type sugar mimics. The corresponding glycomimetics-containing peptide derivatives of the CSF114-type sequence were tested in competitive and solid-phase non-competitive ELISA on Multiple Sclerosis patients' sera.

Toward biomarkers in multiple sclerosis: new advances.

Multiple sclerosis is an autoimmune disease that commonly affects young adults. If initially characterized by acute relapses, it is later followed by only incomplete remission. Over years, progressive disability and irreversible deficit lead to chronic neurological deficits in the majority of patients. The clinical course is protracted and unpredictable, and no biological marker is useful in predicting the evolution of autoaggression and disability. It is difficult to diagnose and to monitor disease progression after the initial symptoms or even during the major clinical manifestations, and it is difficult to treat. In this review, the authors report recent advances in the field, focusing on the search of new antigens as a marker of the disease, in their relevance to the pathophysiology and diagnosis of the disease.

Bradykinin antagonists modified with dipeptide mimetic beta-turn inducers.

Bradykinin (BK) is involved in a wide variety of pathophysiological processes. Potent BK peptide antagonists can be developed introducing constrained unnatural amino acids, necessary to force the secondary structure of the molecule. In this paper, we report a structure-activity relationship study of two peptide analogues of the potent B2 antagonist HOE 140 by replacing the D-Tic-Oic dipeptide with conformationally constrained dipeptide mimetic beta-turn inducers.