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A growing industrial interest toward the peptide drug market fueled the need for the development of effective and cGMP compliant manufacturing methods for these complex molecules. Solid-phase strategies are considered methods of election for medium-length peptide syntheses not only on the research scale but for multigram-scale production, as well. The possibility to use microwave-assisted technology on the multigram scale, recently introduced, prompted us to evaluate the possibility to conveniently set up a safe and fully cGMP-compliant pilot process to produce eptifibatide, a generic peptide active pharmaceutical ingredient. Accordingly, we developed an optimized process on the laboratory scale (1-5 mmol), which was subsequently successfully scaled up to 70 mmol, obtaining all the information required by regulatory agencies to validate the process and qualify the pilot scale plant. The process consists of 5 steps: (1) automated microwave-assisted solid-phase synthesis of eptifibatide linear precursor; (2) cleavage from the resin with concomitant amino acid side-chains deprotection; (3) disulfide-bond formation in solution; (4) purification by flash column chromatography; (5) ion-exchange solid-phase extraction. Since the direct scale-up of a multigram-scale cGMP compliant peptide API production procedure is a challenge that requires an accurate understanding of each involved step, we initially performed a quality management risk assessment, which enabled a smooth and effective achievement of a successful final result.

We investigated several strategies, based on the use of microwave-assisted solid-phase peptide synthesis (MW-SPPS) and scalable to kilogram-scale manufacturing, for the preparation of Eptifibatide, a disulfide-bridged cyclo-heptapeptide drug approved as an antithrombotic agent. Following the very fast microwave-assisted Fmoc/tBu synthesis of the linear precursor, we explored both the solution (off-resin) and the solid-phase (on-resin) disulfide formation. In order to optimize the oxidation in solution, we focused our attention on the mild disulfide formation procedure based on the use of air, observing some drawbacks, such as the formation of unwanted oxidation byproducts, such as dimers, or the use of large volumes of an environmentally unfriendly solvent (CHCN). In order to overcome these difficulties, we studied four different on-resin strategies, with the final aim to develop a fully automated, single reactor procedure, exploring different strategies to protect the thiol side-chain functional group on the C-terminal Cys residue and to form the Eptifibatide ring. The main difference among these strategies is represented by the final cyclization mode that was obtained either by direct formation of an S-S disulfide bridge or by head to MPA on cysteine side-chain amide bond formation. In conclusion, the optimization of the latter strategy enabled us to devise an optimized scalable fully automated solid-phase microwave-assisted cGMP-ready process to prepare Eptifibatide.

Somatostatin analogues are useful pharmaceuticals in peptide receptor radionuclide therapy. In previous studies, we analyzed a new bicyclic somatostatin analogue (BCS) in connection with Cu(II) ions. Two characteristic sites were present in the peptide chain: the receptor- and the metal-binding site. We have already shown that this ligand can form very stable imidazole complexes with the metal ion. In this work, our aim was to characterize the intramolecular interaction that occurs in the peptide molecule. Therefore, we analyzed the coordination abilities of two cyclic ligands, i.e., P1 only with the metal binding site and P2 with both sites, but without the disulfide bond. Furthermore, we used magnetic circular dichroism (MCD) spectroscopy to better understand the coordination process. We applied this method to analyze spectra of P1, P2, and BCS, which we have described previously. Additionally, we analyzed the MCD spectra of P3 ligand, which has only the receptor binding site in its structure. We have unequivocally shown that the presence of the Phe-Trp-Lys-Thr motif and the disulfide bond significantly increases the metal binding efficiency.

We report herein a novel ChemMatrix&reg, Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-&alpha, and N-&epsilon, amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix&reg, Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix&reg, Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients&rsquo, sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.

The development of conformationally constrained analogues of bioactive peptides is a relevant goal in peptide medicinal chemistry. Among the several classes of conformationally constrained peptides, the so-called stapled peptides, which bear a side-chain-to-side-chain bridge, are particularly interesting since they offer the possibility to stabilize specific conformational elements, such as a-helices or beta-turns. We describe an efficient and reproducible microwave-assisted strategy to prepare side-chain-to-side-chain clicked peptides, performing the copper-catalyzed azide-alkyne cycloaddition on solid phase, using as a model peptide a portion of the H1-relaxin B chain, which contains the binding cassette motif of this important bioactive peptide. All the relevant parameters, that is, resin, solvent, catalytic system, microwave energy and reaction time were optimized using a systematic one-factor-at-a-time (OFAT) approach. This method will be useful for the preparation of libraries of conformationally constrained relaxin analogues.

Understanding the metal ion-protein interactions is crucial in transport, accumulation, and excretion of metals. Furthermore, it seems to be highly important to investigate the role of such complexes in many diseases, such as cancer and neurodegenerative disorders. Unexpectedly, such studies in the systems comprising peptides and Ni2+ ions are still scarce and should be given more importance – particularly considering the toxicity and carcinogenicity of nickel compounds on the living organisms. We report herein the results of a study on the interactions of Ni2+ ions and four pentapeptide analogs: EYHHQ (p1), EHYHQ (p2), HEYHQ (p3), and HEYQH (p4). Potentiometric titration was used to study acid-base properties as well as binding properties of the investigated peptides. Furthermore, stoichiometry of the peptide complexes with Ni2+ ions was evaluated by steady-state fluorescence spectroscopy and potentiometric titration method. Additionally, based on the density functional theory (DFT) calculations, we propose the most likely structures of the complexes of the peptides p1-p4 with Ni2+ ions. It was found that all the peptides form 1:1 and 1:2 thermodynamically stable complexes with Ni2+ cation. In particular, we observed that the interaction between the metal ion and histidine-containing peptide sequences does not depend only on histidines splicing, but also on their orientation in respect to other amino acids in the sequence. Also, the closer proximity of His residue in these short peptides does not increase complex stability, due to stacking interaction between the neighboring imidazole rings.

This work describes the synthesis of thirteen cyclooctapeptides dicarba-analogues of Somatostatin, containing l- or d-allylglycine (Agl) residues at the termini of the peptide chain, through on resin Ring Closing Metathesis (RCM) of the linear octapeptides. We investigated the influence of the stereochemistry of some strategic amino acids on the propensity to give the cyclic compounds in mild conditions (refluxing DCM). Systematic individual replacement of Phe6,7,11 residues with the corresponding enantiomers, strongly favoured the ring closure by conventional heating. The yield of the cyclic products was strictly correlated to the position of this amino acid on the peptide chain. In particular substitution of Phe6 by Tyr in peptides which did not give the cyclic compounds, allowed the ring formation. The effect of the phenolic −OH function of Tyr side chain on the proximity of the terminal Agl residue was studied by NMR techniques. All the linear precursors gave cyclic somatostatin dicarba-analogues, in good to high yields and in short reaction times, by microwave-assisted RCM, performed with the 2nd generation Grubbs catalyst. The unsaturated dicarba-tether resulted in a mixture of E and Z stereoisomers in a variable ratio, depending on the sequence and the cyclization method. The E isomer was largely the most abundant in all but one the described product.

To date, the insect Tenebrio molitor (TM) seems suitable for partial replacement of fish meal (FM) in aquafeeds for a variety of marine and freshwater fish species, considering fish growth performance and diets nutrient utilization. Nevertheless, very few studies were conducted to evaluate the effect of the inclusion of TM meal, as alternative protein source, on total and free amino acid (TAA and FAA, respectively) composition of gilthead sea bream (Sparus aurata L., SB) and rainbow trout (Oncorhynchus mykiss W., RT) muscle. Three isonitrogenous and isocaloric diets were formulated both for SB and RT and tested in two separate trials: a control diet (TM0), and two diets in which TM larvae meal was included at 25% (TM25) and 50% (TM50) as fed as substitute of the conventional protein source (FM and corn gluten meal in SB feed; FM, corn gluten and wheat meal in RT feed). Thirty-one specimens of SB and 15 RT were randomly killed after 163 and 90 days of growing trials, respectively. Then, fish fillets were subdued to TAA and FAA analyses together to TM and feed samples. The TAAs composition of TM was mainly characterized by alanine (Ala), glutamine (Glx), glycine (Gly), asparagine (Asx), and leucine (Leu), being their sum almost 52% of TAAs. Contrariwise, TM was deficient in arginine (Arg). The fish diets were also affected by TM inclusion, as well as TAA and FAA profile of SB and RT. For instance, Ala, Leu, and Lys as TAA were higher in SB-TM50, while histidine (His) and phenylalanine (Phe) were lower in SB-TM25 and SB-TM50 than in SB-TM0. RT-TM50 group had a significantly higher level of total Ala, taurine (Tau), tyrosine (Tyr), cysteine (Cys), Leu and proline (Pro) than RT-TM0 group while Glx, His, Met, and Thr declined in RT-TM50 compared to the other two groups. Even the FAA profiles of the diets and fish muscle were affected by TM inclusion. In the present study, SB-TM50 contained higher Arg, valine (Val), Leu, Phe, Gly, His and Pro than SB-TM0. In addition, both SB-TM25 and SB-TM50 were poorer in Tau than SB-TM0. Concerning RT, we observed an overall significant reduction in the essential AA (Ala, Ile, Leu, and Lys) in RT-TM50. Moreover, the RT-TM50 had the lowest Gly and His content among the tested diets. Since the relation among AA profile of the diet and fish digestion is not still clear, further investigation should be done in order to confirm these preliminary data.

The human Pin1 WW domain catalyzes the cis-trans isomerization of the proline peptide bond. In this study, the conformation and binding of Cu(II) ions by Pin1 were investigated. It has been found that the affinity of peptide fragments of the human Pin1 WW domain for Cu(II) ions depends on its conformation. In particular, we analyzed three peptides derived from human Pin1: the nonapeptide hPin1(14-22) (with sequence Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-NH2, peptide 1) the undecapeptide hPin1(13-23) (with sequence Lys-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-Tyr-NH2, peptide 2) and its derivative Ala13Ala23hPin1(13-23) (with sequence Ala-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-Ala-NH2, peptide 3) to study the role of presence in the sequence of the flanked residues at the N- and C-terminus, i.e., Lys13 and Tyr23. The presence of heat-capacity peaks found by DSC measurements for the systems studied strongly suggests that the conformational equilibria of the peptides studied strongly depend on the temperature. NMR spectroscopy and molecular dynamics simulations were instrumental to verify the conformational preferences of three peptides. The absence of likely or oppositely charged groups at the ends of a short chain fragment destroys chain reversal because the charged groups probably screen the nonpolar core from the solvent. ITC experiment was used to study the interactions with Cu(II) ions. It was found that the most stable complexes with Cu2+ ions are formed with peptide 2, which has the most bent conformation.

The role of pathologic auto-antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35-55). In this scenario, we analyzed the anti-MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1-117). To assess the presence of a B-cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1-117 sequence of MOG, including MOG(35-55). For this purpose, we cloned, expressed in Escherichia coli and on-column refolded MOG(1-117), and we applied an optimized microwave-assisted solid-phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid-phase immunoenzymatic assay testing both naive and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35-55), we hypothesize the presence of both linear and conformational epitopes on MOG(35-55) sequence.

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09 x 10(-7) m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies.

Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T-cells and B-cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto-antigens present in the intestinal mucosa. The most important auto-antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide-based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto-antigenic epitopes present in the tTG N-terminal portion (1-230). A library of 23 overlapping peptides spanning tTG(1-230) was generated by Fmoc/tBu solid-phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac-tTG(1-15)-NH2 , Ac-tTG(41-55)-NH2 , Ac-tTG(51-65)-NH2 , and Ac-tTG(151-165)-NH2 , are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen-antibody interaction.

Surface-enhanced Raman scattering has been employed to characterize the adsorption of an oligopeptide containing histidine residues on colloidal nanoparticles of metals as Ag and Cu obtained by laser ablation. The title molecule consists of two histidine and glycine residues alternating along the chain and terminating with an acetyl on one side and an amide group on the other. Histidine residues are found to act as docking sites of the molecule to the surface of the metal nanoparticles. Semiempirical parameterized model number 3 (PM3) calculations performed on molecule/metal model complexes suggest possible different adsorption geometries depending on the metallic substrate. This investigation could provide useful information to address the interaction of protein systems with metal ions, which is often related to fundamental biological process in living systems and can play an important role in different neuropathological diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Introduction: New low-cost strategies and enhancement of the already described methods to manufacture peptide molecules on an industrial scale are highly requested, particularly for peptides such as octreotide, which, along with goserelin and leuprolide, dominate the global peptide market. A number of patents related to the production of octreotide can be found, concerning both solution and solid-phase synthesis. Thus, there is a need to revise the existing synthetic approaches in order to organize them in a more comprehensible way. Area covered: The octreotide patent landscape could help improvement of the methods for manufacturing of octreotide in industrial scale, leading to the appearance of innovative approaches. Expert opinion: The pharmaceutical value of octreotide can be seen from its high market percentage among other peptide drugs. The complex chemical structure of octreotide represents the main challenge for its industrial production. Two synthetic steps are crucial in the preparation of octreotide: (i) threoninol attachment or on resin formation working in solid-phase and (ii) disulphide bond formation to achieve cyclic structure. Analysis of various patents filed to date allows us to see the trend in simplification of the synthetic approaches from the labor intensive syntheses in solution to the more versatile and rapid solid-phase methods.

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidyl-anthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved N-terminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA.

Taking into account that microwave technology applied to SPPS is proposed as a valid support to the enhancement of efficiency of coupling reactions in short times, we compared the efficacy of a new automatic monomode microwave instrument versus a traditional automatic peptide synthesizer in the synthesis of different difficult peptide sequences. We verified that microwave-assisted solid phase synthesis is convenient, in terms of yield, purity, and time consumed to prepare two peptides which were previously shown to be difficult to obtain by conventional strategy, i.e., the antibiotic Gramicidin A and a glycopeptide.

Coupling reagents resembling the structure of Merrifield resin were designed and prepared from 2-chloro-4,6-dibenzyloxy-1,3,5-triazine and the different tertiary bases N-methylmorpholine, N-methylpiperidine, and DABCO. As previously observed for DMTMM, the appropriate N-(4,6-dibenzyloxy-1,3,5-triazin-2-yl) ammonium chloride salts were not suitable as efficient coupling reagents because of their low stability. On the other hand, the stability of the N-(4,6-dibenzyloxy-1,3,5-triazin-2-yl) ammonium tetrafluoroborates was suitable enough for prolonged storage and convenient application in SPPS. Moreover, we observed that the superactive intermediates formed during activation of Fmoc–Aib–OH with 4,6-dibenzyloxy-1,3,5-triazine-based coupling reagents lead to an increase in its concentration inside the polystyrene resin. Therefore, we hypothesize that this increase can enhance efficiency of 4,6-dibenzyloxy-1,3,5-triazine-based coupling reagents in SPPS.

The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

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-alpha 2-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-alpha 2-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.

Multiple sclerosis (MS) is a complex disease that seems to depend on several pathophysiological processes. Because of its varied clinical presentation, natural history, and response to therapeutic interventions, MS can be considered to be a group of diseases that have not been yet characterized, thus resulting in difficult evaluation of prognosis. In the last few years, the role of autoAbs in MS has been reevaluated, and, therefore, their identification as specific biomarkers became a relevant target. In this paper, we demonstrate that an aberrant N -glucosylation is a fundamental determinant of autoAb recognition in MS. Thus, we developed CSF114(Glc), an antigenic probe accurately measuring IgM autoAbs in the sera of a patient population, as disease biomarker. The relevance of CSF114(Glc) is demonstrated by its clinical application and correlation with disease activity and prognosis. In fact, CSF114(Glc), a structure-based designed glycopeptide, is able to recognize, by ELISA, the presence of specific IgM autoAbs in the sera of a MS patient population but not in blood donors and other autoimmune conditions. AutoAbs specific for CSF114(Glc) isolated from MS patients recognized myelin and oligodendrocyte antigens by immunohistochemistry but not other nonrelevant tissues. We demonstrate that CSF114(Glc) is a reliable, specific probe in a longitudinal study of untreated MS patients. Development of IgG/IgM anti-CSF114(Glc) Abs paralleled clinical activity and brain lesions positive to MRI. Therefore, a CSF114(Glc)-based immunoassay on sera may have important prognostic value in monitoring MS disease progression guiding optimal therapeutic treatment.

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.

The efficiency of a series of well-known coupling reagents (TBTU, HATU, and PyBOP) and of newin situ activating reagents (TCTU, HCTU, and DMTMM) was compared by synthesizing the 65–74 fragment of the Acyl Carrier Protein (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH2), containing ‘a difficult sequence’, as a test peptide, in a multiple peptide synthesizer. The longer sequence rMOG(35–55) was also synthesized. It was clear that the aminium salts are more efficient than the phosphonium salt (PyBOP) and that the new 6Cl-HOBt based reagents (HCTU and particularly TCTU) are very efficient, while DMTMM appeared to be not suitable for SPPS.

Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T-cells and B-cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto-antigens present in the intestinal mucosa. The most important auto-antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide-based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto-antigenic epitopes present in the tTG N-terminal portion (1-230). A library of 23 overlapping peptides spanning tTG(1-230) was generated by Fmoc/tBu solid-phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac-tTG(1-15)-NH2 , Ac-tTG(41-55)-NH2 , Ac-tTG(51-65)-NH2 , and Ac-tTG(151-165)-NH2 , are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen-antibody interaction.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n=53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n=82) and healthy age-matched controls (n=29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a syntheticN-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P=0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

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.

The chemical analysis of egg-based wall paintings-the mezzo fresco technique-is an interesting topic in the characterisation of organic binders. A revised procedure for a dot-enzyme-linked immunosorbent assay (dot-ELISA) able to detect protein components of egg-based wall paintings is reported. In the new dot-ELISA procedure we succeeded in maximizing the staining colour by adjusting the temperature during the staining reaction. Quantification of the colour intensity by visible reflectance spectroscopy resulted in a straight line plot of protein concentration against reflectance in the wavelength range 380-780 nm. The modified dot-ELISA procedure is proposed as a semi-quantitative analytical method for characterisation of protein binders in egg-based paintings. To evaluate its performance, the method was first applied to standard samples (ovalbumin, whole egg, egg white), then to model specimens, and finally to real samples (Giotto's wall paintings). Moreover, amino acid analysis performed by innovative ultra-performance liquid chromatography was applied both to standards and to model samples and the results were compared with those from the dot-ELISA tests. In particular, after protein hydrolysis (24 h, 114 °C, 6 mol L(-1) HCl) of the samples, amino acid derivatization by use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate enabled reproducible analysis of amino acids. This UPLC amino acid analysis was rapid and reproducible and was applied for the first time to egg-based paintings. Because the painting technique involved the use of egg-based tempera on fresh lime-based mortar, the study enabled investigation of the effect of the alkaline environment on egg-protein detection by both methods.

Solid-phase strategies speed up the production of both short- and long-sequence peptides compared with solution methodologies. Therefore, solid-phase peptide synthesis (SPPS), proposed by Merrifield in the early 1960s, contributed to the 'Peptide Revolution' in the fields of diagnostics, and drug and vaccine development. Since then, peptide chemistry research has aimed to optimize these synthetic procedures, focusing on areas such as amide bond formation (the coupling step), solid supports and automation. Particular attention was devoted to the environmental impact of SPPS: the requirement for large amounts of organic solvents meant high costs for industrial peptide manufacturing that needed to be reduced. SPPS, alone or in hybrid technologies, has become strategic for the production of peptides as active pharmaceutical ingredients on a commercial scale.

A new generation of triazine-based coupling reagents (TBCRs), designed according to the concept of "superactive esters", was obtained by treatment of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) chloride with lithium or silver tetrafluoroborate. The structure of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate was confirmed by X-ray diffraction. Activation of carboxylic acids by using this reagent proceeds via triazine "superactive ester". The coupling reagent was successfully used for the synthesis of Z-, Boc-, and Fmoc-protected dipeptides derived from natural and unnatural sterically hindered amino acids and for fragment condensation, in 80-100% yield and with high enantiomeric purity. The manual SPPS of the ACP(65-74) peptide fragment (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-OH) proceeded significantly faster than with TBTU or HATU, as well as the automated SPPS of the same fragment gave a purer product than by using TBTU or PyBOP. The reagent was also demonstrated to be efficient in on-resin head-to-tail cyclization of constrained cyclopeptides, in SPPS synthesis of Aib peptides, and in the synthesis of esters from appropriate acids, alcohols, and phenols. The high efficiency and versatility of this new generation of TBCRs confirm, for the first time, the usefulness of the concept of "superactive esters" in rational design of the structure of coupling reagents.

Cyclotetrapeptides are constrained cyclic peptides whose synthesis is considered a difficult task. A methodology based on on-resin head-to-tail cyclization by anchoring the side chain of a trifunctional amino acid was investigated. A series of model cyclotetrapeptides containing the RGD sequence cyclo(Xaa-Arg-Gly-Asp) (Xaa = Ala, Phe, Phg, D-Ala, D-Phe, D-Phg) was synthesized with no cyclodimerization by-products. An evaluation and optimization study of all of the parameters directly involved in the ring closure was performed.

In order to meet national energy requirements, Nicaragua has had to direct its attention towards sources of “alternative energy”, such as geothermal. Excellent geothermal prospects exist in this country, for which reason the Revolutionary Government has deemed it convenient to direct its energy policy towards this alternative source. Studies carried out during past years have led to the selection of nine areas in western Nicaragua, four of which were earmarked Very High Priority because they contain high enthalpy geothermal fields, three areas were earmarked High Priority and only two Low Priority . The positive results obtained in the Momotombo geothermal project have become an incentive to continue research and development nation-wide. At present, a power plant is operating with 35 MW at the Momotombo geothermal field; another 35 MW power plant is under construction in the same field; the El Hoyo-Monte Galan project is in the pre-feasibility phase and surveys are under way throughout national territory.