Peptide Science Research Papers - Academia.edu (original) (raw)

FP-A and FP-B are LDPs produced by the plant pathogen Pseudomonas fuscovaginae. As expected from their primary structure, they shared a similar mechanism of action with the better characterized SPs, synthesized by strains of Pseudomonas... more

FP-A and FP-B are LDPs produced by the plant pathogen Pseudomonas fuscovaginae. As expected from their primary structure, they shared a similar mechanism of action with the better characterized SPs, synthesized by strains of Pseudomonas syringae pv. syringae. Indeed, they displayed hemolytic activity on human erythrocytes and were able to induce calcein release from LUVs: the effect was dependent on the concentration of the FPs and the lipid composition of the liposome and, in particular, it increased with the SM content of the membrane. The permeabilizing activity was further investigated on PLMs. FPs were able to open pores on pure POPC membranes. Pore opening was strongly voltage dependent: by switching the potential from negative to positive values, an increase in the absolute amplitude of transmembrane current was induced with simultaneous closure of pores. In 0.1 M KCl both FPs' pores had a conductance of 4 and 9 pS at − 140 mV and + 140 mV, respectively. Studies on ion selectivity indicated that FPs formed cation-selective channels. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method... more

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method and ring formation was achieved via a ureido group incorporating the side chain amino functions of D-Aaa2 (D-Lys, D-Orn) and Aaa4 (Lys, Orn, Dab, Dap). The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Most showed very high agonist potency in the GPI assay. The peptide containing D-Lys in position 2 and Dab in position 4 was 210 times more active than enkephalin, and that containing Orn and Dab, respectively, was 150 times more active than enkephalin. The latter peptide was also very active in the MVD assay, and showed an IC50 MVD/GPI ratio of 0.816. NMR spectra of selected peptides were recorded, and structural parameters were determined. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. With the help of the NMR spectra each peptide was described as an ensemble of conformations. The conformations have been interpreted with regard to the opioid activities, and comparisons have been made with a model proposed earlier for enkephalin analogues.

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative,... more

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1) or TBTU/DIPEA (1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 6...

Peptide azides acquired growing impact because of application in bioconjugation via ‘click chemistry’ or Staudinger ligation. Furthermore, there are many methods established in organic synthesis addressing the reduction of azides to... more

Peptide azides acquired growing impact because of application in bioconjugation via ‘click chemistry’ or Staudinger ligation. Furthermore, there are many methods established in organic synthesis addressing the reduction of azides to amines, but no observation of a reductive transformation of peptide azides during SPPS cleavage was yet reported. In the present study, the reduction of peptide azides during SPPS cleavage was investigated depending on the choice of thioscavenger, reacting as reductive species. First observed for short PNA/peptide conjugates the occurring extensive side reaction was also validated for one of the applied azide amino acid building blocks and was further investigated by applying different cleavage cocktails to a series of peptides varying in hydrophobicity and position of the azide moiety in the oligomer sequence. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

The sequence-dependent, acid- or base-catalysed aspartimide formation is one of the most serious side reactions in solid-phase synthesis of peptides containing aspartic acid. In the present work, we investigated the susceptibility of... more

The sequence-dependent, acid- or base-catalysed aspartimide formation is one of the most serious side reactions in solid-phase synthesis of peptides containing aspartic acid. In the present work, we investigated the susceptibility of 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl (Dmab), an aspartic acid β-carboxy side-chain protecting group, for aspartimide formation. As a model, 15-amino acid-residue galanin fragment analogue containing the Asp-Ala motif was used during Fmoc-based solid-phase synthesis. Our study showed a strong tendency of Dmab-protected peptide to form aspartimide with unusual high efficiency. Furthermore, to investigate the susceptibility of Asp-Ala motif for aspartimide formation during the synthesis using Asp(ODmab), a 5-amino acid-residue galanin fragment LGPDA, different types of resin linkers, variety of Fmoc-deprotection conditions and coupling methods were applied. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Reversed-phase high-pressure liquid chromatography analysis and purification of three hydrophobic, aggregation-prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing... more

Reversed-phase high-pressure liquid chromatography analysis and purification of three hydrophobic, aggregation-prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2-trifluoroethanol (TFE). The addition of 10-16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE-containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711: 181-186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM-peptide purification. Characterization of the potential side reactions using MALDI and ESI-LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE-formic acid possibly induces O-formylation of the Ser residue and N-formylation of the N-t...

A rare γ-pyranopyrazole skeleton: design, one-pot synthesis and computational study † Drawing upon a consecutive amide coupling and intramolecular cyclisation pathway, a one-pot, straightforward synthetic route has been developed for a... more

A rare γ-pyranopyrazole skeleton: design, one-pot synthesis and computational study †
Drawing upon a consecutive amide coupling and intramolecular cyclisation pathway, a one-pot, straightforward synthetic route has been developed for a range of pyrazole fused γ-pyrone derivatives. The reaction mechanism proposed for the chemoselective formation of γ-pyranopyrazole is furthermore fully supported by experimental and computational studies.

To avoid problems arising from the use of the precursor piperidine as a reagent for Fmoc-group removal in solid-phase peptide synthesis (SPPS) several secondary amines were tested as substitutes of piperidine in this reaction. Kinetics of... more

To avoid problems arising from the use of the precursor piperidine as a reagent for Fmoc-group removal in solid-phase peptide synthesis (SPPS) several secondary amines were tested as substitutes of piperidine in this reaction. Kinetics of Fmoc removal as well as manual SPPS yields and by-product formation were compared with piperidine, piperazine, morpholine, pyrrolidine, 4-methyl-piperidine and also their mixtures with DBU used as Fmoc removing reagents. While possessing the same kinetic parameters of Fmoc removal as piperidine, 4-methyl-piperidine was shown to be superior to all tested reagents in terms of the desired product yields. The use of 4-methyl-piperidine in automated SPPS demonstrated its equal efficiency to piperidine in the synthesis process and superiority to piperidine in its behavior in liquid handling modules, i.e., a complete absence of tubing pellets. It makes 4- methyl-piperidine a good substitute for piperidine with no restrictions relevant to the latter as a p...

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein... more

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties.

Biomarkers are decision-making tools at the basis of clinical diagnostics and essential for guiding therapeutic treatments. In this context, autoimmune diseases represent a class of disorders that need early diagnosis and steady... more

Biomarkers are decision-making tools at the basis of clinical diagnostics and essential for guiding therapeutic treatments. In this context, autoimmune diseases represent a class of disorders that need early diagnosis and steady monitoring. These diseases are usually associated with humoral or cell-mediated immune reactions against one or more of the body's own constituents. Autoantibodies fluctuating in biological fluids can be used as disease biomarkers and they can be, thus, detected by diagnostic immunoassays using native autoantigens. However, it is now accepted that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neo-antigens. In this case, post-translationally modified peptides represent a more valuable tool with respect to isolated or recombinant proteins. In fact, synthetic peptides can be specifically modified to mimic neo-antigens and to selectively detect autoantibodies as disease biomarkers. A ‘chemical reverse approach’ to select synthetic peptides, bearing specific post-translational modifications, able to fishing out autoantibodies from patients' biological fluids, can be successfully applied for the development of specific in vitro diagnostic/prognostic assays of autoimmune diseases. Herein, we report the successful application of this approach to the identification of biomarkers in different autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

In the present work we report the synthesis of a new Fmoc-protected L-lysine-based nucleo-amino acid suitable for the solid phase assembly and its oligomerisation to the corresponding nucleo-ε-peptide that we called ε-lysPNA. The ability... more

In the present work we report the synthesis of a new Fmoc-protected L-lysine-based nucleo-amino acid suitable for the solid phase assembly and its oligomerisation to the corresponding nucleo-ε-peptide that we called ε-lysPNA. The ability to bind complementary RNA and the stability in serum of this synthetic nucleo-ε-peptide were studied to explore its possible use in antisense or diagnostic applications. Our interest to the presented oligonucleotide analogue was also supported by the importance of ε-peptides and other ε-amino acid-containing compounds in natural products with biological activity such as the poly-ε-lysines produced by Streptomyces albulus that possess a highly selective antimicrobial activity. Another aspect we intended to evaluate by this work is the possible prebiotic implication of these nucleopeptides, since ε-peptides, and not α-peptides, were mainly obtained among the other thermal prebiotic polypeptides in pyrocondensation of lysine, a diamino acid also detected in Mighei meteorite. Besides this intriguing question, all the remarkable properties emerged from the present investigation on ε-lysPNAs encourage, without doubts, interest in the therapeutic and diagnostic potential of these bioinspired nucleopeptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.