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In this study, N -methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin "amyloidogenic" analogs containing N -methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO - ) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N -methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N -methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N -methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3 - 7 ). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation., Competing Interests: The authors declare no conflict of interest.

This study investigates the propensity of short peptides to self-organize and the influence of aggregates on cell cultures. The dipeptides were derived from both enantiomers of identical aromatic amino acids and tripeptides were prepared from two identical aromatic amino acids with one cysteine or methionine residue in the C-terminal, N-terminal, or central position. The formation or absence of fibrous structures under physiological conditions was established using Congo Red and Thioflavine T assays as well as by microscopic examination using normal and polarized light. The in vitro stability of the aggregates in buffered saline solution was assessed over 30 days. Materials with potential for use in regenerative medicine were selected based on the cytotoxicity of the peptides to the endothelial cell line EA.hy 926 and the wettability of the surfaces of the films, as well as using scanning electron microscopy. The criteria were fulfilled by H- d Phe d Phe-OH, H- d Cys d Phe d Phe-OH, H-CysTyrTyr-OH, H- d Phe d Phe d Cys-OH, H-TyrTyrMet-OH, and H-TyrMetTyr-OH. Our preliminary results suggest that the morphology and cell viability of L919 fibroblast cells do not depend on the stereochemistry of the self-organizing peptides.

Two new rigid bi-aromatic linkers for synthesis of peptide arrays by SPOT methodology were obtained from cellulose treated with 2,4-dichloro-6-methoxy-1,3,5-triazine. Reaction with m-phenylenediamine gave non-cleavable TYPE I linker which enabled attachment of the peptides via resistant to harsh reaction conditions amide, ether, and amine bonds. Reaction with 3-Fmoc-aminobenzoic acid followed by thermal isomerization of the intermediate "superactive" ester producing an amide-like bond gave TYPE II linker that was very stable during peptide synthesis. However, the peptide was cleavable, with fragment of the linker, in the presence of 1 M LiOH solution. The uniform loading of the cellulose and efficient synthesis of the peptide array was achieved by using N-(4,6-dimethoxy-1,3,5-triazin-1-yl)-N-methylmorpholinium 4-toluenesulfonate as the coupling reagent., (Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.)

A collection of N-triazinylammonium sulfonates, designed according to the concept of 'superactive esters', was obtained by treatment of ammonium sulfonates with 2-chloro-4,6-dimethoxy-1,3,5-triazine. The structure of the tertiary amine as well as sulfonate anion influenced their reactivity and stability in N, N-dimethylformamide (DMF) solution. The reagents were successfully used in solution- and solid-phase synthesis of Z-, Boc-, and Fmoc-protected peptides containing natural and unnatural sterically hindered amino acids as well as in [2+1] fragment condensation approaches, yielding the final products in 80-100 % yield and high optical purity. In manual SPPS of the [Aib]2[Aib]4-enkephalin analogue and the ACP(65-74) peptide fragment VQAAIDYINEG, several sulfonates yielded peptides significantly faster than TBTU or HATU. Comparative analyses demonstrated that 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 4-toluenesulfonate was the most versatile reagent in a wide range of coupling procedures. [ABSTRACT FROM AUTHOR]

N-lipidated short peptides and amino acids immobilized on the cellulose were used as catalysts cleaved amide bonds under biomimetic conditions. In order to select catalytically most active derivatives a library of 156 N-lipidated amino acids, dipeptides and tripeptides immobilized on cellulose was obtained. The library was synthesized from serine, histidine and glutamic acid peptides N-acylated with heptanoic, octanoic, hexadecanoic and (E)-octadec-9-enoic acids. Catalytic efficiency was monitored by spectrophotometric determination of p-nitroaniline formed by the hydrolysis of a 0.1 M solution of Z-Leu-NP. The most active 8 structures contained tripeptide fragment with 1-3 serine residues. It has been found that incorporation of metal ions into catalytic pockets increase the activity of the synzymes. The structures of the 17 most active catalysts selected from the library of complexes obtained with Cu2+ ion varied from 16 derivatives complexed with Zn2+ ion. For all of them, a very high reaction rate during the preliminary phase of measurements was followed by a substantial slowdown after 1 h. The catalytic activity gradually diminished after subsequent re-use. HPLC analysis of amide bond splitting confirmed that substrate consumption proceeded in two stages. In the preliminary stage 24–40% of the substrate was rapidly hydrolysed followed by the substantially lower reaction rate. Nevertheless, using the most competent synzymes product of hydrolysis was formed with a yield of 60–83% after 48h under mild and strictly biomimetic conditions. [ABSTRACT FROM AUTHOR]

We have shown for the first time that a natural protein (human insulin) can be acylated at the N‐terminus with a β‐amino acid (H‐β3hAla‐), in a process catalyzed by the β‐peptidyl aminopeptidase 3‐2W4‐BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases. [ABSTRACT FROM AUTHOR]

Reaction of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) with bis-tetrafluoroborates of quinine and/or quinidine in the presence of sodium bicarbonate gave a pair of pseudo-enantiomeric coupling reagents: N,N'-bis-(4,6-dimethoxy-1,3,5-triazin-2-yl)quinine bis-tetrafluoroborate (2DMT/quinine/2BF4-) and N,N'-bis-(4,6- dimethoxy-1,3,5-triazin-2-yl)quinidine bis-tetrafluoroborate (2DMT/quinidine/2BF4-). The reagents activate the opposite enantiomers of racemic N-protected amino acids. By their reaction with two equivalents of racemic carboxylic components and diverse esters of amino acids, protected dipeptides were obtained with a predictable configuration and 95-99% enantiomeric homogeneity. [ABSTRACT FROM AUTHOR]

The enantioselective condensing reagent 4,6-dimethoxy-1,3,5-triazine (DMT)/strychnine/BF $\rm{{_{4}^{-}}}$ was obtained by treatment of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) with strychnine tetrafluoroborate. The reagent was useful under typical conditions of solid-phase peptide synthesis (SPPS) with enantiomerically homogeneous substrates. By SPPS, desired dipeptides were obtained in 84-94% yield using 4 equiv. of racemic Fmoc-Ala, Fmoc-Phe, and/or Fmoc-Tyr for 1 equiv. of Wang resin loaded with Gly, Ala, Leu, Phe, Glu( tBu), and/or Pro, respectively. For all three Fmoc-protected amino acids, the configuration of the enantiomer preferred under SPPS conditions was independent of the structure of the acylated component and identical to that established in condensations proceeding in solution. In all cases, the enantiomer ratios L/ D (er) were in a similar range, and varied from 9 : 92 to 2 : 98 for alanine, and from 90 : 10 to 100 : 0 for aromatic amino acids. The synthesis of Ac- L-Lys(Ac)- D-Ala- D-Ala-OH from racemic Fmoc-Ala gave an L/ D ratio of 10 : 90 for the esterification of Wang resin, and 0 : 100 for the formation of peptide bonds. [ABSTRACT FROM AUTHOR]

Abstract: Optimization of coupling reactions of glycosylamines with Fmoc-protected aspartic acid, by microwave approach, is described. Different reaction conditions, quantities of substrates and solvents were tested to develop simple and reproducible methodologies. The best results were obtained using new triazine-based coupling reagents with a monomode microwave Discover® BenchMate™ instrument (CEM). The N-glycosyl amino acids were then deprotected to achieve final products for SPPS. [Copyright &y& Elsevier]