BOP‐OXy,BOP‐OBt,and BOP‐OAt: novel organophosphinic coupling reagents useful for solution and solid‐phase peptide synthesis

Stand‐alone coupling reagents derived from bis(2‐oxo‐3‐oxazolidinyl)phosphorodiamidic chloride show efficient performance in solution and SPPS. In particular, the Oxyma Pure (Luxembourg Biotech., Tel Aviv, Israel) derivative shows the additional advantage of being highly soluble in DMF and even fairly soluble in CH₃CN, which can extend its use for the synthesis of complex peptides. These new stand‐alone coupling reagents have the advantage of not bearing any counteranion such as PF₆ or BH₄, whose presence can jeopardize the purification of final peptides prepared in solution. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

COMU: A third generation of uronium‐type coupling reagents

COMU is a third generation of uronium‐type coupling reagent based on ethyl 2‐cyano‐2‐(hydroxyimino)acetate (Oxyma) as well as a morpholino carbon skeleton. The presence of the morpholino group has a marked influence on the solubility, stability and reactivity of the reagent. COMU performed extremely well in the presence of only 1 equiv. of base, thereby confirming the effect of the hydrogen bond acceptor in the reaction. The by‐products of COMU are water soluble and easily removed, making it an excellent choice of coupling reagent for solution‐phase peptide synthesis. Finally, COMU shows a less hazardous safety profile than benzotriazole‐based reagents, such as HATU and HBTU, which in addition exhibit unpredictable autocatalytic decompositions and therefore a higher risk of explosion. Furthermore, in contrast to benzotriazole‐based reagents, COMU is significantly less likely to cause allergic reaction. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of linear and cyclic opioid‐based peptide analogs containing multiple N‐methylated amino acid residues

A series of six novel opioid peptide analogs containing one to three N‐methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid‐phase method. Introduction of two consecutive N‐methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine‐based coupling reagent, 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium toluene‐4‐sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by‐products than observed with the standard coupling reagents such as TBTU or HATU.Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies

Novel Cl-HOBt based coupling reagents have been evaluated forracemization extent in solid-phase peptide synthesis. The resultsshow that all the coupling protocols based on the use of the novel reagents enable incorporation of the racemization prone residue serine with less than 2% racemization. Moreover, serine racemization obtained is less than 0.5% with protocolswhere a pre-activation step is avoided.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies

Summary Novel Cl-HOBt based coupling reagents have been evaluated for racemization extent in solid-phase peptide synthesis. The results show that all the coupling protocols based on the use of the novel reagents enable incorporation of the racemization prone residue serine with less than 2% racemization. Moreover, serine racemization obtained is less than 0.5% with protocols where a pre-activation step is avoided.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Mapping discontinuous protein‐binding sites via structure‐based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure‐based approach and an experimental, high‐throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein–protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well‐characterised murine IgG1 antibody HyHEL‐5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL‐5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright © 2012 John Wiley & Sons, Ltd.     

BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Piezoelectric property of bundled peptide nanotubes stapled by bis‐cyclic‐β‐peptide

Cyclic tetra‐β‐peptides (CP4s) and a bis‐CP4 were synthesized to prepare peptide nanotubes (PNTs) by molecular stacking of cyclic peptides. The addition of bis‐CP4 to the PNT preparation afforded PNT bundles increasing the direct and converse piezoelectiric coefficients, which is ascribable to bis‐CP4 stapling PNTs into the parallel alignment of PNT dipoles.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Analogues of the frog skin peptide alyteserin‐2a with enhanced antimicrobial activities against Gram‐negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH₂) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly¹¹→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC₅₀ = 24 µm ). Antimicrobial potency was increased further by the additional substitution Ser⁷→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC₅₀ = 38 µm ). However, the peptide containing d ‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm ) but appreciably lower haemolytic activity (LC₅₀ = 185 µm ) and cytotoxicity against A549 human alveolar basal epithelial cells (LC₅₀ = 65 µm ). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

DynaDock: A new molecular dynamics‐based algorithm for protein–peptide docking including receptor flexibility

Molecular docking programs play an important role in drug development and many well‐established methods exist. However, there are two situations for which the performance of most approaches is still not satisfactory, namely inclusion of receptor flexibility and docking of large, flexible ligands like peptides. In this publication a new approach is presented for docking peptides into flexible receptors. For this purpose a two step procedure was developed: first, the protein–peptide conformational space is scanned and approximate ligand poses are identified and second, the identified ligand poses are refined by a new molecular dynamics‐based method, optimized potential molecular dynamics (OPMD). The OPMD approach uses soft‐core potentials for the protein–peptide interactions and applies a new optimization scheme to the soft‐core potential. Comparison with refinement results obtained by conventional molecular dynamics and a soft‐core scaling approach shows significant improvements in the sampling capability for the OPMD method. Thus, the number of starting poses needed for successful refinement is much lower than for the other methods. The algorithm was evaluated on 15 protein–peptide complexes with 2–16mer peptides. Docking poses with peptide RMSD values <2.10 Å from the equilibrated experimental structures were obtained in all cases. For four systems docking into the unbound receptor structures was performed, leading to peptide RMSD values <2.12 Å. Using a specifically fitted scoring function in 11 of 15 cases the best scoring poses featured a peptide RMSD ≤2.10 Å. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Efficient synthesis of polypeptide‐α‐thioester by the method combining polypeptide expression and chemical activation for the semi‐synthesis of interferon‐γ having oligosaccharides

In order to synthesize interferon‐γ glycoform having an oligosaccharide at the 97 position by a semi‐synthetic method, interferon‐γ‐polypeptide‐(1–94)‐α‐hydrazide was prepared by the specific Cys‐cyanylation of polypeptide‐(1–94)‐Cys‐His₆ expressed from E. coli and subsequent hydrazinolysis in 22% yield (two steps). This polypeptide‐α‐hydrazide was then converted into corresponding polypeptide‐α‐thioester under NaNO₂/acid conditions followed by thiolysis in 83% yield. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Re‐evaluating the stability of COMU in different solvents

COMU is uronium‐type coupling reagent based on OxymaPure. It showed several advantages over classical benzotriazole‐based coupling reagents such as higher solubility, water‐soluble byproduct, and monitoring the reaction by changing of color. Although COMU is well known to perform excellent in solution, but its hydrolytic stability in DMF limits its use in automatic peptide synthesizer. Herein, we evaluated the hydrolytic stability of COMU in γ‐valerolactone (GVL), acetonitrile (ACN) and N‐formylmorpholine (NFM) and compared its stability against DMF. The stability of COMU after 24 h was found to be 88 and 89% in GVL and ACN, respectively, when compared in DMF (14%). Further, the demanding Aib‐ACP decapeptide and JR decapeptide were successfully synthesized using COMU dissolved in GVL or ACN while Fmoc amino acids were dissolved in DMF. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Morpholinone mediated oxazolone‐free C‐terminus amide coupling permitting a convergent strategy for peptide synthesis

3‐Substituted‐5‐phenylmorpholinones have been demonstrated to act as N‐protected C‐terminus activated α‐amino acids capable of undergoing solution phase N‐terminus peptide extension following standard coupling procedures. The N‐acylated morpholinones do not undergo epimerisation of the stereocentre of the C‐terminus amino acid residue as oxazolone formation is sterically prevented, although C‐terminus peptide coupling is still possible. This convergent approach to peptide synthesis is exemplified by the preparation of L‐ala‐L‐ala‐L‐ala and L‐ala‐D‐ala‐L‐ala. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.