A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Studies leading to optimization of butanedioldimethacrylate-crosslinked polystyrene supports (BDDMA–PS) forsolid phase peptide synthesis are delineated. BDDMA–PScopolymers with different crosslink densities were prepared andfunctionalised with chloromethyl groups. The reactivity of theLys(2-Cl-Z)-OH residue bound to these polymers through a benzylester linkage was investigated by following the kinetics ofacylation by the HOBt active ester of Boc-Alanine. From theresults it was observed that the rate of peptide bond formationwas maximum for a 2% BDDMA crosslinked resin. This resin wascompared with a 2% DVB-crosslinked polystyrene resin (DVB–PS). Synthesis of an extremely insoluble, hydrophobic,antiparallel -sheeted difficult sequencepeptide LMVGGVVIA ( 34–42), C-terminal fragment of -amyloid protein, (1–42), wascarried out on both 2% DVB–PS and 2% BDDMA-crosslinkedpolystyrene supports. The synthesis of the peptide was carriedout using Boc amino acid strategy. Greater extent of swellingof the resino peptide, increased coupling efficiency during theassembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA–PS polymer.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Summary Studies leading to optimization of butanediol dimethacrylate-crosslinked polystyrene supports (BDDMA-PS) for solid phase peptide synthesis are delineated. BDDMA-PS copolymers with different crosslink densities were prepared and functionalised with chloromethyl groups. The reactivity of the Lys(2-Cl−Z)−OH residue bound to these polymers through a benzyl ester linkage was investigated by following the kinetics of acylation by the HOBt active ester of Boc-Alanine. From the results it was observed that the rate of peptide bond formation was maximum for a 2% BDDMA crosslinked resin. This resin was compared with a 2% DVB-crosslinked polystyrene resin (DVB-PS). Synthesis of an extremely insoluble, hydrophobic, antiparallel β-sheeted difficult sequence peptide LMVGGVVIA (β 34–42), C-terminal fragment of β-amyloid protein, β (1–42), was carried out on both 2% DVB-PS and 2% BDDMA-crosslinked polystyrene supports. The synthesis of the peptide was carried out using Boc amino acid strategy. Greater extent of swelling of the resino peptide, increased coupling efficiency during the assembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA-PS polymer.     

Suppressing the epimerization of endothioamide peptides during Fmoc/t‐Bu‐based solid phase peptide synthesis

Despite a number of intriguing utilities associated with thioamide‐containing peptides and proteins in the context of biophysics, pharmacology and chemical biology, it has hitherto remained as one of the underexplored territories of peptidomimetics. The synthesis of long mono to multiply substituted endothioamide peptides is invariably accompanied with severe epimerization, oxoamide formation and various other undesired side reactions, resulting in messy product profiles. This has completely restrained their use as novel chemical tools for biological studies. During the chain elongation of an N‐terminally located thioamide peptide using the Fmoc/t‐Bu chemistry, it becomes vulnerable to the repetitive basic treatments as required for such chemistry. The incompatibility of thioamide moiety with bases as well as strong coupling reagents leads to epimerization as well as other side reactions due to its nucleophilicity, resulting in the loss of the stereochemical identity of the thioamidated amino acid residue. An easy‐to‐implement and efficient protocol to synthesize long (>10‐mer) endothioamide peptides, significantly suppressing epimerization and other side reactions using 10% piperidine/dimethylformamide for 1 min, is reported herein. The novelty of the protocol is shown through the efficient synthesis of a number of 10–12‐mer mono to multiply thioamide‐substituted peptides with broad substrate scopes. The utility of the protocol in the context of protein engineering and chemical protein synthesis is also shown through the synthesis of a thioamide version of the 16‐mer peptide from the B1 domain of protein G. Such a protocol to synthesize long endothioamide peptides would open up avenues toward engineering and accessing novel thiopeptide and thioprotein‐based chemical tools, the synthesis of which had been a serious hurdle thus far. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Solid‐phase synthesis and screening of a library of C‐terminal arginine peptide aldehydes against Murray Valley encephalitis virus protease

Murray Valley encephalitis virus is a member of the flavivirus group, a large family of single‐stranded RNA viruses, which cause serious disease in all regions of the world. Unfortunately, no suitable antivirals are available, and there are commercial vaccines for only three flaviviruses. The solid‐phase synthesis of a library of 400 C‐terminal arginine peptide aldehydes and their screening against Murray Valley encephalitis virus protease are demonstrated. The library was utilised to elucidate several tripeptide sequences that can be used as inhibitors in further SAR studies. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Facile preparation of the N‐acetyl‐glucosaminylated asparagine derivative with TFA‐sensitive protecting groups useful for solid‐phase glycopeptide synthesis

In this study, a novel N‐acetyl‐glucosaminylated asparagine derivative was developed. This derivative carried TFA‐sensitive protecting groups and was derived from commercially available compounds only in three steps. It was applicable to the ordinary 9‐fluorenylmethoxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (SPPS) method, and the protecting groups on the carbohydrate moiety could be removed by a single step of TFA cocktail treatment generally used for the final deprotection step in Fmoc‐SPPS. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

p‐Methoxyphenol: A potent and effective scavenger for solid‐phase peptide synthesis

During the final step of t‐Boc/Bzl, solid‐phase peptide synthesis (SPPS)‐protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support . These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during S_N1‐type reactions. Five peptide sequences were synthesised for evaluating p‐methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin–peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p‐cresol (reported in the literature as an effective acyl ion eliminator) and p‐methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p‐methoxyphenol can be used as a potent scavenger during SPPS by t‐Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.     

Further applications of classical amide coupling reagents: Microwave‐assisted esterification on solid phase

Ester linkage (s) is a key chemical connector in organic chemistry, including natural products, peptides, and synthetic polymers. We herein describe a straightforward method for the efficient formation of ester linkage (s) on solid‐phase. This method simply involves the use of amide coupling reagents under microwave irradiation. The robustness of this method relies on the use of classical solid‐phase coupling reagents, heating by microwave irradiation, and a short time period, which results in high yields and the minimization of racemization.     

Conventional and microwave‐assisted SPPS approach: a comparative synthesis of PTHrP(1–34)NH2

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. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Advances in Fmoc solid‐phase peptide synthesis

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications. Copyright © 2015 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.     

Isopeptide method: development of S‐acyl isopeptide method for the synthesis of difficult sequence‐containing peptides

A novel strategy for a more efficient synthesis of difficult sequence‐containing peptides, the S‐acyl isopeptide method, was developed and successfully applied. A model pentapeptide Ac–Val–Val–Cys–Val–Val–NH₂ was synthesized via its water‐soluble S‐acyl isopeptide using an S‐acyl isodipeptide unit, Boc–Cys(Fmoc–Val)–OH. An S‐acyl isopeptide possessing excellent water solubility could be readily and quantitatively converted to the native peptide via an S N intramolecular acyl migration reaction at pH 7.4. Thus, the S‐acyl isopeptide method provides a useful tool in peptide chemistry. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self‐assembly

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water‐soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N′‐suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self‐assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing‐directed self‐assembly rather than non‐specific aggregation or precipitation. The progress of suberoyldiglycylglycine self‐assembly was observed to be via the formation of oligomers in the solution, which then self‐assembled to form layered β‐sheet type macrostructures. Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5–7 days. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and application of Nα‐Fmoc‐Nπ‐4‐methoxybenzyloxymethylhistidine in solid phase peptide synthesis

The 4‐methoxybenzyloxymethyl (MBom) group was introduced at the N^π‐position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)‐assisted solid phase peptide synthesis (SPPS) with 9‐fluorenylmethyoxycarbonyl (Fmoc) chemistry. The N^π‐MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side‐chain‐induced racemization during incorporation of the His residue even in the case of MW‐assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His‐containing peptide segments from acid‐sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc‐His(π‐MBom)‐OH with the aid of purification procedure by crystallization to effectively remove the undesired τ‐isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large‐scale production without any obstacles. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Preventing aspartimide formation in Fmoc SPPS of Asp‐Gly containing peptides — practical aspects of new trialkylcarbinol based protecting groups

In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β‐trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp‐Gly motif. The N^α‐Fmoc aspartic acid β‐tri‐(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP‐2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.