Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

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.     

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.     

Soft shell resins for solid-phase peptide synthesis.

Soft shell (SS) resins with a lightly or noncross-linked shell layer were prepared by reducing the amount of cross-linking agent, divinylbenzene (DVB), during seed suspension polymerization from polystyrene (PS) resin. These SS resins have a lower swelling volume than that produced by normal cross-linking. Despite its lower swelling, however, SS (10-00) resin, which consists of the 1% DVB-cross-linked core and the noncross-linked surface layer, showed higher efficiency in peptide synthesis compared with 1% DVB-PS resin and other SS resins.     

Facile production of mono-substituted urea side chains in solid phase peptide synthesis

A method is reported for the straightforward generation of urea-containing peptides during Boc solid phase peptide synthesis. Primary amine side chains are converted to mono-alkyl ureas in two steps via an intermediate p-nitrophenyl carbamate. Use of p-methoxybenzyl amine as an ammonia equivalent affords mono-alkyl final products from standard resin cleavage methods, without the need for additional steps. The reaction is highly efficient and applicable to variable length side chains and peptides.     

Monitoring of solid phase peptide synthesis by FT-IR spectroscopy

Aggregation phenomena of growing peptides on the resin have seldom been investigated. We report here how conformations are determined by FT-IR spectroscopy. Therefore the sequence 80–99 of HIV 1-protease was synthesized. After every coupling a resin sample was taken out of the reaction column and a FT-IR spectrum recorded. The results were compared with the UV monitoring obtained from another synthesis of the same peptide. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Polymer-supported biopolymer synthesis: 1. High load solid (gel) phase strategy for peptide synthesis with a phenolic poly(acryloylmorpholine)-based support

An efficient, low-cost, reaction strategy for the solid (gel) phase synthesis of peptides and protected peptide segments has been developed. The strategy involves the use of a new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A. Illustrative syntheses of N-terminal Boc- and Z-protected[Leu]- enkephalin derivatives, including C-terminal acid hydrazides and esters, are described. The strategy, which is effective for the synthesis of peptides at high matrix loadings, is adopted readily for large-scale application.     

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.     

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.     

High-yield, solid-phase synthesis of humanin, an Alzheimer's disease associated, novel 24-mer peptide which contains a difficult sequence.

Humanin is a novel, 24-mer residue bioactive peptide, which antagonizes Alzheimer's disease (AD) related neurotoxicity and offers a hope for developing new therapeutics against AD. Access to adequate amounts of pure humanin is a prerequisite for further, thorough, investigation of the pharmacological properties and therapeutic potency of the peptide. Until now, humanin has been obtained mainly by molecular biology techniques. In this work the Fmoc solid-phase synthesis of humanin on an in-house prepared 2-Cl-tritylamidomethyl polystyrene resin is described fully. Special precautions, i.e. prolonged deprotection steps, should be taken to achieve a high overall yield, since humanin seems to contain a 'difficult sequence' (R4G5F6S7C8L9) near its highly lipophilic, biologically important region L9L10L11L12.     

Solid-phase peptide synthesis using nanoparticulate amino acids in water.

Solid-phase peptide synthesis has many advantages compared with solution peptide synthesis. However, this procedure requires a large amount of organic solvents. Since safe organic solvent waste disposal is an important environmental problem, a technology based on coupling reaction of suspended nanoparticle reactants in water was studied. Fmoc-amino acids are used widely, but most of them show low solubility in water. We prepared well-dispersible Fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol). Leu-enkephalin amide was prepared successfully using the nanoparticulate Fmoc-amino acid on a poly(ethylene glycol)-grafted Rink amide resin in water.     

An alternative solid phase peptide fragment condensation protocol with improved efficiency.

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.     

Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths.

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.     

Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis.

Methodology is described here for the efficient parallel synthesis and cyclization of linear peptide libraries using intramolecular S-alkylation chemistry in combination with Multipin solid phase peptide synthesis (Multipin SPPS). The effective use of this methodology was demonstrated with the synthesis of a 72-member combinatorial library of cyclic thioether peptide derivatives of the conserved four-residue structural motif DD/EXK found in the active sites of the five crystallographically defined orthodox type II restriction endonucleases, EcoRV, EcoRI, PvuII, BamHI and BglI.     

Polymer-supported biopolymer synthesis: 5. Ultra-high load solid (gel) phase peptide synthesis—the stepwise elaboration of quasi-homogeneous peptide gel networks?

An approach to ultra-high load solid (gel) phase peptide synthesis is described in which a bead-form phenolic core polymer, crosslinked poly[N-{2-(4-hydroxyphenyl)ethyl}-acrylamide], is used as a support matrix at near theoretical maximum loading. Consecutive repeating units of the core polymer carry peptide chains undergling stepwise elongation. Synthesis proceeds through a series of solvated networks, which consist mainly of protected peptide. The solvated networks are deemed to be quasi-homogeneous, insofar as each has a regular covalent framework and each is believed to be uniformly distributed throughout the gel beads. Illustrative synthesis of two fully-protected acylpeptide hydrazide segments, corresponding to dynorphin(6–12) and to β_h-endorphin (18–26), are described.     

Rapid solid-phase peptide synthesis using thermal and controlled microwave irradiation.

A rapid and efficient microwave-assisted solid-phase synthesis method is described for the preparation of the nonapeptide WDTVRISFK, using conventional Fmoc/Bu(t) orthogonal protection strategy. The synthesis protocol is based on the use of cycles of pulsed microwave irradiation with intermittent cooling of the reaction during the removal of the Fmoc protecting group and during the coupling. The desired nonapeptide was obtained in highest yield and purity by employing MicroKan technology. The chemical reactions were carried out in a single-mode microwave reactor, equipped with a fiber-optic probe to monitor the reaction temperature continuously.     

Fmoc-based solid phase chemical synthesis of 71-meric neuregulin 1-beta1, an epidermal growth factor-like domain.

The human neuregulin 1-beta1 (NRG1-beta1, amino acid residues 176-246) was chemically synthesized by Fmoc-based solid phase peptide synthesis (SPPS) followed by folding in a redox buffer. The biological activity of the synthesized NRG1-beta1 was confirmed by ligand-induced tyrosine phosphorylation on Chinese hamster ovary (CHO) cells expressing ErbB-4.     

Backbone amide linker (BAL) strategy for Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of peptide aldehydes.

A rapid and efficient strategy has been developed for the general synthesis of complex peptide aldehydes. N(alpha)-Benzyloxycarbonylamino acids were converted to protected aldehyde building blocks for solid-phase synthesis in four steps and moderate overall yields. The aldehydes were protected as 1,3-dioxolanes except for one case where a dimethyl acetal was used. These protected amino aldehyde monomers were then incorporated onto 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyryl-resin (BAL-PEG-PS) by reductive amination, following which the penultimate residue was introduced by HATU-mediated acylation. The resultant resin-bound dipeptide unit, anchored by a backbone amide linkage (BAL), was extended further by routine Fmoc chemistry procedures. Several model peptide aldehydes were prepared in good yields and purities. Some epimerization of the C-terminal residue occurred (10% to 25%), due to the intrinsic stereolability conferred by the aldehyde functional group, rather than any drawbacks to the synthesis procedure.