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.     

Solid phase synthesis of hydrophobic difficult sequence peptides on BDDMA-PS support.

This article illustrates the successful and efficient solid phase assembly of hydrophobic difficult sequence peptides following both t-Boc and Fmoc chemistry. The peptides were synthesized on an optimized 1,4-butanediol dimethacrylate-crosslinked polystyrene support (BDDMA-PS). Four difficult sequence test peptides, VAVAG, VIVIG, QVGQVELG and VQAAIDYING, were synthesized in relatively good yield and purity without any aggregation problems. The peptides were assembled on chloromethylated and 4-hydroxymethylphenoxymethyl (HMP) BDDMA-PS resins. The peptides were fabricated using Boc amino acid 1-hydroxybenzotriazolyl and Fmoc amino acid pentafluorophenyl active esters in coupling reactions. The peptides after synthesis were cleaved from the polymeric support by exposing the peptidyl resin to 90% trifluroacetic acid/5% thioanisole/5% EDT mixture. The HPLC and MALDI TOF MS studies of the peptides revealed the high homogeneity of the synthesized peptides. Chloromethylated resin having a functional group loading of 1.14 mmol Cl/g was used for the synthesis. The yield and homogeneity of these peptides synthesized using the new support were high when compared with the conventional DVB-PS resin.     

Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis

Benzotriazole‐based coupling reagents have dominated the last two decades of solid phase peptide synthesis. However, a growing interest in synthesizing complex peptides has stimulated the search for more efficient and low‐cost coupling reagents, such as COMU which has been introduced as a nonexplosive alternative to the classic benzotriazole coupling reagents. Here, we present a comparative study of the coupling efficiency of COMU with the benzotriazole‐based HBTU and HCTU for use in in situ neutralization Boc‐SPPS. Difficult sequences, such as ACP(65–74), Jung–Redeman 10‐mer, and HIV‐1 PR(81–99), were used as model target peptides on polystyrene‐based resins, as well as polyethylene glycol‐based resins. Coupling yields obtained using fast in situ Boc‐SPPS cycles were determined with the quantitative ninhydrin test as well as via LC‐MS analysis of the crude cleavage products. Our results demonstrate that COMU coupling efficiency was less effective compared to HBTU and HCTU with HCTU ≥ HBTU > COMU, when polystyrene‐based resins were employed. However, when the PEG resin was employed in combination with a safety catch amide (SCAL) linker, more comparable yields were observed for the three coupling reagents with the same ranking HCTU ≥ HBTU > COMU. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

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.     

N,O-bisFmoc derivatives of N-(2-hydroxy-4-methoxybenzyl)-amino acids: Useful intermediates in peptide synthesis

2-Hydroxy-4-methoxybenzyl-amino acid residues inhibit interchain association in solid phase peptide synthesis. They are easily introduced through their N,O-bisFmoc derivatives. Preparation of a range of these derivatives is described.     

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.     

Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1.

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(psiMe, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.     

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.     

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.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

Summary The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

Comparative evaluation of four trityl-type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis.

Four trityl-type (i.e. non-substituted trityl-, o-Cl-trityl-, o-F-trityl- and p-CN-trityl-) amidomethyl polystyrene resins were evaluated comparatively, in terms of the stability of the trityl-ester bond in slightly acidic dichloromethane solutions, and the p-CN-trityl-amidomethyl polystyrene resin was found to be the most stable of them. The above resins were applied, in parallel with Wang benzyl-type resin, well known for its stability in mild acidic conditions, to the Fmoc solid phase synthesis of the 43-amino acid residue long bioactive peptide thymosin beta-4. Independent of their differences in acid sensitivity, the resins seemed to function equally well under the conditions used, since pure thymosin beta-4 was obtained with a final yield of approximately 30% from each resin. The trityl-type amidomethyl polystyrene resins were also applied, in parallel with the Wang resin, to the Fmoc solid phase synthesis of a bioactive peptide containing proline at its C-terminus, i.e. the N-terminal tetrapeptide of thymosin beta-4, AcSDKP. In this case, the best yield (87%) was obtained with the o-Cl-trityl-amidomethyl polystyrene resin, which may be the resin of choice, of those studied, for the Fmoc solid phase peptide synthesis.     

Preparation of polymer-bound trityl-hydrazines and their application in the solid phase synthesis of partially protected peptide hydrazides

Summary Polymer-bound N-tritylhydrazines 4 were easily prepared by reacting polymeric tritylchlorides 3 with hydrazine. Subsequently, compounds 4 have been successfully applied to the solid phase synthesis of partially protected peptide hydrazides using 1-hydroxybenzotriazolyl esters of Fmoc- or Trt-amino acids. The synthesized peptide hydrazides can be quantitatively split off from the resins by mild acidic treatment, while the benzyl- and tert-butyl protecting groups remain unaffected.     

The solid phase coupling of peptide segments catalyzed by the subtilisin-sodium dodecyl sulfate complex

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using Met residue cleavage by BrCN. Deceased.     

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.     

Expediting the Fmoc solid phase synthesis of long peptides through the application of dimethyloxazolidine dipeptides.

This paper describes the step-wise Fmoc solid phase synthesis of a 95-residue peptide related to FAS death domain. Attempts to prepare this peptide employing conventional amino acid building blocks failed. However, by the judicious use of dimethyloxazolidine dipeptides of serine and threonine, the peptide could be readily prepared in remarkable purity by applying single 1 h coupling reactions.     

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.     

Molecular interactions in bacterial cellulose composites studied by 1D FT-IR and dynamic 2D FT-IR spectroscopy

Specific strain-induced orientation and interactions in three Acetobacter cellulose composites: cellulose (C), cellulose/pectin (CP) and cellulose/xyloglucan (CXG) were characterized by FT-IR and dynamic 2D FT-IR spectroscopies. On the molecular level, the reorientation of the cellulose fibrils occurred in the direction of the applied mechanical strain. The cellulose-network reorientation depends on the composition of the matrix, including the water content, which lubricates the motion of macromolecules in the network. At the submolecular level, dynamic 2D FT-IR data suggested that there was no interaction between cellulose and pectin in CP and that they responded independently to a small amplitude strain, while in CXG, cellulose and xyloglucan were uniformly strained along the sample length.     

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.     

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.