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.     

Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation.

Established methodology for the preparation of peptide thioesters requires the use of t-butoxycarbonyl chemistry owing to the lability of thioester linkers to the nucleophilic reagents used in Fmoc solid phase peptide synthesis. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc-based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non-nucleophilic base, 1,8-diazabicyclo[5.4.0]undec-7-ene, together with 1-hydroxybenzotriazole in dimethylformamide, were used as the N(alpha)-Fmoc deprotection reagent. Native chemical ligation of the resulting thioester product to an N-terminal cysteine-containing peptide was successfully performed in aqueous solution to produce a fragment peptide of human alpha-synuclein. The formation of aspartimide (cyclic imide) in a base-sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, this could be controlled by the judicious protection of sensitive residues using the 2-hydroxy-4-methoxybenzyl group.     

Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of beta-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

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.     

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.     

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.     

Conductance measurements in solid phase peptide synthesis. I. Monitoring coupling and deprotection in Fmoc chemistry

Potentiated conductance shows promise for monitoring the progress of both coupling and deprotection reactions in solid phase peptide synthesis using Fmoc chemistry. Sterically hindered base may be added to the aprotic polar solvents used for synthesis, this induces ionisation of acids formed in the coupling and deprotection reactions, thus giving rise to a conductance signal in the solvent. Changes in this signal allow reactions occurring at the resin to be followed with no degradation of coupling efficiency.     

Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The Na-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5-0.6 mmol/g resin). After final removal of the Na-protecting groups, treatment with TFA (50-95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.     

An imidazoline pseudodipeptide suitable for solid phase peptide synthesis.

This paper describes the synthesis of two diastereoisomers of an imidazoline dipeptide mimetic (a 4,5 dihydroimidazole-4-carboxylic acid), suitably protected for incorporation into solid phase peptide synthesis (SPPS) using the Fmoc protocol, from a phenylalanine-derived thioimidate and an alpha,beta-diaminopropanoic acid ester, followed by protecting group manipulation.     

Direct Fmoc/tert-Bu solid phase synthesis of octamannosyl polylysine dendrimer-peptide conjugates

The mannose binding proteins on the surface of the dendritic cells are responsible for capture of pathogens in the early stages of immune response. Conjugation to mannose dendrimers is a rarely explored but potentially powerful strategy for enhancing immunogenicity of synthetic peptides relying on direct delivery to dendritic cells. We describe a general protocol for preparation of pure, monodisperse third-generation mannosylated poly-L-lysine dendrimer-peptide conjugates using direct, machine-assisted Fmoc/t-Bu solid phase peptide synthesis. The glycodendrons were elaborated onto the N- or C-terminus of sequences derived from HIV-1 gp41, SARS-CoV S2 protein, and Influenza Hemagglutinin (consisting of 15-44 residues). The products were obtained in a homogeneous state after cleavage from the resin, deprotection, and a single purification on semipreparative RP-HPLC.     

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.     

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.     

Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7‐selective peptide – protoxin II

Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N‐methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N‐methylmorpholine with 2,4,6‐collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage‐gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Solid phase synthesis of C-terminal peptide amides: development of a new aminoethyl-polystyrene linker on the Multipin solid support.

A new aminoethyl-polystyrene linker, stable at low concentrations of TFA, has been developed for the solid phase synthesis of peptide amides. The described linker is stable under conditions which remove Bu(t) protecting groups (30-50% TFA in DCM) and the desired product can be finally cleaved off the solid support in 95% TFA (5% H2O). Model peptide amides and other N-alkylated peptide amides have been successfully synthesized in good yield and purity.     

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.     

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis

Expressed protein ligation (EPL) is a protein semisynthesis technique that allows the site-specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-thioester to a recombinantly generated fragment containing an N-terminal Cys. Similarly, c-Crk-II was assembled by the sequential ligation of three recombinant polypeptide building blocks, allowing the incorporation of (15)N isotopes in the central domain of the protein. These examples showcase the scope of the protein ligation strategy for selective introduction of isotopic labels into proteins, and the protocols described will be of value to those interested in using EPL on other systems.     

Acid-labile handles for Fmoc solid-phase synthesis of peptide N-alkylamides

Summary By analogy to established methodology for the preparation of C-terminal peptide amides by 9-fluorenylmethyl-oxycarbonyl (Fmoc) chemistry, in conjunction with the acidolyzable 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL, 1) handle, the present paper reports on 5-(4-(N-Fmoc-N-alkyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid [(R)PAL, 2] handles that can be used for synthesis of peptide N-alkylamides. The key step in the preparation of these handles was the NaBH₃CN-mediated reductive amination (60 to 85% yields; R=CH₃, CH₃CH₂, C₆H₅CH₂CH₂, 4-NO₂C₆H₅) of 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid (4), an aldehyde precursor to PAL. The (R)PAL handles (2a and b) were applied to the preparation of LHRH analogues. After anchoring of handles to PEG-PS supports, peptide chain assemblies were carried out, and treatments with TFA-thioanisolephenol-1,2-ethanedithiol (87:5:5:3) for 90 min at 25 °C, followed by aqueous workups, provided the expected products in excellent yields and purities as supported by HPLC and mass spectrometric characterization.Taken in part from the Ph.D. Thesis of M.F. Songster, University of Minnesota, 1996. Preliminary reports of this work were presented at the 14th American Peptide Symposium, Columbus, OH, June 18–23, 1995 (poster P047), and at the Fourth International Symposium on Solid Phase Synthesis and Combinatorial Chemical Libraries, Edinburgh, Scotland, UK, September 12–16, 1995.     

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis ofpeptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin(TCP-resin) with 1,4-phenylenediamine, subsequent coupling withdesired amino acids by the standard Fmoc protocol, and oxidationof the intermediate para-aminoanilides (pAA) with Oxone®. This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.