C-terminal N-alkylated peptide amides resulting from the linker decomposition of the Rink amide resin: a new cleavage mixture prevents their formation.

Decomposition of the resin linkers during TFA cleavage of the peptides in the Fmoc strategy leads to alkylation of sensitive amino acids. The C-terminal amide alkylation, reported for the first time, is shown to be a major problem in peptide amides synthesized on the Rink amide resin. This side reaction occurs as a result of the Rink amide linker decomposition under TFA treatment of the peptide resin. The use of 1,3-dimethoxybenzene in a cleavage cocktail prevents almost quantitatively formation of C-terminal N-alkylated peptide amides. Oxidized by-product in the tested Cys- and Met-containing peptides were not observed, even if thiols were not used in the cleavage mixture.     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.     

A cleavage cocktail for methionine-containing peptides.

A new cocktail has been developed for cleavage and deprotection of methionine-containing peptides synthesized by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis methodology. The cocktail (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%, 1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide 1.5% w/w) was designed to minimize methionine side-chain oxidation. Application of the new cocktail (Reagent H) is demonstrated with the synthesis of a model pentadecapeptide from the active site of DsbC, a periplasmic protein involved in protein disulphide bond formation. The model peptide, which contains one methionine and two cysteine residues, was cleaved with several cleavage cocktails, including Reagent H. The crude peptides obtained with the widely used cocktails K, R and B were found to be 15% to 55% in the methionine sulphoxide form, whereas no methionine sulphoxide was detected in the crude peptide obtained by cleavage and deprotection with Reagent H. Also, no methionine sulphoxide was detected when 1.5% w/w NH4I was added to cocktails K, R and B; however, the yield of the desired peptide was less than with Reagent H. A second 28 amino acid model peptide of the active site of DsbC was also cleaved and deprotected with Reagent H. The reduced dithiol form of the peptide was found to be the major component (51% yield) of the crude peptide obtained by cleavage for 3 h. When the cleavage time was extended to 10 h, the peptide was converted to the intramolecular disulphide form (35% yield). A proposed mechanism for the in situ oxidation of cysteine with Reagent H is presented.     

Solid-phase synthesis of peptides containing phosphoserine using phosphate tert.-butyl protecting group

In this paper, we report the solid-phase synthesis of peptides containing O-phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino-acids linked to p-alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid-labile side-chain protecting groups (including phosphate residue) were used. Thus, both removal of side-chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.     

Solid-phase synthesis and applications of N-(S-acetylmercaptoacetyl) peptides

The reagent pentafluorophenyl S-acetylmercaptoacetate was used to modify the N-terminus of resin-bound side-chain-protected peptides. The modification was carried out in an automated cycle in the final stage of fluorenylmethoxycarbonyl (Fmoc)/polyamide-mediated solid-phase synthesis. Side-chain deprotection and cleavage from the resin with aqueous trifluoroacetic acid gave the N-(S-acetylmercaptoacetyl) peptides. The S-acetylmercaptoacetyl peptides were transformed into reactive thiol-containing peptides by incubation with hydroxylamine at neutral pH. The S-deacetylation was performed in the presence of a sulfhydryl-reactive compound (or intramolecular group) to enable immediate capture of the sensitive thiol. Three applications were investigated. An S-acetylmercaptoacetyl peptide, containing a sequence of a meningococcal membrane protein, was incubated with hydroxylamine in the presence of 5-(iodoacetamido)fluorescein to give the corresponding fluorescein-labeled peptide in 62% yield. The same peptide was also S-deacetylated in the presence of bromoacetylated poly-L-lysine to afford a peptide/polylysine conjugate. Finally, a peptide corresponding to a sequence of herpes simplex virus glycoprotein D was prepared. This peptide, containing an N-terminal-S-acetylmercaptoacetyl group and an additional C-terminal S-(3-nitro-2-pyridinesulfenyl)cysteine residue, was converted into a cyclic disulfide peptide (20%).     

Chemistry of alpha-hydroxymethylserine: problems and solutions

Further improvements related to the synthesis of peptides containing HmS are presented. Efficient synthetic protocols have been developed to synthesize "difficult" sequences containing a C-terminal HmS residue, MeA-HmS or consecutive HmS. Preparative methods for orthogonal N- and/or C-protected HmS(Ipr) derivatives are described. Their compatibility with standard solution or solid-phase peptide chemistry protocols allows synthetic flexibility toward HmS-containing peptides. In the synthesis of the sterically hindered dipeptides with the C-terminal HmS(Ipr) residue, HATU proves the highest efficiency, as compared with the fluoride and PyBroP/DMAP coupling methods. The HATU method also outperforms the fluoride activation in the solid-phase assembly of HmS homosequence. Specific protocols are described to overcome an undesired cyclization to diketopiperazines that occurs during the removal of Fmoc from dipeptides with the C-terminal HmS(Ipr) or HmS residues, thus precluding their C-->N elongation. The successful protocols involve: (i) the 2+1 condensation using mixed anhydride activation yielding the desired product with the highest optical integrity or (ii) use of the 2-chlorotrityl resin as a solid support sterically suppressing the undesired cleavage due to diketopiperazine formation. The latter approach allows the mild conditions of peptide cleavage from solid support, preserving the isopropylidene protection and minimizing the undesired N-->O-acyl migration that was observed under prolonged acid treatment used for cleaving the HmS peptide from the Wang resin.     

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.     

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.     

Ethylcarbamoyl protection for cysteine in the preparation of peptide-conjugate immunogens.

During the solid-phase synthesis of over 100 peptides, we have observed that the ethylcarbamoyl group is useful for the side chain protection of cysteine in peptides containing a single cysteine residue. The ethylcarbamoyl group is stable to the conditions of acidolytic cleavage, purification and long term storage. Brief treatment of peptides containing an S-ethylcarbamoyl-cysteine residue with aqueous sodium hydroxide gives the deprotected cysteine peptide that can be coupled to carrier molecules such as proteins to give immunogen conjugates.     

Fmoc‐Sec(Xan)‐OH: synthesis and utility of Fmoc selenocysteine SPPS derivatives with acid‐labile sidechain protection

We report here the synthesis of the first selenocysteine SPPS derivatives which bear TFA‐labile sidechain protecting groups. New compounds Fmoc‐Sec(Xan)‐OH and Fmoc‐Sec(Trt)‐OH are presented as useful and practical alternatives to the traditional Fmoc‐Sec‐OH derivatives currently available to the peptide chemist. From a bis Fmoc‐protected selenocystine precursor, multiple avenues of diselenide reduction were attempted to determine the most effective method for subsequent attachment of the protecting group electrophiles. Our previously reported one‐pot reduction methodology was ultimately chosen as the optimal approach toward the synthesis of these novel building blocks, and both were easily obtained in high yield and purity. Fmoc‐Sec(Xan)‐OH was discovered to be bench‐stable for extended timeframes while the corresponding Fmoc‐Sec(Trt)‐OH derivative appeared to detritylate slowly when not stored at ?20 °C. Both Sec derivatives were incorporated into single‐ and multiple‐Sec‐containing test peptides in order to ascertain the peptides' deprotection behavior and final form upon TFA cleavage. Single‐Sec‐containing test peptides were always isolated as their corresponding diselenide dimers, while dual‐Sec‐containing peptide sequences were afforded exclusively as their intramolecular diselenides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of glyoxylyl peptides using an Fmoc-protected alpha,alpha'-diaminoacetic acid derivative.

The synthesis of glyoxylyl peptides by coupling the masked glyoxylic acid derivative (FmocNH)(2)CHCO(2)H, 1, to a peptidyl resin assembled using Fmoc/tert-butyl chemistry has been described recently. Deprotection and cleavage of the peptide from the solid support using TFA was followed by unmasking of the glyoxylyl group in solution in the presence of DBU. [] The glyoxylyl peptide was thus generated using non-oxidizing conditions by comparison with the method based on the periodic oxidation of a seryl-precursor. However, base treatment of the (FmocNH)(2)CHCO(2)-peptide led to the formation of a byproduct besides the desired glyoxylyl peptide. This paper describes an optimized procedure for unmasking the Fmoc-protected alpha,alpha'-diaminoacetic acid moiety in solution which suppressed byproduct formation. Also presented is a series of experiments that permitted a structure and a mechanism of formation for the byproduct to be suggested.     

A safety catch linker for Fmoc-based assembly of constrained cyclic peptides.

A new safety-catch linker for Fmoc solid-phase peptide synthesis of cyclic peptides is reported. The linear precursors were assembled on a tert-butyl protected catechol derivative using optimized conditions for Fmoc-removal. After activation of the linker using TFA, neutralization of the N-terminal amine induced cyclization with concomitant cleavage from the resin yielding the cyclic peptides in DMF solution. Several constrained cyclic peptides were synthesized in excellent yields and purities.     

Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate

Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101.     

Site-specific racemization in aging alpha A-crystallin

Of all aspartyl residues in bovine alpha A-crystallin, only Asp-151 exhibits pronounced racemization. Asp-151 is also one of the sites where peptide bond cleavage occurs in in vivo aging alpha A-crystallin. This aspartyl residue is followed by an alanyl residue and resides in a flexible carboxyl terminal extension of alpha-crystallin. Both in vivo and in vitro racemization studies indicate that the pronounced and site-specific racemization of Asp-151 proceeds via formation of a succinimide intermediate. The in vivo racemization of aspartyl residues in alpha A-crystallin is discussed with regard to the proposed tertiary structure of alpha-crystallin.     

Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl phosphate protection.

The synthesis of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO3Me2)-OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the -Tyr(PO3Me2)-residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)-containing peptides using t-butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc-Tyr(PO3tBu2)-OH was prepared in high yield from Fmoc-Tyr-OH by a one-step procedure which employed di-t-butyl N,N-diethyl-phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)-containing peptides, Ala-Glu-Tyr(P)-Ser-Ala and Ser-Ser-Ser-Tyr(P)-Tyr(P).     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

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.     

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.     

Selective 'in synthesis' labeling of peptides with biotin and rhodamine

A new method is described for the selective 'in synthesis' labeling of peptides by rhodamine or biotin at a single, predetermined epsilon-amino group of a lysine residue. The alpha-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group. The labeling technique involves the previous preparation of a suitable Lysine derivative, easily obtained from commercially-available protected amino acids. This new derivative, where the reporter group (biotin, or rhodamine) acts now as permanent protection of lysyl side chain functions, is then inserted into the synthesis program as a conventional protected amino acid, and linked to the preceding residue by aid of carbodiimide. A simpler, alternative method is also described for the selective 'in synthesis' labeling of peptides with N-terminal lysyl residues. Several applications of labeled peptides are reported.     

Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide-protein conjugation through cysteine residues

The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.