Peptide Thioester Synthesis via an Auxiliary-Mediated N–S Acyl Shift Reaction in Solution

The 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group attached to a main chain amide in a peptide is easily transformed into an S-peptide via an intramolecular N–S acyl shift reaction under acidic conditions, and the S-peptide produces a peptide thioester through an intermolecular thiol–thioester exchange reaction. In order to develop a method for efficiently preparing peptide thioesters based on the N–S acyl shift reaction, the factors involved in this process were analyzed in detail. The general features of the transformation at the Dmmb group attached amide bond in a trifluoroacetic acid (TFA) solution and the generation of a peptide thioester were examined by ¹³C-NMR spectral measurements, reversed-phase (RP) HPLC analyses, mass measurements, and amino acid analyses. The methoxy group of the Dmmb group was not essential for the N–S acyl shift reaction, but played a role in stabilizing the thioester form. The addition of water to the TFA solution accelerated the N–S acyl shift reaction mediated by the Dmmb group and also suppressed the acid-catalyzed cleavage of the Dmmb group. A peptide thioester was produced from the S-peptide via an intermolecular thiol–thioester exchange reaction with minimal epimerization of the amino acid residue that constituted the thioester bond. Undesirable side reactions, such as the hydrolysis of the thioester bond and an S–N acyl shift reaction occurred during the synthetic process, which is a subject of further investigation.     

Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl auxiliary group

An efficient method of peptide thioester synthesis is described. The reaction is based on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl (Dmmb) auxiliary‐assisted N‐S acyl shift reaction after assembling a peptide chain by Fmoc‐solid phase peptide synthesis. The Dmmb‐assisted N‐S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S‐peptide with sodium 2‐mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on‐resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41‐mer peptide thioester, a phosphorylated peptide thioester and a 33‐mer peptide thioester containing a trimethylated lysine residue. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Tandem ligation at X-Cys and Gly-Gly positions via an orthogonally protected auxiliary group

4,5-dimethoxy-2-mercaptobenzylamine (Dmmb) has been protected by acetamidomethyl (Acm) and incorporated into a peptide thioester for use in tandem native chemical ligation. Upon ligation between the thioester and a Cys-peptide, Acm was removed from Dmmb using silver acetate, and a second ligation reaction was done at the Dmmb position. Dmmb removal using TFMSA-TFA effected overall tandem ligation at X-Cys and Gly-Gly.     

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.     

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of the Suzuki-Miyaura cross-coupling reaction for the modification of phenylalanine peptides

We have demonstrated an exceptionally simple and a useful methodology for modification of unusual phenylalanine peptides by adapting the building block approach using the Suzuki-Miyaura cross-coupling reaction as a key step. This strategy gave a good overall yield of various modified tri- and pentapeptides that may be useful to prepare various biologically active peptides in a short period of time.     

Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-alpha production by the RAW 264.7 macrophage cell line.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

High-sensitivity isoelectric focusing of biotinylated peptides: a new method

A novel technique to selectively analyze prelabeled peptides by isoelectric focusing (IEF) is presented. The conditions are described for biotinylation of peptides, their separation in polyacrylamide gels by IEF, and their fixation to the gel matrix with glutaraldehyde. The gels are developed by a color reaction catalyzed by an avidin-coupled enzyme. The technique is suitable for peptides with at least one free amino group or guanidino group after N-terminal biotinylation. The presence of other peptides or proteins does not interfere with the detection. The sensitivity is below 10 pmol, representing a 1000-fold improvement over existing techniques for analyzing low molecular weight peptides by IEF.     

Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine,Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH₂, measured on sodium channels whose inactivation was slowed by the α-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH₂ with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH₂ did not cause appreciable fast block, but the divalent cation IFM-NH(CH₂)₂NH₂ was as effective as the pentapeptide ac-KIFMK-NH₂. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH₂ showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.     

Synthesis and use of a pseudo-cysteine for native chemical ligation.

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.     

Synthesis of cyclic peptides from unprotected precursors using removable N alpha-(1-(4-methoxyphenyl)-2-mercaptoethyl) auxiliary.

A new method to cyclize unprotected peptides is presented. The method involves the use of a 1-phenyl-2-mercaptoethyl derivative on the N-terminal glycine. This template acts as an auxiliary thiol-containing group in order to drive cyclization with a counterpart thioester moiety on the same molecule. Subsequent facile removal of the derivative generates products with only native peptide structure. The successful, high-yield cyclization of the peptide GSPYSSDTTPA is described.     

Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.     

Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases

The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.     

A single amino-acid polymorphism in pocket A of HLA-A*6602 alters the auxiliary anchors compared with HLA-A*6601 ligands

In this study we have sequenced peptides eluted from a truncated recombinant HLA-A*6602 molecule, and compared their features with data reported for peptides presented in the A*6601 molecule. A striking change in the amino-acid binding preferences was observed at peptide position P1, which interacts with pocket A of the HLA peptide-binding region. For A*6601, aspartic acid and glutamic acid, both of which possess polar acidic side-chains, have been described as auxiliary anchors. This is in marked contrast to A*6602, where we observed serine, which has a neutral polar side-chain, as auxiliary anchor at P1. Accordingly, this shift in the physico-chemical properties of the auxiliary anchor may be best explained by the HLA amino-acid polymorphism at position 163, where arginine (hydrophilic, alkaline) in A*6601 has been replaced by glutamic acid in A*6602. This amino-acid exchange results in a shift towards higher acidity in pocket A, apparently resulting in the loss of preference for acidic auxiliary anchors, and leading to the preference for the neutral amino acid serine. The change of the auxiliary anchor residue at P1 is likely to alter the spectrum of peptides presented by A*6602 compared with A*6601, which may result in allogenicity in the case of a mismatch in allogeneic stem cell transplantation.     

Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)14,17, GlyS23]-apocytochrome c-(1-23) (I), CF3CO-[GlyS60]-apocytochrome c-(24-60) (II), and CF3CO-apocytochrome c-(61-104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61-104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24-104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys(Cam)14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

Identification of formaldehyde-induced modifications in proteins: reactions with model peptides

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.     

Regulation of activities of NK cells and CD4 expression in T cells by human HNP-1, -2, and -3

Human neutrophil defensin alpha (HNP) is a group of cationic peptides of diverse physiological roles. Recent studies revealed the nature of HNPs as the dominant HLA-DR binding peptides on malignant cancer cells, which may block the major histocompatibility complex for antigen presentation. Here we show that HNPs may inhibit T cells by downregulating CD4 expression, a molecule of critical importance for T cell's interaction with the target cell. HNPs also inhibited tumor-cell-lysis activities of NK cells by downregulating CD16-CD56 expression. More importantly, HNPs were markedly elevated in 14 cancer tissues out of 15 self-paired human colorectal cancers and their adjacent noncancerous tissues. The subset compositions of HNPs extracted from cancer tissues and neutrophils were identical. Immunohistochemical studies indicated that HNPs mainly distributed in the infiltrated neutrophils in the interstitium. The elevated HNPs in cancer tissues may create a microenvironment unfavorable for adaptive immune reaction, implicating the cancer evasion.     

Fluoride-18 radiolabeling of peptides bearing an aminooxy functional group to a prosthetic ligand via an oxime bond

We have developed a novel F-18 prosthetic ligand named fluoro-PEG-benzaldehyde (FPBA) 1. [(18)F]-FPBA 1 is formed in situ from its radiolabeled precursor [(18)F]6. Compound 6 is efficiently synthesized in four steps starting from commercially available 6-bromo-3-pyridine carbaldehyde 2. [(18)F]-FPBA was evaluated as a prosthetic ligand to radiolabel three cyclic peptides bearing an aminooxy functional group at the N-terminus position. Acetal [(18)F]6 is purified by either solid-phase extraction (SPE) or reverse-phase HPLC with the overall radiochemical yields (RCY) and radiochemical purity (RCP) in very close agreement. The SPE purification process has the advantage of shorter reaction times (71-87 min for entire reaction sequence), while the use of the reverse-phase HPLC purification process allows the use of up to fifty times less of the expensive synthetic peptides (~ 50 nmol) in the oxime coupling reaction. We have demonstrated an efficient methodology in the production of [(18)F]-FPBA 1 and demonstrated its use as a prosthetic ligand for the labeling of peptides possessing an aminooxy functional group.     

Vesicles from peptidic side-chain polymers synthesized by atom transfer radical polymerization

Block copolymers can adopt a wide range of morphologies in dilute aqueous solution. There is a significant amount of interest in the use of block copolymer vesicles for a number of applications. We show that a series of oligo(valine) and oligo(phenylalanine) peptides coupled to a methacrylic group can be prepared by conventional peptide coupling techniques. These can be successfully polymerized by atom transfer radical polymerization (ATRP) in hexafluoroisopropanol (HFIP) giving access to poly(ethylene oxide)- b-poly(side-chain peptides). Many of these polymers self-assemble to form vesicles using an organic to aqueous solvent exchange. One example with a divaline hydrophobic block gives a mixture of toroids and vesicles. Circular dichroism demonstrates that secondary structuring is observed in the hydrophobic region of the vesicle walls for the valine side-chain containing polymers.