Dehydroalanine-containing peptides: preparation from phenylselenocysteine and utility in convergent ligation strategies

This protocol describes the methodology for the synthesis of dehydroalanine (Dha)-containing peptides and illustrates their use in convergent ligation strategies for the preparation of peptide conjugates. A nonproteinogenic amino acid, Fmoc-Se-phenylselenocysteine (SecPh), can be prepared in high yield over four synthetic steps and be conveniently incorporated into peptides by standard solid-phase peptide synthesis techniques. Globally deprotected peptides containing phenylselenocysteine can be converted to dehydrated peptides following a chemoselective, mild oxidation with hydrogen peroxide or sodium periodate (i.e., the phenylselenocysteine side chain is converted to that of Dha). Dha residues are electrophilic handles for the preparation of glycopeptides, lipopeptides or other peptide conjugates; one such transformation will be outlined here. The preparation of Dha-containing peptides, including the synthesis of SecPh, peptide elongation and oxidative treatment of phenylselenocysteine-containing peptides can be completed by one person in approximately 3-5 weeks. However, once SecPh is in hand, the time required for the preparation of peptides is significantly shorter and comparable to that for any peptide synthesis.     

SYNTHESIS OF PEPTIDE-OLIGONUCLEOTIDE PHOSPHOROTHIOATE CONJUGATES BY CONVERGENT OR STEPWISE SOLID-PHASE STRATEGIES

A convergent strategy was employed to link eight 10–27-mer peptides to oligonucleotide phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.     

Synthesis of peptide-oligonucleotide phosphorothioate conjugates by convergent or stepwise solid-phase strategies

A convergent strategy was employed to link eight 10-27-mer peptides to oligonucleotide phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.     

Synthesis of C-linked triazole-containing AFGP analogues and their ability to inhibit ice recrystallization

C-Linked antifreeze glycoprotein (C-AFGP) analogues have been shown to have potent ice recrystallization inhibition (IRI) activity. However, the lengthy synthesis of these compounds is not amenable to large-scale preparation for the many commercial, industrial, and medical applications that exist. This paper describes the synthesis of triazole-containing AFGPs using a convergent solid-phase synthesis (SPS) approach in which multiple carbohydrate derivatives are coupled to a resin-bound synthetic peptide in a single step. Modified "Click" conditions using dry DMF as solvent with catalytic Cu(II), sodium ascorbate, and microwave radiation afforded the synthesis of AFGP analogues 9-12 in 16-54% isolated yield. Compound 9 demonstrated no IRI activity, while compounds 10, 11, and 12 were moderate inhibitors of ice recrystallization. These results suggest that, while the triazole group is a structural mimetic of an amide bond, the amide bond in C-AFGP analogue 3 is an essential structural feature necessary for potent IRI activity.     

Synthesis of tolerogenic monomethoxypolyethylene glycol and polyvinyl alcohol conjugates of peptides

Recent studies from this laboratory showed that tolerogenic peptide conjugates are very effective reagents for obtaining epitope-specific immunosuppression of antibody responses to immunopathogenic sites on multideterminant complex protein antigens. This paper describes the procedure for synthesis of well-defined conjugates of peptides to monomethoxypoly-ethylene glycol (mPEG) or to polyvinyl alcohol (PVA). The first step involves succinylation of the hydroxyl groups on the polymers by reaction with succinic anhydride. The polymer is then coupled via the carboxyl of the succinyl group to the -NH₂ of the completed peptide on the synthetic resin, while maintaining intact all the side-chain protecting groups on the peptide. The mPEG or PVA-peptide conjugates are cleaved from the resin and purified by standard procedures. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is coupled to the N-terminal of an otherwise unaltered peptide molecule.     

Synthesis of oligonucleotide 2'-conjugates via amide bond formation in solution

An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide.     

Synthesis of peptide amides by Fmoc-solid-phase peptide synthesis and acid labile anchor groups

The preparation and use of new anchor groups for the synthesis of peptide amides by solid-phase peptide synthesis employing the Fmoc-method is described. Based on the structure of the 4,4'-dimethoxybenzhydryl group (Mbh) handles were developed, which could be cleaved by mild acid treatment to give carboxamides. The syntheses and application of Fmoc-amino-acid-(4-carboxylatomethyloxyphenyl-4'-methoxyphenyl) methyl amide and Fmoc-(4-carboxylatopropyloxyphenyl-4'-methoxyphenyl) methyl amide are described in detail. These handles were coupled to resins and a stepwise elongation of peptide chains proceeded smoothly with N alpha-9-fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives using a carbodiimide/HOBt mediated reaction. The final cleavage of side-chain protecting groups and the release of the C-terminal amide moiety was achieved by the treatment with trifluoroacetic acid, dichloromethane in the presence of scavengers. Various peptides, such as the Leu-enkephalin amide and Leu-Gly-Gly-Gly-Gln-Gly-Lys-Val-Leu-Gly-NH2, which is a good substrate for F XIII, were prepared in high yields and purities.     

Chemical modification and cross-linking of neurophysin tyrosine-49

Photoaffinity labeling of the single neurophysin tyrosine, Tyr-49, with Met-Tyr-azido-Phe amide has been reported to inhibit both neurophysin self-association and peptide binding. Accordingly, we investigated the functional consequences of modification, principally by tetranitromethane, of Tyr-49. Tetranitromethane-mediated tyrosine-tyrosine cross-linking permitted synthesis of covalent neurophysin "dimers" and of peptide-protein conjugates, the latter potentially analogous to the photoaffinity-labeled product. The self-association and binding properties of the covalent dimers were found to be similar or enhanced relative to those of the native protein. In contrast to the photoaffinity-labeled product, covalent conjugates of Tyr-49 with the ligand peptides Met-Phe-Tyr amide, Phe-Tyr amide, and Tyr-Phe amide also generally exhibited normal or increased binding affinity for exogenous peptide; a subfraction of the Phe-Tyr amide adducts showed evidence of reduced affinity. Diiodination of Tyr-49 had no significant effect on binding. However, among the products of tetranitromethane treatment in the absence of peptide was a novel inactive non-cross-linked product, representing modification only of Tyr-49 but containing no demonstrable nitrophenol. As evidenced by circular dichroism and nuclear magnetic resonance (NMR), this product was not significantly unfolded and retained the ability to self-associate. These latter results provide the strongest evidence thus far of a role for Tyr-49 in peptide-hormone binding. The disparate effects of different Tyr-49 modifications are collectively interpreted and reconciled with NMR data and the properties of the photoaffinity-labeled protein to suggest potential mechanisms of Tyr-49 participation in binding and the probable orientation of Tyr-49 relative to peptide residue 3 in neurophysin complexes.     

Use of the multipin peptide synthesis technique for the generation of antipeptide sera

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pinsynthesized peptides and β-amide peptides and the protein antigen coated to microtiter plates.     

The N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine: a heterobifunctional cross-linking agent

Synthetic cysteine-containing peptides were unidirectionally conjugated to albumin via disulfide bonds using the S-(3-nitro-2-pyridinesulfenyl) derivative of cysteine. This method employs the N-hydroxysuccinimide ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine, a protected amino acid derivative used in peptide synthesis, as a heterobifunctional cross-linking agent. The disulfide bonds in the conjugates are formed by the reaction of free thiols with S-(3-nitro-2-pyridinesulfenyl) groups. Bovine albumin was conjugated in this manner to several synthetic peptides derived from human fibrin. Amino acid analysis of these conjugates demonstrated incorporations of from 6 to 11 peptide molecules per molecule of protein.     

9-Fluorenylmethyloxycarbonyl/ tbutyl-based convergent protein synthesis

Besides linear solid phase peptide synthesis, segment condensation in solution and chemical ligation, convergent peptide synthesis (CPS) was developed in order to enable the efficient preparation of complex peptides and small proteins. According to this synthetic strategy, solid phase synthesized and suitably protected peptide fragments corresponding to the entire peptide/protein-sequence are condensed on a solid support or in solution, to the target protein. This review summarizes CPS performed utilizing the mild 9-fluorenylmethyloxycarbonyl/tbutyloxycarbonyl-based protecting scheme for the amino acids.     

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.     

Concise Synthesis of Triazole-Linked 5′-Peptide-Oligonucleotide Conjugates by Click Chemistry

A concise synthesis of oligonucleotide 5′-peptide-conjugates via copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition in aqueous solution is described. Synthesis of reagents was accomplished by on-column derivatization of corresponding peptides and oligonucleotides. This method is well suited for the preparation of peptide–oligonucleotide conjugates containing 1,2,3-triazole linkage between the 5′-position of an oligonucleotide and the N-terminus of a peptide.     

Efficient synthesis of an (aminooxy) acetylated‐somatostatin derivative using (aminooxy)acetic acid as a ‘carbonyl capture’ reagent

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of an (aminooxy)acetylated‐somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a ‘carbonyl capture’ reagent in the final cleavage step. This (aminooxy)acetylated compound was further used for the chemoselective ligation (oxime bond formation) with daunorubicin and 4‐fluorobenzaldehyde leading to the formation of conjugates with potential applications in targeted cancer chemotherapy and positron emission tomography. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]⁺ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thils with pyrrolylated peptides.     

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates

Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N′-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N′-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl₂·2H₂O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid–phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid).     

Discovery and development of N-cadherin antagonists

This article describes over 20 years of research on antagonists of the cell adhesion molecule, N-cadherin. Four types of antagonists are discussed: synthetic linear peptides, synthetic cyclic peptides, non-peptidyl peptidomimetics of the disulfide linked cyclic peptide N-Ac-CHAVC-NH(2) and monoclonal antibodies directed against the N-cadherin ectodomain. The biological activities of these antagonists are also discussed. In particular, the ability of N-cadherin antagonists to act as anti-cancer drugs is considered.     

Use of the multipin peptide synthesis technique for the generation of antipeptide sera.

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pin-synthesized peptides and beta-amide peptides and the protein antigen coated to microtiter plates.     

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.     

Peptide and peptidomimetic libraries

Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.