Pyridoxyl amino acid esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.     

Conservation of primary structure of the pyridoxyl peptide of Escherichia coli and Serratia marcescens tryptophan synthase beta2 protein.

Two labeled peptides were recovered from tryptic digests of the NaB3H4-reduced, performic acid-oxidized beta2 protein of Serratia marcescens tryptophan synthase. These two pyridoxyl peptides were identical except for the presence or absence of an NH2-terminal arginyl residue. Tryptic digestion of nonreduced, performic acid-oxidized protein allowed isolation of the peptides that comprise the two halves of the pyridoxyl peptide. The partial primary structure for this region of the protein was shown to be Arg-Glx-Asx-Ler-Leu-His(Gly,Gly,Ala,His)Lys(Pxy)-Thr-Asx-Glx-Val(Leu,Gly,Glx,Ala,Leu,Leu,Ala)Lys. All the data available indicate that the sequence is identical with the homologous region from the Escherichia coli enzyme.     

Group separation of peptides by ligand-exchange chromatography with a Sephadex containing N-(2-pyridylmethyl)glycine

The synthesis of 2-hydroxy-3[N-(2-pyridylmethyl)glycine]propyl Sephadex ether--a new chelating resin--is described. This resin has been employed in the form of its Cu2+ complex to separate peptides, as a group, from alpha-amino acids and NaCl. Ninety-seven ligands of different structures were separated chromatographically at room temperature. It was shown that two structural parameters of the ligands control the separation process, namely, the presence of ligand donor groups and the possibility for them to form chelate rings of suitable size. Separation of peptides from alpha-, gamma-, delta-amino acids, N-acetyl derivatives of amino acids (except N-Ac-Trp), and NaCl is possible if the peptides fulfill the following structural requirements: the peptide molecule must have a free terminal amino group; a carbonyl group (of the peptide linkage) must be situated in the alpha- or beta-position of the free amino group; and the peptide may not contain an imidazole residue (except Gly-Gly-His). A relationship was found between the log k' and the corresponding pKHHL, log KCuCuL, and log KCuBipyCuBipyL values. Interpretation of the different K' values was possible based on the different basicities of the terminal amino groups and on the structures of the different side chains of the peptides.     

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%).     

Chemical structure of the active site of pig heart mitochondrial aspartate aminotransferase labeled with beta-chloro-l-alanine

Formate-induced inactivation of pig heart mitochondrial aspartate aminotransferase by beta-chloro-L-alanine resulted in the modification of the epsilon-amino group of the lysyl residue which is involved in the formation of an aldimine bond with 4-formyl group of the coenzyme, pyridoxal 5'-phosphate. The tryptic peptide isolated from the labeled site of the enzyme was composed of 25 residues and exhibited positive circular dichroism at 325 and 254 nm where the pyridoxyl chromophore of the labeled site peptide absorbs, while the phosphopyridoxyl peptide isolated from the boro-hydride-reduced enzyme did not show any ellipticity in this spectral region. Its comparison with the analogous tryptic peptide from the labeled site of the cytosolic isoenzyme revealed a high degree of homology in their primary structures as well as in spectral properties. Structural analysis of the labeled site peptide and mechanistic consideration of the labeling process indicated that with both isoenzymes the phosphopyridoxyl group is covalently bound to the alpha amino group of the alanyl moiety derived from beta-chloro-L-alanine, the beta carbon of which is covalently linked to the epsilon-amino group of the lysyl residue.     

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.     

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.     

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Labeling of the active site of cytoplasmic aspartate aminotransferase by -chloro-L-alanine

Syncatalytic inactivation of pig heart cytoplasmic aspartate aminotransferase by β-chloro-[U-¹⁴C]L-alanine resulted in the incorporation of radioactivity corresponding to one mole of the label per mole of the monomeric unit of the enzyme. A borohydride-reduced and then carboxymethylated preparation of the labeled enzyme was digested by trypsin. A radioactive peptide was isolated and found to contain a covalently linked pyridoxyl derivative which absorbed at 325 nm. The amino acid sequence of this peptide was Tyr-Phe-Val-Ser-Glu-Gly-Phe -Glu-Leu-Phe-Cys-Ala-Gln-Ser-Phe-Ser-Lys-Asn-Phe-Gly-Leu-Tyr-Asn-Glu-Arg. In the peptide the phosphopyridoxyl group seems to be covalently bound via alanyl moiety derived from β-chloro-L-alanine, the β-carbon atom of which is covalently linked to the ?-nitrogen atom of the lysyl residue(Lys). From a comparison with the amino acid composition of the phosphopyridoxyl peptide isolated from the tryptic digest of a borohydride-reduced holoenzyme, it was concluded that the modified lysul residue was identical to that involved in binding pyridoxal phosphate to the apoenzyme.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.     

Observations on the reaction of 2-hydroxy-5-nitrobenzyl bromide with a peptide-bound tryptophanyl residue

Previous studies on the isolation of peptides containing tryptophanyl residues modified with 2-hydroxy-5-nitrobenzyl bromide demonstrated multiple products of reaction at the same residue as well as technical difficulties in the primary structure analysis of peptides containing the modified tryptophanyl residue. The present study was undertaken to explore the reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophanyl residue in a synthetic peptide, experimental allergenic encephalitogenic peptide. The modification of this peptide was accomplished in sodium acetate, pH 4.75, and reagent removed by gel filtration. Amino acid analysis of the modified peptide suggested that only the tryptophanyl residue had been modified under these experimental conditions. The modified peptide could be separated into multiple derivatives by high-performance liquid chromatography. Although it is clear that some of the observed heterogeneity reflects a difference in the degree of substitution at the single tryptophanyl residue, several of the derivatives appear to have the same extent of substitution. It is suggested that the heterogeneity observed is a reflection of the establishment of a new diastereoisomeric center in the peptide. These results are consistent with previous observations from other laboratories and provide a basis for the explanation of apparent heterogeneity of peptides obtained from modified proteins.     

Microwave Synthesis of Prion Protein Fragments up to 111 Amino Acids in Length Generates Biologically Active Peptides

Misfolded conformers of the prion protein are aetiologically implicated in neurodegenerative conditions termed prion diseases (also known as transmissible spongiform encephalopathies). Two constitutively expressed N-terminal peptides corresponding to human residues 23–90 and 23–111 are thought to serve normal physiological roles related to neuronal protection with membrane binding possibly playing a part in their mechanism of action. These peptides, along with several derivatives up to 111 residues in length, have been produced by microwave assisted peptide synthesis. HPLC and MS characterisation showed that the peptides were manufactured in good yields at high purity. Peptides were assayed by fluorescence spectroscopy for synthetic lipid-membrane binding activity and by dichlorodihydrofluorescein diacetate assay for the amelioration of reactive oxygen species production. Results of these assays were similar to those reported for the wild type recombinant PrP, demonstrating that these synthetic peptides are useful for biological and chemical assays of PrP activity. Further, the longest peptide 1–111 was dimerised via a single internal cystine residue with good yield. The high yields and low purification burden of the microwave assisted synthesis method lends itself to the production of difficult to produce peptides for such studies.     

The Synthesis of Phosphopeptides Using Microwave-assisted Solid Phase Peptide Synthesis

A series of phosphorylated peptides were synthesised using microwave mediated solid phase peptide synthesis. Acidic cleavage of peptides from the solid support using microwave irradiation often resulted in reattachment of the phosphate benzyl protecting group to the peptide chain. However for most phosphopeptide sequences performing the cleavage reaction at room temperature in order to minimize this undesired alkylation was successful. Notably for phosphopeptides containing a methionine residue flanking the phosphorylated residue (for serine and threonine) the trifluoroacetic acid mediated cleavage afforded the benzylated side product as a major component. This detrimental process was not observed for a corresponding tyrosine containing sequence.     

A viable synthesis of N-methyl cysteine

While a number of methods exist for the production of N-methyl amino acid derivatives, the methods for the production of N-methyl cysteine (MeCys) derivatives are suboptimal as they either have low yields or lead to significant sulfhydryl deprotection during the synthetic protocol. This article focuses on the generation of MeCys and its subsequent use in Fmoc solid-phase peptide synthesis for the generation of N-methyl cystine containing peptides. Various methods for amino methylation of cysteine, in the presence of acid labile or acid stable sulfhydryl protecting groups, are compared and contrasted. Production of MeCys is best attained through formation of an oxazolidinone precursor obtained via cyclization of Fmoc--Cys(StBu)--OH. Following oxazolidinone ring opening, iminium ion reduction generates Fmoc--MeCys(StBu)--OH with an overall yield of 91%. The key to this procedure is using an electronically neutral Cys-derivative, as other polar Cys-derivatives gave poor results using the oxazolidinone procedure. Subsequently, the Fmoc--MeCys(StBu)--OH building block was used to replace a Cys residue with a MeCys residue in two peptide fragments that correspond to the active sites of glutaredoxin and thioredoxin reductase. The examples used here highlight the use of a MeCys(StBu) derivative, which allows for facile on-resin conversion to a MeCys(5-Npys) residue that can be subsequently used for intramolecular disulfide bond formation with concomitant cleavage of the peptide from the solid support. (c) 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 61-68, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.     

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.     

Use of the Sonogashira coupling reaction for the "two-step" labeling of phenylalanine peptide side chains with organometallic compounds

The Pd-catalyzed Sonogashira coupling of ferrocene alkyne derivatives as metal probes to iodophenylalanine containing peptides is described. 4-Iodophenylalanine was incorporated into dipeptides and the neuropeptide [Leu5]-enkephalin (Enk) by solid phase peptide synthesis, thereby creating a functional group suitable for the Sonogashira coupling. The reaction with two different ferrocene alkynes resulted in the corresponding ferrocene-labeled derivatives, which were obtained in good yield and purity. All new compounds were comprehensively characterized, including elemental analysis, 1D and 2D NMR, EI-, FAB- or ESI-MS, IR and UV-vis spectroscopy, and electrochemistry of the ferrocene label. Unlike well-established conjugation methods for peptide side chains such as lysine and cystein, the phenyl group in Phe is not readily available for derivatization. This work presents a versatile procedure for the regioselective introduction of an organometallic label into biologically relevant peptides as exemplified for enkephalin.     

Affinity labeling of ras oncogene product p21 with guanosine diphospho- and triphosphopyridoxals

Purified v-rasH p21 overproduced in Escherichia coli was treated with guanosine diphospho- and triphosphopyridoxals (GP2- and GP3-PL), affinity labeling reagents specific to a lysyl residue located in the guanine nucleotide binding site. GP2-PL and GP3-PL inhibited [3H]GDP binding to p21 competitively. Incubation of p21 with GP2-PL and GP3-PL followed by reduction with NaBH4 resulted in 40 and 50% loss of [3H]GDP binding activity, respectively, whereas the addition of excess GDP completely protected p21 from the inactivation. The tryptic digest of p21 which was modified with GP2-PL or GP3-PL in the presence or absence of protective GDP and subsequently reduced by NaBH4 was analyzed by reverse phase high performance liquid chromatography. The profile of the effluent monitored by the fluorescence from the pyridoxyl moiety showed the existence of peptides which were specifically labeled only in the absence of GDP. Structural analyses of these peptides allowed us to identify the labeled residue as Lys-16. These results suggest that Lys-16 is located in the guanine nucleotide binding site, close to the beta- or gamma-phosphate group of the nucleotide.     

Synthesis of a template-associated peptide designed as a transmembrane ion channel former.

We describe the design and the Fmoc/tBu solid phase synthesis of a 20 residue long peptide containing five regularly distributed lysines. Cyclization of this peptide was achieved using BOP as coupling agent. After side-chain deprotection, all the basic residues were iodoacetylated and then allowed to react either with a C-terminal free COOH peptide or with peptides bearing a cysteamide group. The final pentameric templates were identified by mass and amino acid analysis which gave data compatible with the expected values.