NMR studies of poly-gamma-benzyl-l-glutamate in trifuloroacetic-deuterochloroform solutions: temperature dependence

The helix–random-coil transition process for poly-γ-benzyl-L -glutamate (PBG) in solvent mixtures trifluoroacetic acid/deuterochloroform (TFA/CDCl₃) at different temperatures has been studied by nmr. The chemical shift behavior of the α-CH resonances of the peptide chain and of the TFA carboxylic protons is reported.     

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.     

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

The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc-method is described. The new anchor group was developed based on our experience with 4,4'-dimethoxybenzhydryl (Mbh)-protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N-9-fluorenylmethoxycarbonyl- ([5-carboxylatoethyl-2.4-dimethoxyphenyl)- 4'-methoxyphenyl]-methylamin is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc-amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid-dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C-terminal hexapeptide of secretin are given as examples.     

Reduction of a 4-pyrrole phenylacyl-containing peptide with trifluoroacetic acid-triisopropylsilane-phenol-H2O during solid-phase peptide synthesis and its protein kinase C alpha inhibitory activity

Cleavage of 4-pyrrole phenylacyl TentaGel-appended peptide (5) containing Arg(Pbf) with trifluoroacetic acid/triisopropylsilane/phenol/H2O (90/2/4/4) gives the 4-pyrrole phenylacyl peptide (3). However, cleavage of 4-pyrrole phenylacyl Rink-appended peptide (7) containing Arg(Pbf) using the same reagents furnishes the 4-pyrrolidine phenylacyl peptide (8), which contains the reduced pyrrole ring. Compound 8 displays a Ki of 2.32 microM, approximately fivefold less potent than compound 3.     

Convenient reduction of S-oxides in synthetic peptides, lipopeptides and peptide libraries

Summary The oxidation of thioether bonds in peptides is still one of the major side reactions during peptide synthesis and it is easily detected by electrospray mass spectrometry. A fast reduction of oxidized methionine, biotin and tripalmitoyl-S-glyceryl-cysteine in synthetic peptides is possible using mixtures of trimethylsilylbromide and ethanedithiol as additives in trifluoroacetic acid. Simple procedures have been worked out which are especially suitable for handling the reduction of large numbers of peptides, prepared by multiple peptide synthesis, and of peptide libraries.     

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.     

A differential dilatometer

To obviate the difficulties resulting from partial solubility of membrane proteins in detergents or from the use of noxious solvent mixtures containing phenol or chloral, a simple procedure was devised for acrylamide gel electrophoresis of membrane proteins in 13 m formic acid. Polyacrylamide gels are equilibrated in 13 m formic acid and used in the electrophoresis assembly with 13 m formic acid as the electrolyte. Particulate proteinaceous preparations are dissolved in trifluoroacetic acid or in 24 m formic acid containing glycine to increase the density and to facilitate the solubilization of the protein. Protein samples (10 to 100 μg) migrate as polycations.     

Synthesis of 2,3-O-(propane-1,2-diyl)- and 2,3-O-(3-hydroxypropane-1,2-diyl)-cyclomaltoheptaose

2¹,3¹-O-(Propane-1,2-diyl)cyclomaltoheptaose has been prepared from 2-O-allylcyclomaltoheptaose by mercuration in trifluoroacetic acid, followed by reduction with sodium borohydride. 2-O-(2,3-Epoxypropyl)cyclomaltoheptaose, prepared from 2-O-allylcyclomaltoheptaose by oxidation with dimethyldioxirane, was converted into 2¹,3¹-O-(3-hydroxypropane-1,2-diyl)cyclomaltoheptaose by treatment with trifluoroacetic acid. Both derivatives containing fused 1,4-dioxane rings are mixtures of stereoisomers, in which the methyl and hydroxymethyl group, respectively, that is linked to this ring, occupies an axial or an equatorial position.     

Fluorescent N-methylanthranilyl (Mantyl) tag for peptides: its application in subpicomole determination of kinins.

Highly fluorescent N-methylanthranilyl (Mantyl) peptide derivatives were prepared by a one-step reaction with N-methylisatoic anhydride (MIA) for quantitative detection in HPLC. Reactions were carried out in an organic medium of acetonitrile-triethylamine, in aqueous alkaline sodium carbonate and sodium phosphate buffers. 4-Dimethylaminopyridine (DMAP) catalyzed specific mantylation of -NH2 groups of peptides in the organic reaction medium. The DMAP had no effect in the aqueous buffered reaction systems. Proline amino-terminal peptides reacted equally well with MIA. Mantyl-bradykinin had excitation and fluorescence maxima at 350 nm and 426 nm in water and water/acetonitrile (ACN)/trifluoroacetic acid (TFA) solvent mixtures, respectively. Fluorescence intensity increased with an increase in ACN concentration and decreased with an increase in acid content. Mantyl kinins were completely resolved on a C18 reversed phase HPLC column using an ACN-0.1% TFA gradient and their behavior on the column was similar to having an extra amino acid. Di-Mantyl derivatives obtained with Lys-BK and Met-Lys-BK did not exhibit fluorescence appreciably higher than Mantyl-BK. Fluorescence detection of Mantyl kinins was about 50-100 times more sensitive (lower limits of 0.1 to 0.5 picomole) than UV detection of the phenylisothiocyanate-derivatized kinins under typical HPLC conditions.     

Peptide synthesis on a phenolic resin support

Protected peptides assembled on a phenolic resin support were cleared by peroxide-catalysed hydrolysis. In genenal peptide phenyl ester resins were more labile to nucleophiles than were corresponding Merrifield resin derivatives; transesterification with dimethylaminoethanol providing on alternative cleavage method for peroxide-sensitive peptides. Losses of radiolabelled peptide from both Merrifield and phenolic resins were determined during acid deprotection, base wash and coupling steps in the synthesis of a tetrapeptide. Using 40% (v/v) trifluoroacetic acid in dichloromethane for Boc-deprotection the phenolic resin gave improved results compared to the Merrifield resin. The merits of the procedure for the preparation of protected peptide acids suitable for subsequent condensation reactions were exemplified by the synthesis of an octapeptide sequence of a modified lysozyme.     

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and -chymotrypsin, were determined.     

Novel peptide derivatives of bleomycin A5: synthesis, antitumor activity and interaction with DNA

A series of novel amino acid and peptide derivatives of bleomycin (BLM) A(5) were synthesized. All the compounds possessed significant antitumor activities in vitro against HL-60, BGC-823, PC-3MIE8, and MDA-MB-435 cell lines. Their antitumor activities against MDA-MB-435 were 10-fold higher than BLM A5. The DNA cleavage studies indicated that the hydrophobic amino acid or peptide derivatives of BLM A5 could induce higher cleavage ratio of double to single strand DNA than BLM A5. From the DNA binding studies, we found that the derivatives containing either D-conformation amino acid or basic amino acid could facilitate DNA binding of BLM.     

Analysis of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acids at sub-picomole levels by high-performance liquid chromatography: simultaneous manual sequencing of picomole quantities of several polypeptides

A single-column high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives, generated during polypeptide sequence analysis by the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate/phenylisothiocyanate double coupling technique, is described. Recovery of the serine and threonine derivatives was improved by substituting boron trifluoride-diethyl etherate for trifluoroacetic acid in the thiazolinone cleavage reactions. Residues, including the S-carboxymethyl derivative of cysteine, were assigned after a single injection and a cycle time of 30 min. Quantities of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives as low as 100 fmol were detected. Interference of sequencing artefacts with residue assignment was avoided. This technique allows simultaneous manual sequencing of several proteins or peptides at the level of a few picomoles.     

Synthesis of insulin-like growth factor I using N-methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin

Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.     

The degradation of glycoproteins with lithium borohydride. Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue

By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous beta-elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 2-4 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.     

High-performance liquid chromatography of methotrexate analogs containing terminal lysine or ornithine and their dansyl derivatives

A procedure utilizing a reverse-phase semipreparative high-performance liquid chromatography column and a binary solvent system consisting of trifluoroacetic acid and 1-propanol has been developed for the semipreparative scale purification and analytical identification of four newly synthesized analogs of methotrexate. The methotrexate analogs containing a lysine or an ornithine residue in place of a terminal glutamate residue together with their respective dansyl derivatives were purified in milligram quantities by the procedures described.     

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.     

Facile solid-phase synthesis of C-terminal peptide aldehydes and hydroxamates from a common Backbone Amide-Linked (BAL) intermediate.

C-Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin-bound Weinreb amide precursor. Thus, 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyramido-polyethylene glycol-polystyrene (BAL-PEG-PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin-bound methoxylamine, which was efficiently acylated by different Fmoc-amino acids upon bromo-tris-pyrrolidone-phosphonium hexafluorophosphate (PyBrOP) activation. Solid-phase chain elongation gave backbone amide-linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C-terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68-83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16-53% and initial purities in the range of 30-40%. Under the LiAlH4 cleavage conditions used, those model peptides containing t-Bu-protected aspartate residues underwent partial side chain reduction to the corresponding homoserine-containing peptides. Similar results were obtained when working with high-load aminomethyl-polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.     

Synthesis and purification of amyloidogenic peptides

The polypeptide hormone amylin forms amyloid deposits in patients with type 2 diabetes mellitus. Amyloid-forming peptides are often very difficult to synthesize and purify. Amylin and fragments of amylin are no exception. In this paper we describe the efficient synthesis and purification of two amyloidogenic fragments of human amylin. One fragment corresponds to residues 17 to 37 of the full-length hormone and the other corresponds to residues 24 to 37. These fragments have previously been identified in vivo and have been shown to form amyloid in vitro. The strategy used to elucidate appropriate conditions for the synthesis and purification of these peptides is generally applicable to other peptides that are difficult to synthesize. These peptides were prepared using solid-phase peptide synthesis with Fmoc alpha-amino protection. The effects of varying the solvent, side-chain-protecting group and choice of cleavage conditions were examined. The use of NMP as the main solvent and cleavage with trifluoroacetic acid, phenol, ethanedithiol, thioanisole, and water proved to be optimal. 1,1,1,3, 3,3-Hexafluoro-2-propanol (HFIP) was found to be the best solvent for solubilizing the crude peptides. A wide range of HPLC conditions for the purification of the peptides were examined and an acetonitrile-based solvent system with HCl as the ion pairing agent provided efficient purification.     

Two-step hard acid deprotection/cleavage procedure for solid phase peptide synthesis

A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilyl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nin-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.