Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The Na-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5-0.6 mmol/g resin). After final removal of the Na-protecting groups, treatment with TFA (50-95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.     

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.     

Separation of 17 DL-amino acids and chiral sequential analysis of peptides by reversed-phase liquid chromatography after labeling with R(-)-4- (3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole

Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in dl-amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all dl-amino acids tested. The resulting thiohydantoin derivatives of individual dl-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of dl-amino acid. The method using the reagent was adopted to identification of dl-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala(2)]-leucine enkephalin, [D-Ala(2)]-deltorphin II, d-Phe-Met-Arg-Phe-amide, and Phe-D-Met-Arg-Phe-amide. D-Ala, D-Phe, and D-Met in the peptides were positively identified with the proposed procedures. [L-Ala(2)]-leucine enkephalin, beta-lipotropin, Asp-Ser-Asp-Pro-Arg, and Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide were also analyzed as the references without D-amino acid.     

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.     

Further analogues of plant peptide hormone phytosulfokine-alpha (PSK-alpha) and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor of structure H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In studies on the structure/activity relationship of PSK-alpha the synthesis was performed of a series of a further 23 analogues modified in position 1, 3 or 4 as well as simultaneously in positions 1 and 3 of the peptide chain. Peptides were synthesized by the solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK-alpha according to the test of Matsubayashi et al. Among these peptide analogues, [H-Phe(4-Cl)1]-PSK-alpha (IV), [H-Phe(4-I)1]-PSK-alpha (VII), and [Phe(4-Cl)3]-PSK-alpha (XI) retained 30% PSK-alpha activity. Analogue [Tyr(PO3H2)3]-PSK-alpha (IX) showed 10% of PSK-alpha activity.     

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.     

Site-specific synthesis of Amadori-modified peptides on solid phase.

Glycation of peptides and proteins is a slow chemical reaction of reducing sugars modifying the amino groups. The first intermediates of this nonenzymatic glycosylation are the Amadori products that can undergo further chemical reactions, finally leading to advanced glycation end products (AGEs). The formation of AGEs was not only linked to aging of tissues and organs in general but also to several diseases such as diabetes mellitus and Alzheimer's disease. Because of the importance of these modifications and their potential use as diagnostic markers, a global postsynthetic approach on solid phase was developed. The peptides were synthesized by Fmoc/(t)Bu-chemistry, with the lysine residue to be modified being protected with the very acid-labile methyltrityl group. Incubation of the peptides with D-glucose in DMF at elevated temperatures resulted in product yields of 35%. Neighboring residues with bulky protecting groups reduced the yields only slightly. The major by-products were the unmodified peptide and an oxidation product. Whereas the unmodified peptide eluted before the glycated peptide, all other by-products eluted later in RP-HPLC, allowing simple purification.     

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Efficient solution-phase synthesis of multiple O-phosphoseryl-containing peptides related to casein and statherin.

The multiple Ser(P)-containing peptides, H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA, H-Asp-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA and H-Glu-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA were prepared by the use of Boc-Ser(PO3Ph2)-OH in the Boc mode of solution phase peptide synthesis followed by platinum-mediated hydrogenolytic de-protection of the Ser(PO3Ph2)-containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH2Cl2 used for removal of the Boc group from intermediate Boc-protected peptides.     

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.     

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.     

鲑鱼降钙素及其类似物的合成

鲑鱼降钙素及其类似物的合成潘和平王良友陈正英王芳王会信（军事医学科学院基础医学研究所,北京１００８５０）ＳｙｎｔｈｅｓｉｓｏｆＳａｌｍｏｎＣａｌｃｉｔｏｎｉｎａｎｄＩｔｓＡｎａｌｏｇｓＰａｎＨｅ－ＰｉｎｇＷａｎｇＬｉａｎｇ－ＹｏｕＣｈｅｎＺｈｅｎｇ－...     

Chemical Cleavage of Bovine β-Lactoglobulin by BNPS-Skatole for Preparative Purposes: Comparative Study of Hydrolytic Procedures and Peptide Characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine β-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Solid-phase synthesis of tyrosyl H-phosphonopeptides and methylphosphonopeptides

Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible post-translational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce solid-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh₃)₄. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of ¹H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by ¹H-tetrazole and followed by oxidation led to the methylphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

Microwave‐assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use –Alloc and –OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and –OAllyl may be removed using Pd(PPh₃)₄ and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent–intensive. Herein, we report the development of a method in which the –Alloc/–OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent–intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5‐min deprotections at 38°C, all –Alloc and –OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity

Six hybrid N-ureidoethylamides of octapeptides in which an N-terminal cyclic structure related to enkephalin was elongated by a C-terminal fragment of deltorphin were synthesized on MBHA resin. The synthetic procedure involved deprotection of Boc groups with HCl/dioxane and cleavage of the peptide resin with 45 % TFA in DCM. d-Lys and d-Orn were incorporated in position 2, and Lys, Orn, Dab, or Dap in position 5. The side chains of the dibasic amino function were protected with the Fmoc group. This protection was removed by treatment with 55 % piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate. Using various combinations of dibasic amino acids, peptides containing a 17-, 18-, 19- or 20-membered ring structure were obtained. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid activities were observed, depending on the size of the ring. Extension of the enkephalin sequence at the C-terminus by a deltorphin fragment resulted in a change of receptor selectivity in favor of the δ receptor. The conformational propensities of selected peptides were determined using the EDMC method in conjunction with data derived from NMR experiments carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained earlier with corresponding N-(ureidoethyl)pentapeptide amides.     

Synthesis of esculentin-1 antibacterial peptide fragments on 1,4-butanediol dimethacrylate cross-linked polystyrene support

Peptide segments corresponding to antibacterial esculentin-1 (1–15), (33–44), (9–27), and their modified forms were synthesized on 1,4-butanediol dimethacrylate cross-linked polystyrene (PS-BDODMA) support. Hydroxymethyl and aminomethyl 2% PS-BDODMA supports were used for the synthesis. The HMPB linker was appended to the aminomethyl resin using HBTU in presence of HOBt and the first amino acid was incorporated using MSNT. The conventional Fmoc synthetic protocol was used for the synthesis of peptides. The peptides were cleaved from the support using TFA. The peptides were purified by HPLC, and characterized by amino acid analysis and MALDI TOF MS. The secondary structures of the peptides were revealed by CD measurements. The synthesis of these peptides illustrates the utility of the new support for the synthesis of long-chain bioactive peptides. The synthetic peptides were tested for antimicrobial activity against Escherichia coli Mos blue, E. coli 2, Bacillus brevis, B. megaterium, Pseudomonas HTL, and Vibrio mimicus. The antibacterial activity of the peptides was explained on the basis of the helicity and charged nature of the sequences.     

Synthesis of peptides of the preS1-region of the viral hepatitis B envelope protein and localization of antigenic determinants]

We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides.     

Chemical synthesis of human beta-endorphin(1-27) analogs by peptide segment coupling. Leucine and glycine residues bearing thiocarboxyl functions as junctions for peptide segment coupling.

[Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2, two analogs of human beta-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides. Msc-[Gly8]beta hEP(1-8)SH and Msc-[L-Leu8]beta hEP(1-8)SH, were synthesized by standard solid phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[alpha-(Boc-L-Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 in the segment coupling reaction were approximately 18%. Less than 1% of racemization of Leu-8 occurred during coupling of Msc-[L-Leu8]beta hEP(1-8)SH to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of beta-endorphin(1-27) and its analogs influenced the receptor binding activity.