Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83–99)

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83–99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Summary Studies leading to optimization of butanediol dimethacrylate-crosslinked polystyrene supports (BDDMA-PS) for solid phase peptide synthesis are delineated. BDDMA-PS copolymers with different crosslink densities were prepared and functionalised with chloromethyl groups. The reactivity of the Lys(2-Cl−Z)−OH residue bound to these polymers through a benzyl ester linkage was investigated by following the kinetics of acylation by the HOBt active ester of Boc-Alanine. From the results it was observed that the rate of peptide bond formation was maximum for a 2% BDDMA crosslinked resin. This resin was compared with a 2% DVB-crosslinked polystyrene resin (DVB-PS). Synthesis of an extremely insoluble, hydrophobic, antiparallel β-sheeted difficult sequence peptide LMVGGVVIA (β 34–42), C-terminal fragment of β-amyloid protein, β (1–42), was carried out on both 2% DVB-PS and 2% BDDMA-crosslinked polystyrene supports. The synthesis of the peptide was carried out using Boc amino acid strategy. Greater extent of swelling of the resino peptide, increased coupling efficiency during the assembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA-PS polymer.     

Synthesis and characterization of two potential impurities (des-ethyl-Ganirelix) generated in the Ganirelix manufacturing process

Controlling certain diseases using peptide drugs has remarkably increased in the past two decades. In this regard, a generic formulation is an upfront solution to fulfill market demands. Ganirelix, a leading peptide active pharmaceutical ingredient (API) primarily used as a gonadotropin-releasing hormone antagonist (GnRH), has established a potential market value worldwide. But its generic formulation mandates detailed impurity profiles from a synthetic source and contemplates the sameness of a reference-listed drug (RLD). Post-chemical synthesis and processing of Ganirelix, some commercial sources have revealed two new potential impurities among many known, which show the deletion of an ethyl group from the hArg(Et)₂ residue at the sixth and eighth positions, named des-ethyl-Ganirelix. These impurities are unprecedented in traditional peptide chemistry, and such monoethylated-hArg building blocks are not easily accessible commercially to synthesize these two impurities. Here, we have outlined the synthesis, purification, and enantiomeric purity characterization of the amino acids and their incorporation in the Ganirelix peptide sequence to synthesize these potential peptide impurities. This methodology will enable the convenient synthesis of side-chain substituted Arg and hArg derivatives in peptide drug discovery platforms.     

Protein evolution: The deciphering of latent facets — correlation of synthesis profiles of ribosomally directed proteins and enzyme directed peptides

Key facets pertaining to the evolution of proteins have been probed, using as springboard, the relevant data bases constructed from (i) 60 ribosomally directed proteins, whose 3D structures are known and having 10,000 residues and (ii) from 73 enzyme directed peptides, comprising of 524 residues. The preference profiles, both in terms of the choice of neighbours and the placement of the peptide bonds, have been delineated with respect to each of the 20 coded amino acids. By and large, the preference profile from both the sets are similar, thus giving importance to the nature of the side chains of the coded amino acids. The predictive power of the preference profile has been tested with good results, thus demonstrating the evidence of common preference pathways for peptide formation during evolution. The ribosomally directed protein synthesis, controlled by the genome, proceeds by the addition of single residues at a time. On the other hand, the enzyme directed peptide synthesis largely operates in a more energy conscious block mode, where each constituent of a large enzyme ensemble is engaged in precisely assembling the modules and transfering them to the adjacent one, thus realizing a sequence specific peptide synthesis. Of significance is the fact that, in spite of such divergence in assembly, predictions for neighbour preferences in ribosomally directed protein synthesis work well when applied to enzyme directed peptide synthesis. The findings here are significant since they provide (i) a clear picture of directed peptide synthesis in the absence of direct genomic control, (ii) evidence for the preferred formation of peptide bonds using protein templates, (iii) they also provide evidence for the presence of protein like structures, with catalytic activity, prior to the freezing of the genetic code arising from dominance of the information system and (iv) a logical approach to the evolution of a hierarchical pattern. Presented at the National Symposium on Evolution of Lite.     

Development of non-phosphorylated cyclic thioether peptide binding to the Grb2-SH2 domain

One of the critical intracellular signaling pathways involves specific interactions between growth factor receptors and the adaptor protein Grb2. These interactions normally involve specific tyrosine phosphorylated regions in receptors and other cognate proteins. Following the lead of our recent findings that a phage library based non-phosphorylated disulfide linked 11-mer peptide inhibited such interactions, we report here the synthesis of novel redox-stable cyclic peptide analogs. These include thioether cyclized and backbone cyclized structures. The thioether analog was prepared under mild conditions from an N-terminally chloroacetylated and C-terminally cysteine extended peptide precursor. The thioether peptide showed equipotent binding affinity for the Grb2-SH2 domain (IC50 = 10–15 M) when compared to the disulfide cyclized lead-peptide. The bioactive thioether linked peptide was demonstrated to offer advantages to the disulfide cyclized peptides under physiological conditions.     

Inclusion volume solid-phase synthesis

Solid-phase synthesis of peptides was carried out using only the volume of the solvent included in the swollen solid-phase resin beads [inclusion volume synthesis]. This approach enables (i) the use of higher concentrations of activated amino acids, resulting in increased coupling rates, (ii) drastically decreased consumption of solvents, and (iii) the construction of multiple peptide synthesizers having virtually no reaction vessels.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Aims: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease‐catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent‐tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. Methods and Results: The tripeptide Ac‐Phe‐Gly‐Phe‐NH₂ was synthesized using Ac‐Phe‐OEt and Gly‐Phe‐NH₂ substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1·5 or 0·5 mol l^?1 NaCl. Purification and identification of the peptide product was achieved by RP‐HPLC and ESI‐MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Conclusion: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. Significance and Impact of Study: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Novel amino-Li resin for water-based solid-phase peptide synthesis

We report the first application of a novel amino-Li resin to water-based solid-phase peptide synthesis (SPPS) applying the Smoc-protecting group approach. We demonstrated that it is a suitable support for the sustainable water-based alternative to a classical SPPS approach. The resin possesses good swelling properties in aqueous milieu, provides significant coupling sites, and may be applicable to the synthesis of difficult sequences and aggregation-prone peptides.     

Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1′-Carbonyldiimidazole

A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1′-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.     

Optimization of butanediol dimethacrylate crosslinked polystyrene: A novel polymeric support for solid phase peptide synthesis

Studies leading to optimization of butanedioldimethacrylate-crosslinked polystyrene supports (BDDMA–PS) forsolid phase peptide synthesis are delineated. BDDMA–PScopolymers with different crosslink densities were prepared andfunctionalised with chloromethyl groups. The reactivity of theLys(2-Cl-Z)-OH residue bound to these polymers through a benzylester linkage was investigated by following the kinetics ofacylation by the HOBt active ester of Boc-Alanine. From theresults it was observed that the rate of peptide bond formationwas maximum for a 2% BDDMA crosslinked resin. This resin wascompared with a 2% DVB-crosslinked polystyrene resin (DVB–PS). Synthesis of an extremely insoluble, hydrophobic,antiparallel -sheeted difficult sequencepeptide LMVGGVVIA ( 34–42), C-terminal fragment of -amyloid protein, (1–42), wascarried out on both 2% DVB–PS and 2% BDDMA-crosslinkedpolystyrene supports. The synthesis of the peptide was carriedout using Boc amino acid strategy. Greater extent of swellingof the resino peptide, increased coupling efficiency during theassembly of amino acids and relatively high purity of synthesised peptide were observed in the case of 2% BDDMA–PS polymer.     

非核糖体多肽合成酶研究进展

细菌和真菌采用非核糖体系统合成一些重要的多肽类物质.近年来的研究表明,在该系统中发挥关键作用的是一类分子巨大的非核糖体多肽合成酶.它们由顺序排列的组件构成,酶分子结构本身即蕴涵着多肽合成的信息.对非核糖体多肽合成酶结构和功能的了解,使人们期望可以通过对这类酶的修饰和重组来合成一些新的多肽类物质.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

Synthesis and Characterisation of a Multiphosphorylated Phosphophoryn Repeat Motif; H-[Asp-(Ser(<Emphasis Type="Italic">P</Emphasis>))<Subscript>2</Subscript>]<Subscript>3</Subscript>-Asp-OH

Protein phosphorylation is a critical mechanism in the regulation of cellular biochemical pathways and phosphopeptides can play an important role in determining function. However, the use of phosphopeptides especially multiphosphorylated peptides is hampered by their low abundance, difficulty in isolation from biological samples and in their chemical synthesis. Here we describe methodologies for the Fmoc synthesis, purification and mass spectral analysis of the multiphosphorylated sequence H-[Asp-(Ser(P))₂]₃-Asp-OH from phosphophoryn a protein involved in dentine mineralization. Critical steps in the synthesis of phosphophoryn using Fmoc-Ser(PO₃Bzl,H)-OH as the building block were double acylation steps for each residue, alternating HBTU and HATU as the acylating agents and synthesis on a chlorotrityl resin which was essential for complete removal of the benzyl-side chain protecting groups. The synthetic phosphophoryn was only effectively purified by anion exchange and size exclusion chromatography as both alkaline and acid buffers failed to aid in purification by reversed phase HPLC. MALDI-TOF analysis of phosphophoryn was achieved with good sensitivity (20 fmol/ml) and resolution using the DNA matrix 3-hydroxypicolinic acid, whereas typical protein/peptide matrices failed to provide mass spectra. The synthetic phosphophoryn peptide was found to bind calcium, binding 6 mol of calcium per mole of peptide. In conclusion the methodology described here can be easily adopted for the synthesis and analysis of a wide variety of multiphosphorylated peptides.     

Multiple peptide synthesis

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.     

Synthesis of multiple antigenic peptides (MAPs)—strategies and limitations

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid‐phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS‐made building blocks (indirect approach). Although MAPs and MAP‐like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all‐SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24‐residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12‐residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. (c) 1994 John Wiley & Sons, Inc.     

A Novel Oxazolidine Linker for the Synthesis of Peptide Aldehydes

A reliable method for solid-phase synthesis of peptide aldehydes by using a new oxazolidine linker is described. Based on a comparative study using the usual cleavage protocol as is used for the Fmoc-based peptide synthesis, we found that this new linker is more appropriate for the synthesis of peptide aldehydes compared with the precedent acetal, semicarbazone or threonine linker. Whereas N-Acylated oxazolidines might be partially deprotected to non-N-acylated intermediates in the TFA cocktail containing several soft nucleophiles which cause significant side reactions, the new oxazolidine linker could produce the desired peptide aldehydes by simple Et₂O washing and subsequent aqueous workup in high chemical yields and purity. We demonstrate the new method is useful especially for the preparation of highly functionalized long-chain peptide aldehydes which require several scavenger chemicals in the final deprotection step. This paper is dedicated to the memory of the late Prof. R. Bruce Merrifield, who passed away May 14, 2006.     

Synthesis of a Glycosylated Peptide Thioester by the Boc Strategy and Its Application to Segment Condensation

The synthesis of a chitobiosylated peptide thioester by the t-butoxycarbonyl (Boc) strategy is demonstrated. Boc-Asn carrying benzyl-protected chitobiose was introduced during application of the Boc mode solid-phase method. HF treatment of the resulting protected peptide resin gave the desired chitobiosylated peptide thioester. This thioester was used to prepare the peptide sequence derived from extracellular matrix metalloproteinase inducers (emmprin) (34-94), (34-118) and (22-118) by the thioester segment condensation method. The conformation of these glycopeptides is characterized by circular dichroism (CD) spectral measurement.