Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.     

Total chemical synthesis of dengue 2 virus capsid protein via native chemical ligation: Role of the conserved salt-bridge

The dengue capsid protein C is a highly basic alpha-helical protein of ～100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55′. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C (^WTDEN2C) and its salt bridge-void analog ^E87ADEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.     

Design,synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure–activity relationship in the N-terminal domain of these peptides, three analogues (1–3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.     

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.     

Solid phase synthesis of hydrophobic difficult sequence peptides on BDDMA-PS support.

This article illustrates the successful and efficient solid phase assembly of hydrophobic difficult sequence peptides following both t-Boc and Fmoc chemistry. The peptides were synthesized on an optimized 1,4-butanediol dimethacrylate-crosslinked polystyrene support (BDDMA-PS). Four difficult sequence test peptides, VAVAG, VIVIG, QVGQVELG and VQAAIDYING, were synthesized in relatively good yield and purity without any aggregation problems. The peptides were assembled on chloromethylated and 4-hydroxymethylphenoxymethyl (HMP) BDDMA-PS resins. The peptides were fabricated using Boc amino acid 1-hydroxybenzotriazolyl and Fmoc amino acid pentafluorophenyl active esters in coupling reactions. The peptides after synthesis were cleaved from the polymeric support by exposing the peptidyl resin to 90% trifluroacetic acid/5% thioanisole/5% EDT mixture. The HPLC and MALDI TOF MS studies of the peptides revealed the high homogeneity of the synthesized peptides. Chloromethylated resin having a functional group loading of 1.14 mmol Cl/g was used for the synthesis. The yield and homogeneity of these peptides synthesized using the new support were high when compared with the conventional DVB-PS resin.     

Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths.

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.     

Design and synthesis of new types of oligonucleopeptides

Summary Design and synthesis of oligonucleopeptides (ONPs), structural analogues of oligonucleotides, where the phosphodiester backbone is substituted by a peptide chain, are described. Oligonucleopeptides, in which the number of ordinary bonds between the nucleobases is six and the number of bonds between the backbone and nucleobase is two or four were constructed using two different approaches. The first way is based on incorporation of thyminylalanine residues into the peptide chain alternatively with glycine residues. Experimental studies of the stability of oligonucleotide-oligonucleopeptide complexes as well as model estimations of their potential surfaces indicated the low DNA binding efficiency of this type of reagents. The second approach consists of synthesis of ω-ornithine peptides followed by modification of the backbone with thyminylacetaldehyde attached to an α-amino function of ornithine residues through Schiff bases. ONPs were synthesized using the solid-phase method.     

Solid (gel) phase synthesis of fully-protected acylpeptide hydrazides with hydrazine-labile sidechains

A simple, efficient strategy for the preparation of fully-protected acylpeptide hydrazides with hydrazine-labile sidechains is described. The method involves controlled hydrazinolytic cleavage of target peptides from assemblies elaborated by solid (gel) phase synthesis on phenolic peptide resins.     

Design and synthesis of new types of oligonucleopeptides

Design and synthesis of oligonucleopeptides (ONPs),structural analogues of oligonucleotides, where the phosphodiester backbone is substituted by a peptidechain, are described. Oligonucleopeptides, in whichthe number of ordinary bonds between the nucleobasesis six and the number of bonds between the backboneand nucleobase is two or four were constructed usingtwo different approaches. The first way is based onincorporation of thyminylalanine residues into the peptidechain alternatively with glycine residues.Experimental studies of the stability ofoligonucleotide–oligonucleopeptide complexes as wellas model estimations of their potential surfacesindicated the low DNA binding efficiency of this typeof reagents. The second approach consists of synthesis of-ornithine peptides followed by modification of thebackbone with thyminylacetaldehyde attached to an-amino function of ornithine residues through Schiffbases. ONPs were synthesized using the solid-phase method.     

DNA-instructed acyl transfer reactions for the synthesis of bioactive peptides

We present a method which allows for the translation of nucleic acid information into the output of molecules that interfere with disease-related protein-protein interactions. The method draws upon a nucleic acid-templated reaction, in which adjacent binding of reactive conjugates triggers the transfer of an aminoacyl or peptidyl group from a donating thioester-linked PNA-peptide hybrid to a peptide-PNA acceptor. We evaluated the influence of conjugate structures on reactivity and sequence specificity. The DNA-triggered peptide synthesis proceeded sequence specifically and showed catalytic turnover in template. The affinity of the formed peptide conjugates for the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) is discussed.     

Exendin-4的固相化学合成及鉴定

Exendin-4是首个获准上市的肠促胰素类似物药物,可模拟人体自身激素GLP-1的功能,不仅改善血糖,还能促进胰岛β细胞新生、增殖,抑制β细胞凋亡,改善β细胞功能,促进胰岛素分泌,增加机体对胰岛素的敏感性。根据Exendin-4的潜在市场价值,以Fmoc固相合成策略为基础,NMP为反应溶剂,以HOBt/DIC为缩合剂,添加盖帽程序,优化合成工艺条件。肽树脂裂解采用TFA/Phenol/TIS裂解液,并应用高效液相色谱和四级杆-飞行时间串联质谱对其进行分析鉴定和纯化,最终获得Exendin-4,产率为21%,纯度为99.4%。活性测定结果显示,合成的Exendin-4显著提高胰岛瘤细胞的活性,并呈一定剂量依赖性。     

Efficient and systematic synthesis of a small glycoconjugate library having human complex type oligosaccharides

To prepare a small library of homogeneous glycoconjugates with varying oligosaccharide structures, a combinatorial strategy was employed. The target glycopeptide was divided into two peptide segments (A and B) and both were prepared by solid phase peptide synthesis. These peptides, which can be coupled by native chemical ligation through an amide bond, were subsequently coupled to two kinds of human complex type oligosaccharides. This process systematically afforded the desired glycoconjugate library.     

Traceless and Site-specific Attachment of Proteins onto Solid Supports

Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.     

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.     

Facile production of mono-substituted urea side chains in solid phase peptide synthesis

A method is reported for the straightforward generation of urea-containing peptides during Boc solid phase peptide synthesis. Primary amine side chains are converted to mono-alkyl ureas in two steps via an intermediate p-nitrophenyl carbamate. Use of p-methoxybenzyl amine as an ammonia equivalent affords mono-alkyl final products from standard resin cleavage methods, without the need for additional steps. The reaction is highly efficient and applicable to variable length side chains and peptides.     

Convenient solid phase synthesis method for the preparation of cysteine C-terminally derivatized peptides

Summary This paper describes a novel solid phase peptide synthesis method for the systematic C-terminal modification of cysteine-containing peptides. In this method, cysteine is linked to chloromethylated polystyrene resin by its thiol functionality, followed by protection of the N-terminus and derivatization of the carboxylic acid to esters or amides. We report here on examples of the methodology and its application to the synthesis of Ac-Asp-cyclo(Cys-Gly-Pro-Cys)-NHBzl, a cyclic peptide amide. The method has been applied to the synthesis of complex esters as well as amides.Abbreviations Ac acetyl - AcN acetonitrile - Ac₂O acetic anhydride - AcOH acetic acid - Boc t-butyloxycarbonyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - cHex cyclohexyl - DBU 1,8-diazabicyclo[5.4.0]-undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA diisopropylethylamine - DMF dimethylformamide - DMS dimethylsulfide - HOB 1-hydroxybenzotriazole - MBzl 4-methyl benzyl - MeOH methanol - TEA triethylamine - TEAP triethylammonium phosphate - TFA trifluoroacetic acid     

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.     

Total chemical synthesis of human matrix Gla protein

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five gamma-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 microg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1-84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in alpha-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification.     

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.     

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.