Solid phase synthesis of peptide aldehyde protease inhibitors. Probing the proteolytic sites of hepatitis C virus polyprotein.

The solid phase synthesis of a set of peptide aldehydes derived from the NS5A/NS5B junction of hepatitis C virus (HCV) viral polyprotein is demonstrated using an oxazolidine linker and the Multipin method. Deletion of the P6 and P5 residues results in a dramatic loss of inhibitory activity.     

A simple oxazolidine linker for solid-phase synthesis of peptide aldehydes

A very simple and cheap linker has been used for solid-phase synthesis of peptide aldehydes. Protected amino acid aldehydes are immobilized on 2-Cl(trt) resin as oxazolidine formation via diethanolamine. After classical Fmoc SPPS, treatment of the resin with AcOH/DCM/H(2)O (8:1:1) affords peptide aldehydes in high yield and purity.     

New methodology for automated SPOT synthesis of peptides on cellulose using 1,3,5‐triazine derivatives as linkers and as coupling reagents

Two new rigid bi‐aromatic linkers for synthesis of peptide arrays by SPOT methodology were obtained from cellulose treated with 2,4‐dichloro‐6‐methoxy‐1,3,5‐triazine. Reaction with m‐phenylenediamine gave non‐cleavable TYPE I linker which enabled attachment of the peptides via resistant to harsh reaction conditions amide, ether, and amine bonds. Reaction with 3‐Fmoc‐aminobenzoic acid followed by thermal isomerization of the intermediate “superactive” ester producing an amide‐like bond gave TYPE II linker that was very stable during peptide synthesis. However, the peptide was cleavable, with fragment of the linker, in the presence of 1 M LiOH solution. The uniform loading of the cellulose and efficient synthesis of the peptide array was achieved by using N‐(4,6‐dimethoxy‐1,3,5‐triazin‐1‐yl)‐N‐methylmorpholinium 4‐toluenesulfonate as the coupling reagent.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of lucifensin by native chemical ligation and characteristics of its isomer having different disulfide bridge pattern

The antimicrobial 40‐amino‐acid‐peptide lucifensin was synthesized by native chemical ligation (NCL) using N‐acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid‐phase synthesis is imcompatible. Two models of ligation were developed: [15 + 25] Ala‐Cys and [19 + 21] His‐Cys. The [19 + 21] His‐Cys method gives lower yield because of the lower stability of 18‐peptide‐His‐Nbz‐CONH₂ peptide, as suggested by density functional theory calculation. Acetamidomethyl‐deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1?6], [C3?5], and [C2?4], which limits its ability to form α‐helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one‐pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A convenient method for the synthesis of norstatine derivatives from N-Fmoc or N-Boc amino acids

Summary A new and convenient method for synthesizing norstatine derivatives has been developed. The key step is the preparation of propargylic alcohols from N-Fmoc or N-Boc amino aldehydes, followed by transformation to carboxylic acids after protection of the alcohols. The N-Boc-protected derivatives are coupled to a dipeptide sequence to afford peptide mimetics (norstatines).     

An enzymatic method for the determination of hemoglobinA<Subscript>1C</Subscript>

Fructosyl peptide oxidase is a flavoenzyme that catalyzes the oxidative deglycation of N-(1-deoxyfructosyl)-Val-His, a model compound of hemoglobin (Hb)A_1C. To develop an enzymatic method for the measurement of HbA_1C, we screened for a proper protease using N-(1-deoxyfructosyl)-hexapeptide as a substrate. Several proteases, including Neutral protease from Bacillus polymyxa, were found to release N-(1-deoxyfructosyl)-Val-His efficiently, however no protease was found to release N-(1-deoxyfructosyl)-Val. Neutral protease also digested HbA_1C to release N-(1-deoxyfructosyl)-Val-His, and then the fructosyl peptide was detected using fructosyl peptide oxidase. The linear relationship was observed between the concentration of HbA_1C and the absorbancy of fructosyl peptide oxidase reaction, hence this new method is a practical means for measuring HbA_1C.     

Synthesis of sequential oligopeptides and polypeptide having the sequence of L-alanyl-L-leucylglycine

A series of sequential oligopeptides having simple nonpolar side chains, Nps-(L -Ala-L -Leu-Gly)_n- OEt has been prepared by a stepwise fragment-condensation method using Nps-L -alanyl-L -leucylglycine N-hydroxysuccinimide ester, which was prepared by the Nps-N-carboxy α-amino-acid-anhydride method. The success of the synthesis of the peptide having a high-molecular weight, such as octadecapeptide, results from the highest solubility of the tripeptide unit, L -alanyl-L -leucylglycine. The sequential polypeptide having the same tripeptide sequence was also prepared by polycondensation of the tripeptide N-hydroxy-succinimide ester.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

3-Thiopropionic acid as a highly versatile multidetachable thioester resin linker

Summary This paper describes a practical new use of 3-mercaptopropionic acid as a highly versatile multidetachable linker for solid-phase synthesis. Our approach is based on the stability of the alkylthioester functionality to optimized Boc-SPPS protocols and HF treatment, as well as on the mild activation of the thioester functionality toward nucleophilic or reductive displacement. This allows several C-terminal modifications to be introduced into a synthetic molecule during the cleavage step. We have shown that unprotected peptides can be efficiently cleaved from a propyl thioester-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer resin using a great variety of nucleophiles to give the corresponding C-terminally modified peptides (esters, thioesters, carboxylic acids, thioacids, amides, hydroxamic acids, hydrazides, alcohols). The nucleophilic cleavage reaction is both rapid and exceptionally clean in all the cases tested. Abbreviations: HBTU,N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphateN-oxide); DIEA,N,N-diisopropylethylamine; DMF,N,N-dimethyl formamide; ES-MS, electrospray mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; HMBA, hydroxymethylbenzoic acid; HPLC, high performance liquid chromatography; PBS: phosphate buffer saline; PEGA, polyethylene glycolpoly-(N,N-dimethylacrylamide); TFA, trifluoroacetic acid; SPPS, solid-phase peptide synthesis. Standard IUPAC single and triple letter codes for amino acids are used throughout     

Solid Phase Synthesis of Phosphopeptides Incorporating 2,2-Dimethyloxazolidine Pseudoproline Analogs: Evidence for <Emphasis Type="Italic">trans</Emphasis> Leu-Pro Peptide Bonds in Stat3 Inhibitors

To answer the question of whether the conformation of the Leu-Pro bond is cis or trans in Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH₂ when complexed with the SH2 domain of Stat3, we substituted 2,2-dimethyloxazolidines derived from serine (Ser(Ψ^Me,Mepro)) and threonine (Thr(Ψ^Me,Mepro)) for proline. The 2,2-dimethyloxazolidine and 2,2-dimethylthiazolidine pseudoproline (ΨPro) analogs induce predominantly cis Xxx-ΨPro peptide bonds. As these ΨPro analogs are acid-labile, the phosphopeptides were synthesized using Fmoc-based SPPS using unprotected phosphotyrosine and 4-hydroxybenzoate as the linker that allowed release from the support by alkaline ammonolysis, conditions that kept the oxazolidine rings intact. Incorporation of Ser(Ψ^Me,Mepro) resulted in 69% cis Leu-ΨPro bond content in aqueous solution whereas that for Thr(Ψ^Me,Mepro) analog was 63%. Affinities for Stat3 were 3–5 fold lower than the lead compound and were inversely correlated with cis content. Thus we conclude that the Leu-Pro peptide bond is trans when the peptide is bound to Stat3.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Rational design of highly stabilized and selective adrenomedullin analogs

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM₁R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM₁R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM₁R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM₁R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.     

Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity

The peptide linker between variable domains of heavy (V_H) and light (V_L) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of V_H-(GGGGS)_n-V_L (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.     

Preparation of an unprotected phosphotyrosine building block and its application in solid-phase synthesis of phosphopeptides

Summary A new and facile synthesis of tyrosine phosphorylated peptides has been developed.N ^α-Fmoc-Tyr(tBu)-OPfp was treated with TFA, phosphorylated with phosphorous oxychloride and the resulting phosphoric acid dichloride was hydrolysed to giveN ^α-Fmoc-Tyr(PO₃H₂)-OPfp1 in an overall yield of 98%. Compound1 was used in solid-phase peptide synthesis of phosphopeptides2, 3 and4, which are fragments of murine adipocyte lipid binding protein. The advantage of using the Pfp ester was the absence of pyrophosphates and other byproducts.     

DNA or RNA Oligonucleotide 2′-Hydrazides for Chemoselective Click-Type Ligation with Carbonyl Compounds

An efficient method for the synthesis of DNA or RNA oligonucleotide 2′-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2′-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH₃CN. The resulting oligonucleotide 2′-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH₃CN reduction to afford novel 2′-conjugates.     

β-Alanine containing cyclic peptides with turned structure: The “pseudo type II β-turn.” VI

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L -Pro-L -Phe-β-Ala)₂. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L -Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d₆ solutions as a mixture of conformational families. One of these corresponds to a C₂ symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ?,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.     

Thiocarbamate‐linked peptides by chemoselective peptide ligation

Peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide‐based macromolecules. We show here that the phenylthiocarbonyl group can be easily introduced into peptides on α or ε amino groups using phenylthiochloroformate and standard solid‐phase method. It reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. S,N‐shift of the alkylaminocarbonyl group from the Cys side chain to the α‐amino group did not occur. The method was used for linking two peptide chains through their N‐termini, for the synthesis of a cyclic peptide or for the synthesis of di‐ or tetravalent multiple antigenic peptides (MAPs). Thiocarbamate ligation is thus complementary to thioether, thioester or disulfide ligation methods. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

The gdh and gdhr genes, encoding B₁₂-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly₄Ser)₄ linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.