Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

Regioselective enzymatic acylation of ribavirin to give potential multifunctional derivatives

Lipase-catalyzed synthesis of potential multifunctional ribavirin derivatives was performed in acetone. Divinyl dicarboxylates with different chain lengths (C₄, C₆, C₉, C₁₀) were used as acyl donors and the reactions were catalyzed by lipase immobilized on acrylic resin from Candida antarctica (CAL-B). Ribavirin was regioselectivly acylated at the primary hydroxyl groups and the corresponding vinyl esters (C₄, C₆, C₉, C₁₀) were prepared in respective yields of 48%, 65%, 54%, 55%.     

Progress in the preparation of peptide aldehydes via polymer supported IBX oxidation and scavenging by threonyl resin.

Peptide aldehydes are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes such as caspases or the proteasome. A novel access to peptide aldehydes is described using a combination of solid phase peptide synthesis with polymer-assisted solution phase synthesis based on the oxidation of peptide alcohols with a mild and selective polymer-bound IBX derivative. The oxidation is followed by selective purification via scavenging the peptide aldehyde in a capture-release procedure using threonine attached to an aminomethyl resin. Peptide aldehydes are obtained in excellent purity and satisfying yield. The optical integrity of the C-terminal residue is conserved in a high degree. The procedures are compatible with the use of common side-chain protecting groups. The potential for using the method in parallel approaches is very advantageous. A small collection of new and known peptide aldehydes has been tested for inhibitory activity against caspases 1 and 3.     

Synthesis of vasoactive intestinal peptide (VIP) via the mixed anhydride method

Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.     

How to predict product yield in kinetically controlled enzymatic peptide synthesis

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters alpha and beta for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH(2) from Mal-L-Phe-L-AlaOMe and L-LeuNH(2). The experimental results demonstrate that this theory adequately describes the real process. (c) 1992 John Wiley & Sons, Inc.     

Solid phase synthesis of peptidyl aldehydes from C-terminal thiazolidinyl peptides

Peptidyl aldehydes are potent transition stateanalogue inhibitors of cysteine and serineproteinases. The aldehyde function has recently beenused for chemoselective peptide ligation. Thepreparation of peptidyl aldehydes on a solid supportrequires that the aldehyde be masked during peptideelongation and generated in a final step under mildconditions. We report here the preparation of peptidylaldehydes by copper salt-mediated neutral hydrolysisof the corresponding C-terminal thiazolidinyl peptideswhich were elongated on a solid support.     

Solid phase synthesis of peptidyl aldehydes from C-terminal thiazolidinyl peptides

Summary Peptidyl aldehydes are potent transition state analogue inhibitors of cysteine and serine proteinases. The aldehyde function has recently been used for chemoselective peptide ligation. The preparation of peptidyl aldehydes on a solid support requires that the aldehyde be masked during peptide elongation and generated in a final step under mild conditions. We report here the preparation of peptidyl aldehydes by copper salt-mediated neutral hydrolysis of the corresponding C-terminal thiazolidinyl peptides which were elongated on a solid support.     

Thia-analogues of amino acids synthesis of peptide derivatives containing 3-thia-analogues of amino acids

Summary N-Protected dipeptides containing L-3-thia-analogues of phenylalanine, p-nitro-phenylalanine, lysine and leucine respectively were prepared applying an enantioselective enzymatic reaction step. Racemic synthetic intermediates of the type acyl-NH-CH(R¹)-CO-D,L-NH-CH(S-R²)-COOBzl were selectively deprotected at the C-terminus by enzymatic hydrolysis using thermitase or trypsin.Abbreviations Ac acetyl - AcOEt ethyl acetate - AcOH acetic acid - Boc tert.-butyloxycarbonyl - Bz benzoyl - Bzl benzyl - DMF dimethyl-formamide - EtOH ethanol - THF tetrahydrofuran - Z benzyloxycarbonyl Dedicated to Prof. D. Cavallini at the occasion of his 75th birthday.     

Regioselective enzymatic acylation of multi-hydroxyl compounds in organic synthesis

With current developments in enzyme-catalyzed reactions and techniques available for rational redesign of natural biocatalysts, the enzymatic biosynthesis can become one of the most valuable synthetic methods. Enzymatic regioselective catalysis in organic media has played a key role in pursuing asymmetric synthesis for active chiral compounds. Here, we shortly describe some historical issues of the rapidly growing area, enzymatic catalysis in synthetic organic chemistry and then review researches that have been carried out in the regioselective enzymatic catalysis for the past two decades. An application of this technology to the modification of some potential target drug compound will be also presented.     

Backbone amide linker (BAL) strategy for Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of peptide aldehydes.

A rapid and efficient strategy has been developed for the general synthesis of complex peptide aldehydes. N(alpha)-Benzyloxycarbonylamino acids were converted to protected aldehyde building blocks for solid-phase synthesis in four steps and moderate overall yields. The aldehydes were protected as 1,3-dioxolanes except for one case where a dimethyl acetal was used. These protected amino aldehyde monomers were then incorporated onto 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyryl-resin (BAL-PEG-PS) by reductive amination, following which the penultimate residue was introduced by HATU-mediated acylation. The resultant resin-bound dipeptide unit, anchored by a backbone amide linkage (BAL), was extended further by routine Fmoc chemistry procedures. Several model peptide aldehydes were prepared in good yields and purities. Some epimerization of the C-terminal residue occurred (10% to 25%), due to the intrinsic stereolability conferred by the aldehyde functional group, rather than any drawbacks to the synthesis procedure.     

Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis

Abstract Peptide synthetases are large multienzyme complexes that catalyze the non-ribosomal synthesis of a structurally diverse family of bioactive peptides. They possess a multidomain structure and employ the thiotemplate mechanism to activate, modify and link together by amide or ester bonds the constituent amino acids of the peptide product. The domains, which represent the functional building units of peptide synthetases, appear to act as independent enzymes whose specific linkage order forms the protein-template that defines the sequence of the incorporated amino acids. Two types of domains have been characterized in peptide synthetases of bacterial and fungal origin: type I comprises about 600 amino acids and contains at least two modules involved in substrate recognition, adenylation and thioester formation, whereas type II domains carry in addition an insertion of about 430 amino acids that may function as a N-methyltransferase module. The role of other genes associated with bacterial opérons encoding peptide synthetases is also discussed.     

Two-step sequential synthesis of pyrimidine derivatives containing a sugar branch via combining of enzymatic Michael addition/acylation

A new strategy for the enzymatic synthesis of pyrimidine derivatives containing a sugar branch was developed via combining of Michael addition and acylation. The first-step reaction of pyrimidines and vinyl 3-propionyloxy propionate was catalyzed by Amano lipase M from Mucor javanicus in DMSO. The initial reaction rates of different pyrimidines decreased in the order of fluorouracil, uracil, thymine, in agreement with their nucleophilicity. The succeeding regioselective acylation of d-glucose and d-mannose with the Michael adducts was catalyzed by alkaline protease from Bacillus subtilis in pyridine. The d-glucose and d-mannose were all acylated at C-6 position. Moderate yield was obtained for each step.     

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.     

Influence of peptide conformation on oligosaccharide binding characteristics--a study using apamin-based chimeric peptide

Interactions between proteins and heparin play a crucial role in most of the cellular process. Unraveling the forces that govern the formation of these complexes is vital for understanding the specificities involved in these biomolecular events. In the present study, a detailed analysis has been undertaken to evaluate the effect(s) of peptide conformation on heparin-binding, using a chimeric peptide, apaK6—a chimera of a highly stable neurotoxic peptide from honey-bee venom and a de novo designed lysine-rich peptide. The dissociation constants of these peptide–heparin complexes were found to be in the submicromolar range. Comparison of the results obtained from the titration of the disulfide-reduced and disulfide-intact chimeric peptide with various sulfated oligosaccharides, derived from heparin, suggest that the initial structure of the peptide has pronounced effect on the binding affinity, binding modes and also on binding preferences. The results of this study indicate that the heparin-binding specificity of an isolated peptide and that exhibited by the same peptide when present in a globular protein could be significantly different, especially if the isolated peptide undergoes conformational change(s) upon binding to the sulfated oligosaccharides. In addition, such dependency of the binding specificity on the preformed structures could be utilized for the design of high-affinity and sequence-specific heparin-binding polypeptides.     

融合自组装双亲短肽对重组过氧化氢酶酶学性质的影响

【目的】利用融合自组装双亲短肽策略对源自枯草芽孢杆菌(Bacillus subtilis)的过氧化氢酶Kat A进行改性,以强化重组过氧化氢酶在工业中的应用适应性。【方法】将自组装双亲短肽S1vw通过连接肽PT-linker融合在Kat A的N端,构建重组质粒p HT254-S1vw-PT-kat A,将其与携带天然酶基因的p HT254-kat A分别转入枯草芽孢杆菌WB800N中进行分泌表达,之后将分离纯化得到的纯酶进行酶学性质研究。【结果】成功构建出工程菌并将胞外粗酶液通过乙醇沉淀、DEAE阴离子交换层析、疏水层析和凝胶过滤层析4步纯化,最终获得电泳纯的重组酶蛋白。酶学性质研究结果显示,融合酶S1vw-PT-Kat A和天然酶Kat A的最适反应温度均为30°C,最适反应p H值均为11.0。然而,融合酶在p H 12.0下孵育30 min的相对酶活为77.3%,是相同处理条件下天然酶相对酶活的14.9倍,在65°C和70°C下孵育30 min的相对酶活分别为19.8%和17.5%,是相同处理条件下天然酶相对酶活的1.8倍和1.7倍。此外,融合酶在4°C储存14 d后相对酶活为8...     

Pulsed column reactor as a novel tool for continuous enzymatic synthesis of peptide in an aqueous/organic biphasic medium

We propose a novel effective method for a continuous peptide synthesis in an aqueous/organic biphasic medium using a pulsed column reactor. N-Formyl-l-aspartyl-l-phenylalanine methyl ester was enzymatically synthesized continuously. With this extractive method using a pulsed column reactor, we can synthesize peptides with a stable performance even if a peptide (or a peptide-amino acid complex) is precipitated due to its high hydrophobicity.     

Solid‐phase synthesis and screening of a library of C‐terminal arginine peptide aldehydes against Murray Valley encephalitis virus protease

Murray Valley encephalitis virus is a member of the flavivirus group, a large family of single‐stranded RNA viruses, which cause serious disease in all regions of the world. Unfortunately, no suitable antivirals are available, and there are commercial vaccines for only three flaviviruses. The solid‐phase synthesis of a library of 400 C‐terminal arginine peptide aldehydes and their screening against Murray Valley encephalitis virus protease are demonstrated. The library was utilised to elucidate several tripeptide sequences that can be used as inhibitors in further SAR studies. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

A natural peptide and its variants derived from the processing of infectious pancreatic necrosis virus (IPNV) displaying enhanced antimicrobial activity: a novel alternative for the control of bacterial diseases

The larger segment of the infectious pancreatic necrosis virus (IPNV) codifies most of the structural and non-structural proteins of the virus in two overlapping open reading frames (ORFs). The longer of the two ORF is expressed as a polyprotein which generates a number of variable length peptides of unknown function during processing. Since an appealing hypothesis would be that these peptides are generated by the virus to act as antimicrobial agents that favor viral infectivity in their fish host, we decided to test this possibility by selecting a master peptide and using it to generate substitution variants that may enhance their antimicrobial potential. A 20-residue master peptide (p20) was selected from the well-described maturation process of the structural viral protein VP2; several variants were then designed and chemically synthesized, ranging in size from 16 to 20 residues. The synthesized peptides were tested for in vitro activity against several prototype bacterial pathogens using standardized laboratory procedures. Chemically synthesized p20 and all its variants displayed broad activity against the tested bacteria and none of them were toxic to eukaryotic cells at least 10× the concentration used against the bacteria. Interestingly, when p20 was tested against the very aggressive bacterial pathogen Piscirickettsia salmonis, a common co-infectant of IPNV in salmonid fish, the specific activity of the novel peptide was significantly higher than that displayed for bactericidal fish farm antibiotics such as oxolinic acid, flumequine and florfenicol, which are commonly used to control Piscirickettsiosis in the field. It is potentially significant that the approach presented in this report provides a novel alternative for generating new and ideally more efficient and friendly safeguards for bacterial prophylaxis.     

The carboxy-terminal end of the peptide deformylase from Mycobacterium tuberculosis is indispensable for its enzymatic activity

The peptide deformylase in bacteria is involved in removal of N-formyl group from newly synthesized proteins. The gene encoding this enzyme from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The enzyme activity of the recombinant protein (mPDF) was insensitive to modulation by common monovalent/divalent cations. Kinetic analysis, using N-formylmethionine-alanine as the substrate, yielded K(cat)/K(m) of approximately 1220 M(-1)s(-1). Actinonin, a naturally occurring antibiotic, and 1,10-ortho-phenanthroline strongly inhibited the enzyme activity. The mPDF was very stable at 30 degrees C with a half-life of approximately 4h and exhibited resistance to oxidizing agents, like H(2)O(2). Thus, the mPDF achieved distinction in its behavior among any reported iron-containing peptide deformylases. Furthermore, amino acid sequence analysis of mPDF revealed the presence of an unusually long carboxy-terminal end (residues 182-197), which is atypical for any gram-positive bacteria. Our results, through deletion analysis, for the first time established the role of this region in mPDF enzyme activity.