9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide α-thioesters

Peptide thioesters play a key role in convergent protein synthesis strategies such as native chemical ligation, traceless Staudinger ligation, and Ag(+) -mediated thioester ligation. The Boc-based solid-phase synthesis provides a very reliable access to peptide thioesters. However, the acid lability of many peptide modifications and the requirements of most parallel peptide synthesizers call for the milder Fmoc-based solid-phase synthesis. The Fmoc-based synthesis of peptide thioesters is more cumbersome and typically proceeds with lower yields than the synthesis of peptide acids and peptide amides. The success of native chemical ligation and related technologies has sparked intensive research effort devoted to the development of new methods. The recent progress in this rapidly expanding field is reviewed.     

Facile peptide thioester synthesis via solution-phase tosylamide preparation

Preparation of peptide thioester is essential for native chemical ligation and block condensation. Our novel methodology involves conversion of the carboxylic acid of a peptide into a thioester using p-toluenesulfonyl isocyanate, followed by alkylation, then thiol substitution. Our methodology can also be used for the preparation of glycopeptide thioesters. Furthermore, it is possible to carry out the reaction as a sequential peptide chemical ligation.     

多肽片段连接方法及肽硫酯和肽醛的合成*

本文综述了多肽和蛋白质合成中的片段连接方法,这是近年来多肽和蛋白质合成领域中方法学上的重要进展.该方法使用非保护的多肽片段,无需酶或化学活化试剂,在缓冲溶液中能够高产率地获得多肽和蛋白质.还介绍了与多肽片段连接有关的肽硫酯和肽醛的合成方法.     

Peptide o‐Aminoanilides as Crypto‐Thioesters for Protein Chemical Synthesis

Fully unprotected peptide o‐aminoanilides can be efficiently activated by NaNO₂ in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o‐aminoanilides as a new type of crypto‐thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o‐aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.     

Synthesis,Characterization and Evaluation of Peptide Nanostructures for Biomedical Applications

There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.     

A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin

The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC₅₀) for the extent of modification and ratios of IC₅₀ values (IC₅₀arginine/IC₅₀lysine and IC₅₀lysine/IC₅₀arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification.     

Investigation of Parameters that Affect Resin Swelling in Green Solvents

The influence of various physical and chemical factors on the swelling of polystyrene and PEG based resins in greener organic solvents has been systematically investigated. In general, chemical factors: the nature of the functionality/linker and the degree of loading were found to have a far larger influence on the swelling of the resins than physical parameters such as bead size. The results are interpreted in terms of Hansen solubility parameters for the solvents and there is evidence that some solvents interact with the polymeric core of a resin whilst others interact with the functionality. The results are extended to a study of the changes in resin swelling observed during both deprotection and chain elongation reactions during solid phase peptide synthesis.     

蛋白质的化学全合成

含有非天然氨基酸的蛋白质（如翻译后修饰蛋白质、修饰有探针分子的蛋白质等）是化学生物学中重要的生理活性分子。这些分子难以通过生物表达来获取，而必须使用化学方法来合成。半胱氨酸肽片段连接方法是目前应用于蛋白质化学全合成中的一种重要方法，该方法能够在温和的水溶液中高效地实现肽片段的连接，从而生成天然或者非天然的蛋白质。本文系统地综述了半胱氨酸肽片段连接方法的基本原理，详细讨论了近年来人们对该方法的一些重要改进。最后又介绍了该方法在几类重要的蛋白质分子合成中的代表性应用。     

树状多肽的合成及应用

树状多肽作为树枝状大分子的一种,具有不同于链状多肽的独特理化特性,在生物学领域中具有广泛的应用.本文简要综述了树状多肽的合成方法,及其在诱导免疫反应、生物分子模拟、免疫诊断试剂、人工酶、药物及基因载体等方面的应用.     

超高效液相色谱-质谱联用法监测人心房钠尿肽的化学合成

多肽作为神经递质、激素、受体等参与和调节许多重要的生命过程。随着对多肽结构与功能认识的深入,化学合成多肽在生物医药、生命分析等领域显示了重要价值。人心房钠尿肽（ANP）是一种由28个氨基酸组成的多肽类激素,其含量和代谢异常与心血管疾病、癌症等密切相关。针对ANP重要的生理功能,该文开展了ANP化学合成及其过程的监测与优化,设计了集固相合成线性直链多肽和液相氧化成环为一体的策略。基于高效液相色谱的分离性能和多级质谱的结构鉴定能力,建立了超高效液相色谱-串联质谱（UHPLC-MS/MS）分析新方法,对固相合成产物进行了分离分析,并对液相氧化方法进行了筛选和优化,最终分离纯化得到了目标多肽,为人心房钠尿肽的高效化学合成及氧化成环反应提供借鉴。     

Emerging Methods and Design Principles for Cell‐Penetrant Peptides

Biomolecules such as antibodies, proteins, and peptides are important tools for chemical biology and leads for drug development. They have been used to inhibit a variety of extracellular proteins, but accessing intracellular proteins has been much more challenging. In this review, we discuss diverse chemical approaches that have yielded cell‐penetrant peptides and identify three distinct strategies: masking backbone amides, guanidinium group patterning, and amphipathic patterning. We summarize a growing number of large data sets, which are starting to reveal more specific design guidelines for each strategy. We also discuss advantages and disadvantages of current methods for quantifying cell penetration. Finally, we provide an overview of best‐odds approaches for applying these new methods and design principles to optimize cytosolic penetration for a given bioactive peptide.     

On-resin peptide ligation via C-terminus benzyl ester

Here, we reported a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and C-terminal peptide ligation on a Rink Amide resin.     

半胱氨酸肽片段连接法合成ω-芋螺毒素MVIIA

ω-芋螺毒素MVIIA是已上市的镇痛药Ziconotide的有效成分.采用标准Fmoc保护策略在聚苯乙烯树脂上合成ω-MVIIA比较困难,是固相合成中的"困难肽".本研究将ω-MVIIA分为N-端15肽硫酯和C-端10肽两个片段采用标准Fmoc保护策略分别合成,再通过半胱氨酸肽片段连接得到全长的ω-芋螺毒素MVIIA肽链.该方法提高了合成ω-芋螺毒素MVIIA产率.该研究为"困难肽"的合成提供了较好的参考方法.     

Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O−N and N−S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.     

Total chemical synthesis by native chemical ligation of the all-D immunoglobulin-like domain 2 of Axl

Axl is a tyrosine kinases receptor playing crucial role in several cellular responses. The deregulation of Axl signaling has been associated to many high impact diseases ranging from cancer to multiple sclerosis. We report the successful procedure for the chemical synthesis of the Ig2 domain of Axl, one of the key extracellular regions of the receptor involved in ligand binding. The protein was synthesized in its d-enantiomeric form (D-Axl-2), opening the way to the selection of D-peptides selectively targeting Axl receptor through the mirror-image phage display peptide library screening approach.     

Congeneric lantibiotics from ribosomal in vivo peptide synthesis with noncanonical amino acids

Expanded repetoire: Synthetic amino acids translated into propeptides dramatically increase the chemical diversity of the two-component lantibiotic lichenicidin. This opens new routes towards novel and unique peptide antibiotic sequences, which could display features important for medical applications.     

Recent advances in enzyme-mediated peptide ligation

Artificial synthesis and site-specific modification of peptides and proteins have evolved into an indispensable tool for protein engineers and chemical biologists. Chemical and enzymatic approaches to peptide ligation are important alternatives of recombinant DNA technology for protein synthesis and modification. In the past decades, several natural peptide ligases have been discovered. Additionally, protein engineering for improving the ligation efficiencies of the natural peptide ligase and reversing the functionality of protease have provided more powerful peptide ligases. Herein, we briefly summarized the advances of enzyme-mediated peptide ligation and their application in protein synthesis and modification.     

合成多肽疫苗研究进展

综述了合成多肽疫苗的研究进展,系统地介绍了目前寻找抗原肽的各种方法以及各类可用作疫苗接种的合成多肽抗原的制备和应用.     

Synthesis of Peptide Cysteine Dimedone Using Fmoc-Cys(Dmd)-OH: Glutathione Cysteine Dimedone as a Probe in Investigating the Sulfenic Acid Mediated Oxidation of Glutathione

Dimedone is the most widely used chemical probe for detection of cysteine sulfenic acid in peptides and proteins. The reaction of dimedone with cysteine sulfenic acid results in the formation of unique cysteine dimedone motif containing thioether bridge. Based on the structure of cysteine dimedone residue in polypeptide, a new building block of Fmoc-Cys(Dmd)-OH was developed for solid phase synthesis of peptide cysteine dimedone. Mass spectrometric sequencing of synthetic peptides have confirmed successful incorporation of cysteine dimedone in peptide chain using HBTU/HOBt as a coupling agent. The new method permits synthesis of peptides containing both cysteine thiol and cysteine dimedone in the same sequence which was difficult to achieve by conventional methods. The synthetic peptide of glutathione cysteine dimedone was used as a standard in probing the air-mediated oxidation of thiol to disulfide form of glutathione. The co-elution of standard peptide and reaction mixture of oxidation of glutathione in presence of dimedone using RP-HPLC have confirmed the formation of glutathione cysteine sulfenic as an intermediate in the air-mediated oxidation of glutathione. The synthetic peptides of cysteine dimedone may find application in the field of redox proteomics and generation of antibodies against modified cysteine residue.     

多肽酰肼连接法合成C端香豆素修饰的泛素

C端用香豆素修饰的泛素分子（Ub-AMC）是研究蛋白质泛素化过程的重要探针.该探针分子的制备目前主要依靠生物表达结合化学修饰的方法,合成效率较低.本文使用多肽酰肼连接反应,发展出化学全合成Ub-AMC分子的新路线.该方法通过N到C顺序两次连接实现了目标分子的组装,再通过自由基脱硫反应得到天然结构的Ub-AMC分子,有望实现较大量的合成.通过酶学活性实验,证实了通过新方法合成的Ub-AMC具有预期的生物活性.