Progress in Chemical Synthesis of Peptides and Proteins

For the proteins that cannot be expressed exactly by cell expression technology (e.g., proteins with multiple posttranslational modifications or toxic proteins), chemical synthesis is an important substitute. Given the limited peptide length offered by solid-phase peptide synthesis invented by Professor Merrifield, peptide ligation plays a key role in long peptide or protein synthesis by ligating two small peptides to a long one. Moreover, high-molecular-weight proteins must be synthesized using two or more peptide ligation steps, and sequential peptide ligation is such an efficient way. In this paper, we reviewed the development of chemical protein synthesis, including solid-phase peptide synthesis, chemical ligation, and sequential chemical ligation. © 2017, The Author(s).

Thermodynamic Analysis and Synthesis Gas Generation by Chemical-Looping Gasification of Biomass with Nature Hematite as Oxygen Carriers

Thermodynamic parameters of chemical reactions in the system were carried out through thermodynamic analysis. According to the Gibbs free energy minimization principle of the system, equilibrium composition of the reactions of chemical-looping gasification (CLG) of biomass with natural hematite (Fe2O3) as oxygen carrier were analyzed using commercial software of HSC Chemistry 5.1. The feasibility of the CLG of biomass with hematite was experimental verified in a lab-scale bubbling fluidized bed reactor using argon as fluidizing gas. It was indicated the experimental results were consistent with the theoretical analysis. The presence of oxygen carrier gave a significant effect on the biomass conversion and improved the synthesis gas yield obviously. It was observed that the gas content of CO and H2 was over 70% in CLG of biomass. The reduced hematite particles mainly existed in form of FeO. It was showed that the reduction of natural hematite with biomass proceeds in a stepwise manner from Fe2O3 →Fe3O4→ FeO. Reduction product of natural hematite can be restored the lattice oxygen by oxidation with air.

Recent advances in chemical protein synthesis:method developments and biological applications

The central dogma of modern biology underscores the pivotal roles proteins play in diverse biological processes,the study of which necessitates advanced methods to produce proteins with precision and versatility.Chemical protein synthesis,a powerful approach utilizing chemical reactions for the de novo construction of structurally accurate proteins,has emerged as a transformative tool for studying proteins and generating protein derivatives/mimics inaccessible by natural biological machinery,including post-translationally modified proteins,proteins comprised of unnatural amino acids,as well as mirror-image proteins.This review summarizes recent strides in synthetic method developments for chemical protein synthesis,including innovative techniques in solid-phase peptide synthesis,the challenges presented by difficult sequences in either synthesis or folding and the exploration of novel ligation reactions using both chemical and enzymatic methods.Furthermore,the review also delves into newly developed protocols for site-selective protein modifications and the generation of stapled or macrocyclized peptides/miniproteins,highlighting the power of chemical methods to make structurally diverse proteins.Recent applications of synthetic proteins in investigating post-translational modifications(phosphorylation,lipidation,glycosylation,ubiquitination,etc.),mirror-image biological processes and drug development are further discussed.Together,these topics provide a comprehensive overview of the current landscape of chemical protein synthesis.

An improved installation of 2-hydroxy-4-methoxybenzyl(iHmb)method for chemical protein synthesis

The 2-hydroxy-4-methoxybenzyl(Hmb)backbone modification can prevent amide bond-mediated sidereactions(e.g.,aspartimide formation,peptide aggregation)by installing the removable Hmb group into a peptide bond,thus improving the synthesis of long and challenging peptides and proteins.However,its use is largely precluded by the limited Hmb’s installation sites.In this report,an improved installation of Hmb(iHmb)method was developed to achieve the flexible installation and the convenient removal of Hmb.The iHmb method involves two critical steps:(1)oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl(Hmab)to give 2-hydroxy-4-methoxy-5-diazonium-benzyl(Hmdab)by combining soamyl nitrite(IAN)/HBF_(4),and(2)reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol(EDT).The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids(e.g.,valine and isoleucine).The practicality and utility of the iHmb method was demonstrated by one-shot solid-phase synthesis of a challenging aspartimide-prone peptide,the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue.Furthermore,the iHmb method can be utilized to facilitate chemical protein ligation,as exemplified by the synthesis of the single-spanning membrane protein sarcolipin.The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins.

Rational Design and Cellular Synthesis of Proteins with Unconventional Chemical Topology

Chemical topology refers to the three-dimensional arrangement(i.e.,connectivity and spatial relationship)of a molecule's constituent atoms and bonds.The molecular mechanism for translation defines the linear configuration of all nascent proteins.Nontrivial protein topology arises only upon post-translational processing events and often imparts functional benefits such as enhanced stability,making topology a unique dimension for protein engineering.Utilizing the assembly-reaction synergy,our group has developed several methods for the effective and convenient cellular synthesis of a variety of topological proteins,such as lasso proteins,protein rotaxanes,and protein catenanes.The work opens the access to new protein classes and paves the road toward illustrating the topological effects on structure-function relationship of proteins,which lays solid foundation for exploring topological proteins’practical application.

Chemical protein synthesis elucidates key modulation mechanism of the tyrosine-O-sulfation in inducing strengthened inhibitory activity of hirudin

Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.

Chemical Synthesis of Proteins Containing 300 Amino Acids

Chemical synthesis of proteins containing up to 300 amino acids may cover 30%—50%of all the proteins encountered in biomedical studies and may provide an alternate approach to the usually used recombinant expression teclmology,vastly expanding the chemical space of the latter.In the present review article,we tried to survey the recent progresses made for more rapid synthesis of increasingly long peptides and more efficient ligation of multiple peptide segments.The developments of seminal methods by many research groups have greatly contributed to the recent breakthroughs in the successful total synthesis of a number of functionally important proteins,such as oligoubiquitins,bacterial GroEL/ES chaperones,and mirror-image DNA polymerases.Through these studies,a potential bottleneck has also been recognized for the chemical synthesis of large proteins,namely,how to ensure that each peptide segment from a large protein avoids unfavorable aggregation when dissolved in aqueous solution.Many new methods,such as removable backbone modification(RBM)strategy have been developed to overcome this bottleneck,while more studies need to be carried out to develop more effective and less costly methods that ultimately,may lead to fully automatable chemical synthesis of customized proteins of 300 amino acids bearing any artificial designs.

Chemical Synthesis of diSUMO Photoaffinity Probes for the Identification of PolySUMO Chain-Specific Interacting Proteins

Small ubiquitin-like modifiers(SUMOs)are protein modifiers that can form polymeric chains.They are important signals in cellular processes,and their study and profiling require the development of molecular tools.Herein,the authors have reported an efficient chemical protein synthesis approach for the generation of dimeric SUMO-2-based photoaffinity probes through the ligation of four readily synthesizable peptides.Proteomic studies using this diSUMO-2 probe on HeLa cell nuclear lysate found it to capture a significantly different selection of proteins compared with its monoSUMO counterparts.This resulted in the identification of several previously unknown SUMO chain-specific interacting proteins such as 40S ribosomal protein S3,which showed a significantly higher affinity for polySUMO chains than monomeric SUMO.Collectively,these results emphasize the need to develop SUMO chain-based probes in other species,and to shed light on the important role of polySUMOylation in diseases.

Recent advances in the chemical synthesis and semi-synthesis of poly-ubiquitin-based proteins and probes

Ubiquitination, a key and extensive posttranslational modification of proteins, has profound effects on a variety of physiological and pathological processes. The inherent complexity of ubiquitin conjugates makes it highly challenging to study the functional and structural mechanisms of ubiquitination. To address these challenges, accesses to sufficient poly-ubiquitin chains or ubiquitinated proteins are urgently needed. Over the last decade, synthetic protein chemists have developed several novel peptide ligation methods for the preparation of ubiquitin conjugates with precise control over the atomic structure. In this review, we summarize the recent breakthroughs and potential challenges in the chemical synthesis and semi-synthesis of ubiquitin conjugates with respect to the preparation of poly-ubiquitin-based proteins and ubiquitin-based probes.

Chemical protein synthesis-assisted high-throughput screening strategies for D-peptides in drug discovery

D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability,improved oral bioavailability,as well as high binding affinity and specificity.Recently,D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years.One D-peptide etelcalcetide has even entered the market that targets the calcium(Ca2+)-sensing receptor(CaSR) to fight secondary hyperparathyroidism.Effective discovery and optimization of D-peptide ligands that can bind to various disease-related targets with high specificity and potency is of great importance for the development of D-peptide drugs.This review surveys the recent method development in this area especially the chemical protein synthesis-assisted high-throughput screening strategies for D-peptide ligands and their application in drug discovery.

伪蛋白生物材料的分类、合成及其应用

伪蛋白是一种新型合成生物可降解材料,以氨基酸为基本结构单元,它与蛋白质不同的是除了含有肽键外,还含有其他连接基团,这种结构使其既具有蛋白质的优良特性,又具有良好的力学性能、热性能、可调节的物理化学性能以及优异的细胞相容性和3D微孔结构等性质,因此伪蛋白能够有效应用于载体、伤口愈合、组织工程等生物医药领域。本文首先综述了目前所研究的伪蛋白的类型,包括非功能性和功能性伪蛋白两类;之后详细阐述了缩聚合成伪蛋白的方法,包括溶液缩聚、界面聚合、熔融缩聚、开环聚合四种方法;接着归纳了伪蛋白生物材料在药物载体、基因载体、组织工程、创面敷料等方面的应用;最后,对伪蛋白生物材料在合成路线及应用方面未来的发展进行了探讨和展望。

化学链合成氨研究进展与展望

氨主要被用作生产氮肥的原料,近年来被认为是一种具有重要应用前景的能源载体。目前工业合成氨主要采用Haber-Bosch工艺,该过程严重依赖化石能源,是高能耗、高碳排放过程,为生态环境带来巨大压力。因此,开发温和条件下可再生能源驱动的绿色合成氨过程具有重要的实用意义和社会价值。化学链合成氨过程是将合成氨反应解耦为两步或多步反应,具有规避催化中普遍存在的Scaling relations关系、避免氮气和氢气竞争吸附、可常压操作等特点,被认为是一种具有应用前景的合成氨技术。此外,该过程适合于分布式、小型化的生产模式,便于与可再生能源利用过程相耦合。因此,近年来化学链合成氨受到广泛关注。本综述首先简述了化学链过程的定义及其在化石能源转化领域的研究概况,随后重点介绍化学链合成氨的发展历史、载氮体分类及近期研究进展,最后对载氮体材料的设计思路及化学链合成氨过程的前景进行展望。根据载氮体不同,分别介绍了二元金属氮化物、多元金属氮化物、亚氨基化物及氮氧化物等载氮体材料在分别以N_(2)-H_(2)和N_(2)-H_(2)O为原料的化学链合成氨过程中材料种类、反应热力学及动力学、合成氨速率等方面研究成果。此外,由于近期外场驱动合成氨取得了较好进展,根据外场驱动力不同,分别介绍了以电、光、等离子体、微波等为外加能量形式的化学链合成氨过程。并从热力学及动力学两方面分别介绍了载氮体材料的改进策略。最后,对化学链合成氨技术存在的问题及外来研究方向进行展望。

连接-脱硫策略在蛋白质化学合成中的发展与应用

蛋白质化学合成是获取特定序列与结构蛋白质分子的关键技术,为研究蛋白质的结构与功能提供了物质基础.传统的固相多肽合成受限于分步氨基酸偶合和脱保护反应效率,难以一次性合成分子量较大的蛋白质.自然化学连接和酰肼连接技术作为无保护肽片段间连接的策略,具有高效选择性,极大地推动了蛋白质化学合成的发展.然而,这些连接策略需依托蛋白质中丰度较低的半胱氨酸,难以适用于无半胱氨酸或者半胱氨酸不合适作为连接位点的蛋白质的合成.连接-脱硫策略的提出首次将连接位点拓展至丙氨酸,并促进了硫代氨基酸的发展,使得蛋白化学合成不再受限于连接位点的选择.在此基础上,自由基脱硫、光化学脱硫、P-B脱硫和铁催化脱硫等新兴脱硫技术的进步为蛋白质化学合成提供了多样化的选择,拓展了其应用范围.连接-脱硫的化学方法不断地演进创新,丰富了蛋白质化学合成方法库,为蛋白质工程与化学生物学领域的深入研究提供了重要支持.本综合评述以时间线的形式,全面回顾了连接-脱硫化学方法在蛋白质化学合成中的发展历程.从早期基于半胱氨酸位点的自然化学连接和酰肼连接技术,到连接-脱硫策略的开创性发展,再到硫代氨基酸和多样化脱硫策略的探索,这些技术不仅丰富了多肽合成的策略,也展示了它们在合成蛋白质中的广阔应用前景和发展潜力.本文旨在为蛋白质化学合成领域的科研工作者提供深刻的见解和有价值的信息,激发该领域的进一步探索与创新.

Total chemical and semisynthetic approaches for the preparation of ubiquitinated proteins and their applications

Protein ubiquitination is an important post-translational modification(PTM) in eukaryotic organisms that regulates a variety of cellular processes, such as protein degradation, signal transduction, apoptosis, and DNA damage tolerance. To decipher mechanistically the diverse biological functions of ubiquitination, homogeneous ubiquitinated proteins are greatly needed.Although direct isolation from cell source and in vitro enzymatic methods can be used to produce such proteins, these methods often suffer from problems of low yield or heterogeneous products. Comparably, total chemical and semisynthetic approaches offer good alternatives to produce the ubiquitinated proteins with high purity and selectivity. This review summarizes the recent developments of protein ubiquitination strategies and the use of the synthesized proteins to help garner structural and functional insight into the inner workings of the ubiquitin system.

Racemic X-ray structure of L-type calcium channel antagonist Calciseptine prepared by total chemical synthesis

Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by using hydrazide based native chemical ligation. The crystal structure of Calciseptine was determined by racemic protein crystallography technique. Compared to the structure of its homologous family protein, we found that Calciseptine is adopting a typical three-finger structure.

Chemical synthesis and structure determination of venom toxins

Venom toxins are widely spread in nature, adopting diverse structures and functions. They often function by blocking or modulating important membrane protein targets thus can be promising therapeutic candidates and biophysical probes. In this review, we briefly discuss the total chemical synthesis of venom toxins including the different refolding strategies reported during the past decade as well as innovative approaches for structure determination.

Reaction characteristics investigation of CeO_(2)-enhanced CaSO_(4) oxygen carrier with lignite

Calcium sulfate(CaSO_(4))has been verified as a promising oxygen carrier(OC)in the chemical looping combustion(CLC)for its high oxygen capacity,abundant reserve and low cost,but its low reactivity and deleterious sulfur species emission from the side reactions of CaSO_(4) should be well considered for its wide application in CLC.In order to promote the reactivity of CaSO_(4) and increase its potential to inhibit the gaseous sulfur emission,a CeO_(2)-enhanced CaSO_(4) OC mixed OC of core–shell structure was prepared using the combined template synthesis method.Reaction characteristics of the prepared CaSO_(4)-CeO_(2) mixed OC with a typical lignite was first conducted and systematically investigated,and an improved reactivity of the prepared CaSO_(4)-CeO_(2) mixed OC was demonstrated than its single component CaSO_(4) or CeO_(2) due to the fast transfer and exchange of oxygen from the CaSO_(4) substrate to coal via the doped CeO_(2).Furthermore,the solid products formed from the mixed CaSO_(4)-CeO_(2) OC with the selected coal were collected and analyzed.Especially,evolution and redistribution of the sulfur species of different forms were focused.At the latter reaction stage of YN reaction with the CaSO_(4)-CeO_(2) mixed OC,the SO_(2) emitted from the side reactions of CaSO_(4) was greatly diminished and the doped CeO_(2) was proven effective to directionally fix the SO_(2) released to turn into different solid sulfur compounds,which were determined as Ce_(2)O_(2)S,Ce_(2)S_(3) and Ce_(2)(SO_(4))_(3)·5H_(2)O and formed through the different pathways.In addition,good regeneration of the reduced CaSO_(4)-CeO_(2) mixed OC could be reached in spite of the unavoidable interaction between the included minerals in coal and the reduced mixed OC.Overall,the combined template method-made CaSO_(4)-CeO_(2) mixed OC reported herein was not only endowed with enhanced reactivity for coal conversion,but also owned the potential to directionally fix the gaseous sulfur emission,which is quite applicable as OC for simultaneous decarbonatization and desulfurization in the real CLC process.

Chemical synthesis and structural analysis of guanylate cyclase C agonist linaclotide

Guanylate cyclase C(GC-C) is an important receptor protein expressed by intestinal epithelial cells, and its dysregulation leads to severe intestinal diseases. Linaclotide is a 14-amino acid peptide approved by the FDA for the treatment of irritable bowel syndrome with constipation(IBS-C), which activates guanylate cyclase C to accelerate intestinal transit. Drug molecule design based on structural information plays a crucial role and the activity of linaclotide still need to improve, while the structure of linaclotide remains unknown. In this work, linaclotide and its D-enantiomer were obtained through Fmoc solid phase peptide synthesis method and co-crystalized through racemic crystallization. The crystal structure showed that linaclotide has a tight, three-beta turns structure immobilized by three pairs of disulfide bonds.

Chemical synthesis and racemic crystallization of rat C5a-desArg

The deletion of the C-terminal arginine of the anaphylatoxin protein C5a reduces it receptor binding affinity.Understanding how C-terminal arginine affects the structure and bioactivity of C5a is important for the development of C5a C-terminal mimics as drug candidates.Herein,we report the total chemical synthesis of rat C5a and its D-enantiomer with its C-terminal arginine deleted,namely L-rC5a-desArg and D-rC5a-desArg.The structure of rC5a-desArg was then determined by racemic crystallography for the first time.The C-terminal residues of rC5a-Arg were found to expand from the fourth helix in a continuous helical confo rmation.This C-terminal conformation is significantly different from that of the previously reported full-length of C5a,indicating that the deletion of C-terminal arginine residue could result in the destruction of a positively charged surface formed by two adjacent Arg residues in C5a.

Total synthesis of TRADD death domain with arginine N-GlcNAcylation by hydrazide-based native chemical ligation

TNFR1-associated death domain protein(TRADD)with arginine N-GlcNAcylation is a novel and structurally unique posttranslational modification(PTM)glycoprotein that blocks the formation of death-inducing signaling complex(DISC),orchestrating host nuclear factorκB(NF-κB)signaling in entero-pathogenic Escherichia coli(EPEC)-infected cells.This particular glycosylated modification plays an extremely vital role for the effective colonization and pathogenesis of pathogens in the gut.Herein we describe the total synthesis of TRADD death domain(residues 195-312)with arginine235 NGlcNAcylation(Arg-GIcNAc TRADD(195-312)).Two longish peptidyl fragments of the wild-type primary sequence were obtained by robust,microwave-assisted,highly efficient,solid-phase peptide synthesis(SPPS),the N-GlcNAcylated sector was built by total synthesis and attached specifically to resinbound peptide with an unprotected ornithine residue via silver-promoted on-resin guanidinylation,ArgGlcNAc TRADD(195-312)was constructed by hydrazide-based native chemical ligation(NCL).The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of other proteins with Arg-GIcNAc modification and to pave the way for the related chemically biological study.