How to synthesize native and modified proteins in the test tube With contributions from a panel of experts representing a range of disciplines, Total Chemical Synthesis of Proteins presents a carefully curated collection of synthetic approaches and strategies for the total synthesis of native and modified proteins. Comprehensive in scope, this important reference explores the three main chemoselective ligation methods for assembling unprotected peptide segments, including native chemical ligation (NCL). It includes information on synthetic strategies for the complex polypeptides that constitute glycoproteins, sulfoproteins, and membrane proteins, as well as their characterization. In addition, important areas of application for total protein synthesis are detailed, such as protein crystallography, protein engineering, and biomedical research. The authors also discuss the synthetic challenges that remain to be addressed. This unmatched resource: Contains valuable insights from the pioneers in the field of chemical protein synthesis Presents proven synthetic approaches for a range of protein families Explores key applications of precisely controlled protein synthesis, including novel diagnostics and therapeutics Written for organic chemists, biochemists, biotechnologists, and molecular biologists, Total Chemical Synthesis of Proteins provides key knowledge for everyone venturing into the burgeoning field of protein design and synthetic biology.

This volume provides updated protocols for chemical protein synthesis. Chapters guide readers through development methods, strategies, and applications of protein chemical synthesis. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Chemical Protein Synthesis aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

Each review within the volume critically surveys one aspect of that topic and places it within the context of the volume as a whole. The most significant developments of the last 5 to 10 years are presented using selected examples to illustrate the principles discussed. The coverage is not intended to be an exhaustive summary of the field or include large quantities of data, but should rather be conceptual, concentrating on the methodological thinking that will allow the non-specialist reader to understand the information presented. Contributions also offer an outlook on potential future developments in the field.

Presenting a wide array of information on chemical ligation – one of the more powerful tools for protein and peptide synthesis – this book helps readers understand key methodologies and applications that protein therapeutic synthesis, drug discovery, and molecular imaging. • Moves from fundamental to applied aspects, so that novice readers can follow the entire book and apply these reactions in the lab • Presents a wide array of information on chemical ligation reactions, otherwise scattered across the literature, into one source • Features comprehensive and multidisciplinary coverage that goes from basics to advanced topics • Helps researchers choose the right chemical ligation technique for their needs

Organic chemists working on the synthesis of natural products have long found a special challenge in the preparation of peptides and proteins. However, more reliable, more efficient synthetic preparation methods have been developed in recent years. This reference evaluates the most important synthesis methods available today, and also considers methods that show promise for future applications. This text describes the state of the art in efficient synthetic methods for the synthesis of both natural and artificial large peptide and protein molecules. Subjects include an introduction to basic topics, linear solid-phase synthesis of peptides, peptide synthesis in solution, convergent solid-phase synthesis, methods for the synthesis of branched peptides, formation of disulfide bridges, and more. The book emphasizes strategies and tactics that must be considered for the successful synthesis of peptides.

A set of techniques has been developed for the routine synthesis and analysis of proteins by total chemical synthesis. Although the synthesis of proteins has been a goal of organic chemistry for nearly a century, straightforward and reproducible synthesis has remained elusive. Recently, the introduction of chemical ligation techniques for the chemoselective reaction of unprotected peptides in solution has greatly enhanced our ability to synthesize proteins of moderate size. In this thesis, an extension to the chemoselective ligation strategy has been developed which yields a native amide bond at the site of ligation. Native chemical ligation has been used to synthesize several proteins from two unprotected peptide fragments; including the chemokine interleukin 8 and barnase, a 110-residue ribonuclease from Bacillus amyloliquefaciens. Routine synthetic access to protein molecules allows unnatural analogues to be synthesized which may give insight into the molecular basis of protein function. In the second part of this thesis, a system has been developed to systematically study the effect of chemical modifications on the structure and function of a protein. Instead of generating a single protein analogue per synthesis, an array of self-encoded protein analogues can be prepared in a single synthetic procedure. This combinatorial approach was demonstrated by the synthesis of nineteen analogues of the cCrk N-terminal SH3 domain that were folded and analyzed for ligand binding as a single mixture. To read out the composition of the binding and non-binding proteins, a chemical cleavage site was incorporated at the site of modification. Cleavage of this bond created a unique set of peptides that can be identified in a single step by MALDI mass spectrometry. The resulting pattern of peaks relates the position of the chemical modification to its effect on protein function. Taken as a whole, the work presented in this dissertation represents a complete system for the investigation of proteins through chemical synthesis. Using these techniques, proteins of interest can be synthesized, analyzed for functional properties, and then systematically modified to give insight into the molecular basis of protein function.

This dissertation, "Protein Chemical Synthesis by Serine and Threonine Ligation" by Yinfeng, Zhang, 张银凤, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Landmark advances in the field of synthetic protein chemistry have enabled the preparation of complex, homogeneous proteins, including those that carry specific posttranslational modifications (PTMs). In addition, chemical synthesis will allow one to incorporate unnatural elements to generate new biologics with altered properties and functions. Native chemical ligation (NCL) is a milestone in the chemical synthesis of proteins (Kent et al., Science, 1994, 266, 776-779), in which a C-terminal peptide thioester and an N-terminal cysteine (Cys)-containing peptide-both in side-chain unprotected forms-are selectively coupled to generate a natural peptidic linkage at the site of ligation. This method requires a cysteine at the optimal convergent ligation site. However, Cys is one of the least abundant amino acids in natural proteins. Therefore, the development of new ligation methods at other amino acids will be necessary and important in this regard. Along these lines, our laboratory has developed a novel thiol-independent approach-serine/threonine ligation (STL). It uses the N-terminal serine or threonine of a peptide segment to chemoselectively react with another peptide segment with a C-terminal salicylaldehyde ester to form an N, O-benzylidene acetal linked product, followed by acidolysis to afford the final product at the natural Ser/Thr site. To extend the application of STL in chemical protein synthesis, we have developed a robust method for the preparation of peptide salicylaldehyde esters via Fmoc-based solid phase peptide synthesis. Furthermore, we have successfully applied this ligation method in the convergent synthesis of peptide drugs of significant therapeutic importance, including Teriparatide (Forteo), Corticorelin (oCRH), Exenatide (Byetta) and Tesamorelin (hGHRH). Of significance, we have demonstrated the effectiveness of our STL in the assembly of a more complex target of biological interest: human erythrocyte acylphosphatase ( 11 kDa). In summary, we have developed a new serine/threonine ligation, which can be effectively used to synthesize peptides and proteins. As there are countless serine and threonine residues in natural proteins, particularly those carrying posttranslational modifications, this method is anticipated to offer new opportunities in synthetic protein chemistry and chemical biology. DOI: 10.5353/th_b5295527 Subjects: Proteins - Synthesis

How to synthesize native and modified proteins in the test tube With contributions from a panel of experts representing a range of disciplines, Total Chemical Synthesis of Proteins presents a carefully curated collection of synthetic approaches and strategies for the total synthesis of native and modified proteins. Comprehensive in scope, this important reference explores the three main chemoselective ligation methods for assembling unprotected peptide segments, including native chemical ligation (NCL). It includes information on synthetic strategies for the complex polypeptides that constitute glycoproteins, sulfoproteins, and membrane proteins, as well as their characterization. In addition, important areas of application for total protein synthesis are detailed, such as protein crystallography, protein engineering, and biomedical research. The authors also discuss the synthetic challenges that remain to be addressed. This unmatched resource: Contains valuable insights from the pioneers in the field of chemical protein synthesis Presents proven synthetic approaches for a range of protein families Explores key applications of precisely controlled protein synthesis, including novel diagnostics and therapeutics Written for organic chemists, biochemists, biotechnologists, and molecular biologists, Total Chemical Synthesis of Proteins provides key knowledge for everyone venturing into the burgeoning field of protein design and synthetic biology.