Preface

Chemical protein synthesis enables the generation of proteins of any architecture and facilitates the site-selective introduction of unnatural amino acids, biophysical probes, posttranslational modifications, and other functionalities. The preparation of these protein analogues enables important biochemical and biophysical studies and contributes to the fundamental understanding of proteins. In this book, we present a salient collection of articles that cover the modern methods and applications of the field, along with descriptions of various case studies in which chemical protein synthesis is leveraged to shed light on fundamental questions in protein science.

In Chapter 1, Stephen Kent, the pioneer and leading figure in modern chemical protein synthesis, introduces the essential role of chemical protein synthesis in developing a fundamental understanding of the principles that give rise to the structures and biological functions of protein molecules. Strong emphasis was given to analytical control and documentation of the synthetic process in order to meet standards, similar to those used to characterize small organic molecules. Characterization of a synthetic protein molecule should rigorously characterize the molecular homogeneity, correct covalent structure, and defined folded structure.

New exciting advances in synthetic methodology have greatly reshaped the landscape of chemical protein synthesis over the past 10?years. In Chapter 2, Bradley L. Pentelute et al. describe the new technology of automated flow-based solid-phase peptide synthesizer (AFPS) that can incorporate each amino acid residue in as little as 40?seconds. This technology offers a template for accelerating syntheses of long polypeptide sequences using an automated flow instrument. This approach may open the chemical biology field to routine manufacture of custom-made, fully synthetic proteins. In Chapter 3, Vangelis Agouridas, Oleg Melnyk, and coworkers describe the development of N,S(Se)-acyl shift systems for linking peptide segments together through the most widely used method of chemical protein synthesis, namely, native chemical ligation. This strategy is especially useful for systems that require latent thioesters. Many biologically active proteins have been successfully synthesized with the SEA (bis(2-sulfanylethyl) amido) and SEAlide (N-sulfanylethylanilide) chemistries. In Chapter 4, Lei Liu et al. describe the development and optimization of hydrazide-based native chemical ligation, as well as the optimization of the preparation, activation, and ligation of peptide hydrazides. The hydrazide-based method exhibits several advantages including easy preparation, high stability, good handling properties, and controllable activation. It has been successfully used in the synthesis of peptide pharmaceutics, posttranslationally modified proteins, photo-activatable protein tools, membrane proteins, and mirror-image proteins.

To further expand the scope and utility of native chemical ligation, Richard J. Payne et al. describe in Chapter 5 the efforts made to develop various thiolated amino acids, which, with the help of mild, radical-mediated desulfurization conditions, can enable ligation at 16 of the 20 proteinogenic amino acids. They also describe their seminal studies on selenocysteine (Sec), which have enabled rapid chemical protein synthesis through the diselenide-selenoester ligation (DSL) and novel strategies such as "selenol ligation auxiliary." In Chapter 6, Suwei Dong et al. describe the recent development on new strategies that aim to facilitate peptide ligations at sterically demanding sites. These ligation reactions are usually challenging, as the strong activating conditions may result in competitive hydrolysis. Furthermore, in Chapter 7, Michael S. Kay et al. describe the recent studies on how to control segment solubility in large protein synthesis. Various strategies that utilize solvent choice, isoacyl dipeptides, and semipermanent solubilizing tags are surveyed and analyzed in depth. This chapter provides very useful guides for researchers to overcome the often-encountered solubility problems in chemical protein synthesis.

With the advent of fast peptide synthesis and ligation, other limiting factors have received attentions for chemical protein synthesis. In Chapter 8, Oliver Seitz et al. describe the current state of the art in the development of methods that allow minimizing the number of high-performance liquid chromatograpohy (HPLC) purification steps. These methods typically rely on rapid and HPLC-free assembly of proteins using capture and release purification handles, opening opportunities for the chemical synthesis of protein arrays under parallel conditions. In Chapter 9, Vincent Aucagne et al. describe solid-phase peptide chemical ligation (SPCL), which entails a ligation-based assembly on a solid support to avoid the laborious intermediate chromatographic purifications. The development of new polymers, new linkers, new ligations reactions with improved kinetics, and new masking/unmasking strategies has broadened the scope of SPCL to more ambitious targets. On a different front, to expand the flexibility of peptide condensation, ligation strategies other than native chemical ligation need to be developed. In Chapter 10, Xuechen Li et al. describe the approach of serine/threonine ligation (STL) between peptide C-terminal salicylaldehyde esters and N-terminal Ser/Thr residues. This approach takes advantage of the high abundance of Ser/Thr residues in the protein sequence and has paved the way for broad applications in proteins synthesis and related constructs.

Modern total chemical synthesis of proteins can now reach approximately 200-400 amino acids. To amplify the ability to make proteins containing unnatural amino acids and posttranslational modifications, an important strategy is protein semisynthesis where a biologically produced protein or protein fragment is selectively modified through covalent chemical reactions. In Chapter 11, Philip A. Cole et al. describe the development of methods for protein semisynthesis including expressed protein ligation, cysteine modification reactions, and enzyme-catalyzed protein/peptide ligations. These methods have been successfully employed to augment our understanding on protein posttranslational modification. As a unique example of protein semisynthesis methods, in Chapter 12, Tilman M. Hackeng et al. describe bio-orthogonal imine chemistry for protein modification. Details were provided for how to incorporate carbonyl or a-nucleophiles moieties into proteins, and how to carry out different imine chemistries such as oxime and hydrazone ligation.

Next are the applications of chemical protein synthesis to solve various biochemical, biophysical, and biomedical problems. In Chapter 13, Vladimir Torbeev describes how to use chemical protein synthesis to decipher the mechanism of protein folding. Many interesting methods have been developed including modification of protein backbone amides, insertion of ß-turn mimetics, inversion of chiral centers in protein backbone and side chains, modulating cis-trans proline isomerization, covalent tethering to facilitate folding of designed proteins, and foldamers and foldamer-peptide hybrids. These methods facilitate biophysical studies on intrinsically disordered proteins (IDPs) that perform important functions such as gene transcription and chromatin remodeling. They are also helpful to studies on improper protein folding related to many diseases such as Alzheimer's and Parkinson's disorders.

One of the most exciting applications of chemical protein synthesis is the development of chemical tools to study the biological functions and mechanisms of proteins bearing posttranslational modifications. In Chapter 14, Ashraf Brik et al. described their contributions in developing innovative methods to construct a variety of ubiquitin conjugates of high purity, in sufficient quantity to facilitate detailed biophysical and biochemical analysis. As a "game changer" to overcome the inherit limitations of the enzymatic approaches, chemical protein synthesis has enabled readily construction of versatile ubiquitin conjugates to study and target ubiquitin-processing enzymes. In Chapter 15 Yasuhiro Kajihara et al. describe the cutting-edge examples of glycoprotein synthesis employing various synthetic methodologies and ligation strategies. The combination of both chemical and enzymatic methodologies gives insight into how glycans function in their biological environment and may eventually lead to homogeneous glycoprotein pharmaceuticals.

In addition to protein posttranslational modification, chemical protein synthesis also plays important roles in the biochemical studies on some unique protein families. In Chapter 16, Christian Becker et al. delineate the chemical synthesis of membrane proteins, a unique protein family comprising about 30% of the proteins encoded by the human genome. Access to membrane proteins by chemical synthesis has helped to decipher important aspects of membrane protein functions due to the unique opportunities that chemical synthesis provides in terms of manipulations of the backbone and side chains of proteins. In Chapter 17, Norman Metanis et al. describe chemical synthesis of selenoproteins,...