Total Chemical Synthesis of Proteins

Ashraf Brik, Philip Dawson, Lei Liu

• Publisher: Wiley-VCH
• Year: 2021
• Print ISBN: 9783527346608
• eBook ISBN: 9783527823581
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Physical & Theoretical Chemistry

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.

1
Characterization of Protein Molecules Prepared by Total Chemical Synthesis

Stephen B. H. Kent

Department of Chemistry, The University of Chicago, Chicago, IL, 60637, USA

“Nevertheless, the chemical enigma of Life will not be solved until organic chemistry has mastered another, even more difficult subject, the proteins, in the same way as it has mastered the carbohydrates.”

Source: Emil Fischer (Nobel Lecture 1902)

1.1 Introduction

Proteins are the “natural prodicts” of the twenty‐first century. Protein molecules are ubiquitous in the biological world, with numerous diverse functions that range from acting as structural materials such as the keratins, to integral membrane proteins that serve as ion channels or as active molecular transporters in cells, to proteins that act as hormonal messengers in higher animal species, and to proteins that regulate gene expression [1]. The most important function of protein molecules is as enzymes, the potent and specific catalysts of the chemical reactions of biological metabolism, without which life would be impossible [2]. Thanks to modern DNA sequencing methods applied to genome [3] and metagenome [4] sequencing, vast numbers of proteins are being discovered at the nucleic acid level as open reading frames that code for a protein's polypeptide chain.

The central dogma of protein science is that the amino acid sequence of the polypeptide chain encodes the folded structure of the protein molecule in its natural environment, and that it is the folded structure of the protein molecule that gives rise to its biological function(s) [5]. Proteins range in mass from less than 5 kDa to more than 100 kDa. The median size of globular protein molecules is ∼35–45 kDa, comprising a polypeptide chain of ∼300–400 amino acid residues. Proteins are typically made up of two or more domains, autonomous units of folding, each of ∼120–160 amino acid residues [6].

Protein molecules are not simply really big peptides. In its native environment, each globular protein molecule has a defined folded structure that gives rise to the functional properties of that protein, including biochemical and biological activities. Synthetic proteins are organic molecules of high molecular mass, comprised of linear polypeptide chains (typically of 50–300 or more amino acid residues) that fold to form complex, dynamic structures. The large size of proteins together with the intricacy of their covalent and folded structures creates special challenges in the characterization of these synthetic molecules. For this reason, it is important for researchers to rigorously characterize protein molecules prepared by total chemical synthesis to meet standards similar to those used in synthetic organic chemistry. Analytical methods and criteria for the rigorous characterization of synthetic proteins have recently been enunciated [7].

1.2 Chemical Protein Synthesis

“The chemical ligation approach …. breaks the conceptual shackles imposed by the peptide bond, frees us from the linear paradigm of the genetic code, and opens the world of proteins to the entire repertoire of chemistry.”

Stephen Kent (23rd European Peptide Symposium 1994)

Chemistry, enabled by total synthesis, has an essential role to play in developing a fundamental understanding of the principles that give rise to the structures and biological functions of protein molecules. Thanks to modern chemical ligation methods [8-10], the total synthesis of protein molecules in the research laboratory is both practical and increasingly robust.

Characterization of a protein prepared by total chemical synthesis should begin with the synthetic process itself. Analytical control of the bond‐forming steps, purification and analysis of each synthetic intermediate, and folding of the full‐length synthetic polypeptide chain to form a defined tertiary structure are integral to verifying the structure of the final synthetic protein product. The steps involved in a typical total chemical synthesis of a protein molecule are listed below. Key aspects pertinent to characterization of the synthetic protein are in bold:

• Establish a verified amino acid sequence of the polypeptide chain of the target protein molecule.
  • Use a database such as UniProt (www.uniprot.org); include posttranslational processing where that is known to give the mature protein molecule.
  • Resolving data base/literature ambiguities in the reported amino acid sequence of a protein can be problematic. It is easy to make a mistake and to end up making an incorrect target sequence.
• Design a (convergent) synt...

Table of contents

• Cover
• Table of Contents
• Total Chemical Synthesis of Proteins
• Copyright
• Preface
• 1 Characterization of Protein Molecules Prepared by Total Chemical Synthesis
• 2 Automated Fast Flow Peptide Synthesis
• 3 N,S‐ and N,Se‐Acyl Transfer Devices in Protein Synthesis
• 4 Chemical Synthesis of Proteins Through Native Chemical Ligation of Peptide Hydrazides
• 5 Expanding Native Chemical Ligation Methodology with Synthetic Amino Acid Derivatives
• 6 Peptide Ligations at Sterically Demanding Sites
• 7 Controlling Segment Solubility in Large Protein Synthesis
• 8 Toward HPLC‐free Total Chemical Synthesis of Proteins
• 9 Solid‐phase Chemical Ligation
• 10 Ser/Thr Ligation for Protein Chemical Synthesis
• 11 Protein Semisynthesis
• 12 Bio‐orthogonal Imine Chemistry in Chemical Protein Synthesis
• 13 Deciphering Protein Folding Using Chemical Protein Synthesis
• 14 Chemical Synthesis of Ubiquitinated Proteins for Biochemical Studies
• 15 Glycoprotein Synthesis
• 16 Chemical Synthesis of Membrane Proteins
• 17 Chemical Synthesis of Selenoproteins
• 18 Histone Synthesis
• 19 Application of Chemical Synthesis to Engineer Protein Backbone Connectivity
• 20 Beyond Phosphate Esters: Synthesis of Unusually Phosphorylated Peptides and Proteins for Proteomic Research
• 21 Cyclic Peptides via Ligation Methods
• Index
• End User License Agreement