Chemical Approaches to the Synthesis of Peptides and Proteins

Paul Lloyd-Williams, Fernando Albericio, Ernest Giralt

• Publisher: CRC Press
• Year: 2020
• ISBN:
  9781000151688
• Edition: 1
• Topic: Art
• Subtopic: Culinary Arts

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.

Chapter 1

Introduction

Proteins play a crucial role in almost all fundamental processes in the living cell. Although the genetic information is encoded in nucleic acids, the mechanisms of DNA replication and of gene expression are controlled by proteins. The enzymes that mediate the chemical reactions necessary for life are proteins, as are several of the hormones that control the biochemical balance in complex organisms. The storage and transport of a variety of materials ranging from macromolecules to electrons are also controlled by proteins. In animals, the immune system uses different types of proteins to identify and reject foreign invaders, and muscles, tendons, skin, bones, nails, and hair are all comprised of several kinds of structural proteins. Although they carry out an almost bewildering range of functions in living things, all proteins are made up of the same basic building blocks, being biopolymers of the 20 DNA-encoded amino acids. Beyond this, however, they are not just unstructured chains of their constituent monomers but, rather, adopt characteristic, highly organized three-dimensional arrangements in solution that are intimately related to their biological function.

Peptides are simply smaller versions of proteins. Their three-dimensional structures tend to be less well defined, but many, such as the peptide-hormones vasopressin, oxytocin, and calcitonin, the neuroactive peptides found in the brain, and the toxins of certain animals and bacteria, are biologically important. There are currently several peptide or peptide-based drugs in widespread clinical use, and peptide molecules show much promise as potential therapeutic agents against infectious disorders such as malaria or foot-and-mouth disease. While there is no clear dividing line between peptides and proteins, an acceptable working distinction is that proteins are large peptides, where large is a relative term and may mean anything from perhaps 30 to several hundred amino acid residues.

Since the difference between peptides and proteins is essentially one of size or of length of the amino acid backbone, the problems involved in the chemical synthesis of proteins are basically those of the synthesis of peptides or of peptide chemistry. Having said that, the difficulties one may encounter in the synthesis of a protein of, say, 150 residues are not the same as those that the synthesis of a peptide of, say, ten residues might present. The synthesis of proteins has challenged chemists for over a century,¹ since the first simple peptides were synthesized by Theodor Curtius² and Emil Fischer.³ Although the total syntheses of oxytocin,⁴ of insulin,^{5, 6, 7, 8} and of ribonuclease A^9,10 were milestones in synthetic organic chemistry, they did not lead to general methods for protein synthesis but, rather, were isolated examples of success in what remained a dauntingly difficult field. However, in the last 30 years the picture has changed dramatically and peptide chemistry has undergone a revolution, brought about, in the main, by two fundamental developments.

Bruce Merrifield’s¹¹ invention of solid-phase peptide synthesis (SPPS) together with the enormous improvements in liquid chromatographic techniques, particularly the advent of high-performance liquid chromatography,¹² have changed peptide chemistry from being a specialist area in which a few research groups were active, into a field where virtually any scientist whose research leads to the need for synthetic peptides may attempt to synthesize them. The refinement of Merrifield’s original method has led to the development of automatic peptide synthesizers. These remove much of the drudgery from modern peptide synthesis by carrying out the tedious, repetitive operations required to couple each of the amino acids of any given peptide. Although the chemist must decide the overall synthetic strategy at the outset, many modern synthesizers simply require that the desired sequence be programmed into a dedicated computer. The machine then carries out all of the various chemical steps needed to elaborate the peptide chain automatically, often without the operator having to intervene.

Modern synthetic methods, whether manual or machine assisted, using solid supports or in solution, allow many peptides to be synthesized without undue difficulty. However, large peptides and proteins, or those peptides having a high incidence of the more sensitive amino acids, are a different matter. Side reactions can always occur even in quite simple sequences, but for larger molecules much more serious complications can manifest themselves. In solid-phase synthesis, incomplete deprotection and coupling reactions tend to become more pronounced as the length of the peptide chain increases. The failure of these key steps can present insurmountable obstacles. Synthesis in solution is only rarely a practical alternative for large peptides, since it is slow, labor-intensive, and dogged by the problem of poor solubility of the synthetic intermediates. However, although the chemical synthesis of complex peptides is neither a routine nor a trivial matter, and in many cases may require the best efforts of the most-seasoned practitioners, today it can be attempted with at least a reasonable expectation of success.

In addition to chemical synthesis, biotechnological techniques now provide an alternative and often very efficient means of producing proteins. In favorable cases they are currently the best way of producing useful amounts of material for research and industrial purposes. It is even possible to adapt the methods so that modified protein structures can be produced. However, despite their power and potential, they also have their drawbacks and disadvantages. The isolation of the desired protein from the fermentation medium can be difficult, and biotechnology is not really appropriate for the generation of the large number of analogues that are routinely needed for structure-activity relationship studies. Such variation of structural characteristics is fundamentally important if therapeutic agents are to be produced and is best done by chemical synthesis. One of the most exciting contemporary areas of research is “de novo” protein design. This involves the design and synthesis of nonnatural protein analogues, either in order to mimic the natural molecules or to investigate specifically some structural or mechanistic aspect of protein function. For this, chemical synthesis is obviously the method of choice. Although there will certainly be further improvements in biotechnology, chemical approaches to the synthesis of peptides and proteins are unlikely to be superseded or made obsolete in the foreseeable future.

1.1 AMINO ACIDS AND PEPTIDE AND PROTEIN STRUCTURE

The 20 DNA-encoded or proteinogenic α-amino acids are shown in Table 1.1. All except proline have the same basic structure that incorporates a primary amino group and differs only in the nature of the side chain. Proline, on the other hand, is unique in having a cyclic structure with a secondary amine. With the exception of glycine, all are chiral, due to the presence of at least one stereogenic carbon atom, and belong to the L-stereochemical series. The chiral a-amino acids all have the (S) configuration at the α-carbon atom, except cysteine in which it is (R) as a consequence of the manner in which the Cahn-Ingold-Prelog convention functions.¹³ Two amino acids, threonine and isoleucine, have a second stereogenic center at the β-carbon atom, giving rise to four possible diastereomers for each. In the case of L-Thr this second atom has the (R) configuration, so that L-Thr has the stereochemistry 2S, 3R, and in L-Ile the β-carbon atom has the (S) configuration so that L-Ile has the stereochemistry 2S, 3S.

The biosynthesis of peptides and proteins with the 20 proteinogenic amino acids is carried out ribosomally under nucleic acid control. However, certain nonprotein-ogenic amino acid residues are sometimes found in peptides and proteins, and this is a consequence of posttranslational enzymatic modification of DNA-encoded residues. In collagen, for example, proline can be hydroxylated giving 4(R)-hydroxy-L-proline. The proteins involved in blood coagulation often contain γ-carboxy-glutamic acid, formed by the carboxylation of glutamic acid. The hydroxyl groups of Ser, The, and Tyr can be sulfated or phosphorylated in a variety of biologically active peptides.

In several types of lower organisms, such as algae, sponges, yeasts, and fungi, peptides are often biosynthesized enzymatically¹⁴ rather than on the ribosomes. Apart from the proteinogenic amino acids, these peptides may contain other, modified a...

Table of contents

• Cover
• Half Title
• Series Page
• Title Page
• Copyright Page
• About The Authors
• Preface
• Table of Contents
• Chapter 1 Introduction
• Chapter 2 Solid-Phase Peptide Synthesis
• Chapter 3 Peptide Synthesis in Solution
• Chapter 4 Convergent Approaches to the Synthesis of Large Peptides and Proteins
• Chapter 5 Formation of Disulfide Bridges
• Chapter 6 Peptide Libraries
• Index