Chemistry of Peptide Synthesis

FUNDAMENTALS OF PEPTIDE SYNTHESIS

Chemical and Stereochemical Nature of Amino Acids
Ionic Nature of Amino Acids
Charged Groups in Peptides at Neutral pH
Side-Chain Effects in Other Amino Acids
General Approach to Protection and Amide-Bond Formation
N-Acyl and Urethane-Forming N-Substituents
Amide-Bond Formation and the Side Reaction of Oxazolone Formation
Oxazolone Formation and Nomenclature
Coupling, 2-Alkyl-5(4H)-Oxazolone Formation and Generation of Diastereoisomers from Activated Peptides
Coupling of N-Alkoxycarbonylamino Acids without Generation of
Diastereoisomers: Chirally Stable 2-Alkoxy-5(4H)-Oxazolones
Effects of the Nature of the Substituents on the Amino and Carboxyl Groups of the Residues that are Coupled to Produce a Peptide
Introduction to Carbodiimides and Substituted Ureas
Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids
Carbodiimide-Mediated Reactions of N-Acylamino Acids and Peptides
Preformed Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids
Purified Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids
Obtained Using a Soluble Carbodiimide
Purified 2-Alkyl-5(4H)-Oxazolones from N-Acylamino and N-Protected Glycylamino Acids
2-Alkoxy-5(4H)-Oxazolones as Intermediates in Reactions of
N-Alkoxycarbonylamino Acids
Revision of the Central Tenet of Peptide Synthesis
Strategies for the Synthesis of Enantiomerically Pure Peptides
Abbreviated Designations of Substituted Amino Acids and Peptides
Literature on Peptide Synthesis

METHODS FOR THE FORMATION OF PEPTIDE BONDS

Coupling Reagents and Methods and Activated Forms
Peptide-Bond Formation from Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids
Factors Affecting the Course of Events in Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids
Intermediates and Their Fate in Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids
Peptide-Bond Formation from Preformed Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids
Peptide-Bond Formation from Mixed Anhydrides of N-Alkoxycarbonylamino Acids
Alkyl Chloroformates and Their Nomenclature
Purified Mixed Anhydrides of N-Alkoxycarbonylamino Acids and Their Decomposition to 2-Alkoxy-5(4H)-Oxazolones
Peptide-Bond Formation from Activated Esters of N-Alkoxycarbonylamino Acids
Anchimeric Assistance in the Aminolysis of Activated Esters
On the Role of Additives as Auxiliary Nucleophiles: Generation of Activated Esters
1-Hydroxybenzotriazole as an Additive that Suppresses N-Acylurea Formation by Protonation of the O-Acylisourea
Peptide-Bond Formation from Azides of N-Alkoxycarbonylamino Acids
Peptide-Bond Formation from Chlorides of N-Alkoxycarbonylamino Acids: N-9-Fluorenylmethoxycarbonylamino-Acid Chlorides
Peptide-Bond Formation from 1-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline–Mediated Reactions of N-Alkoxycarbonylamino Acids
Coupling Reagents Composed of an Additive Linked to a Charged Atom Bearing Dialkylamino Substituents and a Nonnucleophilic Counter-Ion
Peptide-Bond Formation from Benzotriazol-1-yl-Oxy-tris(Dimethylamino)Phosphonium Hexafluorophosphate–Mediated Reactions of N-Alkoxycarbonylamino Acids
Peptide-Bond Formation from O-Benzotriazol-1-yl-N,N,N’,N’ TetramethyluroniumHexafluorophosphate– and Tetrafluoroborate-Mediated Reactions of N-Alkoxycarbonylamino Acids
Pyrrolidino Instead of Dimethylamino Substituents for the Environmental Acceptability of Phosphonium and Carbenium Salt–Based Reagents
Intermediates and Their Fate in Benzotriazol-1-yl-Oxyphosphonium and Carbenium Salt–Mediated Reactions
1-Hydroxybenzotriazole as Additive in Couplings of N-Alkoxycarbonylamino Acids Effected by Phosphonium and Uronium Salt–Based Reagents
Some Tertiary Amines Used as Bases in Peptide Synthesis
The Applicability of Peptide-Bond Forming Reactions to the Coupling of N-Protected Peptides Is Dictated by the Requirement to Avoid Epimerization: 5(4H)-Oxazolones from Activated Peptides
Methods for Coupling N-Protected Peptides
On the Role of 1-Hydroxybenzotriazole as an Epimerization Suppressant in Carbodiimide-Mediated Reactions
More on Additives
An Aid to Deciphering the Constitution of Coupling Reagents from Their Abbreviations

PROTECTORS AND METHODS OF DEPROTECTION

The Nature and Properties Desired of Protected Amino Acids
Alcohols from which Protectors Derive and Their Abbreviated Designations
Deprotection by Reduction: Hydrogenolysis
Deprotection by Reduction: Metal-Mediated Reactions
Deprotection by Acidolysis: Benzyl-Based Protectors
Deprotection by Acidolysis:tert-Butyl-Based Protectors
Alkylation due to Carbenium Ion Formation during Acidolysis
Deprotection by Acid-Catalyzed Hydrolysis
Deprotection by Base-Catalyzed Hydrolysis
Deprotection by beta-Elimination
Deprotection by beta-Elimination: 9-Fluorenylmethyl-Based Protectors
Deprotection by Nucleophilic Substitution by Hydrazine or Alkyl Thiols
Deprotection by Palladium-Catalyzed Allyl Transfer
Protection of Amino Groups: Acylation and Dimer Formation
Protection of Amino Groups: Acylation without Dimer Formation
Protection of Amino Groups: tert-Butoxycarbonylation
Protection of Carboxyl Groups: Esterification
Protection of Carboxyl, Hydroxyl, and Sulfhydryl Groups by tert-Butylation and Alkylation
Protectors Sensitized or Stabilized to Acidolysis
Protecting Group Combinations

CHIRALITY IN PEPTIDE SYNTHESIS

Mechanisms of Stereomutation: Acid-Catalyzed Enolization
Mechanisms of Stereomutation: Base-Catalyzed Enolization
Enantiomerization and Its Avoidance during Couplings of N-Alkoxycarbonyl-L-Histidine
Mechanisms of Stereomutation: Base-Catalyzed Enolization of Oxazolones Formed from Activated Peptides
Mechanisms of Stereomutation: Base-Induced Enolization of Oxazolones Formed from Activated N-Alkoxycarbonylamino Acids
Stereomutation and Asymmetric Induction
Terminology for Designating Stereomutation
Evidence of Stereochemical Inhomogeneity in Synthesized Products
Tests Employed to Acquire Information on Stereomutation
Detection and Quantitation of Epimeric Peptides by NMR Spectroscopy
Detection and Quantitation of Epimeric Peptides by HPLC
External Factors that Exert an Influence on the Extent of Stereomutation During Coupling
Constitutional Factors that Define the Extent of Stereomutation During Coupling: Configurations of the Reacting Residues
Constitutional Factors that Define the Extent of Stereomutation During Coupling: The N-Substituent of the Activated Residue or the Penultimate Residue
Constitutional Factors that Define the Extent of Stereomutation During Coupling: The Aminolyzing Residue and its Carboxy Substituent
Constitutional Factors that Define the Extent of Stereomutation During Coupling: The Nature of the Activated Residue
Reactions of Activated Forms of N-Alkoxycarbonylamino Acids in the Presence of Tertiary Amine
Implications of Oxazolone Formation in the Couplings of N-Alkoxycarbonlyamino Acids in the Presence of Tertiary Amine
Enantiomerization in 4-Dimethylaminopyridine-Assisted Reactions of N-Alkoxycarbonylamino Acids
Enantiomerization During Reactions of Activated N-Alkoxycarbonylamino Acids with Amino Acid Anions
Possible Origins of Diastereomeric Impurities in Synthesized Peptides
Options for Minimizing Epimerization during the Coupling of Segments
Methods for Determining Enantiomeric Content
Determination of Enantiomers by Analysis of Diastereoisomers
Formed by Reaction with a Chiral Reagent

SOLID-PHASE SYNTHESIS

The Idea of Solid-Phase Synthesis
Solid-Phase Synthesis as Developed by Merrifield
Vessels and Equipment for Solid-Phase Synthesis
A Typical Protocol for Solid-Phase Synthesis
Features and Requirements for Solid-Phase Synthesis
Options and Considerations for Solid-Phase Synthesis
Polystyrene Resins and Solvation in Solid-Phase Synthesis
Polydimethylacrylamide Resin
Polyethyleneglycol-Polystyrene Graft Polymers
Terminology and Options for Anchoring the First Residue
Types of Target Peptides and Anchoring Linkages
Protecting Group Combinations for Solid-Phase Synthesis
Features of Synthesis Using Boc/Bzl Chemistry
Features of Synthesis Using Fmoc/tBu Chemistry
Coupling Reagents and Methods for Solid-Phase Synthesis
Merrifield Resin for Synthesis of Peptides Using Boc/Bzl Chemistry
Phenylacetamidomethyl Resin for Synthesis of Peptides Using Boc/Bzl Chemistry
Benzhydrylamine Resin for Synthesis of Peptide Amides Using Boc/Bzl Chemistry
Resins and Linkers for Synthesis of Peptides Using Fmoc/tBu Chemistry
Resins and Linkers for Synthesis of Peptide Amides Using Fmoc/tBu Chemistry
Resins and Linkers for Synthesis of Protected Peptide Acids and Amides
Esterification of Fmoc-Amino Acids to Hydroxymethyl Groups of Supports
2-Chlorotrityl Chloride Resin for Synthesis Using Fmoc/tBu Chemistry
Synthesis of Cyclic Peptides on Solid Supports
REACTIVITY, PROTECTION, AND SIDE REACTIONS
Protection Strategies and the Implications Thereof
Constitutional Factors Affecting the Reactivity of Functional Groups
Constitutional Factors Affecting the Stability of Protectors
The e-Amino Group of Lysine
The Hydroxyl Groups of Serine and Threonine
Acid-Induced O-Acylation of Side-Chain Hydroxyls and the O-to-N Acyl Shift
The Hydroxyl Group of Tyrosine
The Methylsulfanyl Group of Methionine
The Indole Group of Tryptophan
The Imidazole Group of Histidine
The Guanidino Group of Arginine
The Carboxyl Groups of Aspartic and Glutamic Acids
Imide Formation from Substituted Dicarboxylic Acid Residues
The Carboxamide Groups of Asparagine and Glutamine
Dehydration of Carboxamide Groups to Cyano Groups During Activation
Pyroglutamyl Formation from Glutamyl and Glutaminyl Residues
The Sulfhydryl Group of Cysteine and the Synthesis of Peptides Containing Cystine
Disulfide Interchange and Its Avoidance during the Synthesis of Peptides Containing Cystine
Piperazine-2,5-Dione Formation from Esters of Dipeptides
N-Alkylation during Palladium-Catalyzed Hydrogenolytic Deprotection and Its Synthetic Application
Catalytic Transfer Hydrogenation and the Hydrogenolytic Deprotection of Sulfur-Containing Peptides
Mechanisms of Acidolysis and the Role of Nucleophiles
Minimization of Side Reactions during Acidolysis
Trifunctional Amino Acids with Two Different Protectors

VENTILATION OF ACTIVATED FORMS AND COUPLING METHODS

Notes on Carbodiimides and Their Use
Cupric Ion as an Additive that Eliminates Epimerization in Carbodiimide-Mediated Reactions
Mixed Anhydrides: Properties and Their Use
Secondary Reactions of Mixed Anhydrides: Urethane Formation
Decomposition of Mixed Anhydrides: 2-Alkoxy-5(4H)-Oxazolone Formation and Disproportionation
Activated Esters: Reactivity
Preparation of Activated Esters using Carbodiimides and Associated Secondary Reactions
Other Methods for the Preparation of Activated Esters of N-Alkoxycarbonylamino Acids
Activated Esters: Properties and Specific Uses
Methods for the Preparation of Activated Esters of Protected Peptides, Including Alkyl Thioesters
Synthesis using N-9-Fluorenylmethoxycarbonylamino Acid Chlorides
Synthesis using N-Alkoxycarbonylamino-Acid Fluorides
Amino-Acid N-Carboxyanhydrides: Preparation and Aminolysis
N-Alkoxycarbonylamino-Acid N-Carboxyanhydrides
Decomposition during the Activation of Boc-Amino Acids and Consequent Dimerization
Acyl Azides and the Use of Protected Hydrazides
O-Acyl and N-Acyl N’-Oxide Forms of 1-Hydroxybenzotriazole Adducts and the Uronium and Guanidinium Forms of Coupling Reagents
Phosphonium and Uronium/Aminium/Guanidinium Salt–Based Reagents: Properties and Their Use
Newer Coupling Reagents
To Preactivate or not to Preactivate: Should That Be the Question?
Aminolysis of Succinimido Esters by Unprotected Amino Acids or Peptides
Unusual Phenomena Relating to Couplings of Proline
Enantiomerization of the Penultimate Residue During Coupling of an N^µ-Protected Peptide
Double Insertion in Reactions of Glycine Derivatives: Rearrangement of Symmetrical Anhydrides to Peptide-Bond-Substituted Dipeptides
Synthesis of Peptides by Chemoselective Ligation
Detection and Quantitation of Activated Forms

MISCELLANEOUS

Enantiomerization of Activated N-Alkoxycarbonylamino Acids and Esterified Cysteine Residues in the Presence of Base
Options for Preparing N-Alkoxycarbonylamino Acid Amides and 4-Nitroanilides
Options for Preparing Peptide Amides
Aggregation during Peptide-Chain Elongation and Solvents for its Minimization
Alkylation of Peptide Bonds to Decrease Aggregation: 2-Hydroxybenzyl Protectors
Alkylation of Peptide Bonds to Decrease Aggregation: Oxazolidines and Thiazolidines (Pseudo-Prolines)
Capping and the Purification of Peptides
Synthesis of Large Peptides in Solution
Synthesis of Peptides in Multikilogram Amounts
Dangers and Possible Side Reactions Associated with the Use of Reagents and Solvents
Organic and Other Salts in Peptide Synthesis
Reflections on the Use of Tertiary and Other Amines
Monomethylation of Amino Groups and the Synthesis of N-Alkoxycarbonyl-N-Methylamino Acids
The Distinct Chiral Sensitivity of N-Methylamino Acid Residues and Sensitivity to Acid of Adjacent Peptide Bonds
Reactivity and Coupling at -Methylamino Acid Residues
APPENDICES
Useful Reviews
Year, Location and Chairmen of the Major Symposia
On the "Primary Sequence" of Peptides and Proteins
Index

Biography

N. Leo Benoiton

”This book provides the mechanistic basis for developing rational strategies in peptide synthesis. ...The author’s unpretentious writing style belies the authoritative and sophisticated textual content. Over the course of well cross-referenced chapters, this treatise quickly transitions from offering a novice the principles of peptide science to engaging expert biochemists. The narrative flows unerringly as it guides the reader through 207 mechanism-based figures. Many such figures brilliantly superimpose the complex realities of deviant side reaction pathways onto the intended reaction course. In so doing, the study allows for the development of predictive skills in identifying a priori dead-end pathways.”
“Benoiton, in the fashion of a true scholar, relishes communicating the science of peptide synthesis, a field that he has pioneered. He understands the peptide bond as only an enzyme might and, in the course of this text, has ascribed explicit personalities to amino acids, their quirks notwithstanding. This extraordinary book belongs in all academic and research environments involved in peptide chemistry.”
— Kennerly S. Patrick, South Carolina College of Pharmacy, Medical University of South Carolina, in the Journal of Medicinal Chemistry, Vol. 49, No. 8 (2006)

”Leo Benoiton is an experienced peptide chemist who has made fundamental contributions to the chemistry of peptide synthesis. ...The publication of such a book is timely, and could make an important contribution to the field ... useful chemistry related to peptide synthesis is well discussed in this book, particularly in highly specialized or advanced topics such as partial epimerization (racemization), coupling methods/activation, and the molecular origins of the aggregation/insolubility of protected peptides. ...”
— Stephen Kent, Institute for Biophysical Dynamics, University of Chicago, in Angewandte Chemie, Int’l Edition, Vol. 45, No. 26 (2006)

“ Despite its apparent simplicity as depicted in most textbooks, the synthesis of peptides in the many structural manifestations has many problems and pitfalls that often befuddle the synthetic chemist who has never made a peptide before. Having watched over 300 undergraduate and graduate students, postdoctoral associates, and more senior scientists, struggle with doing peptide synthesis and then having to send them to the library to read the primary literature so that they would finally “get it”, the appearance of N. Leo Benoiton’s Chemistry of Peptide Synthesis is a godsend. In fewer than 300 pages Professor Benoiton has distilled the essence of peptide synthesis—the strategies; the tactics; the pitfalls; the mechanisms; and so forth, as has done so in the “simple” language of synthetic and mechanistic organic chemistry. Of course one can always quibble with this point or that, argue that more could have been said or discussed about some aspect of peptide synthesis, but if you are new in peptide synthesis and want to get the “full picture” or are a more experienced peptide synthetic chemist and are branching into new aspects, there is no better place either to start your education in peptide synthesis or to become introduced to and familiar with some unfamiliar aspects of synthetic peptide chemistry than this book.
“The book consists of 8 chapters in what might be called a classical organization of the field. Chapter 1 is a short but masterful exposition of the properties of amino acids and of how these properties impact in peptide synthesis and synthetic tactics. Make sure you read this chapter before you proceed. Chapters 2, 3, and 4 carefully discuss the basic strategies and tactics in peptide synthesis primarily as developed in solution phase synthesis. Chapter 2 discusses methods for peptide bond formation; Chapter 3, protecting groups and methods of deprotection and Chapter 4, problems in chirality in peptide synthesis, how to study it, how to quantify, etc. and how to minimize side reactions. I particularly enjoyed Chapter 4—not only because the author and his group have had a major impact on our understanding of issues of chirality that arise in peptide synthesis, but also because these issues are rarely discussed in any detail in current synthetic peptide chemistry chapters. Chirality nonetheless appears as a problem more often than admitted in most peptide synthesis papers. So when it does, here is the place to read all about it. Chapter 5 provides a basic introduction to the use of solid phase peptide synthesis. It concentrates very much on the fundamentals of preparing linear peptides on a solid support. Chapter 6 provides an excellent discussion of the reactivity, protection, deprotection, and side reactions which result from the presence of the several function groups that are found in the 20 standard amino acids that make up proteins and related polypeptides. A clear exposition is given of the side reactions that can result and how they can be eliminated or minimized. This chapter is a must-read for neophytes in peptide synthesis even if they already have considerate experience in other areas of synthetic chemistry. A careful read of this chapter will prevent many problems of this chapter will prevent many problems that can arise in peptide synthesis. Chapters 7 and 8, the last two chapters in the book, discuss a number of issues that can arise in the synthesis of peptides dealing with coupling reagents, activated esters, anhydrides, mixed anhydrides, etc. These chapters contain many useful ideas and thoughts of the author that are invaluable for their usefulness and for their careful considerations.
Perhaps more could have been said about cyclic peptides: disulfides, lactams, head-to-tail cyclic peptides, lactones, etc., and the difficulties that can occur in their synthesis. And virtually nothing is said about peptide ligation chemistry or synthesis of peptide conjugates. In all fairness, however, as the author clearly states in his introduction, this book was written to expose the fundamentals of peptide synthesis.  In this respect, the author has produced a superb, scholarly treatment of the subject. This book will be very useful for many years to come, and I highly recommend it to those with a need to apply peptide synthesis, or those who wish to educate themselves to the field of peptide synthesis. All readers will learn a wealth of information and obtain many useful insights along the way. It is essential reading for all of my students.”
 —Victor J. Hruby, Regents Professor of Chemistry, Department of Chemistry, University of Arizona, Tucson, USA, written in PeptideScience, Vol. 88, No. 6 (November 2007)