Evaluation of the Synthetic Product

In 1987, an article appeared in the International Journal of Peptide and Protein Research commemorating the 25th anniversary of the development of solid phase peptide synthesis (Barany et al., 1987). While that article dealt with many aspects of peptide synthesis, one statement in particular stands out as exemplifying the rationale for this chapter. It states: “No synthetic endeavor can be considered complete until the product has been adequately purified and subjected to a battery of analytical tests to verify its structure.” The characterization or evaluation of a synthetic peptide is the one step in its production and experimental utilization that will validate the experimental data obtained. Unfortunately, it is also the one step that many investigators all too often give too little attention. If the synthetic product, upon which the theory and performance of the experimental investigation is based, is not the intended product, the conclusions will be incorrect. Without proper characterization, the investigator will either have to be lucky, or be wrong. Worse yet, he or she will not know which is the case. Although today the synthesis of a given peptide is often considered routine, the product should never be taken for granted. Peptide synthesis chemistry, although quite sophisticated, is complex and subject to a variety of problems. These problems, which can manifest themselves as unwanted side reactions and decreased reaction efficiency, are subject to a variety of factors such as reagent quality, incompatible chemistries, instrument malfunctions, sequence specific effects, and operator error. Although every effort is made to eliminate their causes and to plan for potential problems in the design and synthesis steps, it is not always successful and the eventual outcome of a synthesis is not always predictable. One must never assume that the final product is the expected one until that has been proven to be the case. To do otherwise may seriously jeopardize the outcome of the research. Used and performed properly, the evaluation stage is where the fruits of the synthesis are scrutinized and the decision is made to use the peptide as intended, submit it to further purification, or resynthesize it and possibly change elements of the design or synthesis protocols.

Applications of Synthetic Peptides

The tremendous advances in the development of methods for the design and synthesis of peptides. pseudo-peptides and related compounds, as well as the corresponding advances in our understanding of peptide and protein structure, conformation, topography, and dynamics provides unique opportunities to apply designed synthetic peptides for an enormous variety of problems in chemistry, biology, and medicine. In addition, if these advances can be coupled to the advances in molecular biology and the human genome project, on the one hand, and asymmetric synthesis and catalysis, on the other, it should be possible to provide hitherto unavailable, indeed unthinkable, approaches to diverse areas of drug design, behavioral neuroscience, molecular immunology, chemotherapy, and a wide variety of other uses. Already it is clear that peptide therapy has enormous potential in such diverse areas as growth control, blood pressure management, neurotransmission, hormone action, satiety, addiction, pain, digestion, reproduction, and so forth. Nature has “discovered” that it can control nearly all biological processes by various kinds of molecular recognition, and that peptides and proteins are uniquely suited for this control because of their enormous potential for diversity and their unique physico-chemical properties. This finding may, perhaps, be most readily understood if one recognizes that, considering only the 20 normal eukaryotic amino acids, the number of unique chemical entities for a pentapeptide is 3,200,000 (205), for a hexapeptide it is 64,000,000 (206), and so on. Considered from this perspective, perhaps it is not unexpected that Nature has “discovered” that peptides and proteins can do it all, from providing structure and motion, to catalysis, to information transduction, to growth and maturation, and so on. The ability of the immune system in higher animals, including humans, to recognize literally millions of foreign materials made by Nature as well as humans, and to get rid of them as part of its survival strategy, is just one example that illustrates the potential of peptide-based drugs, therapeutics, and modulators of biological function. Despite the enormous potential of peptides and small proteins for these areas, surprisingly little advantage has been taken of the potential of these molecules as drugs and tools for use in basic and clinical research.

Principles and Practice of Solid-Phase Peptide Synthesis

Peptides play key structural and functional roles in biochemistry, pharmacology, and neurobiology, and are important probes for research in enzymology, immunology, and molecular biology. The amino acid building blocks can be among the 20 genetically encoded L-residues, or else unusual ones, and the sequences can be linear, cyclic, or branched. It follows that rapid, efficient, and reliable methodology for the chemical synthesis of these molecules is of utmost interest. A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the sweet dipeptide L-Asp-L-Phe-OMe (aspartame) to clinically used hormones such as oxytocin, adrenocorticotropic hormone, calcitonin, and gonadotropin releasing hormone (GnRH) super-agonists. Synthesis can lead to potent and selective new drugs by judicious substitutions that change functional groups and/or conformations of the parent peptide. These include introduction of N- or C-alkyl substituents, unnatural or D-amino acids, side-chain modifications including sulfate or phosphate groups or carbohydrate moieties, and constraints such as disulfide bridges between half-cystines or side-chain lactams between Lys and Asp or Glu. Commercially important products that evolved from such studies include protease inhibitors, such as captopril and other angiotensin converting enzyme (ACE) inhibitors, peptidomimetic HIV protease inhibitors, and the somatostatin analog lanreotide. Most of the biologically or medicinally important peptides which are the targets for useful structure-function studies by chemical synthesis comprise under 50 amino acid residues, but occasionally a synthetic approach can lead to important conclusions about small proteins (full or domains) in the 100-200 residue size range. Methods for synthesizing peptides are divided conveniently into two categories: solution (classical) and solid-phase pep tide synthesis (SPPS). The classical methods have evolved since the beginning of the twentieth century, and they are described amply in several reviews and books (Wünsch, 1974; Finn and Hofmann, 1976; Bodanszky and Bodanszky, 1984; Goodman et al, 2001). The solid-phase alternative was conceived and elaborated by R. B. Merrifield beginning in 1959, and has also been covered comprehensively (Erickson and Merrifield, 1976; Birr, 1978; Barany and Merrifield, 1979; Stewart and Young, 1984; Merrifield, 1986; Barany et al., 1987, 1988; Kent, 1988; Atherton and Sheppard, 1989; Fields and Noble, 1990; Barany and Albericio, 1991; Fields et al., 1992; Gutte, 1995; Fields, 1997; Lloyd-Williams et al., 1997; Chan and White, 2000; Kates and Albericio, 2000).

Peptide Design Considerations

Peptides have become an increasingly important class of molecules in biochemistry, medicinal chemistry, and physiology. Many naturally occurring, physiologically relevant peptides function as hormones, neurotransmitters, cytokines, and growth factors. Peptide analogs that possess agonist or antagonist activity are useful as tools to study the biochemistry, physiology, and pharmacology of these peptides, to characterize their receptor(s), and to study their biosynthesis, metabolism, and degradation. Radiolabeled analogs and analogs bearing affinity labels have been used for receptor characterization and isolation. Peptide substrates of proteases, kinases, phosphatases, and aminoacyl or glycosyl transferases are used to study enzyme kinetics, mechanism of action, and biochemical and physiological roles and to aid in the isolation of enzymes and in the design of inhibitors. Peptides are also used as synthetic antigens for the preparation of polyclonal or monoclonal antibodies targeted to specific sequences. Epitope mapping with synthetic peptides can be used to identify specific antigenic peptides for the preparation of synthetic vaccines, to determine protein sequence regions that are important for biological action, and to design small peptide mimetics of protein structure or function. A number of peptide hormones or analogs thereof, including arginine vasopressin, oxytocin, luteinizing hormone releasing hormone (LHRH), adrenocorticotropic hormone (ACTH), and calcitonin, have already found use as therapeutic agents, and many more are being investigated actively. Peptide-based inhibitors of proteolytic enzymes, such as angiotensin converting enzyme (ACE) and human immunodeficiency virus (HIV) protease, have widespread clinical use, and inhibitors of renin and elastase are also being investigated for therapeutic use. Finally, peptides designed to block the interaction of protein molecules by mimicking the combining site of one of the proteins, such as the fibrinogen receptor antagonists, show great therapeutic potential as well. With the development of solid-phase peptide synthesis by Bruce Merrifield (1963) and the optimization of supports, protecting groups, and coupling and deprotection chemistries by a large number of researchers, it has become possible to obtain useful amounts of peptides on a more or less routine basis.

Synthetic Peptides: Beginning the Twenty-first Century

This second edition of Synthetic Peptides is being published at the beginning of the twenty-first century and marks nearly 100 years since the beginnings of the chemical synthesis of peptides. From the first decade of the twentieth century up to the present time, the evolution of the development, analysis, and use of synthetic peptides has been steady and remarkable. It has been about 10 years since the first edition of this book was published. Much remains unchanged, such as the basic principles of peptide structure, the basic chemistry for assembling a peptide chain, and many of the techniques used to evaluate synthetic peptides. However, during that time we have seen a switch from primarily the use of Boc chemistry for routine synthesis to that of Fmoc chemistry. Mass spectrometry has also matured with the development of more user-friendly and affordable instrumentation to the point that it is now the premier analytic method for synthetic peptides. Methods for the production of very long peptides, such as chemoselective ligation, are maturing, although they are still not an everyday thing for most peptide chemists, and better chemistries for producing peptides with “posttranslational modifications,” such as phosphates, sugars, and specific disulfide bonds, are now well within reach. As a result, you will find many sections of this book largely unchanged, but you will also find many new sections that document the developments of the last ten years with the inclusion of new information and methodologies. However, a good feeling for what the beginning of the twenty-first century offers can best be appreciated by considering the developments that have led to this point. Emil Fischer introduced the concept of peptides and polypeptides and presented protocols for their synthesis in the early 1900s (Fischer, 1902, 1903, 1906). Although others also made contributions in those days, most notably Theodor Curtius, the work of Fischer and his colleagues stands out, and he is generally regarded as the father of peptide chemistry.