9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids

It is a privilege to be able to contribute to this volume in which Professor Zervas’ friends, students, and colleagues have joined together to honor him and his remarkable contributions to peptide chemistry. Although I did not know him until after his days in the Bergmann Laboratory at the Rockefeller Institute, I can claim the distinction of now working in those very same rooms that they occupied back in the mid-1930s. Like all peptide chemists, I am greatly indebted to Professor Zervas, having depended so heavily on the carbobenzoxy group and on the various modified urethan protecting groups which have been direct extensions of the revolutionary advance that Bergmann and Zervas made.

Solid-phase peptide synthesis.

The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins – mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and Nα-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form β-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of β-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).

Microwave heating in solid-phase peptide synthesis

The last decade has witnessed a renaissance of peptides as drugs. This progress, together with advances in the structural behavior of peptides, has attracted the interest of the pharmaceutical industry in these molecules as potential APIs. In the past, major peptide-based drugs were inspired by sequences extracted from natural structures of low molecular weight. In contrast, nowadays, the peptides being studied by academic and industrial groups comprise more sophisticated sequences. For instance, they consist of long amino acid chains and show a high tendency to form aggregates. Some researchers have claimed that preparing medium-sized proteins is now feasible with chemical ligation techniques, in contrast to medium-sized peptide syntheses. The complexity associated with the synthesis of certain peptides is exemplified by the so-called “difficult peptides”, a concept introduced in the 80's. This refers to sequences that show inter- or intra-molecular β-sheet interactions significant enough to form aggregates during peptide synthesis. These structural associations are stabilized and mediated by non-covalent hydrogen bonds that arise on the backbone of the peptide and—depending on the sequence—are favored. The tendency of peptide chains to aggregate is translated into a list of common behavioral features attributed to “difficult peptides” which hinder their synthesis. In this regard, this manuscript summarizes the strategies used to overcome the inherent difficulties associated with the synthesis of known “difficult peptides”. Here we evaluate several external factors, as well as methods to incorporate chemical modifications into sequences, in order to describe the strategies that are effective for the synthesis of “difficult peptides”. These approaches have been classified and ordered to provide an extensive guide for achieving the synthesis of peptides with the aforementioned features.

The road to the synthesis of “difficult peptides”

A library of peptides required for a project investigating the factors relevant for blood-brain barrier transport was synthesized on solid phase. As a result of the high N-methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N-methylamino acids with N-methylamino acids generally occurs in low yield. (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1-hydroxy-7-azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N-methylamino acid at the N-terminal position, loss of Ac-N-methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N-methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high-performance liquid chromatography (HPLC) profiles of N-methyl-rich peptides show multiple peaks because of slow conversion between conformers.

Solid-phase synthesis and characterization of N-methyl-rich peptides.

Mass spectrometric evaluation of synthetic peptides for deletions and insertions.

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Mass spectrometric evaluation of synthetic peptides for deletions and insertions.