5

Solid-phase synthesis means synthesis on a solid support. It is a technology that dates back to the early 1960s — an era when ion exchange on functionalized polystyrene beads was the prominent method for purification and analysis of small charged molecules. R. B. Merrifield, an immunologist working in the laboratory of D. W. Woolley at the Rockefeller Institute in New York, was required to synthesize analogues of biologically active peptides. In the process, Merrifield perceived that the approach of the day, made up of successive coupling and deprotection reactions in solution, followed by extractions at each step to eliminate unconsumed reactants and secondary products, was very labor intensive and repetitive, and he concluded that there was a need for a rapid and automatic method for the synthesis of peptides. He suggested that the synthesis be carried out with the first residue attached to an insoluble support (Figure 5.1), so that purification could be achieved by simple filtration instead of extraction. According to his proposal, a protected amino acid is anchored to an insoluble functionalized support by a bond that resists all chemistries employed during assembly of the peptide. The amino group is deprotected, and additional residues are introduced successively. Each reaction is followed by filtration, which removes unconsumed reactants and secondary products that are dissolved in the solvent. Final deprotection detaches the peptide from the support. Three advantages were envisioned by Merrifield: high yields achieved by forcing the reactions to completion, less manipulation and consequently less time, and minimized losses of material because reactions and purification would take place without removing the peptide support from the reaction vessel. Successful implementation of the method was announced in 1962, with the first publication appearing the following year. For the first 10 years or so, the method faced considerable opposition

by the traditionalists, but this resistance gradually dissipated. Twenty years of development and refinement by Merrifield and colleagues and others culminated in the award in 1984 of the Nobel Prize in Chemistry to Merrifield for development of a methodology for chemical synthesis on a solid matrix. The method has since found no end of applications. It is interesting to note that an analogous approach was employed by others in 1963 to synthesize a dipeptide, the amino-terminal residue being fixed to a polystyrene support through an amide bond.1,2

Initial attempts to develop the solid-phase method (see Section 5.01) involved attachment of the first residue to a functionalized copolymer of styrene and divinylbenzene, with the trade name Dowex 50, as the benzyl ester with benzyloxycarbonyl for protection of α-amino groups and selective deprotection by acidolysis with hydrogen bromide in acetic acid (see Section 3.5). The method was inefficient, however, because of the significant loss of the chain from the support at each step. The anchoring linkage was stabilized to acidolysis by nitration of the phenyl ring of the benzyl moiety (see Section 3.19), and this allowed the first successful synthesis of a peptide; namely, L-leucyl-L-alanylglycyl-L-valine. Unreacted amino groups were capped by acetylation with triethylammonium acetate after each coupling. The chain was detached from the resin by saponification. The structure was established by comparison with an authentic sample prepared in solution using 4-nitrophenyl esters (see Section 2.9) for peptide-bond formation. It was obvious, however, that the combination of two benzyl-based protectors was not going to be satisfactory. Fortunately, the tert-butoxycarbonyl protector (see Section 3.6) had just become available, and an improved methodology was developed. Figure 5.2 shows the scheme employed in 1964 by Merrifield to prepare bradykinin, which contains nine residues — the first biologically active peptide synthesized by his method. The first residue was attached to a polystyrene-divinylbenzene copolymer as the benzyl ester by reaction of Boc-Nω-nitroarginine with the chloromethylated polymer (see Section 5.7) in the presence of triethylamine. The Boc-protector was removed by acidolysis with hydrogen chloride in acetic acid, and the amine hydrochloride that was generated was neutralized with triethylamine. Liberated tert-butoxycarbonyl produces isobutene and carbon dioxide.