A Practical Guide to Solid Phase Peptide Synthesis (SPPS)

Solid phase synthesis is a process by which chemical transformations can be carried out on solid support in order to prepare a wide range of synthetic compounds. This idea was first developed by Bruce Merrifield to synthesize polypeptides and earned him the Nobel Prize in 1984. Solid phase chemistry offers many advantages over conventional synthesis in terms of efficiency as well as convenient work-up and purification procedures. In solution phase peptide synthesis, particularly in longer sequences, the repetition of coupling and deprotection cycles can become very labor intensive and require the isolation of all peptide intermediates.

I. A Brief Historical Perspective The chemistry of peptide synthesis – first developed in the early 1900’s by Emil Fischer – arguably marks the birth of organic synthesis as we know it today. Whereas the lion share of organic synthesis continues to be performed by solution-phase methods, i.e., with each independent chemical reaction followed by a purification step and characterization of the resulting synthetic intermediate, two peculiarities of peptide chemistry spurred the development of a more efficient synthetic strategy. In contrast to most total synthesis efforts, the synthesis of peptides – at least until the final deprotection step – is an iterative process, with alpha-amino (alphaN) deprotection and amide couplings performed in succession until the desired full-length target peptide is obtained. In addition, most peptides of biological interest are grossly insoluble in most organic solvents irrespective of side-chain protection tactics. The net result of these two characteristic features of peptides was that the first sixty-odd years of peptide synthesis forged little ground until the landmark work in the late 1950’s of R.B. Merrifield at Rockefeller University.

During the course of his Ph.D. work, Merrifield (a biochemist) proposed an entirely new paradigm in organic synthesis. As is often the case when an outsider looks into an insular field and pursues a tack that is anathema to the existing experts in the field, Merrifield’s idea of performing all synthetic manipulations using the C-terminus of the target peptide linked to an insoluble solid support was met with much criticism. However, it was not long before the chemistry of solid-phase peptide synthesis (SPPS) was honed to a point where traditional solution-phase methodologies were no match with regard to speed and versatility. The original “Merrifield” version of SPPS – more accurately referred to as Boc/Benzyl chemistry – was roughly finalized in the late 1960s, and employs a graduated acid lability system for manipulation of all protecting groups (Scheme 1). In this strategy, the alpha-amino t-butoxycarbonyl (Boc) protection is removed with TFA, while side-chain protections and the peptide- resin anchorage (the linker) require much harsher acidic conditions for cleavage. This final step is accomplished using liquid HF, a much stronger acid than TFA (acidity functions of -11 and 0.1, respectively).