A 65-page guide on solid phase peptide synthesis SPPS offers practical advice based on more than 60 years of combined experience. It covers the two main synthetic strategies, Boc/Benzyl and Fmoc/tButyl chemistries, with detailed experimental sections and helpful hints for newcomers.
A 65-page guide on solid phase peptide synthesis SPPS provides practical advice drawn from more than 60 years of combined experience in making peptides. The guide has two purposes: it gives a brief introduction to the development and most common applications of SPPS, and it describes peptide synthesis, cleavage, and purification in detail with helpful hints for newcomers.
Solid phase synthesis is a process that allows chemical transformations to occur on a solid support. This approach was first developed by Bruce Merrifield for polypeptide synthesis, earning him the Nobel Prize in 1984. Solid phase chemistry offers advantages over conventional synthesis in terms of efficiency, work-up, and purification. In solution phase peptide synthesis, especially for longer sequences, repeated coupling and deprotection cycles become labor intensive and require isolation of all peptide intermediates.
The chemistry of peptide synthesis began in the early 1900s with Emil Fischer. For many years, peptide synthesis relied on solution-phase methods, where each reaction step involved purification and characterization. However, peptide synthesis is an iterative process: alpha-amino deprotection and amide couplings are repeated until the full-length target peptide is obtained. Additionally, most biologically relevant peptides are poorly soluble in organic solvents, regardless of side-chain protection. These two characteristics limited progress until the late 1950s, when R.B. Merrifield at Rockefeller University proposed a new approach.
During his Ph.D. work, Merrifield suggested performing all synthetic manipulations with the C-terminus of the target peptide linked to an insoluble solid support. This idea was initially criticized but soon proved faster and more versatile than traditional solution-phase methods. The original version of SPPS, called Boc/Benzyl chemistry, was finalized in the late 1960s. It uses a graduated acid lability system: the alpha-amino Boc protecting group is removed with TFA, while side-chain protections and the peptide-resin anchor require harsher acidic conditions using liquid HF. HF is a much stronger acid than TFA, with acidity functions of -11 and 0.1, respectively.
Solid phase peptide synthesis simplifies reactions by allowing purification through filtration. Excess reagents can be used and then removed by simple washing. The chemistry of SPPS is not fundamentally different from solution-phase peptide synthesis. The only chemical distinction is that in SPPS, the C-terminal protecting group is rendered insoluble by being incorporated into a polymer. All side-chain and alphaN protecting groups, as well as coupling chemistries used in solid phase, have also been successfully applied in solution phase, and vice versa.
During the 1970s, several groups worked on developing milder methods for SPPS that avoided liquid HF for final deprotection and cleavage. A variety of milder graduated acid lability systems were devised.
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