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Solid-phase peptide synthesis (SPPS) is the accepted method for creating peptides and proteins in the lab in a synthetic manner. This allows the synthesis of natural peptides which are difficult to express in bacteria, incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.
SPPS was pioneered by Bruce Merrifield -- the overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield, if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents.
There are two majorly used forms of SPPS -- FMOC and t-BOC. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by these two groups and added onto a deprotected amino acid chain.
Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.
SPPS is limited by yields, and typically peptides and proteins in the range of 70~100 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.
Fmoc SPPS
This method was introduced by R.C. Sheppard in 1971. Fmoc stands for (F)luorenyl-(m)eth(o)xy-(c)arbonyl which describes the Fmoc protecting group. To remove an Fmoc from a growing peptide chain, basic conditions (usually 20% piperidine in DMF) are used. Removal of side-chain protecting groups and peptide from the resin is achieved by incubating in trifluoroacetic acid (TFA). Fmoc deprotection is usually slow because the anionic nitrogen produced at the end is not a particularly favorable product, although the whole process is thermodynamically driven by the evolution of carbon dioxide.
tBoc SPPS
When R. B. Merrifield invented SPPS in 1963, it was according to the tBoc method. t-Boc (or Boc) stands for (tert)-(B)utyl (o)xy (c)arbonyl. To remove Boc from a growing peptide chain, acidic conditions are used (usually neat TFA). Removal of side-chain protecting groups and the peptide from the resin at the end of the synthesis is achieved by incubating in hydrofluoric acid (which can be dangerous); for this reason Boc chemistry is generally disfavored. However, when synthesizing nonnatural peptide analogs which are base-sensitive (such as depsi-peptides), tBoc is necessary.
"Solid phase peptide synthesis"