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Solid-phase peptide synthesis
(SPPS), pioneered by Robert Bruce
Merrifield, resulted in a paradigm shift within the peptide synthesis community. It
is now the accepted method for creating peptides and proteins in the lab in a synthetic manner. SPPS allows the synthesis of natural peptides which are difficult
to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins,
which consist of D-amino acids.
Small solid beads, insoluble yet
porous, are treated with functional units ('linkers') on which peptide chains
can be built. The peptide will remain covalently attached to the bead until
cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus 'immobilized' on the solid-phase
and can be retained during a filtration process, whereas liquid-phase reagents
and by-products of synthesis are flushed away.
The general principle of SPPS is
one of repeated cycles of coupling-wash-deprotection-wash. The free N-terminal
amine of a solid-phase attached peptide is coupled (see below) to a single
N-protected amino acid unit. This unit is then deprotected, revealing a new
N-terminal amine to which a further amino acid may be attached. The superiority
of this technique partially lies in the ability to perform wash cycles after
each reaction, removing excess reagent with all of the growing peptide of
interest remaining covalently attached to the insoluble resin.
The overwhelmingly important
consideration is to generate extremely high yield in each step. For example, if
each coupling step were to have 99% yield, a 26-amino acid peptide would be
synthesized in 77% final yield (assuming 100% yield in each deprotection); 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 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 either of these two groups and added onto a deprotected amino acid
Automated synthesizers are
available for both techniques, though many research groups continue to perform
SPPS is limited by yields, and typically peptides and proteins in the range of 70 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.
Since its introduction over 40
years ago, SPPS has been significantly optimized. First, the resins themselves
have been optimized. Furthermore, the ¡®linkers¡¯ between the C-terminal amino acid and
polystyrene resin have improved attachment and cleavage to the point of mostly
quantitative yields. The evolution of side chain protecting groups has limited the frequency
of unwanted side reactions. In addition, the evolution of new activating groups
on the carboxyl group of the incoming amino acid have improved coupling and
decreased epimerization. Finally, the process itself has been optimized. In
Merrifield¡¯s initial report, the deprotection of the ¥á-amino group resulted in
the formation of a peptide-resin salt, which required neutralization with base
prior to coupling. The time between neutralization of the amino group and
coupling of the next amino acid allowed for aggregation of peptides, primarily
through the formation of secondary structures, and adversely affected coupling.
The Kent group showed that concomitant neutralization of the ¥á-amino group and
coupling of the next amino acid led to improved coupling. Each of these improvements has helped SPPS become the robust technique
that it is today.
Tel 031) 292-6331, CP 010-4015-6331