Polyeptide synthesis became a more practical part of
present-day scientific research following the advent of solid-phase polyeptide
synthesis techniques.
The concept of solid-phasepolyeptide synthesis (SPPS) is to
retain chemistry that has been proven in solution but to add a covalent
attachment step that links the nascent peptide chain to an insoluble polymeric
support (resin). Subsequently, the anchored peptide is extended by a series of addition
cycles It is the essence of the solid-phase polyeptide
synthesis approach that reactions are driven to completion by the use of
excess soluble reagents, which can be removed by simple filtration and washing
without manipulative losses. Once chain elongation has been completed, the
crude peptide is released from the support.
In the early 1960s, Merrifield proposed the use of a
polystyrene-based solid support for peptide synthesis. Polyeptides could be
assembled stepwise from the C to N terminus using Nalpha-protected amino acids.
From the 1960s through the 1980s, Boc-based SPPS was fine-tuned (Merrifield,
1986). This strategy has been utilized for synthesis of proteins such as interleukin-3
and active enzymes including ribonuclease A and all-L and all-D forms of HIV-1
aspartyl protease.
In 1972, Carpino introduced the 9-fluorenylmethoxycarbonyl
(Fmoc) group for Nalpha protection (Carpino and Han, 1972). The Fmoc group
requires moderate base for removal, and thus offered a chemically mild
alternative to the acid-labile Boc group and in the late 1970s, the Fmoc group
was adopted for solid-phase polypeptide synthesis applications. This milder
conditions of Fmoc peptide chemistry as compared to Boc peptide chemistry—which
include elimination of repetitive moderate acidolysis steps and the final
strong acidolysis step—were envisioned as being more compatible with the
synthesis of peptides that are susceptible to acid-catalyzed side reactions. In
particular, the modification of the indole ring of Trp was viewed as a
particular problem during Boc-based peptide synthesis (Barany and Merrifield,
1979), which could be alleviated using Fmoc chemistry. One example of the
potential advantage of Fmoc chemistry for the synthesis of
multiple-Trp-containing peptides was in the synthesis of gramicidin A.
Gramicidin A, a pentadecapeptide containing four Trp residues, had been
synthesized previously in low yields (5% to 24%) using Boc chemistry. The mild
conditions of Fmoc chemistry dramatically improved the yields of gramicidin A,
in some cases up to 87% (Fields et al., 1989, 1990). A second
multiple-Trp-containing peptide, indolicidin, was successfully assembled in
high yield by Fmoc chemistry (King et al., 1990). Thus, the mild conditions of
Fmoc chemistry appeared to be advantageous for certain peptides, as compared
with Boc chemistry.
The milder conditions of Fmoc peptide synthesis chemistry,
along with improvements in the basic chemistry, have led to a shift in the
chemistry employed by peptide laboratories. Possible reasons for the improved
results were any combination of the following (Angeletti et al., 1997):
1. The greater
percentage of peptides synthesized by Fmoc chemistry, where cleavage conditions
are less harsh.
2. The use of
different side-chain protecting group strategies that help reduce side
reactions during cleavage.
3. The use of
cleavage protocols designed to minimize side reactions.
4. More rigor and
care in laboratory techniques.
The next step in the development of solid-phase
polypeptide synthesis techniques includes applications for peptides
containing non-native amino acids, post-translationally modified amino acids, and
pseudoamino acids, as well as for organic molecules in general. Several areas
of solid-phase synthesis need to be refined to allow for the successful
construction of this next generation of biomolecules. The solid support must be
versatile so that a great variety of solvents can be used, particularly for
organic-molecule applications. Coupling reagents must be sufficiently rapid so
that sterically hindered amino acids can be incorporated. Construction of
polypeptides that contain amino acids bearing post-translational modifications
should take advantage of the solid-phase approach. Finally, appropriate
analytical techniques are needed to assure the proper composition of products.
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