In 1972, Carpino introduced the 9-fluorenylmethoxycarbonyl
(Fmoc) group for Nalpha protection for Fmoc peptide synthesis. The Fmoc group
requires moderate base for removal, and thus offered a chemically mild
alternative peptide synthesis to the acid-labile Boc group.
In the late 1970s, the Fmoc peptide synthesis was adopted for solid-phase applications.
Fmoc-based peptide synthesis strategies utilized t-butyl (tBu)–based side-chain
protection and hydroxymethylphenoxy-based linkers for peptide attachment to the
resin. This was thus an “orthogonal” scheme requiring base for removal of the
Nalpha-protecting group and acid for removal of the side-chain protecting
groups and liberation of the peptide from the resin. The milder conditions of
Fmoc peptide synthesis chemistry as compared to Boc 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.
One of the subsequent challenges for practitioners of Fmoc peptide
synthesis chemistry was to refine the technique to allow for construction
of proteins, in similar fashion to that which had been achieved with Boc
chemistry. Fmoc chemistry had its own set of unique problems, including
suboptimum solvation of the peptide/resin, slow coupling kinetics, and
base-catalyzed side reactions. Improvements in these areas of Fmoc chemistry
(Atherton and Sheppard, 1987; Fields and Noble, 1990; Fields et al., 2001)
allowed for the synthesis of proteins such as bovine pancreatic trypsin
inhibitor analogs, ubiquitin, yeast actin-binding protein 539-588, human
beta-chorionic gonadotropin 1-74, mini-collagens, HIV-1 Tat protein, HIV-1
nucleocapsid protein Ncp7, and active HIV-1 protease.
The milder conditions of Fmoc peptide chemistry, along with
improvements in the basic chemistry, have led to a shift in the chemistry
employed by peptide laboratories. This trend is best exemplified by a series of
studies (Angeletti et al., 1997) carried out by the Peptide Synthesis Research
Committee (PSRC) of the Association of Biomolecular Resource Facilities (ABRF).
The PSRC was formed to evaluate the quality of the synthetic methods utilized
in its member laboratories for peptide synthesis. The PSRC designed a series of
studies from 1991 to 1996 to examine synthetic methods and analytical
techniques. A strong shift in the chemistry utilized in core facilities was
observed during this time period—i.e., the more senior Boc methodology was
replaced by Fmoc chemistry. For example, in 1991 50% of the participating
laboratories used Fmoc chemistry, while 50% used Boc-based methods. By 1994,
98% of participating laboratories were using Fmoc chemistry. This percentage
remained constant in 1995 and 1996. In addition, the overall quality of the
peptides synthesized improved greatly from 1991 to 1994. 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 Fmoc peptide
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 Fmoc peptides 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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