![]() ![]() ![]() In the late 1970s, the Fmoc group was adopted for solid-phase applications. The Fmoc group requires moderate base for removal, and thus offered a chemically mild alternative to the acid-labile Boc group. In 1970, Carpino introduced the 9-fluorenylmethoxycarbonyl (Fmoc) group for N α protection ( Carpino and Han, 1970). 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. ![]() From the 1960s through the 1980s, Boc-based SPPS was fine-tuned ( Merrifield, 1986). The first instrument for automated synthesis of peptides, based on Boc SPPS, was built by Merrifield, Stewart, and Jernberg ( Merrifield et al., 1966). The peptide bonds of the assembled chain were stable to these manipulations. SPPS was thus based on “relative acidolysis,” where the N α-protecting group (Boc) was labile in the presence of moderate acid (trifluoroacetic acid TFA), while side-chain-protecting benzyl (Bzl)-based groups and the peptide/resin linkage were stable in the presence of moderate acid and labile in the presence of strong acid (HF). SPPS was later modified to use the t-butyloxycarbonyl (Boc) group for N αprotection ( Merrifield, 1967) and hydrogen fluoride (HF) as the reagent for removal of the peptide from the resin ( Sakakibara et al., 1967). SPPS of a tetrapeptide was achieved by using Cbz as an α-amino-protecting group, coupling with N,N'-dicyclohexylcarbodiimide (DCC), and liberating the peptide from the support by saponification or by use of HBr ( Merrifield, 1963). Peptides could be assembled stepwise from the C to N terminus using N α-protected amino acids. In the early 1960s, Merrifield proposed the use of a polystyrene-based solid support for peptide synthesis. Generalized approach to solid-phase peptide synthesis. Once chain elongation has been completed, the crude peptide is released from the support. It is the essence of the solid-phase 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. ![]() Subsequently, the anchored peptide is extended by a series of addition cycles ( Fig. The concept of solid-phase peptide 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). Peptide synthesis became a more practical part of present-day scientific research following the advent of solid-phase techniques. Solution synthesis continues to be especially valuable for large-scale manufacturing and for specialized laboratory applications. Classical, or solution-phase methods for peptide synthesis have an elegant history and have been well chronicled. DuVigneaud successfully applied early “classical” strategies to construct a peptide with oxytocin-like activity ( Vigneaud et al., 1953). Bergmann and Zervas created the first reversible N α-protecting group for peptide synthesis, the carbobenzoxy (Cbz) group ( Bergmann and Zervas, 1932). The first peptide synthesis, as well as the creation of the term “peptide,” was reported by Fischer and Fourneau ( Fischer and Fourneau, 1901). The concept is a straightforward one, whereby peptide elongation proceeds via a coupling reaction between amino acids, followed by removal of a reversible protecting group. The stepwise assembly of peptides from amino acid precursors has been described for nearly a century. Rapid, efficient, and reliable methodology for the chemical synthesis of these molecules is therefore of utmost interest. More than 400 peptides have entered clinical studies so far. In the year 2008, the peptide therapeutics market reached the multi-billion dollar level ( Saladin et al., 2009). DEVELOPMENT OF SOLID-PHASE PEPTIDE-SYNTHESIS METHODOLOGYĪ number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin ( Pontiroli, 1998). ![]()
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