For peptide preparation, solid-phase peptide synthesis (SPPS) has many advantages over traditional solution-phase organic synthesis. With the exception of large-scale manufacturing of short sequences (i.e., less than 5 amino acid residues), SPPS is faster, more efficient, and more economical than solution-phase peptide synthesis. Some of the benefits of SPPS include:
- Excess reagents and products can be easily washed away.
- The use of excess reagents increase reaction rates and drive reactions to completion.
- Intermediates do not require isolation or characterization.
- Access to a wider range of solvents with low volatility and high polarity.
- Tethered peptide provides a ‘pseudo-dilute’ microenvironment which can inhibit intermolecular reactions making some modifications easier to accomplish.
Solution-Phase Peptide Synthesis Scheme
SPPS is a more rapid and direct approach to peptide synthesis because intermediates are not isolated from the resin during the elongation phase. During this phase, the resin remains in a single reaction vessel throughout the synthesis until the peptide is cleaved from the resin and purified (see scheme below). Excess reagents and solvents can be easily washed away with iterative solvent washes. Highly polar solvents like N,N-dimethylformamide (DMF) and N-methylpyrolidone (NMP) can be utilized during the coupling and deprotection steps. The same solvents in a solution-phase approach are difficult to remove and the intermediates are problematic to isolate. While preactivation of the amino acids with coupling reagents is suitable in more volatile solvents like dichloromethane (DCM), the coupling steps are often more efficient with some amount of DMF or NMP present.
Solid-Phase Peptide Synthesis Scheme
SPPS process begins with the immobilization of an appropriately protected amino acid (Fmoc/Boc-AA-OH) to a linker-bound polystyrene-based resin. The linker determines the conditions in which the peptide is cleaved from the resin and the functionality of the C-terminus (typically an acid or amide). Elongation of the peptide is accomplished by deprotection of the main chain group (Fmoc or t-Boc) followed by the coupling of the next appropriately protected amino acid (e.g., Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, etc.) Deprotection of the main-chain protection group is carried out with base (typically, piperidine-DMF or DBU) for FMOC groups and acid (TFA) for t-Boc groups. The coupling (i.e., amide bond formation) of amino acids is carried out by way of ‘coupling reagents’ that activate the carboxylic acid of the amino acid. Following elongation of the peptide to the desired length and constitution, the peptide is cleaved from the resin, purified by reverse-phase HPLC, and lyophilized to form a fluffy white powder.