Solid-Phase Peptide Synthesis (SPPS) — Efficient Strategies and Innovative Techniques
Product Manager：Harrison Michael
Solid-phase peptide synthesis (SPPS) is a revolutionary biochemical technique that has become the primary method for synthesizing peptides and proteins since its introduction in the 1960s. Robert Bruce Merrifield first proposed the concept of SPPS in 1963, and this groundbreaking work not only significantly improved the efficiency and controllability of peptide synthesis but also earned him the Nobel Prize in Chemistry in 1984.
In SPPS, peptide chain elongation is achieved by sequentially adding amino acid monomers. Unlike solution-phase synthesis, in SPPS, the peptide chain is anchored to an insoluble resin support, simplifying the purification steps after each reaction. The synthesis process begins at the C-terminal amino acid, which is attached to the resin support via its α-amino group. Protected amino acids are then sequentially coupled to the growing peptide chain. After each coupling, unreacted reagents and by-products are removed through simple filtration, thereby reducing the need for complex purification steps typical of traditional solution-phase synthesis.
The core of SPPS lies in two key steps: deprotection and coupling. First, the protecting group on the peptide chain is removed, allowing the next amino acid to be coupled to the free amino group. Common deprotection reagents include trifluoroacetic acid (TFA) and diisopropylethylamine (DIPEA), which effectively remove the protecting group without damaging the peptide chain. After deprotection, the amino acid is activated to facilitate its coupling to the growing peptide chain. Common coupling reagents include HBTU and HATU, which promote the formation of peptide bonds between the activated amino acid and the free amino group on the peptide chain.
The main advantages of SPPS are its efficiency and automation potential. Modern automated synthesizers can synthesize peptide chains dozens of amino acids long in a short time, making the synthesis of complex peptides and small proteins feasible. Additionally, because the synthesis occurs on a solid support, reactants and by-products can be removed through simple washing steps, greatly simplifying post-synthesis purification.
Throughout the development of SPPS, two major strategies have emerged: the Fmoc and Boc approaches. The difference between these two methods lies in the amino acid protecting groups and deprotection conditions they use. The Fmoc strategy uses 9-fluorenylmethyloxycarbonyl (Fmoc) as the protecting group and employs basic conditions, such as piperidine, for deprotection. The Boc strategy uses t-butyloxycarbonyl (Boc) as the protecting group and requires acidic conditions, such as trifluoroacetic acid, for deprotection. The Fmoc strategy has become more popular due to its milder deprotection conditions and fewer by-products.
The Boc strategy was one of the earliest methods developed for solid-phase peptide synthesis, using Boc as the amino-protecting group. During each amino acid coupling, the Boc group is removed under acidic conditions, typically using TFA, to expose the amino group for the next peptide bond formation. In contrast, the Fmoc strategy was developed to avoid potential peptide chain damage caused by acidic conditions. The Fmoc strategy employs Fmoc as the protecting group, which is removed under basic conditions, usually with a piperidine solution, to expose the amino group for the next reaction.
Taking the Fmoc strategy as an example, the synthesis begins with selecting an appropriate resin as the solid support, such as polystyrene resins like Wang or Rink amide resins, which have functional groups that can form covalent bonds with amino acids. The first step in the synthesis is anchoring the C-terminal amino acid to the resin through a stable amide bond. Next, a 20% piperidine solution is used to remove the Fmoc protecting group from the amino acid, exposing the free amino group. The next amino acid, pre-activated with a carboxyl group activator such as HBTU, HATU, or DIC, is then added to form a peptide bond with the free amino group. This cycle is repeated for each amino acid until the entire peptide chain is synthesized. After each reaction, solvents (such as DMF and DCM) and by-products used during synthesis can be removed through simple washing steps.
Although the Boc strategy’s synthesis process is similar to that of the Fmoc strategy, there are some differences in the details. The Boc strategy also begins by coupling the C-terminal amino acid to the resin support, forming an amide bond. Next, strong acidic reagents (such as TFA) are used to remove the Boc protecting group from the amino acid, exposing the free amino group. Then, the next Boc-protected amino acid is added, and in the presence of a carboxyl group activator, a peptide bond is formed. Unlike the Fmoc strategy, the Boc strategy uses acidic conditions for deprotection, which may affect certain acid-sensitive amino acids or peptide chains. Nevertheless, the Boc strategy still offers unique advantages in specific peptide syntheses.
In both Fmoc and Boc strategies, the reaction conditions and reagent choices for each step must be precisely controlled to ensure efficient and accurate peptide chain synthesis. Ultimately, the target peptide is obtained by cleaving the peptide chain from the resin and purifying it. Both strategies have demonstrated distinct advantages in specific applications, providing researchers with flexible and powerful tools for peptide synthesis.
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