“Peptide synthesis” encompasses the preparation of a wide range of substances, from simple small-molecule peptides to large proteins. Bruce Merrifield’s pioneering introduction of solid-phase peptide synthesis (SPPS) fundamentally transformed peptide synthesis strategies by simplifying the cumbersome and highly demanding purification steps required in traditional liquid-phase peptide synthesis (LPPS).
Basic Principles of SPPS
Peptide synthesis involves a large number of repetitive steps, and the use of a solid support offers significant advantages. In such a system, high concentrations of excess reagents can drive coupling reactions to completion, while surplus reagents and by-products can be separated from the insoluble growing peptide chain (resin) simply by filtration and washing. All operations are carried out in the same solvent, eliminating the need for material transfer.
- Anchoring of the initial amino acid: The initial amino acid is anchored to the resin through its C-terminus, forming either an ester bond or an amide bond.
- Linear assembly: The peptide chain is elongated stepwise from the C-terminus to the N-terminus through condensation reactions.
- Protecting-group strategy: Permanent protecting groups are used for side chains to improve stability; non-side-chain amino acid protection is generally achieved using carbamate-type protecting groups, which can be removed under mild conditions, thereby avoiding racemization caused by overly harsh conditions.
- Cyclic operations: Coupling, washing to remove excess reagents, deprotection, and initiation of the next coupling cycle.
- Final cleavage: The peptide chain is cleaved from the resin while simultaneously removing the side-chain protecting groups.
Fmoc/t-Bu Strategy in Solid-Phase Synthesis
Two major protecting-group strategies are mainly used in solid-phase synthesis: Boc/Bzl and Fmoc/t-Bu. The former requires deprotection under relatively strong acidic conditions, such as TFA, and is mainly used for side-chain protection. Strong acidic conditions often damage sensitive amino acid sequences; therefore, the Fmoc/t-Bu strategy is generally adopted for amino acid protection, in which Fmoc is base-labile and t-Bu is weak-acid-labile. Solid-phase synthesis methods using the Fmoc-based strategy have become the preferred approach for routine peptide synthesis.
Solid Supports
Solid supports can be characterized by both the “matrix polymer” and the “linker molecule,” although the term “resin” is often incorrectly used to refer to the entire system. In solid-phase chemistry, the matrix polymer is as important as the liquid-phase environment. At present, there are well over one hundred commercially available resins, some of which are equipped with the same linker molecules. Therefore, the most suitable linker must be selected carefully during synthesis.
Cross-linked polystyrene (PS)-based resins are commonly used solid supports for SPPS. Resin beads with a particle size distribution of 200-400 mesh (approximately 50 μm in diameter) and a loading of 0.5-0.8 mmol/g exhibit good swelling properties in DMF and DCM, which promotes diffusion of reactants into the polymer matrix and ensures adequate access to linker sites buried within the beads. For longer peptide chains (more than 25 amino acids) or sequences that are more difficult to synthesize, lower-loading resins of 0.1-0.2 mmol/g should be used. Cross-linked polyamide (PA)-based resins and polystyrene-polyethylene glycol (PS-PEG) composite resins are more hydrophilic solid supports. Their microscopic and macroscopic physical properties differ from those of PS resins. These supports generally have lower loading capacities and may serve as alternatives to PS for the synthesis of complex sequences and large peptides.
Resin Handling
Why must resins be treated before use? This is analogous to textile dyeing and finishing. Because cotton fibers have irregular oval cross-sections, they must be swollen before dyeing to increase the surface area so that dyes can penetrate effectively. Resin is a polymeric material with long chains that may become entangled or structurally collapsed, and its reactive sites are located inside the resin. Therefore, swelling treatment before use is essential, and the reaction conditions must also be taken into consideration.
Selection of the Reactor
SPPS reaction vessel: The reactor is generally made of glass and equipped with a PTFE or glass frit syringe. The size of the reactor should be selected according to the amount of resin used.
| Container Length (cm) | Container Diameter (cm) | Maximum Resin Weight (g) | Working Volume (mL) |
| 5 | 2 | 0.5 | 10 |
| 11 | 2.6 | 2 | 40 |
| 15 | 3.4 | 4 | 90 |
SPPS Reactor Types
Selection of Solvents
Ninety-nine percent of the coupling sites are not located on the surface of the resin beads but rather inside the resin. Therefore, resin beads bearing the growing peptide chain must be fully swollen to ensure optimal penetration of the protected activated amino acids into the polymer matrix, thereby improving coupling efficiency.
The basic swelling procedure is as follows: before starting solid-phase synthesis, place the resin in a suitable solvent such as DMF or DCM and allow it to swell for 20-30 minutes. For cross-linked polystyrene (PS)-based resin beads, DCM provides the best swelling characteristics. During the coupling step, however, DMF or NMP is preferred because these solvents provide better solubility for the reactants. Alcohols and water are not suitable for cross-linked polystyrene (PS)-based resins. However, in subsequent washing steps, methanol or isopropanol may be used to shrink the resin beads and expel entrapped excess reactants. After this treatment, the beads must be reswollen with DCM and DMF. Resin swelling procedure: Place the resin in a suitable reactor, add DCM until the resin beads are fully immersed, and gently mix the resin suspension with a PTFE stirring rod. Allow it to swell for 20-30 minutes, then remove the solvent by vacuum filtration.
Stirring and Mixing
During SPPS, reaction kinetics are governed primarily by diffusion phenomena; therefore, vigorous stirring of the reaction vessel is unnecessary. In addition, most resin beads used in peptide synthesis are relatively fragile, and strong agitation can damage or fracture them. Gentle mixing may be achieved using the rotor of a rotary evaporator or by employing devices capable of mild motion such as rocking or vortex-like mixing.
Washing
Washing is used to remove soluble by-products and excess reagents generated during coupling and deprotection. The basic washing procedure involves introducing solvent into the reactor and then removing it under vacuum; when necessary, the resin may be stirred gently in the washing solvent with a stirring rod.
Standard washing and reswelling procedure: Add DMF to the reaction vessel, allow it to stand for 10 s, then remove the solvent by vacuum filtration, carefully rinsing all parts of the reactor with DMF. Remove the solvent under vacuum, then add fresh solvent again for washing, repeating the operation 1-2 times. After washing is complete, wash the resin with methanol using a procedure similar to the DMF washing step, repeating 1-2 times. Then switch to DCM for washing 1-2 times, followed by DMF washing 1-2 times.
Linkers and Resin Selection in Fmoc Solid-Phase Synthesis
The first step in SPPS is to anchor the C-terminal amino acid residue (with its N-terminus protected) to the solid support through either an ester bond or an amide bond, depending on whether the target peptide has a C-terminal carboxylic acid or a C-terminal amide group. At present, most linker molecules are commercially available and preloaded onto different matrices, including polystyrene (PS), polyamide (PA), and polystyrene-polyethylene glycol (PS-PEG) composite matrices. C-terminal peptide acids are generated through ester linkages, whereas C-terminal peptide amides are generated through amide linkages. These preloaded linkers simplify experimental preparation and ensure batch-to-batch consistency. Different matrices have different characteristics: PS matrices are highly hydrophobic and suitable for routine peptide synthesis; PS-PEG matrices are more hydrophilic and advantageous for long-peptide synthesis; and polyamide (PA) is used for specialized applications.
Peptide Amides
For the synthesis of C-terminal peptide amides, the following resins are commonly used. The attachment of the first amino acid to these resins is basically consistent with the Fmoc synthesis strategy, and under TFA treatment these resins leave an amide bond upon cleavage to complete the release of the peptide (the resin functions similarly to a protecting group). Because these resins contain free amino groups, they are usually Fmoc-protected and must be deprotected before use. It should be noted that when amino acids with substantial steric hindrance at the C-terminus are coupled to the resin, incomplete conversion may occur, and a second coupling step may be required.
Peptide Acids
Compared with the nucleophilicity of amino groups, hydroxyl groups are less nucleophilic. Therefore, anchoring amino acids onto a solid support in the form of esters is more challenging. It is recommended to directly purchase resins preloaded with the protected C-terminal amino acid to avoid issues such as epimerization, dipeptide formation, and substitution-level variability. When synthesizing C-terminal peptide acids using the Fmoc/tBu strategy, the anchoring reaction must be carried out under anhydrous conditions, and amino acids containing moisture must be dried in advance.
Hydroxyl Resins
Among hydroxyl resins, the less sterically hindered Wang resin more readily forms ester bonds, and the symmetrical anhydride method is commonly used. If anchoring is difficult, the esterification step may be repeated with fresh reagents; some amino acid derivatives may require three esterification cycles to achieve satisfactory results. After anchoring is completed, the unreacted hydroxyl groups on the resin are recommended to be capped with benzoic anhydride or acetic anhydride. Standard operating procedure for hydroxyl resins: First place the resin in the reactor and swell it with a suitable solvent. Meanwhile, dissolve the target Fmoc-protected amino acid (10.0 eq.) in DCM at 0°C, add DIC (5.0 eq.) and allow activation for 10 minutes; DMF may be added if necessary to aid dissolution. Transfer the activated solution to the resin, add DMAP/DMF to catalyze the reaction, and stir for 1 hour; then wash once each with DMF and DCM. Dry under vacuum for 18 hours and determine the loading by the Fmoc release assay. If the loading is below 70%, the esterification step should be repeated.
Once the target loading is achieved, cap the residual hydroxyl groups with benzoic anhydride/acetic anhydride (5 eq.) and pyridine (1 eq.) in DMF for 30 minutes. After final washing and Fmoc deprotection, peptide-chain elongation may begin. This procedure ensures coupling efficiency through low-temperature activation, strict stoichiometric control, and loading verification. Key steps include activation monitoring, catalytic enhancement, and hydroxyl capping to prevent by-product formation.
Trityl-Series Resins
These resins exhibit high acid lability, and the steric hindrance of their linker groups can significantly suppress diketopiperazine (DKP) formation. They are particularly recommended for the synthesis of peptides with C-terminal proline (Pro) or glycine (Gly). These resins can be cleaved under extremely mild acidic conditions, thereby protecting both the peptide segment and the resin. Commercial products are generally supplied as chlorotrityl resin or trityl alcohol precursors.
Typical procedure for trityl resin: Place 1 g of trityl resin (loading 1.0-2.0 mmol chloride/g) into an SPPS reactor and allow it to swell. Add 3 equivalents of the Fmoc amino acid and 7.5 equivalents of DIPEA, then stir at room temperature in anhydrous DCM for 30-60 minutes. Wash the resin sequentially with DMF 2-3 times, then add 10 mL of a DCM/MeOH/DIPEA mixture to cap the residual chloride groups, and continue the reaction for 15 minutes. Finally, wash three times each with DMF and DCM, dry under vacuum, and determine the loading by the Fmoc release assay. Through this procedure, precise control of the amino acid-to-DIPEA ratio, together with methanol quenching, ensures single-site attachment. It should be noted that the activity of trityl chloride must be maintained under anhydrous conditions.