A transparent, step-by-step view of our SPPS workflow - from resin swelling and amino acid coupling to resin cleavage, HPLC purification, and MS verification. Every stage is executed under rigorous quality control to deliver peptides of verified purity and identity.
Each cycle of coupling and deprotection is verified before proceeding - nothing is left to chance.
Weigh 2.0 g of blank Wang resin into a clean, dry reaction tube, add 15 mL of DMF, and allow the resin to fully swell at room temperature for approximately 30 minutes. Complete swelling ensures uniform reaction sites and consistent coupling efficiency throughout the synthesis.
At room temperature, remove the solvent from the previous step by frit filtration. Add 1 mmol (5 molar equivalents) of the first C-terminal amino acid, 5 equivalents of DMAP, and 5 equivalents of DIC, using DMF as the solvent. Allow the reaction to proceed at room temperature for 3 hours. After completion, wash the resin with DMF 4–6 times (5–6 mL per wash).
Capping step: Add pyridine and acetic anhydride at a 1:1 volume ratio and react for 30 minutes, then wash again with DMF 4–6 times (5–6 mL each). This caps the active sites on any unreacted blank resin, preventing side reactions in later cycles.
Remove the previous solvent by filtration, then add 10 mL of 20% piperidine in DMF to the resin and stir under N₂ for 10 minutes. Filter off the solution, add a further 10 mL of 20% piperidine/DMF, stir under N₂ bubbling for 5 minutes, filter, and repeat this operation twice. Finally wash with DMF 4 times and methanol 2 times (5–6 mL per wash).
Withdraw a small portion of resin, wash three times with methanol, then add one drop each of ninhydrin, KCN, and phenol solutions and heat at 105–110 °C for 5 minutes. Interpret the result as follows:
Fmoc removal is complete - free amine groups are confirmed. Proceed to the next coupling cycle.
Protecting groups have not been fully removed - repeat the deprotection procedure above before continuing.
Weigh 3 molar equivalents of the second C-terminal amino acid, 3 equivalents of HBTU, and 3 equivalents of HOBT into the reaction tube. Dissolve completely in an appropriate volume of DMF, then add 10 molar equivalents of neat DIEA and react at room temperature for 40 minutes. Wash with DMF 4–6 times (5–6 mL per wash) and test a small resin sample with the ninhydrin reagent - the result should be colorless.
Then add 10 mL of 20% piperidine/DMF to remove Fmoc in two stages (10 minutes, then 5 minutes), followed by DMF washing 4 times and methanol washing 2 times (5–6 mL each). Test a small resin sample again with the ninhydrin reagent - if the result turns blue, the coupling is confirmed and the next cycle may proceed.
The coupling–deprotection cycle is then repeated in the same manner, amino acid by amino acid, until the final N-terminal amino acid of the target sequence has been incorporated. The last Fmoc protecting group is removed, and the resin is drained dry under filtration - the full-length peptide, still anchored to the solid support, is now ready for cleavage.
Finally, cleave the peptide from the resin using a trifluoroacetic acid cleavage cocktail for 2 hours:
Filter the reaction mixture to obtain the peptide solution in TFA. Evaporate the cleavage solution as thoroughly as possible under a nitrogen stream, then precipitate with diethyl ether and centrifuge; wash the precipitate with ether 3–5 times to obtain a white solid. Dissolve the crude product in pure water and complete the final two stages:
The dissolved peptide is desalted and purified by HPLC, then lyophilized (freeze-dried) to yield the crystalline final product.
A sample of the final product is analyzed by MS to confirm molecular weight, verifying peptide identity and integrity.
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