Solid Phase Synthesis

1. Loading the Resin

1. To a 100mL peptide synthesis vessel, add 2-chlorotrityl chloride (CTC) resin (1.1 mmol/g, 0.360 g, 0.400 mmol). Add 20 mL DMF and allow them to swell for 30 min under N_2.
2. Drain the beads under vacuum and add 10 mL DMF.
3. Add 500 mg Fmoc-Ala-OH (1.60 mmol) and 2.5 mL i-Pr₂EtN, and mix under N₂ for 15 min.
4. Drain the solvent under vacuum and repeat the loading with Fmoc-Ala-OH for 15 min.
5. Drain the solvent under vacuum and wash the beads with 10 mL DMFunder N₂, and drain under vacuum 3x.

2. Deprotection of the Fmoc Group

1. Add 10 mL 20% 4-methylpiperidine in DMF and stir the beads under N₂ for 15 min.
2. Drain the solvent under vacuum and repeat the deprotection.
3. Wash the beads with 10 mL DMF under N₂ and drain under vacuum 3x.

3. Performing the Kaiser Test

1. Perform the Kaiser test by adding 1-2 drops of solution A (0.5 mL 0.01 M KCN, 24.5 mL pyridine), solution B (1 g ninhydrin, 20 mL n-butanol), and solution C (20 g phenol, 10 mL n-butanol) each into two test tubes. One test tube will be the control while the other will monitor the reaction.
2. Add a few beads of the resin from the reaction vessel to reaction test tube and heat up the two test tubes to 110 °C.
3. If the deprotection is complete, the contents of the test tube will turn a dark blue/purple color. If the deprotection is incomplete or failed, the solution will remain yellow. Compare the reaction test tube with the control test tube.

4. Coupling the Next Building Blocks

1. Drain the solvent under vacuum.
2. Wash the beads with 10 mL N-methyl-2-pyrrolidone under N₂ and drain the solvent under vacuum.
3. To begin the next coupling, add 10 mL NMP, 620 mgFmoc-Phe-OH (1.6 mmol), 610 mg HBTU (1.6 mmol), and 2.5 mLi-Pr₂EtN, and allow the resin to bubble under N₂ for 30 min.
4. Drain the solvent under vacuum.
5. Wash the beads with 10 mL DMF under N₂ and drain under vacuum 3x.
6. Perform the Kaiser test (see steps 3.1-3.3) to look for the completion of the coupling. The beads and solution in the test tube should be yellow.

5. Cleaving the Peptide Off the Resin

1. Cleave the remaining Fmoc group using steps 2.1-2.3.
2. After the solvent is drained under vacuum, add 40 mL cleavage solution (95% TFA, 2.5% H₂O, 2.5% TIPS) to the resin and bubble under N₂ for 3 h.
3. Place a new receiving flask on the peptide synthesizer and drain theTFA solution containing the desired peptide under vacuum into the new flask.

6. Precipitation and I solation of the Peptide

1. Separate the TFA solution into 4 conical vials and add 25 mL cold ether (−20 °C) to each vial to precipitate the peptide.
2. Centrifuge the vials (3,000 rpm, 0-4 °C) for 20 min. Decant the remaining TFA and ether solution from the conical vials and concentrate the peptide precipitate to afford the desired dipeptide as a white solid.