Solid Phase Peptide Synthesis

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Two biHis.jpg

This protocol uses Fmoc chemistry (#note 1). The reaction mixture and calculation is based on 0.1mmol of resin, with a target peptide yield of 50-100mg.


Stock Solutions

  • 0.6M HoBt in DMF (0.81g/10ml)
  • 0.6M PyBOP in DMF (3.12g/10mL) (#note 2)
  • 30/70(v/v) Piperidine/DMF solution (#note 3), (#note 4)

Ninhydrin Reagents

  • 15g phenol in 4.5mL ethanol
  • 0.0002M KCN in pyridine (#note 5)
  • 500mg ninhydrin in 10mL ethanol

Other Reagents

  • Dichloromethane (DCM)
  • Dimethylformamide (DMF)
  • 4-Methylmorpholine (NMM)


Preparing resin

Weigh out the desired amount of resin and place in a peptide synthesis vessel with a glass frit. Swell the resin in DCM or DMF for at least an hour, preferrably two. The resin can be swelled overnight for use the following morning.


Initial deprotection is only necessary if the resin is attached to a protecting group; however, this is quite likely to be the case.

  1. Swell the resin with 30% piperidine (~ 3ml) for 2 min, stirring occasionally
  2. Swell the resin with 30% piperidine (~ 3ml) for 20 min, stirring occasionally
  3. Wash with at least 30 ml DMF using slow flow to remove piperidine


  1. Weigh out 3 resin equivalents (0.3 mmol) of amino acid.
  2. Add 500μL HoBt and 500μL PyBOP to amino acid, vortex to dissolve.
  3. Add 5.4 eq (60μL) NMM. Can use up to 6 equivalents.
  4. Mix by inversion and allow to react for 30-40 seconds. The solution should turn slightly yellow.
    • no reaction time needed for Asp, Glu, Cys, or His
  5. Add amino acid mixture to resin. Allow to react for 30 minutes, stirring occasionally.
  6. Try the ninhydrin test. If unsuccessful, repeat the test in 30 minute intervals until coupling is complete. If test shows no change after 2 hours, repeat coupling procedure using the same amino acid.

Ninhydrin Test

  1. Aliquot 20μL of resin from the reaction tube into a filter tube. (#note 6)
  2. Wash the resin aliquot with DMF and DCM, fast flow. Be sure to rinse well.
  3. Dry the aliquot until it is a powder, then transfer it into a glass culture tube.
  4. Add 40μL phenol/ethanol + 40μL KCN/pyridine + 20μL Ninhydrin/EtOH to the resin aliquot.
  5. Heat the tube at 95 °C for 2-5 minutes. A bluish color indicates an unfinished reaction. A completed reaction looks pink.


  1. Wash the resin with 30mL of DMF (slow flow)
  2. Repeat deprotection procedure to remove the N-terminal Fmoc group
  3. Add 5ml of 10% acetic anhydride/10% NMM in DMF. React for 30 min.
  4. Use the ninhydrin test to check for completion.
  5. Wash resin with 30 mL DMF, slow flow.
  6. Wash resin with DMF, DCM, and hexane in fast flow.
  7. Dry the resin, store in a vial at 4°C until ready to cleave from resin.

Cleavage and Deprotection


1. In Fmoc chemistry, the peptide is synthesized from C-terminus to N-terminus. Keep this in mind when determining the amino acid order of addition.
2. The coupling reagents HoBt and PyBOP lose effectiveness if stored for extended periods of time. For best results, remake for each new synthesis.
3. Piperidine is a restricted substance due to its potential use in the synthesis of a controlled substance. If your lab has never ordered it before, request it far in advance; there may be significant paperwork to submit before order processing.
4. Many of the reagents and solvents can dissolve/corrode plastics. Either pipet quickly, or use glass or organic-compatible pipetmen to prevent equipment damage. Similarly, be careful of storing solutions in plastic containers.
5. KCN is insoluble in pure pyridine. To make the solution, dissolve an excess amount of KCN in water, then dilute the solution to the desired concentration using pyridine.