How to Synthesize a Peptide HEADING_TITLE

In solid phase peptide synthesis, the peptide is assembled stepwise from the C-terminal of the peptide which is attached to the peptide synthesis resin.

This is a generalized procedure for preparing a peptide utilizing Fmoc-amino acids, HBTU and Rink Amide resin or pre-loaded Wang resin.  Procedures for loading the first amino acid onto unloaded Wang resin are available in the Wang Resin Technical Bulletin. Protocols for utilizing other resins and coupling reagents are avaliable on our Protocols page.

Weighing and Swelling the Resin

  1. Accurately weight the Rink amide resin or pre-loaded Wang resin into the reaction vessel.  
  2. Add N,N-dimethylformamide (DMF, approximately 10 mL/gram of resin).  Be sure to rinse down any resin sticking to the sides of the reaction vessel.
  3. Let the mixture stand at room temperature for 10 - 15 minutes to swell the resin beads.
  4. Drain the DMF.

Deprotection and Coupling

  1. Remove the Fmoc-protecting group using the standard Fmoc deprotection protocol.
  2. Preactivate and couple the next amino acid in the sequence using the standard HBTU procedures.
  3. Test for completion of the coupling step using the Kasier (ninhydrin) test.  If proline in the amino acid that is being coupled to, Kaiser test results may not be accurate.  Special tests for unprotected proline residues may be neccessary. 
  4. Repeat the deprotection and coupling steps until the peptide sequence is completed.

 

Cleaving the Peptide From the Resin

Cleaving the peptide from Wang resin or Rink Amide resin also removes the protection groups from the sidechains of the peptide.  To prevent the byproducts of deprotection and cleavage from reacting with the peptide and forming undesired impurities, scavengers are added.  Many of the popular cleavage mixtures are described in Technical Bulletins found on the Protocols page under "Solid Phase Peptide Synthesis Cleavage Cocktails"

  1. Remove the N-terminal Fmoc protecting group using the  standard Fmoc removal procedures.
  2. Wash the resin at least 3 times with DMF to remove all piperidine.  This step is important if the cleavage reaction is performed on an automated instrument.  Piperidine reacts with dichloromethane (DCM) forming crystals that can clog valves and tubes.
  3. Wash the resin at least 3 times with DCM to remove the DMF.  Residual DMF can make isolation of the cruce cleaved peptide difficult.
  4. Cleave the peptide from the resin and isolate it using the procedures described in the Wang resin or Rink Amide resin technical bulletins.

Purification

Crude peptides can be purifed by HPLC on preparative or semi-prepartive columns.  General guidlines for HPLC purification are in the technical bulletin HPLC Purification of Peptides.