Monitoring of Peptide Coupling and Capping HEADING_TITLE

Coupling Tests

Monitoring of the coupling steps is critical in successful peptide synthesis and a number of qualitative tests have been developed.  One commonly used historically and today is the Kaiser test based on the reaction of ninhydrin with amines.  Primary amines produce an intense blue color while secondary amines, such as N-terminal proline, produce a less intense red-brown color.  In critical applications, an alternative test for N-terminal proline is recommended.  Two such tests are the isatin test and the chloranil test, which both produce a blue color with unprotected N-terminal proline.

The testing protocol should be followed carefully.  Excess heating can cause loss of Fmoc protecting groups through reaction with pyridine (contained in the test reagents), resulting in a false positive result. 

Other coupling tests include the bromophenol blue test and the 2,4,6-trinitrobenzenesulfonic acid test.  These tests are based on the acid-base reaction of an indicator with the basic amino group.  These tests will detect secondary amines such as proline or N-methyl amino acids as well as primary amines.  The resin samplew should be thoroughly washed before performing the test to remove any acidic or basic reagents that may interfere.

If Coupling is Not Complete

If the monitoring test indicates that there is still unreacted N-terminal amine following a coupling reaction, then a second coupling step should be performed.  If the monitoring test indicates that a significant amount of unreacted amine remains, the peptide is likely aggregating.  Changing the coupling conditions, such as switching to a different solvent (NMP, DMSO, or DCM/DMF instead of DMF) or a different coupling reagent (HATU or HCTU instead of HBTU) may be beneficial.  If unreacted amine is still present after the second coupling, acetic anhydride should be used to cap it.  Capping prevents deletion peptide impurities from forming by blocking any further reactions at the unreacted sites.  Capping can also make purification of the crude peptide product easier.  The desired peptide will have different HPLC retention characteristics than the capped impurities, making it easier to isolate.

Tests for Monitoring Solid Phase Reactions 

The Kaiser Test is a very sensitive test for primary amines.  It is commonly utilized in solid phase peptide synthesis to determine if coupling reactions are complete.  Ninhydrin reacts with the deprotected N-terminal amine group of the peptide-resin to produce an intense blue color.  The Kaiser test is not reliable for detecting secondary amines.  Thus, if the N-terminal amino acid is proline, pipecolic acid, or tetrahydroisoquinoline-3-carboxylic acid, another test such as the Isatin Test or the Chloranil Test is used.

The Fmoc protecting group is not completely stable in the conditions utilized in the Kaiser test and false positive tests can result.  Alternative tests for monitoring solid phase peptide coupling reactions that have been reported include the bromophenol blue test[1] and the 2,4,6-trinitobenzene-sulphonic acid test.[2]  These tests have the added advantage that they also produce positive results with N-terminal proline residues.

Kaiser Test (Ninhydrin Test)[3]

Kaiser Test Solutions

Reagent A:

  1. Dissolve 16.5 mg of KCN in 25 mL of distilled water.
  2. Dilute 1.0 mL of above solution with 49 mL of pyridine (freshly distilled from ninhydrin).
  3. Pour it into a small reagent bottle and label it “A”.

Reagent B:

  1. Dissolve 1.0 g of ninhydrin in 20 mL of n-butanol.
  2. Pour into a small reagent bottle and label it as “B”.

Reagent C:

  1. Dissolve 40 g of phenol in 20 mL of n-butanol.
  2. Pour it into a small reagent bottle and label it “C”.

Kaiser Test Procedure:

  1. Take 10-15 beads of resin in a test tube and label it S.
  2. Take tube S and another empty tube designated R (reference)
  3. To each tube add:

            2 to 3 drops of Reagent A
            2 to 3 drops of Reagent B
            2 to 3 drops of Reagent C

      4. Heat both the tubes at 110°C for 5 minutes.                                                                                                 5. Compare the color with reference.

      Colorless or faint blue color: complete coupling, proceed with synthesis

        Dark blue solution but beads are colorless: nearly complete coupling, extend coupling or cap unreacted chains 

      Solution is light blue but beads are dark blue: coupling incomplete, recouple

        Solution is intense blue and all beads are blue: failed coupling, check amino acid, reagents, then recouple

Isatin Test for Unprotected Proline[4]

Isatin Test Solution

  1. Add 2 g of isatin to 60 mL of benzyl alcohol. 
  2. Stir at room temperature for 2 hours.
  3. Filter to remove any undissolved isatin. 
  4. Dissolve 2.5 g of Boc-Phe-OH in the filtrate.

Isatin Test Procedure

Place a small amount of the test sample (4-5 mg of resin-peptide) in a small test tube.  Add 2 to 3 drops of the isatin solution and heat at 100 °C for 5 minutes.  If the beads turn blue, the coupling reaction is incomplete. 

Chloranil Test for Secondary Amines[5]

Chloranil Test Solutions

Reagent A

Mix 1 mL of acetaldehyde with 49 mL of N,N-dimethylformamide (DMF).

Reagent B

Dissolve 1 g of p-chloranil in 49 mL of DMF.

Chloranil Test Procedure

  1. Place 1 to 5 mg of resin in a small test tube.
  2. Add 1 drop of Reagent A.
  3. Add 1 drop of Reagent B.
  4. Let the mixture stand at room temperature for 5 minutes.
  5. Observe the color of the beads.  If the beads are blue, secondary amine is present.

Bromophenol Blue Test[6]

Bromophenol Blue Test Solution

            Dissolve 3 mg of bromophenol blue in 100 mL of DMF

Bromophenol Blue Test Procedure

 

  1. Transfer 10-15 beads to a small test tube.
  2. Carefully rinse the beads with fresh DMF.  With a pipette, withdraw the rinse liquid without removing the beads.
  3. Add 1 to 2 drops of the test solution.
  4. Observe the color of the beads.

 

If the beads are blue to blue-green, the coupling reaction is not complete and a second coupling may be required.  If the beads are yellow-green, then the coupling reaction is nearly complete.  If the beads are yellow with a trace of green, the coupling reaction is complete.

2,4,6-Trinitrobenzenesulphonic Acid Test

2,4,6-Trinitrobenzene-sulphonic Acid Test Solutions

            Reagent A

            1% 2,4,6 trinitrobenzenesulphonic acid in DMF

         Reagent B

            10% DIPEA in DMF

2,4,6-Trinitrobenzene-sulphonic Acid Test Procedure

 

  1. Remove a few resin beads and wash them thoroughly with DMF.
  2. Suspend the beads in fresh DMF.
  3. Add 1 drop of Reagent A and 1 drop of Reagent B.
  4. Allow the sample to stand at room temperature for 5 minutes.
  5. Wash the beads with DMF and observe the color.

 

Red beads indicate the coupling in not complete.

Standard Capping Procedure

  1. Filter and wash the resin several times with DMF.
  2. Suspend the resin in a DMF solution containing acetic anhydride (50 equivalent based on resin substitution) and pyridine (50 equivalents based on resin substitution).  DIPEA may be substituted for the pyridine.
  3. Gently shake at room temperature for 30 minutes.
  4. Filter and wash the resin with DMF.
  5. Perform a Kaiser test.  If the Kaiser test is not negative, repeat the capping procedure.

 

 


[1] Krchňák, V.; Vágner, J.; Šafár, P.; Lebl, M. Collect. Czech. Chem. Commun., 1988, 53, 2541-2548.

[2] Hancock, W.S.; Battersby, J.E. Anal. Biochem., 1976, 71, 260-264.

[3] Wellings, D. A.; Atherton, E. “Methods in Enzymology Volume 289: Solid-Phase Peptide Synthesis” Ed. Fields, G. B.  Academic Press, San Diego, 1997, p. 54.

[4] Wellings, D. A.; Atherton, E. “Methods in Enzymology Volume 289: Solid-Phase Peptide Synthesis” Ed. Fields, G. B.  Academic Press, San Diego, 1997, pp. 54-55.

[5] Vojkovsky, T. Pept. Res., 1995, 8, 236-237.

[6] Based on procedures reported in Krchňák, V.; Vágner, J.; Šafár, P.; Lebl, M. Collect. Czech. Chem. Commun., 1988, 53, 2541-2548.