TNBSA test

Notes. The TNBSA test was found to be efficient for primary amines, including sterically hindered amines as seen in our probes with the tertiary amine of Aib. 

Transfer a few beads of resin with a Pasteur pipet into an Eppendorf tube and add two drops of DIPEA followed by five drops of TNBSA solution. Inspect the beads by eye or under a microscope if possible. If the beads are colorless after 1 min, then acylation is complete and the next step can be carried out. Any trace of orange color in the beads indicates the presence of free amino groups and incomplete coupling. In these cases steps 2-5 should be repeated until a negative TNBSA test is returned. 

Remove the N-a Fmoc group of the coupled amino acid by carrying out step 1. Confirmation of the removal of the Fmoc group can be determined by performing a TNBSA test that should result in a positive orange color change. 

Add this mixture to a vessel containing the resin-bound peptide (0.06 mmol, 0.25 g amino-peptide resin). After shaking for 2 h remove the solution by filtration and wash the resin with DCM and DMF (3 x 30 mL). Completeness of reaction is monitored using the TNBSA test. If necessary double coupling can be performed. 

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