We were synthesizing a short peptide (12 residues long) with FITC labelled at the N-terminus by a standard peptide synthesizer. Everything went well until the last step of FITC incorporation. Our staff said it may be the problem of having a positively charged residues at the termini but we had never had this problem before (many other sequences were used). Any idea why this may be the case?
Hi dear Hsu,
For FITC coupling at N-terminus of peptides, you must know two things:
(1) before coupling FITC, you should add a non-alpha amino acid (such as a spacer) on the N-terminus, or otherwise after assembling FITC onto a peptide, it can be subsequently removed during TFA cleavage because of Edman degradation;
(2) For coupling of FITC, please use excess of DIEA base (e.g. 10 eq.). This will ensure an efficient coupling of FITC.
It could help to know what chemistry you are using in the SPPS.
Are you familiar with this side reaction using FITC?
Jullian et al., (2009) N-terminus FITC labeling of peptides on solid support: the truth behind the spacer. Tetrahedron Letters 50, 260-263
Dear Kurt, The synthesis was done by the staff of our core facility and I will need to find out what chemistry was used for the SPPS. But I'll certainly read your reference first to see whether that is related to our problem. Cheers, Danny
Hi dear Hsu,
For FITC coupling at N-terminus of peptides, you must know two things:
(1) before coupling FITC, you should add a non-alpha amino acid (such as a spacer) on the N-terminus, or otherwise after assembling FITC onto a peptide, it can be subsequently removed during TFA cleavage because of Edman degradation;
(2) For coupling of FITC, please use excess of DIEA base (e.g. 10 eq.). This will ensure an efficient coupling of FITC.
As Kurt Berndt mentioned, you should read Jullian et al. and use a spacer before coupling FITC. We use beta-alanine as a linker amino acid, this works perfectly fine.
its better to do final FITC labelling not on the synthesizer but manual coupling and for a prolonged time using DIC/DIEA.
Agree with Prachi Anand on that. Always I was doing FITC coupling outside of synthesizer and monitoring by LC-MS/MALDI (small test cleave).
>...prolonged time using DIC/DIEA
Hopefully, this is a misprint? Because DIC is a sort of "red herring" in case of isothiocyanate coupling.
> I was using HCTU/DIEA
In combination with slowly reacting FITC this will produce tetramethylguanidinium by-product (M+98) predominantly. Isothiocyanates are self-sufficient and need only tertiary amine for coupling to proceed. NO extra coupling agent, please!!!
It worked pretty well in my case. I used them both but very low concentration of both.
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