DBD-NCS [=4-(N,N-Dimethylaminosulfonyl)-7-isothiocyanato-2,1,3-benzoxadiazole] [for HPLC Labeling and Edman Degradation]

The compound 1 is an HPLC fluorescence labeling reagent, which has a 2,1,3-benzoxadiazole skeleton and an isothiocyano group, and easily reacts with an amino group to form the corresponding thiourea. The resultant thiourea is stable enough to reach the detector without any decomposition under reversed phase HPLC. An excellent chromatogram can be obtained by fluorescence detection at the excitation and emission wavelengths of 384 nm and 520 nm, respectively. The detection limit for its quantity is an order of sub-picomol (S/N = 3). 1 itself does not fluoresce but shows an excellent stability in forms of both crystal and solution, and its derivatives are also stable. This compound can be used for amino acid sequence analysis (Edman Degradation) by binding with the N-terminal amino acid of peptides or proteins, followed by acid reatment.

Application example:
[Method by Manual Edman Degradation]
Peptide (insulin Chain B 500 pmol)
　　　│
　　　│　・ Dissolve in 20 µL of 50% pyridine / H₂O.
　　　│　・ Add 5 µL of 1% triethylamine / CH₃CN and 10 µL of 20 mM HPLC labeling reagent 1 / pyridine,
　　　│　　　and react the mixture at 50 °C for 15 min under the atmosphere of inert gas.
　　　│　・ After cooling to room temperature, wash the reactant solution 3 times
　　　│　　　with 200 µL of heptane / dichlorormethane (6/4).
　　　│　・ Dry the washed solution at 50 °C for 15 min by using a centrifugation evaporator.
　　　│　・ Add 30 µL of 1% BF₃·Et₂O / CH₃CN to the mixture and incubate the mixture at 50 °C for 5 min.
　　　│　・ Further dry the reactant solution under nitrogen gas.
　　　│　・ Add 20 µL of H₂O, and then extract 2 times with 100 µL of benzene / AcOEt (1/4).
　　　│
　　　│―――――(Aqueous phase) A peptide will be eluted out.
　　　│
　　(Organic phase)
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　　　│　・ Dry the extracted organic phase under nitrogen gas.
　　　│　・ Dissolve the mixture in 2 µL of CH₃CN.
　　　│　・ Add 8 µL of 0.4 M HCl and hydrolyze the mixture at 50 °C for 5 min.
　　　│　・ Treat the reactant with 5 µL of 4 M HCl and 0.5 M NaNO₂ at room temperature for 10 min
　　　│　　　and oxidize it.
　　　│　・ Neutralize the reactant with 23 µL of 1 M NaNO₂, and remove an excessive oxidant
　　　│　　　by adding 20 µL of 0.15 M methionine.
　　　│　　
　　Use 20 µL of this solution as an HPLC sample solution.

TCI Products Pamphlet
HPLC Labeling Reagents ( PDF 10MB )