Sanger’s reagent: N-terminal labelling template

The N-terminal amine of a peptide attacks 1-fluoro-2,4-dinitrobenzene through a Meisenheimer complex and fluoride leaves; 6 M HCl breaks every peptide bond but not the aryl C–N bond, so the DNP-amino acid names the N-terminal residue.

Mechanism step by step

FDNB labels the free α-amino group by SNAr; acid hydrolysis then releases the yellow DNP-amino acid

1. Addition (slow) – The free α-NH₂ attacks the carbon bearing F (R = the rest of the peptide)
2. Meisenheimer complex – The negative charge spreads onto the ortho nitro group
3. Loss of fluoride – Aromaticity returns
4. Deprotonation – Hydrogencarbonate removes H⁺ from nitrogen: the DNP-peptide

Key points

• SNAr: the ortho and para NO₂ groups hold the negative charge of the Meisenheimer complex. Addition is slow, so fluoride is the best leaving group.
• The aryl C–N bond survives 6 M HCl, which cuts every peptide bond. The yellow DNP-amino acid is identified by chromatography.
• The method is destructive: one end residue per sample. Lys ε-NH₂ and Tyr OH are labelled too.
• Sanger used FDNB to find the N-termini of insulin’s two chains (1945), on the way to the first protein sequence.

Drawn with 184 atoms, 14 arrows, 8 shapes – every one editable in MolDraw.

Sources

• Nelson & Cox, Lehninger Principles of Biochemistry, 8th ed., 2021, ch. 3 Textbook
• Berg, Tymoczko, Gatto & Stryer, Biochemistry, 9th ed., 2019 Textbook
• Sanger, Biochem. J. 1945, 39, 507 Original paper