A novel semisynthetic scheme was developed to couple
amine-reactive labeling
reagents to the muramyl
peptide tracheal
cytotoxin (TCT) without affecting a critical
amine group. Tracheal
cytotoxin, N-acetylglucosaminyl-1, 6-anhydro-N-acetylmuramyl-Ala-gamma-Glu-A2pmAla (A2pm,
diaminopimelic acid), is released by Bordetella pertussis, the etiologic agent of
whooping cough. This
glycopeptide reproduces the specific ciliated cell damage observed in the respiratory tract during B.
pertussis infection. To examine binding of TCT to target respiratory cells, we have produced labeled TCT analogs. Structure-function studies have shown that the primary
amine of the A2pm side chain is essential for TCT toxicity in respiratory tissue. The methodology described here allows coupling of
amine-reactive
reagents to TCT without affecting this essential
amine. The terminal
N-acetylglucosamine ring is opened by oxidation with
periodic acid, a dihydrazide linker is coupled to the oxidized ring, and pH control is used to selectively derivatize the free
hydrazide with an
N-hydroxysuccinimide ester, while the A2pm side-chain
amine remains free. Using this method, we have coupled the
Bolton-Hunter reagent to TCT, producing a biologically active 125I-labeled TCT analog.