Muramyl Peptide Probes Derived from Tracheal Cytotoxin ofBordetella pertussis

Abstract

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-γ-Glu-A2pmAla (A2pm, diaminopimelic acid), is released byBordetella pertussis,the etiologic agent of whooping cough. This glycopeptide reproduces the specific ciliated cell damage observed in the respiratory tract duringB. pertussisinfection. 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 terminalN-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 anN-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 active125I-labeled TCT analog.