Disulfides cleavable cross-linkers

The use of periodate as a cleavage agent does have advantages, however. Unlike the use of cleavable cross-linkers that contain disulfide bonds which require a reductant to break the conjugate, cleavage of diol-containing cross-links with periodate typically preserves the indigenous disulfide bonds and tertiary structure of proteins and other molecules. As a result, with most proteins bioactivity usually remains unaffected after periodate treatment. 

Reported structures for homobifunctional aryl azides include a biphenyl derivative and a naphthalene derivative (Mikkelsen and Wallach, 1976), a biphenyl derivative containing a central, cleavable disulfide group (Guire, 1976), and a compound containing a central l,3-diamino-2-propanol bridge between phenyl azide rings that are nitrated (Guire, 1976). The only commercially available homobifunctional photoreactive cross-linker is BASED. 

N-Succinimidyl-4(4-azidophenyl)l,3 -dithiopropionate (SADP) is a photoreactive heterobifunctional cross-linker that is cleavable by treatment with a disulfide reducing agent (Pierce). The cross-linker contains an amine-reactive NHS ester and a photoac-tivatable phenylazide group, providing specific, directed coupling at one end and nonselective insertion at the other end. 

However, since SMCC forms nonreversible thioether linkages with sulfhydryl groups, it only can be used in the preparation of immunotoxins if intact A-B toxins are employed in the conjugate. In such conjugates, the A chain still has the potential for reductive release from the B-chain subunit after cellular docking and internalization. Immunotoxins prepared with A-chain or single-subunit toxins will not display cytotoxicity if cross-linked with SMCC, since the cross-linker does not create cleavable disulfide bonds upon conjugation. 

P. E. Nielsen, J. B. Hansen and O. Buchardt, Photochemical cross-linking of protein and DNA in chromatin. 1. Synthesis and application of a photosensitive cleavable derivative of 9-aminoacridine with 2 photoprobes connected through a disulfide-containing linker, Biochem. J., 1984, 223, 519-526. 

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