Synthesis of a cleavable protein-crosslinking reagent for the investigation of ribosome structure.

This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents, which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2-mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega-activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3'-dithiobispropionimidate, dimethyl 4,4'-dithiobisbutyrimidate, and dimethyl 6-6'-dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega-bromonitriles. Experiments with the bispropionimidate and a test protein, pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can recovered after treatment with 2-mercaptoethanol. The reagents are suitable for studies of ribosomal structure.