2-Iminothiolane disulfide

Proteins modified with 2-iminothiolane are subject to disulfide formation upon sulfhydryl oxidation. This can cause unwanted conjugation, potentially precipitating the protein. The addition of a metal-chelating agent such as EDTA (0.01-0.1M) will prevent metal-catalyzed oxidation and maintain sulfhydryl stability. In the presence of some serum proteins (i.e., BSA) a 0.1M concentration of EDTA may be necessary to prevent metal-catalyzed oxidation, presumably due to the high contamination of iron from hemolyzed blood. 

The 4,4 -dipyridyl disulfide can be used in aqueous solutions, but it has been found that modification of proteins with this reagent yields rapid disulfide bond formation. Only when 2-iminothiolane is used in tandem with 4,4 -dipyridyl disulfide can 4-dithiopyridyl groups be introduced into proteins (King et al., 1978) (Section 4.1, this chapter). This is due to disulfide interchange reactions predominating without the addition of 2-iminothiolane. 

Another way of utilizing SPDP is to again activate the antibody to create the pyridyl disulfide derivative, but this time thiolate the toxin component using 2-iminothiolane (Chapter 1,... 

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

Bifunctional reagents, such as bis-imido esters, have been widely used because they react under mild condidons specifically with the amino groups of proteins. Bifunctional imido esters that introduce a disulfide bond in the cross-link are especially useful since these bonds can be readily cleaved by reduction, allowing individual proteins to be regenerated. The reagent most commonly used for ribosomal proteins is 2-iminothiolane (Traut et al, 1980). 

Goff, D.A. Carroll, S.F. Substituted 2-iminothiolanes reagents for the preparation of disulfide cross-linked conjugates with increased stability. Bioconjugate Chem. 1990, 1, 381-386. 

When 2-iminothiolane is used to modify proteins in tandem with 4,4 -dipyridyl disulfide, a protected sulfhydryl can be introduced in a single step (King et al., 1978). 

FIGURE 54.4 Preparation of iminothiolane-modified poly(ethylene glycol)-Woc -poly(L-lysine) [PEG-f>-(PLL-IM)]. (Reprinted with permission Biomacromolecules, 10(1), Matsumoto, S., Christie, R., Nishiyama, N., Miyata, K., Ishii, A., Oba, M., Koyama, H., Yamasaki, Y, and Kataoka, K., Environment-responsive block copolymer micelles with a disulfide cross-linked core for enhanced siRNA delivery, 119. Copyright 2009 American Chemical Society.)... 

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