Thiolation 2-iminothiolane

Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation.

Nacharaju P, Boctor FN, Manjula BN et al (2005) Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane an approach to mask A, B, and D antigens to generate universal red blood cells. Transfusion 45 374-383... 

The amine groups on these fragments also may be modified with thiolating agents, such as SATA or 2-iminothiolane, to create sulfhydryl residues suitable for coupling to maleimide-activated enzymes (Section 1.1, this chapter) (Figure 20.13). Amine groups further may be utilized... 

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.

Thiolation of Gelonin (or Other Single-Polypeptide Toxins) with 2-Iminothiolane (Traut s Reagent)... 

Concentrate the thiolated toxin to lOmg/ml using centrifugal concentrators. Immediately use the modified protein in the conjugation reaction to prevent inactivation of 2-iminothiolane-modified molecules by recyclization (Chapter 1, Section 4.1). 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KC1, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

The binding of proteins such as tetanus toxoid (TTd) onto dextran can also be achieved via thiolation. Therefore, dextran is converted selectively at the terminal reducing ends. This is possible either after amination and modification of the terminal amino function with 2-iminothiolan hydrochlo-... 

Bernkop-Schnurch, A., Hornof, M. and Zoidl, T. (2003) Thiolated polymers- thiomers modification of chitosan with 2-iminothiolane. Int. J. Pharm. 260 229-237. 

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