2-Iminothiolane

Figure 1.60 Methyl 4-mercaptobutyrimidate forms 2-iminothiolane, which can react with a primary amine to create a sulfhydryl group. The modification preserves the positive charge of the original amine.

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. 

Dissolve 2-iminothiolane in 0.025M sodium borate to a concentration of 0.02M. 

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. 

Add 2-iminothiolane (Thermo Fisher) to this solution to give a molar excess of 20-40 X over the amount of antibody present (MW of Traut s reagent is 137.63). Addition of solid 2-iminothiolane may be done despite the fact that the compound is relatively insoluble in aqueous solution. As the reagent reacts, it will be completely drawn into solution. Alternatively, a stock solution of Traut s may be made in DMF and an aliquot added to the antibody solution (not to exceed 10 percent DMF in the final solution). 

Figure 20.5 Antibodies may be modified with 2-iminothiolane at their amine groups to create sulfhydryls for conjugation with SMCC-activated enzymes. The maleimide groups on the derivatized enzyme react with the thiols on the antibody to form thioether bonds.

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.

Protocol for the Conjugation of SPDP-activated Antibodies with 2-Iminothiolane-Modified Toxins... 

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

Dissolve 2-iminothiolane (Thermo Fisher) in degassed, nitrogen-bubbled deionized water at a concentration of 20mg/ml (makes a 0.14M stock solution). The solution should be used immediately. Add 70 pi of the 2-iminothiolane solution to each ml of the gelonin solution (final concentration is about 10 mM). 

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). 

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.

Figure 23.4 Avidin may be modified with 2-iminothiolane to produce sulfhydryl groups. Subsequent reaction with a maleimide-activated enzyme produces a thioether-linked conjugate.

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

Alagon, A.C., and King, T.P. (1980) Activation of polysaccharides with 2-iminothiolane and its uses. 

Jue, R., Lambert, J.M., Pierce, L.R., and Traut, R.R. (1978) Addition of sulfhydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercap-tobutyrimidate). Biochemistry 17, 5399-5405. 

Singh, R. et al. (1996) Formation of N-subsdtuted 2-iminothiolanes when amino groups in proteins and peptides are modified by 2-iminothiolane. Anal. Biochem. 236(1), 114-125. 

Tarendno, A.L., Phelan, A.W., and Plummer Jr., T.H. (1993) 2-Iminothiolane A reagent for the introduction of sulfhydryl groups into oligosaccharides derived from asparagine-linked glycans. Glycobiology 3, 279-285. 

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