2-Iminothiolane reagents

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. 

Figure 1.61 Traut s reagent can undergo side reactions after the modification of an amine-containing molecule. The terminal thiol group can recyclize to create another iminothiolane derivative that effectively ties up the thiol.

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). 

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

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.

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. 

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). 

Mix 4 mg of KLH or a respective amount of a glycerol-containing solution of KLH (volume maximal 60 pi) with 190 pi Soln. A. Weigh 0.6 mg Traut s reagent (2-iminothiolane) into an Eppendorf tube and add the carrier protein solution. Shake at RT for 30 min. 

Traut s reagent is fully water-soluble and reacts with primary amines in the pH range 7—10. The cyclic imidothioester is stable to hydrolysis at acid pH values, but its half-life in solution decreases as the pH increases beyond neutrality. However, even at pH 8 in 25 mM triethanolamine the rate of sulfhydryl formation without added primary amine was found to be negligible. On addition of dipeptide amine, the reagent reacted quickly as evidenced by the production of Ellman s reagent color. The rate of reaction also can be followed by 2-iminothiolane s absorbance at 248 nm (Xmax e = 8 840 M 1 cm 1). As the cyclic imidate reacts with amines, its absorbance at... 

This produces a neutral six-coordinate species with the requisite inertness and good tumor uptake (89). Attachment of this species to the appropriate antibody is accompHshed by means of the carbon-attached pendant group which terminates in a maleimide moiety. This can be induced to couple with a thiol that exists as a part of a favorably positioned cysteine residue in the antibody or has been introduced by reacting an exposed lysine residue in the antibody with 2-iminothiolane (Traut s reagent). 

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