Thiolating reagents

PDPH also may be used as a thiolation reagent to add sulfhydryl functional groups to carbohydrate molecules. The reagent can be used in this sense similar to the protocol described for AMBH (Chapter 1, Section 4.1). After modification of an oxidized polysaccharide with the hydrazide end of PDPH, the pyridyl group is removed by treatment with DTT, leaving the exposed sulfhydryl (Figure 5.15). 

Thus, this reagent can be used to label fluorescently proteins and other biomolecules containing free sulfhydryl residues. If there are no —SH groups available, their creation can be accomplished by reduction of indigenous disulfides or through the use of various thiolation reagents (Chapter 1, Section 4.1). 

Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups... 

Add 1-10 mg of a protein or antibody containing an available thiol group to the particle suspension. Alternatively, add the protein to be coupled to the particle suspension in an amount equal to 1-10 X molar excess over the calculated monolayer for the protein type to be coupled. The optimal amount of protein to be added should be determined experimentally. Creating thiol groups on proteins or peptides may be done from disulfides by reduction. Alternatively, a thiolation reagent may be used to add thiols to the protein surface for coupling (see the protocols in Chapter 1, Section 4.1). 

Figure 20.13 The thiolation reagent SATA can be used to create sulfhydryl groups on Fab fragments. After deprotection of the acetylated thiol of SATA with hydroxylamine, conjugation with a maleimide-activated enzyme can take place, producing thioether linkages.

Iodoacetate derivatives have been used for decades to block or crosslink sulfhydryl groups in proteins and other molecules (Chapter 1, Section 5.2). At mildly alkaline pH values (pH 8-8.5), iodoacetyl derivatives are almost entirely selective toward the cysteine —SH groups in proteins. Disulfide reduction or thiolation reagents can be used to create the required sulfhydryl groups on proteins containing no free sulfhydryls. 

As has been mentioned above, thermal decarboxylation of heterocycle 36 can be coupled with electrophilic trapping with either bromine or nitro groups to yield the substituted analogues. The same heterocyclic system 59 can be thiolated by treatment either with elemental sulfur (followed by alkylation with an alkyl halide) to afford 60, or the electrophilic thiolating reagent 58 to generate 57 (Scheme 5) <2005HCA1208>. 

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