Amines with SMPT

succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyl disulfide end similar to SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21, Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT (Thermo Fisher). 

Since SMPT is not soluble in aqueous solutions it must be first dissolved in organic solvent and an aliquot of this stock solution transferred to the reaction solution. The reagent is soluble 

Sulfo-LC-SMPT is not as stable as SMPT. The sulfo-NHS ester is more susceptible to hydrolysis in aqueous solutions and the pyridyl disulfide group is more easily reduced to the free sulfhydryl. Stock solutions of sulfo-LC-SMPT may be prepared in water, but should be used immediately to prevent loss of amine coupling ability. 

Dissolve the protein or macromolecule to be thiolated at a concentration of lOmg/ml in 50mM sodium phosphate, 0.15M NaCl, pH 7.2. Other non-amine-containing buffers such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The effective pH for the NHS ester modification reaction is in the range of 7.0-9.0, but conditions close to neutrality will limit ester hydrolysis. 

Add 25 pi of the stock solution of SMPT in acetonitrile to each ml of the protein to be modified. If sulfo-LC-SMPT is used, add 50 pi of the stock solution in water to each ml of protein solution. 

Since SMPT is not soluble in aqueous solutions it must be first dissolved in organic 

Even when an SMPT/acetonitrile ahquot is added to an aqueous solution and stored at room temperature, SMPT will only lose about 5% of its activity after 16 h. By contrast, other NHS esters usually have half-lives of only 2—6 h in aqueous environments. 

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Figure 1.67 SMPT can be used to modify the amine groups of proteins to form disulfide intermediates. The disulfides can be reduced with DTT to create free thiols for subsequent conjugation purposes.

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