Sulfo-LC-SMPT

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). 

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. 

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. 

SMPT or sulfo-LC-SMPT has been used to develop conjugates for in vivo delivery of siRNA to hepatocytes (Rozema et al., 2007), in preparing an anti-CD25-immunotoxin conjugate (Mielke et al., 2007), and in preparing conjugates for selective depletion of donor lymphocytes in stem cell transplantation (Solomon et al., 2005). 

Dissolve SMPT (Pierce) at a concentration of 7.7 mg/ml in acetonitrile (makes a 20 mM stock solution). Alternatively, the water-soluble Sulfo-LC-SMPT may be used and dissolved at a concentration of 6 mg/ml in water (makes a 10 mM solution). This should be done just prior to adding an aliquot to the thiolation reaction. Since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester, it should be used quickly to prevent significant loss of activity. If a sufficiently large amount of protein will be modified to allow accurate weighing of Sulfo-LC-SMPT, the solid may be added directly to the reaction mixture without preparing a stock solution in water. 

A water-soluble analog of SMPT, called sulfo-LC-SMPT or sulfosuccinimidyl-6-... 

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