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). 

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.

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. 

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. 

Figure 5.3 SMPT can form crosslinks between an amine-containing molecule and a sulfhydryl-containing compound through amide and disulfide linkages, respectively. The hindered nature of the disulfide group provides better stability toward reduction and cleavage.

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). 

SMPT often is used in place of SPDP for the preparation of immunotoxin conjugates. The hindered disulfide of SMPT has distinct advantages in this regard. Thorpe et al. (1987) showed that SMPT conjugates had approximately twice the half-life in vivo as SPDP conjugates. Antibody-toxin conjugates prepared with SMPT possess a half-life in vivo of up to 22 hours, presumably due to the decreased susceptibility of the hindered disulfide toward reductive cleavage. 

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

Dissolve SMPT (Thermo Fisher) in DMF at a concentration of 4.8mg/ml. Add 27 pi of this solution to each ml of the antibody solution. Mix gently. The final concentration of SMPT in the reaction mixture is 0.13mg/ml, which translates into about a 4.8-fold molar excess of crosslinker over the amount of antibody present. 

Remove unreacted SMPT and reaction by-products by gel filtration on a desalting resin. Pool fractions containing SMPT-activated antibody (the first peak eluting from the column) and concentrate them to lOmg/ml using centrifugal concentrators with a MW cutoff of 10,000. 

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