Pyridyl Disulfide Reagents

LC-SPDP (Chapter 5, Section 1.1) is an analog of SPDP containing a hexanoate spacer arm within its internal cross-bridge. The increased length of the extended crosslinker is important in some conjugations to avoid steric problems associated with closely linked macromolecules. However, for the preparation of immunotoxins, no advantages were observed for LC-SPDP over SPDP (Singh et al., 1993). 

This multi-step crosslinking method employing SPDP on both molecules has been used to prepare a number of immunotoxin conjugates (Edwards et al., 1982 Thorpe et al., 1982 Colombatti et al., 1983 Wiels et al., 1984 Vogel, 1987 Reiter and Fishelson, 1989). While 

For instance, if toxin A chain-antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of 

Another way of utilizing SPDP is to again activate the antibody to create the pyridyl disulfide derivative, but this time thiolate the toxin component using 2-iminothiolane (Chapter 1, 

The following methods are generalized to provide an overview of how SPDP can be used in these conjugation techniques. The appropriate optimization for a particular toxin conjugate should be done. 

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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). 

With mixing, add an aliquot of the crosslinker to the dendrimer solution to provide the desired molar excess of reagent. For many applications, less than 10 pyridyl disulfide groups are needed per dendrimer molecule therefore, molar ratios in the range of 5-20 X excess of crosslinker over the amount of dendrimer present typically are used. 

The most common type of heterobifunctional reagent used for the activation of lipid components includes the amine- and sulfhydryl-reactive crosslinkers containing an N-hydroxysuccinim-ide (NHS) ester group on one end and either a maleimide, iodoacetyl, or pyridyl disulfide group on the other end (Chapter 5, Section 1). Principle reagents used to effect this activation process include SMCC (Chapter 5, Section 1.3), MBS (Chapter 5, Section 1.4), SMPB (Chapter 5, Section 1.6), SIAB (Chapter 5, Section 1.5), and SPDP (Chapter 5, Section 1.1). Other... 

Figure 28.14 A trifunctional label transfer reagent containing a thiol-reactive pyridyl disulfide group, a photo-reactive phenyl azide, and a biotin affinity tag. This compound can be used to label bait proteins through available thiol groups and capture interacting prey proteins by photoreactive conjugation.

The 4 mM soln of the crude peptide [H-(Gly-Pro-Hyp)5-Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu-Gly-Cys(Acm)-Cys-Gly-Gly-OH] 28 in degassed argon-sat. DMF/AcOH (95 5) was added dropwise to a 100 mM soln of di[5-nitro(2-pyridyl)]disulfide (5 equiv) in DMF/AcOH (95 5) with exclusion of air oxygen. The reaction was monitored spectroscopically at 430 nm, and after completion (1 to 2 h), the solvent was removed under reduced pressure. The resulting residue was dissolved in H20 and the excess reagent was filtered off. The H20 was removed under reduced pressure and the residue was reprecipitated from TFE with methyl /ert-butyl ether and purified by preparative HPLC to give 29 yield 13% the product was characterized by MALDI-TOF-MS, HPLC, and amino acid analysis. 

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