Pyridyl Disulfides

The heterobifunctional PEGs are very useful in linking two entities in cases where a hydrophilic, flexible, and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimides, vinyl sulfones, pyridyl disulfides, amines, carboxylic acids, and /V-hydroxysuccinimide (NHS) esters. 

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. 

Figure 1.123 2,2 -Dipyridyl disulfide reacts with thiols to form an active pyridyl disulfide intermediate.

Figure 5.2 SPDP can react with amine-containing molecules through its NHS ester end to form amide bonds. The pyridyl disulfide group then can be coupled to a sulfhydryl-containing molecule to create a cleavable disulfide bond.

Dissolve a protein or macromolecule containing primary amines at a concentration of 10 mg/ml in 50 mM sodium phosphate, 0.15 M NaCl, pH 7.2. Other non-amine-containing buffers such as borate, HEPES, and bicarbonate also may be used in this reaction. Avoid sulfhydryl-containing components in the reaction mixture as these will react with the pyridyl disulfide end of SPDP. The effective pH for the NHS ester modification reaction is in the range of 7-9, but hydrolysis will increase at the higher end of this range. 

Figure 5.14 PDPH reacts with thiol-containing compounds through its pyridyl disulfide end to form reversible disulfide linkages. Its hydrazide end then may be subsequently conjugated with an aldehyde-containing molecule to form hydrazone bonds. Glycoproteins may be crosslinked using this approach after periodate activation to...

Figure 5.15 PDPH may be used to add a sulfhydryl group to an aldehyde-containing molecule. After reacting its hydrazide end with the aldehyde to form a hydrazone bond, the pyridyl disulfide may be reduced with DTT to create a free thiol.

Figure 5.32 APDP can modify sulfhydryl-containing compounds through its pyridyl disulfide group to form disulfide bonds. Its phenyl azide end then can be photolyzed with UV light to couple with nucleophiles via a ring-expansion process. The disulfide group of the crosslink can be selectively cleaved using Dll.

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. 

Figure 9.24 AMCA-HPDP reacts with sulfhydryl groups through its pyridyl disulfide end to form reversible disulfide bonds.

Figure 10.3 Antibody molecules oxidized with sodium periodate to create aldehyde groups on their polysaccharide chains can be modified with PDPH to produce thiols after reduction of the pyridyl disulfide. Direct labeling of the sulfhydryls with "Tc produces a radioactive complex.

Figure 11.9 Biotin-HPDP reacts with sulfhydryl-containing molecules through its pyridyl disulfide group, forming reversible disulfide bonds. The biotin group may be released from modified molecules by reduction with DTT.

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