Amines with SPDP

The following procedure is similar to the method of Cumber etal. (1985), but with some modifications. 

Dissolve the protein or macromolecule to be thiolated 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. The effective pH for the NHS ester modification reaction is in the 

SPDP-Modtied Protein Containing Active PyridyIdisulfide Groups 

Add 25 p,l of the stock solution of either SPDP or LC-SPDP in DMSO to each milliliter of the protein to be modified. If Sulfo-LC-SPDP is used, add 50 jxl of the stock solution in water to each milliliter of protein solution. 

Mix and react for at least 30 min at room temperature. Longer reaction times, even overnight, will not adversely affect the modification. 

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Figure 7.9 Amine-containing dendrimers can be activated with SPDP to create thiol-reactive derivatives. Alternatively, the pyridyl dithiol group may be reduced to create free thiols on the dendrimer surface for subsequent conjugation.

In a somewhat similar scheme, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with a dextran polymer. The carboxylate then was aminated with ethylene diamine to form an amine-terminal spacer (Figure 25.15). This dextran derivative finally was reacted with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1) to create the desired sulfhydryl-reactive polymer (Section 2.4, this chapter). The SPDP-activated polymer then could be used to prepare an immunoconjugate composed of an antibody against human colon cancer conjugated with the drug mitomycin-C. 

Figure 25.17 An amine-functionalized dextran derivative may be further reacted with SPDP to create a sulfhydryl-reactive product.

Noguchi et al. (1992) used an amine-terminal spacer arm derivative of dextran to react with SPDP (Chapter 5, Section 1.1) in the creation of a pyridyldisulfide-activated polymer (Fig. 391). Brunswick etal. (1988) used a different amine-terminal spacer arm derivative of dextran and subsequently coupled iodoacetate to form a sulfhydryl-reactive polymer (Fig. 392). Heindel et al. (1991) used a unique approach. They... 

SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate, is one of the most popular heterobifunctional crosslinking agents available. The activated NHS ester end of SPDP reacts with amine... 

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.

Modification of Amine-Dendrimers with Suifo-NHS-LC-SPDP... 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

Dissolve the purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

Figure 14.10 The crosslinker SPDP can be reacted with amine particles to create thiol-reactive pyridyl disulfide groups on the surface. Thiol-containing proteins or other thiol molecules can be reacted with these activated particles to result in disulfide linkages, which are reversible by reduction.

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

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