Amine reactive conjugates

Carbohydrate molecules containing amine groups, such as D-glucosamine, easily may be conjugated to other macromolecules using a number of amine reactive chemical reactions and crosslinkers (Chapter 2, Section 1 and Chapter 3). Some polysaccharides containing acetylated... 

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. 

Finally, the small amine-reactive and photoreactive crosslinker, NHS-ASA (Chapter 5, Section 3.1), can be iodinated to provide a non-cleavable radioactive conjugate. 

Figure 14.25 The preparation of highly controlled fluorescent silica nanoparticles can be done by first polymerizing APTS that has been covalently modified with an amine-reactive dye to form fluorescent core particles. The core then is capped by a shell of silica by polymerization of TEOS. The shell layer can be further derivatized with silane coupling agents to provide functional groups for conjugation.

Figure 18.25 Amino-PEG -carboxylate compounds contain a primary amine on one end and a carboxylate group on the other end. They can be used to add water-soluble spacer arms to molecules or surfaces. Using an amine-reactive group, the amino-PEG -carboxylate compound can be coupled via an amide bond, thus leaving the carboxylate end free for further conjugation reactions. Avoid the use of single-step EDC conjugation reactions, as this will polymerize the amino-PEGK-carboxylate by reacting with both ends.

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