NHS-PEG-Maleimide Coupling to Amine-Dendrimers

If a NHS-PEG-maleimide compound is used for this type of activation and coupling, the intermediate maleimide-activated dendrimer should be quickly purified of excess crosslinker and reaction by-products and immediately used to couple ligand. This is due to the fact that the maleimide hydrolyzes in aqueous solution at a higher rate than an SMCC-type crosslinker, because of the extreme hydrophilicity of the PEG spacer arm compared to the cyclohexane spacer of SMCC. 

Alternatively, shorter, discrete crosslinkers containing PEG spacers of known chain length are now available (Thermo Fisher, Quanta BioDesign see Chapter 18). These reagents are designed to contain an exact number of PEG units, typically from between 2 repeating units 

Dissolve 10mg of an amine-containing dendrimer into 1ml of 50mM sodium phosphate, 0.15 M NaCl, pH 7.5 (coupling buffer) with mixing. 

Dissolve NHS-PEGg-maleimide (MW 601.6) into DMSO at a concentration of 20mM. Short, PEG-type crosslinkers often exist as a thick oily mass, and preparing the solution typically involves dissolving an entire vial of the compound into DMSO to determine accurately the required concentration. Use only dry DMSO to avoid hydrolysis of the NHS ester. 

Add 50 pi of the NHS-PEGg-maleimide solution to the 1ml dendrimer solution and mix thoroughly to dissolve. This represents approximately a 14-fold molar excess of crosslinker over the quantity of dendrimer present, if a G-3 PAMAM dendrimer is used with an ethylenediamine core. The optimum molar ratio of crosslinker-to-dendrimer should be determined experimentally for best performance of the resultant conjugate in its intended application. If enough material is available, doing a series of experiments at different mole ratios of crosslinker-to-dendrimer will help to optimize the resultant conjugate. 

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