We recommend that coupling reactions between BG-PEG-NH
2 and NHS esters or activated carboxylic acids take place in anhydrous N,N-dimethyl formamide (DMF) using triethylamine as a base.
Typical reactions use approximately equimolar quantities of the BG-PEG-NH
2 and the desired NHS-label at 5-20 mM final concentration in the presence of a 1.5 fold molar excess of triethylamine in DMF. The reaction is generally performed at 30°C overnight.
Example Reaction: Coupling of BG-PEG-NH
2 to (+)-biotin-N-hydroxysuccinimide ester: Coupling of BG-PEG-NH 2 to (+)-biotin-N-hydroxysuccinimide ester. Triethylamine (0.9 µl, 6.2 µmol, 1.5 eq.) was added to a solution of BG-PEG-NH 2 (2.0 mg, 4.1 µmol) in 200 µl DMF in an Eppendorf tube. A solution of (+)-biotin-N-hydroxysuccinimide ester (1.4 mg, 4.1 µmol, 1.0 eq.) in 200 µl DMF was added to the mixture, which was shaken at 30°C overnight. The solvent was evaporated under vacuum. The redissolved solid was absorbed on silica gel and purified by flash column chromatography (CH 3Cl/MeOH 5:1) to give a colorless viscous solid after solvent evaporation.
The purification strategy will depend on the label used and the reaction scale. Good results have been obtained with both HPLC and silica get chromatography.