Synthesis of two photolabile poly(ethylene glycol) derivatives for protein conjugation

Two photolabile end functionalised mono methyl poly(ethylene glycol) (PEG) (5000 g/mol) derivatives were prepared. First, a diazopyruvyl derivative (PEG-DAP 4) that undergoes photo induced Wolff rearrangement to give a reactive ketene amide was prepared. Secondly, a commercially available N-hydroxysuccinimide ester of a hexanoyl substituted nitroaryl azide was coupled to PEG to provide the second derivate (PEG-ANPAH 11). Both derivatives were purified by filtration and trituration. They displayed the expected 1H NMR spectra and were sufficiently pure as determined by combustion analysis. Analysis by triple detection GPC suggested that the small, dipolar diazopyruvyl endgroup of PEG-DAP 4 did not induce aggregation while the more hydrophobic hexyl nitroaryl azide of PEG-ANPAH 11 did have the propensity to aggregate. Photoactivated conjugation of both reagents was observed to occur with lysozyme, with indications that PEG-DAP 4 may produce a mono-PEG conjugate. Lysozyme conjugation with PEG-ANPAH 11 appeared to give conjugates with increasing number of conjugated PEG molecules. The conjugation of ribonuclease with PEG-DAP 4 was observed to give fewer multi-PEG species while the mono-conjugate was produced.