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 H-1 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. (c) 2005 Elsevier B.V. All rights reserved.