Data on the removal of peroxides from functionalized polyethylene glycol (PEG) and effects on the stability and sensitivity of resulting PEGylated conjugates

Abstract

Cyanine-5 (Cy5) is a fluorescent dye active in the far-red region of the visible spectrum (λ ex = 646 nm, λ em = 662 nm) [1] . While Cy5 displays fluorescence in its oxidized form, it can be readily converted to its non-fluorescent hydrocyanine equivalent (H-Cy5) when exposed to reducing agents. H-Cy5 can then be converted back to its fluorescent oxidized form when exposed to reactive oxygen species (ROS), allowing it to act as a highly sensitive, high wavelength fluorescent ROS sensor [2] . However, H-Cy5 is a small, poorly water-soluble molecule, and is rapidly taken up into cells in vivo , preventing its use for sensing extracellular ROS, which are implicated in inflammation, wound healing, and other processes [3] , [4] , [5] , [6] . A solution to this lies in the conjugation of Cy5 to a polyethylene glycol (PEG) polymer, increasing its solubility [7] . This conjugate (Cy5-PEG) can be reduced to H-Cy5-PEG to allow the highly sensitive detection of ROS in an aqueous extracellular environment. However, after PEG conjugation, a significant decrease in stability and sensitivity is observed, likely owing to the presence of ROS contaminants in commercial samples of PEG. It has been reported that these ROS impurities can be removed from PEG through a simple freeze-drying procedure [8] . Here, we demonstrate that a simple, straightforward method for the purification of PEG can allow the synthesis of a highly functional, water-soluble ROS sensor that could be used for extracellular ROS sensing.

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“…Using a previously described method for peroxide removal, a new H‐Cy5‐PEG derivative that displayed greatly improved stability in aqueous solutions, comparable to that of the unconjugated H‐Cy5, was prepared (Figure 1d). [ 70 ] The excitation and emission spectra of the oxidized Cy5 and Cy5‐PEG were also evaluated and found to be no shifts, suggesting that PEGylation causes no interference with the fluorescent ROS‐sensing moiety of the dye (Figure 1b).…”