Having established that DTT and nitrogen aid in stabilization of 1 and 2, we questioned if these conditions would affect the activity of PyNP-04. Therefore, we performed phosphorolysis experiments at 80 °C with either a in nitrogen-saturated buffer or the model substrate 5iodouridine (c) in buffer with 5 mM DTT, as the latter nucleoside allowed us to obtain UV spectra that could be deconvoluted despite the heavy background absorption of DTT at lower wavelengths (see Figure of ref. ). In contrast to the comparable activity observed in nitrogensaturated buffer, the application of DTT caused a ≈40% drop in enzymatic activity with our model substrate c (160 U mg -1 to 95 U mg -1 ; Figure ). However, due to the prevention of oxidation by DTT, we considered this decrease in enzymatic activity tolerable and proceeded with these conditions. In summary, the application of hypoxic conditions combined with the reducing agent DTT improved the stability of 1 and 2 in aqueous solution with ). Please see the externally hosted Supporting Information for raw data. no detectable degradation after 24 h at 80 °C, while only slightly affecting the enzymatic activity.