As expected, GPC traces of the resulting product (PSarGHS) after incubation demonstrate a slight but noticeable difference between the respective elution volumes. The minor difference in the apparent molar mass suggests that substitution occurs only with one available nucleophilic group (Figure ). The presence of a new signal at 3.80 ppm in the 1 H NMR spectra also substantiates a successful reaction (Figure ). An overlay of the 1 H NMR spectra of PSarGHS and GHS showed that the shifting of the signals 1 and 5 depends on the used buffer solution. After incubation in the TRIS-HCl buffer, signal 1 shifted towards lower The presence of side reactions such as cyclization or rapid hydrolysis of resulting thioester bonds cannot be excluded. Mitamura et al. reported on the rapid hydrolysis of the thioester bond in GHS conjugated products and reports the presence of a thiocarboxylic acid and an elimination product of the thioester in mass spectra . Here, MALDI-ToF mass spectrometry was performed to confirm the successful GHS conjugation. Interestingly, compared to PSaroxid, the MALDI-ToF MS of PSarGHS is much better resolved again gives a clear and narrow m/z distribution. While we could not attribute signals of the fully desired GHS conjugate PSarGHS, the most intense distribution can be attributed to a thiocarboxylic acid fragment with a K + (α) ion doping (Figure ). We suppose that resulting PSarGHS is not ionized efficiently itself but rather degrades during MALDI-ToF analysis. However, also the presence of the signals attributed to partly oxidized (ε) and oxidized (δ) PSar with amine ωtermini as well as partly hydrolyzed PSar bearing sodium (γ) and potassium (β) ion doping is observed (Figure ). The presence of the PSaroxid clearly shows that the conversion is not the complete under the chosen conditions.