In our kinetic model, we do not consider the formation of the acylpyridinium ion formed due to the reaction between O-acylurea and pyridine. Hence, all constants related to O-acylurea also consider the acylpyridinium ion, which is why they monotonically change with pyridine concentration (Table , Figure ). S was 20 % without pyridine and increased to 97 % with 35 mM pyridine, indicating the N-acylurea gets much less favored with increasing pyridine. Still, no significant increase was observed between 10 mM pyridine and 35 mM pyridine (Figure ), suggesting the effect of pyridine concentration wears off. Next, we tested the effect of temperature on the suppression of N-acylurea by fueling 100 mM C3 with 50 mM EDC at 5, 21, and 35°C. An increase in temperature tends to increase reaction rates while decreasing selectivity. Indeed, with increasing temperature, the overall reaction cycle became faster (Figure , Table , Figure ). The anhydride concentration peaked at around 5 mM at all the temperatures, but the half-lives of the anhydride decreased from 16 min (5 °C) to 4 min (35 °C), respectively. Moreover, the concentration of Nacylurea doubled from 10 mM (5 °C) to 20 mM (35 °C) when increasing the temperature (Figure ). From the kinetic model, it became clear that the amount of EDC used to make anhydride decreased slightly while the fraction of EDC used for N-acylurea increased significantly (Figure ). We calculated the selectivity factor, which increased from 20 % (21 o C) to 50 % (5 o C) and a marginal decrease to 18 % (35°C) (Figure ). We conclude that the formation of the unwanted N-acylurea can be suppressed at lower temperatures. Similarly, we tested the effect of pH on the suppression of N-acylurea by fueling 100 mM C3 with 50 mM EDC in 200 mM MES buffer at 21 o C at pH 5, 6, and 7. With increasing pH, the overall reaction cycle became much slower because the rate of EDC consumption dropped by orders of magnitude (Table , Figure ). Moreover, with increasing pH from 5 to 7, the anhydride hydrolysis decreased, resulting in an increased anhydride half-life from 7.5 to 19.2 min (Table , Figure ). Due to the decreased reactivity of EDC, the maximum anhydride concentration dropped drastically (Figure ). Nevertheless, the amount of EDC used to produce anhydride varied minimally, with similar values for pH 5 and 7, around 46%, and a slight increase to 60% at pH 6 (Figure ). However, the formed N-acylurea concentration increased drastically from pH 5 to 7 (Figure , Figure ). In line with these data, the selectivity decreased from 58 % to -3 % with increasing pH (Figure ), which implies that at higher pH, more EDC is used to make the unwanted N-acylurea than the wanted anhydride. Overall, pH 5 showed the best ability to suppress N-acylurea and keep the reaction cycle efficient.