Adding 20 mM EDC to a mixture of 40 mM C6P and 40 mM C8 initiated the reaction network (Figure ). We investigated the kinetic profile of the reaction using HPLC and confirmed the compound identity by LCMS (Figure , Supplementary Figure , and Supplementary Table ). We observed that the 20 mM EDC was consumed within the first hour after its addition, which resulted in the formation of two new, transient peaks in our chromatogram that were identified to be the acyl phosphate (C8C6P) and the symmetric acid anhydride of C8 (C8C8, see Supplementary Figure ). The C8C8 was short-lived and fully decayed in 2.5 hours. Instead, the C8C6P decayed over four days. In a short time window, all EDC was consumed while C8C8 was still present. In that time, the concentration of C8C6P increased, from which we conclude that some of the C8C6P was synthesized through the C8C8 intermediate (black arrow in Figure ), which is in line with very recent work by von Delius and coworkers. Additionally, we observed a decrease in the concentration for both C8C8 and C8C6P. We detected up to 100 micromolar of the by-product N-acylisourea (Supplementary Figure ). We further confirmed the absence of detectable amounts of byproducts that could be produced by EDC and alkyl phosphates, e.g., dialkyl pyrophosphate. We used the HPLC data to write a kinetic model that predicts the concentration of all species involved in the chemical reaction cycle (See Methods section in the Supplementary Information). The model solves a set of ordinary differential equations and fits it to the experimental data, minimizing an error function using a least mean square method. We validated the kinetic constants we obtained from fitting one set of typical reaction conditions by predicting the kinetic profiles of all compounds involved at different fuel levels and starting acid and phosphate concentrations. The set of rate constants reasonably fitted the experimentally measured kinetic profiles at varying fuel and starting acid/phosphate concentrations well (See Supporting Table ). The model further predicts that the half-life of C8C6P is t1/2 = 16 hours.