Non-enzymatic hydrolysis of creatine ethyl ester

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 °C over the pH range of 1.6–7.0 using 1H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE+), and the diprotonated form (H2CEE2+). The values of pKa1 and pKa2 of H2CEE2+ were found to be 2.30 and 5.25, respectively. The rate law is found to beRate=-dCCEE/dt=k++[H2CEE2+][OH-]+k+[HCEE+][OH-]+k0[CEE][OH-]Rate=-dCCEE/dt=k++[H2CEE2+][OH-]+k+[HCEE+][OH-]+k0[CEE][OH-]where the rate constants k++, k+, and k0 are (3.9 ± 0.2) × 106 L mol−1 s−1, (3.3 ± 0.5) × 104 L mol−1 s−1, and (4.9 ± 0.3) × 104 L mol−1 s−1, respectively. Calculations performed at the density functional theory level support the hypothesis that the similarity in the values of k+ and k0 results from intramolecular hydrogen bonding that plays a crucial role. This study indicates that the half-life of CEE in blood is on the order of one minute, suggesting that CEE may hydrolyze too quickly to reach muscle cells in its ester form.