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 pK_a1 and pK_a2 of H₂CEE²⁺ were found to be 2.30 and 5.25, respectively. The rate law is found to beRate = - dC_CEE / dt = k_{+ +} [H₂ CEE^{2 +}] [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.