Carboxyphosphate (CP) is an important intermediate involved in reactions catalyzed by acetyl-CoA carboxylase, pyruvate carboxylase, N5-Carboxyaminoimidazole ribonucleotide synthetase, propionyl-CoA carboxylase, urea amidolyase, and carbamoyl phosphate synthetase. Despite its important role, properties for CP have never been reported due to its short estimated half-life (t1/2 70 ms). Thus, the high level ab initio methods, MP2 and CCSD(T), along with the DFT functionals: B3LYP, BB1K, M05-2X, M06-2X, and MPW1K were used to investigate the structure and energetics of CP in both vacuum and the PCM continuum solvation model of water. It was found that CP adopts a novel pseudo-cyclic structure featuring an intramolecular charge-assisted hydrogen bond (CAHB) that is reminiscent of chair cyclohexane. This structure is found to be the most stable in both vacuum and implicit solvation for both mono and dianionic charge states. Additionally, the M06-2X/aug-cc-pVTZ level of theory was shown to give consistent agreement with ab initio methods for both geometric and energetic properties. The strengths of the CAHBs observed in mono- and dianionic CP were estimated to be within the range of -17.8 to -25.4 and -15.7 to -20.9 kcal/mol, respectively. This classifies them as short-strong but not low-barrier and makes them the dominant stabilizing feature for these conformations. pKa values were computed to distinguish between different possible protonation states of CP. The predicted pKa values were found to be be -3.43±0.81, 4.04±0.35, and 8.14±1.92 for the first, second and third acid dissociations of CP, respectively, indicating it is most likely to be present as a dianion or trianion in aqueous solution but more work is required to predict its charge state in the enzymatic pocket
