Molecular Simulation Study On The Degradation Mechanism Of N-Glycolylneuraminic Acid

The work was mainly focused on the exploration of content decrease mechanism on non-human red meat N-glycolylneuraminic acid,which was a kind of red meat carcinogenic factors,during food processing factors（solvent effects,electric effects,interaction with hydroxyl radical（OH·）and inulinase,etc）.Based on the geometry optimization at the M062X/6-311+G（d,p）level,the structural properties of Neu5Gc were explored with the concept of density functional theory and wave function analysis methods.The molecular electronic structure,reaction parameters,antioxidant mechanism of Neu5Gc was considered under different dielectric constant solvents（benzeneε=2.27,acetic acidε=6.25,ethanolε=24.85,lactic acidε=22.00,formic acidε=51.1,waterε=78.35）.In order to study the structural stability changes of Neu5Gc molecules under the external electric field（0,0.005,0.01,0.015,0.02,0.025au）with different strengths in the Cartesian X-axis positive direction and above solvents,the PBE1PBE/TZVP level was applied to perform vibration and absorption spectroscopy calculations.According to the above analysis,the effect and possible products of OH·on Nu5Gc in vitro was simulated by ultraviolet assistant Fenton oxidation system and detected by Liquid-phase mass spectrometry（LC-MS）,firstly.The fitted possible reaction paths were calculated with the transition state theory and reaction potential energy surface theory.Secondly,the LC-MS,molecular docking,molecular dynamics simulation and intermolecular interaction calculations were used to study the effect and molecular mechanism of two inulinases on the reduction content of Neu5Gc standards.The electronic structure properties and reactivity parameters calculations show that there are four intramolecular hydrogen bonds exist in the Neu5Gc molecule in the gas phase.The atom in molecular theory results indicates that they are all weak hydrogen bonds.The HOMO orbitals are mainly distributed around the amide group,and the LUMO orbitals are mainly distributed around the carboxyl group.The polar solvent can enhances the nucleophilic activity of the free radicals on the Neu5Gc hydroxyl hydrogen atom.The calculation of the antioxidant mechanism indicates that the carboxyl hydrogen atom is prone to occur hydrogen transfer mechanism（HAT）,and the alcohol hydroxyl hydrogen atom is prone to occur the sequential proton loss electron transfer mechanism（SPLET）.Theoretical spectrum analysis shows that the infrared and Raman spectrum of Neu5Gc in solvent are affected by induced dipole effect and intermolecular interaction with solvents,thus make the peaks of the spectrum was blue-shifted.The Infrared hydroxyl systolic vibration frequency of Neu5Gc in ethanol,formic acid and water phase were all increased,the maximum UV absorption wavelength value was 183.26 nm under acetic acid,which mainly contributed by the transition from HOMO to LUMO+1 orbital.The Infrared and Raman spectrum of Neu5Gc under electric field varies different due to the field induced dipole effect.At 0.025 au,the largest distribution peaks was caused by C-H bond validations,and the maximum UV absorption wavelength was 189.63 nm,which was mainly the transition from HOMO-4 and HOMO-1 to LUMO+1 orbital.The results of OH·simulated degradation in vitro showed that the best degradation rate was 76.23±2.17%at the follow treatment conditions:Hydrogen peroxide（H₂O₂）was 2%and the 254nm UV lamp was irradiation for 15 minutes.In the gas phase,benzene phase and water phase,the station points structures of all 19channels one-step hydrogen abstraction reactions between OH·and Nu5Gc were optimized at the M062X/6-31+G（d,p）level,enthalpy correction values was gained at the same time with the frequency calculation.Then,the M062X/def2TZVP level for calculate the reaction energy barrier.The results show that the hydrogen extraction energy barrier of H24 in Neu5Gc with 13.20 kJ/mol was the lowest at gas phase,while in water phase occurs at H29 with 12.97 kJ/mol,and the lowest activation energy was 1.72 kJ/mol at H38 position in the benzene phase.In temperature range of298K-473K,the fastest reaction rate was 7.97E-15cm³/molecule/s at H38 under 453K.Based on the comprehensive hydrogen abstraction process and the product secondary mass spectrometry,the possible pathway of Neu5Gc degradation by OH·was derived.The potential energy surface of the deduced pathway was calculated at the M062X/6-31G（d,p）level for station potions structure optimization,the calculated results indicates that when alcoholic hydroxyl dehydration and carbon-carbon bond break of Neu5Gc caused by OH·,the highest energy barrier can up to 79.94 kcal/mol.Therefore,the possible reason for Neu5Gc degradation by OH·can be the one step hydrogen extraction reaction on Neu5Gc non-hydroxyl hydrogen atoms,which was thermodynamic and kinetic both more favorable than other pathways.The average reduction rates of the exo-and endo-inulinases on Neu5Gc content were 55.33±5.1%and 43.81±4.3%,respectively.Exo-inulinase and endo-inulinase（protein database numbers 1Y9G and 3SC7,respectively）were selected for docking with Neu5Gc,and the subsequent molecular dynamics simulations showed that the active site residues of two inulinases can stably Neu5Gc to its active pocket through hydrogen bonding and hydrophobic interactions.The virtual mutation of amino acid residues and intermolecular weak interaction showed that the hydroxyl oxygen and amide nitrogen of Neu5Gc can interacted with the inulinase active residue ASP（with nucleophilic property）and the GLU（with acid-base property）,and formed stable hydrogen bonds.The binding energy of Neu5Gc to the two exo-and endo-inulinase was-22.71 and-8.463 kca/mol,respectively.Combined the molecular simulation results and the mass spectrometry analysis,indicated that the inulinase may reduce the Neu5Gc content by the role of active sites catalytic degradation effects.