N- and S-Oxidation Model of the Flavin-containing MonoOxygenases

Transcription

1 N- and S-Oxidation Model of the Flavin-containing MonoOxygenases Peter J. Walton, Mario Ӧeren, Peter Hunt, Matthew Segall Optibrium, StarDrop, Auto-Modeller, Card View, Glowing Molecule and Augmented Chemistry are trademarks of Optibrium Ltd.

2 Background Flavin-containing MonoOxygenase (FMO) 5 active isoforms (FMO1 5) FMO3 major isoform found in adult liver Flavin Moiety Enzyme class found in multiple tissues Phase I enzyme involved in compound metabolism Metabolites commonly subject to Phase II metabolism PDB ID: 2XVE 2

3 Role of Flavin-containing Monooxygenases Works in conjunction with Cytochrome P450 in Phase I metabolism FMO3 polymorphism responsible for trimethylaminuria Decreased rate of metabolism leads to increased drug exposure Variety of reaction types possible: N/S-Oxidation 99% cases Demethylation, Bayer-Villiger Oxidation, Desulfuration Trimethylamine Chlorpromazine Voriconazole Fenthion Tozasertib 3

4 N/S Oxidation N/S-Oxidation involves transfer of Oxygen from peroxide of FAD OOH Debate in the literature regarding mechanism: Radical S N 2 Our focus: Better understanding of the mechanism substrate oxidation by FAD peroxide. 4

5 Model System

6 Simplifying the System Full FAD too large for practical calculations Tail buried in groove and does not interact with substrate Compared bond stretching of the full FAD OOH vs the simplified analogue Behaviour of simplified system at peroxide site analogous to larger molecule DFT Energy (kj mol -1 ) DFT Energy vs r O-O for FAD and Simplified FAD O-O Bond Length (Å) FAD Simplified FAD B3LYP/def2-SVP 6

7 Finding the Transition State We searched for both S N 2 and radical transition state Despite multiple attempts we were unable to find a transition state for a radical mechanism Transition state found for closed-shell system, S N 2 mechanism indicative of B3LYP/def2-SVP Bach R. D., J. Phys. Chem. A 2011, 155, 40,

8 Analysis of the S N 2 Mechanism

9 Thermodynamic Feasibility 900 N-O bond length (Å) kj mol -1 Interaction Energy (kj mol -1 ) O-O N-O 233 kj mol O-O bond Length (Å) 73 kj mol -1 B3LYP/def2-SVP QTAIM 9

10 Electron Density Reactant e Bohr -3 B3LYP/def2-SVP 10

11 Electron Density Pre-transition State e Bohr -3 B3LYP/def2-SVP 11

12 Electron Density Transition State e Bohr -3 B3LYP/def2-SVP 12

13 Electron Density Post-transition State e Bohr -3 B3LYP/def2-SVP 13

14 Electron Density Comparison of reactant and post-transition state structures B3LYP/def2-SVP 14

15 Properties Derived from Electron Density

16 Charge Analysis Isolated FAD OOH Transition State Hirshfeld Charge 16

17 Charge Analysis Transition State FAD O- Anion Hirshfeld Charge 17

18 Charge Analysis Transition State FAD O. Radical Charges seen in transition state more consistent with S N 2 mechanism Hirshfeld Charge 18

19 Orbital Investigation Orbital shape and orientation conducive to successful interaction Lone pair of trimethylamine (TMA) large component of HOMO O O σ* component of the LUMO of FAD OOH accessible HOMO of TMA LUMO of FAD OOH B3LYP/def2-SVP 19

20 Full S N 2 Mechanism

21 Constrained Optimisation Fixed N O bond length = 5.00 Å Pre-optimised Structure Post-optimised Structure B3LYP/def2-SVP 21

22 Constrained Optimisation Fixed N O bond length = 1.85 Å Pre-optimised Structure Post-optimised Structure B3LYP/def2-SVP 22

23 Constrained Optimisation Fixed N O bond length = 1.70 Å Pre-optimised Structure Post-optimised Structure B3LYP/def2-SVP 23

24 Full Concerted Mechanism Hydrogen transfer during optimisation shows no secondary local minima B3LYP/def2-SVP 24

25 Application to Other Molecules 121 kj mol kj mol -1 Observed Non-Observed 156 kj mol kj mol kj mol kj mol kj mol-1 28 kj mol kj mol kj mol kj mol kj mol kj mol -1 B3LYP/def2-SVP 25

26 Summary Identified transition state for closed-shell (S N 2) mechanism Extensive tests further support S N 2 mechanism Modelling of reaction coordinate provides a greater understanding of the N/S-Oxidation process Further tests indicate that this mechanism is applicable to larger substrates This enables further efforts to predict FMO metabolism 26

27 Thank You! Mario Ӧeren Peter Hunt Matthew Segall Jonathan Hirst 27