One of the remaining questions on the reactivation of AChE is the possibility of a different approach of 2-PAM towards the phosphorus than the classical approach with the oxime is trans position relative to the catalytic serine (see Figure V-8A). The alternate approach that could be envisaged is an approach with the oxime in trans position relative to the phosphoryl bond. In a view of AChE’s active site with the oxyanionic hole on top and the phosphoryl bond completely vertical, the oxime would approach from underneath the phosphorus (see Figure V-8B). The pre-reactive position for 2-PAM trans from the phosphoryl
is 9.42 kcal.mol-1 less stable trans the classical approach in B3LYP-D3/def2-SV(P). This large
energy difference is confirmed by both the B3LYP-D3/def2-TZVP, and DLPNO-CCSD(T)/def2- TZVPP single points.
Figure V-8. Pre-reactive positions for 2-PAM in: A) trans from the serine position and B) trans from the phosphoryl
position
To completely rule out the possibility of the alternate approach, the energy difference between the reactant and product with both approaches was established. In the subsequent discussion, the reactant for both approaches are used as the respective 0 for this approach. The structure of the product of the alternate approach is not known as some form of rearrangement is to be expected. To not bias the energy difference from this approach a mono-dimensional scan forcing 2-PAM to close the distance with the phosphorus what performed. Besides the distance constraint between the phosphorus and the oxygen of the oxime all atoms are free to move and the expected rearrangement does occur. As the oxime is brought closer to the phosphorus the oxime slides up and breaks linearity with the axis of
the phosphoryl bond. The methyl substituent of VX slides opposite to the catalytic serine
simultaneously in what could be the result of a Berry pseudo-rotation (see Figure V-9).[20]
Figure V-9. Energy profile for the reactivation of VX-inhibited AChE by 2-PAM when Glu202 is unprotonated
following two possible approaches for the reactivator: trans from the serine (1/2) and trans from the phosphoryl group (1A/2A)
The energy profile in Figure V-9 contains the energetics of the reactivation of VX- inhibited AChE by 2-PAM through the classical approach in the case of unprotonated Glu202.
This reactivation has an energy difference of 18.30 kcal.mol-1 in B3LYP-D3/def2-SV(P) (17.00
kcal.mol-1 in B3LYP-D3/def2-TZVP and 9.55 kcal.mol-1 in DLPNO-CCSD(T)/def2-TZVPP) and an
energy barrier of 20.14 kcal.mol-1 in B3LYP-D3/def2-SV(P) (18.90 kcal.mol-1 in B3LYP-D3/def2-
TZVP and 17.49 kcal.mol-1 in DLPNO-CCSD(T)/def2-TZVPP). When the reactivation occurs
through the alternate approach the endothermicity is increased by 10.18 kcal.mol-1 in B3LYP-
D3/def2-SV(P) (5.49 kcal.mol-1 in B3YLP-B3/def2-TZVP and 5.68 kcal.mol-1 in DLPNO-
CCSD(T)/def2-TZVPP) to 28.12 kcal.mol-1 in B3LYP-D3/def2-SV(P) (22.49 kcal.mol-1 in B3LYP-
D3/def2-TZVP and 15.13 kcal.mol-1 in DLPNO-CCSD(T)/def2-TZVPP). The endothermicity of the
reactivation through the alternate approach exceeds the energy barrier for the reactivation through the classical approach. The energy barrier for the alternate approach should be at least as high in energy as the product for the alternate approach and is thus also higher than the barrier for the classical approach. Based on transition state theory, and using the endothermicity as a minimum boundary for the energy barrier, the alternate approach is expected to have a rate constant at least 420 times inferior to the classical approach. The choice was made not to spend tremendous computational resource and time to locate the transition state(s) for the reactivation from the alternate approach because the established
energy difference and energy barrier difference make this approach very unfavourable compared to the classical approach in the case of unprotonated Glu202. Calculations were also performed to check the feasibility of the alternate approach when Glu202 is protonated.
Figure V-10. Energy profile for the reactivation of VX-inhibited AChE by 2-PAM when Glu202 is protonated
following two possible approaches for the reactivator: trans from the serine (1P/2P) and trans from the phosphoryl group (1PA/2PA)
The energy profile in Figure V-10 presents the energetics of the react through both the classical and the alternate approach in the case of protonated Glu202.
The reactivation through the classical approach has an energy difference of 2.97
kcal.mol-1 in B3LYP-D3/def2-SV(P) (-0.19 kcal.mol-1 in B3LYP-D3/def2-TZVP and -6.87 kcal.mol-
1 in DLPNO-CCSD(T)/def2-TZVPP) and an energy barrier of 4.42 kcal.mol-1 in B3LYP-D3/def2-
SV(P) (8.84 kcal.mol-1 in B3LYP-D3/def2-TZVP and 1.89 kcal.mol-1 in DLPNO-CCSD(T)/def2-
TZVPP).
When the reactivation occurs through the alternate approach the endothermicity is
increased by 8.93 kcal.mol-1 in B3LYP-D3/def2-SV(P) (5.59 kcal.mol-1 in B3LYP-D3/def2-TZVP
and 4.27 kcal.mol-1 in DLPNO-CCSD(T)/def2-TZVPP) to 11.90 kcal.mol-1 in B3LYP-D3/def2-SV(P)
(5.40 kcal.mol-1 in B3LYP-D3/def2-TZVP and -2.60 kcal.mol-1 in DLPNO-CCSD(T)/def2-TZVPP).
The increase in endothermicity make the alternate approach much less favourable than the classical approach. Once again, the choice was made not to investigate the energy barrier for the reactivation through the alternate approach as there is compelling evidence that this approach is less favourable in the case of protonated Glu202 as well.
Now that the reactivation in both protonation states of Glu202 has been modelled in QM/MM, the question of the deprotonation of the reactivator in the active site remains to be studied for a full overview of the reactivation process.