DFT Study Of The Monooxygenation Reactions Of The Substrates Containing Nitrogen Or Sulfur Atom By Cytochrome P450

Cytochrome P450 is one of the most versatile enzymes in nature.It uses dioxygen and two reducing equivalents to catalyze the monooxygenation reactions of wide variety of substrates so that it can function as oxidant which safeguards organisms against exnobiotics,metabolizes drugs,and leads to biosynthesis of vital compounds.There still remain mechanistic uncertainties and long-standing unresolved issues which pose tantalizing challenges to mechanistic chemistry although a few decades of considerable effort has been dedicated to elucidate the mechanisms of P450 catalyzed reactions.In this thesis,we employed density functional theory to investigate some intriguing unresolved issues that emerge from the area of P450,such as N-dealkylation,N-oxygenation and sulfoxidation.The thesis is composed of four parts:1.A major transformation performed by P450 enzymes is C-H hydroxylation that is believed to occur by reaction of the substrate with the high-valent oxo-ferryl species,known as CompoundⅠ(CpdⅠ).It was concluded that C-H hydroxylation proceeds by the rebound mechanism with two-state reactivity(TSR).While TSR accounts for the unusual results encountered in this mechanism and while theory has repeatedly produced this picture,a lingering question remains:is there any good experimental probe for TSR? In this part,we perform DFT calculations of C-H hydroxylation of N,N-dimethylaniline(DMA) by CpdⅠwith its high spin(HS) and low spin(LS) states and reveal some new exciting mechanic feature:The computational barrier of the C-H hydroxylation of DMA is very small which is in accord with the experimental observation that DMA is the most reactive substrate in P450 oxidation.The barrier of the bond activation transition state in LS is lower than that in HS.With a large energy difference between HS and LS hydrogen atom transfer (HAT) transition states,the reaction was proposed to proceed mostly via the LS pathway.The calculations demonstrate that the kinetic isotope effects(KIEs) of the two processes are very different and only KIE_LS fits the experimental datum.As such, KIE can be a sensitive probe of spin state reactivity. 2.A major mechanism,which is still under dispute,concerns the cytochrome P450-catalyzed oxidation of amine which may lead to N-dealkylation.and/or N-oxidation.Here we perform a DFT calculation of full N-dealkylation and N-oxygenation of trimethylamine(TMA) by CpdⅠof cytochrome P450.The calculations reveal that a hydrogen atom transfer(HAT) mechanism is at work for C-H hydroxylation of TMA in the first step of the N-dealkylation and the reaction barrier given in this calculation is a very low value for C-H hydroxylation.The reaction involves two processes nascent from the two spin states of CpdⅠbut exhibits a LS selective scenario same as in DMA C-H hydroxylation due to the obvious energy difference between HS and LS HAT transition states.Computed KIEs of the two processes are very different and only KIE_LS matches the experimental data.As such,we further confirm the conclusion that the KIE can be a sensitive probe of spin state reactivity.The reaction of C-N bond dissociation of the carbinolamine to generate N-dealkylated amine undergoes in a nonenzymatic process in water since the O-Fe bond energy in the hydroxylated product cluster is rather small.Similar to the N-dealkylation,the N-oxidation of TMA by P450 proceeds spin selectively,i.e., mostly via the LS pathway in a concerted single-step reaction pattern.With the smaller activation barrier relative to N-oxygenation,the C-H hydroxylation is preferred in the amine oxidations by P450.The tremethylamine-N-oxide can acts both as the oxygen donor and substrate via the reverse N-oxygenation pathway with a barrier of 25.8 kcal/mol.3.One of the major and a "hot" mechanistic problems in P450 chemistry is what is actually the oxidant species that carries the oxidative processes of this enzyme and what is the root cause of its immense versatility as a primary oxidant in nature:is it the high valent iron-oxo species,so-called CompoundⅠor the precursor species,so called Compound 0? We undertook the challenge posed by the experimental data and carried out theoretical calculations,designed to addresses the CompoundⅠ--Compound 0 conundrum in sulfoxidation of thiafatty acids by P450.Our results demonstrate that CompoundⅠleads to an extremely fast process,while Compound 0 will be at least six orders of magnitude slower.We suggest that most likely,thiafatty acids promote CompoundⅠformation even in the T→A mutant of P450_BM3.In this manner we provide a clear answer to a major mechanistic puzzle and suggest some new experiments.4.A preliminary DFT calculations were carried out for the study of regiochemistry by the enzyme cytochrome P450 by taking p-methylsulfoxy-N,N-dimethylaniline(p-MS-DMA) as substrate.It has been revealed that when taking CpdⅠas the primary oxidant,the N-dealkylation is much faster than sulfoxidation and the N-oxygenation has not competitive reactivity.In the presence of CpdⅠ,Cpd 0 is clearly silent for sulfoxidation.While in the absence of CpdⅠ,Cpd 0 is still an inferior oxidant compared with CpdⅠ.