【作者】 李春森；

【导师】 吴玮；

【作者基本信息】 厦门大学 ， 物理化学， 2007， 博士

【摘要】 细胞色素P450酶是自然界中最万能的生物酶之一。它利用生物体内的分子氧和两种还原等价物,催化多种底物发生单加氧化反应,具有保护生物体免受外源化学物质侵害,代谢药物和合成重要生命物质的功能。虽然经过多年可观的努力,P450还存在许多未解决的具有急切挑战性的问题。本文采用密度泛函理论方法研究了P450领域一些具有诱惑力的未解之谜,如N-脱烷基化、N-氧化和S-氧化等反应,论文主要由四个部分构成:1.C-H键的羟化反应是P450酶催化的一类重要反应,一般认为它是由被称作CompoundⅠ（CpdⅠ）的高价铁-端氧活性物种与底物相互作用,通过rebound反应机制发生,展现出双态反应性（two-state reactivity,TSR）。虽然TSR可以解释这种机制下所发生的不寻常的反应现象,虽然理论上也一再重复得到TSR的反应图景,但是一直存在一个悬而未决的疑问,那就是:是否存在很好的实验探针可以清楚地探测到TSR?在论文的这个部分中,我们采用DFT方法计算研究了底物N,N-二甲基苯胺（N,N-dimethylaniline,DMA）在简并的高自旋（high spin,HS）和低自旋（low spin,LS）CpdⅠ作用下发生的C-H键羟化反应,发现了一些新的令人兴奋的反应机制特征:计算所得到的DMA发生C-H键羟化反应的能垒很小,与实验上得出的“DMA是P450氧化反应中最具有反应活性的底物”的结论一致。由于LS的氢原子转移（hydrogen atom transfer,HAT）过渡态能垒明显比HS的低,反应极可能是通过LS反应路线进行,体现出反应的低自旋选择性。同时,高低自旋态的动力学同位素效应（Kinetic isotope effect,KIE）值差别巨大,且只有KIE_LS与实验值一致,因此我们认为KIE可以作为P450反应自旋态的准确探针。2.P450作用下胺底物生成N-脱烷基产物或N-氧化产物的单加氧化反应是P450的一个主要催化反应,它的反应机制仍然处于争议中。我们对底物三甲基胺（trimethylamine,TMA）的完整N-脱烷基化反应及N-氧化反应进行了DFT理论计算研究,发现HAT的反应机制在TMA的N-脱烷基化反应第一步反应——C-H键羟化反应——中是可行的,而且这一反应能垒很低。由于参与反应的活性氧化剂CpdⅠ的高低自旋态简并,因此反应体现出双态路线,但是由于LS的反应能垒比HS的低很多,因此与DMA底物的研究结果一致,TMA的C-H键羟化反应也是通过LS反应路线进行,是低自旋选择的。计算得到只有KIE_LS与实验值相一致,因此KIE作为P450反应态探针的结论仍然成立。羟化产物发生降解的C-N键解离反应是非酶促的过程,它在水分子作用下由醇胺降解为二级胺。与N-脱烷基反应类似,N-氧化反应也是低自旋选择的,但与C-H键羟化反应相比,二者竞争下C-H键羟化反应更易发生。N-氧化反应的逆反应能垒不高（25.8kcal/mol）,氧化三甲胺可能作为P450反应的氧源参与反应。3.我们采用DFT方法计算研究了CpdⅠ和Cpd 0催化下的二甲硫醚发生硫氧化的反应机制过程,结果表明CpdⅠ催化硫氧化反应具有低自旋选择性,反应能垒仅为7.1 kcal/mol,说明CpdⅠ在硫氧化反应中活性很高,催化的反应速率极快。而Cpd 0通过分步的O-O键均裂/O_d进攻的硫氧化反应需要跨越第一步至少17.8 kcal/mol的能垒,相对CpdⅠ而言,Cpd 0的活性显得很弱,对应的反应速率约比CpdⅠ的慢6个数量级。此外Cpd 0通过O-O键均裂产生活性自由基物种的反应还受到其它平行反应的有效竞争,因此我们认为,在原态的P450中对S-氧化起作用的活性物种是CpdⅠ,甚至在突变的P450中,Cpd 0的作用也可以忽略。实验上T268A突变的P450_BM3催化硫醚脂肪酸发生S-氧化时,底物自身对CpdⅠ的产生起到促进作用,因此突变前后S-氧化产物不变。这样,我们对P450催化S-硫化的反应机制难题给出了清楚的答案并且建议了新的值得尝试的实验。4.我们对P450催化底物p-methylsulfoxy-N,N-dimethylaniline（p-MS-DMA）发生的区域选择氧化进行了初步的DFT计算,研究表明当CpdⅠ作为主要反应活性氧化剂时,N-脱烷基化反应比S-氧化反应快很多,N-氧化反应在三者中不具备竞争活性。在CpdⅠ存在时,Cpd 0对S-氧化处于“哑声”状态。CpdⅠ不存在时,Cpd 0相对CpdⅠ而言,仍然是参与催化S-氧化的较差的氧化剂。更多还原

【Abstract】 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.更多还原