p450

PAPER /dalton |Dalton TransactionsTheoretical study of N -dealkylation of N -cyclopropyl-N -methylanilinecatalyzed by cytochrome P450:insight into the origin of the regioselectivity†Dongmei Li,a ,b Yong Wang,a Chuanlu Yang c and Keli Han*aReceived 25th June 2008,Accepted 1st October 2008First published as an Advance Article on the web 19th November 2008DOI:10.1039/b810767jThe mechanism of N -dealkylation of N -cyclopropyl-N -methylaniline (3)catalyzed by cytochrome P450(P450)was investigated using density functional theory.This reaction involves two steps.The first one is a C a –H hydroxylation on the N -substituent to form a carbinolaniline complex,and the second is a decomposition of the carbinolaniline to yield cyclopropanone (or formaldehyde)and N -methylaniline (or N -cyclopropylaniline).Our calculations demonstrate that the first step proceeds in a spin-selective mechanism (SSM),mostly on the low-spin (LS)doublet state.The rate-limiting C a –H activation is an isotope-sensitive hydrogen atom transfer (HAT)step.The environmental effect switches the regioselectivity of this reaction from a competition between N -decyclopropylation andN -demethylation to a clear preference for N -demethylation.This preference is consistent with former experimental studies.However,it is not in accord with the normal D E-BDE correlation since the BDE of C a –H on the methyl group is higher than that on the cyclopropyl group.Insight into the origin of the preference for N -demethylation reveals that tertiary amine 3is different from normal hydrocarbons,possessing a unique p Ph -p C-N conjugated system.The electron delocalization effect of the p Ph -p C-N conjugated system in 3makes the transition state pose a polar character,and the bulk polarity and hydrogen bonding capability of the protein pocket can exert a remarkable effect on the regioselectivity of N -dealkylation of 3.Decomposition of carbinolaniline is a water-assisted proton-transfer process in the nonenzymatic environment.The ring-intact cyclopropanone formed in the reaction sheds some light on the inability of 3to inactivate P450during its N -decyclopropylation.IntroductionCytochrome P450(P450)is a family of heme-monooxygenases found in most forms of life including bacteria,plants,and mammals.Due to its key role in many biochemical processes,the mechanism of P450-catalyzed reactions has been extensively studied.1–10One of the most intriguing reactions is N -dealkylation of alkylamine,which has been of great interest and debated for many years.Two alternative mechanisms have survived the years of discourse.One is a formal hydroxylation of a C–H bond on the carbon adjacent to the heteroatom (HAT,hydrogen atom transfer),11–14the other is a one-electron oxidation of the heteroatom itself (SET,single electron transfer).5,15The cyclopropylamine structure is found in numerous natural products,drugs and drug candidates,many of which undergo P450-catalyzed N -dealkylation with loss of the cyclopropyl group and other acyclic alkyl groups.Some cyclopropylamines are also suicide substrates for P450.16–18The cyclopropyl group’s unique electronic structure compared to ordinary aliphatic groups 19and unique ability to inactivate P45016–18promoted cyclopropylaminesa State Key Laboratory of Molecular Reaction Dynamics,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China.E-mail:klhan@;Fax:+86-411-84675584;Tel:+86-411-84379293bGraduate School of the Chinese Academy of Sciences,Beijing 100039,China cDepartment of Physics,Ludong University,Yantai 264025,China†Electronic supplementary information (ESI)available:Tables S1–S5:SCF energies,Mulliken charge and spin densities,and data for KIE calculations.See DOI:10.1039/b810767jbeing widely applied as probes of P450and other oxidative enzymes in many mechanistic studies 20–30involved in the “HAT-SET controversy”.The HAT mechanism was early and widely accepted on the observation that the alkyl group lost from an ordinary amine during N -dealkylation appeared as an aldehyde or ketone,which arises from the decomposition of a carbinolamine intermediate.31,32Hanzlik once invoked this mechanism to explain the observation that N -benzyl-N -cyclopropylamine (1)inactivated P450as it underwent P450-catalyzed oxidation.16However,the subsequent observation that N -benzyl-N -(1¢-methylcyclopropyl)amine (2),the 1¢-methyl derivative of 1,which lacks an a -H,also inactivated P450nearly as efficiently as 117,18challenged the HAT mechanism and proposed the SET mechanism.Since then,both mechanisms have been debated by many different authors.However,for many years,the fate of the cyclopropyl group lost during N -dealky-lation and the nature of the reactive intermediate involved in the inactivation of P450remained obscure.More recently,Hanzlik and co-workers performed product studies on the oxidation of cy-clopropylamines by P450and other enzymes using dual [14C/13C]labeling NMR.They first reported that oxidation of N -cyclo-propyl-N -methylaniline (3)by horseradish peroxidase,a well-known SET oxidant,led exclusively to ring-opened products.21,22In sharp contrast,P450-catalyzed oxidation of 3led to ring-intact metabolite cyclopropanone hydrate and no ring-opened products,and 3did not inactivate P450.23The characterization of the cyclopropanone hydrate provided a direct evidence for the HAT mechanism and ruled out the SET mechanism.To helpD o w n l o a d e d b y D A L I A N U N I VE R S I T Y OF T E C H N O L OG Y o n 31 J u l y 2010P u b l i s h e d o n 19 N o v e m b e r 2008 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 810767Jreconcile the different behaviors of 3with SET versus HAT,Cerny and Hanzlik undertook a detailed exploration of the interaction of 1with P450,both as a substrate and as an inactivator.24They reported for the first time that 3-hydroxypropionaldehyde (3HP),the long anticipated “signature metabolite”for SET oxidation of a cyclopropylamine,was indeed a major metabolite of 1.They also found that N -benzyl-N -cyclopropyl-N -methylamine (4)did not inactivate P450and did not give rise to 3HP without first undergo-ing N -demethylation to 1.Thus,they suggested that HAT at C a –H bonds in secondary and tertiary cyclopropylamines (e.g.,1,4and 3)led to nonrearranged carbinolamine intermediates and thereby to “ordinary”N -dealkylation products including the ring-intact cyclopropanone hydrate.Alternatively,HAT at the N–H bond (N-HAT)of secondary cyclopropylamines (e.g.,1)gave a neutral aminyl radical,which could undergo rapid ring-opening leading either to enzyme inactivation or 3HP formation (Scheme 1).Therefore,it’s necessary for us to model the N -dealkylation of cyclopropylamines and identify the fate of the cyclopropyl group that is lost during the reaction in the aspect of calculation,which can shed light on the formation of cyclopropanone hydrate and 3HP or the enzyme inactivation.Herein,we use density functional theory (DFT)to focus on the HAT of tertiary cyclopropylamine 3,which leads to the ring-intact cyclopropanone hydrate.The N-HAT of secondary cyclopropylamine 1,which leads to the ring-opened 3HP and inactivation of the enzyme is underway.Scheme 1Proposed mechanisms for interaction of 1,3,4with P450.Many DFT studies have been performed to elucidate the mechanism of C–H hydroxylation.9,10An excellent linear corre-lation between the energy barrier (D E)of HAT by the active species of P450(Compound I,Cpd I in brief)and the C–H bond dissociation energy (BDE)was established for a series of hydrocarbons.33Experimental studies showed that the barrier ofHAT by metal-oxo complexes or non-heme iron(IV)-oxo catalysts exhibited a linear correlation with the C–H bond energy as well as with the strength of the newly formed O–H bond.34,35DFT calculations on the energies of HAT were demonstrated to be able to predict the regioselectivity of substrate metabolism.36While rate constants and selectivities for HAT from normal hydrocarbons were generally expected to parallel the strength of the C–H bond,this trend was not observed for tertiary amines.Hydrogen abstractions from tertiary amines by the t -butoxyl radical (t BuO ∑,which had been used as the chemical model for P450)were reported to be entropy controlled,and the activation energy did not vary significantly with the C–H BDE.37,38Moreover,product analysis by Shaffer et al.23revealed a preference for N -demethylation over N -decyclopropylation in the metabolism of 3by P450.Bhakta and Wimalasena 25,26also observed a high product ratio of p -Cl-N -cyclopropylaniline to p -Cl-methylaniline in the N -dealkylation of p -Cl-3,although the C a –H bond strength on the cyclopropyl group was weaker than that on the methyl group in 3(or p -Cl-3).Earlier theoretical studies on the hydroxylation mechanism of alkanes revealed a two-state reactivity (TSR)scenario,39–43originating from the high-spin (HS)quartet and low-spin (LS)doublet states of Cpd I.However,recent work by Li et al.44indicated that C a –H hydroxylation of N,N -dimethylaniline (5)was in a spin-selective manner (SSM).Moreover,the results of Wang et al.45showed that the mechanism switched from SSM to TSR in the C a –H hydroxylation of a series of p -substituted 5.From the above description,we can see that the mechanism of 3N -dealkylation poses some intriguing puzzles.First,since the D E-BDE correlation is predictive for normal hydrocarbons,33,46why do the activation energies for tertiary amines not parallel the C–H BDEs 37,38and why do experiments 23,25,26show an observation that N -demethylation is preferred over N -decyclopropylation?Is this an intrinsic property of the substrate undergoing N -dealkylation?Second,DFT calculations show significant differences between the C–H hydroxylation of 5and alkanes,44different p -substituted 5also possess different mechanisms,45so what kind of HAT mecha-nism does 3N -dealkylation proceed by,TSR or SSM?The present work tries to answer the aforementioned questions by performing DFT calculations on N -dealkylation of 3by Cpd I and provides some essential and complementary insights into the experiments.Computational methodAll DFT calculations presented here were performed using the Gaussian03program 47and employed the unrestricted hybrid density functional method UB3L YP .48–51We employed an iron-oxo porphyrin without side chains and a thiolate axial ligand (SH -)to model Cpd I.41,42,44,45,52–58Geometries for all the species were fully optimized without symmetry constraints.For geometry optimization we used the double-z LACVP basis set 59,60on iron and the 6–31G*basis set on the remaining atoms (B1in brief).Single-point energy calculations were performed with higher basis sets,the triple-z LACV3P+*basis set 59,60on iron and the 6–311+G*basis set on all other atoms (B2in brief).These basis sets have been fully tested and proved to be reliable.9,10,44,45Transition states were ascertained by vibrational analysis with only one imaginary frequency mode.The weak polarization effect of the protein environment and the strong polarization effect of the nonenzymatic environment were modeled using theD o w n l o a d e d b y D A L I A N U N I VE R S I T Y OF T E C H N O L OG Y o n 31 J u l y 2010P u b l i s h e d o n 19 N o v e m b e r 2008 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 810767JPCM solvation method with dielectric constants of e =5.62(chlorobenzene)and e =78.39(water)separately.The effect of hydrogen bonding to the sulfur ligand was mimicked by adding two ammonia molecules to the system which point toward the sulfur of the proximal ligand at a fixed distance of r NH–S =2.660A˚.44,45,52–56,61,62The kinetic isotope effect (KIE)of the C a –H hydroxylation step of the reaction was estimated by replacing hydrogen by deuterium in the substrate and was determined using the frequency data based on two expressions.63Semiclassical KIEs were estimated with the Eyring equation:KIE k k G G RT E HD D H ==()-()ÈÎÍ͢˚˙˙ππexp D D (1)In this equation R is the gas constant and T is the temperature(310.15K).Correction to the semiclassical KIEs using the Wigner model can be achieved by multiplying the KIE E by the tunneling ratio (Q t H /Q t D )as follows:KIE KIE Q Q W E t H t D=¥,,(2)Q h k T t B =+()124u /(3)Here,h is the Planck’s constant,k B is the Boltzmann constant,and u is the value of the imaginary frequency in the transition state.Results and discussionC a –H hydroxylation on the N -substituent of 3by Cpd IThere are two N -dealkylation pathways of 3(Scheme 2,Path A:N -decyclopropylation;Path B:N -demethylation).Fig.1shows the energy profiles for C a –H hydroxylation on the N -substituent of 3by Cpd I.In Path A,C a –H hydroxylation starts with hydrogen transfer from the substrate to the oxygen of Cpd I in both HS and LS states,leading to a pair of transition states (4,2TS 1A ).This C a –H bond activation step is then followed by an O-rebound process,which leads to C a –O bonding and formation of the carbinolaniline–heme complex (4,2PC 1A ).A closer inspection of Fig.1a reveals a few notable points.First,reaction on the LS state is a concerted one,going to the carbinolaniline–heme complex without a distinct rebound step.The radical intermediate on the HS state,4IM 1A ,corresponding to the usual iron-hydroxo/cyclopropyl radical complex,behaves as a shoulder on the potential energy surface and once the cyclopropyl radical snaps out of the weak OH–C a interaction,thesubsequentScheme 2N -dealkylation of 3catalyzed byP450.Fig.1Energy profiles for C a –H hydroxylation (a )on the cyclopropyl group and (b )on the methyl group.Values outside the parentheses are relative potential energies in the gas phase,while the values in parentheses involve corrections due to bulk polarity,NH–S hydrogen bonding,and ZPE.RC:reactant complex;TS:transition state;IM:intermediate;PC:product complex.All values are in kcal/mol relative to the reactant 4RC 1A .rebound process is barrierless.Second,hydrogen-transfer is the rate-determining step in the C a –H hydroxylation process.The activation energies are about 11.2/9.3kcal/mol on HS/LS states,and they decrease to 10.8/7.1kcal/mol when environmental effects and ZPE corrections are taken into account.These barriers are much lower than those of alkane hydroxylation,10,33which are derived at least in part from the weaker C a –H bond of 3.Finally,the lowest-lying route occurs on the LS state,which has a 1.9kcal/mol lower reaction barrier than the HS state.This barrier difference increases to 3.7kcal/mol when the bulk polarity,the NH–S hydrogen bonding and ZPE corrections are added.With this large barrier difference,the reaction will proceed predominantly via the LS state.Therefore,C a –H hydroxylation on the cyclopropyl group of 3proceeds via the SSM mechanism similar to that of 544,45rather than the TSR mechanism similar to that of alkane.39–42,61For Path B,C a –H hydroxylation proceeds in a similar concerted way,an initial hydrogen-transfer process involving two states (i.e.HS and LS)followed by a barrier-free rebound to form the corresponding carbinolaniline–heme complex.The HS/LS activation energies are 11.4/9.7kcal/mol,and they decrease toD o w n l o a d e d b y D A L I A N U N I VE R S I T Y OF T E C H N O L OG Y o n 31 J u l y 2010P u b l i s h e d o n 19 N o v e m b e r 2008 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 810767J10.4/5.8kcal/mol when environmental effects and ZPE correc-tions are added.The large barrier difference between HS and LS states makes the C a –H hydroxylation on the methyl group also proceed in an SSM way,predominantly via the LS state.Thus,it is seen that although these two reaction pathways possess different activation energies and different transition states,the “chemical mechanism”does not change.The transition states in Fig.2are hydrogen-transfer species,having no unusual features other than the very large imagi-nary frequencies.On 4TS 1A /2TS 1A ,the angles of C–H–O are 164.2◦/163.1◦,thus the configurations of the C–H–O portions are almost linear.The structures are seen to involve hydrogen-transfer of the radical type,as are evidenced from the large spin densities (r sub =0.68/-0.54)and the small charges (Q sub =0.09/0.13)on the organic moieties.The transition states of C a –H hydroxylation on the methyl group in Fig.2b,4TS 1B /2TS 1B ,also share linear structures (j C–H–O =173.7/175.3)with large spin densities (r sub =0.65/-0.51)and nearly zero charges (Q sub =0.08/0.12)on the organic moieties.Thus,the C a –H activation steps in Path B are also HAT processes.The geometric data in Fig.2alsosuggestFig.2Optimized structures for the C a –H bond activation transition states,4,2TS 1A and 4,2TS 1B .Values outside the parentheses correspond to 4TS 1,while the values in parentheses correspond to 2TS 1.Beside the structures,we indicate the imaginary frequencies of the TS 1species,the spin densities on the organic moiety (r sub )and the migrating H-atom (r H ),and the charge on the organic moiety (Q sub ).that the source of the barrier difference between HS and LS states can be attributed to the degrees of C a –H bond cleavage in the TS 1species.In 2TS 1A and 2TS 1B ,the hydrogen atoms are closer tothe carbon atoms (r C–H,A =1.248A˚,r C–H,B =1.228A ˚)than to the oxygen atoms (r O–H,A =1.310A˚,r O–H,B =1.368A ˚)and are,therefore,more reactant-like in character.On the contrary,the 4TS 1A and 4TS 1B possess more product-like geometries with r C–H,A =1.333A˚(r C–H,B =1.320A˚)and r O–H,A =1.219A ˚(r C–H,B =1.249A ˚).This means that the degrees of C a –H bond cleavage are larger in 4TS 1A and 4TS 1B than those in 2TS 1A and 2TS 1B .Indeed,the barriers for C a –H hydroxylation on the two states reflect the difference in the degrees of C–H bond cleavage in the TS 1species.Fig.3shows the effects of the environment on the relative energies of the transition states for C a –H bond activation.We can see that in the gas phase,the HS transition states are higher than the LS ones by ca.2kcal/mol,and the two pathways are competitive since the energy difference between the two lower lying TS 1species is only 0.4kcal/mol.The bulk polarity of the protein environment increases the energy variations of the four TS 1species and causes a clear preference for the LS transition state in Path B,2TS 1B ,by 1.4kcal/mol.Addition of the NH–S hydrogen bonding effects further accentuates this trend and leads 2TS 1B lower than its counterpart in Path A by 2.0kcal/mol.ZPE corrections decrease the energy gap between 2TS 1A and 2TS 1B ,from 2.0kcal/mol to 1.2kcal/mol,but still predict a preference for Path B.It follows,therefore,that the environmental effects in the protein pocket switch the regioselectivity of the N -dealkylation reaction from a competition between Path A and Path B to a clear preference for Path B.The preference for N -demethylation over N -decyclopropylation is also a common theme in P450-catalyzed cyclopropylamine N -dealkylation ob-served in experimental studies.23,25,26However,the BDE of C a –H on the methyl group is 3.5kcal/mol higher than that on the cyclopropyl group at the UB3L YP/6–311+G*level.Thus,it appears that for P450-catalyzed N -dealkylation of 3,BDE considerations cannot adequately predict which pathway will be preferred,the protein pocket can exert a remarkable effect on the regioselectivity of N -dealkylation of 3.Fig.3Relative energies of the TS 1species at different levels.All values are in kcal/mol relative to 2TS 1A .Insight into the origin of the preference of Path B,which is dominated by the bulk polarity of the hydrophobic heme pocket and the hydrogen bonding to the sulfur ligand of Cpd I,can be gained from Fig.4.Fig.4shows some highly occupied orbitals of 3and TS 1s,along with the dipole moments of the TS 1species.It is seen that in 3the C a –H hybrid orbital is coupled with the lone pair p orbital of N so as to form a p C–N orbital,which isD o w n l o a d e d b y D A L I A N U N I VE R S I T Y OF T E C H N O L OG Y o n 31 J u l y 2010P u b l i s h e d o n 19 N o v e m b e r 2008 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 810767JFig.4Highly occupied orbitals showing electronic delocalization in 3and TS 1species.Below the TS 1species we specify the dipole moments (m )in Debye units.in turn conjugated with the p orbital on the phenyl group and forms a conjugated p Ph -p C–N orbital.The interaction between this p Ph -p C–N conjugated system and Cpd I induces electron flux from the phenyl ring moiety to the C–N moiety,which makes the electron population hold large delocalization and results in the transition state possessing “polar”character.The polar character of the TS 1species is reflected in the dipole moments shown in Fig.4.For each pathway,a larger dipole moment is possessed by the LS transition state.The LS transition state in Path B,2TS 1B ,is the most polar TS 1species possessing the largest dipole moment.It seems that the dipole moment comes into play especially through the bulk polarity and the NH–S hydrogen bonding effects as described in Fig.3.Consequently,environmental effects exert their influence on the TS 1species in the order of their dipole moments,such that 2TS 1B ,possessing the largest dipole moment,is stabilized more than all the other species,followed by 2TS 1A .The HS species,4TS 1B and 4TS 1A having smaller dipole moments,are stabilized less than the LS species.It is expected,therefore,that the bulk polarity and hydrogen bonding capability of the protein pocket can exert a remarkable effect on the regioselectivity of the N -dealkylation reaction of 3and cause a preference for N -demethylation over N -decyclopropylation,owing to the polar character of the TS 1species caused by the electron delocalization effect of the unique p Ph -p C–N conjugated system in 3.We also calculated the KIEs of the model reactions.The semiclassical values for the LS states are 5.1/4.5,and the Wigner-corrected values of 5.9/5.0indicate that tunneling effects are negligible on the LS states.However,the semiclassical KIEs on the HS state are 6.1/6.3,and Wigner-corrected KIEs of 8.6/8.8are much higher than the semiclassical values,indicating that tunneling effects are important for the HS states.These values are larger than the KIEs of a series of N -alky-N -cyclopropyl-p -chloroanilines studied in experiments 25–28probably because of the different N -substituents and the p -chloro of the benzene ring,but they are in line with the KIEs (ca.4–12)observed for aliphatic hydroxylation 64–67and O -dealkylation,68,69and are large enough to be consistent with a HAT mechanism for the loss of the cyclopropyl and the methyl group.Decomposition of carbinolanilineA carbinolaniline–heme complex is formed at the end of C a –H hydroxylation on the N -substituent.The second step of Path A (Path B)is the carbinolaniline decomposition to yield N -methylaniline (N -cyclopropylaniline)and cyclo-propanone (formaldehyde).Fig.5shows the energy profiles for the carbinolaniline decomposition process in the enzymatic en-vironment.The activation energies are about 35.5/30.5kcal/mol (36.3/31.8kcal/mol)on HS/LS states when environmental effects and ZPE corrections are taken into account.There is no significant spin density change through the reactant complex,transition state and product complex,thus this process is a proton-transfer one.From the discussion mentioned above we have seen that the C a –H hydroxylation step of Path A (Path B)is an exothermal process,and the carbinolaniline would decompose easily with this excess energy in the gas phase.However,in the enzymatic environment,this large excess energy is distributed to the protein pocket so fast that there is no or,at least,not sufficient energy left for the carbinolaniline to pass the high barrier of the decomposition process.Furthermore,carbinolaniline can escape from the heme pocket and be released to the nonenzymatic environment easily because of its low binding energy to the heme (HS:2.9kcal/mol,LS:6.4kcal/mol in Path A;HS:1.3kcal/mol,LS:7.3kcal/mol in Path B).Thus,decomposition of carbinolaniline will be performed in the nonenzymatic environment,consistent with the conventional concept.13Fig.5Energy profiles for the decomposition of carbinolaniline complex in the enzymatic environment.Values outside the parentheses are relative potential energies in the gas phase,while the values in parentheses involve corrections due to bulk polarity,NH–S hydrogen bonding,and ZPE.RC:reactant complex;TS:transition state;PC:product complex.All values are in kcal/mol relative to the reactant 4RC 1A .As the second step of the N -dealkylation,decomposition of the carbinolaniline requires a proton transfer from the hydroxyl oxygen to the aniline nitrogen,while the C–N bond between the alpha carbon and aniline nitrogen gradually breaks.We examined two different processes for the second step:one associated with a direct proton-transfer,and the other associated with a water-assisted proton-transfer.For the direct proton-transfer processD o w n l o a d e d b y D A L I A N U N I VE R S I T Y OF T E C H N O L OG Y o n 31 J u l y 2010P u b l i s h e d o n 19 N o v e m b e r 2008 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 810767Jinvolving transition state TS 2A (TS 2B ),the proton is transferred directly from the hydroxyl oxygen to the aniline nitrogen.For the water-assisted proton-transfer process involving transition state TS ¢2A (TS ¢2B ),the water molecule hydrogen-bonding with the aniline nitrogen gradually transfers a proton to the aniline nitrogen through the hydrogen bond,while the hydroxyl proton gradually transfers to the water oxygen.We can see from Fig.6that the activation energy of direct proton-transfer is 31.5kcal/mol in Path A (39.6kcal/mol in Path B)when the bulk solvation effect and ZPE correction are taken into account,whereas it decreases to 14.2kcal/mol (14.3kcal/mol in Path B)when this process is assisted by a molecule of water.It follows that the reaction process involving water-assisted proton transfer should dominate the carbinolaniline decomposition in the nonenzymatic environ-ment.Similar results were also reported for N -demethylation of N,N -dimethylanilines.45The large barrier decrease of the water-assisted proton-transfer process can be derived from the fact that decomposition of carbinolaniline is a pericyclic reaction,as is depicted in Fig.7.The transition state of the direct proton-transfer process is a rhomboid,whereas it becomes a hexagon after adding the water molecule.The smaller tension of the hexagon compared to that of the rhomboid leads to the decrease of thebarrier.Fig.6Energy profiles for nonenzymatic decomposition of carbino-lamine:(i),(iii)direct proton-transfer;(ii),(iv)water-assisted proton-trans-fer.Values outside the parentheses are relative potential energies,while the values in parentheses involve corrections due to bulk solvation and ZPE.RC:reactant complex;TS:transition state;PC:product complex.At the end of N -decyclopropylation,we can see from Fig.6a that the cyclopropyl ring lost during the reaction is not open,forming a ring-intact cyclopropanone (in PC ¢2of Fig.6a),and it will readily form the gem-diol hydrate of cyclopropanone (i.e.,cyclopropanone hydrate)that has been detected experimentally.23This further suggests that N -decyclopropylation of 3,unlike secondary amines,will not inactivate P450,which agrees precisely with experimental observations.23Fig.7Optimized structures for transitions states for the nonenzymatic decomposition of carbinolaniline:(i),(iii)direct proton-transfer;(ii),(iv)water-assisted proton-transfer.ConclusionsP450-catalyzed N -dealkylation of 3involves two steps,i.e.,C a –H hydroxylation on the N -substituent to form a carbinolaniline and decomposition of the carbinolaniline.The C a –H hydroxylation step of the reaction proceeds predominantly via the LS state in the SSM mechanism.The rate-limiting C a –H activation is an isotope-sensitive HAT step.With the lower barrier possessed by Path B,we predict a preference for N -demethylation over N -decyclopropylation,which is in accord with experimental studies but does not follow the normal D E-BDE correlation.Insight into the origin of the regioselectivity reveals that there is a unique p Ph -p C–N conjugated system in 3which cannot be found in normal hydrocarbons.The electron delocalization effect of the this p Ph -p C–N conjugated system makes the transition state possess a polar character,and regioselectivity of N -dealkylation of 3switches from a competition between N -decyclopropylation and N -demethylation to a clear preference for N -demethylation under the effect of the bulk polarity and hydrogen bonding capability in the protein pocket.Decomposition of carbinolaniline is a nonen-zymatic water-assisted proton-transfer process.The identification of cyclopropanone at the end of the N -decyclopropylation,which means intactness of the cyclopropyl group,further suggests that no enzyme inactivation will occur during the N -decyclopropylation of 3.References1P .R.Ortiz de Montellano,Cytochrome P450:Structure,Mechanism and Biochemistry ,Plenum,New Y ork,2nd edn,1995.2F .P .Guengerich,J.Biol.Chem.,1991,266,10019–10022.3F .P .Guengerich,Chem.Res.Toxicol.,2001,14,611–650.4M.J.Coon,Annu.Rev.Pharmacol.Toxicol.,2005,45,1–25.5F .P .Guengerich and T.L.Macdonald,FASEB J.,1990,4,2453–2459.6T.D.Porter and M.J.Coon,J.Biol.Chem.,1991,266,13469–13472.D o w n l o a d e d b y D A L I A N U N I VE R S I T Y OF T E C H N O L OG Y o n 31 J u l y 2010P u b l i s h e d o n 19 N o v e m b e r 2008 o n h t t p ://p u b s .r s c .o r g | d o i :10.1039/B 810767J。