lipid peroxidation 
 plays important roles in the pathophysiology 
 of various human diseases , such as atherosclerosis , asthma , alzheimer s 
 disease , and nonalcoholic fatty liver 
 disease . increasing research effort has 
 been devoted to the oxidation products of lipids as they not only 
 serve as important biomarkers for disease but also exert a variety of biological activities . the rate - determining step of free radical - mediated lipid peroxidation 
 is the hydrogen atom transfer from a lipid substrate to a propagating 
 peroxyl radical . previously we determined 
 the propagation rate constants ( kp ) of 
 free radical peroxidation of different lipids including polyunsaturated 
 fatty acids , cholesterol , and 7-dehydrocholesterol ( 7-dhc ) 
 in solution and in liposomes . the kp for 7-dhc was exceptionally large at 2260 
 m s in solution , a value that 
 is 200 times that for cholesterol , and 
 20 times that for the simple 1,3-cyclohexadiene when normalized to 
 the number of reactive hydrogen atoms . peroxidation of 7-dhc leads to the formation of over a dozen 
 oxysterols , and although a detailed reaction 
 mechanism has 
 been proposed , the origin of some of the oxysterols is still ambiguous . briefly , the reaction of 7-dhc involves hydrogen 
 atom transfer from c-9 or c-14 of the sterol to a propagating peroxyl 
 radical ( h-9 or h-14 mechanism ) , 
 leading to the formation of two different pentadienyl radical intermediates 
 ( c5c6c7c8c9 and c5c6c7c8c14 ) 
 ( scheme 1 ) . for convenience , we designate the 
 c5c6c7c8c9 radical as endo - b since it is endocyclic in ring b of the sterol 
 and the c5c6c7c8c14 radical as exo - b since one arm of this radical is exocyclic 
 to ring b. the formation of both endo - b and exo - b is followed by precedented 
 free radical transformations such as oxygen addition , 5-exo cyclization , and intramolecular radical substitution ( shi ) that lead to the oxysterol products 
 isolated and identified . to further understand the high 
 reactivity of 7-dhc and better define 
 the reaction mechanisms , we synthesized conjugated and methylene - skipped 
 nonconjugated cholestadien-3-ols ( abbreviated as  cholestadienols  ) 
 that would readily give either the endo - b or exo - b pentadienyl radicals upon 
 abstraction of the reactive hydrogen atom(s ) ( scheme 2 ) . here we report the following : ( i ) the structure  reactivity 
 studies on the free radical oxidation of different cholestadienols 
 that include 7-dhc ( 5,7-dienol ) , 8-dhc ( 5,8(9)-dienol ) , 6,8(9)-dienol , 
 5,8(14)-dienol , and 6,8(14)-dienol ; ( ii ) analysis of oxidation products 
 of these dienols and elucidation of the reaction mechanism ; ( iii ) 
 the effect of an antioxidant , -tocopherol , on the product distribution 
 of the free radical oxidation of the dienols . hydrogen atoms on bis - allylic carbon centers 
 are normally more reactive than monoallylic hydrogen atoms toward 
 reaction with peroxyl radicals because of the lower bond dissociation 
 enthalpy of the reactive c  h bond . thus linoleate ( 31 m s per 
 h - atom ) is much more prone to give up a hydrogen atom to a peroxyl 
 radical than oleate ( 0.22 m s per h - atom ) . to compare the reactivities of 
 monoallylic and bis - allylic hydrogen atoms in the ring system of cholesterol , 
 we prepared a series of cholestadienols with double bonds spanning 
 ring b and/or c as shown in scheme 2 and measured 
 their propagation rate constants using the linoleate peroxyl radical 
 clock . the 6,8(9)-dienol 
 was synthesized from tbs - protected 7-dhc via selective 
 hydroboration / oxidation at the c5 double bond , mesylation / elimination of the resulting alcohol at c6 , 
 and deprotection ( scheme s1 ) . rate constants for hydrogen 
 atom transfer from the dienols to a linoleate peroxyl radical were 
 shown ( see table 1 ) . in the  slow  linoleate peroxyl radical clock , oxidation 
 of methyl linoleate gave trans , cis - hydroxyoctadecadienoates 
 ( hodes ) and trans , trans - hodes as the primary products 
 and the ratio of trans , cis-/trans , trans - hodes is proportional to the hydrogen atom transfer rate constant 
 ( kh ) for a given hydrogen atom donor ( r  h ) 
 times the concentration of that donor ( [ r  h ] ) . thus , under pseudo - first - order reaction conditions , by 
 varying the concentrations of r  h while keeping the concentration 
 of linoleate constant , kp can be solved 
 from the plot of trans , cis-/trans , trans - hodes vs [ r  h ] . the propagation rate constants of the cholestadienols 
 are summarized in table 1 and scheme 2 , and the clocking plots are shown in figure s1 . as seen in the table , the reactivities 
 of the cholestadienols decrease in the order of 7-dhc ( 5,7-dienol ) , 
 6,8(9)-dienol , 8-dhc ( 5,8-dienol ) , 5,8(14)-dienol , and 6,8(14)-dienol . at 37 c in 
 benzene or chlorobenzene ; 
 errors are 2. in order to unequivocally elucidate 
 the h-9 and h-14-mechanisms of 7-dhc 
 peroxidation and to identify the reactive hydrogen atoms in the cholestadienols 
 under study , peroxidation products of these dienols were analyzed 
 by hplc - ms . free radical oxidation of the sterol dienols ( 0.010.08 
 m ) in benzene at 37 c was set up as described previously using 
 ( 2,2-azobis(4-methoxy-2,4-dimethylvaleronitrile ) 
 ( meoamvn ) as the radical initiator . the product 
 mixture was treated with pph3 to reduce hydroperoxides , 
 and this product mixture was analyzed by normal phase hplc - ms . typical 
 chromatograms of oxidation product mixtures of individual dienols 
 are shown in figures 1a  c and s2 , and the structures of major oxysterols identified 
 are shown in figure 1d . based on the proposed 
 reaction mechanisms , oxidation of 8-dhc should exclusively give the 
 products formed from the h-9 mechanism ( endo - b ) while oxidation of the 5,8(14)-dienol 
 should only give products from the h-14 mechanism ( exo - b ) after initial 
 hydrogen abstraction at the bis - allylic positions . comparison of the 
 oxysterol profile from the peroxidation of 7-dhc with the profiles 
 from these two unconjugated dienols shows conclusively that compounds 1 , 2a , and 2b are formed from the h-9 mechanism , while compound 4 is formed from 
 the h-14 mechanism and compound 3 can 
 be formed from either mechanism with the h-14 mechanism being the primary route . under the chromatographic conditions used 
 here , compound 2b coelutes with a minor product of 7-dhc 
 peroxidation , 3,5-dihydroxycholesta-7,9(11)-dien-6-one 
 ( dhcdo ; see figure 2 ) , as shown previously . however , these two compounds can be differentiated 
 by observing the srm transition 433415 , which dhcdo lacks 
 ( see also figure s4 ) . although 7-dhc-5,6-epoxide 
 can only form from 7-dhc via peroxyl radical addition , 
 we note that there is an oxysterol derived from the h-14 mechanism ( 4.68-min peak in figure 1c ) that might have 
 a small contribution to the peak intensity of the epoxide ( 4.81 min ) 
 in figure 1a . normal phase hplc - apci - ms - ms analysis 
 of oxysterols formed from 
 free radical oxidation of 7-dhc ( a ) , 8-dhc ( b ) , and cholesta-5,8(14)-dienol 
 ( c ) . d7 - 3,5-dihydroxycholest-7-en-6-one ( dhceo ) was added to each sample 
 as a standard for proper comparison of the retention times between 
 different chromatograms . the parent ion of each chromatogram and the 
 ms / ms transition were marked in the corresponding ms panel . human tissues and cells are rich in antioxidants , and -tocopherol 
 is the major hydrophobic chain - breaking phenolic antioxidant present in vivo . studies of lipid oxidation 
 in the presence of -tocopherol have biological relevance , and 
 we therefore carried out the free radical oxidation of the series 
 of cholestadienols , including 7-dhc , in the presence of -tocopherol 
 ( 1.0 m ) . under these conditions ( i.e. , high concentration 
 of -tocopherol but low rate of initiation ) , the tocopheryl 
 radical becomes the main chain - propagating radical even though at 
 a much slower rate ( 0.10 m s),i.e . typical hplc - ms chromatograms 
 of product mixtures and the oxysterol products identified in these 
 oxidations are shown in figures 2 and s3 . oxidation of 7-dhc gave three partially 
 overlapping peaks with the mass of the dehydration ion of [ 7-dhc + 
 1o + h ] and these products are the only products formed 
 from the oxidation of 5,8(14)-dienol in the presence of -tocopherol , 
 suggesting that these three products were derived from exo - b and the h-14 mechanism . epoxidation 
 of 7-dhc was not observed under these conditions , likely because of 
 rapid trapping of peroxyl radicals by -tocopherol before addition 
 to a c = c bond can occur . normal phase hplc - apci - ms - ms analysis 
 of oxysterols formed from 
 free radical oxidation of 7-dhc ( a ) , 8-dhc ( b ) , and cholesta-5,8(14)-dienol 
 ( c ) in the presence of 1.0 m -tocopherol . d7 - 3,5-dihydroxycholest-7-en-6-one 
 ( dhceo ) was added to each sample as a standard for proper comparison 
 of the retention times between different chromatograms . the parent 
 ion of each chromatogram and the ms / ms transition were marked in the 
 corresponding ms panel . three of the 7-dhc products are identical to the products 
 of peroxidation 
 of 8-dhc , suggesting that they are formed from endo - b and the h-9 mechanism . two products observed 
 in the srm panel of 415  397 were identified to be 3,5,9-trihydroxycholest-7-en-6-one 
 ( thceo ) and dhcdo ( figure 2 ) , by comparing 
 retention times and srm transition characteristics with standards 
 isolated from free radical oxidation of 7-dhc ( figures s4 and s5 ) . these two 
 compounds were minor products of the free radical oxidation of 7-dhc 
 in the absence of -tocopherol while thceo was identified as 
 one of the major 7-dhc - derived oxysterols observed in brain tissue 
 of dhcr7-ko mouse , a genetic animal model for smith  lemli the product eluting in the peak 
 in the srm panel of 399  381 was identified to be 7-oxo-5,8-dien-3-ol 
 ( 7-keto-8-dhc ) by comparing its srm transition characterisics and 
 retention time with those of the known compound that was synthesized 
 from dehydration of 7-hydroperoxy-5,8-dien-3-ol , which was prepared in turn by photooxidation 
 of 7-dhc . in 
 the absence of -tocopherol , product analysis by hplc - ms 
 suggests that 7-dhc undergoes free radical oxidation via three pathways designated h-9 or h-14 abstraction and peroxyl radical addition . the formation of the 7-dhc-5,6-epoxide 
 suggests that the addition of a propagating peroxyl radical to 7-dhc 
 can compete with abstraction of a hydrogen atom by the same radical 
 ( figure 1 ; scheme 3 ) . peroxyl radical addition at c-5 of 7-dhc eventually leads via an shi process to the formation of the epoxide 
 and an alkoxyl radical that can propagate the radical chain reaction . 
 in the presence of -tocopherol ( 1.0 m ) , however , the addition 
 pathway is completely suppressed due to the competition from hydrogen 
 atom transfer from tocopherol to the peroxyl radical ( kh(-tocopherol ) = 3.5  10 m s ) ( figure 2 ) . potential sites in the pentadienyl radicals endo - b and exo - b for addition of 
 molecular oxygen . for each pentadienyl radical , molecular oxygen can add to three 
 positions that have high spin density , c-5 , c-7 , or c-9 for endo - b and c-5 , c-7 , or c-14 for exo - b ( figure 3 ) . thus , in 
 the h-9 mechanism , three peroxyl radical regioisomers 
 could form as shown in scheme 4 . in our previous 
 studies of 7-dhc peroxidation , products derived from the bis - allylic 
 peroxyl radical were not observed likely due to the rapid -fragmentation 
 of this radical to give back its precursor pentadienyl radical . the rate constant of such a fragmentation in an acyclic system ( linoleic 
 acid ) was found to be 2.6  10 s. in the presence of -tocopherol , 
 however , a product derived from the bis - allylic peroxyl radical , 7-keto-8-dhc , 
 was observed as one of the major products formed . on the other hand , 
 even in the presence of 1.0 m -tocopherol , products formed 
 from direct trapping of the 5- or 9-peroxyl radical were not observed 
 but rather those formed from 5-exo cyclization were 
 detected ( see below ) ( figure 2 ; scheme 4 ) , suggesting that the 5-exo cyclization 
 of these peroxyl radicals is fast enough to compete with hydrogen 
 atom transfer from -tocopherol ( pseudo - first - order rate constant 
 = kh(-tocopherol)[-tocopherol ] 
 = 3.5  10 s ) . this large peroxyl 
 radical 5-exo cyclization rate constant is surprising 
 since the rate constant for the same reaction in an acyclic system 
 was estimated to be ca . 800 s and the corresponding 
 carbon radical cyclization is only 4.1  10 s at 37 c . an alternative mechanism 
 to be considered for this transformation is the concerted addition 
 of oxygen to endo - b , leading directly 
 to the five - membered peroxide - allyl radical ( figure 4 ) , although a concerted transformation of this type has , to 
 our knowledge , no precedent . we note that oxygen - radical complex species 
 have been suggested as intermediates in the oxidation of acyclic dienols 
 such as linoleic acid , which proceeds via extended 
 chain pentadienyl radical intermediates . an oxygen - radical complex of the ring constrained endo - b pentadienyl radical would appear to be well 
 on its way to the product allyl radical . we suggest that the 
 ketone products observed in the h-9 
 mechanism were formed via abstraction of 
 the remaining bis - allylic or allylic hydrogen atom at the -position 
 of hydroperoxide , followed by -fragmentation of the peroxide 
 bond ( scheme 4 ) . an alternative pathway could 
 be that the hydroperoxide was reduced by -tocopherol to give 
 an alkoxyl radical , which is followed by elimination of the -hydrogen 
 atom . ketones formation has been observed in free radical oxidation 
 reactions , either from termination reactions of two peroxyl radicals via the  russell mechanism  or from decomposition 
 of hydroperoxides ( via alkoxyl radical ) , such as those 
 observed in the oxidation of linoleate . however , complete conversion 
 of a peroxide to a ketone has not been previously reported . the formation of thceo requires the reduction of the cyclic 
 peroxide 
 bond , which may be achieved by proton - coupled electron transfer from 
 the most reducing agent in the reaction , -tocopherol , as shown 
 in scheme 4 . we suggest that even though cyclic 
 peroxides are normally considered stable to pph3 and -tocopherol , 
 the strain of the 5,9-cyclic peroxide and electron - withdrawing effect 
 of the carbonyl group at c6 may lead to higher reactivity of this 
 peroxide toward reduction . one such example is that isoprostanes bicyclic 
 endoperoxides can be readily reduced by pph3 to give diols . thceo was found to be one of the major 7-dhc - derived 
 oxysterols in the brain of dhcr7-ko mouse . we reported previously that thceo is a metabolite 
 of compounds 1 and 2b in neuro2a and human 
 fibroblast cells , and our results here 
 suggest that thceo could form directly from 7-dhc via the h-9 mechanism in the presence of -tocopherol . in the h-14 mechanism , oxygen can be added to 
 three different positions of the exo - b pentadienyl radical ( scheme 5 ) . however , 
 after reduction by pph3 , only three products corresponding 
 to the molecular weight of [ 7-dhc + 1o ] were observed , possibly because 
 5-exo cyclization is not favored for any of these 
 peroxyl radical intermediates ( figure 2 ) . we 
 speculate that these three products are the hydroxydienols shown in 
 scheme 5 . the peak eluting at 7.8 min in figure 2 was tentatively assigned to the known compound 4 based on its hplc retention time . proposed radical intermediates formed from cholesta-6,8(9)-dienol 
 ( a ) or cholesta-6,8(14)-dienol ( b ) via peroxyl radical 
 addition ( kadd ) or hydrogen atom transfer 
 mechanism ( kh ) . note that the peroxyl 
 radical addition pathways are expected to be completely suppressed 
 in the presence of 1.0 m -tocopherol as observed in the reaction 
 of 7-dhc . in free radical oxidation of 6,8(9)-dienol 
 or 6,8(14)-dienol with 
 or without -tocopherol , additional products that were not derived 
 from the h-9 or h-14 mechanism were 
 observed ( figures s2 and s3 ) . we speculate 
 that these products were derived from peroxyl radical addition to 
 the double bond ( to give an epoxide ; only in the absence of -tocopherol ) 
 or from hydrogen atom abstraction at the allylic position c-11 ( for 
 6,8(9)-dienol ) or c-17 ( for 6,8(14)-dienol ) ( figure 5 ) . we found that cholestadienols displayed much higher 
 reactivity 
 than the acyclic dienes toward free radical oxidation even though 
 some of them do not possess a bis - allylic methylene group . even the 
 least reactive dienol tested , the 6,8(14)-dienol , has a large propagation 
 rate constant of 412 m s , 
 a value that is ca . 7 times that of the methylene - skipped 
 acyclic diene , linoleate ( 62 m s ) . the overall reactivity trend for the cholestadienols suggests 
 that the dienols that lead to the formation of the pentadienyl radical endo - b are more reactive than those leading 
 to the pentadienyl radical exo - b . it 
 is known that cisoid 1,3-dienes have higher enthalpies 
 than transoid 1,3-dienes , which would imply a smaller activation energy of hydrogen abstraction 
 from the allylic positions of the cisoid dienes , 
 thus supporting our results on the reactivities of the conjugated 
 dienes : 5,7-dienol ( 7-dhc ) > 6,8(9)-dienol  6,8(14)-dienol . it is unusual to observe that the ring - b conjugated dienes , 7-dhc 
 and 6,8(9)-dienol , display significantly higher reactivity than the 
 dienols bearing a bis - allylic methylene group , 8-dhc and 5,8(14)-dienol , 
 since the bis - allylic c  h bond normally is more reactive than 
 the monoallylic c  h bond in acyclic structures ( i.e. , in polyunsaturated 
 fatty acids ) . we reported previously 
 that , in 7-dhc , the torsion angles between the active c  h bond 
 ( at c9 or c14 ) and the diene plane are close to 90 , so that 
 the two hydrogen atoms are well aligned for removal from the -face 
 of the steroid ring ; i.e. , maximum delocalization 
 stabilization is achieved in the hydrogen atom transfer transition 
 state ( figure s6a ) . similar molecular mechanics calculation suggests that , 
 in the 6,8(9)-dienol , the torsion angle for h5c5c6c7 
 is 79.2 , also close to 90 ( figure 
 s6b ) . in 8-dhc , the torsion angels for the c h bonds 
 at c7 are 111.5 ( c5c6c7h7 ) and 108.6 ( h7c7c8c9 ) 
 for h and 133.5 ( c5c6c7h7 ) and 129.8 ( h7c7c8c9 ) 
 for h , respectively ( figure 
 s6c ) . although the diene in 8-dhc appears to be in the same 
 plane , the reactive c  h bonds are not positioned well for hydrogen 
 atom transfer . in the 5,8(14)-dienol , the diene does not appear to 
 be in the same plane , which would require extensive molecular reorientation 
 in order to achieve maximum delocalization ( figure 
 s6d ) . further theoretical investigation would be worthwhile 
 to elucidate the differences in the reactivities of these cholestadienols . elevated 
 dienyl cholesterol precursors have been observed in a 
 number of cholesterol biosynthesis and metabolism disorders , such 
 as 7-dhc and 8-dhc in slos , 7-dhc in cerebrotendinous 
 xanthomatosis ( ctx ) , 8-dhc in x - linked 
 dominant chondrodysplasia punctata ( cdpx2 ) , 8(9),14-dienol in hem dysplasia , etc . the high reactivity of cholestadienols 
 suggests that oxidative stress should be considered as a factor in 
 disorders where dienyl sterol intermediates are accumulated . oxidation 
 of the reactive sterols would lead to the formation of oxysterols 
 with diverse structures and functions , metabolites that may ultimately play significant roles in the pathophysiology 
 of these diseases . in this study , we found 
 that dienes in the rigid cyclic systems 
 ( cholestadienols ) display much higher reactivities toward free radical 
 oxidation than the known acyclic dienyl fatty acids . by comparing 
 the oxysterol profiles from the oxidation of several custom - designed 
 cholestadienols with those of 7-dhc , the free radical oxidation mechanism 
 of 7-dhc was refined , with or without the presence of -tocopherol . 
 from the reactions carried out in the presence of -tocopherol , 
 evidence was presented that supports either an unusually fast 5-exo peroxyl radical cyclization or a concerted addition 
 of molecular oxygen to a pentadienyl radical , both of which are unprecedented . also notable was the complete conversion of a hydroperoxide to a ketone 
 during the process of free radical oxidation . the knowledge of the 
 reactivity and oxidation product profiles of these cholestadienols 
 revealed in this study contributes to the overall understanding of 
 the pathobiology of related cholesterol biosynthesis / metabolism disorders . the initiator , meoamvn , 
 was purchased from wako chemicals , dried under vacuum , and then stored 
 at 40 c . 7-dehydrocholesterol ( > 98% ) , tert - butyldimethylsilyl chloride , boranethf ( 1.0 m in thf ) , and 
 methanesulfonyl chloride were purchased from sigma - aldrich co. and 
 were used without further purification . was purified on silica gel prior to use ( 10% ethyl acetate in 
 hexane ) and was stored under argon . benzene ( hplc grade ) was passed 
 through a column of neutral alumina and stored over molecular sieves . the synthesis 
 of 6,8(9)-dienol was described in the supporting 
 information , and the nmr spectra matched those reported in 
 the literature , in which the compound was synthesized by a different 
 method . briefly , for each series of 
 reactions , the same amount of a stock solution of methyl linoleate 
 in benzene was added to each reaction vial , followed by the addition 
 of a stock solution of the specific sterol , an appropriate amount 
 of benzene , and a stock solution of meoamvn in benzene in order to 
 make the total reaction volume 100 l . the final concentrations 
 for methyl linoleate , the sterol under study , and meoamvn are 0.302 
 m , 0.010.08 m , and 0.0010.002 m , respectively . the 
 reaction was carried out at 37 c for 1 h and was quenched by 
 the addition of bht ( 50 l ; 0.1 m in benzene ) and pph3 ( 50 l ; 0.1 m in benzene ) . the resulting mixture was blown 
 dry under nitrogen and redissolved in hexanes ( 500 l ) for the 
 analysis of hodes by hplc - uv as previously described . for oxysterol analysis , the reactions were carried out 
 similarly as described above , but the resulting reaction mixtures 
 were diluted to 1 ml with benzene before being analyzed by hplc - apci - ms / ms 
 using the method that was described previously . the oxidation was carried 
 out similarly as the clocking experiments , but with an additional 
 reaction component ( 1.0 m -tocopherol ) and a longer reaction 
 time ( 2 h ) . the reactions were quenched similarly and diluted to 1 
 ml with benzene for the analysis of oxysterols using the same hplc - ms 
 method .
7-dehydrocholesterol ( 7-dhc ) is the 
 most oxidizable lipid molecule 
 reported to date , with a propagation rate constant for free radical 
 peroxidation that is 200 times that of cholesterol . to better understand 
 the high reactivity of 7-dhc and elucidate the reaction mechanism 
 , 
 we synthesized conjugated and skipped nonconjugated cholestadienols 
 that would give one of the two putative pentadienyl - radical intermediates 
 formed in 7-dhc peroxidation . 
 the additional dienols include 6,8(9)-dienol , 
 5,8(14)-dienol , 6,8(14)-dienol , and the biologically important 8-dehydrocholesterol 
 ( 8-dhc ; 5,8(9)-dienol ) . 
 we found that all of the dienols are significantly 
 ( at least 40 times ) more reactive than cholesterol . among them , dienols 
 leading to the formation of the pentadienyl radical in ring b ( termed endo - b ) of the sterol are more reactive than 
 those leading to the pentadienyl radical spanning rings b and c ( termed exo - b ) . by comparing the oxysterol profile 
 formed from 7-dhc and those formed from 8-dhc and 5,8(14)-dienol 
 , 
 products formed from abstraction of the hydrogen atoms at c-9 and 
 c-14 ( h-9 or h-14 mechanism ) were 
 clearly differentiated . when the oxidation was carried out in the 
 presence of the good hydrogen atom donor -tocopherol , 
 the oxysterol 
 profile of 7-dhc peroxidation differed distinctly from the profile 
 observed in the absence of the antioxidant and resembles more closely 
 the profile observed in biological systems . 
 this study suggests that 
 oxidative stress and the accumulation of oxysterols should be considered 
 as two key factors in cholesterol biosynthesis or metabolism disorders , 
 where dienyl sterol intermediates are accumulated .