cellular redox status 
 is a dynamic state that balances reducing 
 and oxidizing species , and if the balance is thrown off in the cell , 
 dysfunction arises . the hydroxyl radical 
 ( ho ) is a powerful oxidizing species found in the 
 cell and causes redox imbalance due to its particularly high redox 
 potential ( 2.31 v vs nhe , ph 7 ; ho(+h)/h2o ) . the fenton reaction 
 can generate ho when a redox - active metal effects 
 cleavage of h2o2 , a metabolic product that can 
 easily diffuse within and between cells . hydrogen peroxide is weakly reactive , but fe(ii ) species have the 
 capability to generate ho plus fe(iii ) via the 
 fenton reaction ( reaction 1 ) . furthermore , reducing 
 agents in the cell such as ascorbate ( asc ) allow this reaction to 
 be catalytic in iron by reducing fe(iii ) back to fe(ii ) ( reaction 2 ) . the unfortunate events that occurred 
 at fukushima , japan in 2011 the high - energy photons released upon radioactive decay are capable 
 of yielding ho via radiolysis of water ( reactions 3 and 4 ) . the iron - fenton reaction 
 and x - ray radiolysis of water represent two methods for generating 
 hydroxyl radicals that can oxidize biomolecules such 
 as dna . dna damage 
 plays a significant role in aging , and the development of diseases 
 such as cancer.1234 dna is vulnerable to oxidative insults from ho , and the damage is particularly deleterious because it can cause 
 mutations that are heritable to daughter cells . notable progress has 
 been made in the discovery of dna oxidation pathways resulting from 
 radical oxygen species , such as ho , during the 
 past three decades . in dna , the most susceptible 
 site is 2-deoxyguanosine ( dg ) because the guanine 
 heterocycle has the lowest redox potential ( 1.29 v vs nhe , ph 7 ; dg(h)/dg ) among the four dna bases . the nucleoside dg is a prime target for direct oxidation 
 or electron transfer - mediated oxidation that can result from ho . oxidative damage to dg yields products that result 
 from the initial product - forming reaction occurring at one of three 
 sites that include the c5 or c8 positions of the heterocyclic ring , 
 or the 2-deoxyribose unit ( figure 1 ) . these 
 products are identified by their characteristic mass changes from 
 the parent nucleoside dg ( m ) . sugar oxidation is a 
 commonly observed reaction pathway of dg damage resulting 
 from ho . products 
 observed along this pathway include 
 5,8-cyclo-2-deoxyguanosine ( cyclo - dg ) 
 diastereomers derived from h atom abstraction at 
 c5 , and release of the free base guanine ( gua ) after hydroxylation of c1. direct base release in dna leads 
 to strand breaks due to the lability of the abasic site so formed . base oxidation of dg leads to two lesions resulting from reaction at c8 that include 
 8-oxo-7,8-dihydro-2-deoxyguanosine ( dog ) , observed 
 in high yield under aerobic oxidizing conditions , or the ring - opened 
 product 2,6-diamino-4-hydroxy-5-formamidopyrimidine-2-deoxyribonucleoside 
 ( fapy - dg ) observed under anaerobic reducing conditions . both dog and fapy - dg are mutagenic compounds 
 found in vivo , while dog is a biomarker monitored to assay the extent of cellular 
 oxidative stress . because dog has a lower redox potential ( 0.74 v vs nhe , ph 7 ; dog(h)/dog ) than dg , it is readily 
 oxidized to an electrophilic intermediate that reacts with water at 
 c5 to ultimately yield the hydantoin lesions spiroiminodihydantoin-2-deoxyribonucleoside 
 ( dsp ) and 5-guanidinohydantoin-2-deoxyribonucleoside 
 ( dgh ) . additionally , oxidation 
 of dg leads to products resulting 
 from initial chemistry occurring at the c5 position of the purine . 2,5-diaminoimidazolone-2-deoxyribonucleoside ( diz ) and its hydrolysis product 2,2,4-triamino-2h - oxazol-5-one-2-deoxyribonucleoside ( dz ) are four - electron oxidation products resulting from exposure to 
 ho or one - electron oxidants under aerobic conditions . recent studies concerning dg oxidation 
 have identified 5-carboxamido-5-formamido-2-iminohydantion-2-deoxyribonucleoside 
 ( d2ih ) as a product from fenton chemistry with copper 
 or lead , activation of khso5 with nickel or manganese complexes , 
 co3 oxidations , and the epoxidizing 
 agent dimethyldioxirane . these observations have led us to the question of whether d2ih is in fact a major product from dg oxidation 
 by the hydroxyl radical generated either from the fe(ii)-mediated 
 fenton reaction or by x - ray radiolysis of water . toward this 
 goal , we investigated the formation and yield of dg - oxidation 
 products from the fe(ii)-mediated fenton reaction 
 or x - ray radiolysis of water while monitoring the effect of reductant 
 concentration ( ascorbate or n - acetylcysteine ) on 
 their yields in the nucleoside , short oligodeoxynucleotide , and -dna 
 contexts . these studies identify d2ih to be a major guanine 
 oxidation product observed when reactions were conducted with low 
 millimolar amounts of reductant , conditions that mimic the presence 
 of reducing agents such as glutathione , urate , or ascorbate in cells . the results of these studies led to proposed 
 mechanisms for the formation of d2ih and to discussions 
 of its possible biological significance . the nucleoside dg ( 3 mm ) 
 was initially chosen for oxidation by the fe(ii)-mediated 
 fenton reaction or x - ray irradiation in the absence or presence of 
 ascorbate ( asc ) or n - acetylcysteine ( nac ) . all reactions 
 were conducted with 75 mm napi buffer ( ph 7.4 ) at 22 c . the reaction mixtures were initially analyzed by reversed - phase uplc - esi - ms . this allowed identification of guanine ( gua ) , 8-oxo-7,8-dihydroguanine ( og ) , dog , 
 and the diastereomers of cyclo - dg after comparison to 
 known standards . the void volume from this analysis was collected 
 and reanalyzed with a hypercarb column equipped with an esi - ms detector . this method allowed detection of gh , dz , and the diastereomers of d2ih , dgh , and dsp . because diz hydrolyzes to dz during preparation of the void volume , the hypercarb column allowed the separation of the 
 diastereomer products of d2ih and dsp for 
 which the absolute configurations for the peaks had been previously 
 determined . the two diastereomers of dgh are interconvertible and 
 were not quantified individually . further 
 support for the identification of d2ih , dsp , and dz was obtained by esi - ms / ms fragmentation 
 of the free bases from in - source fragmentation of the n - glycosidic bond of the parent nucleosides . hrms was conducted to further support the 
 identification of each product ( supporting information figures s3s13 ) . product quantification was conducted with 
 reversed - phase hplc while 
 monitoring peak elution at 240 nm ; product peak areas were integrated 
 and normalized based on the product s extinction coefficient 
 at 240 nm ( 240  nm ) . the extinction coefficient for d2ih has not been experimentally 
 determined , and due to its instability toward n - glycosidic 
 bond hydrolysis it was not determined in these studies . however , our previous time - dependent density 
 functional theory studies on the uv  vis and ecd properties 
 for dsp and d2ih allow us to use these previous 
 results to determine the relative difference in extinction coefficient 
 between these two compounds at the wavelength monitored . these calculations determined the 240  nm for d2ih to be 30% less than that of dsp ; 
 therefore , the 240  nm value used to quantify d2ih was calculated to be 2290 lmolcm . the fenton 
 reaction was initially conducted with the fe(ii)/edta catalyst and 
 10 mm h2o2 under aerobic conditions to give 
 5% conversion to product . we elected to conduct reactions to a low 
 overall yield so that primary oxidation products could be identified 
 and compared . in this reaction , the highest absolute product yields 
 observed were dz ( 1.5% ) and gua ( 1.5% ) in 
 equal amounts , with the mass balance completed by low yields of cyclo - dg ( 0.2% ) , dog ( 0.4% ) , dsp ( 0.9% ) , and d2ih ( 0.4% , table 1 ) . in the next reaction , the same conditions were applied with the 
 addition of 2 mm asc that led to a 7-fold increase in product formation , 
 as well as a significant change in product distribution . in the presence 
 of asc , the major dg product detected was d2ih ( 13.4% ) with lower yields of the 2-deoxyribose oxidation products cyclo - dg ( 0.9% ) and gua ( 7.1% ) , as well as og ( 0.6% ) and gh ( < 0.1% ) ; also , lower yields 
 of dz ( 2.7% ) , dog ( 4.9% ) , dsp ( 5.6% ) , and dgh ( 0.4% ) were observed ( table 1 ) . these asc - dependent studies revealed two key 
 observations : ( 1 ) the reaction yields dramatically increased ( 7-fold ) 
 with asc because the fe(ii)/edta complex can redox cycle in the presence 
 of a reductant ( reaction 2 ) , and ( 2 ) d2ih was the major product observed when asc was present . because dsp and dgh are further oxidation products of 
 the precursor dog , the yield 
 of d2ih ( 13.4% ) was compared to the combined yields of dog , dsp , and dgh ( 10.9% ) , and it 
 was found that the yield of d2ih was higher than that 
 of dog and its oxidation products . reported values represent the average 
 of three trials that have errors of 8% of the value . low o2 represents a partial 
 reduction in the o2 concentration by bubbling argon into 
 the sample for 10 min that was later found to be insufficient to completely 
 remove o2 . in 
 the next set of reactions , first , a reaction was conducted with dg in the presence 
 of fe(ii)/edta and h2o2 under low o2 conditions achieved by bubbling argon into the sample for 10 min 
 that was insufficient to completely remove o2 . in this 
 reaction , the overall conversion of dg to product was 
 10% , and the major product observed was dsp ( 3.9% ) , with 
 low yields of dog ( 1.9% ) , dgh ( 1.9% ) , d2ih ( 1.0% ) , and the sugar oxidation product cyclo - dg ( 1.2% , table 1 ) . the addition of 2 mm asc 
 to the low o2 reaction increased the dg conversion 
 by 2-fold ( 20% ) and changed the product distribution , giving dog ( 6.6% ) and dsp ( 6.0% ) in high yield and low 
 yields of d2ih ( 3.3% ) , dgh ( 2.0% ) , and cyclo - dg ( 1.9% , table 1 ) . these low 
 o2 studies indicated the yield of d2ih was 
 at a minimum under these conditions . additionally , the major products 
 observed under low o2 conditions were dog and 
 its oxidation product dsp . many literature sources point to 
 the presence of fapy - dg as a guanine oxidation product 
 formed under anaerobic , reducing conditions . in our present 
 low o2 studies , this elusive compound was not observed , 
 even after exhaustive searching of the lc - ms data . this led us to 
 probe deeper into the formation of fapy - dg , using the 
 following conditions to make this compound from dg by 
 the iron - fenton reaction . first , the reaction had to be rigorously 
 purged with argon for > 30 min prior to addition of h2o2 with 2 mm asc , and the hplc mobile phase was changed 
 from 
 running buffered [ 20 mm nh4oac ( ph 7 ) ] water and mecn to 
 unbuffered ddh2o and mecn . these changes allowed observation 
 of a broad hump after the void volume in the reversed - phase hplc chromatogram 
 with a uv  vis signature consistent with fapy - dg ( max = 270 and 218 nm ) , and a defined peak that eluted after dg with a similar 
 uv  vis profile . the broad hump 
 was consistent with literature reports because fapy - dg exists as anomers of two different sugar configurations . lc - esi - ms of all these new peaks only found masses consistent 
 with the fapy free base , as well as a peak with mass fapy + 18 ( figure s24 ) . these observations 
 identify that fapy - dg is not stable to laboratory manipulation 
 as are d2ih , dog , dsp , or dgh , and it appears to be sensitive to nucleophilic buffers 
 in the hplc mobile phase , e.g. , nh4oac . further test reactions 
 that had argon bubbled through them for a few seconds up to 1 min 
 before h2o2 addition did not furnish the fapy peaks . the fapy peaks only appeared after 
 purging the system with argon for > 30 min . to reiterate , in cases 
 with low o2 and asc present , dog was the major 
 product observed , and when o2 was at ambient concentrations 
 with asc present , the major product was d2ih . these observations 
 identify fapy - dg to be very challenging to obtain , as 
 it is only observed under extreme anoxic conditions ( 100% 
 argon and 0% o2 ) , conditions under which cells 
 can not survive . if fapy - dg was formed in a cell , detection 
 of the nucleoside would be problematic due to the hydrolytic instability 
 of the n - glycosidic bond . this later feature is in 
 contrast to d2ih , dog , dsp , 
 and dgh that are all at least stable enough to allow 
 their characterization under very mild conditions ( 22 c in water 
 buffered at ph 7 ) . finally , fapy - dg has a low redox 
 potential ( 1.1 v 
 vs nhe ) rendering it subject to further 
 oxidation ; however , this is also the case for dog ( 0.7 
 v vs nhe ) that has an even lower redox 
 potential , and we detect and quantify this intermediate species and 
 its further oxidation products dsp and dgh . thus , the conditions outlined do not simply over - oxidize fapy - dg causing its loss . we conclude that fapy - dg is not a major contributor to the product distribution of guanosine 
 oxidation by a hydroxyl radical under cellularly relevant conditions . in cells , two main small - molecule reductants are 
 found in high concentrations , ascorbate and glutathione , found at 
 concentrations of 0.83 mm and 110 mm , respectively . knowing that d2ih yields increased 
 when asc was present during the fe(ii)-mediated fenton oxidation of dg ( table 1 ) , we then studied the role 
 of increasing reductant concentrations on the relative yields of the 
 products . in addition , the nature of the reductant was studied , in 
 which we chose n - acetylcysteine ( nac ) as a model 
 for glutathione . note that protection of the amine group of cysteine 
 with an acetyl group prevents amine adduct formation with dg oxidation intermediates . titration of 
 asc from 05 mm into the fe(ii)-mediated fenton oxidation of dg provided the following trends in the relative product distributions 
 ( figure 2a ) : ( 1 ) as the asc concentration increased , 
 the reaction yield increased , supporting the role of asc in allowing 
 the iron catalyst to redox cycle between fe(ii ) and fe(iii ) and thereby 
 increasing the production of ho ( reactions 1 and 2 ) . ( 2 ) the amount of 
 2-deoxyribose oxidation decreased ( 10% ) as the concentration 
 of asc increased . this observation negates the involvement of asc 
 in sugar oxidation , and it is likely that asc inhibits this pathway . ( 3 ) the relative yields of dog , dsp , and dgh slightly increased ( 6% ) with more asc . ( 4 ) the 
 asc concentration was shown to be critical in the relative yields 
 of the c5 products , dz and d2ih . when asc 
 was not present , the major c5-oxidation product was dz ( also a major product overall ) ; however , as asc was titrated into 
 the reaction , the relative yield of dz decreased and d2ih increased until it was the major product of the reaction 
 at asc > 2 mm ( figure 2a ) . within the range 
 of physiological asc concentrations ( 13 mm),d2ih was always the major oxidation product 
 detected ( figure 2a ) . moreover , when the study 
 was conducted with variable nac concentrations , the same trends were 
 observed ( figure 2b ) . again , the most notable 
 observation is that d2ih was the major oxidation product 
 observed in reactions conducted with > 2 mm thiol concentrations 
 ( figure 2b ) , even when considering a comparison 
 of d2ih ( 37% ) with the combined yields of dog , dsp , and dgh ( 30% , 
 figure 2a and 2b with 
 2 mm reductant ) . effect of reductant concentration on relative product 
 distributions 
 observed from the fe(ii)-mediated fenton reaction . the reactions were 
 conducted with 3 mm dg , 10 m fe(ii)/edta , 10 mm 
 h2o2 in 75 mm napi buffer ( ph 7.4 ) 
 at 22 c while varying the concentration of asc ( a ) or nac ( b ) 
 from 05 mm . data points represent the average of three trials 
 with an error in each value of 310% . in the next set of studies , dg oxidation product distribution versus the h2o2 concentration ( 010 mm ) was monitored with a 
 static amount of reductant ( 0.8 mm asc or 3 mm nac , figure 3a and 3b , respectively ) . key observations from these studies include the following : ( 1 ) as 
 expected , the reaction yield increased as more h2o2 was added to the reaction ( supporting 
 information , figures s16 and s17 ) . ( 2 ) with increased h2o2 concentrations , the flux of ho increased , and the relative yield of dsp increased 
 at the expense of dog . this observation supports dog as the intermediate leading to dsp . ( 3 ) both dz and d2ih increased as a function 
 of h2o2 concentration ( 010 mm ) ; moreover , 
 the amount of d2ih increased 4-fold when asc was the 
 reductant while dz increased 3-fold ( figure 3a ) . in the h2o2-dependent 
 studies with nac , d2ih increased 13-fold while dz increased 3-fold when h2o2 increased 
 from 0 to 10 mm ( figure 3b ) . these studies 
 demonstrate that the yield of d2ih was maximal under 
 conditions that boost formation of ho . the reactions 
 were conducted with 3 mm dg , 10 m fe(ii)/edta , 
 and with either 0.8 mm asc ( panel a ) or 3 mm nac ( panel b ) in 75 mm 
 napi ( ph 7.4 ) at 22 c , while varying the concentration 
 of h2o2 from 010 mm . data points represent 
 the average of three trials with an error in each value of 511% . next , oxidation of dg was conducted with an x - ray 
 source at a dose rate of 25 gy / min for 30 min ( total dose = 750 gy ) . the major oxidation products observed were dz ( 14.7% ) , dsp ( 10.8% ) , and gua ( 12.4% ) and in low absolute yields were d2ih ( 2.9% ) , dog ( 1.4% ) , and dgh ( 1.4% ) . next , asc ( 2 mm ) 
 was added to the reaction to give the following changes in the yields . ( 1 ) the absolute conversion of dg to product decreased 
 with asc ( 2.5-fold ) , as expected , because asc quenches radical reactions . ( 2 ) the major products were now d2ih ( 6.3% ) , dsp ( 3.9% ) , and gua ( 4.1% ) , with 
 the mass balance completed by low yields of dz ( 1.1% ) , dog ( 2.5% ) , cyclo - dg ( 0.5% ) , and dgh ( < 0.1% ) . the most striking observation was that d2ih was the major oxidation product when asc was present during x - ray 
 mediated oxidations ; however , this is in contrast to studies without 
 asc where dz was the major oxidation product quantified 
 ( table 2 ) . when considering the yield of d2ih ( 6.3% ) in comparison to the combined yields of dog , dsp , and dgh ( 6.4% ) , the initial 
 split between d2ih and the c8 products was nearly equal 
 with x - ray irradiation when reductant was present . because the x - ray 
 reactions required the reaction vessel to be open to the atmosphere , 
 studies were not conducted under anaerobic conditions . reported values represent the average 
 of three trials , and the error in each value was 410% . reactions with 
 and without asc suggest a major role for the reductant 
 in the product distributions for x - ray mediated oxidations ( table 2 ) . therefore , studies were conducted with the reductant 
 asc or nac titrated ( 05 mm ) into the x - ray - mediated reaction 
 mixture while monitoring the product yields ( figure 4a and 4b ) . from these studies , the 
 following observations were made in x - ray - mediated oxidations : ( 1 ) 
 as the reducing agent concentration increased , the reaction yield 
 significantly decreased ( supporting information , figures s19 and s21 ) . ( 2 ) the relative yield of dog increased ( 6-fold ) as a function of reductant concentration ( figure 4a and 4b ) , while the yield 
 of dsp decreased ( 1.5-fold ) and dgh decreased 
 ( 3-fold ) . this result supports the conclusion that the reductant quenched 
 the further oxidation of dog to the hydantoins . ( 3 ) the 
 yields of the sugar oxidation products were not significantly changed 
 by the increase in reductant concentration ( figure 4a and 4b ) . ( 4 ) the reductant had the 
 most dramatic effect on the c5-pathway products , dz and d2ih . when the reductant was not present , dz was 
 the major product and d2ih was observed in very low yield ; 
 however , when asc or nac was increased to relevant concentrations , d2ih was the major oxidation product observed with very little dz detected . effect of reductant concentration on the dg - oxidation 
 product yield from the x - ray reaction . the reactions were conducted 
 with 3 mm dg in 75 mm napi ( ph 7.4 ) at 22 
 c with a 30 min exposure to 25 gy / min x - ray source ( total dose 
 = 750 gy ) while varying the concentration of asc ( panel a ) or nac 
 ( panel b ) from 05 mm . data points represent the average of 
 three trials with an error in each value of 310% . in the next set of reactions , dg was irradiated with 
 x - rays at a rate of 25 gy / min while varying the time ( i.e. , total 
 dose ) and keeping the asc concentration static ( 2 mm ) . as the total 
 dose of x - rays increased from 25 to 750 gy , the following trends were 
 observed . ( 1 ) as expected , the overall reaction yield increased ( supporting information figure s22 ) . ( 2 ) the yield 
 of dsp and dgh increased at the expense 
 of dog with increasing x - ray dose . ( 3 ) the yield of sugar 
 chemistry increased with increasing x - ray dose . ( 4 ) the yield of dz and d2ih both increased as a function of the 
 x - ray dose , and d2ih was always the major c5-oxidation 
 pathway product , as well as being the major product of the reaction 
 overall ( figure 5 ) . effect of x - ray irradiation 
 time on dg - oxidation product 
 distributions . the reactions were conducted with 3 mm dg , 2 mm asc in 75 mm napi ( ph 7.4 ) at 22 c while 
 varying the exposure time ( 030 min ) to an x - ray source that 
 was delivered at a rate of 25 gy / min . data points represent the average 
 of three trials with an error in each value of 310% . under all of the conditions studied , 
 products derived from sugar 
 oxidation were observed . oxidation at the 5-carbon yields 
 the 5 carbon - centered radical that attacks c8 of the heterocyclic 
 ring of dg to furnish an intermediate radical that loses 
 another electron and proton to furnish cyclo - dg ( scheme 1 ) . oxidation at the 1 , 2 , 3 , 
 4 , or 5 carbons also yields a carbon - centered radical 
 that reacts with o2 , and many of these pathways ultimately 
 cause cleavage of the n - glycosidic bond releasing gua ( scheme 1 ) . the yield of cyclo - dg in 
 the fenton reaction was maximal under anaerobic reducing conditions 
 and was observed to be at a minimum under aerobic nonreducing conditions 
 ( table 1 ) . in contrast , base release to give gua was observed to be maximal under aerobic and reducing 
 conditions and not observable under anaerobic conditions ( table 1 ) . these results further support the role of o2 in effecting base release from 2-deoxyribose oxidation . however , under anaerobic conditions 
 the 5-carbon radical was not trapped by o2 , but 
 rather added into the heterocyclic ring at c8 leading to cyclo - dg , and again this reaction was not sensitive to the presence of the 
 reductant . also observed in very low yield were og and gh , two products that are derived from further oxidation of gua ( table 1 ) . the observation of gh and not sp under these conditions ( ph 7.4 
 and 22 c ) is consistent with a previous observation that oxidations 
 of the free base og only give gh and not sp . in the x - ray - mediated oxidation 
 of dg , o2-dependent studies could not be conducted ; 
 therefore , only product dependence on the reductant was studied . in 
 this study , the products derived from sugar oxidation were not greatly 
 affected by the presence of the reductant ( table 2 ) . other products resulting from oxidation of the 2-deoxyribose 
 sugar in dg were not observed by lc - esi - ms 
 ( figure s3 ) . for the sake of brevity , mechanistically , 
 these 
 products form when ho adds at the c8 carbon to 
 yield 8-ho - dg ( scheme 2 ) . this intermediate can be further oxidized to 
 yield dog , or be reduced and ring opened to yield fapy - dg ( scheme 2 ) . under 
 all conditions , dog was observed ; however , we did not 
 detect fapy - dg in these studies without exhaustive removal 
 of o2 ( figure s24 ) . in both 
 the fenton and x - ray oxidations , the yield of dog was 
 at a maximum when the reductant was present , which appears to be inconsistent 
 with previous results ; however , the current 
 studies were conducted under aerobic conditions ( tables 1 and 2 ) , while the previous studies 
 were conducted under anaerobic conditions . because dog can undergo further oxidation to hydantoin products ( see below ) , the presence of the reductant suppresses the 
 further oxidation and addresses why dog concentrations 
 are highest under oxidations conducted with the reductant . in conclusion , 
 ho - mediated oxidation of dg via fe(ii)-fenton 
 chemistry and x - ray irradiation effects oxidation at c8 of dg to yield dog as the dominant product along this pathway 
 under low oxidant flux with o2 and the reductant present 
 ( figures 3a , 3b , and 5 ) . furthermore , as the oxidant flux increases , the 
 yield of dog decreases due to further oxidation to dsp . the low redox potential of dog renders 
 this nucleoside 
 labile toward further oxidation to yield the hydantoins dsp and dgh ( scheme 3 ) . two - electron oxidation of dog followed by water attack 
 at c5 yields 5-ho - dog that bifurcates along two pathways 
 to either dsp or dgh . acyl migration 
 to dsp dominates under conditions of higher ph ( ph > 
 5.8 ) and unencumbered contexts such as nucleosides , single - stranded 
 dna , and g - quadruplexes , while the yield of dgh is highest 
 at low ph ( ph < 5.8 ) or sterically encumbered contexts such as 
 double - stranded dna . these and 
 previous observations explain why dsp was the major hydantoin 
 observed in all unencumbered nucleoside studies . as previously stated , 
 the yield of dog was highest when the reductant was present 
 at relevant concentrations ( figures 2 and 4 ) . in contrast , the yield of dsp was 
 highest in the absence of the reductant . this observation supports dog being the stable intermediate that leads to dsp , as further oxidation of dog to dsp was 
 quenched with added reductant ( figures 2 and 4 ) . these data , in which dog was obtained 
 in greater yield than the hydantoins under conditions with physiologically 
 relevant reductant , further explain why concentrations of dog are generally much greater than hydantoins in vivo . oxidations of dog have detected a four - electron 
 oxidation 
 product dehydroguanidinohydantoin-2-deoxyribonucleoside 
 ( dgh ) , or formally a six - electron 
 oxidation product of dg ( scheme 3 ) . this compound has a short half - life 
 and decomposes to yield oxaluric acid that further hydrolyzes to liberate 
 oxalate and ultimately 2-deoxyribosyl - urea . these analytical conditions allow identification of oxaluric 
 acid ( unpublished result from our laboratory ) ; however , under the 
 current ho - mediated oxidations , neither dgh nor oxaluric acid were detected . in these 
 studies , the products dz and d2ih result from 
 oxidation of dg followed by initial product forming chemistry 
 occurring at c5 . previous ho - mediated oxidations 
 of dg detected dz as a major product under 
 aerobic and nonreducing conditions , which we observed in these studies 
 ( tables 1 and 2 ) . mechanistically , dz was proposed to form from one - electron 
 oxidation of dg ( dg / dg(h ) , 
 pka 3.9 ) to a neutral radical that couples with o2/o2 to ultimately yield a hydroperoxy intermediate 
 ( 5-hoo - dg ) . next , 5-hoo - dg decomposes through 
 a multistep process to diz followed by hydration to dz ( scheme 4 ) . additional support for the role of o2/o2 in the product defining 
 step leading to dz was observed in the reactions under 
 anaerobic conditions , for which dz was not detected ( table 1 ) . further , as the reducing agent was titrated into 
 the reactions , the yield of dz dramatically decreased , 
 an observation consistent with a previous report . when the oxidations were conducted under aerobic conditions 
 with 
 relevant concentrations of the reductant ( asc or nac ) , d2ih was the major product detected ( figures 3 , 4 , and 5 ) . in scheme 5 a mechanism formation of ho via the fe(ii)-fenton reaction 
 or x - ray radiolysis of water ( reactions 1  4 ) effects the one - electron oxidation of dg followed by proton loss to yield dg . next , dg couples with o2 to ultimately yield 5-hoo - dg(24 ) that is the intermediate susceptible 
 to reduction . when the reductant was not present , 5-hoo - dg decomposes to yield diz ; however , when the reductant 
 was present , the hydroperoxyl group can be reduced to the alcohol 5-ho - dg . next , 5-ho - dg undergoes acyl migration 
 to reduced - dsp ( dspred ) that hydrates at c8 and ring opens to yield d2ih ( scheme 5a ) . this proposed mechanism provides 
 a role for o2 and the reducing agent in the formation of d2ih that was observed in the product distribution studies 
 ( figures 2 and 4 ) . the 
 proposed mechanistic steps from 5-ho - dg to d2ih were previously proposed from dg oxidations with khso5 catalyzed by a mn  porphyrin complex . because d2ih was also observed under anaerobic conditions 
 ( table 1 ) there must be an alternative pathway 
 for its formation that is not o2 dependent . in scheme 5b , such a mechanism is proposed in which ho adds to c5 yielding 5-ho - dg that proceeds through a second one - electron 
 oxidation and deprotonation to yield 5-ho - dg ; acyl migration 
 and hydration then yield d2ih ( scheme 5b ) . the alternative 
 pathway must be a minor reaction channel to d2ih , because 
 added reductant significantly increases d2ih formation 
 supporting the mechanism in scheme 5a . product formation upon oxidation of dg initially occurs 
 on the sugar leading to base release or cyclo - dg ( scheme 1 ) , on c5 of the heterocyclic base leading to d2ih and dz ( schemes 4 and 5 ) , or on c8 of the heterocyclic base 
 leading to fapy - dg and dog ( scheme 2 ) , in which dog is further oxidized 
 to dsp and dgh ( scheme 3 ) . inspection of the product distributions allows ranking 
 the initial dg site of reactivity toward oxidation under 
 each condition . the focus will be on reactions conducted under aerobic 
 conditions with and without the addition of asc ( tables 1 and 2 ) . the iron - fenton reaction without 
 the addition of asc focused on the sugar , c5 , and c8 carbons in a 
 ratio of 1.3:1.5:1.0 , respectively , with the c5 products showing the 
 highest yield . a comparison of the c5 and c8 product distributions 
 show that c5 products are 150% of the c8 products . when asc 
 ( 2 mm ) was added to the mixture , the ratio of reactivity was 1.0:1.9:1.3 
 for the sugar , c5 , and c8 carbons , respectively ( table 1 ) . the addition of asc to the iron - fenton reaction suppressed 
 oxidation of the sugar , and initial reaction at the c5 was still greater 
 than c8 . this observation is consistent with the proposed 
 mechanistic role of asc in product formation , in which it reduces 
 intermediate species that have already reacted at c5 or c8 ( scheme 5 ) . the x - ray mediated oxidation of dg without asc yielded 
 sugar , c5 , and c8 products in a 1.0:1.4:1.0 ratio , respectively ( table 2 ) . initial reactivity at c5 was the dominant pathway , 
 and it was 140% greater than the c8 pathway , similar to that 
 observed for the iron - fenton mediated oxidation of dg . when asc was added to the mixture the ratio of reactivity at the 
 sugar , c5 , and c8 sites on dg was 1.0:1.5:1.4 , respectively . overall , 
 asc decreased the sugar oxidation products ( table 2 ) and the c5 and c8 products were found in nearly the same 
 yield . the reason c5 and c8 products were observed in similar yields 
 has to do with dz . the presence of asc dramatically diminished 
 the yield of dz in the x - ray mediated oxidations , leading 
 to less c5 products and similar yields of c5 and c8 products . these 
 comparisons support a greater level of oxidation occurring at the 
 heterocyclic ring of the dg nucleoside compared to the 
 sugar particularly with asc present . use of a hypercarb column 
 allowed separation of the d2ih and dsp diastereomers 
 for which the absolute configurations 
 and elution order on this hplc column are known . in the current studies , the r and s isomers of d2ih were observed in a 1:2 ratio , respectively , 
 for both oxidation reactions studied , while the r and s isomers of dsp were observed 
 in a 1:1 ratio . furthermore , these ratios did not change under any 
 of the reaction conditions studied . the observation that the d2ih isomers were not in a 1:1 ratio points to steric hindrance 
 during the defining point of the reaction that determines product 
 stereochemistry and was likely caused by the 2-deoxyribose sugar . in contrast to this result , the diastereomer ratio of d2ih found from the copper - mediated fenton oxidation was 2:1 . in the other d2ih studies , the 
 products resulting from base oxidation of dg in oligonucleotides 
 of known sequence in single- ( odn1 ) 
 and double - stranded ( odn2 ) contexts were determined , as well as products 
 observed from -dna . the product analysis was achieved using 
 hf to hydrolyze the bases from the sugar  phosphate backbone . further , products resulting from oxidation of other 
 nucleotides , leading to thymine glycol , for example , were observed 
 but were not quantified ; masses consistent with guanine cross - links 
 with thymine and cytosine were observed , but their yields are not 
 reported due to a lack of established extinction coefficients to obtain 
 their yields ( figure s24 ) . these limitations 
 in product analysis prevent an ideal comparison ; however , we can use 
 these data to understand context - dependent product distributions leading 
 to c5 products ( 2ih and z ) vs c8 products 
 ( og , sp , and gh ) when iron - fenton 
 or x - ray mediated oxidations were conducted with asc ( 2 mm ) present 
 under aerobic reaction conditions . the overall context - dependent trends were very similar for 
 iron - fenton 
 and x - ray irradiation reactions ( figure 6 a 
 and b ) . the relative yield of z diminished dramatically 
 when comparing oxidations between nucleoside to odn and dna contexts . 
 in the duplex contexts no z was observed , an observation 
 similar to previous studies of the added reductant during the oxidations . the og free base was observed in 
 all contexts and comprised the smallest relative yield in the odn 
 and dna contexts and largest yield in the nucleoside studies from 
 both iron - fenton and x - ray oxidations . the yield of sp and gh showed strong context - dependent yields , in which sp was greatest in nucleoside and single - stranded contexts 
 and gh was observed to be greatest in duplex contexts . lastly , the relative yield of 2ih remained nearly the 
 same in all three contexts ( 4050% , figure 6a and b ) . without complete mass balances for oxidations 
 in the odn and dna contexts , it can not be definitively stated that d2ih is the major oxidation product of dg ; nonetheless , d2ih is clearly a major product observed from oxidation of dg in the nucleoside , single- and double - stranded odn , and 
 -dna contexts . context - dependent yields 
 of dg base oxidation products from the 
 iron - fenton and x - ray irradiated samples . the odn and -dna 
 distributions were obtained after hf hydrolysis of the oxidized samples . 
 in the odn and -dna samples , products were also observed from 
 oxidation of the other nucleotides . because of the hf hydrolysis to 
 liberate the oxidized free bases , the extent of sugar oxidation leading 
 to free base could not be determined . the current results are compared to literature studies that 
 reported d2ih as shown in table 3 . the ball 
 laboratory exclusively observed d2ih when dg was exposed to the epoxidizing reagent dimethyldioxirane ( dmdo ) , 
 and they confirmed the structure of d2ih via complementary 
 nmr methods . the meunier laboratory observed d2ih ( m+34 ) when dg was oxidized 
 with mn - tmpyp / khso5 . studies in the karlin 
 and rokita laboratories observed a product mass in high yield consistent 
 with d2ih ( m+34 ) when an oligodeoxynucleotide 
 was allowed to react with a dicopper(ii)-complex . in our laboratory , d2ih was detected in high yield 
 with the nicr / khso5 system , and in studies utilizing the 
 copper - mediated fenton oxidation of dg . further , the bohme laboratory conducted the lead - mediated fenton 
 oxidation of dg to yield d2ih as the major 
 product . in all of the metal - catalyzed 
 studies , the transition metal was proposed to play a role in the product - forming 
 step leading to d2ih . however , studies conducted in the 
 shafirovich laboratory observed d2ih during co3 oxidations of dg , demonstrating 
 that a transition metal was not required for d2ih formation . in the current report , d2ih was 
 the major product of  free  ho - mediated 
 oxidation of dg with relevant amounts of reductant under 
 aerobic conditions . the x - ray studies demonstrate once again that d2ih can be formed under conditions that do not require the 
 involvement of a transition - metal catalyst . ( + + ) a major product , ( + ) a minor 
 product , ( n.d . ) not detected or not reported . the current studies 
 monitored oxidation products from dg resulting from ho generated via the fe(ii)-mediated 
 fenton reaction or x - ray radiolysis of water . products resulting from 
 oxidation and initial reaction at the sugar , c5 , or c8 carbons were 
 quantified ( figures 25 ) . under aerobic conditions without a reductant , the major 
 products observed include dz , dsp , and sugar 
 oxidation leading to gua . under anaerobic reaction conditions 
 the major product observed was dsp . when reactions were 
 conducted with cellularly relevant ( mm ) amounts of the reductant ( asc 
 or nac ) and o2 , the major product observed in all cases 
 was d2ih . the isomers of d2ih are two - electron 
 oxidation products of dg that have not been previously 
 detected in oxidations with ho generated by the 
 fe(ii)-mediated fenton reaction or x - ray radiolysis of h2o . the high yields of d2ih observed and the observation 
 that this lesion is highly prone to piperidine cleavage in a dna oligomer , 
 unlike dog , lead to a hypothesis 
 that oxidations in dna imposed by electron transfer agents yield c5 
 oxidation products ( i.e. , d2ih or dz ) and 
 not the c8 oxidation product dog . oxidations in the context of single- and double - stranded 
 odns and -dna yielded d2ih as a product of dg oxidation in significant yield that was competitive with 
 the yield of dog . more importantly , the current results 
 highlight d2ih as a major product observed in reactions 
 that include relevant amounts of reductant in the presence of o2 , suggesting that d2ih should be investigated 
 more closely for its biochemical properties . a 200-l solution of dg ( 3.0 mm , 0.6 moles , 0.16 mg ) in a buffer ( 75.0 mm napi , ph 7.4 ) was mixed with fe(ii)/edta ( 0.10 mm , 0.02 moles , 
 0.008 mg ) , h2o2 ( 10.0 mm , 2 moles , 0.5 
 mg ) , and asc or nac ( 2.0 mm , 0.40 moles , 0.03 mg ) and incubated 
 for 1 h at 22 c . the fe(ii)/edta complex was freshly made by 
 mixing fe(nh4)2(so4)2 and 
 na2edta in a 1:2 ratio 30 min prior to reaction . the fe(ii)/edta 
 complex was removed by column chromatography using ion - exchange resin 
 prior to hplc analysis following the method outlined below . a 200-l solution containing dg ( 3.0 mm , 0.6 moles , 0.16 mg ) in buffer ( 75.0 mm 
 napi , ph 7.4 ) and asc or nac ( 2.0 mm , 0.40 moles , 
 0.03 mg ) was irradiated with an x - ray rs 2000 biological research 
 irradiator source at 22 c for variable times ( 130 min ) . single - stranded 
 odn1 or double - stranded odn2 were oxidized in 20 mm napi ( ph 7.4 ) with 100 mm nacl at 37 c . iron - fenton oxidations 
 were conducted in a 200-l reaction volume with odn ( 100 m , 
 0.01 moles , 0.05 mg ) to which were added fe(ii)edta ( 0.10 mm , 
 0.02 moles , 0.008 mg ) , asc ( 2.0 mm , 0.40 moles , 0.03 
 mg ) , and h2o2 ( 1.0 mm , 0.2 moles , 0.05 
 mg ) ; the reaction was allowed to proceed for 30 min . the stock solutions 
 of fe(ii)edta , asc , and h2o2 were all freshly 
 prepared . to irradiate the odns , they were placed in the same buffer 
 system as the iron - fenton reaction followed by x - ray irradiation with 
 a dose rate of 25 gy / min for 30 min ( total dose = 750 gy ) . for the 
 -dna oxidations , everything was identical to the odn oxidations 
 with the exception that the -dna ( 1 m , 0.1 nmol , 0.03 
 mg ) was different . the oxidized odn or -dna samples were 
 hydrolyzed to the 
 free bases for hplc analysis . the hydrolysis was performed on the 
 oxidized and lyophilized dna by adding 50 l of 70% hf in pyridine 
 for 30 min at 37 c . after the reaction , the excess hf was neutralized 
 by adding 1 ml of ddh2o and 80 mg of caco3 to 
 the sample . the insoluble salts were removed by centrifugation , and 
 the supernatant was then lyophilized to dryness . next , the lyophilized 
 samples were dissolved in ddh2o and submitted to lc - ms 
 and hplc analysis as described for the nucleoside studies below . product identification was initially 
 achieved by uplc - esi - ms ( 100 mm  2.1 mm , 1.7 m ) 
 and uplc - esi+-ms with a hypercarb column ( 100 mm  2.1 mm , 5 
 m ) . the following masses were observed : guam / z [ m + h ] calcd 152.1 , found 
 152.1 . r and s diastereomers of cyclo - dgm / z [ m + h ] calcd 266.2 , found 266.1 . ( s)-d2ih and ( r)-d2ih(42)m / z [ m + h ] calcd 302.3 , found 302.1 ; esi - ms / ms m / z [ m + h ] lit . 186 , 158 , and 141 ; found 186 , 158 , and 141 . hrms ( esi - tof ) m / z [ m + na ] calcd for c5h7n5o3na 208.0447 , found 
 208.0449 . the hrms value was obtained on the free base 2ih , due to the nucleoside s instability toward acid . dghm / z [ m + h ] calcd 
 274.3 , found 274.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c9h15n5o5na 296.0971 , found 296.0980 . ( s)-dsp and ( r)-dsp(62)m / z [ m + h ] calcd 
 300.2 , found 300.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c10h13n5o6na 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit . 184 , 156 , 141 , 113 , 99 , and 86 ; found 184 , 156 , 
 141 , 113 , 99 , and 86 . dzm / z [ m + h ] calcd 247.2 , found 247.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c8h14n4o5na 269.0862 , found 
 269.0870 ; esi - ms / ms m / z [ m + h ] lit . 247 , 203 , 
 and 131 ; found 247 , 203 , and 131 ( figures s3s13 , supporting information ) . the dgh diastereomers were characterized by nmr , dz was characterized by nmr , cyclo - dg was characterized by x - ray crystallography , the dsp diastereomers have been 
 characterized by x - ray crystallography and nmr , and the diastereomers of d2ih have been previously characterized by nmr . the oxidation 
 products from 
 the fe(ii)-fenton reaction or x - ray irradiation reaction were quantified 
 by c18 reversed - phase hplc and hypercarb hplc . first , the reaction 
 mixture was injected on a reversed - phase hplc ( 250 mm  4.6 mm , 
 5 m ) to quantify gua , og , dog , and cyclo - dg . the void volume from this run was collected , 
 lyophilized to dryness , and then dissolved with the hypercarb column 
 starting mobile phase ( 0.1% acetic acid ) . next , the reconstituted 
 void volume was injected on a hypercarb column ( 150 mm  4.6 
 mm , 5 m ) to quantify gh , ( r)-d2ih , ( s)-d2ih , dgh , ( r)-dsp , ( s)-dsp , and dz . product 
 peaks were quantified by their absorbance intensity at 240 nm followed 
 by normalization of these intensities by each compound s extinction 
 coefficient at 240 nm . the values for 240  nm ( ddh2o ) are dg 14  080 , dog 14  300 , cyclo - dg 14  080 , gua 14  080 , og 14  300,dsp 3280,d2ih 2290,dgh and gh 2410 , and dz 1780 .
exogenously 
 and endogenously produced reactive oxygen species attack 
 the base and sugar moieties of dna showing a preference for reaction 
 at 2-deoxyguanosine ( dg ) sites . in the present 
 work , 
 dg was oxidized by ho via the 
 fe(ii)-fenton reaction or by x - ray radiolysis of water . 
 the oxidized 
 lesions observed include the 2-deoxynucleosides of 8-oxo-7,8-dihydroguanine 
 ( dog ) , spiroiminodihydantoin ( dsp ) , 
 5-guanidinohydantoin ( dgh ) , oxazolone ( dz ) , 5-carboxamido-5-formamido-2-iminohydantoin ( d2ih ) , 5,8-cyclo-2-deoxyguanosine ( cyclo - dg ) , and the free base guanine ( gua ) . 
 reactions conducted 
 with ascorbate or n - acetylcysteine as a reductant 
 under aerobic conditions identified d2ih as the major 
 lesion formed . 
 studies were conducted to identify the role of o2 and the reductant in product formation . from these studies , 
 mechanisms are proposed to support d2ih as a major oxidation 
 product detected under aerobic conditions in the presence of the reductant . 
 these nucleoside observations were then validated in oxidations of 
 oligodeoxynucleotide and -dna contexts that demonstrated 
 high yields of d2ih in tandem with dog , dsp , and dgh . 
 these results identify dg oxidation to d2ih to occur in high yields leading to 
 a hypothesis that d2ih could be found from in cells stressed 
 with ho. further , the distorted ring structure 
 of d2ih likely causes this lesion to be highly mutagenic .