the guanine heterocyclic 
 ring is rich in chemical reactivity toward 
 oxidants and adduct - forming species . in dna , 2-deoxyguanosine 
 ( dg , mass = m ) is the chief site for base oxidation due 
 to its low redox potential leading to many products that are characterized 
 by their mass signatures .
the principal products 
 characterized with one - electron oxidants in aqueous solutions can 
 be grouped based on the site of reaction on the radical intermediate 
 resulting from one - electron oxidation of dg .
the 2-deoxyribonucleoside products arising from initial 
 reactivity at c5 of dg include a four - electron oxidation 
 product , an imidazolone ( diz , m-39 ) that hydrolyzes to 
 an oxazolone ( dz , m-21 ) , and 
 the two - electron oxidation product 5-carboxamido-5-formamido-2-iminohydantoin 
 ( d2ih , m+34 ; scheme 1 ) .
when the initial reaction occurs at c8 of dg , either 
 2,6-diamino-4-hydroxy-5-formamidopyrimidine ( fapy - dg , 
 m + 18 ) is observed under reducing conditions or 8-oxo-7,8-dihydroguanine 
 ( dog , m+16 ) is observed under oxidizing conditions , in 
 which the latter compound is a key marker for monitoring oxidative 
 stress in cells ( scheme 1 ) .
the 
 two - electron oxidation product dog is stable but is highly 
 susceptible to further oxidation leading to two hydantoin compounds . 

the yield for the thermodynamically preferred product spiroiminodihydantoin 
 ( dsp , m + 32 ) is greatest in nucleoside reactions at 
 ph > 6 or in unencumbered reaction contexts ( i.e. , single - stranded 
 dna or g - quadruplexes ) , while the yield of 5-guanidinohydantoin ( dgh , m + 6 ) is greatest in nucleoside reactions at ph < 
 6 or in sterically demanding duplex contexts ( scheme 1 ) .
the hydantoins are also readily formed from direct four - electron 
 oxidation with o2 .
this list of products represents those that are consistently observed 
 from many oxidant systems conducted by several laboratories ; however , 
 other compounds have been reported in lower yield . in the cellular context
, oxidation reactions 
 with dg can have other nucleophilic participants such 
 as amines or phenols . 
 in most cases , when amines or phenols participate in the reaction , 
 products
are observed with similar backbone structures as characterized 
 with water serving as the nucleophile .
for example , when dg is oxidized in the presence of lysine , products retaining the dg heterocycle and those with a core similar to dgh or dsp have been observed .
the adducts characterized by 
 our laboratory and others show lysine competing with water for sites 
 of covalent bond formation , where a single lysine is observed at c8 
 analogous to dog , at either c5 or c8 of a spirocyclic 
 core similar to dsp , and last a bis - adduct at c5 and 
 c8 with a spirocyclic ring structure has been identified ( figure 1 ) .
the lysine 
 adducts are all stable allowing their quantification and characterization . 
 as a last example
, the polyamine spermine has been adducted to dg and dog under oxidative conditions . in the dg oxidation studies , a spermine adduct at c8
is observed 
 retaining the dg heterocyclic core , while dog oxidations yield an adduct at c5 
 that does not undergo acyl migration to create a spirocycle , but instead 
 the polyamine can generate an unstable hemiaminal intermediate that 
 decomposes leaving a ribosylurea lesion at the adduct formation site .
moreover , the facile formation of spermine adducts 
 to dog oxidation intermediates has been harnessed for 
 quantification of dog from dna samples .
in contrast to amines , phenols ( i.e. , tyrosine ) are more 
 redox active than amines and better able to compete with dg for oxidant during reactions .
the products observed when phenols 
 participate with dg during oxidation have more variability 
 in their structures compared to amine adducts .
these model studies aid in understanding 
 the chemical nature of dna  protein cross - links that are detrimental 
 to cellular processes . in the current work ,
oxidations that allowed dg to 
 react with nh3 were conducted while the full product and 
 diastereomer distributions were monitored .
after careful characterization 
 of the nucleoside reactions , similar oxidations were conducted in 
 the single - stranded and duplex oligodeoxynucleotide ( odn ) contexts . 
 the purified ammonia adducts were also studied with respect to their 
 decomposition pathways leading to end products that have not been 
 previously determined .
lastly , polymerase insertion studies were conducted 
 to test the hypothesis that amine adducts might have altered base - pairing 
 preferences ; every substitution of an oxo group on a base for an amino 
 group converts a hydrogen bond acceptor into a potential hydrogen 
 bond donor .
these results provide a fundamental understanding of the 
 stability and base - pairing properties of amine adducts to dg that result during oxidation , which may occur in vivo due to the 
 high concentration of nucleophilic amines in the vicinity of the genome .
adducts 
 observed when dg and lysine are allowed to 
 react in the presence of oxidant .
the unencumbered nucleoside dg ( 1 mm ) was chosen for 
 the initial oxidation reactions where nh4cl ( 20 mm ) provided 
 the source of nucleophilic nh3 .
all reactions were conducted 
 with 75 mm napi buffer at ph 7.4 at 22 c .
the oxidants 
 chosen include the photooxidants riboflavin and rose bengal and the 
 one - electron oxidant na2ircl6 .
reactions were 
 conducted in triplicate to achieve 70% conversion to products 
 in each reaction , and the products ( scheme 2 ) were analyzed by a dual hplc method . the first round of hplc analysis 
 utilized a reversed - phase column to identify dog ( m+16 ) 
 and 8-amino - dg ( m+15 ) , while all other products eluted 
 in the void volume .
the void volume from the previous run was collected 
 and analyzed on a hypercarb hplc column that allowed analysis of the 
 hydantoins , ammonia adducts , and dz .
however , diz hydrolyzes 
 to dz that was detected on the hypercarb column ; thus , 
 formation of diz is inferred from quantification of dz .
moreover , the hypercarb hplc column also allowed separation 
 and quantification of all spirocyclic diastereomeric pairs of products . 

the dgh diastereomers are interconvertible , and thus , their diastereomer ratios are not 
 reported ( see the supporting information for complete experimental details ) .
lastly , a test reaction with 
 na2ircl6 was conducted in which half was directly 
 analyzed by the hypercarb hplc column and the other half was analyzed 
 by the dual hplc method outlined above .
the product distributions 
 observed from these comparative studies were within 3% of 
 one another .
the photooxidant riboflavin led to the largest number 
 of different 
 products and was used to obtain suitable amounts of material for characterization . 

esi - ms ( figure s1 , supporting information ) , and the adducts observed that did not involve participation of 
 nh3 during product formation included dz ( m 
 - 21 ) , dog ( m + 16 ) , dgh ( m + 6 ) , and dsp ( m + 32 , scheme 2 ) .
confirmation 
 of their structures was achieved by esi - ms / ms fragmentation 
 of the free bases while monitoring the daughter fragments .
the esi - ms / ms experiments were conducted on hplc purified nucleosides 
 in which the n - glycosyl bond was cleaved in the ionization 
 source to liberate free bases that were further fragmented in the 
 cid chamber to generate the daughter fragments used in identification 
 of the structures .
the ms / ms fragmentation spectra were compared to 
 literature values for the two dsp diastereomers ( figure 2 ) and dz to further confirm their structural 
 assignments ( figures s2 and s3 , supporting information ) .
the structure for dsp has been further established 
 by x - ray crystallography and nmr .
ammonia adducts observed included two pairs 
 of chromatographic peaks for the diastereomers of spirodi(iminohydantoin)-2-deoxyribonucleosides 
 ( dsi ) , whose names are based on the site of nh3 attachment to guanine , thus 5-dsi ( m + 31 ) and 8-dsi ( m + 31 ) ( scheme 2 , see reference 
 cited for the correct dsi iupac nomenclature ) . to confirm the identities of the 5- and 8-dsi constitutional isomers and their respective 
 diastereomers , esi - ms / ms of the free bases were conducted 
 ( figure 2 ) .
comparison of the esi - ms / ms spectrum for 5-dsi with dsp gave 
 a pair of peaks for 5-dsi that established c5 as the 
 site of nh3 attachment ( m / z [ m + h ] = 140 and 96 , figure 2 ) .
as for 8-dsi , comparison of its esi - ms / ms 
 spectrum with those obtained from dsp and 5-dsi identified new masses that are best explained by nh3 attachment 
 at c8 ( m / z [ m + h ] = 
 141 and 97 , figure 2 ) . on the basis of the 
 proposed mechanism ( scheme 2 ) , a product that 
 includes bis - addition of nh3 at c5 and c8 of an oxidized 
 guanine to yield a spirocycle ( 5,8-dsi , m+30 ) is possible ; however , this adduct was not observed 
 for reasons that will be elaborated on below .
additionally , nh3 adducts with a dgh core were not observed most 
 likely due to the fact that the reactions were performed at ph 7.4 
 where yields of dgh are minimal .
lastly , the yields of dog ( m + 16 ) and 8-amino - dg ( m + 15 ) were very 
 low ( < 1% ) as determined by lc - esi - ms ( figure s1 , supporting information ) ; hence , their quantities 
 are not reported .
the nucleosides dog and c8 amine - adducted dg have redox potentials that are 600 mv below the 
 parent nucleoside dg causing them to be much more susceptible 
 to further oxidation .
further , one - electron oxidized dog was observed to have an even lower redox potential than 
 the parent compound dog , which means that once oxidation of dog , and likely 8-amino - dg , occurs product formation is inevitable due to 
 the 70% conversion to product that yielded the spirocyclic 
 compounds dsp , 5-dsi , or 8-dsi .
esi - ms / ms spectra for dsp ( top ) , 5-dsi ( middle ) , and 8-dsi ( bottom ) .
the data 
 provided was collected on the first eluting diastereomer of each spirocycle 
 from a hypercarb hplc column .
data for the second eluting diastereomer 
 for dsp , 5-dsi , and 8-dsi can 
 be found in the supporting information ( figures 
 s2 , s4 , and s5 ) .
the heavy lines represent the fragment observed and 
 the thin lines represent the portion of the molecule lost upon fragmentation .
relative product distributions 
 observed with each oxidant system 
 were determined by integration of the hplc peak areas measured at 
 240 nm followed by normalization via each molecule s unique 
 extinction coefficient ( 240 nm ) .
extinction coefficients for 5-dsi and 8-dsi are not known but were determined from experiments that 
 are reported below . in the riboflavin - mediated oxidations ,
this result was anticipated because riboflavin is a type i photooxidant 
 and effects oxidation by electron transfer from dg yielding 
 o2 under aerobic reaction conditions .
oxidation of dg by one - electron 
 and proton transfer yields an intermediate radical ( dg ) that only couples with o2 to initially yield diz that 
 is prone to hydration leading to dz ( scheme 2 ) , the species quantified .
the adducts 
 derived from the nucleophile trapping of electrophilic dg oxidation intermediates show nh3 participation through 
 the products 5-dsi ( 29% ) that was the highest yielding 
 nh3 adduct followed by nearly 3-fold less 8-dsi ( 11% ) .
the participation of h2o as the trapping nucleophile 
 was identified by the lower yields of dsp ( 11% ) and dgh ( 4% ) .
the nucleophilicity of nh3 is much greater 
 than h2o ; therefore , as expected , adducts resulting from 
 nh3 participation dominated over those derived from h2o .
relative product distributions observed when dg reacted 
 with nucleophilic h2o or nh3 in oxidation reactions . 

the oxidants include the photooxidants riboflavin and rose bengal , 
 as well as na2ircl6 .
reactions were conducted 
 with 1 mm dg , 20 mm nh4cl , in 75 mm napi buffer ( ph 7.4 ) at 22 c .
( 1 ) photoactivation of riboflavin 
 ( 200 m ) was achieved with 350 nm light for 3 h , ( 2 ) photoactivation 
 of rose bengal ( 100 m ) was achieved with 350 nm light for 3 
 h , and ( 3 ) oxidation with na2ircl6 ( 10 mm ) was 
 achieved by bolus addition of the salt .
results represent the average 
 of triplicate trials and the error is 10% of each reported 
 value .
the second oxidant studied was 
 rose bengal , a type ii photooxidant 
 ( o2 ) that furnished 5-dsi as the 
 major product ( 62% ) followed by a 3-fold lower amount of dsp ( 31% ) , and low yields of dz ( 5% ) and dgh ( 2% ) were observed to complete the mass balance ( figure 3 ) . as expected , this oxidant did not yield 8-dsi based on its mechanism of oxidation .
oxidation of dg by o2 proceeds by 4 + 2 cycloaddition 
 to the imidazole ring , followed by ring opening to yield 8-hoo - g that eliminates water giving the proposed electrophile dog .
next , dog is trapped by nucleophiles at 
 c5 leading to dsp with h2o or 5-dsi with nh3 ( scheme 3 ) .
support for 
 exclusive nucleophilic attack at c5 under o2 oxidations was derived from h2o studies 
 followed by mapping the labeled site by esi - ms / ms ; the current observation that the only nh3 adducts were the 5-dsi diastereomers further 
 support this previously proposed mechanism , and further confirms our 
 structural assignments for these peaks .
comparisons of the product 
 distributions from the photooxidants riboflavin and rose bengal show 
 a dramatic difference with respect to the major product observed ( figure 3 ) . in the riboflavin oxidation ,
dz was 
 the major product while in the rose bengal oxidation , 5-dsi was the major product , and these observations can be ascribed to 
 each oxidant s unique mechanism of oxidation ( schemes 2 and 3 ) .
product distributions from this oxidant 
 included 
 nearly equivalent distributions of dsp ( 50% ) and 5-dsi ( 42% ) with the mass balance completed by dgh ( 8% , figure 3 ) . in this oxidation reaction
, 
 the distribution did not yield nh3 adducts as the major 
 products , which was unexpected on the basis of nh3 being 
 the better nucleophile compared to h2o .
comparison 
 of the product distributions observed with the one - electron 
 oxidants riboflavin and na2ircl6 identified 
 both nh3- and h2o - adducted compounds .
the current 
 results differ from those previously reported by our laboratory when 
 lysine was adducted to dg in analogous oxidation reactions . previously , in riboflavin oxidations spirodihydantoins 
 with lysine at c8 were 2-fold greater than those with lysine at c5 
 ( figure 1 ) , and in na2ircl6 oxidations a c8 lysine adduct was observed .
in contrast to these 
 results , riboflavin oxidations in the presence of nh4cl 
 gave more c5 adducts ( 29% ) than c8 adducts ( 11% ) , and na2ircl6 oxidations did not yield detectable amounts of c8 
 adducts .
we propose the difference in the current ammonia results 
 compared to the lysine data can be attributed to a difference in the 
 mechanism of product formation for the c8 adduct in the nucleoside 
 context .
the former work from our laboratory proposed that c8 amine 
 adducts result from oxidation of the amine to the aminyl radical that 
 adds at the c8 carbon of dg followed by oxidation leading 
 to product formation ( scheme 4 ) . in nucleoside dg , this proposed 
 mechanism best supports the observation of ammonia adducts at c8 , 
 because upon one - electron oxidation of dg the initial 
 radical cation ( dg ) 
 formed is very acidic ( pka  3.9 ) and rapidly deprotonates to the neutral radical 
 ( dg ) that is not susceptible 
 to nucleophilic attack . thus , dg reacts with o2 yielding diz / dz and not with amines or 
 h2o .
formation of amine adducts at c8 must result 
 from a difference in the amine reactivity .
the key difference between 
 lysine and ammonia resides in their standard reduction potentials . 

in general , primary amines ( 1.0 v vs nhe , ph 10 ) have a lower redox potential than ammonia ( > 1.3 v vs 
 nhe , 
 ph 9 ) ; this trend should scale down to 
 ph 7 , in which the oxidations were conducted .
thus , oxidation of lysine 
 to an aminyl radical that adds to c8 of dg is possible 
 with na2ircl6 ( 0.9 v vs nhe , ph 7 ) and riboflavin 
 ( 1.7 v vs nhe , ph 7 ) ; in contrast , the 
 analogous reaction does not readily occur for nh3 because 
 at ph 7 dg is the dominant site of oxidation due to its 
 lower redox potential leading to products other than 8-dsi . in summary ,
one - electron oxidant driven oxidations of dg in the presence of nh3 lead to spirocyclic adducts at 
 c5 and c8 that are in competition with h2o adducts of the 
 same core structure . as the next step , we set out to determine the decomposition 
 products 
 and pathways through which the ammonia adducts proceed .
hplc was used 
 to provide diastereomerically pure 5-dsi and 8-dsi samples that were subjected to conditions of ph 3 ( 0.1% formic acid ) 
 or ph 10 ( 20 mm napi ) at 22 c for 30 min or 10 h. 
 not only could the decomposition products be determined , but having 
 diastereomerically pure starting material also allowed us to probe 
 the mechanism of decomposition , specifically for 5-dsi .
first , both 5- and 8-dsi were stable 
 at ph 10 during this time frame , while 5-dsi readily 
 hydrolyzed to dsp at ph 3 in 30 min and 8-dsi hydrolyzed to dsp after 10 h. for 5-dsi , deamination of the amine group can occur by two possible mechanisms : 
 ( 1 ) a retro - acyl migration can occur followed by loss of nh3 to yield an electrophilic intermediate ( dog ) susceptible to h2o attack followed by 
 a second acyl migration back to dsp or ( 2 ) acid - catalyzed 
 deamination can occur directly to furnish dsp ( scheme 5 ) .
if the retro - acyl migration proceeds on the diastereomerically 
 pure sample , it is expected that a mixture of dsp diastereomers 
 would be observed , and if direct deamination occurs , then only one dsp diastereomer would be predicted . upon incubation of one 
 diastereomer of 5-dsi at ph 3 ,
only one diastereomer 
 of dsp was observed , supporting the direct deamination 
 mechanism .
moreover , the early eluting 5- and 8-dsi nh3 adducts decomposed to the early eluting dsp isomer 
 and the late eluting adducts deaminated to the later eluting dsp isomer ( figure s6 , supporting information ) .
these results aid in establishing the absolute configuration of 
 the 5-dsi and 8-dsi diastereomers that will 
 be discussed below .
moreover , these results also allude to an explanation 
 as to why the bis - ammonium adduct was not observed in any of the reactions . 
 based on these data , the bis - ammonium adduct , if formed , could rapidly 
 deaminate to yield either 5-dsi or 8-dsi , although this hypothesis could not be further validated .
utilization of the hypercarb 
 hplc column for analyzing the product 
 distributions of these spirocycles allowed determination of the diastereomer 
 ratios .
it has previously been determined that ( r)-dsp elutes first from this 
 column and ( s)-dsp elutes second .
the absolute stereochemistry for the diastereomers 
 of the ammonia adducts had not yet been determined .
the results of 
 these studies identified ( r)-dsp and ( s)-dsp yields to be nearly equal . also , for the 5-dsi and 8-dsi ammonia adducts nearly equal 
 yields for the diastereomers were observed . on the basis of these 
 results , the defining point of the reaction to determine product stereochemistry 
 must not be very sterically impeded in order to lead to such a small 
 diastereomer preference . as stated above , the diastereomerically 
 pure ammonia adduct samples 
 were determined to decompose to give a single diastereomer of dsp ; therefore , on the basis of hplc elution order , the absolute 
 configuration for the diastereomers of the two dsi constitutional 
 isomers can be determined .
the first - eluting 5-dsi and 8-dsi diastereomers decomposed to the first dsp diastereomer ( r ) , and the late - eluting diastereomer 
 decomposed to the late - eluting dsp diastereomer ( s ) ; therefore , for 5-dsi the r diastereomer elutes first and the s diastereomer 
 elutes second , because the r and s assignments are the same between dsp and 5-dsi .
in contrast , the r and s assignments 
 for the 8-dsi diastereomers are opposite those of dsp ; thus , ( s)-8-dsi elutes first and ( r)-8-dsi elutes second and from the hypercarb 
 hplc column ( figure 4 ) .
these examples provide 
 a fascinating case outlining how the movement of ring substituents 
 affects the r and s stereochemical 
 assignments .
assignment of absolute configurations for the diastereomers 
 of dsp , 5-dsi , and 8-dsi based 
 on their 
 elution profile from a hypercarb hplc column and their ecd spectra . 

the r and s assignments for the diastereomers 
 of 8-dsi are the opposite of those for the dsp and 5-dsi isomers with the same geometric configuration 
 of the spirocyclic ring , due to a change in the cahn  ingold 
the diastereotopic ammonia 
 adducts were then probed by electronic 
 circular dichroism spectroscopy ( ecd ) .
previously , ecd was used in 
 tandem with vibrational circular dichrosim spectroscopy , nmr , and 
 x - ray crystallography to establish the absolute configuration for dsp .
the ecd spectra for ( r)- and ( s)-dsp isomers gave three lobes that were mirror images 
 of one another , as expected ( figure 4 ) .
critical 
 for assigning the absolute configuration for dsp was 
 the low energy lobe , in which the s isomer gave a 
 positive rotation at 258 nm , and the r isomer gave 
 a negative rotation at 259 nm . in comparison 
 to these results ,
the 5-dsi diastereomers gave ecd spectra 
 that were different than dsp but showed a similarity 
 in the low energy lobe ( figure 4 ) .
for ( r)-5-dsp , the 
 lobe at 258 nm gave a negative rotation and ( s)-5-dsi gave a positive rotation 
 at 258 nm .
the higher energy lobes observed in the 
 ecd for the 5-dsi diastereomers were different than those 
 measured for dsp ( figure 4 ) .
as 
 a last comparison , the 8-dsi diastereomer ecd spectra 
 were compared to those recorded for dsp ( note : 8-dsi and dsp isomers with the same geometric 
 configuration of the spirocyclic ring give opposite r and s assignments due to a difference in the cahn 
the low energy 
 lobe gave a negative rotation at 262 nm similar to ( r)-dsp , and ( r)-8-dsi gave a positive rotation 
 similar to ( s)-dsp .
furthermore , the 8-dsi diastereomers also gave similar 
 rotations and energies as their analogous dsp diastereomers 
 at the 238 nm lobe , and 8-dsi and dsp gave similar rotations at the highest energy lobe , but the absolute 
 energy was different ( i.e. , 202 nm for 8-dsi and 
 211 nm for dsp ; figure 4 ) .
the similarity in the critical low energy lobe further supports 
 the absolute configuration assignments that were made from the deamination 
 studies monitored by hplc .
future computational studies to model these 
 ecd spectra will help solidify these conclusions , and may address 
 some of the challenges that occurred during modeling of the ecd spectra 
 for the dsp diastereomers .
the 
 ability to deaminate the dsi adducts to dsp allowed the determination of the extinction coefficients 
 for 5- and 8-dsi relative to dsp , in which these were the values used to determine the relative yields . 

peak areas for identically pure 5-dsi samples were measured 
 before and after incubation in formic acid and the change in peak 
 area measured was used to determine the relative 240  nm compared to the value established for dsp .
a similar 
 experiment was conducted for the 8-dsi diastereomers . 
 from these experiments ,
the 240  nm for the 5-dsi and 8-dsi diastereomers were determined 
 to be 3800 and 3500 lmolcm , respectively , which are both slightly greater than that determined 
 for the dsp diastereomers ( 3300 lmolcm ) .
the next step from the nucleoside studies 
 was to explore the context 
 dependence of nh3 adduct formation in single- and double - stranded 
 odns ( ssodn and dsodn ) .
the photooxidant riboflavin was chosen for 
 the studies because it gave the most diverse distribution of products 
 in the nucleoside reactions and might provide the most insight into 
 context effects . for these studies , the ssodns selected for analysis 
 were the 18-mers odn-1 and odn-2 , and the 
 dsodn context was studied via the duplex formed from these two single 
 strands ( odn-12 ) . after oxidation of these odn systems , 
 they were digested with a suite of nucleases and phosphatase to the 
 nucleosides followed by analysis using the previously described hplc 
 methods .
previously , our laboratory demonstrated that the digestion 
 method used provides complete degradation of dsp - containing 
 odns to nucleosides ; it is assumed that 
 the dsi adducts are equally digested to completion .
the 
 nuclease digestion conditions were modified to include ammonium salts 
 for buffer to prevent loss of 5-dsi via deamination to dsp ( scheme 5 ) , controls conducted 
 with sodium salts for buffers did not detect 5-dsi due 
 to deamination to dsp during the nuclease digestion ( 18 
 h at ph 5.4 , scheme 5 ) .
a comparison 
 between the contexts studied when dg was 
 allowed to react with nh3 in the presence of the photooxidant 
 riboflavin is provided in figure 5 .
the first 
 observation from these data was with respect to dz distributions 
 that dramatically decreased when proceeding from the nucleoside to 
 odn contexts ; specifically , the dz distribution in the 
 dsodn context ( 2% ) was 10-fold less than the ssodn context ( 20% ) 
 and 20-fold less than that observed in the nucleoside context ( 40% ) . 

this observation has already been reported in the literature and is 
 proposed to result from the odn context increasing the lifetime of 
 the dg that reacts with 
 nucleophilic h2o to give the c8 product dog while decreasing the lifetime of dg that reacts with o2 to 
 yield dz ( schemes 2 and 6 ) . with respect to 
 the yields of the hydantoins , more dsp relative to dgh was observed in the nucleoside context ( 11% vs 4% , respectively ) 
 versus the dsodn context ( 9% vs 21% , respectively ) .
again , this trend 
 follows those in the literature in which the duplex context favors 
 the less sterically demanding product dgh .
the nh3 adducts 5-dsi and 8-dsi provided some interesting context - dependent product distributions . 

the yield of 5-dsi was greatest for the nucleoside ( 29% ) 
 and decreased by half in all odn contexts studied ( 14% ) . in 
 contrast
, the yield of 8-dsi was smallest for the nucleoside 
 ( 11% ) and increased by more than 3-fold in the ssodn context ( 39% ) , 
 and over half the products observed in the dsodn context were 8-dsi ( 54% ) .
as previously stated , the lifetime of the electrophilic dg is longer in the duplex 
 context and the superior nucleophilicity of nh3 compared 
 to h2o greatly increased the yield of 8-dsi in dsodn oxidations ( scheme 6 ) .
furthermore , 
 the increase in 8-dsi yield from the dsodn oxidations 
 further supports this product resulting from nucleophilic addition 
 of nh3 at c8 leading to 8-dsi , and not the 
 nh3 aminyl radical adding at c8 to yield the same product . 

these results clearly demonstrate a dependence on the reaction context 
 in formation of dg oxidation products and nh3 adducts .
a look at the distribution of diastereomers resulting from 
 oxidation of the odn contexts gave nearly equal amounts of both r and s diastereomers .
this observation 
 parallels our previous work looking at dsp diastereomer 
 formation in single - stranded and duplex contexts .
coupled together , these observations support a state of 
 disorder in the duplex at the time of nucleophilic attack at c5 of 
 oxidized dog or 8-amino - dg ( likely intermediates 
 leading to the spirocycles ) that leads to nearly equal covalent bond 
 formation from both the re and si faces of these electrophiles .
relative product distributions measured 
 when dg was 
 allowed to react with nh3 in the presence of photoexcited 
 riboflavin in various contexts .
reactions were conducted in 75 mm 
 napi buffer ( ph 7.4 ) at 22 c , and 20 mm nh4cl . in 
 the nucleoside studies , 1
mm dg was studied , and in the 
 odn contexts 20 m ssodn or 10 m dsodn was studied .
( 1 ) 
 photoactivation of riboflavin ( 200 m ) was achieved with 350 
 nm light for 3 h in the nucleoside studies and 30 min in the odn studies . 

these conditions achieved 70% conversion to product in the 
 nucleoside reactions and 50% in the odn studies .
it must first be noted 
 that the bis - ammonium adduct could not be 
 quantified due to its instability , but if formed , the adduct is most 
 likely deaminated to either 5-dsi or 8-dsi . keeping this limitation in mind ,
the oxidation of dg by riboflavin initially 
 yields dg that rapidly 
 deprotonates to dg in 
 the nucleoside contexts ( pka  
 3.9 ) , dominating the product - forming 
 step of the reaction .
in contrast , dg in the dsodn context retains more cationic character because 
 the acidic proton is h - bonded with 2-deoxycytosine in a base 
 pair ; therefore , the product - forming steps of the reaction are determined 
 by the radical cation intermediate . as shown in scheme 2 , dg could couple 
 with radicals ( e.g. , o2 ) to 
 ultimately yield dz , while dg reacts with nucleophiles ( e.g. , h2o and nh3 ) at c8 , ultimately yielding a spirocyclic product 
 after completion of the four - electron oxidation .
thus , the yield of 8-dsi was anticipated to be greatest in the duplex context , 
 as indeed it is .
furthermore , these results support the original hypothesis 
 by steenken that the duplex context would favor the radical cation 
 nature of one - electron oxidized dg , which is still the focus of many current research efforts . because of the limitation imposed by the inability to detect the 
 bis - ammonium adduct , or to understand its decomposition pathway ,
any 
 comparison between the ammonia adduct distributions would be based 
 on poorly defined assumptions ; consequently , further discussion of 
 these results is not provided . for the last set of studies , 
 we were inspired by the observation 
 that the a ring of dsp mimics the h - bonding pattern of 
 thymidine , while the a ring of 5-dsi mimics the h - bonding 
 pattern of 2-deoxycytidine ( figure 6 ) . on the basis of this observation , polymerase insertion assays 
 opposite dsp vs 5-dsi
were conducted to 
 determine if these h - bonding schemes applied during the selection 
 of a nucleotide to insert opposite these spirocycles . comparison of h - bonding 
 patterns between dsp vs t and 5-dsi vs dc .
site - specific synthesis of dsp or 5-dsi was achieved by synthesizing dog into an odn by solid - phase 
 synthesis and then oxidizing this strand with or without nh4cl present with the one - electron oxidant na2ircl6 to furnish the desired products . upon ion - exchange hplc purification , 

the absolute stereochemical 
 assignments for the dsp isomers have been established 
 for the odns , and those for 5-dsi are assigned in this work to be analogous based on the mechanism 
 of deamination discussed above ( see figure s7 , supporting information , for more details ) .
standing start 
 polymerase studies were conducted while providing the enzyme only 
 one type of nucleotide per reaction . on the basis of previous studies , 
 insertion of dttp opposite dsp
was not observed ; therefore , to ensure that only one nucleotide 
 was inserted opposite the lesion , and no extension past the lesions 
 occurs , the sequence had an a placed 5 to the spirocycle ( figure 7 ) .
klenow fragment exo- was selected as the polymerase , 
 and after the extension reactions , polyacrylamide gel electrophoresis 
 ( page ) was conducted to determine the amount of datp , dttp , dgtp , 
 or dctp incorporated opposite the dsp or 5-dsi diastereomers .
the polymerase did not select either pyrimidine for 
 insertion opposite the two lesions ( figure s7 , supporting information ) ; thus , only data for purine insertion 
 opposite the lesions is provided in figure 7 . with respect to insertion
opposite the dsp diastereomers , ( r)-dsp was observed 
 to give a slight preference for insertion of datp opposite , and ( s)-dsp gave nearly 
 equal insertion of datp and dgtp . in comparison to the dsp results
, the 5-dsi isomers showed that ( s)-5-dsi gives similar 
 amounts of datp and dgtp insertion , while ( r)-5-dsi yields a slight preference 
 for datp insertion .
unfortunately , these results do not support the 
 hypothesis that h - bonding in the a ring of the spirocycle is the key 
 parameter for nucleotide selection , but do show that stereochemistry 
 may be important for selecting a base pairing partner by this polymerase . sequence 
 studied for polymerase dntp insertion studies and the 
 percent dntp insertions opposite the diastereomers of dsp and 5-dsi .
( a ) sequence for the dsodn construct used 
 in the polymerase insertion assays and ( b ) comparison of the percent 
 purine dntp insertion opposite the lesions dsp and 5-dsi by the dna polymerase klenow fragment exo- .
the present studies mapped 
 the pathways and quantified the products 
 observed when dg was allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal , 
 as well as the one - electron oxidant na2ircl6 .
on the basis of the analysis of the products in the nucleoside 
 context , the major products were oxidant dependent .
the major product 
 for riboflavin was dz , rose bengal yielded the 5-dsi diastereomers , and na2ircl6 gave 
 the dsp diastereomers as the major products ( figure 3 ) .
further analyses determined that 5-dsi and 8-dsi decompose via an acid - catalyzed deamination pathway leading to dsp as an end product ( scheme 5 ) .
oxidations 
 of dg in the nucleoside , ss- and dsodn contexts with 
 photochemically activated riboflavin demonstrated three major context 
 effects : ( 1 ) the yield of dz was highest in the nucleoside 
 studies and decreased dramatically in dsodn contexts ; ( 2 ) when nh3 participated as the nucleophile , 5-dsi was obtained 
 in the highest yield in nucleoside studies , while the yield of 8-dsi was highest in dsodn contexts ; and ( 3 ) when h2o was the nucleophile in nucleoside reactions , dsp presented 
 in the highest yield , while in dsodn contexts dgh was 
 obtained in the highest yield ( figure 4 ) . furthermore , 
 a combination of mapping the decomposition pathways of the dsi compounds to dsp , in which the absolute configuration 
 has been assigned , allowed the determination of the absolute configurations 
 for the diastereomers of 5- and 8-dsi .
these studies provide fundamental chemical insight 
 into formation of amine adducts to dg and their stability . 

further , these studies insert a cautionary note to researchers who 
 purify their odns using ammonium salts and then conduct oxidation 
 reactions after purification .
ammonia is a better nucleophile and 
 out - competes water for the electrophilic intermediates derived from dg or dog oxidation , resulting in a new mass 
 of dg + 31 or dog + 15 , respectively .
a similar observation has been highlighted for 
 oxidations occurring in tris buffer that generate tris adducts to dg .
oxidations were conducted 
 with 2-deoxyguanosine ( dg ) at 1 mm concentration 
 in 75 mm napi buffer ( ph 7.4 ) and 22 c .
reactions 
 with 20 mm nh4cl and without this salt allowed product 
 profile comparisons .
the oxidants and specific reaction conditions 
 were achieved as follows : ( 1 ) riboflavin oxidations were initiated 
 by adding 200 m riboflavin while exposing the samples to 350 
 nm light for 3 h. ( 2 ) rose bengal oxidations were achieved by adding 
 100 m rose bengal and exposing these samples to 350 nm for 
 3 h. the light source for the riboflavin and rose bengal reactions 
 came from a sun lamp that was placed 7 cm above the reaction 
 eppendorf tubes .
the tube lids were left open to allow all wavelengths 
 of light to pass into the reaction samples .
( 3 ) the na2ircl6 oxidations were initiated by a bolus addition of 
 the oxidizing salt with a final concentration of 10 mm , after a 30 
 min reaction the samples were quenched with 50 mm edta ( ph 8) .
the 
 reaction products and their distributions were determined by a dual 
 hplc method following a previously reported set of protocols ( specific 
 details can be found in the supporting information file ) . the first rp - hplc run allowed 
 analysis of dog and 8-amino - dg ( both observed 
 in < 1% yield ) , while all other products eluted in the void volume 
 of this run .
the void volume was collected , dried , and reinjected 
 on a hypercarb hplc column to analyze the diastereomers of dgh , dsp , 5-dsi , and 8-dsi , as 
 well as the product dz , while monitoring their absorbance 
 at 240 nm . to determine product distributions the peak areas were 
 integrated and normalized by each compound s 240 nm ( values provided below ) .
esi - ms , and then each compound 
 was hplc purified for further structural analysis .
the dgh diastereomers were previously characterized by nmr , dz was also previously characterized by nmr , and the dsp diastereomers have 
 been characterized by x - ray crystallography .
structural characterization of 5-dsi and 8-dsi by nmr was not conducted because of the instability of these compounds 
 toward deaminating to dsp , and the lack of nonexchangeable 
 protons on the ring of either ammonia adducts makes them very challenging 
 for structural analysis .
furthermore , if deamination of either 5-dsi or 8-dsi to dsp occurs in 
 the nmr tube during analysis , the peaks observed would be similar 
 and challenging to interpret ; therefore , the best method for obtaining 
 structural data on the diastereomers of 5-dsi and 8-dsi is via the esi - ms / ms that was conducted 
 ( figure 2 ) , and provided satisfactory results 
 to determine their structures .
characterization for each molecule 
 is as follows , the mixture of the dgh diastereomers ( not 
 resolvable ) gave tr = 6 min ; lc  esi - ms m / z [ m + h ] calcd 274.3 , found 274.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c9h15n5o5na 296.0971 , found 296.0979 ; uv  vis 
 240 = 2400 l molcm.(r)-dsp : tr = 11 min ; lc  esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; 
 hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit .
184 , 156 , 141 , 114 , 113 , 99 , 86 , found 184 , 
 156 , 141 , 114 , 113 , 99 , and 86 ; uv  vis 240 = 3,300 lmolcm ; cd  ( c 1.24  10 m in ddh2o ) nm 259 (  8.7 ) , 236 
 ( + 35.1 ) , and 211 ( 37.4 ) .
( s)-dsp : tr = 18 
 min ; lc  esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 
 322.0761 ; esi - ms / ms m / z [ m + h ] lit .
184 , 156 , 
 141 , 114 , 113 , 99 , 86 , found 184 , 156 , 141 , 114 , 113 , 99 , and 86 ; 
 uv  vis 240 = 3300 lmol cm ; cd  
 ( c 1.30  10 m in ddh2o ) nm 258 (  + 8.7 ) , 234 ( 32.9 ) , and 212 
 ( + 40.5 ) .
( r)-5dsi : tr = 9 min ; lc  esi - ms m / z [ m + h ] calcd 
 299.3 , found 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0920 ; esi - ms / ms m / z [ m + h ] found values 183 , 
 166 , 140 , 123 , 113 , 96 , and 86 ; uv  vis 240 = 3800 lmolcm ; cd  ( c 1.50  10 m in ddh2o ) nm 258 (  8.2 ) , 242 ( + 20.8 ) , and 216 ( + 61.8 ) .
( s)-5dsi : tr = 12 min ; lc - esi - ms m / z [ m + h ] calcd 299.3 , found 299.3 ; 
 hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0916 ; esi - ms / ms m / z [ m + h ] found 183 , 166 , 
 140 , 123 , 113 , 96 , and 86 ; uv  vis 240 = 3800 
 lmolcm ; cd  
 ( c 1.45  10 m in ddh2o ) nm 258 (  + 8.2 ) , 242 ( 20.1 ) , and 216 
 ( 62.2 ) .
( r)-8dsi : tr = 7 min ; lc  esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0924 ; esi - ms / ms m / z [ m + h ] found 
 183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv  vis 240 = 3500 lmolcm ; cd  ( c 1.30  10 m in ddh2o ) nm 261 (  + 20.0 ) , 237 ( 43.2 ) , 
 and 203 ( + 18.6 ) .
( s)-8dsi : tr = 14 min ; lc  esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0926 ; esi - ms / ms m / z [ m + h ] found 
 183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv  vis 240 = 3500 lmolcm ; cd  ( c 1.24  10 m in ddh2o ) nm 262 (  13.7 ) , 239 ( + 46.0 ) , and 201 ( 33.1 ) .
dz : tr = 27 min ; lc  esi - ms m / z [ m + h ] calcd 247.3 , found 247.3 ; hrms ( esi - tof ) m / z [ m + na ] for c8h14n4o5na calcd 269.0862 , found 269.0870 ; esi - ms / ms m / z [ m + h ] lit .
131 and 117 , found 131 and 117 ; uv  vis 
 240 = 1800 lmolcm .
the odns were hplc purified 
 using an ion - exchange hplc column , and the purification salts ( naoac ) 
 were removed by dialysis prior to their oxidation following previously 
 reported methods .
the riboflavin oxidations 
 were conducted similarly to those reported for the nucleoside studies , 
 with the following exceptions : the ssodn oxidations were conducted 
 on 20 m samples while the dsodn oxidations were conducted on 
 10 m samples . in addition , the reaction times were decreased 
 for the odn reactions to 30 min .
after the oxidations , the odns were 
 digested with a suite of nucleases and phosphatases to nucleoside 
 samples following a previously established protocol , with the exception that all buffers during the digestion 
 process were comprised of ammonium salts .
next , the digested mixture 
 was analyzed by the hplc method used in the nucleoside studies .
the polymerase 
 insertion assays were conducted on duplex odn samples that had site 
 specific incorporation of dsp or 5-dsi in 
 the template strand .
the site - specific synthesis was commenced on 
 odns that had a dog phosphoramidite synthesized at the 
 desired site of modification within the sequence 5-cgt tax ggc gca act gga aa-3 where x = dog .
the modifications 
 were synthesized by taking 1 nmol of the dog - containing 
 odn and placing it in 100 l of reaction buffer ( 75 mm napi , ph 7.4 ) with or without 2 mm nh4cl . the reaction 
 without nh4cl gave the dsp diastereomers and 
 the reaction with nh4cl gave the 5-dsi diastereomers . 

the individual diastereomers were purified using an ion - exchange hplc 
 column running naoac as the resolving salt and characterized via digestion 
 of the odn to nucleosides followed by hplc analysis ( purification 
 details can be found in the supporting information ) .
the primer template duplex 
 for the insertion studies was made by annealing 125 nm primer ( 5-tt 
 tcc agt tgc gcc-3 ) with 156 nm lesion - containing template 
 ( 5-cgt tax ggc gca act 
 gga aa-3 where x = ( r)-dsp , ( s)-dsp , ( r)-5-dsi , or ( s)-5-dsi to obtain 
 100 nm duplex in klenow fragment exo- buffer ( 50 mm tris , 50 mm nacl , 
 5 mm mgcl2 , 1 mm dtt at ph 8) . to a 25 l reaction 
 20 l of the annealed duplex was added , 1 l of klenow 
 fragment exo- ( 0.2 units/l ) , 0.5 l of dntp ( 500 m 
 stock solution ) , and 8.5 l of klenow buffer to obtain a 100 
 nm duplex solution with 10 m dntp and 0.2 u of polymerase . 

the reaction was incubated at 
 37 c for 30 min , after which loading dye ( 95% dmf plus 0.025% 
 bromophenol blue , and 0.025% xylene cylanol ) was added to the samples 
 and they were heated at 95 c for 20 min to quench the reaction 
 and denature the dna 
the denatured samples 
 were loaded on a 20% page gel and electrophoresed for 2 h at 45 w. 
 upon completion of the electrophoresis , the gel was placed in a phosphor 
 screen overnight and imaged by storage phosphor autoradiography .
 upon oxidation of the heterocyclic 
 ring in 2-deoxyguanosine 
 ( dg ) , the initial electrophilic intermediate displays 
 a wide range of reactivities with nucleophiles leading to many downstream 
 products . in the present study , 
 the product profiles were mapped when 
 aqueous solutions of dg were allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose 
 bengal as well as the diffusible one - electron oxidant na2ircl6 . 
 product characterization identified the 2-deoxyribonucleosides 
 of spiroiminodihydantoin , 5-guanidinohydantoin , and oxazolone resulting 
 from h2o as the nucleophile . 
 when nh3 was the 
 nucleophile , a set of constitutional isomers that are diastereotopic 
 were also observed , giving characteristic masses of dg + 31 . 
 esi+-ms / ms of these nh3 adducts identified 
 them to be spirocycles with substitution of either the c5 or c8 carbonyl 
 with an amine . 
 the nh3 adducts exhibit acid - catalyzed hydrolysis 
 to spiroiminodihydantoin . 
 quantification of the nh3 and 
 h2o adducts resulting from oxidation of dg in the nucleoside , single - stranded , and duplex oligodeoxynucleotide 
 contexts were monitored allowing mechanisms for product formation 
 to be proposed . 
 these data also provide a cautionary note to those 
 who purify their oligonucleotide samples with ammonium salts before 
 oxidation because this will lead to unwanted side reactions in which 
 ammonia participates in product formation . 
Introduction
Results and Discussion
Conclusion
Experimental
Section