nitroarenes are widespread environmental pollutants found in cigarette smoke , coal fly ash , exhausts from diesel and gasoline engines , and air particulate matter .
3-nitrobenzanthrone ( 3nba , 3-nitro-7h - benz[de]anthracen-7-one ) , a member of this class of compounds , is one of the most potent bacterial mutagens known to date   and exhibits strong mutagenic activity in mammalian cells .
human fibroblasts exposed to n - acetoxy-3-aminobenzanthrone , an active metabolite of 3nba , show predominance of base substitution mutations mainly g  t transversions followed by g  a and a  g substitutions .
transgenic mice treated with 3nba exhibit increased mutation frequency in liver , colon , bladder , and bone marrow , with g  t transversions dominating this effect [ 5 , 6 ] .
analysis of 3nba - induced mutations in mouse fibroblasts harboring a functional copy of the human p53 gene also reveals a preponderance of g  t transversions , followed by adenine base substitutions .
in addition to mutations , 3nba causes dna double - strand breaks and induces the formation of micronuclei in human cell lines [ 8 , 9 ] .
female f344 rats treated with 3nba by intratracheal instillation show acute and subacute changes in their lungs just few weeks after treatment and develop squamous cell lung tumors within 9 to 12 months , depending on the amount of 3nba administered .
some epidemiological evidence links environmental 3nba exposure to lung cancer in humans [ 11 , 12 ] .   like other environmental nitroarenes
, 3nba undergoes metabolic transformation to reactive electrophilic intermediates before binding to dna and exerting its genotoxic effects . in rats , mice and humans , cytosolic nad(p)h : quinone oxidoreductase ( nqo1 ) is the main enzyme that reduces 3nba to n - hydroxy-3-aminobenzanthrone ( n - oh-3-aba ) , which after further activation by phase ii enzymes ( n-,o - acetyl transferases , sulfotransferases ) can react with dna forming purine adducts ( figure 1 ) [ 13 , 14 ] .
nadph : cytochrome p450 reductase can also activate 3nba to reactive species in human cells , but its involvement seems to occur to a minor extent than that of nqo1 . a fully reduced 3nba metabolite , namely , 3-aminobenzanthrone ( 3aba ) , has been recovered from urine of humans occupationally exposed to diesel exhaust .
3aba can undergo cyp1a1-mediated ( or cyp1a2- ) oxidation to the same n - hydroxyl intermediate generated by the reductive metabolism , indicating a further mechanism for the activation and the genotoxicity of 3nba [ 17 , 18 ] .
in addition , because 3nba induces increased levels of nqo1 and cyp1a1 proteins in rat lung and kidney tissues , its genotoxicity may be enhanced by autoinduction of its metabolic pathway .   
the reactive metabolite of 3nba , n - oh-3-aba , can react with dna in vitro and in vivo forming purine adducts .
up to five adducts are detected in rodent samples utilizing the tlc - p - postlabeling techniques with enrichment by butanol extraction [ 5 , 1921 ] .
hplc comparison of these spots with synthetically prepared adduct standards   showed that 2-(2-deoxyguanosin - n -yl)-3-aminobenzanthrone ( dg(n ) -3aba ) , n-(2-deoxyguanosin-8-yl)-3-aminobenzanthrone ( dg(c8)-aba ) , and 2-(2-deoxyadenosin - n -yl)-3-aminobenzanthrone   ( da(n ) -3aba ) are the mayor adducts formed in rats organs after 3nba administration .
although the synthesis of the above - mentioned 3aba adducts and their corresponding n - acetyl forms has been reported , their incorporation in oligodeoxyribonucleotides has not been reportted . to understand the molecular mechanisms of 3nba mutagenesis as well as the repair of 3nba - damaged dna , a procedure for the site - specific incorporation 3aba lesions in oligomeric
, we present a synthetic scheme that allows the preparation of oligomeric dna containing the dg(n ) -3aba adduct ( figure 1 ) , one of the major dna lesions generated by nba in vivo .
the synthetic approach is robust permitting the incorporation of the adduct in any sequence context .
we also present results showing that the presence of   dg(n ) -3aba increases the stability of the damaged duplex , a finding previously observed with other dg(n ) lesions , including the n -deoxyguanosine derivative of aaf , 2-(2-deoxyguanosin - n -yl)-2-acetylaminofluorene .
interestingly , both of these kinds of damage are characterized by an unusually long persistence in rat tissues [ 21 , 25 ] , suggesting that lack of thermodynamic destabilization by bulky lesions is a factor that hinders the recognition of the lesions by the nucleotide excision repair ( ner ) pathway .
all general chemicals were purchased from sigma - aldrich ( st . louis , mo ) , and solid - phase dna synthesis reagents from glen research co. ( sterling , va ) .
s - benzyl thiotetrazole was crystallized from toluene and dried in vacuo over p2o5 before use .
electrospray ionization - mass spectrometry ( esi - ms ) and tandem ms ( ms / ms ) experiments were carried out on tsq quantum access ( thermo scientific ) and ltq orbitrap xl ( thermo - fisher scientific ) spectrometers . a 50  :  50  :  1  v / v mixture of acetonitrile  :  water  :  triethylamine was used as the solvent for electrospray ms determinations .
high - resolution mass spectra ( hrms ) were acquired on the ltq orbitrap xl ( thermo - fisher scientific ) equipped with an electrospray ionization ( esi ) source .
the spray voltage was set to 3.0  kv and the temperature of the heated capillary was maintained at 300c .
measurements were done in a positive ion mode with an injection time of 200  ms and an infusion rate of 3  ul / min in 90% acetonitrile / water .
duplex denaturation profiles were recorded on a cary 100 bio uv - vis spectrophotometer ( varian , inc . ,
dna duplexes were dissolved in 25  mm sodium phosphate buffer , ph 6.8 , containing 150  mm nacl and 500  m edta .
determinations proceeded while heating and cooling the sample , using a temperature change rate of 0.2c / min .
thermodynamic parameters were obtained from the analysis of 1/tm versus ln(ct ) plots ( van't hoff plots ) , whose slope and intercept yield h and s , respectively .  

2-(3,5-bis - o - tert - butyldimethylsilyl - o-2-(4-nitrophenylethyl)-2-deoxyguanosin - n -yl)-3-nitrobenzanthrone ( 1)it was synthesized according to a previously published procedure .
the final material was purified by crystallization from methanol , instead of the reported column chromatography method .
the h - nmr spectrum of 1 was essentially identical to that previously published . 
 
the final material was purified by crystallization from methanol , instead of the reported column chromatography method .
the h - nmr spectrum of 1 was essentially identical to that previously published . 

2-(o-2-(4-nitrophenylethyl)-2-deoxyguanosin - n -yl)-3-nitrobenzanthrone ( 3)silylated nucleoside 1 ( 255  mg , 0.25  mmol ) was dissolved in 3  ml of a dimethylformamide  :  triethylamine trihydrofluoride  :  triethylamine mixture ( 6  :  4  :  3 ) and heated at 50c for 1 hour .
the mixture was poured into 50  ml of ethyl acetate , centrifuged , the residue rinsed with ethyl acetate , and dried in vacuo over p2o5 , yielding 131  mg ( 0.19  mmol , 78% ) of 3 as a fine red powder . for nmr spectroscopy ,
h - nmr ( dmso - d6 , tms ,  ( p.p.m . ) ) : 9.87 ( br .
s. , 1h ) , 9.20 ( s. 1h ) , 8.55 ( d , 1h ) , 8.49 ( d , 1h ) , 8.32 ( s , 1h ) , 8.28 ( m , 2  h ) , 7.99 ( t , 1h ) , 7.92 ( d , 2  h , j = 8.0  hz ) , 7.83 ( t , 1h ) , 7.66 ( t , 1h ) , 7.37 ( d , 2  h , j = 8.0  hz ) , 6.29 ( t , 1h , j = 6.0  hz ) , 5.21 ( br s , 1h ) , 4.83 ( br s , 1h ) , 4.66 ( br s , 2  h ) , 4.31 ( br s , 1h ) , 3.81 ( br s , 1h)3.50 - 3.46 ( m , 3  h ) , 2.632.52 ( m , 2  h ) ( supplementary material available at doi : 10.4061/2011/521035 ) .
c - nmr ( dmso - d6 , tms ,  ( p.p.m . ) ) : 128.23 , 159.00 , 154.67 , 153.08 , 146.17 , 146.08 , 139.78 , 137.86 , 134.34 , 133.98 , 132.60 , 130.27 , 129.97 , 129.56 , 128.20 , 127.58 , 127.32 , 124.99 , 124.55 , 123.82 , 123.17 , 123.05 , 116.37 , 87.84 , 83.10 , 70.66 , 66.07 , 61.56 , 45.60 , 34.13 ( supplementary material ) .
silylated nucleoside 1 ( 255  mg , 0.25  mmol ) was dissolved in 3  ml of a dimethylformamide  :  triethylamine trihydrofluoride  :  triethylamine mixture ( 6  :  4  :  3 ) and heated at 50c for 1 hour .
the mixture was poured into 50  ml of ethyl acetate , centrifuged , the residue rinsed with ethyl acetate , and dried in vacuo over p2o5 , yielding 131  mg ( 0.19  mmol , 78% ) of 3 as a fine red powder . for nmr spectroscopy ,
h - nmr ( dmso - d6 , tms ,  ( p.p.m . ) ) : 9.87 ( br .
s. , 1h ) , 9.20 ( s. 1h ) , 8.55 ( d , 1h ) , 8.49 ( d , 1h ) , 8.32 ( s , 1h ) , 8.28 ( m , 2  h ) , 7.99 ( t , 1h ) , 7.92 ( d , 2  h , j = 8.0  hz ) , 7.83 ( t , 1h ) , 7.66 ( t , 1h ) , 7.37 ( d , 2  h , j = 8.0  hz ) , 6.29 ( t , 1h , j = 6.0  hz ) , 5.21 ( br s , 1h ) , 4.83 ( br s , 1h ) , 4.66 ( br s , 2  h ) , 4.31 ( br s , 1h ) , 3.81 ( br s , 1h)3.50 - 3.46 ( m , 3  h ) , 2.632.52 ( m , 2  h ) ( supplementary material available at doi : 10.4061/2011/521035 ) .
c - nmr ( dmso - d6 , tms ,  ( p.p.m . ) ) : 128.23 , 159.00 , 154.67 , 153.08 , 146.17 , 146.08 , 139.78 , 137.86 , 134.34 , 133.98 , 132.60 , 130.27 , 129.97 , 129.56 , 128.20 , 127.58 , 127.32 , 124.99 , 124.55 , 123.82 , 123.17 , 123.05 , 116.37 , 87.84 , 83.10 , 70.66 , 66.07 , 61.56 , 45.60 , 34.13 ( supplementary material ) .
2-(5-o - dimethoxytriphenylmethyl - o-2-(4-nitrophenylethyl)-2-deoxyguanosin - n -yl)-3-nitrobenzanthrone ( 4)nucleoside 3 ( 120  mg , 0.17  mmol ) was coevaporated with pyridine , redissolved in 2  ml of dry pyridine , mixed with 120  mg of dimethoxytrityl chloride ( 0.35  mmol ) , and left for 12 hour at room temperature .
after disappearance of the starting material ( estimated by tlc analysis ) , the reaction mixture was diluted with 20  ml of ethyl acetate , extracted with 10% sodium citrate ( 3  100  ml ) , dried with sodium sulfate , and evaporated in vacuo .
flash chromatographic purification of the residue ( 5% triethylamine in methylene chloride ) yielded 123  mg ( 0.125  mmol , 73% ) of 4 as a deep red transparent glassy solid .
h - nmr ( cdcl3 , tms ,  ( p.p.m . ) ) : 9.93 ( s , 1h ) , 9.21 ( s , 1h ) , 8.68 ( d , 1h , j = 6.5  hz ) , 8.50 ( d , 1h , j = 6.9  hz ) , 8.49 ( d , 1h , j = 8.7  hz ) , 8.39 ( d , 1h , j = 8.7  hz ) , 8.12 ( d , 2  h , j = 8.3  hz ) , 7.98 ( s , 1h ) , 7.90 ( t , 1h , j = 7.8  hz ) , 7.8 ( t , 1h , j = 7.5  hz ) , 7.63 ( t , 1h , j = 7.0  hz ) , 7.47 ( d , 2  h , j = 8.0  hz ) , 7.38 ( d , 2  h , j = 7.2  hz ) , 7.287.26 ( m ) , 6.78 ( d , 4  h , j = 8.2  hz ) , 6.58 ( t , 1h , j = 6.1hz ) , 4.83 ( t , 2  h , j = 7.2  hz ) , 4.67 ( m , 1h ) , 4.21 ( m , 1h ) , 3.76 ( s , 6  h ) , 3.44 ( m , 1h ) , 3.353.30 ( m , 3  h ) , 2.85 ( br s , 1h ) , 2.812.79 ( m , 1h ) , 2.652.64 ( m , 1h ) ( supplementary material ) .
c - nmr ( cdcl3 , tms ,  ( p.p.m . ) ) : 183.2 , 160.8 , 158.8 , 154.0 , 153.5 , 152.6 , 150.4 , 147.0 , 145.8 , 144.6 , 139.0 , 135.8 , 135.7 , 134.3 , 134.2 , 133.2 , 131.2 , 130.7 , 130.3 , 130.2 , 130.1 , 129.3 , 129.1 , 128.3 , 128.2 , 128.0 , 127.9 , 127.6 , 127.2 , 125.9 , 123.9 , 123.2 , 117.6 , 116.2 , 115.0 , 114.5 , 113.5 , 113.4 , 87.0 , 86.9 , 83.9 , 72.9 , 68.1 , 67.2 , 56.1 , 56.0 , 55.4 , 52.9 , 46.0 , 32.2 , 25.7 ( supplementary material ) .
nucleoside 3 ( 120  mg , 0.17  mmol ) was coevaporated with pyridine , redissolved in 2 
ml of dry pyridine , mixed with 120  mg of dimethoxytrityl chloride ( 0.35  mmol ) , and left for 12 hour at room temperature .
after disappearance of the starting material ( estimated by tlc analysis ) , the reaction mixture was diluted with 20  ml of ethyl acetate , extracted with 10% sodium citrate ( 3  100  ml ) , dried with sodium sulfate , and evaporated in vacuo .
flash chromatographic purification of the residue ( 5% triethylamine in methylene chloride ) yielded 123  mg ( 0.125  mmol , 73% ) of 4 as a deep red transparent glassy solid .
h - nmr ( cdcl3 , tms ,  ( p.p.m . ) ) : 9.93 ( s , 1h ) , 9.21 ( s , 1h ) , 8.68 ( d , 1h , j = 6.5  hz ) , 8.50 ( d , 1h , j = 6.9  hz ) , 8.49 ( d , 1h , j = 8.7  hz ) , 8.39 ( d , 1h , j = 8.7  hz ) , 8.12 ( d , 2  h , j = 8.3  hz ) , 7.98 ( s , 1h ) , 7.90 ( t , 1h , j = 7.8  hz ) , 7.8 ( t , 1h , j = 7.5  hz ) , 7.63 ( t , 1h , j = 7.0  hz ) , 7.47 ( d , 2  h , j = 8.0  hz ) , 7.38 ( d , 2  h , j = 7.2  hz ) , 7.287.26 ( m ) , 6.78 ( d , 4  h , j = 8.2  hz ) , 6.58 ( t , 1h , j = 6.1hz ) , 4.83 ( t , 2  h , j = 7.2  hz ) , 4.67 ( m , 1h ) , 4.21 ( m , 1h ) , 3.76 ( s , 6  h ) , 3.44 ( m , 1h ) , 3.353.30 ( m , 3  h ) , 2.85 ( br s , 1h ) , 2.812.79 ( m , 1h ) , 2.652.64 ( m , 1h ) ( supplementary material ) .
c - nmr ( cdcl3 , tms ,  ( p.p.m . ) ) : 183.2 , 160.8 , 158.8 , 154.0 , 153.5 , 152.6 , 150.4 , 147.0 , 145.8 , 144.6 , 139.0 , 135.8 , 135.7 , 134.3 , 134.2 , 133.2 , 131.2 , 130.7 , 130.3 , 130.2 , 130.1 , 129.3 , 129.1 , 128.3 , 128.2 , 128.0 , 127.9 , 127.6 , 127.2 , 125.9 , 123.9 , 123.2 , 117.6 , 116.2 , 115.0 , 114.5 , 113.5 , 113.4 , 87.0 , 86.9 , 83.9 , 72.9 , 68.1 , 67.2 , 56.1 , 56.0 , 55.4 , 52.9 , 46.0 , 32.2 , 25.7 ( supplementary material ) . hrms : observed [ m + h ] 992.3227 , calculated 992.3250 . 

2-(3-o-(cyanoethoxydiisopropylaminophosphino)-5-o - dimethoxytriphenylmethyl - o-2-(4-nitrophenylethyl)-2-deoxyguanosin - n -yl)-3-nitrobenzanthrone ( 5)nucleoside 4 ( 112  mg , 0.11  mmol ) was coevaporated with toluene , dissolved in 2  ml of dry thf under argon , mixed with 200  l of diisopropyl ethylamine , followed by the addition of 104  l ( 0.37  mmol ) of 2-cyanoethoxy diisopropyl chlorophosphoramidite .
after disappearance of the starting material ( assayed by tlc analysis ) , the mixture was dissolved in 30  ml of ethyl acetate and washed with 10% potassium carbonate ( 2  50  ml ) . the organic layer was dried with sodium sulfate and evaporated in vacuo .
the resulting viscous oil was purified by reverse - phase chromatography using an acetonitrile - water gradient ( 60100% , over 15  min ) as the mobile phase , yielding 5 ( 98  mg , 0.082  mmol , 74% ) as a deep - red colored transparent glassy solid . the h - nmr spectrum had a double set of signals , of 5545 intensity ratio , which corresponded to the diastereomers related to the phosphorus atom .
h - nmr ( cdcl3 , tms ,  ( p.p.m . ) , chemical shifts for the major component are ( given when possibe ) 9.79 ( s , 1h ) , 9.82 ( s , 1h ) , 8.71 ( d , 1h ) , 8.52 ( d , 1h ) , 8.50 ( d , 1h ) , 8.37 ( t , 1h ) , 8.10 ( t , 1h ) , 8.00 ( s , 1h ) , 7.92 ( t , 1h ) , 7.73 ( t , 1h ) , 7.63 ( t , 1h ) , 7.437.39 ( m , 4  h ) , 7.297.17 ( m ) ,   7.19 ( t , 1h ) , 7.90 ( d , 2  h ) , 7.60 ( d , 2  h ) , 6,50 ( m , 1h ) , 4.82 ( br t , 2  h ) , 4.72 ( m , 1h ) , 4.28 ( m , 1h ) ,   3.76 ( s , 3  h ) , 3.75 ( t , 3  h ) , 3.663.43 ( m , 4  h ) , 3.443.35 ( m , 2  h ) , 3.30 ( br t , 2  h ) , 2.86 ( sept , 1h ) , 2.71 ( m , 1h ) , 2.45 ( m , 1h ) , 1.53 ( m , 12  h ) ( supplementary material ) . hrms : observed [ m + h ] 1192.4365 , calculated 1192.4328 . 
 
nucleoside 4 ( 112  mg , 0.11  mmol ) was coevaporated with toluene , dissolved in 2  ml of dry thf under argon , mixed with 200  l of diisopropyl ethylamine , followed by the addition of 104  l ( 0.37  mmol ) of 2-cyanoethoxy diisopropyl chlorophosphoramidite .
after disappearance of the starting material ( assayed by tlc analysis ) , the mixture was dissolved in 30  ml of ethyl acetate and washed with 10% potassium carbonate ( 2  50  ml ) . the organic layer was dried with sodium sulfate and evaporated in vacuo .
the resulting viscous oil was purified by reverse - phase chromatography using an acetonitrile - water gradient ( 60100% , over 15  min ) as the mobile phase , yielding 5 ( 98  mg , 0.082  mmol , 74% ) as a deep - red colored transparent glassy solid . the h - nmr spectrum had a double set of signals , of 5545 intensity ratio , which corresponded to the diastereomers related to the phosphorus atom .
h - nmr ( cdcl3 , tms ,  ( p.p.m . ) , chemical shifts for the major component are ( given when possibe ) 9.79 ( s , 1h ) , 9.82 ( s , 1h ) , 8.71 ( d , 1h ) , 8.52 ( d , 1h ) , 8.50 ( d , 1h ) , 8.37 ( t , 1h ) , 8.10 ( t , 1h ) , 8.00 ( s , 1h ) , 7.92 ( t , 1h ) , 7.73 ( t , 1h ) , 7.63 ( t , 1h ) , 7.437.39 ( m , 4  h ) , 7.297.17 ( m ) ,   7.19 ( t , 1h ) , 7.90 ( d , 2  h ) , 7.60 ( d , 2  h ) , 6,50 ( m , 1h ) , 4.82 ( br t , 2  h ) , 4.72 ( m , 1h ) , 4.28 ( m , 1h ) ,   3.76 ( s , 3  h ) , 3.75 ( t , 3  h ) , 3.663.43 ( m , 4  h ) , 3.443.35 ( m , 2  h ) , 3.30 ( br t , 2  h ) , 2.86 ( sept , 1h ) , 2.71 ( m , 1h ) , 2.45 ( m , 1h ) , 1.53 ( m , 12  h ) ( supplementary material ) . hrms : observed [ m + h ] 1192.4365 , calculated 1192.4328 .
it was performed on an abi dna synthesizer using ultramild ce phosphoramidites and s - benzylthio - tetrazole as the activator reagent .
a four - fold molar excess of amidite was used and the coupling time was increased to 5 minutes for introduction of the xenonucleotide moiety .
oligodeoxyribonucleotide cleavage from the solid support and subsequent deprotection was accomplished by overnight incubation with concentrated aqueous ammonia at room temperature .
after evaporation in vacuo , the synthetic products were purified by reverse - phase chromatography .  
purification was performed on a gilson hplc system with uv monitoring at 260 and 290  nm , a 7.5  250  mm zorbax ods column ( 5  m particle size , 300   pore size ) , using a linear acetonitrile gradient ( 5 to 50% , 30  min ) in 50  mm triethylammonium acetate , ph 6.5 , at flow rate of 5  ml / min .
purified oligodeoxyribonucleotides were lyophilized and the 5-dmt group was removed by co - evaporation with an 80% acetic acid solution .
the residue was dissolved in 2  ml 1  m tetrabutylammonium chloride , purified by hplc using the protocol described above , and lyophilized to dryness .
the resulting product was dissolved in water ( 1  ml ) and 100  l 1  m sodium sulfide was added .
the immediate color transition from an orange - red to a deep purple attested that the reduction of nitro group had taken place . after standing for 1  hr at room temperature
, the reaction mixture was purified by hplc yielding the desired oligodeoxyribonucleotide product , which was lyophilized , precipitated from ethanol in sodium form and subsequently desalted using a sephadex g-10 column .
one- and two - dimensional h nmr spectra were recorded using varian ( inova ) or bruker ( avance ) spectrometers operating at 9.4 , 11.75 , or 14.1  t field strengths .  
proton chemical shifts were referenced relative to tetramethyl silane ( for organic solvents ) or sodium 3-(trimethylsilyl ) propionate-2,2,3,3-d4 ( for water solutions ) at 0  ppm .
phase - sensitive noesy ( 300  ms mixing times ) and dqf - cosy spectra of the damaged duplex were collected at 25c with the sample dissolved in 50  mm phosphate buffer solution ( ph 6.9 ) in 100% d2o .
the residual water signal was suppressed by presaturation during the repetition delay of 1.5  s. nmr data were processed and analyzed using nmrpipe   and sparky , running in linux workstations .
datasets consisted of 4096 and 256 complex data points in the t2 and t1 dimensions , respectively .
time domain data were multiplied by a shifted sine - bell window function and zero filled to 4096 points .
a general synthetic method for the introduction of modified dg moieties having aminoaryl or n - acetylaminoaryl substitutions ( xenonucleotides ) is the use of the 2-(n - trifluoroacetylaminoaryl)-deoxyguanosine phosphoramidite during conventional phosphoramidite solid phase synthesis . in this approach
, the key nitroarene precursor is obtained via a palladium - mediated condensation of protected 2-deoxyguanosine with the corresponding bromo nitroarene , followed by deprotection , nitro - reduction and trifluoroacetylation . despite its simplicity and versatility , this method showed some drawbacks in the dg(n ) -3aba case .
first , the direct reduction of the nitro group and ensuing trifluoroacetylation lead to a complex mixture of products , a complication that , in principle , can be prevented by using additional deprotection and protection steps .
second , the use of trifluoroacetyl protection imposes some restrictions on the conditions of solid phase synthesis and oligodeoxyribonucleotide deprotection that reduced final yields . to overcome these problems
, we decided to introduce dg(n ) -3nba directly during oligodeoxyribonucleotide synthesis and generate the amino group of the adduct by a postsynthesis treatment . using standard procedures [ 23 , 30 ] , we readily prepare the protected dg(n ) -3nba nucleoside ( 1 ) .
treatment of 1 with tbaf removes the oxygen protecting groups ( tbdms and p - no2-phenetyl ( npe ) ) , yielding the fully deprotected adduct ( 2 ) ( scheme 1 ) .
this compound , however , produces a complex mixture of products during the subsequent synthesis steps ( data not shown ) , making it an unsuitable intermediate .
a possible explanation is that the unprotected dg(n ) -3nba ( 2 ) has poor solubility in both water and organic solvents , impeding its purification by standard treatments including liquid - liquid extraction and column chromatography .
in addition , this compound seems to exist in several protonation states , a fact easily seen by sharp color changes , from violet - purple to deep blue to deep green , upon transition to higher ph .
therefore , we decided to keep the guanine - o protecting group on during the phosphoramidite preparation and solid phase synthesis .
treatment of ( 1 ) with a tea3hf  :  tea  :  dmf mixture removes the tbdms protection leaving the npe group intact .
nucleoside ( 3 ) is then 5-dimethoxytritylated to give 4 which , in turn , is converted to the corresponding phosphoramidite ( 5 ) using the standard protocol ( scheme 1 ) .
we tested two possible approaches for the postsynthetic deprotection of the oligodeoxyribonucleotide and reduction of the nitro group .
the first one was the removal of the guanine - o protecting group by treatment of the synthesis product , still attached to the solid support , with 1  m dbu / dmf , followed by reduction of the nitro group with 1  m sodium dithionite / sodium citrate at ph 7.0 .
subsequent treatment with concentrated ammonia should remove the other protecting groups and cleave the oligodeoxyribonucleotide from the solid support .
however , during the ammonia treatment we observed considerable color changes , suggesting a partial degradation of the dg(n ) -3aba moiety and the final yield was unacceptably low for large - scale synthesis .
after solid phase synthesis , treatment with concentrated ammonia at room temperature cleaved the tritylated oligodeoxyribonucleotide from the solid support and removed the acyl and cyanoethyl protecting groups .
after solvent evaporation and purification by reverse phase hplc , esi - ms confirmed that the major product was the npe - protected dg(n ) -3nba containing oligomer ( 6 ) .
subsequent treatment with tbaf removed the npe protecting group and addition of 1  m na2s reduced the nitro group of dg(n ) -3nba to the corresponding amino of   dg(n ) -3aba ( 7 ) with excellent yields ( scheme 2 ) .
after purification of the reaction products by an additional hplc run , esi - ms analysis showed that the main product was the desired dg(n ) -3aba containing oligodeoxyribonucleotide ( supplementary material ) .
two - dimensional nmr spectroscopy confirmed the presence of 3aba in the purified sample and allowed the assignment of its aromatic protons ( figure 2 ) .
the aromatic region of an noesy   ( 300  ms mixing time ) spectrum in 100% d2o phosphate buffer , ph 6.9 , revealed a single set of reasonably sharp proton signals , indicating that the damaged duplex assumes a major stable conformation at room temperature ( figure 2(a ) ) .
noe interactions were particularly strong among 3aba protons , readily differentiating them from cross - peaks originated from the aromatic nucleotide protons of the duplex ( figure 2(b ) ) .
the identical region of a dqf - cosy spectrum was very simple since its diagonal only shows signals for protons that have scalar coupling , specifically cytosine - h6 and 3aba protons .
thus , the 3aba(h1 ) proton was absent on the dqf - cosy spectrum ( denoted by an arrow ) but exhibited a strong noe interaction with 3aba(h11 ) , which is very close in space across the bay region of 3aba ( figure 2 ) . from here , identification of the remaining 3aba protons was straightforward , with the exceptions of h4 and h6 whose specific assignments remained ambiguous .
figure 3 displays the expanded base - h1 sugar proton region of the same 300  ms mixing time noesy spectrum .
each base proton ( purine(h8 ) or pyrimidine(h6 ) ) of the duplex exhibited noe interactions to its own and the 5-flanking h1 sugar protons , indicating that the 3aba - damaged duplex adopts a right - handed helical conformation in solution .
the bulky aromatic moiety , however , did induce some perturbations of the ideal b - form conformation , as evidenced by the absence of the a11(h8)-a11(h1 ) intraresidue interaction ( figure 3 , empty box ) and the weak intensity detected for the a11(h8)-c10(h1 ) and a13(h8)-t12(h1 ) cross - peaks .
in addition , several proton signals exhibited unusual chemical shift values , including c6(h1 ) and a11(h1 ) , that resonate at 4.43 and 5.34  ppm , respectively , suggesting that the 3aba moiety resides in the duplex minor groove pointing towards the 5-end of the modified strand .
these chemical shift values are reminiscent of those observed on duplexes containing other bulky adducts positioned in minor groove , such as dg(n ) -aaf , a minor acetylaminofluorene lesion previously characterized by our laboratory , or the extensively studied trans dg(n ) -b[a]p adducts [ 31 , 32 ] .
analysis of uv melting curves shows that the melting temperature of the self - complementary dg(n ) -3aba duplex is about 7.5c higher than that of the unmodified control sample ( figure 4 ) .
van't hoff thermodynamic parameters for the damaged duplex indicate a large entropy increase that overcompensates for the 2.6  kcal / mol enthalpy increase , resulting in 3.0  kcal / mol reduction of gibbs 's free energy at 30c ( table 1 ) . due to duplex symmetry ,
there are two dg(n ) -3aba adducts per duplex molecule , which in principle , can contribute independently to gibbs 's free energy reduction or display additional interactions between themselves . since the 3aba duplex has four undamaged gc base pairs ( that showed regular noe interactions ) separating the damaged nucleotides , the aba moieties are too far apart ( 17   in b - form dna ) to expect any direct interaction between them .
therefore , the duplex stabilizing effect of a single dg(n ) -3aba lesion in the 5-txc sequence context is 1.5  kcal / mol at 30c .   notwithstanding
the absence of a refined three - dimensional model of the dg(n ) -3aba duplex , it is interesting to compare the stability impact of the different dg(n ) minor groove lesions .
our previous study of the dg(n ) -aaf - containing duplex revealed a considerable stabilization on the double helix by the acetylaminofluorene moiety .
the hydrophobic fluorene moiety resides deep in the minor groove of the duplex , displacing water molecules and exposing only 10% of its surface to solvent . as a result , a significant entropy increase more than compensates for the moderate enthalpy destabilization of the duplex , causing a 1.8  kcal / mol reduction of gibb 's free energy per lesion , somewhat larger that the effect observed here for the dg(n ) -3aba adduct .
similar to the aaf case , the dg(n ) -3aba lesion resides in the duplex minor groove and exhibits an almost identical topology along the bay region edge of the adduct ( figure 5 ) .  
it is natural to expect , however , that the 3aba moiety , being bulkier than the aaf residue , would extend beyond the boundaries of the minor groove , exposing a greater fraction of its hydrophobic ring system to water , thus diminishing the overall stabilizing effect relative to the former .
in addition , the 5 lesion - flanking base pair differs on the dg(n ) -aaf and dg(n ) -3aba duplexes and its contribution to the stability increase can be slightly different .
in contrast to the case of the aaf and 3aba lesions , the presence of dg(n ) -(trans)b[a]p reduces the thermodynamic stability of the damaged duplex .
a main structural difference in the latter case is the topology of the lesion ( figure 5 ) , most importantly the sp hybridization of the linkage carbon that results in the exposure of one face of the b[a]p moiety to solvent [ 31 , 32 ] .
in addition , differences on sequence context between the dg(n ) -3aba and dg(n ) -(trans)b[a]p lesion may also play a role in explaining the stability effects of these adducts .  
the dna stabilizing effect of dg(n ) -3aba is shared by only few bulky lesions , specifically the dg(n ) -aaf adduct , the dg(n7 ) and fapy - dg adducts of aflatoxin b1 [ 34 , 35 ] .
in contrast to aaf and aflatoxin b1 , 3-nitrobenzanthrone is an abundant environmental pollutant in developed countries and the properties of 3-nba generated lesions may help explain the incidence of random mutations and sporadic cancers .
the current models of the nucleotide excision repair ( ner ) pathway postulates that the recognition of bulky lesions is mediated by sensing the local instability [ 3638 ] or alteration of conformational mobility   induced by the adducts . in excellent agreement with these concepts , the increased thermodynamic stability of duplex dna containing dg(n ) -aaf adducts
recent studies have found that dg(n ) -3aba persists in rat dna for a long period of time after a single instillation of 3nba .
our observations that dg(n ) -3aba increases duplex stability and adopts a single conformation in solution point to a mechanistic explanation for its persistence in cellular dna .
however , a careful examination of the recognition of the dg(n ) -3aba damage by the ner system is needed before these ideas can be confirmed .
such investigations and the nmr structure determination of dg(n ) -3aba - containing dna are currently in progress .
the environmental pollutant 3-nitrobenzanthrone is one of the most potent bacterial mutagens and has strong mutagenic activity in mammalian cells .
after cellular activation to electrophilic intermediates , it reacts with dna forming purine adducts including the prevalent dg(n ) -3aba lesion .
we reported here an efficient synthetic method for incorporation of this lesion in oligomeric dna in any sequence context , opening the door to future site - specific mutagenesis and dna repair studies .
we also presented evidence showing that dg(n ) -3aba increases the stability of the damaged duplex , a finding previously observed with other dg(n ) lesions and that is associated with the long persistence of the adducts in rat tissues .
our results suggest that the lack of thermodynamic destabilization of dna is a key factor during the recognition of bulky the lesions by the ner pathway .
 3-nitrobenzanthrone ( 3nba ) , an environmental pollutant and potent mutagen , causes dna damage via the reaction of its metabolically activated form with the exocyclic amino groups of purines and the c-8 position of guanine . 
 the present work describes a synthetic approach to the preparation of oligomeric 2-deoxyribonucleotides containing a 2-(2-deoxyguanosin - n2-yl)-3-aminobenzanthrone moiety , one of the major dna adducts found in tissues of living organisms exposed to 3nba . 
 the nmr spectra indicate that the damaged oligodeoxyribonucleotide is capable of forming a regular double helical structure with the polyaromatic moiety assuming a single conformation at room temperature ; the spectra suggest that the 3aba moiety resides in the duplex minor groove pointing toward the 5-end of the modified strand . 
 thermodynamic studies show that the dg(n2)-3aba lesion has a stabilizing effect on the damaged duplex , a fact that correlates well with the long persistence of this damage in living organisms . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions