gemcitabine is a modified 
 cytidine analog having two fluorine atoms 
 at the 2-position in the deoxyribose sugar moiety ( scheme 1 ) . for nearly 20 years , it has been widely used 
 to treat specifically pancreatic cancer .
it has been proposed that gemcitabine inhibits 
 ribonucleotide reductase ( rnr ) activity as well as acting as 
 a replication stop , thereby affecting dna synthesis 
 and elongation .
the two highly electronegative 
 f - atoms at c2 substantially increase the acidity of h3 
 through the inductive effect .
it is well established 
 that in the absence of oxygen , thiyl radicals 
 are able to abstract hydrogen ( h ) atoms to form neutral c - centered 
 radicals .
for example , h - atom abstraction by thiyl radicals has been 
 shown to induce isomerization of cis-2,5-dimethyltetrahydrofuran 
 to the trans-2,5-dimethyltetrahydrofuran . on this basis , stubbe et al .
, in their enzymatic and electron 
 spin resonance ( esr ) studies with gemcitabine , have proposed that 
 inhibition of the rnr activity by gemcitabine should occur via radical 
 formation at the c3 site by direct h - atom abstraction to produce 
 a c3 via a enzymatic thiyl radical ( reaction 1 ) .
subsequently , the c3 intermediate 
 has been proposed to form 3-keto c2 via hf 
 loss .
this c2 is stabilized by an oxy radical resonance 
 contribution ( reaction 2 ) .
c2 
 and its immediate precursor c3 ( reactions 1 and 2 ) play a key role in 
 the rnr inactivation.12 the h - atom abstraction reaction ( reaction 1 ) is not only important in rnr activation but also 
 plays a very key 
 role toward stable product formation in other biologically damage 
 processes in dna , such as oxidative intrastrand cross - link formation and in the formation of sugar radicals that 
 are strand break precursors .
it is noteworthy that pulse radiolysis 
 experiments with a 1,4-anhydro-5-deoxy-6-thio - d - ribo - hexofuranitol 
 detected the formation of ribosyl - based carbon - centered radical(s ) 
 after h - atom abstraction by thiyl radicals .
these studies are supportive of reactions 1 and 2 but unequivocal , and direct detection 
 of c3 and c2 employing esr or pulse 
 radiolysis during rnr - catalyzed deoxygenation of the natural substrates or during inactivation by gemcitabine still remains elusive . in this work , we report the formation of c2 from 
 a likely c3 in a gemcitabine analog which mimics the 
 mechanism proposed above . from the structural formula of gemcitabine 
 ( scheme 1 )
, it is expected that the negative 
 inductive effect ( i ) of two highly electronegative f - atoms 
 at c2 should increase the acidity of h3. from our 
 previous work on nucleoside cation radicals , the gemcitabine cation radical formed upon one - electron oxidation 
 is expected to produce c3 after deprotonation of the 
 acidic proton h3. in this work , esr spectroscopy has been 
 employed to investigate one - electron oxidation of gemcitabine and 
 other 2-modified derivatives , for example , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ( psi-6130 ) ; scheme 2 ) and 2-fluoro-2-deoxycytidine ( 2-fdc , scheme 2 ) , in order to test the influence of 2- 
 substituent on radical site formation .
it is noteworthy that mefdc 
 is a well - known clinically efficacious inhibitor of hepatitis c virus .
the esr results clearly identify the c3 formation 
 in one - electron oxidized gemcitabine and the production of c2 
 in one - electron oxidized mefdc .
these calculations show 
 that in the case of one - electron oxidized mefdc , the lowest energy 
 path is the rapid formation of c2 from c3 
 via f loss .
this f loss is a 
 barrierless reaction between the 2-f - atom and the proximate 
 h3o which was formed via deprotonation of h3 
 in the cation radical .
gemcitabine ( scheme 1 ) and 2-fdc 
 ( scheme 2 ) were obtained 
 from carbosynth ltd .
mefdc ( scheme 2 ) was prepared as described or 
 purchased from adooq bioscience ( irvine , ca ) .
lithium chloride ( licl ) ( ultra 
 dry , 99.995% ( metals basis ) ) was 
 obtained from alfa aesar ( ward hill , ma , usa ) .
2-deoxycytidine 
 ( 2-dc ) was obtained from sigma chemical company ( st louis , 
 mo , usa ) .
deuterium oxide ( d2o ) ( 99.9 atom % d ) was purchased 
 from aldrich chemical co. inc .
( paris , ky , usa ) . cytidine-5,6-d2 ( [ 5,6-d , d]-cyd , 99 atom % d ) was purchased from cdn isotopes ( quebec , 
 canada ) .
homogeneous solutions 
 of gemcitabine were prepared by dissolving 210 mg / ml either 
 in 7.5 m licl in d2o or in h2o .
solutions of 
 other compounds ( 2-dc , 2-f - dc , mefdc , and [ 5,6-d , d]-cyd ) 
 were prepared by dissolving ca .
k2s2o8 ( 616 
 mg / ml ) was added as an electron scavenger so that only the formation 
 of the one - electron oxidized species and its subsequent reactions 
 can be followed by employing esr spectroscopy .
the above - mentioned 
 procedure for preparation of solutions is according to our ongoing 
 studies on various model systems of dna and rna .
the ph of gemcitabine in 7.5 m licl / d2o was adjusted to the range of ca .
the ph of gemcitabine in 7.5 m licl / h2o 
 and the ph of other compounds ( 2-dc ,
2-f - dc , mefdc , 
 and [ 5,6-d , d]-cyd ) in 7.5 m licl / d2o was adjusted at ph 
 ca . 10 .
these ph adjustments were performed by adding l amounts 
 1 m naoh as per our previous efforts .
these solutions have high ionic strength ( 7.5 m licl ) ; therefore , 
 the ph meters would not provide accurate ph measurements of these 
 solutions . instead , as per our previous works ,
ph values reported in this work were obtained using ph papers and 
 are approximate measurements . as 
 per our previous works , these ph - adjusted homogeneous solutions were thoroughly bubbled 
 with nitrogen to remove the dissolved oxygen . immediately , these solutions 
 were drawn into 4 mm suprasil quartz tubes ( catalog no .
buena , nj , usa ) and were rapidly cooled in 
 liquid nitrogen ( 77 k ) .
the rapid cooling of these homogeneous liquid 
 solutions at 77 k leads to the formation of transparent homogeneous 
 glassy solutions .
these glassy solutions were later used for the irradiation 
 and subsequent progressive annealing experiments .
all glassy samples 
 were stored at 77 k in teflon containers in the dark .
all samples 
 were  ( co)-irradiated ( absorbed 
 dose = 1.4 kgy ) at 77 k and stored at 77 k in teflon containers in 
 dark following our previous efforts .
a variable - temperature 
 assembly was employed which passed liquid nitrogen cooled nitrogen 
 gas past a thermister and over the sample as described in our earlier 
 studies .
the glassy samples have been 
 annealed anywhere from ( 140170 ) k for 15 min .
annealing leads 
 to one - electron oxidation of the solute by the matrix radical cl2 thus , forming only the cation 
 radical of the solute , e.g. , gemcitabine .
following our earlier studies , immediately after -irradiation of the glassy sample at 77 
 k , the esr spectrum was recorded at 77 k. also , immediately after 
 each annealing step , the sample was cooled to 77 k by immersing in 
 liquid nitrogen ( 77 k ) , and the esr spectrum was recorded at 77 k 
 which maximizes signal height and allows for comparison of signal 
 intensities . a varian century series x - band ( 9.3 ghz ) esr spectrometer 
 with an e-4531 dual cavity , 9 in .
magnet , and a 200 mw klystron was 
 used , and fremy s salt ( gcenter = 2.0056 , a(n ) = 13.09 g ) was employed for the 
 field calibration .
all esr spectra have been recorded at 77 k and 
 at 40 db ( 20  w ) .
anisotropic simulations of esr spectra 
 have been performed using the win - epr and simfonia programs of bruker 
 as per our previous works .
the simulated spectra thus obtained 
 were compared to experimental spectra , and esr parameters were adjusted 
 for the best fit ( also supporting 
 information figure s3 ) .
gaussview and jmol programs were used to plot the spin densities 
 and molecular structures .
the geometries of all the radicals considered 
 in the present study were fully optimized using the b97x functional and 6 - 31g(d ) basis set .
we note here that b97x 
 functional was developed by the group of head  gordon and found 
 to be very successful for the calculations of various properties of 
 molecules in their different spin states .
the hyperfine coupling constants ( hfccs ) of the radicals were calculated 
 using the same method and basis set , i.e. , b97x/6 - 31g(d ) in 
 the gas phase . in order to treat the effect of solvent on hf loss 
 from the c3 in gemcitabine and in mefdc , we employ 
 the integral equation formalism polarized continuum model ( ief - pcm ) as implemented in gaussian 09 . in 
 addition to pcm , for c3 in both systems
a h3o is placed in the vicinity of the c3-oh bond 
 for c3 in gemcitabine and for c3 in 
 mefdc and have optimized the structures .
the electronic energy profile 
 of f dissociation from c2 site of c3 
 in mefdc as well as the electronic energy profile of f dissociation for each of the two f - atoms from c2 site of 
 c3 in gemcitabine were obtained in the presence of 
 a single water molecule at the same level of theory ( supporting information figure s4c , d ) .
furthermore , employing 
 the wb97x/6 - 31++g(d , p ) method along with the ief - pcm model for the 
 solvent effect , the pka of the c3-oh 
 group for the c3 of gemcitabine and also of the c3-oh 
 group for the c3 of 2-dc was calculated .
in figure 1a , we show the experimentally recorded ( 77 k ) esr spectrum 
 ( green ) of one - electron oxidized gemcitabine at ph ( pd ) ca . 7 in a 
 homogeneous glassy 7.5 m licl / d2o solution .
the one - electron 
 oxidation of gemcitabine was induced by cl2 attack after annealing at 155 k in the dark .
912 showed identical spectra after 
 one - electron oxidation of gemcitabine by cl2 on annealing at 150155 k. thus , only the spectrum 
 obtained from the gemcitabine sample at pd ca .
10 is presented in 
 figure 1b along with the simulated spectrum 
 in blue .
it is evident from figure 1a , b , the 
 line shape , total hyperfine splitting , and the center of the simulated 
 spectra match with those of the experimentally recorded spectra quite 
 well .
each of the spectra in figure 1a , b show 
 two anisotropic -f - atom hyperfine couplings and a -h - atom 
 hyperfine coupling .
the -h - atom hyperfine coupling creates 
 the doublet splitting in the line components in figure 1a , b .
figure 1a is best matched 
 with a simulation 
 employing the two different anisotropic -f - atom ( nuclear spin 
 = 1/2 ) hfcc values of ( 15.0 , 15.0 , 105 ) g and ( 15.0 , 15.0 , 69.0 ) g , 
 one -h hfcc as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0080 , 2.0050 , 2.0020 ) along with a mixed ( lorentzian / gaussian 
 ( 1:1 ) ) line - width of 14 g. the simulated spectrum in blue is superimposed 
 on the experimentally recorded spectrum in figure 1a . on the other hand , the best fit for figure 1b
is obtained employing the two identical anisotropic -f - atom 
 ( nuclear spin = 1/2 ) hfcc as ( 17.0 , 17.0 , 86.0 ) g , one -h hfcc 
 as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0060 , 2.0050 , 
 2.0020 ) along with a mixed ( lorentzian / gaussian ( 1:1 ) ) line - width 
 of 10 g. following our work on the radicals produced in monomers 
 of dna 
 and rna , the a ( i.e. , 
 the azz ) component of each of the two 
 anisotropic -f - atoms ( see table 1 ) as 
 well as the a of the -h are directly measured 
 from the width of the experimentally recorded spectra with an uncertainty 
 of 2 g ( see supporting information figure s3 ) . on the other hand ,
the theoretically obtained values 
 of axx and ayy components of each of the two anisotropic -f - atoms and of the 
 -h - atom in table 1 were adjusted to 
 fit the experimentally recorded spectra with estimated uncertainty 
 of 4 g ( see supporting information figure s3 ) .
7 show two nonequivalent anisotropic -f - atom hfccs , whereas , 
 the one - electron oxidized gemcitabine spectrum at ph ca .
the -h - atom 
 hfcc does not show any observable change in the one - electron oxidized 
 gemcitabine spectrum throughout the ph range ca .
the coupling to two -f - atoms ( c2 ) and one -h - atom 
 ( c4 ) is clear evidence for the generation of c3 
 after one electron oxidation of gemcitabine at 150155 k. the 
 electron - withdrawing effect of the two electronegative f - atoms at 
 c2 increases the acidity of h3 , which leads to deprotonation 
 ( see supporting information table t2 ) and 
 prevents observation of the initially formed cytosine base -cation 
 radical ( c ) in gemcitabine as indicated in reaction 3 .
therefore , the mechanism of c3 
 formation due to one - electron oxidation of gemcitabine is proposed 
 as follows : one - electron oxidation of gemcitabine results in the formation 
 of metastable c , which is unstable even at ca . 

155 k. the metastable c quickly deprotonates at 
 c3 in the sugar moiety producing c3 ( reaction 3 ) via a proton - coupled electron - transfer ( pcet ) mechanism.3 esr 
 spectra obtained from matched gemcitabine samples [ concentration 
 of gemcitabine in each sample = 2 mg / ml in 7.5 m licl / d2o ] in the presence of the electron scavenger k2s2o8 ( 8 mg / ml in each sample ) .
each sample has been -irradiated 
 ( absorbed dose = 1.4 kgy at 77 k ) , subsequently annealed to 155 k 
 for 15 min in the dark at various phs ( a ) ph ca .
7 ( green ) and ( b ) 
 ph ranging ca . 912 ( pink ) . here
the blue spectra that are superimposed on the experimentally 
 recorded spectra are the simulated spectra of c3. 
 see text for the details of simulation .
all esr spectra are recorded 
 at 77 k. the three reference markers ( open triangles ) in this figure 
 and in the subsequent figures show the position of fremy s 
 salt resonance with the central marker at g = 2.0056 .
the spacing 
 separating the markers is 13.09 g. as is evident from the hfcc 
 values of the spectra shown in figure 1 and 
 also in table 1 , the ph of the solution clearly 
 affects the individual -f - atom anisotropic hfcc but not the 
 sum of the two -f - atom anisotropic hfcc along with the c4 
 -proton hfcc ( see section above ) . at ph ca .
912 , the two -f - atom anisotropic hfccs 
 are equivalent ; whereas , at ph ca . 7 ,
the sum of the two -f - atom 
 anisotropic hfccs remain the same , but they individually differ .
the presence of two 2-f - atoms in gemcitabine will lower the 
 pka value of both h3 and c3-oh 
 hydrogens .
for example , the oh in 2,2-difluoroethanol has its pka lowered by 3.5 units in comparison with ethanol .
further , radical formation has also been shown 
 to lower the pka of the alcoholic oh group 
 ( e.g. , pka ( ch3)2choh = 17.1 , pka ( ch3)2coh = 12.03 ) . based on 
 these factors , the pka value of the c3-oh 
 group for c3 in gemcitabine is estimated to be in 
 the range of 79 .
employing dft/b97x/6 - 31++g(d , p ) method 
 along with the ief - pcm model for the solvent effect , the pka value of the c3-oh group for c3 
 in 2-dc has been calculated as 14.2 .
the same level of calculation 
 for the c3-oh group of c3 in gemcitabine which 
 replaces each of the two hydrogens at c2 with fluorine predicts 
 a pka of 6.8 ( see supporting information pages s8 , s9 ) .
therefore , the two 2-f - atoms 
 are predicted to lower the pka of the 
 c3-oh group in c3 by 7 full units .
considering 
 the sensitivity of the calculations for predicting pka to the small changes in free energy , these results are very reasonable .
furthermore , the theoretically 
 calculated pka value 6.8 of the c3-oh 
 group for c3 in gemcitabine is in good agreement with 
 its experimentally estimated value ( 79 ) . the spectrum 
 in figure 1a ( ph ca .
7 ) should 
 therefore be for c3 in gemcitabine with a c3-oh 
 group and the spectrum in figure 1b ( ph ca . 

912 ) should be for c3 with the deprotonated 
 group , i.e. , c3-o ( reaction 4 ) .
thus , we attribute the variation of the two -f - atom 
 anisotropic hfcc to the deprotonation of the c3-oh group at 
 higher phs.4 esr spectral studies 
 of one - electron oxidation of gemcitabine in h2o glasses 
 ( 7.5 m licl / h2o ) were performed and compared with the results 
 found in d2o glasses ( 7.5 m licl / d2o ) .
no observable difference in spectra other than a small 
 line broadening was observed on formation of c3 in 
 h2o glasses versus in d2o glasses .
since c3 
 only has the c3-oh as an exchangeable proton at ph 7 and this 
 does not contribute to a significant hyperfine coupling , the esr spectrum 
 found on formation of c3 is not altered by a change 
 of the solvent from d2o to h2o .
similar experiments to those performed for gemcitabine 
 were carried out for the methyl / fluoro analog mefdc ( scheme 2 ) .
using mefdc , we investigated whether the formation 
 of c3 observed in gemcitabine bearing geminal difluoro unit at c2 ( figure 1 ) is 
 affected by the substitution of one of the f - atoms ( i ) with 
 a methyl ( me ) group ( + i ) .
( a ) esr spectrum ( black ) obtained from mefdc [ concentration 
 = 2 
 mg / ml in 7.5 m licl / d2o ] in the presence of the electron 
 scavenger k2s2o8 ( 8 mg / ml ) , ph ca . 

10 , -irradiated to a dose of 1.4 kgy at 77 k and subsequently 
 annealed to 155 k for 15 min .
( c ) 
 spectrum ( black ) obtained after subtraction of 60% of spectrum ( b ) 
 from spectrum ( a ) .
for comparison , the c spectrum 
 ( blue ) in 2-dc ( supporting information figure s2 and pp s3s5 ) is superimposed .
( d ) spectrum ( black ) 
 assigned to c2 is obtained after subtraction of 50% 
 of spectrum ( c ) from spectrum ( a ) .
the simulated c2 
 spectrum ( for simulation parameters see figure 2 and text ) is superimposed 
 on the experimentally isolated spectrum for comparison .
all the spectra 
 are recorded at 77 k. shown in figure 2a is the esr spectrum 
 ( black ) 
 of a matched sample of mefdc that has been -irradiated ( absorbed 
 dose = 1.4 kgy ) , subsequently annealed to 155 k for 15 min in the 
 dark , and recorded at 77 k. figure 2b 
 was obtained by annealing this 
 sample for 15 min to 170 k. comparison of spectrum 2a with spectrum 
 2b shows clearly that a central doublet decreases along with an increase 
 of the other line components upon annealing .
therefore , the central 
 doublet ( black ) shown in figure 2c is isolated 
 by subtraction of 60% of the spectrum 2b from 
 spectrum 2a .
the doublet due to c spectrum ( blue ) in 2-dc ( see supporting information figure s2 and its discussion ( pp s3s5 ) ) 
 is superimposed on it for comparison . from the spectral similarities 
 of both doublets , the doublet in black shown in figure 2c
subtraction 
 of 50% c spectrum 2c ( black ) from 
 spectrum 2a results in the black spectrum shown in figure 2d .
this overall quintet spectrum arises from 4 isotropic 
 -proton couplings : three methyl -protons ( ca .
25.5 g due to a -proton 
 assigned to the c1-h ( vide infra ) .
the experimental ( black ) 
 spectrum is simulated using the above - mentioned hfcc values along 
 with a 10 g line - width and g - value = 2.0033 ( this g - value is typical for c - centered sugar radicals ) . the simulated spectrum ( red ) in figure 2d 
 matches the overall line components of the experimental spectrum well . 

since the c2 ( reaction 5 ) is 
 the only likely radical structure that would explain the large hyperfine 
 coupling to a methyl group and the additional -proton hyperfine 
 coupling ( assigned to c1 ) , the experimental spectrum in figure 2d has been assigned to c2 ( reaction 5 ) . the spectra 2a and 2b are a composite of c ( black , 
 figure 2c ) and c2 ( black , figure 2d ) in different amounts . under the same constant 
 gain and constant microwave power and upon gradual and stepwise annealing 
 of the sample from 155 k ( spectrum 2a ) to 170 k ( spectrum 2b ) ,
no 
 loss of spectral intensity was observed , and our analyses show an 
 additional ( ca .
20% ) conversion of the c to c2. consideration of the results presented in figures 1 and 2 suggest that for mefdc , c is produced first and on annealing converts to a transient 
 c3 , which is not observed .
in contrast , the line shape , 
 line width , and the overall hyperfine splitting of the c3 
 spectrum in gemcitabine do not change upon annealing to ca .
thus , unlike 
 the rapid conversion of c3 to c2 found 
 in one - electron oxidized mefdc , a similar conversion of c3 
 to c2 is not observed for one - electron oxidized gemcitabine 
 at these low temperatures .
the lack of observation of the transient 
 c3 in one - electron oxidized mefdc and the low temperatures 
 employed in these experiments implies a very low activation barrier 
 for the conversion of c3 to c2 ; whereas , 
 the activation barrier for the conversion of c3 to 
 c2 for one - electron oxidized gemcitabine should be 
 4 kcal / mol .
calculations suggest that the proximity of the 
 lost h3 proton as h3o to the 2-f - atom 
 quite likely provides the driving force for this rapid unimolecular 
 reaction ( see figure 3 ) .
therefore , we propose 
 that c3 in one - electron oxidized mefdc readily converts 
 to c2 via a barrierless f loss 
 ( see reaction 5 , figure 3 , and supporting information s5 ) . the b97x/6 - 31g(d ) 

calculated hfccs of c3 and c2 found 
 in gemcitabine and in mefdc along with experimental hfccs ( in gauss ) 
 are presented in table 1 .
it is evident from 
 table 1 that experimental and theoretically 
 calculated hfccs are in reasonably good agreement .
estimated errors are of 2 g for 
 azz and 4 g for axx and also for ayy .
calculated in the presence 
 of one 
 water molecule only isotropic 
 hfcc values have 
 been considered . to explore the reaction mechanism of c2 
 formation from c3 in one - electron oxidized mefdc and 
 gemcitabine , using the dft b97x/6 - 31g(d ) method
, we considered 
 three possible reaction paths : ( i ) hf loss due to deprotonation of 
 3-hydroxyl group , ( ii ) hf loss due to fluorine dissociation , and additionally , ( iii ) hf loss in the presence of a hydronium ion 
 ( h3o ) .
the electronic energy profile 
 of hf loss from c3 in mefdc and also from c3 
 in gemcitabine in the presence of a water molecule is shown in figure 
 s4 in the supporting information . from supporting information figure s4a ,
it is evident 
 that for c3 of mefdc , the formation of c2 
 via hf loss with deprotonation of 3-oh has a significant barrier 
 of ca . 18 kcal / mol .
for c3 in gemcitabine , the hf 
 loss associated with deprotonation of 3-hydroxyl group was 
 calculated to be ca .
we have also considered the dissociation of 
 fluorine in c3 
 of mefdc and in c3 of gemcitabine and found that stretching 
 the c2-f bond up to 1.8  needs ca .
this shows that dissociation of fluorine for c3 in 
 mefdc occurs at lower energy than dissociation of fluorine for c3 
 in gemcitabine ( supporting information figure 
 s4 ) .
alternatively , we consider the fact that deprotonation 
 of h3 
 will form h3o initially in close proximity 
 of the c2-c3 bond which may then induce hf loss ( reaction 5 ) . employing the b97x/6 - 31g(d ) method and 
 considering the full solvent effect through the polarized continuum 
 model ( pcm ) , this mechanism have been modeled by placing a h3o in the vicinity of the c3-oh bond for c3 
 in gemcitabine and for c3 in mefdc and have optimized 
 the structures . from our calculations , we have observed that for c3 
 in gemcitabine , a minimum structure exists in the electronic energy 
 profile in which the h3o stabilizes the f-atom 
 through forming a hydrogen bond ( 1.57  ) ( see figure 3a and supporting information figure s4c ) .
a second minimum structure was also found in the electronic 
 energy profile in which the h3o stabilizes 
 the f-atom through forming a hydrogen bond of identical length , 
 1.57  ( see supporting information figure s4d ) .
a barrier of 5 kcal / mol and the overall reaction energy 
 of 7 to 8 kcal / mol were found for hf formation in 
 both cases ( see figure 3a and supporting information figures s4c , d ) .
however , for c3 
 in mefdc , the h3o reacts with the 2-f - atom 
 without a barrier and forms hf and c2 ( see figure 3b ) .
the bond distances of c3-o3 
 and o3-h bonds for c3 in gemcitabine are calculated 
 as 1.34  ( primarily c  o single bond character ) and 0.97  , respectively .
the values of 
 the corresponding bond distances of c3-o3 and o3-h 
 bonds for c3 in mefdc are obtained as 1.28  
 ( mainly double bond character and 1.0 
  respectively ) .
thus , these calculations show that the 
 hf loss from c3 
 in gemcitabine has a ca .
5 kcal / mol barrier ( supporting 
 information figure s4c , d ) while for c3 in mefdc , 
 the loss of hf is barrierless , and the c2 production 
 is exothermic in nature as shown in figure 3 .
these findings support our experimental observations that in mefdc , 
 c3 is too unstable to be observed , and only c2 
 is found .
in contrast , in gemcitabine only c3 formation 
 is observed without any conversion to c2 in the same 
 temperature range .
pcm-b97x/6 - 31g(d ) calculated structures of c3 
 in ( a ) gemcitabine and in ( b ) mefdc .
as indicated in ( a ) the c3 
 in gemcitabine involves a barrier of 5 kcal / mol ( detailed electronic 
 energy profiles are provided in supporting information figure s4c , d ) to hf loss in the presence of h3o . in ( b ) , the c3 in mefdc is unstable in the presence 
 of h3o and reacts without a barrier to form 
 c2 via hf loss . the animations ( movies ) of the optimization steps for reaction involving h3o for c3 in both gemcitabine ( a ) 
 and mefdc ( b ) are provided in the supporting information ( s5 ) .
our 
 work has the following two salient findings : ( i ) one electron 
 oxidation leads to cytosine base -cation radical ( c ) in 2-dc and 2-f - dc but to c3 
 in gemcitabine . as expected from the one - electron redox potentials 
 of the bases and the backbone , one - electron oxidation of 2-dc and 2-f - dc leads 
 to c formation as evidenced by the ca .
16 g doublet 
 that is characteristic of c. however , gemcitabine 
 ( scheme 1 ) leads to the formation of c3 
 on one - electron oxidation .
the two highly electronegative f - atoms 
 at 2-position , through their negative inductive effect , lead 
 to a substantial increase in the acidity of h3. therefore , 
 c in gemcitabine is highly unstable toward the 
 loss of h3 as deprotonation at 150155 k. this is evidenced 
 by the free energy changes of the cation radical for the loss of h3 
 as deprotonation to the surrounding solvent ( see supporting information table t2 ) ; this deprotonation shifts 
 the unpaired spin from the cytosine base of metastable c in gemcitabine to sugar at c3 via a pcet 
 process .
( ii ) c2 formation does not occur in 
 gemcitabine 
 but does in its analog mefdc .
it has been proposed in the literature 
 that in gemcitabine both c3 and c2 
 ( reactions 1 and 2 ) play 
 an important role in the rnr inactivation .
conversion of 
 c3 to c2 takes place via an irreversible 
 f loss from c2 during rnr inactivation 
 by gemcitabine .
however , experimental and theoretical results shown 
 in this work have clearly demonstrated that in our system ( supercooled 
 homogeneous glassy solutions ) , c3 in gemcitabine does 
 not convert to c2 on annealing up to 170 k owing to 
 theoretically predicted barrier of greater than 5 kcal / mol .
theoretically , 
 dft calculations support the mechanism involving a h3o induced barrierless conversion of c3 to c2 
 in one - electron oxidized mefdc .
experimentally , c2 
 is observed in one - electron oxidized mefdc upon annealing to ca .
160170 
 k. thus , our study in one - electron oxidized mefdc provides the first 
 evidence of formation of c2 ( via the unstable intermediate 
 c3 ( reaction 5 ) ) in a nonenzymatic 
 system even at low temperature.5
 gemcitabine 
 is a modified cytidine analog having two fluorine atoms 
 at the 2-position of the ribose ring . 
 it has been proposed 
 that gemcitabine inhibits rnr activity by producing a c3 
 intermediate via direct h3-atom abstraction followed by loss 
 of hf to yield a c2 with 3-keto moiety . 
 direct 
 detection of c3 and c2 during rnr 
 inactivation by gemcitabine still remains elusive . to test the influence 
 of 2- substitution on radical site formation , 
 electron spin 
 resonance ( esr ) studies are carried out on one - electron oxidized gemcitabine 
 and other 2-modified analogs , i.e. , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ) and 2-fluoro-2-deoxycytidine 
 ( 2-fdc ) . 
 esr line components from two anisotropic -2-f - atom 
 hyperfine couplings identify the c3 formation in one - electron 
 oxidized gemcitabine , but no further reaction to c2 
 is found . 
 one - electron oxidized 2-fdc is unreactive toward 
 c3 or c2 formation . in one - electron 
 
 oxidized mefdc , esr studies show c2 production presumably 
 from a very unstable c3 precursor . 
 the experimentally 
 observed hyperfine couplings for c2 and c3 
 match well with the theoretically predicted ones . 
 c3 
 to c2 conversion in one - electron oxidized gemcitabine 
 and mefdc has theoretically been modeled by first considering the 
 c3 and h3o+ formation via h3-proton 
 deprotonation and the subsequent c2 formation via 
 hf loss induced by this proximate h3o+ . 
 theoretical 
 calculations show that in gemcitabine , c3 to c2 
 conversion in the presence of a proximate h3o+ has a barrier in agreement with the experimentally observed 
 lack of c3 to c2 conversion . 
 in contrast , 
 in mefdc , the loss of hf from c3 in the presence of 
 a proximate h3o+ is barrierless resulting in 
 c2 formation which agrees with the experimentally 
 observed rapid c2 formation . 
Introduction
Materials and Methods
Results and Discussion
Conclusion