Tautomerism in 8-Nitroguanosine Studied by NMR and Theoretical Calculations

Article abstract of DOI:10.1080/15257770.2015.1114127

The guanine base in DNA, due to its low oxidation potential, is particularly sensitive to chemical modifications. A large number of guanine lesions have been characterized and studied in some detail due to their relationship with tissue inflammations. Nevertheless, one example of these lesions is the formation of 8-nitro-guanosine, but the NMR data of this compound was only partially interpreted. A comprehensive study of the two possible tautomeric forms, through a detailed characterization of this compound, has implications for its base pairing properties. The target compound was obtained through a synthetic sequence of five steps, where all intermediates were fully characterized using spectral data. The analysis of the two tautomers was then evaluated through NMR spectroscopy and theoretical calculations of the chemical shifts and NH coupling constants, which were also compared with the data from guanosine.

Full text of DOI:10.1080/15257770.2015.1114127

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Tautomerism in 8-Nitroguanosine Studied by NMR
and Theoretical Calculations
Thaís M. Barbosa, Roberto Rittner, Katie Alexander, Richard Cosstick &
Raymond J. Abraham
To cite this article: Thaís M. Barbosa, Roberto Rittner, Katie Alexander, Richard
Cosstick & Raymond J. Abraham (2016): Tautomerism in 8-Nitroguanosine Studied by
NMR and Theoretical Calculations, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2015.1114127
To link to this article: http://dx.doi.org/10.1080/15257770.2015.1114127
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Date: 31 January 2016, At: 23:14

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
http://dx.doi.org/./..
Tautomerism in -Nitroguanosine Studied by NMR and
Theoretical Calculations
Thaís M. Barbosaa, Roberto Rittnera, Katie Alexanderb, Richard Cosstickb, and
Raymond J. Abrahamb
aChemistry Institute, University of Campinas, UNICAMP, P.O. Box , , Campinas, SP, Brazil; bDepartment
of Chemistry, University of Liverpool, P.O. Box , Liverpool, UK
ABSTRACT
ARTICLE HISTORY
Received  May 
Accepted  October 
The guanine base in DNA, due to its low oxidation potential, is
particularly sensitive to chemical modifications. A large number
of guanine lesions have been characterized and studied in some
detail due to their relationship with tissue inflammations. Never-
theless, one example of these lesions is the formation of 8-nitro-
guanosine, but the NMR data of this compound was only par-
tially interpreted. A comprehensive study of the two possible tau-
tomeric forms, through a detailed characterization of this com-
pound, has implications for its base pairing properties. The tar-
get compound was obtained through a synthetic sequence of
five steps, where all intermediates were fully characterized using
spectral data. The analysis of the two tautomers was then eval-
uated through NMR spectroscopy and theoretical calculations of
the chemical shifts and NH coupling constants, which were also
compared with the data from guanosine.
KEYWORDS
NMR; nitroguanosine;
theoretical calculations; DNA
lesions
1. Introduction
In 1863 Virchow noted a presence of leucocytes in neoplastic tissues and suggested a
connection between inflammatory process and cancer. Even after all these years the
understanding of the inflammatory microenvironment of malignant tissues has sup-
ported Virchowꢀs hypothesis, and the connection found between cancer and inflam-
mation is starting to have implications for prevention and treatment.[1]
Epidemiological studies have shown that chronic inflammation inclines individu-
als to various types of cancer.[2] This is explained by the fact that inflammatory cells
produce reactive oxygen and nitrogen species that have been proposed to induce
DNA and tissue damage, which contribute to the multistage process of carcino-
genesis.[3] Some examples of cancer that are based on inflammatory process are:
liver cancer, which usually is a consequence of inflammation caused by chronic viral
hepatitis; lung cancer, which results from inflammation caused by years of inhaled
CONTACT Roberto Rittner
rittner@iqm.unicamp.br
Box  -, Campinas, SP, Brazil.
Chemistry Institute, University of Campinas, UNICAMP, P.O.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
©  Taylor & Francis Group, LLC

2
T. M. BARBOSA ET AL.
tobacco smoke, and the association between inflammatory stomach infections (such
as Helicobacter pylori) and gastric cancer.[1,4,5] It is estimated that underlying infec-
tions and inflammatory responses are linked to around 20% of all deaths from cancer
worldwide.[1,2]
One of the reactive nitrogen species (RNS) that can be formed in inflammatory
cells is NO which reacts rapidly with superoxide anion (O2•−) to form peroxynitrite,
which is a strong oxidant and can initiate reactions characteristic of hydroxyl radi-
•
cal (HO•), nitronium ion (NO2+) and nitrogen dioxide radical (NO2 ).[3] Previous
studies of Yermilov and co-workers showed that guanine reacts rapidly with perox-
ynitrite under physiological conditions to form several substances.[6] On the basis
of chromatographic and spectral evidence, 8-nitroguanine was observed to be the
major product from the reaction of peroxynitrite with guanine.[6]
The guanine base in DNA, due to its low oxidation potential, is particularly sen-
sitive, and a large number of guanine lesions have been characterized and studied
in some detail.[7,8] One example is the 8-nitro-2ꢀ-deoxyguanosine, which is know
to labilize the glycosidic bond causing the release of 8-nitroguanine, which can be
detected in urine, and leaves an abasic site in DNA (Figure 1).[4,6,9]
The synthesis and study of oligonucleotides containing site-specific DNA lesions
lead to an understanding of how chemical changes of DNA generate specific muta-
tions. However, the high sensitivity of the 8-nitro-2ꢀ-deoxyguanosine glycosidic
bond explains why the synthesis of oligodeoxynucleotides (ODNs) containing 8-
nitroguanine by a conventional chemical approach has not been achieved. There
is, however, strong evidence to suggest that this lesion is highly mutagenic and thus
deserves a detailed study of its base pairing and other physicochemical properties.[4]
To obtain a better understanding of the mutagenic effect of 8-nitro-2ꢀ-
deoxyguanosine, Cosstick and co-workers described the synthesis of ODNs con-
taining 8-nitro-2ꢀ-O-methylguanosine, in which the glycosidic bond is stabilized by
a ribose sugar (Figure 2).[4,10]
The detailed characterization of these C8-NO2 lesions allowed the understanding
of their biological consequences, particularly with regard to base pairing preferences
and recognition by repair enzymes.[11] There are at least two possible tautomeric
forms (Figure 3) for the 8-NO2-guanosine, both have implications in base pairing
properties.
Figure . Nitration of the guanine base in DNA by reactive nitrogen species and release of
-nitroguanine to leave an abasic site in DNA.[]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Figure . -Nitro-ꢀ-O-methylguanosine is much more resistant to hydrolysis of the glycosidic bond
than -nitro-ꢀ-deoxyguanosine.[]
This class of compounds which has a bulky substituent at the C8 position
is reported to exist in the syn conformation about the glycosyl bond, as steric
hindrance disfavours the anti-conformation; the syn conformation is known to be
also promoted by intramolecular hydrogen bonding and other intermolecular fea-
tures.[4,12]
Two model molecules were reported to evaluate the keto-enol equilibrium in
guanosine (Figure 4).[11] The methyl group in 1-methyl-guanosine prevents C6 eno-
lization, and the N1 atom has a chemical shift of 143 ppm. On the other hand, the
O6-alkylated guanosine is a model for the 6-enol-tautomer, and in this case N1 has
a chemical shift of 214.1 ppm.
2. Experimental section
2.1 Flash column chromatography
The required quantity of silica (particle size 40–63 μM, supplied by BDH) was made
into homogenous slurry with the column eluent and applied to the column over
a base layer of sand and some cotton wool. The material to be purified was then
introduced onto the column in the minimum volume of eluent or adsorbed into
Figure . The two possible tautomeric forms for the -NO-guanosine.

4
T. M. BARBOSA ET AL.
Figure . Model compounds[] (a) for the keto (δ N = . ppm) and (b) enol form (δ N =
. ppm), respectively.
silica and then eluted with the eluent of choice. All fractions were collected and
analysed by TLC.
2.2 Nuclear magnetic resonance spectroscopy (NMR)
All NMR spectra were recorded on a Bruker DPX 400MHz and Bruker Avance III
500 MHz; 1H NMR at 400 MHz or 500 MHz, 13C at 100 or 125 MHz and 15N at
50 MHz. All chemical shifts are reported in ppm and coupling constants (J) in hertz.
1H and 13C were referenced to the DMSO signal (2.54 ppm for 1H and 39.51 ppm
for 13C), whereas 15N was referenced to the TMS signal.
2.3 High resolution mass spectroscopy (HRMS)
The mass spectrum was recorded using a Micromass LCT mass spectrometer. The
sample was diluted in CH3OH and analysed by negative mode electrospray ioniza-
tion (ES−) with a direct infusion syringe pump.
2.4 pH measurements
pH measurements were carried out using a Metrohm 691 pH meter. This was cali-
brated prior to use with buffers purchased from Thermo Russell (pH 4.00, 7.00 and
10.00 0.01 at 25°C.
2.5 Reverse-phase high pressure liquid chromatography (RP-HPLC)
Reverse-phase HPLC was performed on an automated Gilson HPLC system
equipped with an autoinjector, a photodiode array detector and a dual hydraulic

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
pump. Chromatographic data was manipulated using UniPoint Version 3.0 and
absorbances measured at 254 nm.
HPLC solvents:
• 1M TEAB solution was prepared by bubbling CO2 gas through a 1 M aqueous
triethylamine solution until the pH was established between 7.50 and 7.70.
• Buffer A: (0.1 M aqueous TEAB solution, 500 mL) was prepared by diluting
50 mL of 1M TEAB solution with 450 mL of distilled water.
• Buffer B: (0.1 M aqueous TEAB solution, 500 mL) was prepared by diluting
50 mL of 1M TEAB solution with 250 mL of water and 200 mL of HPLC grade
acetonitrile.
HPLC control method:
The eluent was gradually changed from 0% buffer B (100% buffer A) to 60% buffer
B (40% buffer A) over 14 min, then gradually changed to 100% B over 2 min and
kept 100% buffer B for another 2 min. After this, the eluent was gradually changed
to 100% A over 2 min and kept 100% buffer A until the end of the run, which took
a total of 25 min.
2.6 Synthetic steps to obtain 8-nitro-guanosine[4]
Syntheses of ꢀ,ꢀ,ꢀ-tri-O-acetyl-guanosine ()
To a stirred suspension of guanosine (5.0 g, 17.7 mmol) in MeCN (65 mL) was added
Et3N (6.47 mL, 46.5 mmol) and DMAP (0.22 g, 1.8 mmol). The resulting suspension
was cooled in an ice bath prior to dropwise addition of Ac2O (5.84 mL, 61.84 mmol).
Once the addition was completed, the mixture was allowed to reach room tempera-
ture and was stirred for a further 2 h. The reaction was quenched by the addition of
MeOH (6.5 mL) and concentrated in vacuo to a white semi-solid precipitate which
i
was boiled with PrOH and filtered, leaving the product as a white solid. (5.79 g,
80%). 1H NMR (500 MHz, DMSO-d6): δ (ppm) 10.70 (1H, s, NH); 7.91 (1H, s, H8);
ꢀ
ꢀ
6.49 (2H, s, NH2); 5.98 (1H, d, 3JH1 H2ꢀ = 6.1 Hz, H1ꢀ); 5.79 (1H, t, 3JH2 H3ꢀ = 6.0 Hz,
H2ꢀ); 5.50 (1H, dd, 3JH3 H4ꢀ = 4.2, H3ꢀ); 4.37 (1H, dd, 3JH5 H4ꢀ = 3.8 and 2JH5 H5
=
ꢀ
ꢀ
ꢀ
ꢀꢀ
11.5 Hz, H5ꢀ); 4.31 (1H, m, H4ꢀ); 4.26 (1H, dd, 3JH5 ꢀ = 5.7 Hz, H5ꢀ); 2.11 [3H, s,
ꢀꢀ
H4
(CH3)3ꢀ]; 2.04 [3H, s, (CH3)5ꢀ]; 2.03 [3H, s, (CH3)2ꢀ]. 13C NMR (125 MHz, DMSO-
d6): δ (ppm) 170.89 [(CO)5ꢀ]; 170.24 [(CO)3ꢀ]; 170.07 [(CO)2ꢀ]; 157.46 (C6); 154.75
(C2); 151.95 (C4); 136.45 (C8); 117.74 (C5); 85.35 (C1ꢀ); 80.40 (C4ꢀ); 72.93 (C2ꢀ);
71.18 (C3ꢀ); 63.92 (C5ꢀ); 21.34 [(CH3)5ꢀ]; 21.20 [(CH3)3ꢀ] and 21.01 [(CH3)2ꢀ].
Syntheses of -bromo-ꢀ,ꢀ,ꢀ-tri-O-acetyl-guanosine ()
Compound 1 (5.77 mg, 14.1 mmol) was suspended in distilled water (34 mL). Satu-
rated bromine water (ca. 20 mL) was added with vigorous stirring at room temper-
ature at such a rate that the orange colour dissipated between additions. Once the
orange color persisted, the mixture was allowed to stir for a further 30 min before
filtering. The solution was filtered and the solid washed with ice cold iPrOH leaving
the product as a pale orange solid. (5.79 g, 84%). 1H NMR (500 MHz, DMSO-d6): δ
3
(ppm) 10.86 (1H, s, NH); 6.55 (2H, s, NH2); 6.03 (1H, dd, 3JH2 H1 = 4.5 and JH2 H3
ꢀ
ꢀ
ꢀ
ꢀ

6
T. M. BARBOSA ET AL.
3
ꢀ
ꢀ
= 6.4 Hz, H2ꢀ); 5.88 (1H, d, H1ꢀ); 5.65 (1H, t, JH3 H4 = 6.3, H3ꢀ); 4.42 (1H, dd,
3JH5 H4ꢀ = 3.8 and 2JH5 H5ꢀꢀ = 11.9 Hz, H5ꢀ); 4.32 (1H, td, 3JH4 H5ꢀꢀ = 6.1, H4ꢀ); 4.21
(1H, dd, H5ꢀꢀ); 2.11 [3H, s, (CH3)3ꢀ]; 2.07 [3H, s, (CH3)2ꢀ]; 1.99 [3H, s, (CH3)5ꢀ].
13C NMR (125 MHz, DMSO-d6): δ (ppm) 171.02 [(CO)5ꢀ]; 170.40 [(CO)3ꢀ]; 170.33
[(CO)2ꢀ]; 156.33 (C6); 154.73 (C2); 152.84 (C4); 121.03 (C8); 118.11 (C5); 88.55
(C1ꢀ); 80.23 (C4ꢀ); 72.19 (C2ꢀ); 70.84 (C3ꢀ); 63.69 (C5ꢀ); 21.41 [(CH3)5ꢀ]; 21.23
[(CH3)3ꢀ] and 21.17 [(CH3)2ꢀ].
ꢀ
ꢀ
ꢀ
Syntheses of N-dimethoxytrityl -bromo-ꢀ,ꢀ,ꢀ-tri-O-acetyl-guanosine ()
Product 2 (5.79 mg, 11.85 mmol) was added in a 250 mL bottom round flask and
co-evaporated to dryness with anhydrous pyridine before re-dissolving in anhy-
drous pyridine (36.7 mL). The resultant solution was stirred at room tempera-
ture under an atmosphere of N2 and dimethoxytrityl chloride (DMTCl) (4.40 g,
13.0 mmol) was added in ca. 0.5 g portions over 5 min. The mixture was
stirred at room temperature for a further 8 h before removing most of the pyri-
dine under reduced pressure. The isolated solid was partitioned between DCM
(Dichloromethane) (50 mL) and water (50 mL), the DCM layer was further washed
with water (2 × 50 mL) and saturated aqueous NaHCO3 (30 mL) before drying
over Na2SO4, filtering and evaporating to a yellow solid. Purification by flash col-
umn chromatography (100% DCM – 2% MeOH/DCM) gave the product as a yellow
solid (5.9 g, 63%). 1H NMR (500 MHz, DMSO-d6): δ (ppm) 10.86 [1H, s, NH (1)];
7.63 [H, s, NH (2)]; 7.31–7.28 (2H, m, DMT); 7.22-7.19 (3H, m, DMT); 7.15–7.12
ꢀ
(4H, m, DMT); 6.87–6.83 (4H, m, DMT); 5.43 (1H, d, 3JH1 H2ꢀ = 5.8 Hz, H1ꢀ); 5.30
ꢀ
ꢀ
ꢀ
ꢀ
(1H, t, 3JH2 H3 = 5.8 Hz, H2ꢀ); 4.82 (1H, t, 3JH3 H4 = 5.8 Hz, H3ꢀ); 4.03-3.99 (2H,
m, H4ꢀ and H5ꢀ); 3.81 (1H, dd, 2JH5 H5ꢀꢀ = 12.2 Hz, H5ꢀꢀ); 3.72 [3H, s, (CH3)DMT];
ꢀ
3.71 [3H, s, (CH3)DMT]; 2.06 [3H, s, (CH3)3ꢀ]; 2.00 [3H, s, (CH3)5ꢀ]; 1.92 [3H,
s, (CH3)2ꢀ]. 13C NMR (125 MHz, DMSO-d6): δ (ppm) 170.81 [(CO)5ꢀ]; 170.03
[(CO)3ꢀ]; 169.75 [(CO)2ꢀ]; 158.75 (DMT); 158.71 (DMT); 156.03 (C6); 152.42 (C2);
151.91 (C4); 145.97 (DMT); 137.17 (DMT); 130.52 (DMT); 130.45 (DMT); 129.10
(DMT); 128.73 (DMT); 127.56 (DMT); 121.06 (C8); 118.61 (C5); 113.98 (DMT);
113.93 (DMT); 87.64 (C1ꢀ); 79.33 (C4ꢀ); 70.63 (DMT); 70.41 (C2ꢀ); 70.24 (C3ꢀ); 63.59
(C5ꢀ); 55.83 (DMT); 21.43 [(CH3)5ꢀ]; 21.16 [(CH3)3ꢀ] and 20.84 [(CH3)2ꢀ].
Syntheses of N-dimethoxytrityl -nitro-ꢀ,ꢀ,ꢀ-tri-O-acetyl-guanosine ()
Product 3 (1.0 g, 1.26 mmol) was solubilized in a mixture of DMF (100 mL) and
18-crown-6 (3.33 g, 12.60 mmol). KNO2 (1.07 g, 12.6 mmol) was added and the
resulting mixture was stirred at 100°C for about 8 h under an inert atmosphere.
A small sample of the reaction mixture was partitioned between ethyl acetate and
water and the resulting organic layer was analysed by TLC (100% ethyl acetate). This
was performed until the detection of degradation product was observed. The reac-
tion was allowed to cool to room temperature, the Dimethylformamide (DMF) was
evaporated under reduced pressure and the resulting solid was extracted with water
(25 mL) and ethyl acetate (25 mL) three times. The organic layer was then washed

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
with saturated solution of NaHCO3, dried over Na2SO4, filtered and dried over
reduce pressure. Purification by flash column chromatography (ethyl acetate/hexane
(1:1) – ethyl acetate/hexane (8:2)) leads to isolation of product 4 as a yellow solid
(0.1 g, 13%). 1H NMR (500 MHz, DMSO-d6): δ (ppm) 11.16 [1H, s, NH (1)]; 8.03
[H, s, NH (2)]; 7.34-7.31 (2H, m, DMT); 7.26-7.21 ꢀ(3H, m, DMT); 7.14-7.12 (4H, m,
DMT); 6.89-6.87 (4H, m, DMT); 5.56 (1H, d, 3JH1 H2ꢀ = 4.8 Hz, H1ꢀ); 5.46 (1H, dd,
ꢀ
ꢀ
ꢀ
ꢀ
3JH2 H3ꢀ = 7.2 Hz, H2ꢀ); 5.15 (1H, t, 3JH3 H4ꢀ = 7.0 Hz, H3ꢀ); 4.14 (1H, dd, 3JH5 H4
=
3.6 and 3JH5 H5ꢀꢀ = 12.1 Hz, H5ꢀ); 3.98 (1H, td, 3JH4 H5ꢀꢀ = 6.2 Hz, H4ꢀ); 3.87 (1H, dd,
H5ꢀꢀ); 3.73 [3H, s, (CH3)DMT]; 3.72 [3H, s, (CH3)DMT]; 2.05 [3H, s, (CH3)3ꢀ]; 1.99
[3H, s, (CH3)5ꢀ]; 1.78 [3H, s, (CH3)2ꢀ]. 13C NMR (125 MHz, DMSO-d6): δ (ppm)
170.67 [(CO)5ꢀ]; 169.93 [(CO)3ꢀ]; 169.64 [(CO)2ꢀ]; 158.84 (DMT); 158.79 (DMT);
156.84 (C6); 154.18 (C2); 152.12 (C4); 145.50 (DMT); 143.72 (C8); 137.32 (DMT);
136.88 (DMT); 130.51 (DMT); 130.47 (DMT); 129.08 (DMT); 128.71 (DMT);
127.66 (DMT); 116.09 (C5); 114.02 (DMT); 113.96 (DMT); 88.09 (C1ꢀ); 79.12 (C4ꢀ);
71.75 (C2ꢀ); 70.88 (DMT); 69.48 (C3ꢀ); 62.68 (C5ꢀ); 55.88 (CH3 (DMT); 55.86 [CH3
(DMT]; 21.19 [(CH3)5ꢀ]; 21.01 [(CH3)3ꢀ] and 20.66 [(CH3)2ꢀ].
ꢀ
ꢀ
Syntheses of -nitro-guanosine ()
Product 4 (350.0 mg, 0.46 mmol) was dissolved in CHCl3 before the addition of
a solution of TsOH (38.0 mg, 0.23 mmol) in MeOH (4.6 mL). After 30 min, the
solution was neutralized with triethylamine (32.0 μL, 0.23 mmol) and evaporated
to dryness. The resulting solid was washed with ether a couple of times and the
solution was monitored with TLC in 100% ethyl acetate until the spot corresponding
to DMT disappeared. Then, the residue was dissolved in 7M NH3 in MeOH, in a
sealed vessel and stirred at room temperature for 48 h. The reaction was monitored
by TLC using DCM in 15% of methanol and 2 drops of triethylamine, when no more
reactan tprotected with the acetyl groups could be detected, the solvent and NH3
were removed in vacuo. The resulting product contained traces of acid, so it was
washed with isopropanol, evaporated and purified by HPLC. It led to the isolation
of product 5 (6.9 mg, 4.6%). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 11.18 (1H, s,
ꢀ
NH); 7.11 (2H, s, NH2); 6.29 (1H, d, 3JH1 H2ꢀ = 5.2 Hz, H1ꢀ); 5.41 (1H, s, OH); 5.07
ꢀ
ꢀ
ꢀ
(1H, s, OH); 4.95 (1H, t, 3JH2 H3ꢀ = 5.3, H2ꢀ); 4.87 (1H, s, OH); 4.24 (1H, t, 3JH3 H4
ꢀ
ꢀ 3
ꢀ
ꢀ
= 5.0, H3ꢀ); 3.88 (1H, q, 3J
/ JH4 H5ꢀꢀ = 4.7, H4ꢀ); 3.70 (1H, dd, 2JH5 H5ꢀꢀ = 11.7,
H4 H5
H5ꢀ); 3.56 (1H, dd, H5ꢀꢀ). 13C NMR (125 MHz, DMSO-d6): δ (ppm) 157.72 (C6);
156.48 (C2); 153.48 (C4); 144.44 (C8); 116.13 (C5); 91.45 (C1ꢀ); 86.61 (C4ꢀ); 72.05
(C2ꢀ); 71.03 (C3ꢀ); 62.80 (C5ꢀ).
3. Results and discussion
The synthesis of the 8-nitroguanosine derivatives is shown in Figure 5. The tau-
tomerism analysis of 8-NO2-guanosine (see Figure 3 for tautomeric forms) was per-
formed in relation to the guanosine data, since it is reported to exist in the 6-keto
form.[11] The 6-keto-nitroguanosine form is 0.3 kcal mol−1 more stable than the
6-enol-nitroguanosine. This study took into account experimental values of 15N and

8
T. M. BARBOSA ET AL.
Figure . Synthesis of -nitroguanosine and its derivatives using the procedures outlined in Ref. .
Table . Experimental chemical shifts and coupling constants related to N of the reagent, interme-
diates and final product, as well as, the theoretical chemical shift of N for both tautomeric forms.
N Chemical shift (ppm)
Experimental Theoretical -keto form Theoretical -enol form JNH Coupling constant (Hz)
Guanosine
(1)
(2)
(3)
(4)
(5)
.∗
–
.
.
.
–
.
–
–
–
–
.
–
–
–
–
.??
–
.
.
.
–
.
.
∗Ref.[]
13C chemical shifts, as well as 15N coupling constants. In addition to that, theoret-
ical calculations were performed to evaluate the 13C and 15N chemical shifts; the
geometry of the compounds was optimized at B3LYP and MP2 level of theory and
aug-cc-pvDZ basis set, and the chemical shift values were obtained using BLYP level
of theory and IGLO III[13] basis set (Tables 1–3).
1
The H-15N HSQC contour maps of all intermediates involved in the 8-NO2-
guanosine synthesis provided us directly with the 15N chemical shifts, and its slice
gave us the 1JN-H. Figure 6 shows an example of 1H-15N HSQC for 4, however the
broad signal (∼400 Hz) of H-1 from compound 5 precluded its analysis.
The analyses of Table 1 shows that neither NH chemical shifts nor NH coupling
constants change significantly in both experimental and theoretical approach, which
Table . Experimental chemical shifts and coupling constants related to N of protected guanosine
and protected NO-guanosine.
N Chemical shift (ppm)
JNH Coupling constant (Hz)
Guanosine-OAc-DMT
Guanosine-NO2-OAc-DMT
.
.
.
.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Table . Experimental and theoretical C chemical shifts (ppm) for guanosine and -NO-guanosine.
Experimental
Theoretical
-NO-
guanosine
-keto-
guanosine
-enol-
guanosine
-keto-NO
-guanosine
-enol-NO
-guanosine
guanosine
C
C
C
C
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
implies that there is no change in the tautomerism equilibrium among these com-
pounds. Thus, as it was not possible to analyse the 8-NO2-guanosine, it was decided
to synthesize the guanosine with the acetyl and DMT protecting groups and com-
pare their data with the corresponding values for 8-NO2-guanosine with the pro-
tecting groups to detect the NO2 effect (Table 2). The synthetic route to obtain
the guanosine with the protecting groups was the same as described for 8-NO2-
guanosine apart from that only steps 1 and 3 were performed.[4]
The values shown in Table 2 confirm that the presence of the NO2 group does
not affect the tautomeric equilibrium, since no change in chemical shift or in cou-
pling constants can be observed comparing the two compounds. The final analyses
of tautomerism of 8-NO2-guanosine were performed comparing experimental and
theoretical 13C chemical shifts (Table 3). The theoretical chemical shift was calcu-
lated by the following equation:[13]
δ = σ − σ .
(1)
st
i

Figure . H–N HSQC contour map of guanosine-NO-OAc-DMT, 4 (product from step ).

10
T. M. BARBOSA ET AL.
where δ is the chemical shift and σ and σ are the shielding constants of the refer-
st
i
ence (TMS = 176.96 ppm) and the nuclei, respectively, which were calculated at the
same level of theory.
It is clear that the enol tautomer exhibits a larger C6 chemical shift in comparison
with the keto form for both compounds. The 13C spectrum shows only one signal in
the region of 158 ppm. Therefore, it is clear that there is only one tautomer present,
and it is the keto form for the guanosine and for the NO2-guanosine.
4. Conclusion
The 15N chemical shift and coupling constant data, obtained through 1H-15N HSQC
and 1H-15N HMBC contour maps, show that any structural modification could be
detected from the values obtained for guanosine and 8-nitro-guanosine. Thus, it
can be assumed that the NO2 does not affect the tautomeric equilibrium, and it is
predominantly in the keto-form. This idea was enhanced by experimental and the-
oretical data of 13C chemical shifts, which also did not show any modification.
Acknowledgments
The authors thank for scholarship to T.M.B. #2013/08251-2; São Paulo Research Foundation
(FAPESP) and also the Chemistry Institute of University of Campinas (UNICAMP) and the
Chemistry Department of University of Liverpool for the facilities.
References
1. Balkwill, F.; Mantovani, A. Inflammation and cancer: back to Virchow? Lancet 2001, 357,
539–545.
2. Mantovani, A.; Allavena, P.; Sica, A.; Balkwill, F. Cancer-related inflammation, Nature 2008,
454, 436–444.
3. Yermilov, V.; Rubio, J.; Ohshima, H. Formation of 8-nitroguanine in DNA treated with per-
oxynitrite in vitro and its rapid removal from DNA by depurination, Febs. Lett. 1995, 376,
207–210.
4. Bhamra, I.; Compagnone-Post, P.; O¢Neil, I.A.; Iwanejko, L.A.; Bates, A.D.; Cosstick, R. Base-
pairing preferences, physicochemical properties and mutational behavior of the DNA lesion
8-nitroguanine, Nucleic Acids Res. 2012, 40, 11126–11138.
5. Coussens, L. M.; Werb, Z. Inflammation and cancer, Nature 2002, 420, 860–867.
6. Yermilov, V.; Rubio, J.; Becchi, M.; Friesen, M.D.; Pignatelli, B.; Ohshima, H. Formation of
8-nitroguanine by the reaction of guanine with peroxynitrite in vitro, Carcinogenesis 1995,
16, 2045–2050.
7. Steenken, S.; Jovanovic, S.V. How easily oxidizable is DNA? One-electron reduction poten-
tials of adenosine and guanosine radicals in aqueous solution, J. Am. Chem. Soc. 1997, 119,
617–618.
8. Neeley, W.L.; Essigmann, J.M. Mechanism of formation, genotoxicity, and mutation of gua-
nine oxidation products, Chem. Res. Toxicol. 2006, 19, 491–505.
9. Sawa, T.; Tatemichi, M.; Akaike, T.; Barbin, A.; Ohshima, H. Analysis of urinary 8-
nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
chromatography-electrochemical detection coupled with immunoaffinity purification: asso-
ciation with cigarette smoking, Free Radic. Biol. Med. 2006, 40, 711–720.
10. Garrett, E.R.; Mehta, P.J. Solvolysis of adenine nucleosides. 1. Effects of sugars and adenine
substituents on acid solvolyses, J. Am. Chem. Soc. 1972, 94, 8532–8541.
11. Cho, B.P.; Kadlubar, F.F.; Culp, S.J.; Evans, F.E. 15N Nuclear Magnetic resonance studies
on the tautomerism of 8-hydroxy-2ꢀ-deoxyguanosine, 8-hydroxyguanosine, and other C8-
substituted guanine nucleosides, Chem. Res. Toxicol. 1990, 3, 445–452.
12. Suzuki, T.; Kosaka, A.; Inukai, M. Formation of 8-S-L-cysteinylguanosine from 8-
bromoguanosine and cysteine, Bioorg. Med. Chem. Lett. 2013, 23, 3864–3867.
13. Standara, S.; Malinakova, K.; Marek, R.; Marek, J.; Hocek, M.; Vaara, J.; Straka, M. Under-
standing the NMR chemical shifts for 6-halopurines: role of structure, solvent and relativistic
effects, Phys. Chem. Chem. Phys. 2010, 12, 5126–5139.