640 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Fu et al.
On the basis of 1H NMR spectroscopic analysis in the presence of
a known quantity of tert-butanol as internal standard, 3.8 mg of
DHP-dA-1 (0.07% yield) and 3.6 mg of DHP-dA-2 (0.067% yield)
were obtained.
DHP-H6a), 3.82 (1H, m, dG-H4′), 3.84-3.90 (1H, m, DHP-H7b),
3.92-3.97 (1H, m, DHP-H7a), 4.26 (1H, t, J ) 14, DHP-H9b),
4.35 (1H, two sets of d, J ) 14.2, DHP-H9a), 4.35 (1H, m, dG-
H3′), 4.74 (1H, dd, J ) 6.4, 7.6, DHP-H5), 6.11 (1H, d, J ) 2.7,
DHP-H2), 6.17 (1H, m, dG-H1′), 6.68 (1H, two sets of d, J ) 2.7,
DHP-H3), 7.89 (1H, s, dG-H8).
1H NMR spectroscopic data for each of the isolated adducts
follow.
DHP-dA-1. 1H NMR (DMSO-d6) δ 2.23-2.31 (1H, m, dA-H2′′),
2.35-2.47 (1H, m, DHP-H6b), 2.68-2.78 (1H, m, dA-H2′), 2.85-2.93
(1H, m, DHP-H6a), 3.49-3.56 (1H, two sets of d, J ) 4.5, dA-H5′′),
3.60-3.66 (1H, two sets of d, J ) 4.5, dA-H5′), 3.85-3.93 (2H, m,
DHP-H5b, dA-H4′), 4.01-4.25 (3H, m, DHP-H5a, H9a, H9b),
4.38-4.45 (1H, m, dA-H3′), 4.48 (1H, bs, OH), 5.10-5.45 (2H, bs,
overlapped, OH), 5.78 (1H, bs, DHP-H7), 6.04-6.10 (1H, two sets
of d, J ) 2.5, DHP-H2), 6.35-6.38 (1H, dd, dA-H1′), 6.60-6.67 (1H,
two sets of d, J ) 2.5, DHP-H3), 8.07 (1H, bs, dA-N6H), 8.27 (1H,
bs, dA-H8), 8.35 (1H, s, dA-H2).
1
DHP-dG-1. H NMR (DMSO-d6) δ 2.21-2.25 (1H, m, dG-
H2′′), 2.34-2.38 (1H, m, DHP-H6b), 2.62-2.68 (1H, m, dG-H2′),
2.86-2.90 (1H, m, DHP-H6a), 3.49-3.54 (1H, m, dG-H5′′),
3.57-3.60 (1H, m, dG-H5′), 3.80-3.83 (1H, m, dG-H4′), 3.89-3.94
(1H, m, DHP-H5b), 3.99-4.05 (1H, m, DHP-H5a), 4.28 (1H, bd,
DHP-H9b), 4.32 (1H, bd, DHP-H9a), 4.38-4.40 (1H, m, dG-H3′),
4.51 (1H, bs, OH/NH), 4.87 (1H, bt, OH/NH), 5.25-5.28 (2H, m,
DHR-H7, OH/NH), 6.09 (1H, d, J ) 2.6, DHP-H2), 6.20 (1H, dd,
J ∼ 7, dG-H1′), 6.66 (1H, d, J ) 2.6, DHP-H3), 6.73 (1H, bs,
OH/NH), 7.93 (1H, s, dG-H8), 10.31 (1H, s, dG-NH1).
DHP-dA-1. 1H NMR (DMSO-d6/D2O) δ 2.25-2.31 (1H, m, dA-
H2′′), 2.33-2.47 (1H, m, DHP-H6b), 2.67-2.73 (1H, m, dA-H2′),
2.87-2.96 (1H, m, DHP-H6a), 3.48-3.56 (1H, two sets of d, J )
4.5, dA-H5′′), 3.59-3.62 (1H, two sets of d, J ) 4.5, dA-H5′),
3.84-3.92 (2H, m, DHP-H5b, dA-H4′), 4.04-4.23 (3H, m, DHP-
H5a, H9a, H9b), 4.37-4.43 (1H, m, dA-H3′), 5.72 (1H, bs, DHP-
H7), 6.04-6.06 (1H, two sets of d, J ) 2.5, DHP-H2), 6.34 (1H,
dd, dA-H1′), 6.59-6.65 (1H, two sets of d, J ) 2.5, DHP-H3)
8.25 (1H, s, dA-H8), 8.32 (1H, s, dA-H2).
DHP-dG-1. 1H NMR (DMSO-d6/D2O) δ 2.21-2.26 (1H, m, dG-
H2′′), 2.31-2.37 (1H, m, DHP-H6b), 2.61-2.67 (1H, m, dG-H2′),
2.83-2.90 (1H, m, DHP-H6a), 3.80-3.83 (1H, m, dG-H4′),
3.87-3.91 (1H, m, DHP-H5b), 3.99-4.03 (1H, m, DHP-H5a), 4.25
(1H, d, J ) 12, DHP-H9b), 4.29 (1H, d, J ) 12, DHP-H9a),
4.37-4.39 (1H, m, dG-H3′), 5.25-5.27 (1H, m, DHP-H7), 6.08
(1H, d, J ) 2.5, DHP-H2), 6.19 (1H, dd, J ∼ 7, dG-H1′), 6.65
(1H, d, J ) 2.5, DHP-H3), 7.91 (1H, s, dG-H8).
1
DHP-dG-2. H NMR (DMSO-d6) δ 2.21-2.26 (1H, m, dG-
DHP-dA-2. 1H NMR (DMSO-d6) δ 2.23-2.32 (1H, m, dA-H2′′),
2.34-2.45 (1H, m, DHP-H6b), 2.68-2.78 (1H, m, dA-H2′), 2.84-2.94
(1H, m, DHP-H6a), 3.47-3.56 (1H, m, dA-H5′′), 3.57-3.66 (1H, m,
dA-H5′), 3.81-3.94 (2H, m, DHP-H5b, dA-H4′), 4.04-4.25 (3H, m,
DHP-H5a, H9a, H9b), 4.41 (1H, bs, dA-H3′), 4.47 (1H, bs, OH), 5.25
(1H, bs, OH), 5.33 (1H, bs, OH), 5.78 (1H, bs, DHP-H7), 6.06 (1H,
d, J ) 2.0, DHP-H2), 6.35-6.38 (1H, dd, dA-H1′), 6.60-6.67 (1H,
two sets of d, J ) 2.0, DHP-H3), 8.06 (1H, bs, dA-N6H), 8.27
(1H, bs, dA-H8), 8.35 (1H, s, dA-H2).
H2′′), 2.34-2.39 (1H, m, DHP-H6b), 2.62-2.68 (1H, m, dG-H2′),
2.86-2.90 (1H, m, DHP-H6a), 3.49-3.54 (1H, m, dG-H5′′),
3.57-3.60 (1H, m, dG-H5′), 3.81-3.83 (1H, m, dG-H4′), 3.89-3.94
(1H, m, DHP-H5b), 4.00-4.05 (1H, m, DHP-H5a), 4.29-4.34 (1H,
m, DHP-H9b), 4.32-4.34 (1H, m, DHP-H9a), 4.38-4.40 (1H, m,
dG-H3′), 4.51 (1H, t, OH/NH), 4.87 (1H, t, OH/NH), 5.25-5.28
(2H, m, DHP-H7, OH/NH), 6.09 (1H, d, J ) 2.5, DHP-H2), 6.20
(1H, dd, J ∼ 7.1, dG-H1′), 6.66 (1H, d, J ) 2.5, DHP-H3), 6.73
(1H, bs, OH/NH), 7.92 (1H, s, dG-H8), 10.28 (1H, s, dG-NH1).
DHP-dG-2. 1H NMR (DMSO-d6/D2O) δ 2.21-2.23 (1H, m, dG-
H2′′), 2.34-2.36 (1H, m, DHP-H6b), 2.63-2.67 (1H, m, dG-H2′),
2.83-2.90 (1H, m, DHP-H6a), 3.80-3.83 (1H, m, dG-H4′),
3.87-3.92 (1H, m, DHP-H5b), 4.00-4.03 (1H, m, DHP-H5a), 4.26
(1H, d, J ) 12, DHP-H9b), 4.30 (1H, d, J ) 12, DHP-H9a),
4.36-4.38 (1H, m, dG-H3′), 5.25-5.27 (1H, m, DHP-H7), 6.08
(1H, d, J ) 2.5, DHP-H2), 6.19 (1H, dd, J ∼ 7, dG-H1′), 6.65
(1H, d, J ) 2.5, DHP-H3), 7.91 (1H, s, dG-H8).
DHP-dA-2. 1H NMR (DMSO-d6/D2O) δ 2.24-2.32 (1H, m, dA-
H2′′), 2.33-2.44 (1H, m, DHP-H6b), 2.62-2.74 (1H, m, dA-H2′),
2.85-2.96 (1H, m, DHP-H6a), 3.47-3.56 (1H, two sets of d, J )
4.0, dA-H5′′), 3.65 (1H, m, dA-H5′, overlapped with D2O),
3.84-3.92 (2H, m, DHP-H5b, dA-H4′), 4.02-4.23 (3H, m, DHP-
H5a, H9a, H9b), 4.37-4.43 (1H, m, dA-H3′), 5.74 (1H, bs, DHP-
H7), 6.03-6.09 (1H, two sets of d, J ) 2.0, DHP-H2), 6.32-6.35
(1H, dd, dA-H1′), 6.60-6.65 (1H, two sets of d, J ) 2.0, DHP-
H3), 8.25 (1H, s, dA-H8), 8.32 (1H, s, dA-H2).
DHP-dG-3. 1H NMR (DMSO-d6) δ 2.19-2.26 (2H, m, dG-H2′′,
DHP- H6b), 2.54-2.68 (2H, m, dG-H2′, DHP-H6a), 3.48-3.53
(1H, m, dG-H5′′), 3.57-3.59 (1H, m, dG-H5′), 3.81-3.85 (2H,
m, dG-H4′, DHP-H5b), 3.99-4.03 (1H, m, DHP-H5a), 4.23-4.28
(1H, m, DHP-H9b), 4.31-4.37 (1H, m, DHP-H9a), 4.35-4.38 (1H,
m, dG-H3′), 4.81 (1H, m, OH/NH), 5.06-5.08 (1H, m, DHP-H7),
5.14-5.15 (1H, m, OH/NH), 5.26-5.27 (1H, m, OH/NH), 6.09
(1H, d, J ) 2.6, DHP-H2), 6.18 (1H, m, dG-H1′), 6.34 (1H, m,
OH/NH), 6.61 (1H, d, J ) 2.5, DHP-H3), 7.91 (1H, s, dG-H8),
10.40 (1H, s, dG-NH1).
Synthesis of Isotopically Labeled DHP-dG and DHP-dA
13
Adducts. DHP-[15N5]dG and DHP-[15N5, C10]dA adducts were
prepared as described above by reacting DHR (3.0 mg, 0.02 mmol)
with [15N5]dG (5 mg, 0.02 mmol) and [15N5,13C10]dA (5 mg, 0.02
mmol), respectively.
Chemical Reaction of DHR with Calf Thymus DNA. To
prepare DNA standards modified with DHR, 5 mg of calf thymus
DNA in 2 mL of 5 mM Tris-HCl buffer, pH 7.1, containing 0.1
mM EDTA (Tris-EDTA) was reacted with 0, 0.1, 0.5, 1, 5, and 50
µg of DHR in 100 µL of methanol at 37 °C for 3 h. After the
incubation, the reaction mixture was extracted twice with 5 mL of
a chloroform/isoamyl alcohol mixture (24/1, v/v). The DNA in the
aqueous phase was precipitated by adding 250 µL of 3 M sodium
acetate followed by an equal volume of cold 2-propanol and washed
with 70% ethanol. After the DNA was dissolved in Tris-EDTA,
the DNA concentration was determined spectrophotometrically. The
DNA solution was stored at -78 °C prior to hydrolysis and LC-
ES-MS/MS analysis.
The deoxynucleoside adducts were quantified by HPLC-ES-MS/
MS after standard enzymatic hydrolysis of the DNA to deoxy-
nucleosides (43). The hydrolysis mixtures from 100 µg aliquots of
the DHR-modified DNA were analyzed directly, without any adduct
enrichment procedure.
HPLC-ES-MS/MS Analysis of DHP-Derived DNA Adducts
Formed in Vitro and in ViWo. Quantification of DHP-DNA
Standards. Purified samples of the DHP-dG-1 and DHP-dG2
adduct standards were quantified spectrophotometrically on the basis
of the UV molar extinction coefficients 1.70 × 104 and 1.72 × 104
DHP-dG-3. 1H NMR (DMSO-d6/D2O) δ 2.18-2.25 (2H, m, dG-
H2′′, DHP- H6b), 2.55-2.66 (2H, m, dG-H2′, DHP-H6a), 3.78-3.83
(2H, m, dG-H4′, DHP-H5b), 3.98-4.03 (1H, m, DHP-H5a), 4.24
(1H, two sets of d, J ) 14, DHP-H9b), 4.32 (1H, two sets of d,
DHP-H9a), 4.33-4.38 (1H, m, dG-H3′), 5.03-5.08 (1H, m, DHP-
H7), 6.07 (1H, d, J ) 2.7, DHP-H2), 6.19 (1H, m, dG-H1′), 6.60
(1H, d, J ) 2.5, DHP-H3), 7.89 (1H, s, dG-H8).
1
DHP-dG-4. H NMR (DMSO-d6) δ 2.18-2.24 (1H, m, dG-
H2′′), 2.34-2.37 (1H, m, DHP-H6b), 2.56-2.65 (2H, m, dG-H2′,
DHP-H6a), 3.49-3.53 (1H, m, dG-H5′′), 3.54-3.57 (1H, m, dG-
H5′), 3.82 (1H, m, dG-H4′), 3.86-3.92 (1H, m, DHP-H7b),
3.95-4.00 (1H, m, DHP-H7a), 4.25-4.32 (1H, m, DHP-H9b),
4.33-4.36 (1H, m, DHP-H9a), 4.33-4.37 (1H, m, dG-H3′), 4.76
(1H, dd, DHP-H5), 4.85 (1H, bs, OH/NH), 5.25 (1H, d, OH/NH),
6.12 (1H, d, DHP-H2), 6.18 (1H, m, dG-H1′), 6.43 (1H, bs, OH/
NH), 6.70 (1H, two sets of d, DHP-H3), 7.90 (1H, s, dG-H8), 8.56
(1H, s, OH/NH), 10.38 (1H, bs, dG-NH1).
DHP-dG-4. 1H NMR (DMSO-d6/D2O) δ 2.19-2.24 (1H, m, dG-
H2′′), 2.34-2.39 (1H, m, DHP-H6b), 2.56-2.65 (2H, m, dG-H2′,