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908 Chem. Res. Toxicol., Vol. 12, No. 10, 1999
Guengerich et al.
(4) Kin etic An a lysis. For the formation of amination
products of Guo, Guo or dGuo (2.5 mg) was mixed with 9 mg of
2,4-dinitrophenoxyamine in 1 mL of a DMF/H2O mixture (3:1,
v:v) in an amber glass vial under Ar at 45 °C. At different times,
25 µL aliquots were analyzed by HPLC, using a Beckman 10
mm × 250 mm octadecylsilane column and increasing the
concentration of CH3OH in 10 mM NH4CH3CO2 buffer (pH
5.0): 1% during the first 12 min, increasing from 1 to 4.5% from
12 to 15 min, and increasing from 4.5 to 22.5% from 15 to 30
min (all % v:v). The eluate was monitored by collecting data
from 220 to 350 nm with a rapid scanning spectrophotometer.
The A254 traces were used in the kinetic analysis, and spectra
were used for peak verification.
In the analysis of N7-NH2Guo decomposition kinetics, N7-
NH2Guo was repurified by preparative HPLC, using a slight
modification of the system described above for analysis, and
diluted directly into buffers at different pHs. The sample was
held at room temperature under Ar in amber glass, and at
various times, aliquots were analyzed by HPLC using the
system described above.
and biphenyl were prepared by ammonium bisulfite reduction
of their corresponding nitroarenes as described previously (33,
34). 2-[15N]Nitrofluorene was synthesized by nitration of fluo-
rene with Na15NO3. Briefly, 100 mg of fluorene was dissolved
in 5 mL of glacial CH3CO2H and added to a solution of 54 mg of
Na15NO3 in 3 mL of dry CF3CO2H. After the solution had been
mixed, the reaction was allowed to proceed for 24 h at room
temperature. The crude product was precipitated by addition
of 20 mL of H2O, collected by centrifugation, washed with H2O,
and then dried in vacuo. The residue was dissolved in benzene
and further purified by column chromatography on neutral
alumina using the same solvent as the eluant. The yellow band
was collected; the product was crystallized by addition of hexane
and dried in vacuo. The yield of 2-[15N]nitrofluorene was 51%
and was judged to be >98% pure by HPLC: UV (HPLC solvent)
λmax ) 229 nm; MS m/z (relative intensity) 212 (M+, 100), 195
(M+ - OH, 34), 165 (M - 15NO2, 93).
(5) C8,N9-Me2-N7-(2-[15N]Am in oflu or en yl)Gu a . This was
prepared from the reaction of 8,9-Me2Gua with 2-[15N]NHOH-
fluorene and acetylsalicylic acid and isolated by HPLC as
described previously (13).
Syn t h esis. (1) 2,4-Din it r op h en oxya m in e. 1-Chloro-2,4-
dinitrobenzene was reacted with ethyl N-hydroxyacetamidate
in the presence of 1 equiv of KOH to yield ethyl O-(2,4-
dinitrophenyl)hydroxamate in 53% yield (28): mp 110-111.5
°C (uncorrected) (lit. 110-112 °C); UV (C2H5OH) ꢀ308 ) 10 300
(6) C8-(2-[15N]Am in oflu or en yl)Gu o. This compound was
prepared by the reaction of Guo with 2-[15N]NHOH-fluorene and
acetylsalicylic acid and isolated by HPLC (13).
(7) N7-NH2Gu o. The material was prepared by reaction of
Guo with excess 2,4-dinitrophenoxyamine in 3:1 v:v DMF/H2O
at 45 °C under Ar. For the material used to analyze the kinetics
of degradation, 5.8 mg of Guo was incubated with 23 mg of 2,4-
dinitrophenoxyamine. The sample used to obtain the rotational
rotating frame Overhauser enhancement spectroscopy (ROESY)
NMR spectrum was prepared from a mixture of 25 mg of Guo
and 105 mg of 2,4-dinitrophenoxyamine. The reactions were
monitored by analytical HPLC, and after ∼2 days, the solvent
was removed by lyophilization following the addition of H2O.
The residue was dissolved in HPLC buffer using sonication and
purified by preparative HPLC using the Beckman 10 × 250 mm
octodecylsilane column and slight modification of the program
used for analysis. The peak was collected, concentrated by
lyophilization, repurified using the same HPLC system, con-
centrated by lyophilization, and stored in a desiccator at -20
°C.
M-1 cm-1 1H NMR (C2HCl3) δ 1.40 (t, 3H, CH2CH3), 2.25 (s,
;
3H, NdCCH3), 4.22 (q, 2H, CH2CH3), 7.89 (d, 1H, J ) 9.4 Hz,
aryl H-3), 8.41 (dd, 1H, J ) 2.7, 9.3 Hz, aryl H-5), 8.90 (d, 1H,
J ) 2.7 Hz, aryl H-6). The product (2.17 g) was treated with
70% HClO4 in 1,4-dioxane on ice to yield 2,4-dinitrophenoxy-
amine, with the reaction monitored by TLC (silica gel G, 3:1
v:v CH2Cl2/cyclohexane), in 62-84% yield for several prepara-
tions (29). The phenoxyamine was carefully crystallized from
hot C2H5OH: mp 102-105 °C (lit. 112-113 °C); UV (C2H5OH)
1
ꢀ298 ) 10 500 M-1 cm-1; H NMR (C2HCl3) δ 6.39 (s, 2H, NH2),
8.06 (d, 1H, J ) 9.4 Hz, aryl H-3), 8.45 (dd, 1H, J ) 2.7, 9.4 Hz,
H-5), 8.82 (d, 1H, J ) 2.6 Hz, H-6). The phenoxyamine was
mixed with acetophenone to yield the hydroxamate derivative:
mp 169-172 °C (uncorrected) [lit. 175 °C (30)].
(2) [8-13C]Gu o. [8-13C]Gua (120 mg in 80 mL of 1 N HCl, 10
mM) was added to 400 mL of 0.20 M Tris base and the pH
adjusted to 7.0, followed by the addition of 300 mg of ribose
1-phosphate and 9 mg of purine nucleoside phosphorylase (31).
HPLC analysis (25) indicated that the reaction was >90%
complete in 1 h (23 °C). The reaction mixture was applied to a
4.5 cm × 50 cm Sephadex G-10 column (Pharmacia, Piscataway,
NJ ), previously equilibrated with 25 mM CH3CO2H (pH 3.5),
and eluted with the same solvent (room temperature). Fractions
containing Guo, as judged by A254 measurements, were combined
and concentrated by lyophilization. [8-13C]Guo was purified by
preparative HPLC using a 10 mm × 250 mm Beckman octa-
decylsilane column (10 µm) using 4% CH3OH (v/v) in 10 mM
NH4CH3CO2 (pH 5.0), and the material containing [8-13C]Guo
was combined and concentrated by lyophilization.
(3) N2-NH2dGu o (2-Hydr azin odeoxyin osin e) (32). 2-Fluoro-
O6-(trimethylethysilyl)deoxyinosine (18 mg, 49 µmol) was re-
acted with NH2NH2‚H2O (1.0 µmol) in 1.0 mL of DMF at 50 °C
for 24 h under Ar. Glacial CH3CO2H (0.25 mL) was added, and
the mixture was heated at 50 °C for 2 h. The product was
separated by preparative HPLC (5 to 45% CH3OH gradient over
the course of 25 min, v:v, in 10 mM NH4CH3CO2, pH 5.0, flow
rate of 4 mL/min) and concentrated by lyophilization to yield
∼3 mg of recovered product: UV (HPLC solvent) λmax ) 254,
265-290 nm (shoulder); MS m/z (relative intensity) 283 (MH+,
23), 167 (MH+ - deoxyribose, 100); 1H NMR (Me2SO-d6) δ 2.19
(m, 1H, H-2′), 2.50 (m, 1H, H-2′′), 3.48 (m, 2H, H-5, H-5′′), 3.78
(dd, 1H, H-4′), 4.33 (s, 1H, OH), 4.93 (dd, 1H, H-3′), 5.27 (s, 1H,
OH), 6.12 (t, 1H, H-1′), 7.92 (s, 1H, H-8), 8.30 (s, broad, 1H,
NH). In typical HPLC systems, the compound was eluted
immediately prior to dGuo.
Sp ectr a l Ch a r a cter iza tion of Rea ction P r od u cts (2,4-
Din it r op h en oxya m in e a n d Gu o R ea ct ion or Decom p o-
stion of N7-NH2Gu o). (1) N7-NH2Gu o (24): UV λmax 259, 278
nm (shoulder); MS m/z (relative intensity) 299 (M+, 43), 167 (M+
1
- ribose, 100); H NMR (Me2SO-d6) δ 3.57 (m, 1H, H-3′), 3.68
(m, 1H, H-5′′), 3.95 (m, 1H, H-4′), 4.11 (m, 1H, H-3′), 4.41 (dd,
1H, H-2′), 5.73 (d, 1H, H-1′), 6.33 (s, broad, 2H, 2-NH2), 6.94 (s,
broad, 2H, N7-NH2), 9.08 (s, 1H, H-8) (see Figure 3 for NMR).
(2) C8:5′-O-CycloGu o (24): UV λmax 252, 280 nm; MS m/z
(relative intensity) 282 (MH+, 57), 304 (M + Na+, 100), 321 (M
+ K+, 75); CID [m/z 282 (relative intensity)] daughter ion at
m/z 168 (MH+ - ribose, 30); 1H NMR (Me2SO-d6) δ 3.85 (m,
1H, H-5′), 3.92 (m, 1H, H-5′′), 4.13 (m, 1H, H-4′), 4.40 (m, 1H,
H-3′), 4.50 (m, 1H, H-2′), 5.75 (d, 1H, H-1′).
(3) 8-OxoGu o (35, 36): UV λmax 247, 294 nm; MS m/z
(relative intensity) 300 (MH+, 22), 168 (MH+ - ribose, 100); 1H
NMR (Me2SO-d6) δ 3.55 (m, 2H, H-5′, H-5′′), 3.78 (m, 1H, H-4′),
4.09 (m, 1H, H-3′), 4.82 (t, 1H, H-2′), 5.58 (d, 1H, H-1′), 6.48 (s,
broad, NH), 6.58 (s, 1H, NH).
(4) C8-NH2Gu o: UV λmax 256, 291 nm; MS m/z (relative
intensity) 299 (MH+, 91), 321 (M + Na+, 95), 337 (M + K+, 45),
167 (M - ribose, 100); 1H NMR (Me2SO-d6) δ 3.60 (m, 2H, H-5′,
H-5′′), 3.86 (m, 1H, H-4′), 4.07 (m, 1H, H-3′), 4.53 (m, 1H, H-2′),
5.02 (d, 1H, 5′-OH), 5.26 (d, 1H, 3′-OH), 5.50 (m, 1H, 2′-OH),
5.73 (d, 1H, H-1′), 5.96 (s, 2- or 8-NH2), 6.14 (s, 2- or 8-NH2),
6.58 (s, 1H, N1H).
(5) N7-NH2Gu a : UV λmax 244, 286 nm; MS m/z (relative
intensity) 167 (MH+, 100), 149 (MH+ - H2O, 35); CID [m/z 167
(relative intensity)] m/z 150 (MH+ - NH3).
(6) 2-Am in o-5-(N-a m in o-N-for m yla m in o)-6-(N-glycosyl-
a m in o)-4-oxo-3,4-d ih yd r op yr im id in e (N-NH2 F AP Y glyco-
(4) 2-NH OH -F lu or en e, 2-[15N]NH OH -F lu or en e, a n d 4-
NHOH-Bip h en yl. The N-hydroxyamino derivatives of fluorene