150 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Golding et al.
C-2), 146.8 [s, d and d, 1J C-
N-8
) 12.5 Hz, 1J J C-
) 23.9 Hz,
15
15
powder (120 mg, 71%). The procedure was repeated with
[amino-15N] guanosine (containing 15-20% of molecules with
15N at N-1, 75% of molecules with 15N at NH2, and 5% of
molecules with only 14N) (28, 29) to give the 15N-labeled adduct
3c.
N-5
1
15N-8
C-4a], 149.9 (s, C-3a), 153.8 [s and d, J C-
) 8.7 Hz, C-9]
[n.b. C-9, C-4a, C-7, and C-9a were all shifted upfield due to
15N]; MS (FAB negative ion) m/z 693 [(M + 3Na - H)-], 671
[(M + 2Na - H)-], 649 [(M + Na - H)-], 561 [(M + 3Na - ribose
- H)-], 539 [(M + 2Na - ribose - H)-], 517 [(M + Na - ribose
15N-labeled adduct 3b: 1H NMR [300 MHz, (CD3)2SO] δ 3.53
(2H, m, 2 × H-5′), 3.91 (1H, m, H-4′), 4.13 (1H, m, H-3′), 4.48
- H)-]; IR νmax (KBr disk) 3420s, 1714s, 1555s cm-1
.
3
(1H, m, H-2′), 4.86 (2H, d, 3J ) 6.8 Hz, 7-CH2), 5.03 [1H, t, J
An a lyses of th e Glycid a ld eh yd e-Gu a n osin e Rea ction
by 1H NMR. To a heterogeneous suspension of guanosine (3.4
mg, 0.012 mmol) in borax-D2O buffer (pH 10, 0.6 mL) was
added glycidaldehyde (0.09 mg, 0.08 µL, 0.012 mmol). The
reaction was monitored at room temperature by 1H NMR at
intervals: after ca. 10 min, 35 min (see Figure 1), 1 h 40 min,
and 2 days.
) 6 Hz, OH (C-5′)], 5.13 (1H, t, 3J ) 6 Hz, 7-CH2OH), 5.22 (1H,
3
3
d, J ) 6 Hz, 3′-OH), 5.48 (1H, d, J ) 6 Hz, 2′-OH), 5.83 (1H,
d, 3J ) 5.7 Hz, H-1′), 7.24 (1H, d, 3J ) 3.2 Hz, H-6), 8.14 (1H,
s, H-2), 12.4 (1H, br s, NH) [a small amount (5%) of ethenogua-
nosine 5b was present: δ 7.39 (1H, t, J ) 3 Hz, H-6 or H-7),
7.58 (1H, t, J ) 3 Hz, H-6 or H-7), 8.06 (1H, s, H-2), and the
remaining resonances were superimposed on the corresponding
signals for 3b]; 13C NMR [90 MHz, (CD3)2SO] δ 55.0 (s, C-7′),
An a lyses of th e Glycid a ld eh yd e-Gu a n osin e Rea ction
by HP LC (with O. J . Taylor). The retention times of the
adducts 3a and 4a in an HPLC system equipped with an
analytical Ultrasphere ODS 5 µm column, with gradient elution
starting with water and ending with 60% methanol in water,
were 1.21 (adduct 3a ) and 1.46 min (adduct 4a ) relative to
guanosine. Time course monitoring of the reaction was per-
formed by using either a Partisil 5/25 ODS3 or Spherisorb 5
RP18 analytical column eluted with an aqueous methanol
61.2 (s, C-5′), 70.2 (s, C-3′), 73.6 (s, C-2′), 85.1 (s, C-4′), 86.7 (s,
2
15N-8
C-1′), 113.8 (s, C-6), 116.0 [s and d, J C-
) 10 Hz, C-9a],
1
15N-8
124.7 [s and d, J C-
15
d and d, J (CC-
N-8
C-3a), 153.7 [s and d, J C-
) 10 Hz, C-7], 137.3 (s, C-2), 146.7 [s,
1
1
15
) 13 Hz, J C-
) 24 Hz, C-4a], 150.2 (s,
) 9 Hz, C-9] (n.b. C-9, C-4a, and
N-5
1
15
N-8
C-7 were all shifted upfield, whereas C-9a was shifted downfield
due to 15N); MS (FAB negative ion) m/ z 337 [(M - H)-], 205
[(M - ribose - H)-], 359 [(M + Na - H)-]; IR νmax 3395s, 1698s,
gradient at a flow rate of 1 mL min-1
.
1598m cm-1
.
Isola tion of 1,N2-Eth en ogu a n osin e (5a ) fr om th e Gly-
cid a ld eh yd e-Gu a n osin e Rea ction . To a stirred suspension
of guanosine (70 mg, 24.7 mmol) in water (12.8 mL), adjusted
to pH 9.9 with 0.1 M sodium hydroxide solution, was added
glycidaldehyde (18 mg, 16 µL, 0.25 mmol). The mixture was
stirred at room temperature for ca. 20 min and an aliquot (80
µL) was analyzed by HPLC [semipreparative Ultrasphere ODS
5 µm 25 × 1 cm column eluted at a flow rate of 3 mL min-1
with a gradient (Waters Model 6000A programmer, program 9)
starting at 8% methanol in water, increasing to 32% methanol
over 35 min, then maintaining for an additional 10 min]. The
fraction of tR ) 2.16 min (relative to guanosine at 1.00) was
collected and lyophilized to give adduct 5a : 1H NMR [360 MHz,
(CD3)2SO] δ 3.64 (2H, m, 2 × H-5′), 3.93 (1H, m, H-4′), 4.13 (1H,
m, H-3′), 4.62 (1H, m, H-2′), 5.1 [1H, t, 3J ) 5 Hz, OH (C-5′)],
5.38 [1H, d, 3J ) 6 Hz, OH (C-3′)], 5.62 [1H, d, 3J ) 4.5 Hz, OH
(C-2′)], 5.77 (1H, d, 3J ) 6 Hz, H-1′), 7.24 (1H, d, 3J ) 2.5 Hz,
H-7), 7.52 (1H, d, 3J ) 2.5 Hz, H-6), 7.95 (1H, s, H-2), 8.46 (1H,
br s, H-5); MS (FAB positive ion) m/ z 308 (MH+).
15N-labeled adduct 3c: 13C NMR [90 MHz, (CD3)2SO] δ 55.0
(s, C-7′), 61.2 (s, C-5′), 70.2 (s, C-3′), 73.6 (s, C-2′), 85.1 (s, C-4′),
86.7 (s, C-1′), 113.8 [d, 1J C- N-5 ) 11 Hz, C-6], 115.8 (br s, C-9a),
15
1
15N-5
124.7 (s, C-7), 137.3 (s, C-2), 146.7 [s, d and d, J C-
) 12.5
1
15N-8
Hz, J C-
) 23.5 Hz, C-4a], 150.2 (s, C-3a), 153.7 (s, C-9).
P r ep a r a tion of 7,7′-Meth ylen ebis[5,9-d ih yd r o-9-oxo-3-
(â-D-r ibofu r a n osyl)im id a zo[1,2-a ]p u r in e (4a ). To a stirred
solution of guanosine (504 mg, 1.78 mmol) in water (90 mL),
adjusted to pH 11 with aqueous 0.1 M sodium hydroxide, was
added glycidaldehyde (139 mg, 122 µL, 1.92 mmol). The mixture
was stirred for 2 days at room temperature, after which time
the blue solution was lyophilized and then reconstituted in
water. Cooling at 0 °C for ca. 5 h produced a white precipitate,
which was filtered and recrystallized from ca. 10% dimethyl
sulfoxide in water to give 4a (690 mg, 62%): mp 310 °C; 1H
NMR [360 MHz, (CD3)2SO] δ 3.53 (4H, m, 4 × H-5′), 3.94 (2H,
m, 2 × H-4′), 4.15 (2H, m, 2 × H-3′), 4.50 (2H, m, 2 × H-2′),
5.09 [2H, br s, CH2 (C-7)], 5.18 [2H, d, 3J ) 5 Hz, 2 × OH (C-
Rea ction of 1,N2-Eth en ogu a n osin e w ith F or m a ld eh yd e.
To a stirred solution of 1,N2-ethenoguanosine in water was
added an excess of 13 M formaldehyde solution at room
temperature, and the reaction was monitored by HPLC [Ultra-
sphere ODS 25 × 0.46 cm diameter column eluted at 1 mL min-1
with a gradient (Waters program 9) starting at 16% methanol
in water and ending at 40% methanol in water over 20 min].
3
5′)], 5.45 [4H, m, 2 × OH (C-3′), 2 × OH (C-2′)], 5.83 (2H, d, J
) 6 Hz, 2 × H-1′), 7.02 (2H, s, 2 × H-6), 8.08 (2H, s, 2 × H-2);
13C NMR [90 MHz, (CD3)2SO] δ 23.8 (7-CH2), 61.4 (C-5′), 70.3
(C-3′), 73.6 (C-2′), 85.2 (C-4′), 86.9 (C-1′), 114.5 (C-6), 116.1 (C-
9a), 121.8 (C-7), 136.9 (C-2), 146.9 (C-4a), 153.9 (C-3a), 158.4
(C-9); IR (KBr disk) νmax 3420s, 1702s, 1600s, 1527s, 770m cm-1
;
UV λmax (0.1 M NaOH), 310 (ꢀ ) 13 700 dm3 mol-1 cm-1), 280
(14 300), 254 (13 000) nm; MS (FAB negative ion) m/z 625
(M - H)-, 648 [(M + Na - H)- 648. Anal. Calcd for
C25H26N10O10: C, 47.9; H, 4.2; N, 22.4. Found: C, 46.0; H, 4.45;
N, 21.1. Anal. Calcd for C25H26N10O10‚2H2O: C, 45.3; H, 4.6;
N, 21.1.
Id en tifica tion of F or m a ld eh yd e in th e Glycid a ld e-
h yd e-Gu a n osin e Rea ction . To a stirred suspension of
guanosine (53 mg, 0.185 mmol) in water (9.6 mL), adjusted to
pH 10 with 0.1 M sodium hydroxide at room temperature, was
added glycidaldehyde (17 mg, 14 µL, 0.23 mmol). The reaction
was monitored by HPLC [Ultrasphere ODS 25 × 0.46 cm
diameter column eluted at a flow rate of 1 mL min-1 with a
gradient (Waters program 9) starting at 8% methanol in water,
increasing to 32% methanol over 20 min, and then maintaining
for an additional 10 min]. After 2 min 1,N2- ethenoguanosine
(5a ) was detected. The reaction mixture was cooled to -20 °C
after 10 min of reaction. Analysis by HPLC showed the absence
of adduct 4a . 3-Methyl-2-benzothiazolinone hydrazone (80 mg,
0.37 mmol) was added, and the solution was stirred for 1 h at
room temperature. Extraction with dichloromethane (4 × 10
mL), drying (sodium sulfate), filtration, and evaporation gave
a crude yellow product (50 mg), which was purified by prepara-
tive layer chromatography on silica gel (elution with dichlo-
romethane, recycled plate 4×) to give formaldehyde MBTH 6
(3 mg, 8.5%), identified by comparison (TLC, MS, and NMR)
with authentic material (31).
P r ep a r a tion of 15N-La beled Ad d u ct 4b. The procedure
described earlier was followed, except that [1-15N]guanosine (158
mg, 0.56 mmol) was allowed to react with glycidaldehyde (44
mg, 39 µL, 0.61 mmol) at pH 11. The product was isolated as
a fine white powder (220 mg, 63%): 1H NMR [300 MHz, (CD3)2-
SO] δ 3.63 (4H, m, 4 × H-5′), 3.95 (2H, m, 2 × H-4′), 4.15 (2H,
m, 2 × H-3′), 4.53 (2H, m, 2 × H-2′), 5.08 [2H, s, CH2 (C-7)],
5.20 [2H, m, 2 × OH (C-5′)], 5.44 [4H, m, 2 × OH (C-3′), 2 ×
OH (C-2′)], 5.78 (2H, d, 3J ) 5.7 Hz, 2 × H-1′), 6.96 [2H, d, J H-
15N-
8 ) 3.2 Hz, 2 × H-6], 8.08 (2 H, s, 2 × H-2) [n.b. this spectrum
also showed the presence of compound 3b (ca. 5%) characterized
by a doublet at δ 7.13 (3J ) 2.3 Hz) from coupling of H-6 with
15N-8 and a singlet at δ 8.13 due to H-2; the remaining proton
resonances of 3b were superimposed on the corresponding
signals in compound 4b]; 13C NMR [75 MHz, (CD3)2SO] δ 23.6
[s, CH2 (C-7)], 61.3 (s, C-5′), 70.3 (s, C-3′), 73.7 (s, C-2′), 85.1 (s,
2
15N-8
C-4′), 86.9 (s, C-l′), 114.4 (br s, C-6), 116.1 [s and d, J C-
)
Id en tifica tion of F or m a ld eh yd e in th e Con ver sion of
Ad d u ct 3a to 4a . A stirred solution of adduct 3a (15 mg, 0.043
9.9 Hz, C-9a], 121.8 [s and d, 1J C-
) 10.4 Hz, C-7], 136.9 (s,
15
N-8