Cheng and McClelland
1883
guanosine, rather than 2>-deoxyguanosine, were employed
for economical reasons. A further aspect of the present study
involves the measurement of the kinetics of the initial reac-
tion of the two methylated guanine derivatives with nitrenium
ions. These show some interesting effects that are also consis-
tent with the initial reaction occurring at the C-8 position.
4-biphenylyl (455 nm), 4>-methyl-4-biphenylyl (475 nm),
and 4>-methoxy-4-biphenylyl (500 nm). First-order rate con-
stants were obtained by fitting the decay traces to the equa-
tion for a single exponential process. Solutions were flashed
once and replaced with 4 to 5 kinetic runs being performed
with each solution.
Lamp flash photolysis experiments were performed using
an apparatus previously described (20). Aqueous solutions
were prepared containing 2 × 10–4 M of the nucleoside. The
pH was maintained by HCl, acetate buffer, or cacodylate
buffer. In the case of the buffers, serial buffer dilutions were
employed so that rate constants could be extrapolated to zero
buffer concentrations. Shortly before irradiation, a concen-
trated acetonitrile solution of 2-fluorenyl azide was added to
give a final azide concentration of 8 M. The solution was
irradiated with a broad band flash lamp of ca. 100 s dura-
tion. The absorbance decay at 365 nm was monitored and fit
to the exponential equation. For a given solution, 4 to 5 ki-
netic traces were obtained and the rate constants for these
averaged. After irradiation, the pH of the solution was mea-
sured.
Experimental section
2-Fluorenyl azide (12), 4-biphenylyl azide (12), 4>-methyl-
4-biphenylyl azide (14), and 4>-methoxy-4-biphenylyl azide
(14) were from previous studies. N1-Methylguanosine was
prepared by the reaction of guanosine and N,N-dimethyl-
acetamide dimethyl acetal in methanol (15). N2,N2-
Dimethylguanosine was prepared in a multistep procedure:
(i) guanosine was converted to 2>,3>,5>-tri-O-acetylguanosine
by the reaction with acetic acid anhydride in the presence of
4-dimethylpyridine and triethylamine (16); (ii) 2>,3>,5>-tri-O-
acetylguanosine was reacted with phosphorus oxychloride in
acetonitrile containing tetraethylammonium chloride and
N,N-dimethylaniline to give 2-amino-6-chloro-9-(2, 3, 5-tri-
O-acetyl-b-D-ribofuranosyl)purine (17); (iii) this was con-
verted to 2-amino-6-chloro-9-b-D-ribofuranosylpurine by
treatment with saturated methanolic ammonia (18); (iv) the 2-
amino-6-chloro-9-b-D-ribofuranosylpurine was reacted with
sodium benzyloxide in benzyl alcohol to give 2-amino-6-
benzyloxy-9-b-D-ribofuranosylpurine (91); (v) diazotization
with sodium nitrite in 48% aqueous fluoroboric acid af-
forded 2-fluoro-6-benzyloxy-9-b-D-ribofuranosylpurine (19);
(vi) the fluoride was substituted in 20% methanolic
dimethylamine to give 2-dimethylamino-6-benzyloxy-9-b-D-
ribofuranosylpurine (19); (vii) hydrogenolysis with palla-
dium on carbon gave the desired product N2,N2-dimethyl-
guanosine (19). Full experimental details of our preparations
of N1-methylguanosine and N2,N2-dimethylguanosine are
available as supplementary material.2
Laser flash photolysis experiments involved ca. 20 ns pulses
at 308 nm (60–120 mJ per pulse) from a Lumonics excimer
laser. A pulsed Xenon lamp provided the monitoring light.
After passing through a monochromator, the signal from the
photomultiplier tube was digitized and sent to a computer
for analysis. Stock solutions of the nucleosides — guanosine
(0.001 M), N1-methylguanosine (0.001 M), and N2,N2-
dimethylguanosine (0.0004 M) — were prepared in 20% aceto-
nitrile containing 0.002 M NaH2PO4:0.002 M Na2HPO4. For
each nucleoside, a series of solutions of 100 mL volume were
prepared by adding volumes of the stock solutions — 100, 80,
60, 40, 20, 0 mL — to a 100 mL volumetric flask, and filling
to the mark with the 20% acetonitrile solution containing the
phosphate buffer. These 100 mL solutions were divided into
four 25 mL volumetric flasks. A concentrated acetonitrile
solution of the four azides — 2-fluorenyl, 4-biphenylyl, 4>-
methyl-4-biphenylyl, and 4>-methoxy-4-biphenylyl azide —
was added to each of these flasks immediately before irradi-
ation to give a solution of final concentration ~10 M. This
solution was transferred to a 4 × 1 × 1 cm cuvette, and irra-
diated at 308 nm, with the decay of the transient nitrenium
ion being monitored at its ꢁmax (14) — 2-fluorenyl (450 nm),
Results
Laser flash photolysis — Reaction of arylnitrenium
ions and nucleosides
In these experiments, four nitrenium ions were generated
and their decay studied in the presence of the three nucleo-
sides guanosine, N1-methylguanosine, and N2,N2-dimethyl-
guanosine. The nucleosides significantly accelerated the
decay of the nitrenium ion. Plots of the observed first-order
rate constant for the decay were linear in the concentration
of the nucleosides. The slopes of these plots are the second-
order rate constants for the reaction of the nitrenium ion and
the nucleoside. These rate constants are provided in Table 1,
along with pertinent values that had been previously ob-
tained.
Lamp flash photolysis — Decay of intermediate
These experiments were conducted with the intermediate
formed by the reaction of the 2-fluorenylnitrenium ion and
the three nucleosides guanosine, N1-methylguanosine, and
N2,N2-dimethylguanosine. Concentrations of the nucleo-
sides were kept constant at 200 M, a concentration where
greater than 90% of the nitrenium ion reacts with the added
nucleoside. As observed in the previous experiments with 2>-
deoxyguanosine, a second intermediate is observed follow-
ing the initial reaction of the nitrenium ion and the
nucleoside. We monitored the decay of this intermediate at
365 nm, a wavelength where there is a significant difference
between the absorbance of the intermediate and the
absorbance of the final product. Rate constants were ob-
tained in a series of HCl solutions and in acetate and
cacodylate buffers. In the case of the buffers, serial buffer di-
lutions were performed. The buffer does accelerate the rate
of decay (5). This aspect of the kinetics was not investigated
in detail in this study, since our interest was in the rate con-
stant extrapolated to zero buffer concentration. These rate
2 Copies of material on deposit may be purchased from the Depository of Unpublished Data, Document Delivery, CISTI, National Research
© 2001 NRC Canada