Insights into the biological redox chemistry of 2′-deoxyadenosine 5′-monophosphate by electrochemical techniques

Article abstract of DOI:10.1246/bcsj.78.1944

The electrochemical oxidation of 2′-deoxyadenosine 5′-monophosphate at a pyrolytic graphite electrode (PGE) was studied in the pH range 2.65-10.03 and was found to proceed in a single well-defined oxidation peak, I_a, over the entire pH range. The cyclic voltammetric behavior indicated the pH dependence of the oxidation pathway. The kinetic studies of the UV-absorbing intermediate generated during electrooxidation were followed spectrophotometrically, and decay occurred in a pseudo first-order reaction having k values in the range (0.595-0.986) × 10^-3 s^-1 over the entire pH range studied. Coulometric studies indicated that the process of the oxidative pathway involved n values as 5.0 ± 0.5 in the acidic range (pH 3.37) and 4.0 ± 0.5 at physiological pH 7.22. The products of electrooxidation were characterized by GC-MS and on the basis of electrochemical, spectrochemical, and product analysis; a plausible redox mechanism is suggested.

Full text of DOI:10.1246/bcsj.78.1944

1944 Bull. Chem. Soc. Jpn., 78, 1944–1952 (2005)
ꢀ 2005 The Chemical Society of Japan
Insights into the Biological Redox Chemistry of 20-Deoxyadenosine
50-Monophosphate by Electrochemical Techniques
ꢀ
Rajendra N. Goyal and Aikta Dhawan
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247 667, India
Received March 7, 2005; E-mail: rngcyfcy@iitr.ernet.in
The electrochemical oxidation of 20-deoxyadenosine 50-monophosphate at a pyrolytic graphite electrode (PGE)
was studied in the pH range 2.65–10.03 and was found to proceed in a single well-defined oxidation peak, Ia, over
the entire pH range. The cyclic voltammetric behavior indicated the pH dependence of the oxidation pathway. The ki-
netic studies of the UV-absorbing intermediate generated during electrooxidation were followed spectrophotometrically,
and decay occurred in a pseudo first-order reaction having k values in the range ð0:595{0:986Þ ꢁ 10ꢂ3 sꢂ1 over the entire
pH range studied. Coulometric studies indicated that the process of the oxidative pathway involved n values as 5:0 ꢃ 0:5
in the acidic range (pH 3.37) and 4:0 ꢃ 0:5 at physiological pH 7.22. The products of electrooxidation were character-
ized by GC-MS and on the basis of electrochemical, spectrochemical, and product analysis; a plausible redox mechanism
is suggested.
20-Deoxyadenosine 50-monophosphate (20-dAMP) is one of
the deoxyribonucleotides that form a skeletal framework of the
hereditary carrier DNA, and is a phosphate derivative of 20-de-
oxyadenosine, a purine nucleoside.1 It is present in the embryo
of copepod Euchaeta Japonica before hatching.2 20-dAMP
blocks the bitter taste of foods and beverages by a pharmaceu-
tically active oral dose that comes in contact with the taste tis-
sues.3 It is thus used in oral (mouth wash) formulations.4 Fast-
dissolving edible films containing 20-dAMP are formulated to
inhibit the activation of bitter taste G-protein sensory percep-
tion of bitter-tasting medicaments.5 It is reported that 20-dAMP
on selective hydrolysis in the presence of Xanthomonas malto-
philia, Escherichia coli, Pseudomonas Putida, and Lactobacil-
lus Acidophilus leads to the formation of nucleoside 20-deoxy-
adenosine with no accumulation of free bases.6 20-dAMP sup-
presses hematologic cell growth exhibiting an anti-prolifera-
tive effect on leukemic cells.7 It also inhibits RNAse H
activity of human hepatitis B virus polymerase, and prevents
the replication of human hepatitis B virus (HBV).8 Adefovir
dipivoxil (ADV) is esterologically cleaved to the 20-dAMP an-
alog, which upon phosphorylation acts as an anti-Hepatitis B
virus (HBV) agent.9 20-dAMP has been reported to prevent
mitochondrial DNA depletion in a resting culture of deoxy-
guanosine kinase deficient fibroblast.10 It has been reported
to inhibit deoxynucleotide synthesis in chick embryos, due to
which embryos usually die, or give rise to malformed chicks.11
20-Deoxyadenosine 50-monophosphate has potential as
drugs for the prevention and treatment of amyloidosis, includ-
ing Alzheimer’s disease, by displacing the serum amyloid P
(SAP) component, a glycoprotein associated with amyloid de-
posists.12 In view of the importance of dAMP, adducts of a car-
cinogenic metabolite of 6-NC (6-nitrochrysene) with 20-deoxy-
adenosine 50-monophosphate have been synthesized in vitro.13
Its halogenated derivative, 2-chloro-20-deoxyadenosine 50-
monophosphate, has been separated from 2-chloro-20-deoxy-
adenosine by reversed-phase HPLC.14 The molecular confor-
mational changes of 20-deoxyadenosine 50-monophosphate,
induced by hexadecyltrimethylammonium bromide (NTAB)
micelles and dimethyldioctadecylammonium bromide (DAB)
bilayers, have been evaluated by CD spectroscopy.15 Oxygen
radicals produced in cells have been found to cause cell dam-
age to purine nucleotides and DNA by causing hydroxylation
at the C-2 position.16 The oxidatively damaged nucleotides,
particularly hydroxy derivatives, formed are incorporated into
growing DNA, and have been proved to be substrate for DNA
polymerases. The repair of oxidized OH radical adducts of de-
oxyadenosine monophosphate by phenylpropanoid glycosides
has been found to be useful17 for decreasing further oxidation
of OH adducts of dAMP and dGMP. A literature survey re-
vealed that practically no information is available concerning
the oxidation of 20-dAMP. In view of the importance of 20-
dAMP in biological systems, an attempt has been made to
study the electrochemical oxidation behavior of this com-
pound. The present work reports the results of electrooxidation
studies of 20-dAMP carried out at a pyrolytic graphite elec-
trode (PGE).
Experimental
Material. 20-Deoxyadenosine 50-monophosphate (dAMP) (I)
(Chart 1) was obtained from Aldrich, and was used as received.
N,N-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was obtained
from Sigma, and silylation-grade acetonitrile was a product of
Pierce Chemical Co., U. S. A. All other chemicals used were of
analytical grade. Cyclic sweep voltammetric studies were per-
formed on a Cypress system Inc. (Ver. 7.0, Kansas, U. S. A.).
The supporting electrolytes used throughout the study were
phosphate buffers (ꢀ ¼ 1:0 M) of different pH values, which were
prepared according to a method of Christian and Purdy18 from
reagent-grade chemicals. The pH values of the buffer solutions
were measured using a Century digital pH-meter (model CP-
901-P) after due standardization.
All potentials were reported with respect to the Ag/AgCl/KCl
Published on the web October 31, 2005; DOI 10.1246/bcsj.78.1944

R. N. Goyal et al.
Bull. Chem. Soc. Jpn., 78, No. 11 (2005) 1945
sample was injected in GC-MS.
NH
2
Procedure. For recording voltammograms, a stock solution
(1 mM) of 20-dAMP was prepared in doubly distilled water. The
stock solution (2.0 mL) was then mixed with 2.0 mL of phosphate
buffer (ꢀ ¼ 1 M) of the desired pH so that the effective ionic
strength of the solution became 0.5 M. Before recording the vol-
tammograms, the solutions were deoxygenated by passing a
stream of nitrogen gas for 10–12 min.
N
N
N
N
HO
P O
HO
O
O
H
H
H
H
The progress of the electrolysis was monitored by recording
spectral changes at different time intervals. For this purpose, elec-
trolysis was carried out in an H-cell at a potential 100 mV more
positive than the peak potential of the oxidation peak. For record-
ing the UV–vis spectrum, about 2–3 mL of the solution from the
electrolysis cell was transferred each time to a 1 cm quartz cell,
and the spectrum was recorded in the range 200–400 nm. In a sec-
ond set of studies, the potential was turned off by open-circuit re-
laxation when the absorbance at ꢁmax was reduced to 50%. The
change in absorbance with time at selected wavelengths was then
monitored. The values of the rate constant (k) were calculated
OHH
(I)
Chart 1.
(sat) electrode at an ambient temperature of 20 ꢃ 2 ꢄC. The work-
ing, reference, and counter electrodes were pyrolytic graphite
electrode (surface area of ꢅ6 mm2), Ag/AgCl/KCl (sat), and
platinum wire, respectively. The fabrication of the PGE used for
electrochemical studies was carried out in the laboratory by a
reported method.19 After each voltammogram, renewal of the
PGE surface was performed by rubbing it on emery paper,
followed by washing with a jet of distilled water and gently drying
with soft tissue paper. This resulted in a significantly new surface
for each run, and showed a variation of ꢅꢃ10% in peak currents
in repeated runs. Thus, the reported peak-current values are an
average of at least three runs. The number of electrons (n)
involved during electrooxidation were determined by monitoring
the exponential decay of the current–time curve.
from linear logðA ꢂ A Þ versus time plots.
/
The products of the electrooxidation of 20-dAMP were charac-
terized at pH 3.37 and pH 7.22 by exhaustively electrolyzing 15–
20 mg of the compound in a single compartment cell at a potential
100 mV more positive than the oxidation peak potential. The
progress of the electrolysis was monitored by recording the UV
spectra at different time intervals. When the oxidation peak (Ia)
disappeared, the electrolyzed solution was removed from the cell
and lyophilized. The products of electrooxidation were then char-
acterized by GC-mass and HPLC analysis.
UV spectral studies during the electrooxidation of 20-dAMP
and the kinetics of the UV-absorbing intermediate at the PGE
were monitored by withdrawing the sample at different times. A
Perkin Elmer-Lambda 35 UV–vis spectrophotometer was used
for monitoring the spectra in a 1 cm cell. TLC was performed us-
ing glass plates coated with silica gel-G using benzene–methanol
(4:1) as the solvent. Lyophilizer (Harrison) was used for freeze-
drying of an exhaustively electrolyzed solution. GC-mass data
(EI, 70 eV) were obtained on a Perkin Elmer Clares 500 Spec-
trometer, and HPLC analysis was carried out using an Agilent
1100 Series HPLC system.
Results and Discussion
Cyclic Sweep Voltammetry. Cyclic sweep voltammetry
of a 0.5 mM solution of 20-dAMP at a sweep rate of 100
mV sꢂ1 exhibited a well-defined oxidation peak (Ia) over the
entire pH range of 2.65–10.03 when the sweep was initiated
in the positive direction. In a reverse sweep, no cathodic peak
was obtained. A typical cyclic voltammogram of 20-dAMP for
PGE at pH 3.37 and 7.22 are presented in Fig. 1, depicting ox-
idation of the species generated during electrooxidation.
The peak potential of the oxidation peak (Ia) was dependent
on the pH, and shifted to a less-positive potential with an in-
crease in the pH. The Ep versus pH plot for the oxidation peak
Ia was linear Fig. 2 and the dependence of Ep on the pH using a
linear-regression analysis can be expressed by
For analysis by GC-MS, the dried extracted material (50–100
mg) was silylated with BSTFA (50ꢄ mL) and acetonitrile (50 mL)
in a sealed voil and heated at 110 C for about 12–15 min in an
oil bath.20 After cooling at room temperature, 2 mL of the silylated
Fig. 1. Typical cyclic voltammograms observed for 0.5 mM 20-deoxyadenosine 50-monophosphate at sweep rate 100 mV sꢂ1 in
phosphate buffers (A) at pH 3.37 and (B) at pH 7.22 at PGE.

1946 Bull. Chem. Soc. Jpn., 78, No. 11 (2005)
Redox Chemistry of 20-Deoxyadenosine Monophosphate
pffiffi
Fig. 2. Dependence of peak potential (Ep) on pH for the
voltammetric peaks Ia for 0.5 mM 20-deoxyadenosine 50-
monophosphate at sweep rate 100 mV sꢂ1 at PGE.
Fig. 4. Dependence of ip= v on log v for peak Ia of 0.5
mM 20-deoxyadenosine 50-monophosphate at pH 7.22 at
PGE.
Fig. 3. Dependence of peak current (ip) of peak Ia on con-
centration of 20-deoxyadenosine 50-monophosphate at pH
7.22, sweep rate 100 mV sꢂ1 at PGE.
Fig. 5. Dependence of Ep on log v for peak Ia of 0.5 mM
20-deoxyadenosine 50-monophosphate at pH 7.22 at PGE.
curve.23 The plot of current versus time was exponential in na-
ture. The value of n was calculated from the net charge passed
through the solution, and the average experimental value of n
was found to be 5:0 ꢃ 0:5 at pH 3.37 and 4:0 ꢃ 0:5 at pH 7.22.
Spectral Studies. The spectral changes during the electro-
oxidation of 20-dAMP were monitored at pH 3.37, 5.22, 7.22,
and 9.08. The UV-spectrum of 20-dAMP exhibited two well-
defined ꢁmax at 210 and 260 nm over the entire pH range.
The typical results of spectral changes observed at pH 3.37
are depicted in Fig. 6. Curve 1 in Fig. 6 is the initial spectrum
of 20-dAMP just before electrooxidation. Upon applying a po-
tential 100 mV more positive than that of oxidation peak (Ia),
the absorbance decreased systematically in the region 200–218
and 235–290 nm, as presented by (curves 2–10), whereas the
absorbance in the region 218–235 nm increased systematically
(curves 2–10). Two clear isosbestic points were observed at
218 and 290 nm, respectively. The decrease in absorbance at
longer wavelength indicated that the formed products of oxida-
tion of 20-dAMP formed absorbed at smaller wavelength due
to a less-conjugated nature compared with the starting materi-
al. Similar changes were observed at pH 5.22.
EpðIaÞ ¼ 1633:6 ꢂ 58:942pH;
ð1Þ
in the pH range 2.65–10.03, having a correlation coefficient
of 0.993, where Ep is in mV versus Ag/AgCl. This behavior
indicates the involvement of protons in the electrode process.
The peak current for the peak Ia was found to increase with
an increase in the 20-dAMP concentration in the range 0.05–
0.7 mM; at higher concentrations the peak current became con-
stant Fig. 3. This behavior indicated adsorption complications
in the electrode reaction.21 The effect of the sweep rate (v) on
the peak-current function of peak Ia was studied in the sweep
range 20–800 mV sꢂ1 at pH 7.22. A rapid increase in the
pffiffi
pffiffi
ip= v values with increasing sweep rate further indicate the
involvement of adsorption complications, as suggested by
Wopschall and Shain.21 In the present studies, a plot of
ip= v versus log v was found to increase with an increase in
the sweep rate, as presented in Fig. 4. The effect of the sweep
rate on Ep of the peak Ia was also studied in the sweep range
20–800 mV sꢂ1. The value of Ep shifted to more positive po-
tentials with an increase in the sweep rate, and a plot of Ep ver-
sus log v was linear with a correlation coefficient of ꢅ0:992
(Fig. 5). This behavior is consistent with the EC nature of
the reaction in which the electrode reaction is coupled with
an irreversible follow-up chemical step.22
Coulometric Studies. The controlled potential electrolysis
of 20-dAMP was performed in phosphate buffer at pH 3.37 and
pH 7.22 at a potential 100 mV more positive than peak Ia. The
number of electrons (n) involved in the electrooxidation was
determined by the graphical integration of the current time
At pH 7.22, curve 1 in Fig. 7 shows the initial spectrum of
dAMP just before electrooxidation. Upon the application of a
potential of 1.4 V, the absorbance in regions 200–218 and
240–282 nm decreased, as presented by (curves 2–9), whereas
in the regions 218–240 and 282–400 nm it increased systemati-
cally (curves 2–9). Three clear isosbestic points were observed
at 218, 240, and 282 nm, respectively. Similar changes were
also observed at pH 9.08.

R. N. Goyal et al.
Bull. Chem. Soc. Jpn., 78, No. 11 (2005) 1947
Fig. 8. Plot of absorbance versus time and logðA ꢂ A Þ vs
/
time (inset) plot measured at 260 nm for the first-order de-
cay of the UV-absorbing intermediate generated during
the electrooxidation of 0.5 mM 20-deoxyadenosine 50-
monophosphate at PGE, pH 7.22.
Fig. 6. Spectral changes observed during the electrooxida-
tion of 0.5 mM 20-deoxyadenosine 50-monophosphate at
pH 3.37 at PGE. Curves were recorded at (1) 0 min, (2)
5 min, (3) 10 min, (4) 20 min, (5) 30 min, (6) 45 min,
(7) 60 min, (8) 90 min, (9) 120 min, and (10) 180 min
of electrolysis.
Table 1. Observed k Values for the Decomposition of the
UV–Vis Absorbing Intermediate Generated during the
Electrooxidation of 20-Deoxyadenosine 50-Monophosphate
at PGE
pH
ꢁ
max/nm
k/10ꢂ3 sꢂ1
3.37
225
260
0.640
0.805
5.22
7.22
225
260
0.626
0.768
225
260
300
0.736
0.986
0.595
9.08
225
260
300
0.725
0.946
0.622
Fig. 7. Spectral changes observed during the electrooxida-
tion of 0.5 mM 20-deoxyadenosine 50-monophosphate at
pH 7.22 at PGE. Curves were recorded at (1) 0 min, (2)
5 min, (3) 15 min, (4) 25 min, (5) 40 min, (6) 55 min,
(7) 85 min, (8) 115 min, and (9) 175 min of electrolysis.
ic range (Rf ¼ 0:44, 0.49, and 0.54), and four spots at neutral
pH range (Rf ¼ 0:54, 0.63, 0.68, and 0.72).
The products of electrooxidation were further characterized
by HPLC and the GC-MS technique. The details of the char-
acterized products are as follows.
The kinetics of decay of the UV absorbing intermediate
generated was monitored after open-circuit relaxation. The
changes in the absorbance with time at selected wavelengths
were recorded and the resulting absorbance-versus-time pro-
files showed an exponential decay. The values of the pseudo
first-order rate constant (k) were determined at different pH,
In Acidic Range (pH 3.37): An HPLC analysis of the
dried extracted material dissolved in methanol exhibited three
peaks at Rt ꢅ 2:137, 2.284, and 2.328 min. Efforts to separate
the products were not successful due to close retention times
and overlapping of these peaks. The material was then silylat-
ed and analyzed by GC-MS. The GC-MS chromatogram gave
four peaks at Rt ꢅ 5:781, 6.018, 9.986, and 10.563 min having
m=z 449 ðM þ HþÞ, 465 ðM þ HþÞ, 908 (Mþ), and 937 (Mþ),
respectively (Chart 2). The data giving m=z values and the rel-
ative abundance of high-mass fragments in the fragmentation
pattern are summarized in Table 2.
In Neutral Range (pH 7.22): An HPLC analysis of the
dried extracted material dissolved in methanol exhibited four
peaks at Rt ꢅ 2:124, 2.285, 2.363, and 2.497 min. Due to a
close overlapping, these peaks could not be separated. A
GC-MS chromatogram of the silylated material gave four clear
using logðA ꢂ A Þ versus time plot (Fig. 8), and were found
/
to be in the range ð0:595{0:986Þ ꢁ 10ꢂ3
s
ꢂ1, as presented in
Table 1.
Product Characterization. The ultimate products of elec-
trooxidation of 20-dAMP were characterized at pH 3.37 and pH
7.22. For this purpose, exhaustively electrolysed solutions of
20-dAMP at these pH values were lyophilized. The freeze-
dried material was extracted using methanol (2 ꢁ 10 mL)
and methanolic extract was concentrated. The brownish-col-
ored material obtained exhibited three spots in TLC in an acid-

1948 Bull. Chem. Soc. Jpn., 78, No. 11 (2005)
Redox Chemistry of 20-Deoxyadenosine Monophosphate
1. −C-O-N- trimer:
1. −O-O- dimer:
(CH
) Si
]
[Si(CH
NH
)
N
N
N
[Si(CH
]
3 3
)
3 3
3 3
2
2
N
N
N
N
N
N
N
O
O
N
NH
N
O
O
N
N
N
O
(CH
)
Si
N
N
3 3
(CH ) Si O
3 3
Si(CH )
3 3
(CH
)
Si
O
N
N
N
Si(CH
)
3 3
3 3
(CH ) Si
3 3
(CH ) Si
3 3
N
N
N
O
) Si
3 3
(CH
(CH
)
3 3
Si
(Octasilylated)
m/z = 908
(VII)
(CH ) Si
3 3
(Heptasilylated)
m/z = 969
2. −C-O-N- trimer:
(XXVIII)
[Si(CH )
N
]
2
3 3
N
2. −C-N-C- trimer:
N
O
(CH ) Si
3 3
N
HN Si(CH
)
HN Si(CH
N
)
3 3
3 3
N
N
N
N
N
N
(CH ) Si
3 3
N
N
O
N
(CH ) Si
3 3
O
N
N
N
O
N
N
N
N
N
Si(CH
)
O
N
N
3 3
(CH ) Si
3 3
(CH ) Si
3 3
N
N
N
(CH
)
Si
N
3 3
(CH
) Si
3 3
(CH ) Si
3 3
(Hexasilylated)
m/z = 949
(Heptasilylated)
m/z = 937
(XXXII)
(XXII)
3. −C-(N-C)-C- trimer:
3. Hydrated alloxan:
N [Si(CH )
]
3 3
2
O
N
N
N
Si(CH
)
N
)
[Si(CH
N
]
O
Si(CH )
3 3
3 3
3 3
2
(CH ) Si
N
3 3
N
N
N
O
N
Si(CH )
3 3
N
(CH
)
Si
3 3
O
O
N
N
N
N
N
(CH ) Si
3 3
(CH )
3 3
Si
(CH
)
Si
3 3
(Tetrasilylated)
m/z = 448
(XVI)
(Octasilylated)
m/z = 977
(XXXVI)
Chart 3.
4. Urea deoxyribose:
H
N
tion presented above, the following mechanism can be sug-
gested for the electrooxidation of 20-dAMP (I) at PGE.
In Acidic Media: The oxidation of 20-dAMP (I) appears to
occur in a 1eꢂ, 1Hþ step to give a free-radical moiety, IIa. The
removal of 1eꢂ, 1Hþ would occur from the amino group at
position 6, as was reported earlier during the oxidation of pu-
rines.22 The free radical IIa formed has several possible reso-
nating structures (IIa–IIe), as shown in Scheme 1. The free rad-
icals can undergo electrodimerization by any of various mech-
anisms, such as radical–radical coupling or radical–substrate
coupling, as shown in Schemes 1 and 2.
(CH ) Si
3 3
O
(CH ) Si
3 3
N
O
(CH ) Si
3 3
O
(CH ) Si
O
3 3
(Tetrasilylated)
m/z = 464
(XVII)
Chart 2.
A parallel pathway also appears to take place for the electro-
oxidation of 20-dAMP, which involves the initial 2eꢂ, 2Hþ ox-
idation to form either a 2-hydroxy or a 8-hydroxy derivative.
However, based on studies of adenine oxidation24 reported in
the literature, it can be concluded that first 2eꢂ, 2Hþ oxidation
leads to the formation of 2-hydroxy-20-dAMP (III), which on
further 2eꢂ, 2Hþ oxidation gives 2,8-dihydroxy-20-dAMP
(IV). Such an oxidation at position 8 is well-documented for
purines and substituted purines.24 The 1eꢂ, 1Hþ oxidation of
2,8-dihydroxy-20-dAMP (IV) at position-8 to give a free-radi-
cal species (V) has been well reported22,25 for adenosine and
peaks at Rt ꢅ 6:017, 9.986, 17.480, and 17.955 min having
m=z at 970 ðM þ HþÞ, 908 (Mþ), 950 ðM þ HþÞ, and 977
(Mþ), respectively (Chart 3). The product corresponding to
m=z 908 was found to be an –O–O– linked dimer (VII), as ob-
served at pH 3.37. The data giving m=z values and the relative
abundance of high-mass fragments are summarized in Table 3.
The four major components were identified as the dimer/tri-
mer of 20-dAMP.
Redox Mechanism. Based on the voltammetric behavior,
electronic spectral data, coulometry, and product characteriza-

R. N. Goyal et al.
Bull. Chem. Soc. Jpn., 78, No. 11 (2005) 1949
Table 2. Relative Abundances Observed for High-Mass Fragments of the Peaks Observed in
GC-MS at pH 3.37
Abundance of different
fragments/%
Compound
identified
Rt/min
m=z
9.986
908
(1.91%)
878 (1.38), 454 (1.68), 365 (3.82),
294 (5.92)
VII
5.781
6.018
449
(5.46%)
433 (2.73), 381 (2.73), 371 (4.20),
369 (2.77), 360 (0.61)
XVI
XVII
XXII
465
(3.82%)
460 (6.94), 433 (1.86), 414 (2.42),
395 (5.00), 370 (5.35)
10.563
937
(6.62%)
892 (2.46), 877 (2.55), 659 (2.42),
366 (2.55), 293 (7.50), 278 (2.81)
Further hydration of imine alcohol (X) to give 4,5-diol (XI)
would be a slow step,31 and appears to be the step responsible
for the decay observed in the first-order reaction in the absorb-
ance vs time plot. Cleavage of the imidazole ring then occurs
to give the aminopyrimidine derivative (XII), urea deoxyribose
(XIII), and a phosphate moiety. A rupture of the imidazole ring
of purine nucleosides in acidic medium during oxidation has
been extensively reported in the literature.32,33 The hydrolysis
of compound (XII) in two subsequent steps gives hydrated al-
loxan (XV). The silylation of (XV) gave tetrasilylated hydrat-
ed alloxan (XVI) having m=z 448. Corresponding to the forma-
tion of XVI, a clear M þ Hþ peak having m=z 449 and a Mþ ꢂ
15 peak were obtained at Rt ꢅ 5:781 min in the GC-MS chro-
matogram. The formation of urea deoxyribose (XIII) can be
accounted for, on the basis of the peak obtained in GC-MS
at Rt ꢅ 6:018 min having m=z 465, which corresponds to the
M þ Hþ peak of the silylated urea deoxyribose (XVII) with
m=z 464.
The formation of compound XXI appears to be due to 1eꢂ,
1Hþ oxidation of 2-hydroxy-20-dAMP in a side reaction to
give a radical species (VIII), which rapidly combines with
the IIa radical species to give XVIII. Species XVIII upon
2eꢂ, 2Hþ oxidation gives dimer XIX having a hydroxy group
at the C0-2 position. The dimeric species XIX undergoes 1eꢂ,
1Hþ oxidation at the C0-2 position to give a radical species
XX, which rapidly combines with the IIa radical species to
form a trimer (XXI), as shown in Scheme 1. Silylation of
the trimer (XXI) gave compound XXII having m=z 937, which
was identified in GC-MS studies at Rt ꢅ 10:563 min. Interest-
ingly, in compound XXII all of the three deoxyribose phos-
phate units were hydrolyzed during silylation, and a heptasilyl-
ated trimer (XXII) was thus obtained. Compound XXI appears
to be formed as a minor product of oxidation, as judged by the
relative peak area of the peak at Rt ꢅ 10:563 min.
Table 3. Relative Abundances Observed for High-Mass
Fragments of the Peaks Observed in GC-MS at pH 7.22
Abundance of different
fragments/%
Compound
identified
Rt/min m=z
17.955
9.986
6.017
977
(2.53%) 467 (2.56), 190 (0.09)
917 (75.0), 904 (2.82), 627 (1.23), XXXVI
908 878 (1.46), 454 (1.76), 365 (1.24),
(4.85%) 294 (4.78)
VII
970
954 (2.56), 939 (6.08), 924 (2.56), XXVIII
(7.22%) 896 (64.0), 823 (1.47), 587 (5.28),
498 (8.85), 382 (5.45), 293 (1.41)
17.480
950
(8.46%)
934 (7.67), 671 (8.46), 205 (3.83)
XXXII
adenosine 50-monophosphate. The radical species (V) immedi-
ately dimerizes with a similar moiety to form an –O–O– linked
dimer (VI) through the C-8 and C0-8 positions (Scheme 1). The
silylation of the dimer (VI) gave compound VII having m=z
908 corresponding to the peak at Rt ꢅ 9:986 min in a GC-
MS chromatogram. It is interesting to observe that both of
the deoxyribose phosphate moieties of dimer VI hydrolyze
during silylation, which was also observed earlier by several
workers.22,26,27 This structure has eight reactive sites that can
be replaced by (CH3)3Si groups upon silylation. Thus, under
the silylation conditions used, all of the reactive sites were oc-
cupied by the trimethylsilyl group, and an octasilylated –O–O–
dimer (VII) was obtained. The oxidation of 2,8-dihydroxy-
deoxyadenosine (IV) can also proceed in an oxidative pathway
involving a 2eꢂ, 2Hþ step to give the corresponding diimine
species (IX). The diimine species (IX) obtained during the ox-
idation of purines was found to be unstable due to two C=N
bonds, and its short half life of a few ms (ꢅ50 ms) has been
already documented in the literature.28 Hence, the diimine spe-
cies (IX) will be readily attacked by water molecules to give
4,5-diol (XI). A similar attack of water molecules on diimine
has been reported in the case of large a number of purines.29,30
The formation of diol (XI) would occur in two distinct steps.
An attack of the first molecule of water would occur instanta-
neously in a chemical follow-up step to give imine alcohol (X).
In Neutral Media: In contrast to acidic pH, four products
identified by GC-MS at pH 7.22 can be explained as follows.
In the first course of the electrooxidation of 20-dAMP at pH
7.22, the radical species (V) formed by the overall involve-
ment of 5eꢂ, 5Hþ as presented in Scheme 1, dimerized with
a similar moiety to give VI. Thus, product VI was obtained
both in acidic as well as in neutral media. Corresponding to
the formation of VII in the neutral media, a clear peak having
m=z 908 was noticed in the GC-MS chromatogram. The radical

1950 Bull. Chem. Soc. Jpn., 78, No. 11 (2005)
Redox Chemistry of 20-Deoxyadenosine Monophosphate
.
NH
N
NH
2
NH
NH
N
NH
NH
N
2
N
N
-
-1e -1H
+
N
N
N
.
N
N
N
N
N
N
N
.
.
N
R
N
R
N
R
N
N
R
N
N
R
P
N
.
N
R
P
P
P
P
P
(I)
(II )
a
(II )
(II )
b
c
(II )
d
(II )
e
- +
O -2e -2H
H
2
NH
2
NH
NH
N
2
2
N
N
N
-
+
N
-1e -1H
(II )
a
N
N
+
O
HN
N
N
R
N
P
N
R
N
R
N
N
HO
O
N
P
.
P
(III)
N
R
(VIII)
- +
O -2e -2H
H
P
2
(XVIII)
NH
2
NH
NH
N
2
2
-
+
-2e -2H
H
N
N
-
+
-2e -2H
N
N
H
O
N
O
2
N
OH
O
O
NH
N
2
N
R
N
N
R
O
N
R
N
H
HO
P
N
N
P
P
N
(IV)
(IX)
O
HN
N
-
+
-1e -1H
H
O
2
P
R
N
N
H
H
O
HO
H
2
N
N
H
N
N
NH
N
R
2
(XIX)
2
H
N
O
H
N
P
N
(k)
N
N
N
-
+
-1e -1H
O
P
O
.
O
H
O
2
O
N
R
O
N
R
N
R
N
NH
O
H
2
HO
P
P
(V)
N
N
(X)
(XI)
O
HN
N
R
NH
N
2
P
N
O
N
N
O
+
NH CONHR+ P
2
(XIII)
.
NH
N
NH
O
2
2
N
R
N
(XX)
+(II
O
N
H
P
N
N
N
N
N
silylation
O
O
P
)
a
(XII)
NH
N
2
N
R
N
R
OH
N
HO
P
N
H
O
2
(VI)
m/z = 464
N
O
(XVII)
HN
N
silylation
P- R)
N
R
O
P
2 (
N
O
O
HN
O
HN
N
N
R
m/z = 908
O
N
H
P
N
(VII)
N
(XXI)
(XIV)
N
R
P
silylation
3 (P- R)
H
O
2
O
O
O
= P
R
P
m/z = 937
(XXII)
OH
HN
OH silylation
O
m/z = 448
HO
O
N
H
(XVI)
(XV)
Scheme 1. Tentative mechanistic pathway proposed for the electrooxidation of 20-deoxyadenosine 50-monophosphate at pH 3.37.
species (V) could also combine with radical IIa to give dimer
XXIII having an –O–N– linkage between the two units. The
species XXIII on further 2eꢂ, 2Hþ oxidation in presence of
water gave dimer XXIV having a hydroxy group at the C0-2
position. Further, 2eꢂ, 2Hþ oxidation of dimer XXIV in pres-
ence of water would give dimer XXV having a hydroxy group
at the C0-8 position. Finally, XXV underwent 1eꢂ, 1Hþ oxida-
tion at the C0-8 position to give a radical species XXVI which
rapidly combined with the IIa radical species to form a trimer
XXVII, as shown in Scheme 2. It is well documented in the
literature that the C-8 position is more prone to oxidation com-
pared to the C-2 position in purines.27 The silylation of the
trimer (XXVII) gave compound XXVIII having m=z 969.
The peak obtained in GC-MS at Rt ꢅ 6:017 min corresponds
to M þ Hþ having m=z 970, and indicates that all three deoxy-
ribose phosphate moieties of the trimer XXVII are removed
during silylation. Although trimer XXVII has nine silylable
sites, only seven underwent silylation under the conditions
used. The –O–NH– hydrogen did not appear to undergo silyla-
tion under the conditions used, as observed by the observed
fragmentation pattern. The high-mass peaks observed in the
fragmentation pattern are compiled in Table 3.
The formation of products having m=z 949 and 977 can be
explained based on the combination of radical IIe with other
free radicals. The dimer XXIX, having a –C–N–C– linkage be-
tween the two units, can be formed by the combination of IIa
with IIe. This dimer then undergoes 1eꢂ, 1Hþ oxidation at the
–NH– position to give a radical XXX, which further combines
with IIe radical to give a trimer, XXXI. Two deoxyribose phos-
phate moieties and a phosphate group seem to hydrolyze dur-
ing silylation to give XXXII having m=z 949. The structure of
XXXI has eight reactive sites, which can be replaced by
(CH3)3Si groups upon silylation. However, it was observed
that under the silylation conditions used, only one hydrogen

R. N. Goyal et al.
Bull. Chem. Soc. Jpn., 78, No. 11 (2005) 1951
NH
N
NH
N
2
2
N
N
N
N
.
O
.
N
R
N
R
HO
P
P
(V)
(II )
e
+(II
)
a
NH
NH
N
2
2
+(II )
e
+(II )
a
N
N
N
N
NH
N
NH
N
2
2
O
N
NH
NH
N
N
N
N
R
N
R
N
N
N
N
HO
N
P
P
N
N
R
N
R
N
R
N
R
N
P
+
(XXIII)
P
(XXIX)
P
P
(XXXIII)
-
-
+
-2e -2H
-
+
-1e -1H
H
O
-1e -1H
N
2
NH
N
NH
2
.
2
NH
NH
2
N
N
N
.
N
N
N
N
O
N
NH
N
N
R
N
R
N
R
N
N
R
N
N
HO
NH
N
N
P
N
P
N
P
P
(XXXIV)
N
R
N
R
N
(XXX)
HO
P
NH
N
P
2
(XXIV)
+(II )
e
N
-
H
+
-2e -2H
N
+(II )
e
NH
N
NH
2
2
O
NH
N
NH
N
2
2
2
N
N
R
N
N
N
N
N
N
N
P
N
N
O
N
NH
N
N
R
N
R
N
R
N
N
N
R
N
HO
N
R
P
P
N
P
P
P
N
OH
(XXXV)
N
R
N
N
R
HO
N
P
P
silylation
3 (P-R)
(XXV)
(XXXI)
silylation
[2 (P-R)+ P]
NH
N
2
-
+
-1e -1H
m/z = 977
(XXXVI)
N
N
O
NH
m/z = 949
(XXXII)
N
R
N
HO
P
N
.
O
N
R
N
HO
P
(XXVI)
+ (II )
a
NH
N
2
O
N
N
O
= P
R
P
N
O
N
NH
HO
N
HO
P
R
O
N
NH
N
R
N
P
HO
N
(XXVII)
N
R
N
P
silylation
3 (P-R)
m/z = 969
(XXVIII)
Scheme 2. Tentative mechanistic pathway proposed for the electrooxidation of 20-deoxyadenosine 50-monophosphate at pH 7.22.
of the amino group at the C-6 and C00-6 positions were re-
placed by trimethylsilyl groups to give the C–N–C trimer
XXXII having m=z 949. The peak obtained in GC-MS at Rt ꢅ
17:480 min corresponds to M þ Hþ peak having m=z 950. The
silylation of one hydrogen of the amino group in purines and
their nucleosides is not uncommon, and has been reported in
the literature.26 The formation of trimer XXXV appears to
be due to the combination of the radical species IIe with a sim-
ilar moiety to give dimer XXXIII having a C–C linkage. This
compound then undergoes 1eꢂ, 1Hþ oxidation at the amino
group at position 6 or 60, and a free radical, XXXIV, is likely
to be obtained. Oxidation at –NH– at position 6 in the case of a
dimer of purines is well reported in the literature.26 The free
radical XXXIV then combined with radical IIe to give a trimer,
XXXV, which upon silylation gave a peak in the GC-MS chro-
matogram at Rt ꢅ 17:955 min, having m=z 977. An analysis of
the trimer XXXVI indicates that it is octasilylated, and all
three deoxyribose phosphate units seem to hydrolyze during si-
lylation. Moreover, the increase in the absorbance in the higher
wavelength region (282–320 nm) during spectral analysis ap-
pears to be due to the formation of trimer XXXV, which pos-
sesses a highly conjugated ꢂ-system due to (C8–C08) linkage,
and hence absorbs at longer wavelengths.
Scheme 1 and Scheme 2 suggest the most probable routes
for the formation of various products of the electrooxidation,
but it should be noted that other pathways are always possible.
The tentative mechanism suggested here explains all of the
observed voltammetric, coulometric, and spectral data of 20-
dAMP. The formations of oxidative free radicals and dimeric
purine moieties have been detected in oxidized DNAs by many
workers.34 The purine dimers produced by the coupling of free
radicals generated in cellular DNA play a central role in neu-
rodegenerative diseases, such as Alzheimer’s disease, Down’s
Syndrome, and Xeroderma pigmentosum. To monitor the pres-
ence of such products, studies have been conducted on rabbit
anti-purine dimer antiserum using purine dimer 8-8-(20-deoxy-
guanosyl)-20-deoxyguanosine 50-monophosphate.34 Hence,
based on such studies it can be concluded that deoxyadenosine
nucleosides and deoxyadenosine nucleotides are also oxidized
in biosystems in a similar manner, leading to the formation of

1952 Bull. Chem. Soc. Jpn., 78, No. 11 (2005)
Redox Chemistry of 20-Deoxyadenosine Monophosphate
8
L. Young, B. H. Young, K. Younhee, M. R. Hyune, and
several dimers/trimers. Thus, the present studies on the oxida-
tive mechanism of 20-dAMP help us to understand the nature
of the intermediates and the products that can possibly form
during metabolic activities in human physiology, their toxic
behavior and interaction with proteins.
J. Guhung, Biochem. Biophys. Res. Commun., 233, 401 (1997).
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10 J. W. Taanman, J. R. Muddle, and A. C. Muntau, Hum.
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Conclusion
The electrooxidative mechanistic pathway of 20-deoxyade-
nosine 50-monophosphate was also compared with its parent
compound, 20-deoxyadenosine. In acidic media, 20-dAMP
upon electrooxidation led to the formation of –O–O– dimer,
alloxan, urea deoxyribose, and –C–O–N– trimer, different
from the products obtained on 20-deoxyadenosine oxidation.
It is interesting to observe that –O–O– dimer obtained in the
case of 20-dAMP electrooxidation lost both the moieties of de-
oxyribose phosphate during silylation, in contrast to the reten-
tion of both deoxyribose moieties in the case of the –O–O–
dimer of 20-deoxyadenosine. This can be accounted for due
to the presence of bulky deoxyribose phosphate units, com-
pared to simple deoxyribose. The electrooxidation of 20-dAMP
at neutral pH leads to the formation of the –O–O– dimer, –C–
O–N–, –C–N–C–, and –C–(N–C)–C– trimers. The hexasilylat-
ed trimer (XXXII) formed in the present studies was similar to
that obtained in the case of 20-deoxyadenosine. This happened
because of the hydrolysis of phosphate moieties during the
silylation process. Thus, the suggested oxidative pathways
help us to understand the mechanism of electron-transfer reac-
tions in the biological system.
22 R. N. Goyal and A. Sangal, J. Electroanal. Chem., 521, 72
(2002).
23 J. J. Lingane, ‘‘Electroanalytical Chemistry,’’ 2nd ed,
Wiley, New York (1966), p. 222.
24 R. N. Goyal, A. Kumar, and A. Mittal, J. Chem. Soc.,
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25 R. N. Goyal and A. Sangal, J. Electroanal. Chem., 557,
147 (2003).
A. Dhawan is thankful to the University Grant Commission,
New Delhi for the award of Junior Research Fellowship and
financial assistance for the work was provided vide grant
No. 6405-13-414.
26 P. Suramanian and G. Dryhurst, J. Electroanal. Chem.,
224, 137 (1987).
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