Vol. 46 No. 2
SCIENCE IN CHINA (Series B)
April 2003
Pulse radiolysis of one-electron oxidation of rare tricyclic
nucleoside derivative
ZHAO Hongwei ()1, JIANG Zhiqin ()2, DOU Daying ()1,
WU Tieyi (
)1, WANG Wenfeng ()1, DU Xiaoxing ()2
& YAO Side ()1
1. Shanghai Institute of Nuclear Research, Chinese Academy of Sciences, Shanghai 201800, China;
2. Department of Chemistry, Tongji University, Shanghai 200092, China
Correspondence should be addressed to Yao Side (email: [email protected])
Received November 15, 2002
Abstract One-electron oxidation of N-4-desmethylwyosine (dYt), a derivative of rare tricyclic nucleoside wyosine with several oxidative radicals, has been studied by using pulse radiolysis. The
transient spectra of the radicals dYt•+ and dYt(-H)• and the pKa of the radical cation were measured.
The rate constants of reactions of dYt with SO•4− , CO3• − , N3• and •OH radicals have been deter-
mined and related reaction mechanisms were discussed.
Keywords: rare tricyclic nucleoside derivative, one-electron oxidation, pulse radiolysis, radical, transient absorption
spectra.
Among the natural rare nucleosides found in tRNAsPhe, Y-nucleosides are hypermodified and
distinctive. They are the derivatives of guanosine with the unique tricyclic structure. RajBhandary
et al.[1] found a highly fluorescent Y-nucleoside first time in yeast phenylalanine transfer ribonucleic acid tRNAPhe in 1967. Similar structural rare nucleosides were also found later in various
tRNAPhe sources such as Torulopsis utilis, wheat germ and some mammalian liver[2,3]. The rare
Y-nucleosides adjacent to the 3
-end of the anticodon loop of tRNAPhe and are involved in the recognition of the phenylalanine codons. Their glycosidic bonds with mild acid treatment almost loss
the ability of tRNAPhe to show codon recognition property which is necessary for the protein
biosynthesis[4]. Y-nucleosides have the tricyclic ring structure with the rigid conformation and
high electron delocalization, and the major UV absorption band locates in 300320 nm region
with a high molecular extinction coefficient. In addition, they have strong fluorescence with a
long decay time, both the absorption and emission bands are well separated from those of the
normal bases, which can be used to detect the conformation changes in the anticodon loop[5]. They
are also likely to be used as effective natural fluorescent probe to study the structure and sequence
of RNA[6]. Owing to their distinctive biological and chemical properties, the rare Y-nucelosides
have attracted much attention[7].
Considering the potential application of this kind of Y-nucleosides in the biological research,
the photophysical and photochemical properties of N-4-desmethylwyosine (dYt), a derivative of
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PULSE RADIOLYSIS OF RARE NUCLEOSIDE DERIVATIVE
145
natural rare nucleoside wyosine (see fig.1), were studied with 248 nm KrF laser flash photolysis in
our laboratory[8]. The results show that the compound was photoionized and damaged more easily
by UV light than normal guanosine. In order to confirm the radical cation of dYt produced in its
photoionization and obtain more information of its oxidation, the nanosecond pulse radiolytical
studies have been carried out. In the present paper, the one-electron oxidation of dYt by some
oxidative species generated by pulse radiolysis was investigated. The transient spectra of the
radical cation dYt•+, neutral radical dYt(-H)• and the pKa have been obtained. The rate constants
for oxidation of dYt by SO 4− , CO3− , N 3 and •OH radicals have been determined. It may offer
•
•
•
some help to understand the photochemical reactions and oxidative properties of the rare tricyclic
nucleosides derivatives.
Fig. 1. Position of Y-nucleoside within the secondary structure of yeast tRNAPhe anticodon loop and the molecular structures of guanosine, wyosine and N-4-desmethylwyosine (dYt).
1
Experimental
1.1 Materials
The compound of dYt was synthesized according to ref. [9] and identified by UV spectra, 1H
and 13C NMR. K2S2O8, NaN3 and KSCN were purchased from Sigma. Na2CO3, NaOH and HClO4
(AR) were obtained from Shanghai Reagent Chemical Co. KH2PO4 and K2HPO4 were the standard samples from Shanghai Leici Co. Tert-butanol (AR) was distilled before use. All solutions
were prepared with triply distilled water and saturated with high purity N2 or N2O gases before
use.
1.2 Apparatus
The transient absorption measurements were performed on nanosecond time-resolved pulse
radiolysis equipment in Shanghai Institute of Nuclear Research, Chinese Academy of Sciences.
Pulse radiolysis experiments were conducted by using a linear accelerator providing 10 MeV electron pulse with a duration of 8 ns and 2—3 A pulse current. The dose was determined using an
aerated 1D10−2 molCL−1 KSCN solution by taking G[(SCN) 2− ] = 2.9 and ε [(SCN)2− ] =7600
•
•
LCmol−1Ccm−1 at 480 nm. The average dose was 10 Gy per pulse. The source of analyzing light
was a 500 W xenon lamp. The electron beam and analyzing light beam passed perpendicularly
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through a quartz cell with an optical length of 20 mm. The analyzing light was transformed into
the electronic signal after entering a monochromator followed by an R955 photomultiplier. The
signals were collected using an HP54510 transient recorder, then processed with a personal computer and analyzed by relevant software.
1.3 Methods
−
In radiolysis of aqueous solution, hydroxyl radical •OH, hydrated electron eaq
and hydrogen
−
) = 2.8 and G (H•) = 0.6 as primary radical species were generated.
atom H• with G (•OH) = G ( eaq
In N2O-saturated solution, hydrated electron was converted into hydroxyl radical (eq.(2))[10].
Hydroxyl radical could be scavenged by tert-butanol to produce •CH2C(CH3)2OH radical. Because
•
CH2C(CH3)2OH is less reactive and its absorption is weak in the UV region, it should not affect
the experimental results (eq.(3)). The hydrated electron and hydroxyl radical could be transformed
into one-electron oxidants such as sulfate radical anion SO 4− , carbonate radical CO3− and azide
•
•
radical N 3 as shown in eqs. (4)(6), which were used to oxidize the substrate dYt in order to
•
obtain its radical cation. The sample solutions were deaerated by high purity N2 or N2O bubbling
for 20 min to deoxygen or scavenge the hydrated electron. The pH values of the solutions were
adjusted with dilute HClO4, NaOH and phosphate buffer. All experiments were carried out at
room temperature.
H2 O
−
eaq
+ N2 O
•
OH + (CH3)3COH
−
eaq
•
−
eaq
, OH, H , ,
•
•
(1)
N2 + OH− + •OH
(2)
H2O + •CH2C (CH3)2OH
(3)
+ S2 O82−
SO 4 + SO 42 −
(4)
OH + CO32 −
OH − + CO3• −
(5)
OH − + N3•
(6)
•
OH + N3−
•−
2 Results and discussion
2.1 Reaction with SO 4− radical
•
SO 4− is a strong oxidant with the potential 2.43 V/NHE[11,12]. In this paper it was produced
•
from the reaction of peroxydisulfate with hydrated electron generated from the pulse radiolysis.
Fig.2 shows the transient absorption spectra in the pulse radiolysis of N2-saturated aqueous
solution containing 2h10−2 molgL−1 K2S2O8, 2h10−2 molgL−1 tert-butanol and 7h10−5 molgL−1
dYt at pH 6.8. At 1 µs after the pulse, a transient spectrum with λ max of 460 nm appears, which
could be assigned to SO 4− . Following the decay of SO 4− observed at 460 nm, a new transient
•
•
band with the absorption maximum around 320ü340 nm and a weak absorption band at 640 nm
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PULSE RADIOLYSIS OF RARE NUCLEOSIDE DERIVATIVE
147
appear. As can be seen in the inset of fig. 2, the build-up process observed at 340 and 640 nm is
synchronous with the decay of SO 4− at 460 nm, so the transient species is assigned to the radical
•
cation dYt•+.
Fig. 2. Transient absorption spectra from the pulse radiolysis of 710−5 molL−1 dYt and 210−2 molL−1
K2S2O8 aqueous solution, containing 210−2 molL−1 tert-butanol, deoxygenated with N2 at pH 6.8. , 1 µs, ,
10 µs. Inset: The transient absorption curves with the decay of SO•4− observed at 460 nm and the growth traces at
340 and 640 nm.
The transient spectrum of radical cation
may have some changes due to its deprotonation
at different pH in the aqueous solution. As
shown in fig. 3, from the curve of pH dependence of the absorbance, the pKa of dYt•+ is determined to be 3.2, meaning that dYt•+ undergoes
deprotonation to yield its neutral form at pH 3.2. The process can be described as eq. (7).
However, at pH 2.8, the absorbance of dYt•+
decreases. A possible explanation may be that
the molecular structure and distribution of electron density are changed at low pH, leading to
the change of absorption coefficient.
Fig. 3. A curve showing pH dependence of the absorbance
at 320 nm of the transient formed in the aqueous solution due
to SO•4− radical reaction with dYt.
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−H
SO 4− + dYt 
→ dYt + 
→ dYt(-H) + SO 24−
•
•
+
•
(7)
In our present research, by plotting the observed first-order growing rate constant of dYt•+
(kobs ) against the concentration of dYt ( range from 1D10−5 to 2D10−4 molCL−1 ), the absolute
rate constants for oxidation of dYt by SO 4− are calculated to be 1.3 D 109 and 1.1 D 109
•
LCmol−1Cs−1 at 320 and 340 nm, respectively.
2.2 Reactions with CO3− and N 3 radicals
•
•
One-electron redox potentials of CO3− and N3 in the neutral aqueous solution are
•
•
E° ( CO3− / CO32 − )= 1.5 V[13] and E° ( N3 / N 3− ) = 1.9 V[14]. They can be produced by reactions of
•
•
OH with CO32 − and N 3− respectively. Fig. 4 shows the transient spectra obtained from the pulse
•
radiolysis of N2O-saturated aqueous solution containing 2D10−1 molCL−1 Na2CO3 and 1D10−4
molCL−1 dYt at pH 10. At 1 µs after the pulse, a transient spectrum with λ max of 600 nm appears,
which can be assigned to the CO3− . Following the decay of CO3− , a new transient spectrum with
•
•
the absorption maximum around 340 nm appears. As shown in the inset of fig. 4, the build-up
process observed at 340 nm is synchronous with the decay of CO3− at 600 nm. The transient spe•
cies should be the neutral radical dYt(-H)•, because the pH 10 is much higher than the pKa of dYt•+
Fig. 4. Transient absorption spectra from the radiolysis of 110−4 molL−1 dYt and 210−1 molL−1 Na2CO3
aqueous solution saturated with N2O at pH 10. , 1 µs; , 50 µs. Inset: The transient absorption curves with the
decay of CO3• − observed at 600 nm and the growth at 340 nm.
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PULSE RADIOLYSIS OF RARE NUCLEOSIDE DERIVATIVE
149
(eq. (8)). The rate constant of the growing process at 340 nm is determined to be 1.0D109
LCmol−1Cs−1, which is in agreement with the value of 8.5D108 LCmol−1Cs−1 obtained for the
decay of CO3− at 600 nm.
•
−H
CO3− + dYt 
→ dYt + 
→ dYt(-H) + CO32 −
•
•
+
•
(8)
Similar results are obtained for the oxidation of dYt by N 3 . The rate constant of 2.1109
•
Lmol−1s−1 is determined at 340 and 410 nm. The details are not listed here.
2.3 Reaction with •OH
Hydroxy radical causes damage to the organs and tissues through electron transfer, hydrogen
abstraction and •OH adduct. The transient absorption spectra recorded at 1 and 10 µs after the
pulse for N2O-saturated aqueous solution containing 710−5 molL−1 dYt at pH 6.8 are shown in
fig. 5(a). In the presence of nitrous oxide, the hydrated electron is converted into •OH rapidly with
making double yields of hydroxyl radical. In view of the low yields of the other radicals in the
system, their influence can be neglected and the main reaction considered is •OH with dYt. As can
be seen, the absorption spectra of transient products are quite broad in a region of 300680 nm
compared with that observed in the oxidation of dYt by SO 4− , CO3− and N 3 radicals. In addi•
•
•
tion, the rate constant for the •OH oxidation is higher than that for other radicals. Because •OH can
be added to DNA bases with diffusion-controlled rates (5109 Lmol−1s−1 ) to produce hydroxyl additive radicals[15], and to double bond of organic compounds efficiently[16], it can be deduced that the •OH adduct should be the main products for the reaction of •OH with dYt, as shown
in the following equation:
Fig. 5. (a) Transient absorption spectra obtained in the pulse radiolysis of N2O-saturated aqueous solution containing 710−5 molL−1 dYt and 210−3 molL−1 phosphate buffer at pH 6.8. , 1 µs; , 10 µs. Inset: The
transient absorption curves observed at 340, 410 and 640 nm. (b) Kinetic plots for the reaction of dYt with hydroxyl radical at 340 and 410 nm.
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•
Vol. 46
OH + dYt 
→ [dYt-OH] • main products
(9)
By changing the concentration of dYt, the rate constants for the •OH addition to dYt observed at 340 and 410 nm are determined to be 4.9109 and 4.5109 Lmol−1s−1, respectively,
which are in good agreement with the data shown in fig.5(b).
The results for the oxidation of dYt by several oxidants and the addition of hydroxyl radical
in aqueous solution are listed in table 1.
Table 1 Kinetics and spectroscopic properties for the reactions of oxidizing radicals with dYt in aqueous solutions
Radicals
•−
SO4
Products
dYt(-H)
•
λ/nm
pH
k/LCmol−1Cs−1
320
6.8
1.3D109
1.1D109
340
CO3• −
dYt(-H) •
340
10
8.5D108 a)
600
N3•
dYt(-H) •
340
6.8
OH
[dYt-OH]
• b)
2.1D109
2.1D109
410
•
1.0D109
340
6.8
4.9D109
4.5D109
410
•−
a) Kinetics of decay of CO3 radical at 600 nm, b) the main products.
3
Conclusion
The oxidation of dYt by SO 4− , CO3− and N3• via electron transfer to produce dYt•+ has been
•
•
investigated. The radical cation dYt•+ converts into the neutral radical through deprotonation at pH
pKa (dYt•+), which is in accordance with the results of our previous studies by the laser photoly-
sis. It suggests that the •OH adduct is the main products for the reaction of •OH with dYt. The rate
constants for the reactions of dYt with SO 4− , CO3− , N3• and •OH have been obtained. This pa•
•
per may provide some theoretic evidences for further understanding of the oxidation of rare
Y-nucleosides.
Acknowledgements The authors thank Zhang Wenlong, Tu Tiecheng and Chen Yuling for their help. This work was
supported by the National Natural Science Foundation of China (Grant No. 20072025) and Promoted Research Programs of
Chinese Academy of Sciences.
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