Photoinduced reduction of thymine and uracil derivatives by hypophosphite: Unusual high quantum yield of chromophore loss

Article abstract of DOI:10.1039/a802836b

The quantum yield of chromophore loss of thymine, uracil and their corresponding nucleosides and nucleoside-5′-monophosphates undergoing irradiation with 254 nm UV light was found to be sharply enhanced by hypophosphite; thymine and uracil were reduced by hypophosphite to give 5,6-dihydrothymine and 5,6-dihydrouracil respectively.

Full text of DOI:10.1039/a802836b

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Photoinduced reduction of thymine and uracil derivatives by hypophosphite:
unusual high quantum yield of chromophore loss
Kongjiang Wang,*† and Zhifang Chai
Institute of High Energy Physics, The Chinese Academy of Sciences, PO Box 2732, Beijing 100080, PR China
The quantum yield of chromophore loss of thymine, uracil
and their corresponding nucleosides and nucleoside-5A-
monophosphates undergoing irradiation with 254 nm UV
light was found to be sharply enhanced by hypophosphite;
thymine and uracil were reduced by hypophosphite to give
5,6-dihydrothymine and 5,6-dihydrouracil respectively.
sharply. The low reversibility or irreversibility of the irradiated
samples of uracil, uridine and uridine-5-monophosphate in the
concentration range corresponding to photoinduced reduction
by hypophosphite also indicates that hydrates are not the main
products. In dilute solution, dimers of pyrimidine bases and
their derivatives arise through the triplet state.1 f for thymine
dimer formation is small ( < 0.1), while the reversal quantum
yield is about 1.8 This, combined with the reported limiting
concentrations for uracil dimerization ( > 0.1 mm)9 and the
aerated aqueous media (O2 as triplet quencher) in the current
system eliminates the possibility of forming the dimers in large
quantity.
Fig. 2 indicates that major photoproducts have been formed
in aqueous solutions of uracil and thymine containing sodium
hypophosphite, which have been purified‡ and characterized.§
Using the authentic samples (Sigma) as internal standard, the
retention time of the purified products was found to be the same
to 5,6-dihydrothymine (2a) and 5,6-dihydrouracil (2b). The
corresponding peaks were found to disappear by HPLC analysis
after incubating the photoproducts in 0.2 m KOH for 1 h at room
temperature. This is in agreement with the alkaline lability of
dihydrouracil and dihydrothymine.10 However, after heating the
aqueous solution of the photoproducts with or without 0.1 m
HCl in a boiling water bath for 10 min, no obvious change was
observed via HPLC. Both IR and NMR spectra of the purified
samples were found to be the same as those of the authentic
samples. Overall these data indicate that the major product of
In the photochemistry of nucleic acids, there has been much
interest in oxidative damage to DNA and its biological
consequences in living cells.1,2 Here we report the photo-
induced reduction (hydrogenation) of thymine (1a), uracil (1b)
and their derivatives by hypophosphite, which was initially
found in our tentative simulation of the photochemistry of the
primitive sea, by irradiation with a medium pressure mercury
lamp.3 Contrary to the wavelength dependence of the enhance-
ment of the photolysis of nucleic acid monomers by orthophos-
phates and pyrophosphate on UV light for photoionization of
phosphates ( < 210 nm),3,4 no obvious wavelength dependence
has been observed in photoinduced reduction by hypophos-
phite. We present the results obtained by the irradiation with
254 nm UV light.
For actinometry of the low pressure mercury lamp, we
repeatedly used the chromophore loss of uridine in areated
aqueous solution at pH 6.5 A 20% MeCO2H solution of 1 cm
pathlength was employed to filter off the photochemically
significant quantities of 184.9, 194.2 and 222.4 nm UV light.
The quantum yield of chromophore loss (fcl) was determined
from the decrease of absorbance at lmax of the substrates. Fig.
1 shows the concentration dependence of fcl of thymine, uracil
and their corresponding nucleosides and nucleoside-
5A-monophosphates. It is clear from this figure that fcl of
thymine, uracil and their derivatives increases when the
concentration of sodium hypophosphite is about 3 3 1023 m or
higher. fcl for uracil and thymidine is higher than for the other
derivatives. Note that fcl for thymine, thymidine and thymidine-
5A-monophosphate increases so sharply that at 1 m sodium
hypophosphite fcl for thymine, thymidine and thymidine-
5A-monophosphate is 0.016, 0.046 and 0.029, fcl being 0.064,
0.16 and 0.11 for uracil and its derivatives under the same
conditions. However, no significant fcl change for uracil and
thymine has been observed in the presence of 0.1 m concentra-
tions of other anions (SO422, Cl2, CO322, citrate, acetate,
arsenate, cacodylate, orthophosphates, pyrophosphate), 0.1 m
urea and 0.004 m metaphosphate under irradiation with 254 nm
UV light. In contrast to the enhancement of the photolysis of
nucleic acid monomers by orthophosphates and pyrophos-
phate,4 no obvious fcl increase has been observed for cytosine,
cytidine and cytidine-5A-monophosphate in the range 1025–1 m
hypophosphite. For adenine, guanine and their derivatives, fcl
was observed to increase when the concentration rises to more
than 0.01 m, but the relative fcl value is < 5.
600
A
B
40
30
20
10
0
500
400
300
200
100
0
–5 –4 –3 –2 –1
0
–5 –4
–3
–2 –1
0
log ([hypophosphite] / M)
Fig. 1 fcl for thymine (A), uracil (B) and their derivatives as a function of
the molar concentration of sodium hypophosphite undergoing the irradia-
tion with 254 nm UV light (6.05 3 1024 Einstein min21): (2) the base, (8)
the corresponding nucleoside and (Ω) nucleoside-5A-monophosphate.
Generally, 1 3 1024 m aerated pyrimidines or their mixed solutions with
sodium hypophosphite (pH 7 ± 0.3) were irradiated so as to facilitate UV
absorbance and spectrum assay. fcl for thymine, thymidine and thymidine-
5’-monophosphate in the absence of hypophosphite is 9.7 3 1025, fcl for
uracil, uridine and uridine-5A-monophosphate in the absence of hypophos-
phite is 1.8 3 1023, 0.018 and 0.014. fcl in the presence of hypophosphite
has been normalized to fcl in the absence of hypophosphite.
Hydrate formation of uracil, uridine and uridine-5-mono-
phosphate was quantified using both HPLC6 and the method
introduced by Moore and Thomson7 because the latter method
has been demonstrated to bring about total dehydration.6 HPLC
elution demonstrates that hydrate formation of uracil and
uridine in the concentration range corresponding to photo-
induced reduction by hypophosphite ( > 3 3 1023 m) decreases
Chem. Commun., 1998
1543

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40000
30000
20000
10000
0
hypophosphite (pH 7 ± 0.3). The photoproducts were extracted using ethyl
acetate (for photoproducts of thymine) and a mixed solution of butanone
and ethyl acetate (80:20, v/v) (for photoproducts of uracil). The collected
samples were concentrated on a rotary evaporator. The residue was taken up
in water, the products were purified by reversed-phase HPLC (Waters 600,
Waters mBondapak C18 19 3 300) with aqueous solution (uracil) or 2%
MeOH aqueous solution (thymine). The collected samples were lyophilized
to dryness.
Thy
Ura
§ Selected spectroscopic data: 1, UV spectrum of 2a and 2b shows the
disappearance of the characteristic absorption ( > 230 nm) of uracil and
thymine. 1, 2a (C5H8N2O2), Anal. Calc. for C5H8N2O2: C, 46.88; H, 6.25;
N, 21.88; O, 25. Found: C, 47.1; H, 7.1; N, 22.1; O, 23.8%. FTIR (n/cm21
)
3236m, 3088m, 2892w, 1736vs, 1715vs, 1496m, 1392w, 1240s; 819w,
696w, 448w; dH(DSS, 600 MHz) 1.079 and 1.096 (d, Me), 2.647–2.723 (m,
5 H), 3.043–3.099 (m, 6 H), 3.036–3.045 (m, 6 H). m/z (atmosphere
pressure chemical ionization), 129 (M + 1)+. 2, 2b (C4H6N2O2), Anal. Calc.
for C4H6N2O2: C, 42.1; H, 5.26; N, 24.56; O, 28.07. Found: C, 42; H, 5.1,
N, 24.6, O, 28.54%. FTIR (n/cm21) 3236m, 3087m, 2892w, 1736vs,
1715vs, 1678m, 1496m, 1391w, 1239s, 820w, 756w, 453w; dH(DSS, 600
MHz) 2.554–2.589 (t, 5 H), 3.324–3.405 (t, 6 H); m/z 115 (M + 1)+.
0
2
4
6
8
10
12
14
16
18
Retention time, t / min
Fig. 2. HPLC elution profiles of products from irradiated 1 3 1024
m
thymine (Thy) and uracil (Ura) containing 0.2 m hypophosphite. The
products were eluted from the mBondapak C18 column (Waters, 3.9 3 300
mm) at a flow rate of 1.4 ml min21 (Thy) and 1 ml min21 (Ura). The
chromatograms were monitored at 210 nm.
1 J. Cadet and P. Vigny, Bioorganic Photochemistry, Photochemistry and
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3 K. J. Wang, Ph.D. Thesis, 1995, Peking University, pp. 70–73.
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Life Evol. Biosphere, 1998, in the press.
thymine and uracil containing hypophosphite is 5,6-dihydro-
thymine (2a) and 5,6-dihydrouracil (2b). UV absorbance at 254
nm of 1 m sodium hypophosphite is 0.009, eliminating
complications in the reaction mechanism owing to the com-
petitive UV absorbance of hypophosphite. This, combined with
detection of the quantitative oxidated hypophosphite (600 MHz,
13P NMR spectrum), indicates that the enhancement of the
chromophore loss by hypophosphite is indeed the photoinduced
reduction by hypophosphite [eqn. (1)].
O
O
5 G. J. Fisher and H. E. Johns, Photochemistry and Photobiology of
Nucleic Acids, ed. S. Y. Wang, Academic Press, New York, 1976, vol.
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107, 143.
H
O
R
H
H
O
R
H
H
N
N
UV
–
+ H2PO2– + H2O
+ H2PO4 (1)
N
N
H
H
H
1
2
a R = Me
b R = H
The fact that no obvious difference in the quantum yield has
been found between the aerated and nitrogen-saturated samples
favors the singlet pathway as the reaction mechanism.
This work was supported by the Presidential Foundation and
is a major project of the Chinese Academy of Sciences.
7 A. M. Moore and C. H. Thomson, Can. J. Chem., 1957, 35, 163; A. M.
Moore and C. H. Thomson, Science, 1955, 122, 594.
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10 R. D. Batt, K. Martin, J. M. Ploesser and J. Murphy, J. Chem. Soc., 1954,
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A. J. Varghese, Biochemistry, 1971, 10, 4283.
Notes and References
† E-mail: wangkj@lnat.ihepa.ac.cn
‡ The major photoproducts were prepared by 254 nm irradiation of 1 3
1023 m aerated thymine or uracil aqueous solution containing 1 m sodium
Received in Cambridge, UK, 15th April 1998; 8/02836B
1544
Chem. Commun., 1998