Salicylic acid as a photosensitizer for thymidine dimerization induced by UV

Article abstract of DOI:10.1248/cpb.c18-00647

When a neutral solution of a nucleoside mixture was irradiated with UV light having wavelength longer than 300nm, addition of salicylic acid to the solution greatly accelerated the reaction of thymidine. The UV light irradiation of thymidine solution in the presence of salicylic acid resulted in four major product peaks in HPLC. All the products were identified as isomers of cyclobutane thymidine dimers by MS and NMR. The cyclobutane thymidine dimers were generated from thymidine almost exclusively. UV irradiation with the longer wavelength of 350nm induced almost no reaction. The results indicate that salicylic acid is a photosensitizer for thymidine dimerization excited by UV light of wavelength 300 to 350nm.

Full text of DOI:10.1248/cpb.c18-00647

1
30
Chem. Pharm. Bull. 67, 130–134 (2019)
Vol. 67, No. 2
Regular Article
Highlighted Paper selected by Editor-in-Chief
Salicylic Acid as a Photosensitizer for Thymidine Dimerization Induced
by UV
Toshinori Suzuki,* Hiroki Ota, Yuma Namba, and Toshifumi Fujino
School of Pharmacy, Shujitsu University; 1–6–1 Nishigawara, Okayama 703–8516, Japan.
Received August 22, 2018; accepted November 8, 2018
When a neutral solution of a nucleoside mixture was irradiated with UV light having wavelength longer
than 300nm, addition of salicylic acid to the solution greatly accelerated the reaction of thymidine. The UV
light irradiation of thymidine solution in the presence of salicylic acid resulted in four major product peaks
in HPLC. All the products were identified as isomers of cyclobutane thymidine dimers by MS and NMR. The
cyclobutane thymidine dimers were generated from thymidine almost exclusively. UV irradiation with the
longer wavelength of 350nm induced almost no reaction. The results indicate that salicylic acid is a photosen-
sitizer for thymidine dimerization excited by UV light of wavelength 300 to 350nm.
Key words salicylic acid; thymidine dimer; UV light; photosensitizer
Introduction
dG slightly decreased and those of dC and dA did not change,
Exposure to UV light from the sun is associated with the as shown in Fig. 1A. Figure 1B shows the SA dose-dependent
1)
occurrence of skin cancer in humans. UV light directly in- changes on concentrations of nucleosides when the nucleoside
duces various forms of DNA damage including bipyrimidine solution with 0–2mM SA was irradiated with UV light for
2
,3)
photoproducts, cytosine hydrates, and 8-oxo-dGuo. Among 20min. The concentration of dT decreased with increasing SA
these, cyclobutane thymine dimers were found to be the major dose up to 1mM, whereas for 1–2mM SA, the concentration
photoproducts generated by artificial UV light and sunlight of nucleosides was unchanged. Table 1 shows the effects of
2
–4)
within cellular DNA.
In the UV light reaction of thymidine longpass filters on the UV reaction of nucleosides. The mixed
(
dT), a free nucleoside, six configurationally-distinct forms of nucleoside solution without or with 1mM SA in potassium
5
)
cyclobutane thymidine dimer are formed. The cys–sin and phosphate buffer at pH 7.4 was irradiated with UV through no
trans–anti isomers exist as single molecules, whereas both filter, a 300nm longpass filter, or a 350nm longpass filter at
of the trans–syn and cis–anti isomers exist as enantiomeric a temperature of 37°C for 20min. On UV irradiation without
pairs. In the presence of a photosensitizer, UV light with lon- a filter and through the 300nm filter, addition of SA greatly
ger wavelength and visible light can indirectly induce DNA enhanced the reaction of dT. However, on UV irradiation
damage. Many studies have shown that ketone photosensitiz- through the 350nm filter, no reaction was observed.
ers such as acetone and acetophenone enhance the forma-
Table 2 shows the effects of related compounds of SA on
the reaction of nucleosides with UV light with a longer wave-
2)
tion of thymidine dimers from dT through energy transfer.
Salicylic acid (SA) is a good agent for peeling, since it can
6
)
exfoliate the corneum. Many cosmetic products, includ-
ing shampoos, soaps, lipsticks, and solution serums for skin
care, include SA as an ingredient at concentrations up to 3%
7
)
(
ca. 200mM). It is reported that SA is not a photosensitizer
7,8)
and does not show phototoxycity. In the present study, we
investigated the effect of SA on the reaction of dT with UV
light, and report here that salicylic acid is a photosensitizer of
the reaction of dT with UV from 300 to 350nm, resulting in
cyclobutane thymidine dimers almost exclusively.
Results and Discussion
A mixed solution of nucleosides (2′-deoxycytidine (dC),
Fig. 1. (A) Time Courses of Concentration Changes in dC (Circle),
dG (Square), dT (Triangle), and dA (Rhombus) When a Solution of
2
1
′-deoxyguanosine (dG), dT, and 2′-deoxyadenosine (dA);
00 µM each) with SA in 100mM potassium phosphate buffer
at pH 7.4 was irradiated with UV light from a high-pressure
100 µM Each dC, dG, dT, and dA with 1mM SA Was Irradiated with
UV through a 300nm Longpass Filter in 100mM Potassium Phosphate
mercury lamp through a 300nm longpass filter at a tempera- Buffer at pH 7.4 and at a Temperature of 37°C for 0–40min; (B) The SA
Dose-Dependence of Concentrations of Nucleosides When a Solution of
ture of 37°C. The reaction mixture was analyzed by reversed
100 µM dC, dG, dT, and dA with 0–2mM SA Was Irradiated with UV
phase (RP) HPLC. The nucleoside concentrations were de-
termined by absorbance areas of HPLC detected at 260nm.
In the presence of 1mM SA, the concentration of dT greatly
through a 300nm Longpass Filter in 100mM Potassium Phosphate Buffer
at pH 7.4 and at a Temperature of 37°C for 20min
All the reaction mixtures were analyzed by RP-HPLC. Means± standard devia-
decreased as the irradiation time increased, whereas that of tion (S.D.) (n=3) are presented.
*
To whom correspondence should be addressed. e-mail: tsuzuki@shujitsu.ac.jp
©
2019 The Pharmaceutical Society of Japan

Vol. 67, No. 2 (2019)
Chem. Pharm. Bull.
131
a)
Table 1. Effects of Longpass Filters on the Reaction of Nucleosides with UV in the Absence or Presence of SA
dC (µM)
dG (µM)
dT (µM)
dA (µM)
No filter
71.9± 1.5
83.4± 3.5
98.0± 2.5
96.5± 0.8
100.5± 0.4
101.1± 0.6
78.5± 2.6
87.0± 0.9
97.5± 1.8
91.4± 1.8
97.9± 0.5
99.7± 0.8
83.8± 0.9
6.1± 0.5
98.7± 2.3
21.6± 1.3
99.6± 0.1
99.1± 1.0
95.6± 0.7
95.3± 1.3
99.1± 2.0
98.9± 2.4
100.2± 0.1
97.8± 0.5
No filter+SA
3
3
3
3
00nm filter
00nm filter+SA
50nm filter
50nm filter+SA
a) A mixed solution of 100µM dC, dG, dT, and dA in 100mM potassium phosphate buffer at pH 7.4 in the absence and presence of 1mM SA was irradiated with UV light
through no filter, a 300nm longpass filter, or a 350nm longpass filter at a temperature of 37°C for 20min.
a)
Table 2. Effects of Related Compounds of SA on the Reaction of Nucleosides with UV through a 300nm Longpass Filter
Compounds
dC (µM)
dG (µM)
dT (µM)
dA (µM)
Salicylic acid
96.5± 0.8
98.3± 2.1
97.4± 0.9
100.2± 1.8
99.5± 0.4
97.4± 0.3
97.1± 2.9
98.1± 2.4
97.4± 3.0
92.7± 1.4
74.0± 1.0
91.4± 1.8
95.4± 2.0
99.0± 0.5
99.6± 1.6
100.0± 0.6
97.7± 0.4
98.1± 1.0
94.9± 0.3
93.3± 0.3
95.0± 1.7
14.8± 0.0
21.6± 1.3
94.7± 2.2
92.3± 1.1
101.2± 1.4
99.6± 0.7
97.8± 0.4
97.3± 0.5
2.1± 0.3
98.9± 2.4
99.4± 2.0
99.6± 0.2
101.2± 1.6
100.4± 0.6
99.1± 0.3
99.8± 0.7
98.4± 0.4
98.7± 0.6
96.8± 1.2
79.9± 0.9
m-Hydroxybenzoic acid
p-Hydroxybenzoic acid
Benzoic acid
Phenol
Methyl salicylate
Acetylsalicylic acid
o-Aminobenzoic acid
m-Aminobenzoic acid
p-Aminobenzoic acid
Acetophenone
98.1± 2.6
33.8± 1.8
39.9± 2.4
a) A solution of 100µM dC, dG, dT, and dA in 100mM potassium phosphate buffer at pH 7.4 in the absence and presence of 1mM related compounds of SA was irradiated
with UV through a 300nm longpass filter at a temperature of 37°C for 20min.
length than 300nm. A solution of nucleosides (100µM each)
with 1mM of the compounds in 100mM potassium phos-
phate buffer at pH 7.4 was irradiated with UV light through
a 300nm longpass filter at a temperature of 37°C for 20min.
The effects of acceleration on the dT reaction by m-hydroxy-
benzoic acid and p-hydroxybenzoic acid, which are positional
isomers of salicylic acid (o-hydroxybenzoic acid), were rather
smaller than that by salicylic acid. Benzoic acid, phenol,
methyl salicylate, and acetylsalicylate showed no effects. o-
Aminobenzoic acid and p-aminobenzoic acid accelerated the
dT reaction, whereas m-aminobenzoic acid did not. Acetophe-
none, a keton photosensitizer, accelerated the reaction of dG
more than dT.
To obtain information about the reaction products from dT,
a solution of 10mM dT with 1mM SA in 100mM potassium
phosphate buffer at pH 7.4 was irradiated with UV through
a 300nm longpass filter at a temperature of 37°C for 60min.
Fig. 2. RP-HPLC Chromatogram of a Reaction Mixture of dT with SA
Detected at 230nm
Figure 2 shows the RP-HPLC chromatogram of the reac-
The insets are on-line detected UV spectra of dT and SA. A solution of 10mM
tion mixture detected at 230nm. The UV-Vis spectra of SA dT and 1mM SA was irradiated with UV through a 300nm lowpass filter in
1
00mM potassium phosphate buffer at pH 7.4 and 37°C for 60min. The HPLC
and dT (Fig. 2 insets) indicated that SA can absorb UV light
system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-Vis photodiode-
having a longer wavelength than 300nm, whereas dT can- array detector (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil ODS-3
octadecylsilane column of size 4.6± 250mm and a particle size of 5µm (GL
not. Four major product peaks with retention times of 16.5,
1
Sciences, Tokyo) was used. The eluent was 20mM ammonium acetate (pH 7.0)
7.2, 17.6, and 19.7min, referred to as Peak 1, Peak 2, Peak containing methanol. The methanol concentration was increased from 0 to 40%
during 30min in linear gradient mode. The column temperature was 40°C and the
3, and Peak 4, respectively, were detected in addition to the
flow rate was 1mL/min.
peaks of dT and SA. All the product peaks showed similar
UV spectra with λmax of 215–218nm (data not shown). The
product peaks were isolated by RP-HPLC and subjected to 1 and 4, H-NMR showed two sets of an aliphatic proton, a
MS and NMR. All the peaks on electrospray ionization-time- methyl proton, and seven deoxyribose protons. No aromatic
1
13
of-flight (ESI-TOF)-MS showed a signal at m/z 483 in the proton signal was observed. C-NMR showed two sets of two
negative mode. High-resolution (HR)-ESI-TOF/MS values of aliphatic carbons, two carbonyl carbons, a methyl carbon, and
1
the molecular ion for all the peaks agreed with the theoreti- five deoxyribose carbons. For Peaks 2 and 3, H-NMR showed
cal molecular mass for C H N O within 3ppm. For Peaks a set of an aliphatic, a methyl proton, and seven deoxyribose
2
0
27
4
10

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32
Chem. Pharm. Bull.
Vol. 67, No. 2 (2019)
Fig. 3. Structures of Cyclobutane Thymidine Dimers
Fig. 4. (A) The Time Course of Concentration Changes in Peak 1
Closed Circle), Peak 2 (Closed Square), Peak 3 (Closed Triangle), and
Peak 4 (Closed Rhombus) with dT (Open Triangle) When a Solution of
(
13
protons. C-NMR showed one set of two aliphatic carbons, 10mM dT with 1mM SA Was Irradiated with UV through a 300nm
two carbonyl carbons, a methyl carbon, and five deoxyribose Longpass Filter in 100mM Potassium Phosphate Buffer at pH 7.4 and
at a Temperature of 37°C for 0–60min; (B) The SA Dose-Dependence
of Concentrations of Nucleosides When a Solution of 10mM dT with
carbons. Cadet et al. reported the identification of six con-
figurationally-distinct forms of cyclobutane thymidine dimer
0
–2mM SA Was Irradiated with UV through a 300nm Longpass Filter
5
)
generated from dT with UV light. Comparing the NMR
in 100mM Potassium Phosphate Buffer at pH 7.4 and at a Temperature
data of the present study with the reported data, Peak 1 was of 37°C for 60min; (C) The dT Dose-Dependence of Concentration of SA
identified as a single molecule of cis–syn thymidine dimer. When a Solution of 1mM SA with 0–20mM dT in 100mM Potassium
Phosphate Buffer at pH 7.4 Was Irradiated with UV through a 300nm
Longpass Filter at a Temperature of 37°C for 60min
In cis–syn thymidine dimer, the two nucleoside units are not
symmetrically equivalent, since the symmetry is not retained
by the addition of the two D-sugars (it is retained when one of
All the reaction mixtures were analyzed by RP-HPLC. Means± S.D. (n=3) are
presented.
two 2-deoxy-D-ribose moieties in the compound is replaced
5
)
by 2-deoxy-L-ribose). Peaks 2 and 3 were identified as two dT in 100mM potassium phosphate buffer at pH 7.4 was irra-
stereoisomers of cis–anti thymidine dimer, cis–anti (−) and diated with UV through a 300nm longpass filter at a tempera-
cis–anti (+), respectively, in which the two nucleoside units ture of 37°C for 60min. Consumption of SA decreased with
are symmetrically equivalent. Peak 4 was identified as a single increasing dT concentration. In the presence of 10mM dT, the
molecule of trans–anti thymidine dimer in which the two consumption of SA was 0.22mM.
nucleoside units are not symmetrically equivalent, like Peak 1.
The remaining two stereoisomers, trans–syn thymidine dimer of dT with UV in the absence or presence of SA. The solu-
trans–syn (−) and trans–syn (+)), were not detected, probably tion of 10mM dT without or with 1mM SA was irradiated
Table 3 shows the effects of longpass filters on the reaction
(
because of their small production yield or overlap with other with UV directly or through the filters at pH 7.4 and 37°C
peaks. The structures of cyclobutane thymidine dimers are for 60min. On UV irradiation without a filter, addition of SA
shown in Fig. 3.
enhanced the reaction of dT. On UV irradiation through the
Figure 4A shows the time-dependent changes in the con- 300nm longpass filter, addition of SA greatly enhanced the
centrations of Peaks 1–4 and dT when a solution of 10mM dT reaction of dT. The total concentration of cyclobutane thymi-
with 1mM SA in 100mM potassium phosphate buffer at pH dine dimer formed was 3.18mM. Since each thymidine dimer
7.4 was irradiated with UV through a 300nm longpass filter has two thymidine units in the structure, the concentration of
at a temperature of 37°C for up to 60min. The concentrations total thymidine unit in products was 6.36mM, which is almost
of Peaks 1–4 increased with increasing irradiation time. Fig- equivalent to the consumed dT concentration. In the presence
ure 4B shows the SA dose-dependence of concentrations of of 1mM SA, the yields of cyclobutane thymidine dimers by
Peaks 1–4 and dT when the solution of dT with 0–2mM SA UV light through 300nm longpass filter were greater than
was irradiated with UV light for 60min. The concentration of those by UV light without filter. UV-irradiation at wavelength
dT decreased with increasing SA dose up to 1mM, whereas lower than 230nm has been shown to induce efficient photor-
for 1–2mM SA, the concentrations of Peaks 1–4 and dT were eversion, in which the cyclobutane ring of the dimers were
2)
unchanged. Figure 4C shows the dT dose-dependence of con- split generating the parent dT due to direct photo absorption.
centration of SA when a solution of 1mM SA with 0–20mM This photoreversion may explain the relatively low yields of

Vol. 67, No. 2 (2019)
Chem. Pharm. Bull.
133
a)
Table 3. Effects of Longpass Filters on the Reaction of dT with UV in the Absence or Presence of SA
Peak 1 (mM)
Peak 2 (mM)
Peak 3 (mM)
Peak 4 (mM)
dT (mM)
No filter
0.06± 0.01
0.53± 0.01
0.03± 0.00
1.76± 0.04
0.01± 0.00
0.03± 0.00
0.02± 0.00
0.13± 0.01
0.03± 0.00
0.48± 0.01
0.00± 0.00
0.01± 0.00
0.03± 0.00
0.10± 0.00
0.02± 0.00
0.29± 0.01
0.00± 0.00
0.01± 0.00
0.02± 0.00
0.15± 0.01
0.02± 0.00
0.65± 0.02
0.00± 0.00
0.01± 0.00
8.80± 0.07
7.31± 0.03
9.35± 0.03
3.55± 0.17
9.24± 0.03
9.13± 0.02
No filter+SA
3
3
3
3
00nm filter
00nm filter+SA
50nm filter
50nm filter+SA
a) A solution of 10mM dT in 100mM potassium phosphate buffer in the absence or presence of 1mM SA was irradiated with UV through no filter, a 300nm longpass filter,
or a 350nm longpass filter at pH 7.4 and at a temperature of 37°C for 60min.
cyclobutane thymidine dimers when the irradiation was car- solution (1mL) of nucleosides or dT containing sodium salicy-
ried out without filter. On UV irradiation through the 350nm late or other additives in 100mM potassium phosphate buffer
longpass filter, almost no reaction was observed.
(pH 7.4) in a glass vial (12mm i.d.) without a cap at 37°C.
p-Aminobenzoic acid was widely used as an ingredient in Longpass filters LU0300 (cut-on 300nm) or LU0350 (cut-
sunscreens, since it reduced erythema and hyperplasia of the on 350nm) (Asahi Spectra, Tokyo, Japan) were used as the
9)
skin by UV light. However, p-aminobenzoic acid has been optical filter. The intensity of radiation on the surface of the
found to photosensitize bacterial cell killing and mutation sample solution was measured with a photometer (UIT-150,
1
0,11)
in bacterial and mammalian DNA.
It photosensitizes the Ushio) equipped with a sensor UVD-S254 or UVD-S365. The
2
dimerization of thymine bases in DNA upon irradiation at intensities of the UV light were 57mW/cm for 254nm and
wavelength greater than 300nm. However, in the Interna- 487mW/cm for 365nm without a filter, 1mW/cm for 254nm
tional Agency for Research on Cancer (IARC) classification, and 490mW/cm for 365nm with the 300nm longpass filter,
p-aminobenzoic acid is categorized in Group 3 (not classifi- and 0mW/cm for 254nm and 468mW/cm for 365nm with
able as to its carcinogenicity to humans), since no case reports the 350nm longpass filter.
11)
2
2
2
2
2
12)
or epidemiological studies were available. SA is widely used
HPLC and MS Conditions The HPLC system con-
as an ingredient in skin care products, since it can exfoliate sisted of LC-10ADvp pumps and an SPD-M10Avp UV-Vis
6
)
the corneum. It is reported that salicylic acid is not a pho- photodiode-array detector (Shimadzu, Kyoto, Japan). For the
7,8)
tosensitizer. In an animal experiment using hairless mice, RP-HPLC, an Inertsil ODS-3 octadecylsilane column of size
salicylic acid inhibited the rate of UVB-induced nonmelanoma 4.6×250mm and a particle size of 5µm (GL Sciences, Tokyo,
13)
skin cancer. In the IARC classification for carcinogenicity, Japan) was used. The eluent was 20mM ammonium acetate
SA is not categorized in any group. The present study showed (pH 7.0) containing methanol. The methanol concentration
that the reaction of dT induced by UV (300–350nm) was was increased from 0 to 40% during 30min in linear gradient
greatly accelerated by SA, resulting in the formation of thymi- mode. The column temperature was 40°C and the flow rate
dine dimers. This implies that SA is a photosensitizer for the was 1mL/min. The RP-HPLC chromatogram was detected at
UV reaction of dT, resulting in cyclobutane thymidine dimers. 260nm for nucleosides reactions and at 230nm for dT reac-
Generally, the reaction mechanism of dT dimerization by light tions. ESI-TOF-MS measurements were performed on a Mi-
in the presence of photosensitizer is explained as follows; At croTOF spectrometer (Bruker, Bremen, Germany) in negative
first, ground state photosensitizer is excited to the triplet state mode. The sample isolated by RP-HPLC was directly infused
by light irradiation. Then, the triplet energy of photosensitizer into the MS system by a syringe pump without a column.
is transferred to ground state dT resulting in triplet state dT,
when the triplet energy of photosensitizer is higher than or
nearly equal to that of dT. The dT triplet reacts with ground
Spectrometric Data
Peak 1 (cis–syn)
ESI-TOF-MS (negative mode): m/z 483. HR-ESI-TOF-MS
state dT nearby to yield dT dimers. This reaction mechanism (negative mode): m/z 483.173623 obsd. (Calcd for C H N O
2
0
27
4
10
is proposed not only for ketone photosensitizers but also for 483.173267). UV: λ =215nm (pH 7.0). cis–syn A unit.
max
2
,11)
1
p-aminobenzoic acid.
A similar reaction mechanism would
H-NMR (500MHz, D O): δ (ppm/trimethylsilyl propanoic
2
be applicable to the photosensitization by SA.
acid (TMSP)-d ) 5.95 (dd, 1H, H-1′), 4.47 (ddd, 1H, H-3′),
4
The present study showed that SA is the photosensitizer for 4.40 (d, 1H, H-6), 3.92 (ddd, 1H, H-4′), 3.77 (m, 1H, H-5′ or
thymidine dimerization by UV light. Since SA is used as an 5″), 3.74 (m, 1H, H-5′ or 5″), 2.47 (m, 1H, H-2′ or 2″), 2.21
13
ingredient in many cosmetics, we should pay close attention to (m, 1H, H-2′ or 2″), 1.47 (s, 3H, CH3). C-NMR (125MHz,
its genotoxicity in terms of photosensitization.
D O): δ (ppm/TMSP-d ) 176.0 (C-4), 156.0 (C-2), 89.4 (C-1′),
2
4
8
8.5 (C-4′), 74.5 (C-3′), 65.1 (C-5′), 59.8 (C-6), 51.8 (C-5), 40.4
1
Experimental
(C-2′), 19.9 (CH ). cis–syn B unit. H-NMR (500MHz, D O):
3
2
Materials Nucleosides were purchased from Sigma- δ (ppm/TMSP-d ) 5.40 (dd, 1H, H-1′), 4.42 (ddd, 1H, H-3′),
4
Aldrich (MO, U.S.A.). Sodium salicylate and other compounds 4.36 (d, 1H, H-6), 3.91 (ddd, 1H, H-4′), 3.79 (m, 1H, H-5′ or
are obtained from Nacalai (Kyoto, Japan). Water was purified 5″), 3.73 (m, 1H, H-5′ or 5″), 2.58 (m, 1H, H-2′ or 2″), 2.10
1
3
with a Millipore Milli-Q deionizer.
(m, 1H, H-2′ or 2″), 1.51 (s, 3H, CH3). C-NMR (125MHz,
Irradiation Conditions High-intensity UV light originat- D O): δ (ppm/TMSP-d ) 175.2 (C-4), 155.1 (C-2), 91.6 (C-1′),
2
4
ing from a 250W high-pressure mercury lamp (SP5-250UB, 88.9 (C-4′), 73.7 (C-3′), 64.5 (C-5′), 62.7 (C-6), 49.4 (C-5), 39.2
Ushio, Tokyo, Japan) with or without an optical filter through (C-2′), 20.0 (CH3).
a liquid light guide was irradiated directly to the surface of a

1
34
Chem. Pharm. Bull.
Vol. 67, No. 2 (2019)
Peak 2 (cis–anti (−))
ESI-TOF-MS (negative mode): m/z 483. HR-ESI-TOF/MS HPLC chromatograms detected at 230nm and by the molecu-
lar extinction coefficients at 230nm (ε230nm). The ε230nm value
ucts were evaluated according to integrated peak areas on RP-
(
4
negative mode): m/z 483.173473 obsd. (Calcd for C H N O
2
0
27
4
10
1
−1
−1
83.173267). UV: λmax =216nm (pH 7.0). H-NMR (500MHz, of 2650M cm was used for dT. The ε230nm values of Peaks
D O): δ (ppm/TMSP-d ) 6.12 (dd, 1H, H-1′), 4.37 (ddd, 1H, 1–4 were determined from the integration of proton signals of
2
4
H-3′), 4.00 (d, 1H, H-6), 3.99 (ddd, 1H, H-4′), 3.79 (m, 1H, NMR and the HPLC peak area detected at 230nm relative to
H-5′ or 5″), 3.69 (m, 1H, H-5′ or 5″), 2.23 (m, 1H, H-2′ or that of dT in the mixed solution. The estimated ε values
2
30nm
13
−1
−1
−1
−1
2
(
″), 1.94 (m, 1H, H-2′ or 2″), 1.50 (s, 3H, CH3). C-NMR were 3380M cm for Peak 1, 5130M cm for Peak 2,
125MHz, D O): δ (ppm/TMSP-d ) 175.8 (C-4), 155.1 (C-2), 4240M cm for Peak 3, and 5040M cm for Peak 4.
−1
−1
−1
−1
2
4
8
9.0 (C-4′), 87.6 (C-1′), 73.9 (C-3′), 64.4 (C-5′), 59.7 (C-6),
52.5 (C-5), 40.6 (C-2′), 23.9 (CH3).
Acknowledgments The author thanks Dr. K. Masuda
Peak 3 (cis–anti (+))
ESI-TOF-MS (negative mode): m/z 483. HR-ESI-TOF/MS
(Shujitsu University) for discussing the carcinogenisity of SA.
(
4
negative mode): m/z 483.173490 obsd. (Calcd for C H N O
83.173267). UV: λmax =215nm (pH 7.0). H-NMR (500MHz, interest.
Conflict of Interest The authors declare no conflict of
2
0
27
4
10
1
D O): δ (ppm/TMSP-d ) 6.32 (dd, 1H, H-1′), 4.46 (ddd, 1H,
2
4
H-3′), 4.16 (d, 1H, H-6), 3.96 (ddd, 1H, H-4′), 3.80 (m, 1H, References
H-5′ or 5″), 3.76 (m, 1H, H-5′ or 5″), 2.32 (m, 1H, H-2′ or
1) IARC, IARC monographs on the evaluation of carcinogenic risks to
1
3
humans, “Solar and ultraviolet radiation,” Vol. 100D, Lyon, IARC
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2
(
8
″), 2.23 (m, 1H, H-2′ or 2″), 1.55 (s, 3H, CH3). C-NMR
125MHz, D O): δ (ppm/TMSP-d ) 175.4 (C-4), 155.6 (C-2),
8.3 (C-4′), 86.2 (C-1′), 74.1 (C-3′), 64.6 (C-5′), 58.2 (C-6),
2
4
2
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Cadet J., Vigny P., “The photochemistry of nucleic acids,” Vol. 1,
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Peak 4 (trans–anti)
3
ESI-TOF-MS (negative mode): m/z 483. HR-ESI-TOF/MS
8
8, 1048–1065 (2012).
(
negative mode): m/z 483.173444 obsd. (Calcd for C H N O
2
0
27
4
10
4) Pfeifer G. P., Besaratinia A., Photochem. Photobiol. Sci., 11, 90–97
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4
83.173267). UV: λmax =218nm (pH 7.0). trans–anti A unit.
H-NMR (500MHz, D O): δ (ppm/TMSP-d ) 6.29 (dd, 1H,
H-1′), 4.36 (ddd, 1H, H-3′), 4.29 (d, 1H, H-6), 3.93 (ddd, 1H,
H-4′), 3.68 (m, 1H, H-5′ or 5″), 3.68 (m, 1H, H-5′ or 5″), 2.39
m, 1H, H-2′ or 2″), 2.14 (m, 1H, H-2′ or 2″), 1.44 (s, 3H,
CH3). C-NMR (125MHz, D O): δ (ppm/TMSP-d ) 176.9
C-4), 155.5 (C-2), 88.3 (C-4′), 87.9 (C-1′), 74.3 (C-3′), 64.5
1
2
4
6
7
)
)
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(
13
2
4
8)
Kristensen B., Kristensen O., Acta Derm. Venereol., 71, 37–40
(
(
(1991).
C-5′), 62.2 (C-6), 49.8 (C-5), 39.3 (C-2′), 19.8 (CH ). trans–
3
9)
Gonzenbach H., Hill T. J., Truscott T. G., J. Photochem. Photobiol.
B, 71, 147–153 (1993).
1
anti B unit. H-NMR (500MHz, D O): δ (ppm/TMSP-d ) 5.87
dd, 1H, H-1′), 4.43 (d, 1H, H-6), 4.36 (ddd, 1H, H-3′), 3.91
ddd, 1H, H-4′), 3.73 (m, 1H, H-5′ or 5″), 3.73 (m, 1H, H-5′ or
2
4
(
(
5
3
1
0) Osgood P. J., Moss S. H., Davies D. J., J. Invest. Dermatol., 79,
54–357 (1982).
″), 2.41 (m, 1H, H-2′ or 2″), 2.18 (m, 1H, H-2′ or 2″), 1.45 (s, 11) Aliwell S. R., Martincigh B. S., Salter L. F., J. Photochem. Photo-
3
13
H, CH3). C-NMR (125MHz, D O): δ (ppm/TMSP-d ) 177.3
biol. Chem., 71, 147–153 (1993).
2
4
(
C-4), 156.7 (C-2), 89.6 (C-1′), 88.7 (C-4′), 73.5 (C-3′), 64.5 12) IARC, IARC monographs on the evaluation of the carcinogenic risk
of chemicals to man, “Some aromatic amines and related nitro com-
pounds,” Vol. 16, Lyon, IARC Press, 1978, pp. 249–257.
3) Bair W. B. 3rd, Hart N., Einspahr J., Liu G., Dong Z., Alberts D.,
(C-5′), 59.9 (C-6), 50.7 (C-5), 39.8 (C-2′), 20.6 (CH3).
Quantitative Procedures For nucleosides reactions, the
concentrations of the nucleosides were quantified by integrated
peak areas on RP-HPLC chromatograms detected at 260nm,
and compared with those of the initial solution of nucleosides
mixture. For the dT reactions, the concentrations of the prod-
1
Bowden G. T., Cancer Epidemiol. Biomarkers Prev., 11, 1645–1652
(2002).