Sensitized photochemistry of di(4-tetrazolouracil) dinucleoside monophosphate as a route to dicytosine cyclobutane photoproduct precursors

Article abstract of DOI:10.1039/c3pp25402j

The DNA cis-syn cyclobutane photoproduct formed between two adjacent cytosine residues is highly mutagenic and responsible for the tandem CC to TT transition. However, its instability has prevented its in vitro study, so far. With a view to prepare oligodeoxynucleotides containing the CC cyclobutane lesion, we have synthesized in good yield a ditetrazolouracil cyclobutane dinucleotide photoproduct as a stable precursor of this photoproduct. Our approach also overcomes the low photochemical reactivity of the cytosine-cytosine deoxydinucleoside monophosphate.

Full text of DOI:10.1039/c3pp25402j

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Sensitized photochemistry of di(4-tetrazolouracil)
dinucleoside monophosphate as a route to dicytosine
Cite this: DOI: 10.1039/c3pp25402j
cyclobutane photoproduct precursors†
Frédéric Peyranea and Pascale Clivio*b
The DNA cis-syn cyclobutane photoproduct formed between two adjacent cytosine residues is highly
mutagenic and responsible for the tandem CC to TT transition. However, its instability has prevented its
in vitro study, so far. With a view to prepare oligodeoxynucleotides containing the CC cyclobutane lesion,
we have synthesized in good yield a ditetrazolouracil cyclobutane dinucleotide photoproduct as a stable
precursor of this photoproduct. Our approach also overcomes the low photochemical reactivity of the
cytosine–cytosine deoxydinucleoside monophosphate.
Received 27th November 2012,
Accepted 19th March 2013
DOI: 10.1039/c3pp25402j
www.rsc.org/pps
Introduction
Ultraviolet exposure of DNA leads to the formation of cyclo-
butane photoproducts between adjacent pyrimidines. Among
the cyclobutane pyrimidine dimers (CPDs) formed at the four
possible combinations of dipyrimidine sites, the dicytosine
(CC) CPD is the least abundant.1 However, despite its scarcity,
this highly mutagenic photoproduct is responsible for almost
all of the CC to TT tandem mutations.2 This particular
mutation is often considered as a signature for UV mutagen-
esis due to its rare occurrence in cancers other than those of
the skin.3 Although the precise sequence of events separating
CC CPD formation from CC to TT transition is currently not
known, the double deamination of the initial CC CPD leading
to the stable end product, UU CPD (Fig. 1), is believed to be a
major event.4
In this scenario, an error-free translesion synthesis past UU
CPD due to delayed bypass of CC CPD would lead to a CC to
TT transition.4b This deamination-bypass mechanism was
initially proposed by Taylor and O’Day5a and later supported
by in vivo and in vitro studies on U-, C- and m5C-containing
CPD.5b–h If the A-rule was earlier proposed to incorporate A
opposite a deaminated C of a C-containing CPD,5b it was later
shown that an instructional insertion mechanism occurred.5c
In addition, C- and m5C-containing CPDs do not constitute a
Fig. 1 Structure of the primary and secondary c,s CPDs at the CC site in DNA
(only the imino tautomer of the dihydrocytosine moiety is represented for sec-
ondary CPDs).
block for translesion synthesis, as earlier proposed,5b but are
efficiently bypassed in an error-free manner.5f–h No doubt the
current knowledge of the CC to TT mutation mechanism
derives from these studies. Impairment of either the repair or
the error-free replicative bypass of the CC CPD lesion is also
involved in the mutagenic pathway.3,6,7 In addition, an error-
prone translesion synthesis of CC CPD has been noted.7
Finally, a reduction of the oxidative stress of the cell,8 a sup-
pression of its immunity9 or the flanking sequence of the CC
site10 may also impact on the level of UV-induced CC to TT
tandem mutation. Paradoxically, CC CPD is one of the most
efficiently repaired CPD lesions.11 Therefore, the detailed mole-
cular pathway linking CC CPD to CC to TT double mutation
still needs further studies.
aInstitut de Chimie des Substances Naturelles, CNRS, 1 Avenue de la Terrasse,
91190 Gif-sur-Yvette, France
bInstitut de Chimie Moléculaire de Reims, UMR 7312 CNRS Université de Reims
Champagne Ardenne, UFR Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex,
France. E-mail: pascale.clivio@univ-reims.fr
Interestingly, no in vitro bypass polymerase study has been
carried out with CC CPD due to its instability,7a even though
such studies would be extremely valuable for investigating
the presumed mutagenic bypass of some polymerases.7b
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3pp25402j
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Additionally, chemical synthesis of CC CPD-containing oligo-
deoxynucleotides (ODNs) is also highly challenging.7c All
Experimental section
except one ODN containing a C or a m5C CPD have been pre- Commercial grade reagents were used without further purifi-
pared by UV B irradiation of the corresponding sequences fol- cation. Solvents were dried as follows: THF was freshly distilled
lowed by HPLC purification using conditions to avoid under argon over sodium–benzophenone. Pyridine, N,N-diiso-
deamination.5f–h The low yields reported (ca. 6%) lead us to propylethylamine, triethylamine, acetonitrile and dichloro-
anticipate an even lower yield for the preparation of CC CPD- methane were dried over CaH2, distilled and stored on 4 Å
containing ODNs using this method. In addition, the for- molecular sieves. Methanol was distilled over magnesium
mation yield of C and m5C CPD in ODNs highly depends on methoxide. DMF was azeotropically co-distilled with toluene
the sequence context.12 Therefore, a purely synthetic approach and then distilled over barium oxide. Analytical grade acetone
would be ideal.
was used as a photosensitizer without further purification.
Synthesis of CC CPD-containing ODNs by automated solid Photolysis was performed with a high pressure mercury vapor
phase synthesis necessitates not only a stabilizing group pre- lamp Original Hanau TQ 150 fitted with a quartz cooling
venting deamination of the CC CPD while compatible with envelope. Pyrex tubes containing the solutions to be irradiated
ODN synthesis conditions, but also an efficient photoreactivity under argon were placed against the envelope. Chromato-
of the dicytosine dinucleotide required for the synthesis of the graphy was performed on Chromagel 60 silica (35–70 μm)
CC CPD phosphoramidite. Disappointingly, the dinucleotides (SDS) and LiChroprep RP18 (Merck). NMR experiments were
CpC and dCpdC are quasi-inefficient at producing the corre- performed on Bruker AC-250, AM-300, AMX-400, DRX-600
1
sponding CPD by direct excitation.13,14 However, acetone- spectrometers. H and 13C chemical shifts (δ) are reported in
photosensitized irradiation of dCpdC should lead to a 40% ppm relative to residual solvent peak (CDCl3 δH 7.26, δC 77.2;
conversion into CPD adducts.15 Unfortunately, in our hands CD3CN δH 1.94, δC 1.32; MeOD δH 3.31, δC 49.00; C6D6 δH 7.16,
and others’,16 dCpdC could hardly produce significant yields δC 128.06; D2O δH 4.79) or to dioxane (D2O, δC 67.19). 31P
of CC CPD from acetone-mediated photosensitization.
chemical shifts are reported relative to an external capillary
Therefore, as a first step toward this complex synthetic goal, standard of 85% phosphoric acid. Superscript (a), (b), (c) and
we envisioned to make use of a precursor of cytosine dinucleo- (d) denote interchangeable assignments within a compound.
tide that would be endowed with a high photochemical reactiv- * Indicates that the assignments have been made by HMQC and
ity and whose corresponding photoproducts would be still HMBC experiments. Prochiral H2′ proton was attributed from
stable to hydrolysis. Noticeably, such an approach has been its NOE with the H6 proton of the base. Prochiral C5′ protons
recently applied to the synthesis of a T m5C CPD-containing have not been assigned and the most deshielded one has been
ODN through the protection of the NH2 of C by an acetyl arbitrarily labelled H5′. High resolution mass spectra were
group.17 During our screening in search of the desired cytosine recorded on a Micromass LCT (ESI, CH3OH) and a PerSeptive
dinucleotide derivative, we explored the photosensitized Biosystems Voyager-DE STR (MALDI, 2,5-dihydroxybenzoic
irradiation of N4-acetyl-2′-deoxycytidine without success. We acid).
therefore turned our attention to another masked 2′-deoxycyti-
dine derivative.
2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-O-thexyldimethyl-
silyluridine (5)
The tetrazole moiety can be considered as a latent amino
functionality.18 Therefore, it could be expected that, as for the To a solution of 4 (2.16 g, 4.07 mmol) in DMF (20 mL) was
uracil dinucleotide UpU,19 exposure of the di(4-tetrazoloura- added imidazole (555 mg, 8.15 mmol) followed by thexyldi-
cil)20 dinucleoside monophosphate 2 to UV light in the pres- methylsilyl chloride (1.2 mL, 6.11 mmol). The reaction mixture
ence of a photosensitizer would lead to the corresponding was stirred at room temperature for 24 h, diluted with CH2Cl2,
tetrazolo CPD dinucleotides. 4-Tetrazolopyrimidine nucleo- washed with brine (2×), dried over Na2SO4 and evaporated
sides21 have been mostly developed as potential therapeutic under reduced pressure. Column chromatography (elution
agents and also as a precursor of cytosine derivatives.18b,22 In heptane–EtOAc 50% with 1% triethylamine) yielded the title
addition, upon direct UV excitation, in the uracil series, these compound as a white foam (2.58 g, 94%). δH (300 MHz,
compounds, such as 1, give rise to ring-expanded or -con- CDCl3): 8.46 (1H, br s), 7.91 (1H, d, J = 8.2 Hz), 7.40–6.84 (13H,
tracted products.23 However, the sensitized photochemistry of m), 6.28 (1H, t, J = 6.0 Hz), 5.38 (1H, dd, J = 8.2; 2.2 Hz), 4.52
the 4-tetrazolouracil moiety has never been examined.
(1H, td, J = 4.9; 6.1 Hz), 3.95 (1H, m), 3.81 (6H, s), 3.53–3.32
We herein report the synthesis of 2 and its sensitized photo- (2H, m), 2.42–2.14 (2H, m), 1.56 (1H, m), 0.82–0.78 (12H, m),
chemical behaviour.
0.09 and 0.05 (6H, 2s); HRMS (ESI, (M + Na)+) calcd for
C38H48N2O7SiNa 695.3129, found 695.3136.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-3-O-thexyldimethylsilyl-
β-D-ribofuranosyl]tetrazolo[4,5-c]pyrimidin-2(1H)-one (6)
To a solution of 5 (100 mg, 0.15 mmol) in THF (1.5 mL) was
added DBU (66 μL) at 0 °C and under argon; after 30 min tri-
isopropylbenzenesulfonyl chloride (113 mg, 0.37 mmol) was
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added by portion, and the ice bath was removed. After 30 min, 0.91–0.87 (12H, m), 0.16 (6H, s); δC (75 MHz, CDCl3): 163.6,
NaN3 (19 mg, 0.3 mmol) was added with 150 μL of DMF, and 150.5, 140.3, 102.4, 87.5, 85.4, 72.1, 63.3, 41.5, 34.1, 25.4, 20.4,
stirring was continued at room temperature overnight. The 20.3, 18.6, 18.5, −3.3, −3.5; HRMS (ESI, (M + Na)+) calcd for
reaction mixture was diluted with CH2Cl2, washed with brine C17H30N2O5SiNa 393.1822, found 393.1818.
(2×), dried over Na2SO4 and evaporated under reduced
1-[2-Deoxy-3-O-(4,4′-dimethoxytrityl)-5-O-thexyldimethylsilyl-
β-D-ribofuranosyl]tetrazolo[4,5-c]pyrimidin-2(1H)-one (10)
pressure. Purification was performed by column chromato-
graphy (elution EtOAc–heptane from 10 to 30% with 1% tri-
ethylamine) yielding the title compound as a white foam Compound 8 (690 mg, 1.86 mmol) was co-evaporated with pyri-
(77 mg, 76%). δH (300 MHz, CDCl3): 8.31 (1H, d, J = 7.8 Hz), dine (2×), and then treated with dimethoxytrityl chloride
7.40–6.84 (13H, m), 6.51 (1H, dd, J = 6.4; 4.7 Hz), 6.35 (1H, d, (810 mg, 2.4 mmol) in pyridine (5 mL) overnight. An
J = 7.8 Hz), 4.64 (1H, m), 4.05 (1H, ddd, J = 5.0; 2.7; 2.5 Hz), additional portion of DMTCl (100 mg, 0.3 mmol) was added
3.81 (6H, s), 3.63 (1H, dd, J = 10.9; 2.7 Hz), 3.43 (1H, dd, J = and the crude material was heated to 60 °C to complete the
10.9; 2.5 Hz), 2.56 (1H, dt, J = 13.6; 6.4 Hz), 2.33 (1H, ddd, J = conversion of the starting material. The solvents were removed
13.6; 6.4; 4.7 Hz), 1.58 (1H, m), 0.85–0.79 (12H, m), 0.10 and in vacuo and the crude product was purified by column chrom-
0.01 (6H, 2s); δC (62.5 MHz, CDCl3): 158.9, 150.9, 144.1, 142.5, atography (elution EtOAc–heptane 30% with 1% triethyl-
135.1, 135.0, 134.8, 130.2, 128.3, 128.1, 127.4, 123.8, 113.4, amine). The solid obtained was dissolved in THF (18 mL) and
93.6, 87.2, 86.9, 70.3, 61.7, 55.3, 42.3, 34.2, 24.9, 20.3, 18.6, DBU (834 μL, 5.58 mmol) was added at 0 °C under argon; after
−2.5, −2.9; HRMS (ESI, (M + Na)+) calcd for C38H47N5O6SiNa 30 min triisopropylbenzenesulfonyl chloride was added
720.3193, found 720.3217.
(1.41 g, 4.65 mmol) and the ice bath was removed. After
30 min, the reaction mixture was treated with NaN3 (240 mg,
3.72 mmol) and DMF (1.8 mL) and allowed to react for 16 h.
After the evaporation of the solvents under reduced pressure,
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-
tetrazolo[4,5-c]pyrimidin-2(1H)-one (7)
A solution of 6 (500 mg, 0.72 mmol) in THF (12 mL) was the crude was diluted with CH2Cl2, washed with brine (3×),
treated with 1 mL of TEA–HF. After 60 h at room temperature, dried over Na2SO4 and evaporated under vacuum. Column
the reaction mixture was diluted with EtOAc, washed with an chromatography (elution EtOAc–heptane from 20 to 50% with
aqueous solution of NaHCO3 (2×) and brine. The combined 1% triethylamine) afforded the title compound as a slightly
aqueous phases were extracted with EtOAc, and the organic yellowish foam (794 mg, 61%). δH (300 MHz, CDCl3): 8.00 (1H,
fractions were pooled together, dried over Na2SO4 and evapor- d, J = 7.9 Hz), 7.47–6.84 (13H, m), 6.77 (1H, d, J = 7.9 Hz), 6.63
ated under reduced pressure. Purification performed by (1H, dd, J = 8.8; 5.6 Hz), 4.34 (1H, d, J = 6.1 Hz), 4.16 (1H, br s),
column chromatography (elution EtOAc–heptane from 30 to 3.81 (6H, br s), 3.74 (1H, d, J = 11.4 Hz), 3.38 (1H, dd, J = 11.4;
100% with 1% triethylamine) led to the title compound as a 1.8 Hz), 1.96 (dd, 1H, J = 13.6; 5.6 Hz), 1.67 (ddd, 1H, J = 13.6;
white foam (324 mg, 81%). δH (250 MHz, CDCl3): 8.18 (1H, d, 8.8; 6.1 Hz), 1.54 (m, 1H), 0.84–0.77 (m, 12H), 0.03 and −0.02
J = 7.9 Hz), 7.42–6.84 (13H, m), 6.52 (1H, t, J = 6.0 Hz), 6.41 (6H, 2s); δC (75 MHz, CDCl3): 158.9, 150.7, 145.0, 142.5, 136.2,
(1H, d, J = 7.9 Hz), 4.70 (1H, m), 4.14 (1H, m), 3.81 (6H, s), 136.1, 134.6, 130.3, 130.2, 128.3, 128.2, 127.3, 113.4, 93.9, 87.9,
3.65–3.51 (2H, m), 2.66 (1H, m), 2.44 (1H, m); δC (62.5 MHz, 87.6, 87.1, 75.2, 63.7, 55.3, 41.2, 34.0, 25.3, 20.3, 20.1, 18.5,
CDCl3): 158.9, 150.8, 144.2, 142.5, 135.1, 135.0, 134.8, 130.2, 18.4, −3.3, −3.7; HRMS (ESI, (M
+
Na)+) calcd for
128.2, 127.4, 113.4, 93.7, 87.5, 86.7, 71.0, 62.4, 55.4, 41.9; C38H47N5O6SiNa 720.3193, found 720.3193.
HRMS (ESI, (M + Na)+) calcd for C30H29N5O6Na 578.2016,
found 578.2003.
1-[2-Deoxy-3-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]-
tetrazolo[4,5-c]pyrimidin-2(1H)-one (11)
2′-Deoxy-5′-O-thexyldimethylsilyluridine (8)
Compound 10 (1.0 g, 1.43 mmol) was dissolved in 24 mL of
2′-Deoxyuridine 3 (2.0 g, 8.77 mmol), co-evaporated with pyri- THF and treated with 2 mL of HF–TEA. Stirring was continued
dine, was dissolved in the same solvent (20 mL). DMAP for 72 h and the reaction mixture was diluted with EtOAc,
(107 mg, 0.88 mmol) and imidazole (1.19 g, 17.5 mmol) were washed with aqueous NaHCO3 (3×) and then with brine. The
added. Dropwise addition of thexyldimethylsilyl chloride combined aqueous phases were extracted with EtOAc, and the
(1.89 mL, 9.65 mmol) was performed at 0 °C over a period of combined organic phases were dried over Na2SO4 and evapor-
5 min. After 10 min, the ice bath was removed and stirring was ated under reduced pressure. Purification by column chrom-
maintained at room temperature overnight. The reaction atography (elution EtOAc–heptane from 30 to 100% with 1%
mixture was diluted with EtOAc, washed with HCl 0.1 N (3×) triethylamine) afforded the title compound as a white solid
and brine, dried over Na2SO4 and evaporated under reduced (680 mg, 86%). δH (300 MHz, CDCl3): 7.95 (1H, d, J = 8.1 Hz),
pressure. Purification was performed by column chromato- 7.47–7.22 (9H, m), 6.86–6.81 (5H, m), 6.55 (1H, dd, J = 7.6;
graphy (elution MeOH–CH2Cl2 from 0 to 10%) to yield the title 6.0 Hz), 4.41 (1H, m), 4.02 (1H, m), 3.79–3.72 (7H, m), 3.36
compound as a white foam (2.85 g, 88%). δH (300 MHz, (1H, m), 2.12 (1H, m), 1.89 (1H, m); δC (75 MHz, CDCl3): 158.8,
CDCl3): 8.44 (1H, br s), 7.84 (1H, d, J = 8.1 Hz), 6.34 (1H, t, J = 150.8, 145.0, 142.6, 136.1, 135.3, 130.3, 128.3, 128.1, 127.2,
6.4 Hz), 5.70 (1H, d, J = 8.1 Hz), 4.48 (1H, m), 4.03 (1H, m), 113.4, 93.9, 87.7, 87.5, 74.3, 62.4, 55.3, 40.7; HRMS (ESI,
3.87 (2H, m), 2.42 (1H, m), 2.15 (1H, m), 1.64 (1H, m), (M + Na)+) calcd for C30H29N5O6Na 578.2016, found 578.2028.
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Compound 13
-pN), 3.84 (2H, m, H5′H5′′ Np-), 2.75 (1H, m, H2′′ Np-),(a)
2.67–2.41 (3H, m, H2′ Np-,(a) H2′H2′′ -pN); δC (62.5 MHz, D2O):*
152.6 (C4 Np-, C4 -pN), 145.1 (C2 Np-),(b) 145.0 (C2 -pN),(b)
137.4 (C6 Np-),(c) 137.1 (C6 -pN),(c) 95.4 (C5 Np-, C5 -pN), 88.7
(C1′ Np-),(d) 88.5 (C1′ -pN),(d) 87.9 (JCP = 6.0 Hz, C4′ -pN), 87.2
(JCP = 8.2 Hz, C4′ Np-), 76.6 (JCP = 4.9 Hz, C3′ Np-), 71.6
(C3′ -pN), 66.2 (JCP = 4.9 Hz, C5′ -pN), 62.2 (C5′ Np-), 40.7 (C2′
-pN), 39.9 (JCP = 3.3 Hz, C2′ Np-); δP (121 MHz, D2O): −0.61;
HRMS (ESI, (M − H)−) calcd for C18H21N10O10P 567.1102,
found 567.1083.
Compound 7 (340 mg, 0.61 mmol) was dissolved in THF
(2.7 mL) containing N,N-diisopropylethylamine (160 μL,
0.92 mmol). 2-Cyanoethyl-N,N-diisopropylphosphoramido-
chloridite (167 μL, 0.76 mmol) was added at −75 °C under
argon. Stirring was maintained for 1 h at room temperature
and the reaction mixture was diluted with ice-cold EtOAc,
washed with ice-cold aqueous solutions of NaHCO3 and NaCl,
dried over Na2SO4, and evaporated under reduced pressure.
The crude material was dissolved in 1 mL of CH3CN, and a
solution of 11 (306 mg, 0.55 mmol) in CH3CN (1.15 mL) was
added, followed by a solution of imidazolium triflate (267 mg,
1.23 mmol) in CH3CN (1 mL). After 30 min, TBHP (100 μL,
5–6 M in nonane) was added; after an additional 30 min
period, the reaction mixture was evaporated and purified by
column chromatography using a gradient of EtOAc (50 to 70%)
in heptane and then a gradient of MeOH (2 to 5%) in CH2Cl2
as the eluent affording 483 mg of a white solid (71%). δH
(300 MHz, CDCl3): 8.01 and 7.94 (1H, 2d, J = 7.9 Hz), 7.65 and
7.59 (1H, 2d, J = 7.9 Hz), 7.47–7.21 (18H, m), 6.87–6.79 (9H,
m), 6.50–6.42 (2H, m), 6.37 (1H, d, J = 7.9 Hz), 5.21 (1H, m),
4.36 (1H, m), 4.27 (1H, m), 4.20–3.87 (5H, m), 3.78 and 3.76
(12H, 2s), 3.51 (2H, m), 2.82 (1H, m), 2.71–2.60 (2H, m),
2.52 (1H, m), 2.21 (1H, m), 1.78 (1H, m); δC (75 MHz, CDCl3):
159.0, 150.7, 144.7, 143.9, 142.6, 135.7, 134.8, 134.6, 134.4,
134.3, 130.2, 128.2, 127.5, 127.4, 116.4, 116.3, 113.7, 113.5,
Photosensitized irradiation of compound 2
Degassed acetone (3.7 mL) was added to a degassed solution
of 2 (62 mg, 0.11 mmol) in H2O–CH3CN (7 : 3, 69.5 mL) and
the mixture was irradiated in a pyrex vessel for 3 h. This experi-
ment was performed twice and solutions were combined, evap-
orated under reduced pressure without heating, and the
resulting aqueous solution was lyophilized. Analytic HPLC was
performed on a SYMMETRY C18 (5 μm, 250 × 4.6 mm) column
eluted with ammonium acetate 0.05 M at a flow rate of 1 mL
min−1 using a photodiode array detector (RT 14a = 11.4 min,
RT 14b = 25.8 min, RT 14c = 30.1 min). HPLC purification was
performed on a Hypersil column (HSC18, 5 μm, 250 × 10 mm)
eluted with ammonium acetate 0.05 M at a flow rate of 4 mL
min−1 (230 nm detection). Three fractions were collected and
lyophilized. The first fraction (RT = 10 minutes) corresponding
to the cis-syn CPD adduct 14a (51 mg, 41%) was obtained as a
white solid. The second fraction (RT = 27 min) was identified
as the trans-syn II CPD adduct 14b (20 mg, 16%, white solid).
The third fraction (RT = 34 min) was tentatively assigned as
the trans-syn I CPD photoproduct 14c.
94.4, 94.3, 94.1, 87.8, 87.6, 86.4, 85.5 (JCP = 6.0 Hz), 85.2 (JCP
=
7.6 Hz), 84.9 (JCP = 8.7 Hz), 84.7 (JCP = 7.1 Hz), 78.7 (JCP = 4.9
Hz), 78.3 (JCP = 4.9 Hz), 73.6 (JCP = 8.7 Hz), 67.9, 62.7, 55.4,
39.9, 19.7; δP (121 MHz, D2O): −2.04, −2.32; HRMS (MALDI,
(M + Na)+) calcd for C63H60N11O14PNa 1248.3942, found
1248.3956.
cis-syn Photoproduct 14a
δH (600 MHz, D2O):* see Table 1; δC (62.5 MHz, D2O):* 152.8
(C4 -pN), 151.0 (C4 Np-), 146.5 (C2 -pN), 144.0 (C2 Np-), 91.5
Di(4-tetrazolouracil) dinucleoside monophosphate 2
Compound 13 (467 mg, 0.38 mmol) was dissolved in DBU– (C1′ Np-), 87.1 (C1′ -pN), 85.1 (C4′ Np-, C4′ -pN), 78.5 (JCP
=
CH3CN 0.5 M (1.5 mL) and stirring was continued for 30 min 6.0 Hz, C3′ Np-), 70.4 (C3′ -pN), 67.6 (JCP = 5.5 Hz, C5′ -pN),
at room temperature. After evaporation of the volatile 64.8 (C6 Np-), 63.1 (C5′ Np-), 52.9 (C6 -pN), 39.6 (C5 -pN), 37.3
materials, 80% aqueous AcOH (30 mL) was added and allowed (C2′ -pN), 33.1 (JCP = 5.1 Hz, C2′ Np-), 29.2 (C5 Np-); δP
to react for 2 h, then removed in vacuo. The residue was co- (121 MHz, D2O): −1.41; HRMS (MALDI, (M + Na)+) calcd for
evaporated with water (2×). An additional portion of water was C18H21N10O10PNa 591.1077, found 591.1083.
added, and a wash with CH2Cl2 was performed (3×). The
trans-syn II Photoproduct 14b
organic phase was back-extracted with a small volume of
water, and the combined aqueous phases were evaporated δH (600 MHz, D2O):* see Table 1; δC (75 MHz, D2O):* 154.5 (C4
under reduced pressure; the crude material was submitted to Np-, C4 -pN), 145.4 (C2 Np-), 144.0 (C2 -pN), 93.9 (C1′ -pN),
reverse phase column chromatography (elution CH3CN–H2O 85.8 (JCP = 6.5 Hz, C4′ -pN), 85.5 (C1′ Np-), 85.2 (JCP = 4.6 Hz,
from 0 to 10%). The obtained dinucleotide was dissolved in C4′ Np-), 71.5 (JCP = 5.1 Hz, C3′ Np-), 70.2 (C3′ -pN), 66.2 (JCP
=
water (10 mL) and stirred gently with Dowex 50W-X8 resin 5.1 Hz, C5′ -pN), 64.2 (C6 -pN), 59.2 (C5′ Np-), 54.6 (C6 Np-),
(10 g, H+ form). Filtration and evaporation of the filtrate 39.4 (JCP = 4.6 Hz, C2′ Np-), 38.9 (C2′ -pN), 35.4 (C5 Np-),(a)
afforded the title compound as a colorless film (124 mg, 57%). 35.2 (C5 -pN);(a) δP (121 MHz, D2O): −0.70; HRMS (MALDI,
δH (300 MHz, D2O):* 8.10 (1H, d, J5,6 = 7.9 Hz, H6 Np-), 8.00 (M
(1H, d, J5,6 = 7.9 Hz, H6 -pN), 7.12 (1H, d, J5,6 = 7.9 Hz, H5 591.1072.
-pN), 7.07 (1H, d, J5,6 = 7.9 Hz, H5 Np-), 6.53 (1H, t, J1′,2′
J1′,2′′ = 6.4 Hz, H1′ -pN), 6.45 (1H, t, J1′,2′ = J1′,2′′ = 6.7 Hz, H1′
Np-), 4.86 (1H, m, H3′ Np-), 4.65 (1H, m, H3′ -pN), 4.33 (1H, m, δH (300 MHz, D2O): 5.82 (1H, dd, J1′,2′′ = 9.8 Hz,(a) J1′,2′
H4′ Np-), 4.29 (1H, m, H4′ -pN), 4.23–4.12 (2H, m, H5′H5′′ 5.6 Hz,(a) H1′ -pN), 5.50 (1H, dd, J1′,2′ = 10.3 Hz, J1′,2′′ = 5.0 Hz,
+
Na)+) calcd for C18H21N10O10PNa 591.1077, found
=
trans-syn I Photoproduct 14c
=
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Table 1 Proton chemical shifts (ppm) and coupling constants (Hz) of 14a and
14b
cis-syn CPD 14a
trans-syn II CPD 14b
Np- -pN
6.19 5.44
Np-
-pN
6.17
δ1′
δ2′
δ2′′
δ3′
δ4′
δ5′
δ5′′
δ5
5.57
2.76
2.38
4.75
4.35
3.72a
3.72a
5.22
5.04
10.6
4.5
2.56
2.41
4.32
4.09
4.19
4.02
5.08
5.48
7.6
3.10
2.70
4.90
3.92
3.88
3.76
4.51
5.55
2.2
2.81
2.37
4.86
3.99
4.25a
4.25a
4.59
5.12
2.9
δ6
3J1′,2′
3J1′,2′′
2J2′,2′′
3J2′,3′
3J2′′,3′
3J3′,4′
3J4′,5′
3J4′,5′′
2J5′,5′′
3J5,6
3J5,5
3J6,6
3J5′,P
3J5′′,P
4J6,5
4J2′,P
6.0
8.8
8.8
13.2
13.6
7.6
4.5
5.1
1.8
9.3
11.7
8.5
14.2
8.1
9.1
8.0
2.1
1.6
13.0
8.7
14.3
4.7
8.3
8.2
b
b
d
d
c
d
d
7.6
8.1
5.2
—
8.5
b
8.7
d
d
4.8
9.3
—
—
—
b
3.0
2.1
b
—
—
a Degenerated H5′H5′′ signals. b Not observed. c Not determinable due
to partially overlapped H3′ -pN and H3′ Np- signals, and non-first-
order splitting of H4′ -pN signal. d Not determinable by first-order
splitting.
Scheme 1 i: DMTCl, DMAP, pyridine; ii: TDMSCl, imidazole, DMF; iii: (a) DBU,
TPSCl, THF; (b) NaN3, DMF; iv: HF–TEA, THF; v: P(NiPr2)(O(CH2)2CN)Cl, DIPEA,
THF; vi: TDMSCl, DMAP, imidazole, pyridine; vii: DMTCl, pyridine; viii: imidazo-
lium triflate, CH3CN; ix: TBHP; x: DBU, CH3CN; xi: 80% aqueous AcOH; xii:
Dowex 50W-X8 (H+ form), H2O.
H1′ Np-), 5.28 (1H, dd, J = 8.6 Hz; 8.1 Hz, H6 or H5), 5.18 (1H,
t, J = 8.1 Hz, H6 or H5), 4.80 (3H, m, H3′ Np-, H5 or H6, H5 or
H6), 4.58 (1H, m, H3′ -pN), 4.28 (1H, ddd, J5′,5′′ = 12.2 Hz, J4′,5′
= 4.2 Hz,(c) J5′,P = 1.9 Hz,(c) H5′ -pN), 4.23–4.14 (2H, m, H5′′ -pN,
H4′ Np-), 4.00 (1H, m, H4′ -pN), 3.72 (2H, m, H5′H5′′ Np-), 3.38
(1H, ddd, J2′,2′′ = 14.3 Hz, J1′,2′ = 10.3 Hz, J2′,3′ = 4.8 Hz, H2′ Np-
),(d) 2.76 (1H, dd, J2′,2′′ = 14.3 Hz, J1′,2′′ = 5.0 Hz, H2′′ Np-),(d)
2.16 (2H, m, H2′H2′′ -pN); HRMS (MALDI, (M + Na)+) calcd for
C18H21N10O10PNa 591.1077, found 591.1067.
substituted with NaN3 to furnish the corresponding tetrazolo
derivative 6 in 76% yield. Desilylation of 6 (HF–TEA in THF)
furnished 7 in 81% yield.
Independently, 2′-deoxyuridine 3 was 5′-silylated using
TDMSCl in pyridine in the presence of imidazole and DMAP to
provide 8 in 88% yield (Scheme 1). Dimethoxytritylation of
compound 8 using DMTCl in pyridine, and then azidation
(TPSCl and then NaN3), gave 10 in 61% yield (two steps). Com-
pound 11 was obtained in 86% yield by desilylation of 10
using HF–TEA in THF.
Abbreviations: Np- refers to the 5′-residue and -pN to the
3′-residue of the dinucleoside monophosphate.
Compound 7 was transformed into the phosphoramidite
derivative 12 using 2-cyanoethyl-N,N-diisopropylchloropho-
sphoramidite in THF in the presence of diisopropylamine
(Scheme 1). An acetonitrile solution of 12 was then allowed to
react with alcohol 11 in the presence of imidazolium triflate as
an activator24 to afford the corresponding phosphite triester.
Results and discussion
Synthesis of the di(4-tetrazolouracil) dinucleoside
monophosphate 2
Dinucleotide 2 was synthesized using the phosphoramidite Subsequently oxidation with t-butylhydroperoxide (TBHP) gave
chemistry. Tritylation of 2′-deoxyuridine (3) afforded the 13 in 71% yield from 7 (three steps). Non-nucleophilic base
known 2′-deoxy-5′-dimethoxytrityluridine 4. Silylation of 4 DBU was used to deprotect the phosphate group due to the
using thexyldimethylsilyl chloride (TDMSCl) in DMF in the known instability of the tetrazolo nucleoside toward nucleo-
presence of imidazole afforded 5 in 94% yield (Scheme 1). Posi- philes.25 Full deprotection of 13 was carried out in a one-pot
tion C4 of compound 5 was transiently activated using triiso- procedure (decyanoethylation by DBU followed by detritylation
propylbenzensulfonyl chloride (TPSCl) in the presence of 1,8- by 80% aqueous acetic acid). Purification performed on
diazabicyclo[5.4.0]undec-7-ene (DBU) in THF and then reverse phase silica gel chromatography followed by exchange
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of the DBUH+ counter-ion (Dowex resin, H+ form) led to 2 in Np- and H6 -pN (5.2 Hz) in 14a is a signature of the cis-syn
57% yield (three steps).
stereochemistry (lit. 4.9 Hz).19,26
For the cis-syn CPD of UpU,19 we noticed an inversion of
J H5 -pN and H6 Np- (7.32 Hz) and J H5 -pN and H6 -pN (0 Hz)
since 7.32 Hz is too high to be a 4J and J H5–H6 cannot be
Acetone sensitized photochemistry of 2
The photosensitized irradiation of 2 was conducted at a con- null. In addition, the W arrangement in the cis-syn CPD of
centration of 1.5 mM in a water–acetonitrile (7/3) solution con- UpU is reported to be between H5 Np- and H6 -pN and the
taining 5% acetone. Analysis of the crude irradiation mixture magnitude of J H5–H6 is higher at the 5′-end than at the
by RP HPLC revealed the presence of three photoproducts 3′-end. A reversal of the attribution of 5′- and 3′-end cyclobu-
(14a–c) in 9 : 4 : 2 ratio assuming a similar molar extinction tane protons would fit with our results. Our assignments are
coefficient at 230 nm for each photoproduct.
unambiguously made using short- and long-range H–C coup-
Photoproducts 14a and 14b were isolated by RP HPLC in lings while those of the literature19 were made by comparison
1
41% and 16% yields, respectively. Assignment of their H and with similar compounds.
13C NMR signals was achieved by using 2D H–H and H–C
experiments. Np- and -pN deoxyribose signal residues were
identified from carbon splitting due to carbon–phosphorus
coupling (C3′ and C2′ for Np- and C5′ for -pN).
Minor compound 14c turned out to be particularly unstable
upon storage, giving a mixture of products, in contrast to 14a–
b that were stable. Its identification was performed using only
1H and COSY NMR spectra as well as mass spectroscopy.
Compounds 14a–c were assigned as cyclobutane photo-
products due to the absence of UV absorption above 250 nm
and the unchanged m/z value on the mass spectrum compared
1
to 2. Examination of the H NMR spectrum of each photopro-
duct confirmed the absence of H6 olefinic protons.
The NMR signals of the cyclobutane ring of 14b appeared at δ
Geometrical constraints inherent to the dinucleotide back- 5.55 (t, HA′, J = 8.7 Hz), 5.12 (dd, HB′, J = 8.5; 8.7 Hz), 4.59 (d,
1
bone of 2 limit the available reactive approaches of the two HC′, J = 8.5 Hz), 4.51 (d, HD′, J = 8.7 Hz) on the H spectrum,
modified pyrimidine moieties for cycloaddition. Thus, only cis- and at δ 54.6 (CA′), 64.2 (CB′), 35.4 and 35.2 (CC′ and CD′) on the
syn and trans-syn photoproducts can be formed, depending on 13C spectrum. As for photoproduct 14a, correlations between
the glycosidic bond conformation during the photocycloaddi- H1′ Np- and CA′ and between H1′ -pN and CB′ allowed the
tion reaction.
unambiguous identification of CA′ as C6 Np- and CB′ as C6
In the case of photoproduct 14a, 1H NMR signals of the -pN, respectively. Attribution of HD′ and HC′ as H5 Np- and H5
cyclobutane moiety appeared at δ 5.48 (dd, HA, J = 5.2; 8.5 Hz), -pN was deduced from their coupling with H6 Np- and H6 -pN,
5.22 (dd, HB, J = 7.6; 8.1 Hz), 5.08 (ddd, HC, J = 3.0; 8.1; 8.5 Hz) respectively.
and 5.04 (ddd, HD, J = 3.0; 5.2; 7.6 Hz). Signal of each corre-
The coupling constant between the two H6 (8.7 Hz) is in
sponding carbon was determined by HMQC: δ 52.9 (CA), 29.2 favour of a trans-syn structure.19,26–28 The NOESY experiment
(CB), 39.6 (CC) and 64.8 (CD). Assignment of HA and HD to H6 only showed correlations between H6 and H5 of their respect-
-pN and H6 Np-, respectively, was unambiguously deduced ive base moieties and confirmed a trans-syn stereochemistry.
from HMBC correlations between H1′ -pN and CA and H1′ Np- Finally, a correlation between H6 Np- and both H2′ Np- and
and CD, respectively. These attributions are in accordance with H3′ Np- was in agreement with an anti glycosidic conformation
those of UpU; cyclobutanes were C6 Np- and C6 -pN resonate of the Np- moiety. Compound 14b is the trans-syn II CPD
near 60 ppm and 50 ppm, respectively.19 Assignment of HB isomer, resulting from the photochemical reaction of the
and HC as H5 Np- and H5 -pN was deduced from their coup- 5′-end base and the 3′-end base of 2 in an anti and syn glycosi-
ling with H6 Np- and H6 -pN, respectively. Finally, H6 -pN was dic bond conformation, respectively. This stereochemistry is
shown to correlate with H5 Np-, H5 -pN, and H6 Np- on a further supported by the characteristic NMR chemical shifts of
NOESY spectrum; all of these protons are thus on the same the two H1′ protons of 14b (H1′ Np-: δH 6.19; δC 85.5; H1′ -pN:
side of the cyclobutane ring, establishing its cis stereo- δH 5.44; δC 93.9) which are typical of a trans-syn II stereo-
chemistry. Another correlation between H6 -pN and H2′ -pN chemistry regardless of the cyclobutane substitution.26–28 The
evidenced the anti glycosidic conformation of the -pN moiety. assignment of trans-syn I or trans-syn II structure to the iso-
As a conclusion, compound 14a was assigned as the cis-syn lated trans-syn cyclobutane photoproduct of UpU is not clearly
CPD isomer. It results from the photocycloaddition between stated.19 However, we believe that the large difference between
the two parent bases in an anti–anti glycosidic bond confor- δ of the two H1′ protons (Δδ 0.9) and the deshielding of H1′
mation. A scalar coupling between H6 Np- and H5 -pN indi- Np- with respect to H1′ -pN and of their corresponding carbon
cated a W arrangement of these protons. In agreement with of this trans-syn UpU photoproduct17 is a signature of a trans-
the literature, the value of the coupling constant between H6 syn II structure.26a,27,28
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the trans-syn II photoproduct is produced more efficiently than
the trans-syn I CPD.
Conclusion
In this study, we have demonstrated that cis-syn and trans-syn
II di(4-tetrazolouracil) CPD can be efficiently produced upon
photosensitized excitation of the di(4-tetrazolouracil) dinucleo-
side monophosphate 2. Functionalization and incorporation
of these masked CC CPDs into ODNs by solid phase synthesis
using modified conditions should lead, after reduction of the
tetrazolo motifs, to the rare and delicate CC cyclobutane
photoproducts. In addition, the cis-syn CC CPD analogue will
also be helpful to investigate some biophysical and biological
properties of the major CC CPD where hybridization and/or
shape/size of the base adduct moiety play a central role.
The COSY spectrum of 14c allowed to connect the sugar
protons of each residues. The most deshielded H3′ proton was
attributed to the one of the Np- residue. Consequently, each
proton of the Np- or -pN moiety could be assigned. The high
deshielding of one of the protons at the C2′ position of the Np-
residue (δ 3.38, H2′; δ 2.76, H2′′) was a signature of a trans-syn I
structure in the 2′-deoxy series.26,27 The δ difference between
the two H1′ protons (Δδ = 0.3) was in accordance with this
stereochemistry.26,27 Only the two deshielded protons of the
cyclobutane moiety appeared distinctly (δ 5.28, dd, J = 8.6 Hz;
8.1 Hz and δ 5.18, t, J = 8.1 Hz) and coupled to each other.
They are most probably the two H6 protons and the magnitude
of their coupling is also in accordance with a trans-syn I stereo-
chemistry.26,27 Therefore, it is likely that 14c is the trans-syn I
photoproduct resulting from the cycloaddition between the
5-end base and the 3′-end base of 2 in a syn and anti glycosidic
bond conformation, respectively.
Acknowledgements
We thank Dr A. Sarasin for his interest in this work and the
Ligue Nationale contre le Cancer for a doctoral fellowship
to F.P.
Notes and references
1 (a) D. L. Mitchell, J. Jen and J. E. Cleaver, Sequence speci-
ficity of cyclobutane pyrimidine dimers in DNA treated
with solar (ultraviolet B) radiation, Nucleic Acids Res., 1992,
20, 225–229; (b) T. Douki and J. Cadet, Individual determi-
nation of the yield of the main UV-induced dimeric pyrimi-
dine photoproducts in DNA suggests a high mutagenicity
of CC photolesions, Biochemistry, 2001, 40, 2495–2501;
(c) T. Douki, A. Reynaud-Angelin, J. Cadet and E. Sage,
Bipyrimidine photoproducts rather than oxidative lesions
are the main type of DNA damage involved in the genotoxic
effect of solar UVA radiation, Biochemistry, 2003, 42, 9221–
9226; (d) S. Mouret, C. Baudouin, M. Charveron, A. Favier,
J. Cadet and T. Douki, Cyclobutane pyrimidine dimers are
predominant DNA lesions in whole human skin exposed to
UVA radiation, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,
13765–13770.
While we have not yet a rational explanation for the instabil-
ity of 14c, tetrazolo CPD 14a and 14b are more stable to
hydrolysis than the c,s CPD of CpC whose half life at pH 7 and
at 25 °C is 10 h.13a
2 E. Otoshi, T. Yagi, T. Mori, T. Matsunaga, O. Nikaido,
S.-T. Kim, K. Hitomi, M. Ikenaga and T. Todo, Respective
roles of cyclobutane pyrimidine dimers, (6-4) photo-
products, and minor photoproducts in ultraviolet mutagen-
esis of repair-deficient Xeroderma pigmentosum A cells,
Cancer Res., 2000, 60, 1729–1735.
3 L. Daya-Grosjean and A. Sarasin, The role of UV induced
lesions in skin carcinogenesis: an overview of oncogene
and tumor suppressor gene modifications in Xeroderma
pigmentosum skin tumors, Mutat. Res., Fundam. Mol. Mech.
Mutagen., 2005, 571, 43–56.
In view of incorporating these tetrazolo CPD into ODNs by
solid phase synthesis, we studied the stability of 14a in a
K2CO3–methanol solution (0.05 M) at room temperature since
tetrazolo nucleosides do not withstand nucleophiles.25 HPLC/
MS revealed the rapid and complete conversion of 14a to a
single product (m/z 631, ESI, (M− H)−) corresponding to the
insertion of two methanol molecules. Therefore, incorporation
of these tetrazolo CPD into ODNs will require non-nucleophilic
base deprotection and support cleavage conditions.
Compound 2 presents an atypical photochemical behav-
iour. To the best of our knowledge, it constitutes the first
photoreactive nucleotide for which, upon photosensitization,
4 (a) W. Peng and B. R. Shaw, Accelerated deamination of
cytosine residues in UV-induced cyclobutane pyrimidine
This journal is © The Royal Society of Chemistry and Owner Societies 2013
Photochem. Photobiol. Sci.

View Article Online
Paper
dimers leads to CC→TT transitions, Biochemistry, 1996, 35,
Photochemical & Photobiological Sciences
L. Daya-Grosjean, Analysis of skin cancer risk factors in
immunosuppressed renal transplant patients shows high
levels of UV-specific tandem CC to TT mutations of the
p53 gene, Carcinogenesis, 2006, 28, 724–731.
10172–10181; (b) A. Burger, D. Fix, H. Liu, J. Hays and
R. Bockrath, In vivo deamination of cytosine-containing
cyclobutane pyrimidine dimers in E. coli: a feasible part of
UV-mutagenesis, Mutat. Res., Fundam. Mol. Mech. Mutagen., 10 A. M. Skinner, C. Dan and M. S. Turker, The frequency of
2003, 522, 145–156.
5 (a) J.-S. Taylor and C. L. O’Day, cis-syn Thymine dimers are
not absolute blocks to replication by DNA polymerase I of
CC to TT tandem mutations in mismatch repair-deficient
cells is increased in a cytosine run, Mutagenesis, 2008, 23,
87–91.
Escherichia coli in vitro, Biochemistry, 1990, 29, 1624–1632; 11 S. Mouret, M. Charveron, A. Favier, J. Cadet and T. Douki,
(b) I. Tessman, S.-K. Liu and M. A. Kennedy, Mechanism of
SOS mutagenesis of UV-irradiated DNA: mostly error-free
processing of deaminated cytosine, Proc. Natl. Acad.
Differential repair of UVB-induced cyclobutane pyrimidine
dimers in cultured human skin cells and whole human
skin, DNA Repair, 2008, 7, 704–712.
Sci. U. S. A., 1992, 89, 1159–1163; (c) N. Jiang and 12 V. J. Cannistraro and J.-S. Taylor, Acceleration of 5-methyl-
J.-S. Taylor, In vitro evidence that UV-induced C→T
mutations at dipyrimidine sites could result from replica-
tive bypass of cis-syn cyclobutane dimers or their deamina-
cytosine deamination in cyclobutane dimers by G and its
implications for UV-induced C-to-T mutation hotspots,
J. Mol. Biol., 2009, 392, 1145–1157.
tion products, Biochemistry, 1993, 32, 472–481; 13 (a) K. B. Freeman, P. V. Hariharan and H. E. Johns, The
(d) D.-H. Lee and G. P. Pfeifer, Deamination of 5-methyl-
cytosines within cyclobutane pyrimidine dimers is an
important component of UVB mutagenesis, J. Biol. Chem.,
2003, 278, 10314–10321; (e) K. Takasawa, C. Masutani,
F. Hanaoka and S. Iwai, Chemical synthesis and translesion
replication of a cis-syn cyclobutane thymine-uracil dimer,
Nucleic Acids Res., 2004, 32, 1738–1745; (f) B. Vu,
V. J. Cannistraro, L. Sun and J.-S. Taylor, DNA synthesis
past a 5-methylC-containing cis-syn-cyclobutane pyrimidine
ultraviolet photochemistry of cytidylyl-(3′-5′)-cytidine,
J. Mol. Biol., 1965, 13, 833–848; (b) P. V. Hariharan and
H. E. Johns, Photochemical cross sections in cytidylyl-
(3′-5′)-cytidine, Can. J. Biochem., 1968, 46, 911–918;
(c) G. G. Gurzadyan and H. Görner, Depopulation of highly
excited singlet states of DNA model compounds: quantum
yields of 193 and 254 nm photoproducts of pyrimidine
monomers and dinucleoside monophosphates, Photochem.
Photobiol., 1996, 63, 143–153.
dimer by yeast Pol η is highly nonmutagenic, Biochemistry, 14 (a) T. Douki and J. Cadet, Formation of cyclobutane dimers
2006, 45, 9327–9335; (g) M. J. Horsfall, A. Borden and
C. W. Lawrence, Mutagenic properties of the T–C cyclobu-
tane dimer, J. Bacteriol., 1997, 179, 2835–2839; (h) Q. Song,
S. M. Sherrer, Z. Suo and J.-S. Taylor, Preparation of site-
specific T = mCG cis-syn cyclobutane dimer-containing
template and its error-free bypass by yeast and human poly-
merase η, J. Biol. Chem., 2012, 287, 8021–8028.
6 C. Y. Shin-Darlak, A. M. Skinner and M. S. Turker, A role
for Pms2 in the prevention of tandem CC→TT substi-
tutions induced by ultraviolet radiation and oxidative
stress, DNA Repair, 2005, 4, 51–57.
and (6–4) photoproducts upon far-UV photolysis of
5-methylcytosine-containing dinucleoside monophos-
phates, Biochemistry, 1994, 33, 11942–11950; (b) T. Douki,
T. Zalizniak and J. Cadet, Far-UV-induced dimeric photo-
products in short oligonucleotides: sequence effects, Photo-
chem. Photobiol., 1997, 66, 171–179.
15 S.-T. Kim and A. Sancar, Effect of base, pentose, and phos-
phodiester backbone structures on binding and repair of
pyrimidine dimers by Escherichia coli DNA photolyase, Bio-
chemistry, 1991, 30, 8623–8630.
16 J. Cadet and P. Vigny, The Photochemistry of Nucleic Acids in
Bioorganic Photochemistry, ed. H. Morrison, John Wiley &
Sons, New York, 1990, vol. 1, pp. 1–272.
7 (a) S.-L. Yu, R. E. Johnson, S. Prakash and L. Prakash,
Requirement of DNA polymerase η for error-free bypass of
UV-induced CC and TC photoproducts, Mol. Cell. Biol., 17 J. Yamamoto, K. Nishiguchi, K. Manabe, C. Masutani,
2001, 21, 185–188; (b) J.-H. Yoon, L. Prakash and
S. Prakash, Highly error-free role of DNA polymerase η in
the replicative bypass of UV-induced pyrimidine dimers in
mouse and human cells, Proc. Natl. Acad. Sci. U. S. A., 2009,
F. Hanaoka and S. Iwai, Photosensitized [2 + 2] cyclo-
addition of N-acetylated cytosine affords stereoselectively
formation of cyclobutane pyrimidine dimer, Nucleic Acids
Res., 2011, 39, 1165–1175.
106, 18219–18224; (c) J.-H. Choi and G. P. Pfeifer, The role 18 (a) H. C. Shah, V. H. Shah and N. D. Desai, A novel strategy
of polymerase η in UV mutational spectra, DNA Repair,
2005, 4, 211–220; (d) A. Vaisman and R. Woodgate, Unique
misinsertion specificity of polι may decrease the mutagenic
potential of deaminated cytosines, EMBO J., 2001, 20,
6520–6529.
8 A. M. Skinner and M. S. Turker, High frequency induction
of CC to TT tandem mutations in DNA repair-proficient
mammalian cells, Photochem. Photobiol., 2008, 84, 222–227.
for the synthesis of 2-amino-4,6-diarylnicotinonitrile,
ARKIVOC, 2009, ii, 76–87 and references cited
(b) K. Ciszewski, L. Celewicz and K. Golankiewicz, Novel
synthetic route to 1-substituted cytosines, Synthesis, 1995,
777–779; (c) L. P. Kotra, P. Wang, M. Bartlett,
K. Shanmuganathan, Z. Xu, S. Cavalcanti, M. G. Newton
and C. K. Chu, 4-Azido-2-pyrimidinone nucleosides and
related chemistry, J. Org. Chem., 1997, 62, 7267–7271.
9 S. Queille, L. Luron, A. Spatz, M. F. Avril, V. Ribrag, 19 R. E. Rycyna and J. L. Alderfer, Ultraviolet irradiation of
P. Duvillard, C. Hiesse, A. Sarasin, J. P. Armand and
nucleic acids: formation, purification, and solution
Photochem. Photobiol. Sci.
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conformational analyses of the cis-syn and trans-syn photo- 24 Y. Hayakawa and M. Kataoka, Facile synthesis of oligodeoxy-
dimers of UpU, Biochemistry, 1988, 27, 3142–3151.
20 The proper chemical name is tetrazolo[4,5-c]pyrimidin-2-
one.
nucleotides via the phosphoramidite method without
nucleoside base protection, J. Am. Chem. Soc., 1998, 120,
12395–12401.
21 (a) L. De Napoli, L. Mayol, G. Piccialli, M. Rossi and 25 F. Peyrane and P. Clivio, The chemistry of 4-azido-2-pyrimi-
C. J. Santacroce, Synthesis of novel pyrimidine nucleoside
dinone nucleosides revisited, J. Org. Chem., 2005, 70, 1961–
analogues, J. Heterocycl. Chem., 1986, 23, 1401–1403;
1962.
(b) L. De Napoli, A. Messere, D. Montesarchio and 26 (a) F.-T. Liu and N. C. Yang, Photochemistry of cytosine
C. Santacroce, Synthesis of 4-substituted pyrimidine 2′,3′-
dideoxynucleosides, Nucleosides Nucleotides, 1991, 10,
1719–1728; (c) K. Ciszewski, L. Celewicz and
K. Golankiewicz, Synthesis of 6-substituted tetrazolo[1,5-c]
pyrimidin-5(6)ones: new modification of 3′-azido-3′-deoxy-
thymidine, Biochem. Biophys. Res. Commun., 1992, 187,
1545–1550; (d) T. S. Mansour, C. A. Evans, M. A. Siddiqui,
M. Charron, B. Zacharie, N. Nguyen-Ba, N. Lee and
derivatives. 1. Photochemistry of thymidylyl-(3′→5′)-deoxy-
cytidine, Biochemistry, 1978, 17, 4865–4876;
(b) J. Kemmink, R. Boelens and R. Kaptein, Two-dimen-
1
sional H NMR study of two cyclobutane type photodimers
of thymidylyl-(3′-5′)-thymidine, Eur. Biophys. J., 1987, 14,
293–299; (c) T. M. G. Koning, J. J. G. van Soest and
R. Kaptein, NMR studies of bipyrimidine cyclobutane
photodimers, Eur. J. Biochem., 1991, 195, 29–40.
B. Korba, Structure–activity relationship of pyrimidine 27 (a) W. A. Tabaczynski, D. G. E. Lemaire, B. P. Ruzsicska and
heterosubstituted nucleoside analogues, Nucleosides Nucleo-
tides, 1997, 16, 993–1001.
22 L. P. Kotra, P. Wang, M. Bartlett, K. Shanmuganathan,
Z. Xu, S. Cavalcanti, M. G. Newton and C. K. Chu, 4-Azido-
2-pyrimidinone nucleosides and related chemistry, J. Org.
Chem., 1997, 62, 7267–7271.
J. L. Alderfer, An NMR and conformational investigation of
the trans-syn cyclobutane photodimers of dTpdU, Biopoly-
mers, 1993, 33, 1365–1375; (b) W. A. Tabaczynski,
D. G. E. Lemaire, B. P. Ruzsicska and J. L. Alderfer, An NMR
and conformational investigation of the trans-syn cyclo-
butane photodimers of dUpdT, Biopolymers, 1999, 50, 185–191.
23 F. Peyrane, M. Cesario and P. Clivio, Photochemical ring 28 J. L. F. Kao, S. Nadji and J.-S. Taylor, Identification and
expansion of 4-azidouracil: a route to 5H-1,3,5-triazepin-
2,4-dione in the nucleoside series, J. Org. Chem., 2006, 71,
1742–1745.
structure determination of a third cyclobutane photodimer
of thymidylyl-(3′→5′)-thymidine: the trans-syn-II product,
Chem. Res. Toxicol., 1993, 6, 561–567.
This journal is © The Royal Society of Chemistry and Owner Societies 2013
Photochem. Photobiol. Sci.