Synthesis of the TT pyrimidine (6-4) pyrimidone photoproduct-thio analogue phosphoramidite building block

Article abstract of DOI:10.1039/b305067j

Synthesis of the TT (6-4) pyrimidone photoproduct-thio analogue phosphoramidite building block was analyzed. The reaction was stirred under an argon atmosphere at room temperature and the solvents were evaporated and the residue was purified by reverse-phase chromatography. All the HPLC experiments were conducted at a flow rate of 1 mL min^-1 and the effluent was monitored at 330 nm.

Full text of DOI:10.1039/b305067j

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Synthesis of the TT pyrimidine (6–4) pyrimidone
photoproduct–thio analogue phosphoramidite building block
Sandra Karina Angulo Matus, Jean-Louis Fourrey and Pascale Clivio*
Institut de Chimie des Substances Naturelles, CNRS, 1 Avenue de la Terrasse,
91190 Gif sur Yvette, France. E-mail: pascale.clivio@icsn.cnrs-gif.fr
Received 9th May 2003, Accepted 7th August 2003
First published as an Advance Article on the web 4th September 2003
The phosphoramidite building block synthesis of the thio analogue at the 5,6-dihydropyrimidine C5 position of
the thymidylyl(3Ј–5Ј)thymidine (6–4) photoproduct 1 is presented. This compound was readily obtained from the
appropriately protected dinucleotide P-methyl-5Ј-O-dimethoxytritylthymidilyl(3Ј 5Ј)-4-thiothymidine 2 after
irradiation at 366 nm, then S-sulfenylmethylation of the thiol function of the resulting (6–4) adduct, and
phosphitylation of the 3Ј-hydroxyl group.
direct photolysis of an oligonucleotide containing the Ts4T
Introduction
sequence. However, despite the selective photoactivable prop-
erties of 4-thiothymine, this method does not afford exclusively
the s5(6–4) PP at Ts4T sites.7aFurthermore, the 4-thionucleobase
may also add photochemically to an adjacent 3Ј-pyrimidine.12
In the natural series, the absence of specificity in the type and
site of photoproduct formation has been circumvented by
synthesizing and site specifically incorporating building blocks
containing (6–4)PPs into oligonucleotides.13
Pyrimidine (6–4) pyrimidone photoproducts ((6–4)PPs) are one
of the major causes of DNA damage. They are formed at dipyr-
imidine sites upon UV exposure (Fig. 1).1 Due to their muta-
genic properties2 and their involvement in UV carcinogenesis,3
these lesions attract considerable attention which is currently
intensified by the recent discovery of polymerases that special-
ize in translesion synthesis.4 In addition, the interference of
(6–4)PPs with other metabolic processes such as transcription5
is currently receiving great interest. Beside these natural lesions,
their analogues in which the C5 hydroxyl or amino group is
replaced by a thiol group (Fig. 1) have been proved to be valu-
able tools to investigate some of the properties of the natural
adducts,6 this thiol substituent conferring the unique ability to
be in equilibrium with their thietane precursor. This particular-
ity has been used to provide the first evidence of the formation
mechanism of (6–4)PPs7 and has contributed to the elucidation
of the repair mechanism of (6–4) photolyases.8,9 In addition,
these thio analogues can afford h5(6–5)PPs that could be
suitable analogues for structure–mutagenicity relationship
studies.10 Thus s5(6–4) analogues are attractive compounds and
physico-chemical and biological studies using oligonucleotides
containing the s5(6–4) analogue of TT have therefore
emerged.9,11 So far, such oligonucleotides were prepared by
Envisioning a similar strategy for the preparation of s5(6–4)
PP-containing oligonucleotides, we herein describe an efficient
synthesis of the Ts5(6–4)T photoproduct building block 1.
Results and discussion
Having observed that the 5Ј-dimethoxytrityl (dmt) group was
stable to the light used to photolyse 4-thiothymine (366 nm), we
reasoned that this stability would shorten the synthetic route to
1 compared to the previously described syntheses of (6–4)
phosphoramidite building blocks which required the removal
of this protecting group, due to its instability at 254 nm, and its
subsequent reintroduction.13 Thus, we considered compound 2
as a key intermediate for the synthesis of the s5(6–4) phos-
phoramidite photoproduct (1). We chose to protect the inter-
nucleotide phosphate linkage of 2 with a methyl group for ease
Fig. 1 Pyrimidine (6–4) pyrimidone photoproducts produced at bipyrimidine sites in DNA
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 1 6 – 3 3 2 0
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Scheme 1 Reagents and yields: i, Levulinic acid, DCC, 98%; ii, Trifluoroacetic acid/CH2Cl2, 85%; iii, 1H-Tetrazole; iv, I2/THF/H2O/lutidine;
v, NH2NH2.H2O,pyridine-AcOH, 74%; vi, hν 366 nm; vii, CH3SO2SCH3, K2CO3, 43%; viii, NCCH2CH2OP(Cl)N(iPr)2, EtN(iPr)2, 64%; ix, Conc. aq.
ammonia; x, 40% CH3COOH; xi, Dithiotreitol pH8.
of interpretation of NMR spectra.14 We also reasoned that the
P-methoxy group was structurally close to the P-methyl group
that was previously found not to interfere with the photo-
chemical pathway leading to (6–4) PPs.15
diisopropylphosphoramidite (6)23 in the presence of tetrazole
followed by iodine oxidation and removal of the levulinyl pro-
tection with buffered hydrazine hydrate in pyridine-acetic acid
resulted in the formation of 2 with an overall yield of 74% from
5 (3 steps).24
The synthetic route envisaged to obtain 2, compared to the
corresponding method in the natural series, had to solve a
major difficulty inherent to the presence of the sulfur atom.
Thus, with regards to phosphoramidite chemistry, 4-thiothym-
ine had to be incorporated either under a protected form (S-
pivaloyloxymethyl or S-cyanoethyl)16 for the formation of the
internucleotide phosphotriester linkage or generated by thiol
displacement of a pyrimidine activated at the 4-position12,17
after the formation of the internucleotide phosphotriester
linkage. However, in our case, deprotection of the methyl
phosphotriester could be expected during the removal of the
S-protecting group or the thiol introduction. Having previously
observed that it is not always necessary to protect the thiol
function of 4-thiothymine for the synthesis of dinucleotides at
least for the H-phosphonate chemistry,18 we decided to invest-
igate the synthesis of 2, using the phosphoramidite chemistry,
without protection of the 4-thiothymine derivative sulfur atom.
Another concern was the choice of the 3Ј-hydroxyl transient
protecting group of the 3Ј-unit. Removal conditions of this
group had to be orthogonal with the thiocarbonyl function and
the phosphate protecting group. We decided to use the levulinyl
group as a 3Ј-protecting group due to its clean deprotection
under mild conditions19 that should not lead to the displace-
ment of the sulfur,20 and its compatibility with the methyl
phosphate protection.21
Photolysis of 2 at 366 nm in an aqueous/acetonitrile solution
gave a mixture of photoproducts consisting mainly of the
thietane in equilibrium with the (6–4)PP. Reverse phase (RP)
HPLC separation of these compounds failed as they coeluted
with the remaining starting material. Trapping the (6–4)PP/
thietane equilibrium under a s5(6–4) methyl sulfide yields
77a made its purification possible. In addition, the sulfenyl-
methyl group could provide a good protection for the C5 thiol
function of the (6–4) adduct, in view of its functionalization
into phosphoramidite and its incorporation into oligonucleo-
tides, as this thiol protection is compatible with phosphora-
midite-based oligonucleotide synthesis.20b Thus, treatment of
the crude irradiation mixture of 2 with CH3SO2SCH3 in the
presence of K2CO3 afforded the two phosphorus diastereo-
isomers of compound 7 in pure form after RP HPLC purifi-
cation (43 % yield from 2). The stereochemistry at C5 and C6 of
7, assigned from NOE correlations (H-6 Tp/H-3Ј Tp; H-6 Tp/
CH3 Tp and H-6 Tp/CH3 pT) was found to be C5 R and C6 S as
in the unprotected series.7a It is worth mentioning that irrad-
iation of 2 led to (6–4) photoproducts in a significant higher
yield than in the natural series (16% for TT and 3.9% for TC).13
At this stage, the stability of 7 towards the deprotection con-
ditions used during solid phase oligonucleotide synthesis was
carefully examined by HPLC in the light of the known acid-
and base-sensitivity of 2-pyrimidinone nucleosides.20b,25 The
conditions to remove the phosphate methyl group were also
examined along with the stability of the internucleotide bond to
alkaline conditions. Usually, this group is specifically removed
by the action of strong nucleophiles such as thiophenate26
or odorless disodium-2-carbamoyl-2-cyanoethylene-1,1-dithi-
olate;27 the use of a mixture of pyridine/aqueous ammonia has
Synthesis of compound 2 started from the known 5Ј-O-
dimethoxytrityl-4-thiothymidine 322 (Scheme 1) which was
acylated with levulinic acid and DCC in the presence of DMAP
to give 4 in 98%. This latter was subsequently detritylated on
treatment with 3% trifluoroacetic acid in dichloromethane
giving 3Ј-O-levulinyl-4-thiothymidine (5) in 85% yield. Conden-
sation of 5 with 5Ј-O-dimethoxytritylthymidine 3Ј-methyl-N,N-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 1 6 – 3 3 2 0
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also been reported.28 Since this latter condition should also
allow the deprotection of the nucleobases and the cleavage of
the oligonucleotide from the support, its use would avoid the
necessity to use an additional step for specifically removing the
methyl group. Thus, treatment of a mixture of the two dia-
stereoisomers 7 in a pyridine/concentrated aqueous ammonia
solution (1/1) at room temperature for 20 h led, in 62% yield, to
the formation of one single product of HPLC retention time
(RT) 14 min whose mass spectrum (ESIϪ: m/z 909) confirmed
the removal of the methyl group. Therefore, we decided to util-
ize concentrated aqueous ammonia that had also been reported
to deprotect methylphosphate groups.29 Treatment of 7 with
concentrated aqueous ammonia for 24 h at room temperature
gave, in 90% yield, a single compound having by HPLC analysis
a RT of 14 min. This established that the phosphate methyl
group could be cleanly removed by the use of conc. aqueous
ammonia and that the pyrimidinone motif would withstand the
alkaline conditions necessary for the deprotection of the
nucleobases and cleavage from the support. We then examined
the stability of 7 towards aqueous and non aqueous acidic con-
ditions. Treatment of 7 with a solution of 2% trichloroacetic
acid in CH2Cl2 gave instantaneously the 5Ј-deprotected product
(RT 14 min, ESIϩ: m/z 645) as a major product. Prolonged
treatment (1 h) did not lead to significant decomposition. The
stability of the pyrimidinone motif of 7 in aqueous acidic
conditions was then examined since it has been reported that
2-pyrimidinone deoxynucleosides and oligonucleotides contain-
ing a (6–4) PP could be unstable to this treatment.30,31 Treat-
ment of compound 7 deprotected at the P–OMe position with a
40% aqueous acetic acid solution gave instantaneously the
detritylated product in quantitative yield. Finally the stability
of the methylsulfenyl protecting group towards oxidative con-
ditions was examined. Thus treatment of 7 with the 0.1 M
iodine solution commonly used for oligonucleotide synthesis
led to the formation of a more polar compound that was not
further characterized. However, 7 was proved to be sufficiently
stable upon treatment with a 0.02 M iodine solution.32
Finally, we studied the deprotection conditions of the thiol
function. This last study also ascertained our synthesis for we
transformed 7 into the known compound 8.7a Removal of the
methyl phosphate and dimethoxytrityl groups of 7 was succes-
sively achieved by a treatment with NH4OH then 40% aqueous
acetic acid. Dithiotreitol treatment in phosphate buffer (pH 8)
of the resulting compound led instantaneously to a single
product that co-eluted with an authentic sample of 8 prepared
by photolysis of Tps4T.7a This unambiguously confirmed the
stereochemistry of the asymmetric centers of 7 at positions C5
and C6 indicating that the stereochemical course of the photo-
chemical reaction involved the two pyrimidine bases in an anti
glycosidic conformation as in the natural process. Interestingly,
we did not detect any (6–4)PP derived from a 5Ј-base in a syn
glycosidic bond conformation as observed in the methyl-
phosphonate series.7b,15
to the possible conversion of these oligomers into h5(6–4)-
containing oligonucleotides, functional tethered derivatives
aimed at trapping DNA damage sensor proteins crucial for the
signal transduction pathway leading to DNA repair could be
obtained after thiol derivatization.
Experimental
(2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite
was
obtained from Interchim (Montluçon, France). 5Ј-O-Di-
methoxytrityl-4-thiothymidine 3 was prepared as previously
described.22 Compounds 5, 6 and tetrazole were dried at room
temperature over P2O5 under vacuum overnight prior to use.
CH3CN was dried by heating under reflux with P2O5. N,N-
Diisopropylethylamine and CH2Cl2 were dried by distillation
from calcium hydride and 1,4-dioxane by distillation from
sodium/benzophenone. Thin-layer and column chromato-
graphy were carried out on silicagel 60 F254 60–15 µm and
silicagel 6–35 µm or 35–70 µm respectively from SDS (Peypin,
1
France). H and 13C NMR spectra were recorded on Bruker
AC250 or AM300 instruments. 1H Chemical shifts (δ) are
reported in ppm relative to residual solvent peak in CD3OD
(MeOD δ 3.30) or to TMS (δ 0.00) in CDCl3. 13C Chemical
shifts are reported in ppm relative to solvent peak (CD3OD
δ 49.0, CDCl3 δ 77.7). 31P NMR spectra were recorded on a
Bruker AC 300-P. Chemical shifts are reported relative to an
external capillary standard of 85% phosphoric acid. FAB-
HRMS (m-nitrobenzyl alcohol/glycerol matrix) and ESMS
were carried out using a ZabSpec/T spectrometer (Micromass,
Manchester, UK). CI HRMS (CH4) was carried out using a
Kratos MS 80 spectrometer.
5Ј-O-Dimethoxytrityl-3Ј-O-levulinyl-4-thiothymidine 4
5Ј-O-Dimethoxytrityl-4-thiothymidine 322 (3.60 g, 6.43 mmol)
was dissolved in anhydrous 1,4-dioxane (70 mL), and DMAP
(0.06 g, 0.52 mmol) and DCC (3.31 g, 16.07 mmol) were added.
To this solution, levulinic acid (1.50 g, 12.86 mmol) was added,
and the mixture was stirred for 1 h at room temperature. The
resulting mixture was concentrated, diluted with ethyl acetate
and filtered on silica gel. After evaporation of the solvent, the
residue was purified by flash chromatography using heptane–
ethyl acetate (60:40) containing 0.1% of pyridine to give 4
(4.136 g, 98 %) as a yellow foam. 1H NMR (300 MHz, CDCl3):
δ 1.52 (3H, s, CH3), 2.19 (3H, s, CH3 Lev), 2.48 (2H, m, H-2Ј,
H-2Љ), 2.57 (2H, m, CH2 Lev), 2.76 (2H, m, CH2 Lev), 3.47 (2H,
m, H-5Ј, H-5Љ), 3.79 (6H, s, OCH3 Dmt), 4.16 (1H, br s, H-4Ј),
5.46 (1H, m, H-3Ј), 6.38 (1H, dd, J = 5.6; 8.8 Hz, H-1Ј), 6.82
(4H, d, J = 8.8 Hz, CH Dmt), 7.22–7.38 (9H, m, CH Dmt), 7.67
(1H, s, H-6), 9.68 (1H, br s, NH). 13C NMR (75 MHz, CDCl3):
δ 16.9 (CH3), 28.5 (CH2 Lev), 30.3 (CH3 Lev), 38.3 (C-2Јa), 38.8
(CH2 Leva), 55.8 (OCH3 Dmt), 64.2 (C-5Ј), 76.0 (C-3Ј), 84.9
(C-4Јb), 85.4 (C-1Јb), 87.8 (CIV Dmt), 113.9 (CH Dmt), 120.5
(C-5), 127.8, 128.6, 128.7, 130.6 (CH Dmt), 132.2 (C-6), 135.7,
135.8, 144.7 (CIV Dmt), 148.5 (C-2), 159.3 (CIV Dmt), 172.8
(COCH2 Lev), 191.2 (C-4), 206.8 (COCH3 Lev). HRMS (FAB)
(M ϩ Li)ϩ Calc. for C36H38O8N2SLi 665.2509, found 665.2511.
Having established the identity and stability of 7 toward the
solid phase oxidation and deprotection conditions, it was
subsequently phosphitylated into the corresponding phos-
phoramidite 1 using Hunig’s base and (2-cyanoethyl)-N,N-
diisopropylchlorophosphoramidite in the presence of molec-
ular sieves33 in 64% yield. We have found it convenient to use 1
directly for its incorporation into oligonucleotides and the
results will be reported in due course.
3Ј-O-Levulinyl-4-thiothymidine 5
5Ј-O-Dimethoxytrityl-3Ј-O-levulinyl-4-thiothymidine 4 (3.88 g,
5.88 mmol) was dissolved in 50 mL of 3% CF3COOH in
CH2Cl2. After 1 h 30 min of stirring at room temperature,
methanol (5 mL) containing 10% triethylamine was added. The
mixture was concentrated and the crude product was purified
by flash chromatography on silica gel using a gradient of
methanol in CH2Cl2 (0–2%) to give compound 5 (1.78 g, 85 %)
Conclusion
In this work, we have reported a straightforward and very effi-
cient synthesis of the s5(6–4) thymine–thymine phosphor-
amidite building block designed to allow the preparation
of oligonucleotides containing, site specifically, this lesion
analogue. Such attractive oligonucleotides should have various
biophysical and biological applications. Moreover, in addition
1
as a yellow foam. H NMR (300 MHz, CDCl3): δ 2.07 (3H, s,
CH3), 2.21 (3H, s, CH3 Lev), 2.43 (2H, m, H-2Ј, H-2Љ), 2.59 (2H,
m, CH2 Lev), 2.81 (2H, m, CH2 Lev), 3.91 (2H, m, H-5Ј, H-5Љ),
4.14 (1H, br s, H-4Ј), 5.37 (1H, m, H-3Ј), 6.24 (1H, t, J = 7.0 Hz,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 1 6 – 3 3 2 0
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H-1Ј), 7.70 (1H, s, H-6), 10.77 (1H, br s, NH). 13C NMR (75
MHz, CDCl3): δ 17.8 (CH3), 28.6 (CH2 Lev), 29.7 (CH3 Lev),
38.1 (C-2Јa), 38.4 (CH2 Lev), 62.8 (C-5Ј), 75.4 (C-3Ј), 86.0
(C-4Ј), 87.0 (C-1Ј), 120.3 (C-5), 133.3 (C-6), 148.6 (C-2), 173.2
(COCH2 Lev), 191.1 (C-4), 207.5 (COCH3 Lev). HRMS (CI)
(M ϩ H)ϩ Calcd. for C15H21O6N2S 357.1120, found 357.1152.
Anal. calcd. for C15H20O6N2S: C, 50.55; H, 5.65; N, 7.85; O,
26.93; S, 8.99. Found: C, 50.27; H, 5.43; N, 7.71; O, 26.91; S,
8.92.
stereoisomer had a retention time of 57 min (isomer ꢀ, 43 mg)
whereas the other eluted at 66 min (isomer ꢁ, 35 mg). The
overall yield of 7 from 2 was 42%.
Photoproduct 7ꢀ
1H NMR (400 MHz, CD3OD): δ 1.42 (1H, ddd, J = 14; 8; 2 Hz,
H-2Ј Tp), 1.87 (3H, s, CH3 Tp), 1.98 (3H, s, CH3 pT), 2.16 (1H,
dt, J = 14; 9 Hz, H-2Љ Tp), 2.49 (3H, s, SCH3), 2.54 (1H, m, H-2Љ
pT), 2.82 (1H, dd, J = 14; 7 Hz, H-2Ј pT), 3.33 (2H, d, J = 4 Hz,
H-5Ј and H-5Љ Tp), 3.52 (3H, d, J = 11 Hz, POCH3), 3.62 (1H,
q, J = 9 Hz, H-3Ј Tp), 3.79 (6H, s, OCH3 Dmt), 3.87 (1H, m,
H-4Ј pT), 3.99 (1H, td, J = 4; 9 Hz, H-4Ј Tp), 4.09 (1H, br d, J =
11 Hz, H-5Ј pT), 4.25 (1H, dd, J = 6; 11 hz, H-5Ј pT), 4.55 (1H,
m, H-3Ј pT), 4.77 (1H, s, H-6 Tp), 6.14 (1H, dd, J = 2; 9 Hz,
H-1Ј Tp), 6.45 (1H, d, J = 8 Hz, H-1Ј pT), 6.90 (4H, d, J = 9 Hz,
CH Dmt), 7.20–7.53 (9H, m, CH Dmt), 7.84 (1H, s, H-6 pT).
13C-NMR (50 MHz, CD3OD): δ 15.9 (CH3 pT), 25.2 (CH3 Tp),
25.5 (SCH3), 36.9 and 37.1 (C-2Ј Tp and pT), 55.3 (POCH3),
55.8 (OCH3 Dmt), 57.7 (C-5 Tp), 58.9 (C-6 Tp), 65.1 (C-5Ј Tp),
66.8 (C-5Ј pT), 69.0 (C-3Ј pT), 75.7 (C-3Ј Tp), 81.5 (C-4Ј Tp),
84.5 (C-1Ј Tp), 85.8 (C-4Ј pT), 87.6 (C-1Ј pT), 88.0 (CIV Dmt),
114.3 (CH Dmt), 115.0 (C-5 pT), 128.0, 129.0, 129.3, 131.3,
131.4 (CH Dmt), 136.7 and 136.9 (CIV Dmt), 145.0 (C-6 pT),
146.0 (CIV Dmt), 153.9 (C-2 Tp), 157.4 (C-2 pT), 160.3 (CIV
Dmt), 171.2 (C-4 Tp), 176.7 (C-4 pT). 31P NMR (121 MHz,
CD3OD): δ 0.08. HRMS (FAB) (M ϩ Li)ϩ Calc. for C43H49-
N4O13PS2Li, 931.2635; found, 931.2598.
P-methyl-5Ј-O-dimethoxytritylthymidilyl(3Ј 5Ј)-4-
thiothymidine 2
5Ј-O-Dimethoxytritylthymidine
3Ј-methyl-N,N-diisopropyl-
phosphoramidite 6 (880 mg, 1.24 mmol) and 3Ј-O-levulinyl-4-
thiothymidine 5 (296 mg, 0.83 mmol) were dissolved in
anhydrous acetonitrile (12 mL), and 1H-tetrazole (194.5 mg,
2.77 mmol) was added. After 15 min of stirring at room tem-
perature, a 0.2M iodine solution in tetrahydrofuran-2,6-
lutidine-water (2:1:1, v/v/v) (8 mL) was added, and the mixture
was stirred for 10 min. After concentration, the residue was
diluted with CH2Cl2 and a saturated sodium thiosulfate solu-
tion was added until the solution became colorless, and water
was added. The organic phase was dried over sodium sulfate
and concentrated under reduced pressure. The residue was
dissolved in pyridine (11 mL), and a 0.94 M hydrazine mono-
hydrate solution in pyridine-acetic acid (3.25:2 v/v) (11 mL) was
added. After stirring at room temperature for 40 min, the
mixture was cooled in an ice bath and acetone (6 mL) was
added. The mixture was diluted with CH2Cl2, washed with 2%
aqueous NaHCO,3 and dried with Na2SO4. After coevapor-
ation with toluene, the residue was purified by chromatography
on silica gel with a step gradient of 0–5% methanol in CH2Cl2
containing 0.1% triethylamine to give 2 (544 mg, 74%) as a
yellow foam. 1H NMR (300 MHz, CD3OD): δ 1.86 (3H, s, CH3
Tp), 2.06 (3H, s, CH3 ps4T), 2.21–2.42 (3H, m, H-2Ј and H-2Љ
ps4T, H-2Ј Tpa), 2.50 (1H, m, H-2Љ Tpa), 3.75 (6H, s, OCH3
Dmt), 3.76 (2H, m, H-5Ј and H-5Љ Tp), 3.82 (3H, d, J = 11.8 Hz,
POCH3), 4.07 (1H, m, H-4Ј ps4T), 4.20 (1H, m, H-4Ј Tp), 4.32
(2H, m, H-5Ј, H-5Љ ps4T), 4.41 (1H, m, H-3Ј ps4T), 5.07 (1H, m,
H-3Ј Tp), 6.20 (1H, m, H-1Ј ps4T), 6.27 (1H, m, H-1Ј Tp), 6.81
(4H, d, J = 8.8 Hz, CH Dmt), 7.10–7.40 (9H, m, CH Dmt), 7.52
and 7.74 (1H, 2s, H-6 ps4T), 7.76 (1H, br s, H-6 Tp). 13C NMR
(75 MHz, CD3OD): δ 12.6 (CH3 Tp), 17.4 (CH3 ps4T), 39.6
(C-2Ј Tp), 40.7 (C-2Ј ps4T), 55.7 (P–OCH3 and OCH3 Dmt), 62.5
(C-5Ј Tp), 68.7 (C-5Ј ps4T), 71.4 (C-3Ј ps4T), 80.5 (C-3Ј Tp),
86.0 (C-1Ј Tp, C-4Ј ps4T), 87.1 (C-1Ј ps4T, C-4Ј Tp), 87.7 (CIV
Dmt), 111.9 (C-5 Tp), 114.2 (CH Dmt), 120.6 (C-5 ps4T), 127.7,
128.7, 129.3, 131.2 (CH Dmt), 133.5 (C-6 ps4T), 137.3 (CIV
Dmt), 137.7 (C-6 Tp), 146.3 (CIV Dmt), 149.7 (C-2 ps4T), 152.2
(C-2 Tp), 159.9 (CIV Dmt), 166.3 (C-4 Tp), 192.7 (C-4 ps4T).
31P NMR (121 MHz, CD3OD): δ 0.18. HRMS (FAB) (M ϩ
Li)ϩ Calc. for C42H47N4O13PSLi, 885.2758; found, 885.2769.
Photoproduct 7ꢁ
1H NMR (300 MHz, CD3OD): δ 1.44 (1H, ddd, J = 14; 8; 3 Hz
H-2Ј Tp), 1.83 (3H, s, CH3 Tp), 1.89 (3H, s, CH3 pT), 2.13 (1H,
m, H-2Љ Tp), 2.41 (1H, dd, J = 14; 7 Hz, H-2Љ pT), 2.48 (3H, s,
SCH3), 2.77 (1H, ddd, J = 14; 6; 3 Hz, H-2Ј pT), 3.30 (1H, m,
H-5Ј Tp), 3.47 (1H, dd, J = 11; 2 Hz, H-5Ј Tp), 3.65 (3H, d, J =
11 Hz, POCH3; 1H, m, H-3Ј Tp), 3.77 (6H, s, OCH3 Dmt), 3.84
(1H, m, H-4Ј pTa), 3.96 (1H, m, H-4Ј Tpa), 4.04 (2H, m, H-5Ј
and H-5Љ pT), 4.45 (1H, m, H-3Ј pT), 4.74 (1H, s, H-6 Tp), 6.02
(1H, dd, J = 8; 3 Hz, H-1Ј Tp), 6.41 (1H, dd, J = 7; 3 Hz, H-1Ј
pT), 6.87 (4H, d, J = 10 Hz, CH Dmt), 7.15–7.50 (9H, m, CH
Dmt), 7.78 (1H, s, H-6 pT). 31P NMR (121 MHz, CD3OD):
δ 0.47. HRMS (FAB) (M ϩ Li)ϩ Calc. for C43H49N4O13PS2Li,
931.2635; found, 931.2627.
Stability essays
All the HPLC experiments were conducted at a flow rate of
1 mL minϪ1 and the effluent was monitored at 330 nm.
Alkaline conditions. Pyridine/concentrated aqueous ammonia
solution (1/1): A mixture of 7ꢀꢁ (0.4 mg in 400 µL) was allowed
to stand at room temperature. Analysis was performed on a
Nova-Pak® C18 3.9 × 150mm column, using a 20 min linear
gradient of 0–50% CH3CN in water followed by a 10 min
plateau. Compound 7ꢀꢁ RT: 24 and 25 min. Phosphate depro-
tected 7: RT: 14 min. Concentrated aqueous ammonia: A mixture
of 7ꢀꢁ (0.4 mg in 400 µL) was allowed to stand at room temper-
ature. Conditions of analysis are the same as described above.
Photolysis of 2
A 1mM, 50% aqueous acetonitrile solution of compound 2
(100 mL) was photolyzed at 366 nm at room temperature for 4 h
under argon (bubbling) using an Original Hanau Quarzlampen
Fluotest-Forte ref. 5261. The solution was then concentrated
in vacuo. This experiment was repeated several times. The result-
ing residue from two experiments was dissolved in 1.0 mL of
methanol and 1.5 mL of anh. CH2Cl2. To this solution, K2CO3
(30 mg, 0.22 mmol) then a 5% CH3SO2SCH3 in methanol
solution (200 µL) were added. The reaction was stirred under
an argon atmosphere at room temperature and, after 5 min, the
solvents were evaporated and the residue was purified by
Aprotic acidic conditions. 2% Trichloroacetic acid in CH2Cl2:
Isomer 7ꢁ (1 mg mLϪ1) was allowed to stand at room temper-
ature. Analysis was performed on a Nova-Pak® Silica 3.9 ×
150mm column using an isocratic eluent composed of 3%
CH3OH in CH2Cl2. Compound 7ꢁ RT: 6 min; Compound 7ꢁ
detritylated RT: 14 min.
reverse-phase chromatography on
a
25
×
100mm Prep
Protic acidic conditions. 40% Aqueous acetic acid: Compound
7 deprotected at the P–OMe position (0.2mg/200 µL) was
allowed to stand at room temperature. Analysis was performed
on a Nova-Pak® C18 3.9 × 150mm column, using a 40 min
Nova-Pak HR C18, 6 µm, 60 Å, PrePack® Cartridge using
acetonitrile:water (40:60) as the eluent at a flow rate of 8 mL
minϪ1 with detection set at 330 nm. One phosphorus dia-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 1 6 – 3 3 2 0
3319

View Article Online
G. P. Pfeifer, Biochemistry, 1996, 35, 15693–15703; (b) X. Liu,
A. Conconi and M. J. Smerdon, Biochemistry, 1997, 36, 13710–
13717; (c) P. Vichi, F. Coin, J.-P. Renaud, W. Vermeulen, J. H. J.
Hoeijmakers, D. Moras and J.-M. Egly, EMBO J., 1997, 16,
7444–7456.
linear gradient of 0–50% CH3CN in 0.05 M ammonium acetate.
Compound 7 deprotected at the P–OMe position RT: 35 min.
Compound 7 deprotected at the P–OMe position and detrityl-
ated RT: 14 min.
6 For a review see: P. Clivio, Trends Org. Chem., 2001, 9, 129–136.
7 (a) P. Clivio, J.-L. Fourrey, J. Gasche and A. Favre, J. Am. Chem.
Soc., 1991, 113, 5481–5483; (b) P. Clivio, J.-L. Fourrey, J. Gasche and
A. Favre, Tetrahedron Lett., 1992, 33, 1615–1618.
8 (a) J. Liu and J.-S. Taylor, J. Am. Chem. Soc., 1996, 118, 3287–3288;
(b) P. Clivio and J.-L. Fourrey, Chem. Commun., 1996, 2203–2204.
9 X. Zhao, J. Liu, D. S. Hsu, S. Zhao, J.-S. Taylor and A. Sancar,
J. Biol. Chem., 1997, 272, 32580–32590.
Oxidative conditions. Isomer 7ꢁ (1 mg mLϪ1 of oxidative
solution) was allowed to stand at room temperature. Analysis
was performed on a Nova-Pak® C18 3.9 × 150 mm column,
using 40% CH3CN in water as eluent. 7ꢁ RT: 21 min; Side
product: 15 min.
10 (a) S. K. Angulo Matus, L. Quintero, J.-L. Fourrey and P. Clivio,
Chem. Commun., 2001, 2250–2251; (b) F.-Y. Dupradeau, P. Sonnet,
D. Guillaume, H. M. Senn and P. Clivio, J. Org. Chem., 2002, 67,
9140–9145.
11 M. A. Warren, J. B. Murray and B. A. Connolly, J. Mol. Biol., 1998,
279, 89–100.
12 J.-L. Fourrey, J. Gasche, C. Fontaine, E. Guittet and A. Favre,
J. Chem. Soc., Chem. Commun., 1989, 1334–1336.
13 (a) S. Iwai, M. Shimizu, H. Kamiya and E. Ohtsuka, J. Am. Chem.
Soc., 1996, 118, 7642–7643; (b) T. Mizukoshi, K. Hitomi, T. Todo
and S. Iwai, J. Am. Chem. Soc., 1998, 120, 10634–10642.
14 J.-S. Taylor, I. R. Brockie and C. L. O’Day, J. Am. Chem. Soc., 1987,
109, 6735–6742.
Deprotection of 7 and its identity to 8
Compound 7 was phosphate deprotected then detritylated as
described above. After evaporation of the acetic acid solution,
the crude product was dissolved in a 100 mM dithiotreitol
solution (0.02 M phosphate buffer, pH 8). Analysis was per-
formed on a Nova-Pak® C18 3.9 × 150mm column, using a 40
min linear gradient of (0–50%) CH3CN in 0.05 M ammonium
acetate. Compound 7 deprotected at the P–OMe position and
detritylated RT: 14 min; Completely deprotected 7 RT: 10 min;
Compound 8 RT: 10 min.
15 P. Clivio, J.-L. Fourrey, T. Szabo and J. Stawinski, J. Org. Chem.,
1994, 59, 7273–7283.
Phosphoramidite 1
16 (a) P. Clivio, J.-L. Fourrey, J. Gasche, A. Audic A. Favre and
A. Woisard, Tetrahedron, 1992, 33, 65–68; (b) T. T. Nikiforov and
B. A. Connolly, Tetrahedron Lett., 1992, 33, 2379–2382.
17 (a) T. T. Nikiforov and B. A. Connolly, Tetrahedron Lett., 1991, 32,
3851–3854; (b) Y.-Z. Xu, Q. Zheng and P. F. Swann, J. Org. Chem.,
1992, 57, 3839–3845.
To photoproduct 7 (71 mg, 0.076 mmol) was added N,N-diiso-
propylethylamine (53 µL, 0.31 mmol), CH2Cl2 (1.1 mL) and 4 Å
molecular sieve. Then chloro-(2-cyanoethyl)-bis-N,NЈ-diiso-
propylphosphoramidite (35 µL, 0.153 mmol) was added and the
reaction stirred for 5 min. The mixture was then diluted with
ethyl acetate containing 5% triethylamine washed with 2%
aqueous NaHCO3, and dried over Na2SO4. After evaporation
of the solvent, the phosphoramidite 1 was purified by chrom-
atography on silica gel eluted with ethyl acetate/heptane 95/5
containing 1% triethylamine. Compound 1 was obtained in
64% yield (55 mg) as a white amorphous solid that was directly
used.
18 P. Clivio, J.-L. Fourrey, J. Gasche and A. Favre, J. Chem. Soc. Perkin
Trans. I, 1992, 2383–2388.
19 J. H. Van Boom and P. M. J. Burgers, Tetrahedron Lett., 1976, 52,
4875–4878.
20 (a) J. J. Fox, D. Van Praag, I. Wempen, I. L. Doerr, L. Cheong,
J. E. Knoll, M. L. Eidinoff, A. Bendich and G. B. Brown,
J. Am. Chem. Soc., 1959, 81, 178–187; (b) B. A. Connolly and
P. C. Newman, Nucleic Acids Res., 1989, 17, 4957–4974.
21 G. Kumar and M. S. Poonian, J. Org. Chem., 1984, 49, 4905–4912.
22 A. Roget, H. Bazin and R. Teoule, Nucleic Acids Res., 1989, 17,
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Acknowledgements
23 L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 1983, 24,
We thank CONACYT (Mexico) for a doctoral fellowship to
245–248.
24 T. Murata, S. Iwai and E. Ohtsuka, Nucleic Acids Res., 1990, 18,
7279–7286, with minor modification for the oxidation conditions.21
25 B. Gildea and L. W. McLaughlin, Nucleic Acids Res., 1989, 17,
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S. K. A. M. and the CNRS for financial support (PCV 2000).
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