Synthesis of a convenient thymidine glycol phosphoramidite monomer and its site-specific incorporation into DNA fragments

Article abstract of DOI:10.1080/15257770500267279

□ An original phosphoramidite building block of the thymidine glycol lesion has been prepared taking into account the additional diol function and the high lability of this oxidatively induced nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments by solid support assembling followed by a "one-step" mild final deprotection treatment. Copyright Taylor & Francis Group, LLC.

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Synthesis of a Convenient Thymidine Glycol
Phosphoramidite Monomer and Its Site-specific
Incorporation into DNA Fragments
Didier Gasparutto a , Sonia Cognet a , Solveig Roussel a & Jean Cadet a
a Laboratoire des Lésions des Acides Nucléiques, Service Chimie Inorganique Biologique,
UMR-E3 CEA-UJF, DRFMC, CEA , Grenoble, France
Published online: 16 Aug 2006.
To cite this article: Didier Gasparutto , Sonia Cognet , Solveig Roussel & Jean Cadet (2005) Synthesis of a Convenient
Thymidine Glycol Phosphoramidite Monomer and Its Site-specific Incorporation into DNA Fragments, Nucleosides, Nucleotides
and Nucleic Acids, 24:10-12, 1831-1842, DOI: 10.1080/15257770500267279
To link to this article: http://dx.doi.org/10.1080/15257770500267279
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1831–1842, 2005
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500267279
SYNTHESIS OF A CONVENIENT THYMIDINE GLYCOL
PHOSPHORAMIDITE MONOMER AND ITS SITE-SPECIFIC
INCORPORATION INTO DNA FRAGMENTS
ᮀ
Didier Gasparutto, Sonia Cognet, Solveig Roussel, and Jean Cadet
Labo-
ratoire des Le´sions des Acides Nucle´iques, Service Chimie Inorganique Biologique, UMR-E3
CEA-UJF, DRFMC, CEA Grenoble, France
ᮀ
An original phosphoramidite building block of the thymidine glycol lesion has been prepared
taking into account the additional diol function and the high lability of this oxidatively induced
nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments
by solid support assembling followed by a “one-step” mild final deprotection treatment.
Keywords Oxidative DNA lesion; Thymine glycol; Oligodeoxyribonucleotide; Chem-
ical synthesis; Protecting groups
INTRODUCTION
Various oxidative stress agents including ionizing radiation, ultraviolet-
A light and several chemicals are able to oxidize the sugar and nucleobase
moieties of DNA giving rise to a large set of lesions.[1–3] In that respect, 5,6-
dihydroxy-5,6-dihydrothymine1◦(thymineglycolorthyminediol)(Scheme1)
has been shown to be a major OH and one-electron mediated oxidation
product of thymine. It was shown that the amount of thymine glycol
increases substantially when DNA is exposed to oxidative events.[4] The
lesion 1 is produced in DNA or at the nucleoside level as a mixture of
two pairs of cis and trans diastereomers, which are in equilibrium two by
two in solution through epimerization at C6.[5] Thymidine glycol 1, which
may block DNA polymerases during the replication process,[6] is efficiently
removed by the base excision repair (BER) pathway involving DNA N-glyco-
This paper is dedicated to the memory of Dr. John A. Montgomery.
Received 28 January 2005; accepted 10 April 2005.
The assistance of Christine Saint-Pierre and Colette Lebrun (SCIB/CEA-Grenoble) in the mass
spectrometry measurements is gratefully acknowledged.
Address correspondence to Dr. Didier Gasparutto, CEA Grenoble, LAN/SCIB/DRFMC, 17 avenue
des martyrs, Grenoble, F-38054, France. Fax: 33 4 38 78 50 90; E-mail: dgasparutto@cea.fr
1831

1832
D. Gasparutto et al.
SCHEME 1 Structure of the four 5,6-dihydroxy-5,6-dihydrothymidine isomers (1).
sylases,[7] and also by the nucleotide excision repair (NER) system.[8] It
was found that the two cis thymine glycols were excised in a stereoselective
manner from site-specifically oxidized oligonucleotides by a wide set of DNA
N-glycosylases including Endo III, endo VIII, yNTG1, mNTH and mNEIL1
but not yNTG2.[9] Recently, Friedberg et al. have shown that human DNA
polymerase kappa bypasses and extends behond thymine glycol during
translesional synthesis in vitro by incorporating correct nucleotides.[10]
In order to obtain further biological and structural information on this
major oxidative alteration of DNA, it is necessary to prepare oligodeoxynu-
cleotides (ODNs) that contain 1 at defined sites as probes or substrates.
The presence of hydroxyl groups within the thymine base together with 5,6-
saturation of the ring, confer a high instability to the nucleoside particularly
under alkaline conditions. The latter aspect together with the necessity to
protect the additional hydroxyl functions of the base make the insertion
of 1 into defined sequence oligonucleotides a challenging objective. The
usual approach to prepare thymine glycol-containing ODNs consists in the
post-oxidation, by either osmium tetroxide or potassium permanganate, of
a thymine residue incorporated into a single strand DNA fragment.[7d,11]
However the post-modification approach shows several limitations that con-
cern the stereochemical composition, the chain length, the sequence, the
yield and also the quantity of modified ODNs thus prepared. To overcome

Oligonucleotides that Contain Thymine Glycol Lesions
1833
partly some of these difficulties, Saito and coworkers recently reported a
site-selective post-oxidation of a thymine residue within a DNA fragment in
the presence of bipyridine-tethered complementary ODN.[12]
To our best knowledge, only one total chemical approach for incorpo-
rating 1 into DNA fragments has been yet reported by Iwai.[13] The latter
synthesis, consists in the preparation of a phosphoramidite building block,
that contains one or two t-butyldimethylsilyl (TBDMS) protective groups on
the nucleobase hydroxyl sites. Then, the subsequent incorporation of the
protected oxidized nucleoside into modified ODNs was achieved by solid-
phase assembling. The final support cleavage and deprotection are then
performed by a mild ammonia treatment at room temperature followed by
an additional desilylation step by fluoride ions.
Herein we report the synthesis of a suitable phosphoramidite monomer
8 that contains the levulinyl (Lev) as the nucleobase protective group
(Scheme 2). Prior to the preparation of the phosphoramidite synthon
8, the stability of thymidine glycol 1 was checked at room temperature
under several conditions used during DNA solid support synthesis: 30%
aqueous ammonia, 80% acetic acid and a 0.02 M commercial oxidizing
solution of iodine. Less than 10% of degradation of 1 was observed after
10 h of incubation in the acid and oxidizing solutions. However 1 was
fully decomposed when left for 4 h in aqueous ammonia. The problem
of instability was circumvented using a 0.05 M methanolic solution of
K2CO3 as an alternative mild alkali deprotection system. Under the latter
conditions, no detectable degradation was observed after 4 h at room
temperature. Moreover, the levulinyl-protected thymine glycol derivative
was quantitatively converted into the parent compound 1 upon K2CO3
treatment for 2 h at room temperature. The kinetic and stability studies
show the compatibility of 1 with the latter alkali deprotection conditions,
which may be used in combination with the “Pac-phosphoramidite” chem-
istry.[14] Furthermore, it was expected that the protection of the additional
hydroxyl functions of 1 together with its insertion in the oligonucleotide
chain might increase the stability of the base moiety.
The synthesis of the target phosphoramidite 8 (Scheme 2) started using
5ꢁ-O-DMTr-3ꢁ-O-TBDMS-thymidine (4), prepared by classical dimethoxytrity-
lation (step a) and silylation (step b) of thymidine (2), respectively. Then,
sugar-protected thymidine glycol (5), preferentially in its cis-5R,6S config-
uration,[15] was obtained in a high yield by oxidation with osmium tetrox-
yde in the presence of methylmorpholine-N-oxide in a t-BuOH/THF/H2O
solution.[16] Compound 5 was then converted into a mixture of O6-mono-
levulinyl and O5,O6-dilevulinyl derivatives 6 after treatment with levulinic
acid in the presence of DCC and DMAP in THF. The levulinyl group, which
has been already successfully used for the chemical synthesis of modified
oligonucleotides that contain nucleobase damages,[17,18] has been selected

1834
D. Gasparutto et al.
SCHEME 2 a) DMTr-Cl (2 eq), pyridine, 16 h, 76%; b) TBDMS-Cl (2 eq), imidazole (2.5 eq), pyridine,
24 h, 82%; c) OsO4 (1/19 eq), methylmorpholine-N-oxide (2 eq), t-BuOH, THF, H2O, 45◦C, 24 h, 85%;
d) Levulinic acid (5 eq), DCC (5 eq), DMAP (0.6 eq), THF, 40◦C, 24 h, 71%; e) TBAF (4 eq), THF,
3 h, 60%; f) 2-cyanoethyl-N,N,Nꢀ,Nꢀ-tetraisopropyldiamidite (1.1 eq), diisopropylammonium tetrazolate
(0.5 eq), CH2Cl2, 4 h, 75%; g) oligonucleotide solid-phase assembling and deprotection.
for the following suitable properties: high reactivity toward hydroxyl func-
tions, good stability during acidic and oxidizing treatments, easy removal in
neutral or mild alkali conditions. Due to the weak reactivity of the tertiary hy-
droxyl function at the position 5 of the nucleobase, the O6-mono-protected
residue (Figure 1) was obtained in a large excess (O6-monoLev/O5,O6-
1
diLev = 9/1 from the H-NMR and HPLC analyses). This, in agreement
with previous fundings relative to the lack of reactivity of such tertiary al-
cohol functions,[4d,19] allows the use of the pure O6-monosubstituted com-
pound or in mixture with the O5,O6-dilevulinyl derivative in the subsequent
reaction pathways. Thus, the TBDMSgroup was selectively removed by treat-
ing compounds 6 with 1 M tetrabutylammonium fluoride in THF, yielding
the 3ꢁ-hydroxy derivatives 7. Standard phosphitylation of 7 gave phospho-
ramidite building blocks 8, which have been used for the preparation of
defined-sequence ODNs that contain the thymine glycol lesion.

Oligonucleotides that Contain Thymine Glycol Lesions
1835
FIGURE 1 ESI mass spectrum, in the positive mode, of RP-HPLC purified O6-monolevulinyl derivative
6 (molecular weight: 790 g·mol−1).
Thus, several thymine glycol-containing oligonucleotides (5-, 14-, and
34-mers) were prepared on a DNA synthesizer with few modifications in
the standard procedure: 1) the phenoxyacetyl (pac) group was used for
the amino-protection of the unmodified bases (pac for dA and dG and
isobutyryl for dC, respectively) in combination with anhydride phenoxy-
acetic as the capping reagent, 2) the oxidation step was performed with
either a diluted 0.02 M iodine solution or a 10-camphorsulfonyl-oxaziridine
solution,[20] 3) the final support cleavage and total deprotection of modified
DNA fragments were achieved using a 0.05 M K2CO3 solution in methanol at
room temperature for 4 h as a mild alkali treatment. After RP-HPLC purifica-
tion, the purity and the homogeneity of the modified DNA oligomers were
checked by analytical RP-HPLC, polyacrylamide gel electrophoresis, elec-
trospray and MALDI-TOF mass spectrometry measurements (ESI MS and
MALDI-TOF/MS) (Figure 2). The latter mass spectrometry analyses, which
were in complete agreement with the calculated molecular weights,[21] con-
firm the presence and the integrity of the thymidine glycol residue within
the synthetic oligonucleotides.
In conclusion, the synthesis reported herein provides an improved
method for the preparation of oligonucleotides containing the thymidine

1836
D. Gasparutto et al.
FIGURE 2 MALDI-TOF mass spectrum, in the negative mode, of the purified 34-mer thymine glycol-
containing oligonucleotide (molecular weight: 10,474.8 g·mol−1).
glycol damage at specific positions by a total chemical approach that involves
a mild “one step” final alkali deprotection step. The latter modified DNA
sequences will be used as either substrates or probes to study the biological
properties of thymine glycol base lesions together with their structural
features within DNA.
EXPERIMENTAL SECTION
General Procedures and Materials
The silica gel (70–200 m) used for the low-pressure column chromatog-
raphy was purchased from SDS (Peypin, France). TLC was carried out on
Merck DC Kieselgel 60 F-254 plastic sheets (Darmstadt, Germany). All rea-
gents used were of the highest available purity. Anhydrous solvents were
purchased from SDS. Acetonitrile and methanol (HPLC grade) were ob-
tained from Carlo Erba (Milan, Italy). Buffers for HPLC were prepared us-
ing water purified with a Milli-Q system (Milford, MA). The Hypersil ODS
column (5 m, 4.6 × 250 mm I.D.) was purchased from Interchim (Montlu-
con, France). Functionalized CPG supports and unmodified 2ꢁ-deoxyribo-

Oligonucleotides that Contain Thymine Glycol Lesions
1837
nucleoside 3ꢁ-phosphoramidites, protected with phenoxyacetyl for dAdo,
isopropyl-phenoxyacetyl for dGuo and acetyl for dCyd, were from Glen Re-
search (Sterling, Virginia).
Mass Spectrometry Measurements
All modified and unmodified oligonucleotides were characterized by
electrospray ionization-mass spectrometry measurements (ESI-MS) on an
LCQ ion trap model spectrophotometer (Finnigan). Typically, 0.1 AU260nm
of the sample (approximately 3 g) was dissolved in a solution of acetonitrile
and water (50/50, v/v) containing 1% triethylamine prior to be analyzed in
the negative mode (voltage 5 kV). The modified protected nucleosides were
analyzed by ESI-MS in both the negative and positive modes. For the positive
mode analysis, the sample was dissolved in a solution of methanol and water
(50/50, v/v) that contained 0.5% formic acid.
MALDI mass spectra were obtained with a commercially available time-
of-flight mass spectrometer (Biflex, Bruker) equipped with a 337 nm
nitrogen laser. Spectra were recorded in the linear and positive modes.
For the matrix, a mixture of 3-hydroxypicolinic acid and picolinic acid in a
4/1 (w/w) ratio was dissolved in aqueous acetonitrile solution (50%) that
contained a small amount of Dowex-50W 50X8-200 cation exchange resin
(Sigma). Typically, 1 L of the sample was added to 1 L of the matrix and
the resulting mixture was stirred. Then, the solution was then placed on
the top of the target plate and allowed to dry by itself. The spectra were
calibrated with 1 pmol/ L solution of myoglobin (m/z 16952), using the
same conditions that were described for the analysis of oligonucleotides.
Synthetic Procedures
5ꢁ-O-dimethoxytrityl-thymidine (3) and 5ꢁ-O-dimethoxytrityl-3ꢁO-tert-bu-
tyldimethylsilyl-thymidine (4) were prepared from thymidine (2) nucleoside
by using the chloride derivatives of protecting groups and classical proce-
dures.
5 -O-Dimethoxytrityl-3 -O-tert-butyldimethylsilyl-5,6-dihydroxy-5,6-dihy-
drothymidine (5). 5.6 g of 5ꢁ-O-dimethoxytrityl-3ꢁO-tert-butyldimethylsilyl-
thymidine (4, 8.5 mmol) are dissolved in a mixture of THF (24 mL), tert-
butanol (20 mL), and water (3 mL). Then, 2 g of methylmorpholine-N-
oxide (2 eq, 2 g, 17 mmol) and osmium tetraoxide (0.05 eq, 0.425 mmol)
were added to the solution under stirring. The reaction was placed at 45◦
C for 24 h. The mixture was cooled in an ice-water bath and neutalized
by addition of an aqueous 20% sodium thiosulfate solution (5 mL). The
resulting solvents were evaporated to dryness. The oily residue was dissolved

1838
D. Gasparutto et al.
in ethyl acetate (50 mL), washed with saturated NaHCO3 aqueous solution
(50 mL) and water (50 mL × 2). Then the organic phase was dried over
Na2SO4 and evaporated under reduced pressure. The reaction mixture
was resolved by silica gel column chromatography (0–3% step gradient of
methanol in AcOEt/hexane 25/75) to afford the title compound 5 in a yield
of 85%, 4.98 g, 7.20 mmol), as a white powder. Rf (AcOEt/hexane/MeOH:
25/75/10) = 0.45. ESI-MS (positive mode): m/z = 715.1 [M+Na] ; 303.3
1
(DMTr ). H NMR (200.13 MHz, CD3COCD3): : 7.65–7.30 (m, 9H, H
DMTr); 7.01 (d, 4H 3 J(H,H) = 9 Hz, DMTr); 6.37 (pseudo-t, 1H, 3 J(H,H) =
7 Hz, 1H, H-1ꢁ); 5.17 (m, 1H, H-3ꢁ); 4.85 (s, 1H, H-6); 4.55 (m, 1H, H-4ꢁ);
3.92 (s, 6H, CH3O-DMTr); 3.39 (m, 2H, H-5ꢁ and H-5ꢁꢁ); 2.59 (m, 2H, H-
2ꢁ and H-2ꢁꢁ); 1.51 (s, 3H, CH3-dT); 0.95 (s, 9H, tBu-TBDMS); 0.16 (s, 3H,
CH3-TBDMS); 0.10 (s, 3H, CH3-TBDMS).
5 -O-Dimethoxytrityl-3 -O-tert-butyldimethylsilyl-5,6-O-levulinyl-thymi-
dine Glycol (6). 680 mg of compound 5 (0.98 mmol) was dissolved in dry
CH2Cl2 (5 mL) and evaporated to dryness (×2). Then, the residue was
dissolved in dry THF (30 mL) under stirring and an argon atmosphere.
DCC (1.02 g, 5 eq, 4.95 mmol), DMAP (78 mg, 0.6 eq, 0.6 mmol) and
levulinic acid (500 L, 5 eq, 4.88 mmol) was added and the solution was
placed at 40◦C for 24 h. After a TLC analysis, the mixture was cooled
in an ice-water bath and the reaction stopped by addition of methanol
(2 mL). After 15 min, the resulting DCU formed was eliminated by filtration.
The residue was washed with saturated NaHCO3 aqueous solution (50
mL) and water (50 mL × 2). Then the organic phase was dried over
Na2SO4 and evaporated under reduced pressure. After purification by flash
chromatography on a silica gel column, using a step gradient of methanol
(0 to 2%) in CH2Cl2 as the mobile phase, the title compound 6 was obtained
as a white foam in a 71% yield (550 mg, 0.70 mmol) that contains a mixture
of mono- and di-substituted derivatives (O6-monolev/O5,O6-dilev = 9/1).
Rf (CH2Cl2/MeOH: 94/6) = 0.55 (mono-Lev) and 0.58 (di-Lev). ESI-MS
(positive mode): m/z = 911.1 [M+Na] for di-Lev; 813.1 [M+Na] for di-
1
mono-Lev ; 303.3 (DMTr ). O6-monolevulinyl derivative 6: H-NMR (200
3
MHz, CD3COCD3): = 7.52–7.31 (m, 9H, DMTr); 6.98 (d, 4H J(H,H) =
3
9 Hz, DMTr); 6.24 (pseudo-t, 1H, J(H,H) = 7 Hz, 1H, H-1ꢁ); 4.78 (s, 1H,
H-6); 4.46 (m, 1H, H-3ꢁ); 3.93 (m, 1H, H-4ꢁ); 3.90 (s, 6H, CH3O-DMTr); 3.43
(m, 2H, H-5ꢁ H-5ꢁꢁ); 2.84 (m, 2H, CH2-Lev); 2.68 (m, 4H, H-2ꢁ H-2ꢁꢁ, CH2-
Lev); 2.24 (s, 1H, CH3-Lev); 1.89 (s, 3H, CH3-dT); 0.93 (s, 9H, tBu-TBDMS);
0.14 (s, 3H, CH3-TBDMS); 0.08 (s, 3H, CH3-TBDMS).
5 -O-Dimethoxytrityl-5,6-O-levulinyl-thymidine Glycol (7). 215 mg of
compounds 6 (0.27 mmol) was dissolved in dry THF (5 mL) under stirring
and an argon atmosphere. Then, 1 mL of TBAF in THF (1 M solution, 4 eq,

Oligonucleotides that Contain Thymine Glycol Lesions
1839
1.08 mmol) was added and the solution was stirred at room temperature
for 3 h. After a TLC analysis, the reaction was stopped by addition of water
(1 mL). After 5 min, 15 mL of dichloromethane was added and the residue
was washed with saturated NaHCO3 aqueous solution (20 mL) and water (20
mL × 2). Then the organic phase was dried over Na2SO4 and evaporated
under reduced pressure. After purification by flash chromatography on a
silica gel column, using a step gradient of methanol (0 to 4%) in CH2Cl2
as the mobile phase, the title compound 7 was obtained as a white foam
in a 60% yield (110 mg, 0.165 mmol) (O6-monolev/O5,O6-dilev = 9/1).
Rf (CH2Cl2/MeOH: 94/6) = 0.42 (mono-Lev) and 0.45 (di-Lev). ESI-MS
(positive mode): m/z = 797.2 [M+Na] for di-Lev; 699.2 [M+Na] for
mono-Lev ; 303.3 (DMTr ).
Thymidine Glycol Phosphoramidite Synthon (8). Compound 7 (100
mg, 0.148 mmol) was co-evaporated twice with dry pyridine and subsequently
dissolved in anhydrous CH2Cl2 (15 mL) under an argon atmosphere. Diiso-
propylammonium tetrazolate (12.7 mg, 0.074 mmol, 0.5 eq) and 2-cyano-
ethyl-N,N,Nꢁ,Nꢁ-tetraisopropyldiamidite (49 l, 0.163 mmol, 1.1 eq) were
added to the solution under stirring. The course of the reaction was moni-
tored by TLC (CH2Cl2/MeOH/TEA 95/5/1). After 4 h at room tempera-
ture, the mixture was diluted with ethyl acetate (25 mL) and washed with
saturated NaHCO3 aqueous solution (30 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuum. The resulting residue was puri-
fied by flash chromatography on a silica gel column with a step gradient of
methanol (0–3%) in CHCl3/TEA (99/1, v/v) as the mobile phase. Then, the
collected fractions corresponding to the diastereomers were dried to afford
compound 8 as a white foam (yield 75%, 0.111 mmol, 108 mg). Phospho-
ramidite 8 (O6-monolevulinyl derivative): Rf (CH2Cl2/MeOH/TEA 95/5/1)
= 0.6. 31P-NMR (161.9 MHz, CD3CN) = 149.5–149.9 (two diastereoiso-
mers). ESI-MS (positive mode): m/z = 997.1 [M+Na] , 1013.1 [M+K] .
Stability Studies of the Thymine Glycol Nucleoside (1)
Under the Alkaline Conditions Used for Oligonucleotides
Chemical Synthesis
Aqueous ammonia (32%; 500 L) was added to 0.5 AU230nm of com-
pound 1 in sealed tubes. The solutions were placed at either room tempera-
ture or 55◦C. Then, the reactions were stopped at increasing time intervals
(0, 1, 2, 4, 8, 16, and 24 h, respectively) by freezing in liquid nitrogen and
subsequent lyophilization. Samples were analyzed by HPLC using a C18 Hy-
persil column. The elution was achieved with a 0 to 10% linear gradient of
acetonitrile in 25 mM ammonium formiate buffer over 30 min (flow rate:
1 mL/min; UV detection at 230 nm).

1840
D. Gasparutto et al.
Stability Studies of the Thymine Glycol Nucleoside (1)
Under the Acid Conditions Used for Oligonucleotides
Chemical Synthesis
A similar procedure, as described above for the alkali-stability assay, was
used. This involved incubation of compounds 1 in a 80% acetic acid aqueous
solution for 0, 1, 2, 4, 8, 16, and 24 h, respectively, at room temperature.
Stability Studies of the Thymine Glycol Nucleoside (1)
Under OxidizingConditions Used for Oligonucleotides
Chemical Synthesis
Similarly, compound 1 was incubated in a 0.02 M oxidizing solution of
iodine for 0, 1, 2, 4, 8, 16, and 24 h, respectively, at room temperature.
Solid-phase Synthesis of Oligodeoxyribonucleotides
The synthesis of thymine glycol containing oligodeoxyribonucleotides
was performed at 1 mol scale using the “Pac phosphoramidite” chem-
istry[14], with retention of the 5ꢁ terminal DMTr group (trityl-on mode).
The standard 1 mol DNA cycle was used, on an Applied Biosystems Inc.
392 DNA synthesizer, with slight modifications. The duration of condensa-
tion was increased by a factor of 4 for the modified nucleoside phospho-
ramidite 8 (120 s instead of 30 s for normal nucleoside phosphoramidites).
Under these conditions a coupling efficiency of more than 90% for the
modified monomer 8 was achieved. A 0.3 M solution of phenoxyacetic an-
hydride in tetrahydrofuran and a 0.02 M solution of iodine in water/pyri-
dine/tetrahydrofuran were used for the capping and the oxidation steps,
respectively.
Deprotection and Purification of Modified
Oligodeoxyribonucleotides
Upon completion of the synthesis, the alkali-labile protecting groups of
the thymine glycol containing oligodeoxyribonucleotides were removed by
treatment with concentrated aqueous ammonia (32%) at room temperature
for 4 h. Solvents were removed by evaporation under vacuum. Then, the
crude 5ꢁ-DMTr-oligomers were purified and deprotected on-line by reverse-
phase HPLC using a polymeric support, as previously described.[22] The
modified 34-mer oligonucleotide, used in biochemical studies, was further
purified by preparative polyacrylamide gel electrophoresis and, then, was
desalted using a NAP-25 sephadex column (Pharmacia, Uppsala, Sweden).

Oligonucleotides that Contain Thymine Glycol Lesions
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21. Modified 5-mer [sequence : TG(1)CA]: mass calculated
1512.0 Da; mass found
1511.7 Da.
Modified 14-mer [sequence: ATCGTGAC(1)GATCT]: mass calculated 4287.8 Da; mass found
4286.9 Da. Modified 34-mer [sequence: GGCTTCATCGTTGTC(1)CAGACCTGGTGGATACCG]:
mass calculated
10,474.8 Da; mass found
10475.4 Da.
22. Romieu, A.; Gasparutto, D.; Molko, D.; Cadet, J. A convenient synthesis of 5-hydroxy-2ꢀ-
deoxycytidine phosphoramidite and its incorporation into oligonucleotides. Tetrahedron Lett.
1997, 38, 7531–7534.