Synthesis and characterization of oligonucleotides containing 5',8- cyclopurine 2'-deoxyribonucleosides: (5'R)-5',8-cyclo-2'-deoxyadenosine, (5'S)-5',8-cyclo-2'-deoxyguanosine, and (5'R)-5',8-cyclo-2'-deoxyguanosine

Article abstract of DOI:10.1021/tx9802668

Radiation-induced degradation of purine and pyrimidine nucleosides gave rise to carbon-bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which modification of both the base and 2- deoxyribose has occurred. A solid-phase synthetic method was designed for the incorporation of both 5'R and 5'S diastereoisomers of 5',8-cyclopurine 2'- deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical and biophysical features of such lesions. We report the preparation of the phosphoramidite synthons of (5'R)-5',8-cyclo-2'- deoxyadenosine (2), (5'S)-5',8-cyclo-2'-deoxyguanosine (3), and (5'R)-5',8- cyclo-2'-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then inserted into several oligonucleotides by automated procedures. Analysis of modified DNA oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and endonucleases confirmed the base compositions and the integrity of free radical-induced tandem lesions 2-4 that were chemically inserted.

Full text of DOI:10.1021/tx9802668

412
Chem. Res. Toxicol. 1999, 12, 412-421
Syn th esis a n d Ch a r a cter iza tion of Oligon u cleotid es
Con ta in in g 5′,8-Cyclop u r in e 2′-Deoxyr ibon u cleosid es:
(5′R)-5′,8-Cyclo-2′-d eoxya d en osin e,
(5′S)-5′,8-Cyclo-2′-d eoxygu a n osin e, a n d
(5′R)-5′,8-Cyclo-2′-d eoxygu a n osin e
Anthony Romieu, Didier Gasparutto, and J ean Cadet*
Laboratoire des Le´sions des Acides Nucle´iques, Service de Chimie Inorganique et Biologique,
De´partement de Recherche Fondamentale sur la Matie`re Condense´e, CEA-Grenoble,
F-38054 Grenoble Cedex 9, France
Received December 15, 1998
Radiation-induced degradation of purine and pyrimidine nucleosides gave rise to carbon-
bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which
modification of both the base and 2-deoxyribose has occurred. A solid-phase synthetic method
was designed for the incorporation of both 5′R and 5′S diastereoisomers of 5′,8-cyclopurine
2′-deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical
and biophysical features of such lesions. We report the preparation of the phosphoramidite
synthons of (5′R)-5′,8-cyclo-2′-deoxyadenosine (2), (5′S)-5′,8-cyclo-2′-deoxyguanosine (3), and
(5′R)-5′,8-cyclo-2′-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then
inserted into several oligonucleotides by automated procedures. Analysis of modified DNA
oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and
endonucleases confirmed the base compositions and the integrity of free radical-induced tandem
lesions 2-4 that were chemically inserted.
Ch a r t 1. 5′S a n d 5′R Dia ster eoisom er s of
In tr od u ction
Cyclod Ad o a n d Cyclod Gu o
Since the 1970s, the chemistry of cyclonucleosides has
been an area of intense research. Several cyclonucleosides
bearing a carbon-carbon linkage between the sugar and
the aglycon moieties have been prepared. Interestingly,
some of them were utilized as conformationally fixed
analogues of nucleosides or nucleotides in some enzy-
matic reactions (1). On the other hand, pioneering
experiments have shown that the radiation-induced
degradation of purine and pyrimidine nucleic acid com-
ponents provided such carbon-bridged cyclonucleosides.
Indeed, the 5′R and 5′S diastereoisomers of 5′,8-cyclo-2′-
deoxyadenosine (CyclodAdo)1 and 5′,8-cyclo-2′-deoxygua-
nosine (CyclodGuo) have been found to be generated in
both adenine- and guanine-containing monomers, in DNA
polymers (2-7), and in cellular DNA upon exposure to
ionizing radiation (8). The pyrimidine analogues of the
latter compounds, 5′,6-cyclo-5,6-dihydrothymidine (Cy-
clodHThd) and 5′,6-cyclo-5,6-dihydro-2′-deoxyuridine (Cy-
clodHUrd), have been also detected in γ-irradiated frozen
aqueous solutions of thymidine and 2′-deoxycytidine,
respectively (9). Such modified nucleosides which may
be considered as tandem DNA lesions, the base and the
2-deoxyribose residues both being altered, are likely to
exert a significant biological impact (10). Synthetic
oligodeoxynucleotides (ODNs) that contain such specific
lesions at selected sites in defined sequences are powerful
tools for biochemical (mutagenesis and repair) and con-
formationnal studies of the consequences of cyclonucleo-
side formation in DNA.
* To whom correspondence should be addressed. Telephone: (33)-
4-76-88-49-87. Fax: (33)-4-76-88-50-90. E-mail: cadet@drfmc.ceng.cea.fr.
1
Abbreviations: CyclodAdo, 5′,8-cyclo-2′-deoxyadenosine; Cyclo-
dGuo, 5′,8-cyclo-2′-deoxyguanosine; CyclodHThd, 5′,6-cyclo-5,6-dihy-
drothymidine; CyclodHUrd, 5′,6-cyclo-5,6-dihydro-2′-deoxyuridine; ODN,
oligodeoxyribonucleotide; Bz, benzoyl group; iBu, isobutyryl group;
DIEA, N,N-diisopropylethylamine; DEAD, diethylazodicarboxylate;
DMTr-Cl, 4,4′-dimethoxytrityl chloride; DME, dimethoxyethane; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; FA, ammonium
formate buffer; Ms-Cl, mesyl chloride; TBAF, tetrabutylammonium
fluoride; TBDMS-Cl, tert-butyldimethylsilyl chloride; TIPS-Cl, triiso-
propylsilyl chloride; TEA, triethylamine; FAB-MS, fast atom bombard-
ment mass spectrometry; ESI-MS, electrospray ionization mass spec-
trometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry.
In a previous work, a phosphoramidite building block
of (5′S)-CyclodAdo 1 (Chart 1) was synthesized and
incorporated into ODNs which were found to be not
completely hydrolyzed by exo- and endonucleases (11).
10.1021/tx9802668 CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/09/1999

Oligonucleotides Containing 5′,8-Cyclopurine Nucleosides
Chem. Res. Toxicol., Vol. 12, No. 5, 1999 413
Hz, 1H, H-2′), 2.35 (m, J 2′′3′ ) 4.7 Hz, 1H, H-2′′), 0.89 (s, 9H,
tBu-TBDMS), 0.12 and 0.09 (s, 6H, CH3-TBDMS).
In this paper, we report the insertion of the other 5′,8-
cyclopurine 2′-deoxyribonucleosides, (5′R)-CyclodAdo 2,
(5′S)-CyclodGuo 3, and (5′R)-CyclodGuo 4, using a similar
approach. These modified nucleosides were prepared
according to the methodology developed by Matsuda et
al. (12) for the synthesis of the ribonucleoside analogues.
However, the postfunctionalization of the C-5′ position
by the oxidation-reduction procedure provided only the
5′S diastereoisomers of CyclodAdo and CyclodGuo. There-
fore, the preparation of the 5′R diastereoisomers required
the development of an efficient method for the clean
inversion of C-5′ stereoconfiguration. Characterization
and a few biochemical features of the modified DNA
fragments are also reported.
(5′R)-N6-Ben zoyl-3′-O-(ter t-bu tyldim eth ylsilyl)-5′,8-cyclo-
2′-d eoxya d en osin e (7). Compound 6 (0.806 g, 1.47 mmol) was
dissolved in 40 mL of DMSO/DME (1/1). KO2 (0.381 g, 5.36
mmol) and 18-C-6 crown ether (0.194 g, 0.73 mmol) were added,
and the resulting mixture was stirred at room temperature for
9 h. The reaction was checked for completion by TLC (95/5
CHCl3/CH3OH, v/v) and then neutralized with 1 N HCl. After
10 min, the mixture was evaporated almost to dryness. The
resulting residue was dissolved in 50 mL of dichloromethane
and washed with water (4 × 50 mL). The organic layer was dried
over Na2SO4 and evaporated to dryness. The resulting residue
was purified by chromatography on a silica gel (50 g) column
with a step gradient of methanol (0 to 4%) in chloroform as the
mobile phase. The appropriate fractions were pooled and then
concentrated to dryness giving 0.4 g (0.85 mmol) of (5′R)-N6-
benzoyl-3′-O-(tert-butyldimethylsilyl)-5′,8-cyclo-2′-deoxyadeno-
sine as a yellow powder (yield of 58%). FAB-MS (positive
mode): m/z 490.3 ( 0.1 Da [M + Na]+, 468.4 ( 0.1 Da [M +
H]+. 1H NMR (200 MHz, CDCl3 + D2O): δ 8.72 (s, 1H, H-2),
8.07-7.48 (m, 5H, aromatic H of Bz), 6.60 (d, J 1′2′′ ) 4.4 Hz,
Exp er im en ta l Section
Gen er a l. The silica gel (70-200 µm) used for the low-
pressure column chromatography was purchased from SDS
(Peypin, France). TLC was carried out on Merck DC Kieselgel
60 F-254 plastic sheets. Deuterated solvents (acetone-d6, CDCl3,
D2O, DMSO-d6, and methanol-d4) were purchased from Acros
(Geel, Belgium). CDCl3 was passed through a small plug of Al2O3
immediately prior to use. All reagents were of the highest
available purity. N2-Isobutyryl-2′-deoxyguanosine was synthe-
sized using the transient protection method developed by Ti et
al. (13). Anhydrous solvents were purchased from SDS. The
HPLC-grade solvents (acetonitrile and methanol) were obtained
from Carlo Erba (Milan, Italy). Alkaline phosphatase, nuclease
P1 (Penicilium citrinium), and 0.3 M sodium acetate/ZnCl2
buffer (pH 5.3) were purchased from Sigma (St. Louis, MO).
Bovine pancreas DNase I, snake venom phosphodiesterase, and
calf spleen phosphodiesterase were obtained from Boehringer
Mannheim (Mannheim, Germany). Buffers for high-performance
liquid chromatography (HPLC) were prepared using water
purified with a Milli-Q system (Milford, MA).
1H, H-1′), 4.91 (s, 1H, H-5′), 4.64 (s, 1H, H-4′), 4.32 (q, J 2′3′
)
6.7 Hz, J 2′3′′ ) 4.1 Hz, 1H, H-3′), 2.51 (dd, J 2′2′′ ) -13.5 Hz, 1H,
H-2′), 2.29 (m, 1H, H-2′′), 0.88 (s, 9H, tBu-TBDMS), 0.05 and
0.04 (s, 6H, CH3-TBDMS).
(5′R)-N6-Ben zoyl-3′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-(4,4′-
d im eth oxytr ityl)-5′,8-cyclo-2′-d eoxya d en osin e (8). Com-
pound 7 (0.169 g, 0.36 mmol) was dissolved in dry pyridine (2
mL), and the resulting solution was evaporated to dryness. The
operation was repeated twice. The resulting residue was dis-
solved in 6 mL of dry pyridine, and DMTr-Cl (0.367 g, 1.08
mmol) was added. The mixture was heated at 80 °C and left at
this temperature for 4 h. The reaction was checked for comple-
tion by TLC (97/3 CHCl3/CH3OH, v/v). The solution was cooled
at 5 °C, and methanol (0.5 mL) was added. After 10 min, the
mixture was evaporated to dryness. The resulting residue was
purified by chromatography on a silica gel (30 g) column with a
step gradient of methanol (0 to 1%) in dichloromethane/TEA
(99/1) as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness giving 0.136 g (0.17
mmol) of (5′R)-N6-benzoyl-3′-O-(tert-butyldimethylsilyl)-5′-O-
(4,4′-dimethoxytrityl)-5′,8-cyclo-2′-deoxyadenosine as a yellow
foam (yield of 47%). FAB-MS (positive mode): m/z 770.3 ( 0.1
Da [M + H]+, 303.1 ( 0.1 Da [DMTr]+. 1H NMR (200 MHz,
CDCl3): δ 8.76 (s, 1H, H-2), 8.05-6.82 (m, 18H, aromatic H of
Bz and DMTr), 6.56 (d, J 1′2′′ ) 4.0 Hz, 1H, H-1′), 4.75 (s, 1H,
H-4′), 3.87 (m, 1H, H-3′), 3.75 and 3.73 (s, 6H, CH3O-DMTr),
3.66 (s, 1H, H-5′), 2.27 (dd, J 2′2′′ ) -13.2 Hz, 1H, H-2′), 2.06 (m,
1H, H-2′′), 0.77 (s, 9H, tBu-TBDMS), -0.11 and -0.14 (s, 6H,
CH3-TBDMS).
Ir r a d ia tion System . The far-UV irradiations were per-
formed with a Rayonet photochemical reactor (Southern New
England Ultraviolet Co., Hamden, CT); 14 lamps (λmax ) 254
nm, approximatively 15 W of mainly 254 nm radiation for each
lamp) were used.
High -P er for m a n ce Liqu id Ch r om a togr a p h y Sep a r a -
tion s. All enzymatic digestions were analyzed with the following
chromatographic system: reverse-phase HPLC system (Hypersil
C18 column, 5 µm, 4.6 mm × 250 mm) with acetonitrile and
ammonium formate buffer (FA, 25 mM, pH 6.2) as the eluents
[100% FA (10 min), linear gradient from 0 to 10% acetonitrile
(30 min)] at a flow rate of 1 mL/min. Detection was achieved at
λ ) 260 nm.
(5′S)-N6-Ben zoyl-3′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-m e-
syl-5′,8-cyclo-2′-d eoxya d en osin e (6). (5′S)-N6-Benzoyl-3′-O-
(tert-butyldimethylsilyl)-5′,8-cyclo-2′-deoxyadenosine 5 (0.832 g,
1.78 mmol) was dissolved in dry pyridine (5 mL) and evaporated
to dryness. The resulting white foam was dissolved in 40 mL of
dry dichloromethane and cooled at 5 °C. Then TEA (1 mL, 7.12
mmol) was added followed by dropwise addition of Ms-Cl (0.28
mL, 3.60 mmol). The mixture was left at room temperature for
3 h. The reaction was checked for completion by TLC (97/3
CHCl3/CH3OH, v/v), and the mixture was evaporated to dryness.
The resulting residue was purified by chromatography on a
silica gel (50 g) column with a step gradient of methanol (0 to
2%) in chloroform as the mobile phase. The appropriate fractions
were pooled and then concentrated to dryness giving 0.913 g
(1.67 mmol) of (5′S)-N6-benzoyl-3′-O-(tert-butyldimethylsilyl)-
5′-O-mesyl-5′,8-cyclo-2′-deoxyadenosine as a white solid (yield
of 94%). FAB-MS (positive mode): m/z 546.1 ( 0.1 Da [M +
H]+. 1H NMR (200 MHz, CDCl3): δ 8.70 (s, 1H, H-2), 7.98-
7.49 (m, 5H, aromatic H of Bz), 6.55 (d, J 1′2′′ ) 4.7 Hz, 1H, H-1′),
6.11 (d, J 4′5′ ) 6.5 Hz, 1H, H-5′), 4.89-4.80 (m, 2H, H-3′ and
H-4′), 3.56 (s, 3H, CH3-Ms), 2.64 (dd, J 2′3′ ) 7.3 Hz, J 2′2′′ ) -13.5
(5′R)-N6-Ben zoyl-5′-O-(4,4′-d im eth oxytr ityl)-5′,8-cyclo-2′-
d eoxya d en osin e (9). Compound 8 (0.136 g, 0.17 mmol) was
dissolved in 10 mL of dry THF. A solution of TBAF (0.35 mL,
0.35 mmol) in THF (1 M) was added, and the mixture was
stirred at room temperature for 4 h. The reaction was checked
for completion by TLC (95/5 CHCl3/CH3OH, v/v), and the
mixture was evaporated to dryness. The resulting residue was
purified by chromatography on a silica gel (30 g) column with a
step gradient of methanol (0 to 2%) in dichloromethane/TEA
(99/1) as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness giving 63 mg (0.1
mmol) of (5′R)-N6-benzoyl-5′-O-(4,4′-dimethoxytrityl)-5′,8-cyclo-
2′-deoxyadenosine as a white foam (yield of 59%). FAB-MS
(positive mode): m/z 656.0 ( 0.1 Da [M + H]+, 303.0 ( 0.1 Da
1
[DMTr]+. H NMR (200 MHz, CDCl3): δ 8.99 (bs, 1H, NH-Bz),
8.75 (s, 1H, H-2), 8.08-6.87 (m, 18H, aromatic H of Bz and
DMTr), 6.54 (d, J 1′2′′ ) 4.8 Hz, 1H, H-1′), 4.79 (s, 1H, H-4′), 3.94
(q, J 2′3′ ) 6.7 Hz, J 2′′3′ ) 4.0 Hz, 1H, H-3′), 3.77 and 3.76 (s, 6H,
CH3O-DMTr), 3.23 (s, 1H, H-5′), 2.35 (dd, J 2′2′′ ) -13.8 Hz, 1H,
H-2′), 2.08 (m, 1H, H-2′′).

414 Chem. Res. Toxicol., Vol. 12, No. 5, 1999
Romieu et al.
(5′R)-5′,8-Cyclo-2′-deoxyaden osin e P h osph or am idite De-
r iva tive (10). Compound 9 (63 mg, 0.1 mmol) was dissolved in
dry dichloromethane (5 mL) and evaporated to dryness. The
operation was repeated twice. The resulting residue was then
dissolved in 3 mL of dry dichloromethane and kept under an
argon atmosphere. Then, dry DIEA (33 µL, 0.19 mmol) was
added, followed by dropwise addition of 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (26 µL, 0.11 mmol). The
reaction mixture was stirred for 30 min. The desired product
was checked by TLC (70/30/1 AcOEt/hexane/TEA, v/v/v). The
solution was cooled to 5 °C with an ice bath, and more DIEA
(33 µL) and methanol (100 µL) were added. After 10 min, the
mixture was evaporated to dryness. The resulting residue was
purified by chromatography on a silica gel (25 g) column with a
step gradient of methanol (0 to 2%) in dichloromethane/TEA
(99/1) as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness giving 67 mg (0.08
mmol) of the phosphoramidite synthon 10 as a white foam (yield
of 80%). FAB-MS (positive mode): m/z 856.0 ( 0.1 Da [M +
H]+, 303.0 ( 0.1 Da [DMTr]+. 1H NMR (200 MHz, acetone-d6,
two diastereoisomers): δ 8.75 and 8.59 (s, 1H, H-2), 8.35-6.96
(m, 18H, aromatic H of Bz and DMTr), 6.75 (t, 1H, H-1′), 5.04
and 4.99 (s, 1H, H-4′), 4.38-4.21 (m, 5H, H-3′, CH-iPr and CH2-
OP), 3.88-3.52 (m, 7H, H-5′ and CH3O-DMTr), 2.86-2.41 (m,
4H, H-2′, H-2′′, and CH2CN), 1.40-1.08 (m, 12H, CH3-iPr). 31P
NMR (101 MHz, acetone-d6): δ 151.7 and 150.3 (two diastere-
oisomers).
N2-Isobu tyr yl-5′-p h en ylth io-2′,5′-d id eoxygu a n osin e (12).
A mixture of N2-isobutyryl-2′-deoxyguanosine 11 (2.05 g, 6.1
mmol), diphenyl disulfide (3.88 g, 17.8 mmol), and PBu3 (4.4
mL, 17.9 mmol) in 30 mL of dry DMF was stirred at room
temperature for 22 h. The reaction was checked for completion
by TLC (85/15 CHCl3/CH3OH, v/v). Then, 30 mL of water was
added, and the solution was concentrated under reduced pres-
sure. The residual syrup was dissolved in 200 mL of chloroform
and the title compound precipitated out quickly. The solid was
isolated by filtration, washed with ether (2 × 30 mL), and dried.
Also, filtrates were evaporated to dryness, and the resulting
residue was purified by chromatography on a silica gel (100 g)
N2-Isob u t yr yl-3′-O-(ter t-b u t yld im et h ylsilyl)-5′,8-cyclo-
2′,5′-d id eoxygu a n osin e (14). Compound 13 (0.316 g, 1 mmol)
and imidazole (0.272 g, 4 mmol) were dissolved in dry pyridine
(5 mL) and evaporated to dryness. The resulting residue was
dissolved in 10 mL of dry DMF to which TBDMS-Cl (0.305 g, 2
mmol) was added. The mixture was left at room temperature
for 24 h. The reaction was checked for completion by TLC (93/7
CHCl3/CH3OH, v/v), and the mixture was cooled at 5 °C. Then,
water (10 mL) was added, and after 10 min, the mixture was
evaporated almost to dryness. The residue was taken up in
chloroform (50 mL) and washed with 5% NaHCO3 (40 mL). The
organic layer was dried over Na2SO4 and evaporated to dryness.
The resulting residue was then purified by chromatography on
a silica gel (50 g) column with a step gradient of methanol (0 to
2%) in chloroform as the mobile phase. The appropriate fractions
were pooled and then concentrated to dryness giving 0.317 g
(0.73 mmol) of N2-isobutyryl-3′-O-(tert-butyldimethylsilyl)-5′,8-
cyclo-2′,5′-dideoxyguanosine as a white solid (yield of 73%). FAB-
MS (positive mode): m/z 456.4 ( 0.1 Da [M + Na]+, 434.4 (
0.1 Da [M + H]+. 1H NMR (200 MHz, DMSO-d6): δ 6.39 (d,
J 1′2′′ ) 5.0 Hz, 1H, H-1′), 4.76 (d, J 4′5′ ) 6.2 Hz, 1H, H-4′), 4.64
(q, J 2′3′ ) 7.0 Hz, J 2′′3′ ) 3.5 Hz, 1H, H-3′), 3.38 (dd, 1H, H-5′),
3.10 (d, J 5′5′′ ) -17.9 Hz, 1H, H-5′′), 2.96-2.69 (m, 2H, H-2′
and CH-iBu), 2.22 (m, 1H, H-2′′), 1.24 and 1.21 (s, 6H, CH3-
iBu), 0.98 (s, 9H, tBu-TBDMS), 0.20 and 0.17 (s, 6H, CH3-
TBDMS).
(5′S)-N2-Isobu tyr yl-3′-O-(ter t-bu tyld im eth ylsilyl)-5′,8-cy-
clo-2′-d eoxygu a n osin e (15). Compound 14 (0.317 g, 0.73
mmol) was dissolved in dry 1,4-dioxane (80 mL). SeO2 (0.410 g,
3.69 mmol) was added, and the mixture was stirred under reflux
for 20 h. The reaction was checked for completion by TLC (95/5
CHCl3/CH3OH, v/v). Then, the mixture was cooled at room
temperature and passed through a Celite column. The filtrate
was evaporated to dryness giving 0.326 g (0.73 mmol) of
N2-isobutyryl-3′-O-(tert-butyldimethylsilyl)-5′-oxo-5′,8-cyclo-2′-deoxy-
guanosine as a pink powder. The product contained traces of
colloidal SeO2, but it was suitable for the next reduction step.
The solid residue was dissolved in 25 mL of dry methanol.
NaBH4 (55 mg, 1.45 mmol) was added, and the mixture was
stirred for 1 h at room temperature. The reaction was checked
for completion by TLC (95/5 CHCl3/CH3OH, v/v) and then
neutralized with 1 N HCl. After 5 min, the mixture was
evaporated to dryness. The resulting residue was purified by
chromatography on a silica gel (50 g) column with a step
gradient of methanol (0 to 6%) in chloroform as the mobile
phase. The appropriate fractions were pooled and then concen-
trated to dryness giving 0.139 g (0.31 mmol) of (5′S)-N2-
isobutyryl-3′-O-(tert-butyldimethylsilyl)-5′,8-cyclo-2′-deoxygua-
nosine as a white powder (yield of 42%). FAB-MS (positive
mode): m/z 472.1 ( 0.1 Da [M + Na]+, 450.1 ( 0.1 Da [M +
column with
a step gradient of methanol (0 to 7.5%) in
chloroform as the mobile phase. Both methods of purification
provided 1.83 g (4.3 mmol) of N2-isobutyryl-5′-phenylthio-2′,5′-
dideoxyguanosine 12 as a white solid (yield of 70%). FAB-MS
(positive mode): m/z 430.2 ( 0.1 Da [M + H]+, 222.2 ( 0.1 Da
[BH + H]+. 1H NMR (200 MHz, DMSO-d6): δ 8.35 (s, 1H, H-8),
7.48-7.28 (m, 5H, aromatic H of PhS), 6,34 (t, J 1′2′ ) J 1′2′′ ) 7.5
Hz, 1H, H-1′), 5.59 (d, J OH-3′ ) 3.5 Hz, 1H, OH-3′), 4.50 (m, 1H,
H-3′), 4.05 (m, 1H, H-4′), 3.42-3.35 (m, 2H, H-5′ and H-5′′),
2.97-2.80 (m, 2H, H2′ and CH-iBu), 2.43 (m, 1H, H-2′′), 1.25
and 1.22 (d, J CH
) 1.5 Hz, 6H, CH3-iBu).
3CH
1
H]+. H NMR (200 MHz, CDCl3): δ 6.22 (d, J 1′2′′ ) 5.0 Hz, 1H,
N2-Isobu tyr yl-5′,8-cyclo-2′,5′-dideoxygu an osin e (13). Com-
pound 12 (1.47 g, 3.4 mmol) and P(OEt)3 (6 mL, 35 mmol) were
dissolved in 1.35 L of dry acetonitrile. Because of the poor
solubility of 12, it was necessary to crush it prior to the
solubilization step. Then, argon was bubbled through the
solution for 45 min. The oxygen-free solution was irradiated at
254 nm for 8 h in a 2 L quartz reactor under an argon
atmosphere. The reaction was checked for completion by TLC
(85/15 CHCl3/CH3OH, v/v), and the solution was evaporated to
dryness. The resulting yellow oil was purified by chromatogra-
phy on a silica gel (100 g) column with a step gradient of
methanol (0 to 10%) in chloroform as the mobile phase. The
appropriate fractions were pooled and then concentrated to
dryness giving 0.5 g (1.56 mmol) of N2-isobutyryl-5′,8-cyclo-2′,5′-
dideoxyguanosine as a yellow foam (yield of 46%). FAB-MS
(positive mode): m/z 320.3 ( 0.1 Da [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ 6.35 (d, J 1′2′′ ) 4.8 Hz, 1H, H-1′), 5.50 (d,
J OH-3′ ) 4.3 Hz, 1H, OH-3′), 4.75 (d, J 4′5′ ) 6.5 Hz, 1H, H-4′),
4.44 (m, J 2′3′ ) 7.5 Hz, J 2′′3′ ) 4.3 Hz, 1H, H-3′), 3.39 (dd, J 5′5′′
) -17.7 Hz, 1H, H-5′), 3.03 (d, 1H, H-5′′), 2.85 (m, 1H, CH-
iBu), 2.62 (m, 1H, H-2′), 2.19 (m, 1H, H-2′′), 1.23 and 1.22 (s,
6H, CH3-iBu).
H-1′), 5.29 (s, 1H, OH-5′), 5.27 (d, J 4′5′ ) 6.7 Hz, 1H, H-5′), 4.87
(q, J 2′3′ ) 6.6 Hz, J 2′′3′ ) 4.1 Hz, 1H, H-3′), 4.69 (d, 1H, H-4′),
2.78-2.46 (m, 2H, H-2′ and CH-iBu), 2.21 (m, 1H, H-2′′), 1.28
and 1.24 (d, J CH
) 2.4 Hz, 6H, CH3-iBu), 0.87 (s, 9H, tBu-
3CH
TBDMS), 0.10 and 0.06 (s, 6H, CH3-TBDMS).
(5′S)-N2-Isob u t yr yl-3′-O-(ter t-b u t yld im et h ylsilyl)-5′-O-
(4,4′-d im et h oxyt r it yl)-5′,8-cyclo-2′-d eoxygu a n osin e (16).
The procedure described for 8 (vide supra) was applied. After
purification by silica gel column chromatography, 16 was
obtained as a yellow foam (yield of 60%). FAB-MS (positive
mode): m/z 752.1 ( 0.1 Da [M + H]+, 303.1 ( 0.1 Da [DMTr]+.
1H NMR (200 MHz, CDCl3): δ 7.80-6.82 (m, 13H, aromatic H
of DMTr), 6.04 (d, J 1′2′′ ) 4.4 Hz, 1H, H-1′), 5.12 (d, J 4′5′ ) 5.3
Hz, 1H, H-4′), 4.73 (m, 1H, H-3′), 3.77 (s, 6H, CH3O-DMTr), 2.93
(d, 1H, H-5′), 2.63-2.30 (m, 2H, H-2′ and CH-iBu), 1.91 (m, 1H,
H-2′′), 1.25 and 1.24 (s, 6H, CH3-iBu), 0.78 (s, 9H, tBu-TBDMS),
-0.08 (s, 6H, CH3-TBDMS).
(5′S)-N2-Isobu tyr yl-5′-O-(4,4′-d im eth oxytr ityl)-5′,8-cyclo-
2′-d eoxygu a n osin e (17). The procedure described for 9 (vide
supra) was applied. After purification by silica gel column
chromatography, 17 was obtained as a yellow solid (yield of

Oligonucleotides Containing 5′,8-Cyclopurine Nucleosides
95%). FAB-MS (positive mode): m/z 638.0 ( 0.1 Da [M + H]+,
Chem. Res. Toxicol., Vol. 12, No. 5, 1999 415
deoxyguanosine as white solid (yield of 95%). FAB-MS
a
303.0 ( 0.1 Da [DMTr]+. H NMR (200 MHz, CDCl3): δ 7.81-
(positive mode): m/z 670.3 ( 0.1 Da [M + Na]+, 648.4 ( 0.1 Da
[M + H]+. 1H NMR (200 MHz, CDCl3): δ 6.43 (d, J 1′2′′ ) 4.8 Hz,
1H, H-1′), 6.06 (d, J 4′5′ ) 6.5 Hz, 1H, H-5′), 4.92-4.84 (m, 2H,
H-3′ and H-4′), 3.87 and 3.51 (s, 6H, CH3-Ms), 2.77-2.32 (m,
3H, CH-iBu, H-2′ and H-2′′), 1.29 and 1.26 (s, 6H, CH3-iBu), 1.05
1
6.86 (m, 13H, aromatic H of DMTr), 6.03 (d, J 1′2′′ ) 4.7 Hz, 1H,
H-1′), 5.28 (s, 1H, OH-3′), 5.22 (d, J 4′5′ ) 5.9 Hz, 1H, H-4′), 4.70
(m, 1H, H-3′), 3.79 and 3.78 (s, 6H, CH3O-DMTr), 2.72 (d, 1H,
H-5′), 2.63-2.40 (m, 2H, H-2′ and CH-iBu), 1.99 (m, 1H, H-2′′),
1.25 and 1.24 (s, 6H, CH3-iBu).
i
(m, 21H, Pr-TIPS).
(5′S)-5′,8-Cyclo-2′-deoxygu an osin e P h osph or am idite De-
r iva tive (18). The procedure adopted was analogous to that
described for the preparation of the phosphoramidite synthon
of (5′R)-CyclodAdo 10 (vide supra). After purification by silica
gel column chromatography, 18 was obtained as a white foam
(yield of 72%). FAB-MS (positive mode): m/z 838.1 ( 0.1 Da [M
+ H]+, 303.1 ( 0.1 Da [DMTr]+. 1H NMR (200 MHz, acetone-
d6, two diastereoisomers): δ 7.99-7.00 (m, 13H, aromatic H of
DMTr), 6.20 and 6.16 (d, J 1′2′′ ) 5.0 Hz, J 1′2′′ ) 4.7 Hz, 1H, H-1′),
5.25 and 5.22 (d, J 4′5′ ) 5.5 Hz, J 4′5′ ) 6.5 Hz, 1H, H-4′), 5.08-
4.96 (m, 1H, H-3′), 3.94 and 3.93 (s, 6H, CH3O-DMTr), 3.84-
3.65 (m, 4H, CH-iPr and CH2OP), 3.19 (d, 1H, H-5′), 3.12-2.55
(m, 5H, CH2CN, CH-iBu, H-2′ and H-2′′), 1.37 and 1.22 (m, 18H,
CH3-iPr and CH3-iBu). 31P NMR (101 MHz, acetone-d6): δ 152.4
and 150.5 (two diastereoisomers).
(5′R)-N2-Isobu tyr yl-3′-O-(tr iisop r op ylsilyl)-5′,8-cyclo-2′-
d eoxygu a n osin e (22). P r oced u r e A. Dimesyl derivative 21a
(0.178 g, 0.27 mmol) was dissolved in 10 mL of ethanol. The
resulting solution was warmed to 60 °C and slowly treated with
5 mL of water (to prevent precipitation). Powder NaOH (22 mg,
0.54 mmol) was added and the solution allowed to remain at
room temperature overnight (14 h) while being stirred. The
reaction was checked for completion by TLC (92/8 CHCl3/CH3-
OH, v/v) and then neutralized with 1 N HCl. After 5 min, the
mixture was evaporated to dryness. The resulting residue was
purified by chromatography on a silica gel (50 g) column with a
step gradient of methanol (0 to 6%) in chloroform as the mobile
phase. The appropriate fractions were pooled and then concen-
trated to dryness giving 95 mg (0.19 mmol) of (5′R)-N2-
isobutyryl-3′-O-(triisopropylsilyl)-5′,8-cyclo-2′-deoxyguanosine as
a white solid (yield of 70%). See above for the FAB-MS data
(positive mode). 1H NMR (200 MHz, methanol-d6): δ 6.48 (d,
J 1′2′′ ) 4.8 Hz, 1H, H-1′), 4.74 (s, 1H, H-5′), 4.67 (s, 1H, H-4′),
4.92-4.84 (q, J 2′3′ ) 7.3 Hz, J 2′3′′ ) 4.0 Hz, H-3′), 2.79 (m, 1H,
CH-iBu), 2.57 (dd, J 2′2′′ ) -13.5 Hz, 1H, H-2′), 2.21 (m, 1H,
N 2-Isob u t yr yl-3′-O-(t r iisop r op ylsilyl)-5′,8-cyclo-2′,5′-
d id eoxygu a n osin e (19). Compound 13 (1.14 g, 3.57 mmol) and
imidazole (1.95 g, 28.64 mmol) were dissolved in dry pyridine
(5 mL) and evaporated to dryness. The resulting residue was
dissolved in 15 mL of dry DMF, and 3 mL of TIPS-Cl (14.16
mmol) was added. The mixture was left at room temperature
for 14 h. The reaction was checked for completion by TLC (90/
10 CH2Cl2/CH3OH, v/v), and the mixture was cooled to 5 °C in
an ice bath. Then, 2 mL of pyridine/CH3OH (1/1) was added,
and after 10 min, the mixture was evaporated almost to dryness.
The resulting residue was then purified by chromatography on
a silica gel (75 g) column with a step gradient of methanol (0 to
4%) in dichloromethane as the mobile phase. The appropriate
fractions were pooled and then concentrated to dryness giving
1.57 g (3.32 mmol) of N2-isobutyryl-3′-O-(triisopropylsilyl)-5′,8-
cyclo-2′,5′-dideoxyguanosine as a white solid (yield of 93%). FAB-
i
H-2′′), 1.33 and 1.29 (s, 6H, CH3-iBu), 1.09 (m, 21H, Pr-TIPS).
P r oced u r e B (Mitsu n obu Ester ifica tion ). Compound 20
(0.180 g, 0.36 mmol) was dissolved in 5 mL of dry THF and
evaporated to dryness. The operation was repeated twice. The
resulting residue was dissolved in 10 mL of dry THF. Then,
PPh3 (0.377 g, 1.44 mmol), DEAD (157 mL, 1.44 mmol), and
acetic acid (45 mL, 0.72 mmol) were successively added to the
solution. The mixture was stirred at room temperature for 20
h. After a TLC check (92/8 CHCl3/CH3OH, v/v), the crude
acetylation mixture was concentrated to dryness. The resulting
red residue was purified by chromatography on a silica gel (50
g) column with a step gradient of methanol (0 to 3%) in
chloroform as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness giving 145 mg of (5′R)-
N2-isobutyryl-3′-O-(triisopropylsilyl)-5′-O-6-O-diacetyl-5′,8-cyclo-
2′-deoxyguanosine 21b as a yellow solid. This compound was
dissolved in 10 mL of dry THF, and 55 mg of CH3ONa (1.02
mmol) was added. After being stirred for 1 h and a TLC check
(92/8 CHCl3/CH3OH, v/v), the crude deacetylated mixture was
neutralized with 1 N HCl. After 5 min, the mixture was
evaporated to dryness. The resulting residue was purified by
chromatography on a silica gel (50 g) column with a step
gradient of methanol (0 to 6%) in chloroform as the mobile
phase. The appropriate fractions were pooled and then concen-
trated to dryness giving 113 mg (0.23 mmol) of (5′R)-N2-
isobutyryl-3′-O-(triisopropylsilyl)-5′,8-cyclo-2′-deoxyguanosine as
a white solid (yield of 64%).
MS (positive mode): m/z 498.0 ( 0.1 Da [M + Na]+, 476.0 (
1
0.1 Da [M + H]+. H NMR (200 MHz, CDCl3): δ 6.32 (d, J 1′2′′
)
5.1 Hz, 1H, H-1′), 4.76 (d, J 4′5′ ) 5.9 Hz, 1H, H-4′), 4.56 (m, 1H,
H-3′), 3.54 (dd, 1H, H-5′), 3.18 (d, J 5′5′′ ) -18.0 Hz, 1H, H-5′′),
2.83-2.62 (m, 2H, H-2′ and CH-iBu), 2.30 (m, 1H, H-2′′), 1.28
i
and 1.25 (s, 6H, CH3-iBu), 1.07-1.02 (m, 21H, Pr-TIPS).
(5′S)-N2-Isobu tyr yl-3′-O-(tr iisop r op ylsilyl)-5′,8-cyclo-2′-
d eoxygu a n osin e (20). The procedure described for TBDMS
ether 14 (see above) was applied. After purification by silica gel
column chromatography, the title compound 20 was obtained
as a white solid (yield of 35%). FAB-MS (positive mode): m/z
1
514.4 ( 0.1 Da [M + Na]+, 492.4 ( 0.1 Da [M + H]+. H NMR
(200 MHz, CDCl3): δ 6.29 (d, J 1′2′′ ) 4.4 Hz, 1H, H-1′), 5.37 (d,
J 4′5′ ) 6.5 Hz, 1H, H-5′), 4.95 (m, 1H, H-3′), 4.77 (d, 1H, H-4′),
2.91 (m, 1H, CH-iBu), 2.57 (dd, 1H, H-2′), 2.26 (m, 1H, H-2′′),
1.25 and 1.22 (d, J CH
iPr-TIPS).
) 4.7 Hz, 6H, CH3-iBu), 1.03 (m, 21H,
3CH
(5′R )-N 2-Isob u t yr yl-3′-O-(t r iisop r op ylsilyl)-5′-O-(4,4′-
(5′S)-N2-Isobu tyr yl-3′-O-(tr iisopr opylsilyl)-5′-O-6-O-dim e-
syl-5′,8-cyclo-2′-d eoxygu a n osin e (21a ). Compound 20 (0.130
g, 0.29 mmol) was dissolved in dry dichloromethane (5 mL) and
evaporated to dryness. The operation was repeated twice. The
resulting residue was taken up in 10 mL of dry dichloromethane.
Then, dry DIEA (404 µL, 2.32 mmol) was added followed by
dropwise addition of Ms-Cl (90 µL, 1.16 mmol). The mixture was
left at room temperature for 90 min. The reaction was checked
for completion by TLC (92/8 CHCl3/CH3OH, v/v), and the
mixture was cooled to 5 °C with an ice bath. Then, 0.5 mL of
methanol was added, and after 10 min, the mixture was
evaporated to dryness. The resulting residue was purified by
chromatography on a silica gel (50 g) column with a step
gradient of methanol (0 to 2.5%) in dichloromethane as the
mobile phase. The appropriate fractions were pooled and then
concentrated to dryness giving 0.178 g (0.27 mmol) of (5′S)-N2-
isobutyryl-3′-O-(triisopropylsilyl)-5′-O-6-O-dimesyl-5′,8-cyclo-2′-
d im eth oxytr ityl)-5′,8-cyclo-2′-d eoxygu a n osin e (23). The
procedure described for
8 (see above) was applied. After
purification by silica gel column chromatography, 23 was
obtained as a yellow foam (yield of 65%). FAB-MS (positive
mode): m/z 794.4 ( 0.1 Da [M + H]+, 303.1 ( 0.1 Da [DMTr]+.
1H NMR (200 MHz, CDCl3): δ 7.71-6.86 (m, 13H, aromatic H
of DMTr), 6.26 (d, J 1′2′′ ) 4.0 Hz, 1H, H-1′), 4.61 (s, 1H, H-4′),
3.93 (m, 1H, H-3′), 3.76 and 3.74 (s, 6H, CH3O-DMTr), 3.35 (s,
1H, H-5′), 2.36-1.87 (m, 3H, CH-iBu, H-2′ and H-2′′), 1.25 and
i
1.24 (s, 6H, CH3-iBu), 1.04 (m, 21H, Pr-TIPS).
(5′R)-N2-Isobu tyr yl-5′-O-(4,4′-d im eth oxytr ityl)-5′,8-cyclo-
2′-d eoxygu a n osin e (24). Compound 23 (0.1 g, 0.12 mmol) was
dissolved in 5 mL of dry THF. A solution of TBAF (0.25 mL,
0.25 mmol) in THF (1 M) was added, and the mixture was
stirred at room temperature for 14 h. The reaction was checked
for completion by TLC (95/5 CH2Cl2/CH3OH, v/v), and the
mixture was evaporated to dryness. The resulting residue was

416 Chem. Res. Toxicol., Vol. 12, No. 5, 1999
Romieu et al.
Sch em e 1. Syn th etic Rea ction s Used for th e P r ep a r a tion of th e P h osp h or a m id ite Syn th on of
(5′R)-Cyclod Ad o (2)
purified by chromatography on a silica gel (50 g) column with a
step gradient of methanol (0 to 3%) in dichloromethane/TEA
(99/1) as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness giving 57 mg (0.09
mmol) of (5′R)-N2-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-5′,8-cy-
clo-2′-deoxyguanosine as a white foam (yield of 75%). See above
for the FAB-MS data (positive mode). 1H NMR (200 MHz,
CDCl3): δ 7.71-6.86 (m, 13H, aromatic H of DMTr), 6.25 (d,
J 1′2′′ ) 4.0 Hz, 1H, H-1′), 4.71 (s, 1H, H-4′), 3.90 (m, 1H, H-3′),
3.77 and 3.75 (s, 6H, CH3O-DMTr), 3.15 (s, 1H, H-5′), 2.36-
1.87 (m, 3H, CH-iBu, H-2′ and H-2′′), 1.25 and 1.24 (s, 6H, CH3-
iBu).
were added, and the resulting mixture was incubated for an
additional 1 h at 37 °C. The mixture was taken up in 50 µL of
FA (25 mM, pH 6.2) and analyzed by reverse-phase HPLC with
the chromatographic system described above. The 2′-deoxyri-
bonucleosides were quantified by integration of the peak areas
at 260 nm. The molar extinction coefficients (260 nm, ×103)
reported in the literature for dCTP, dGTP, dATP, and dTTP
(14) were used: 7.30 (dCyd), 11.7 (dGuo), 8.80 (Thd), and 15.40
(dAdo).
Sn a k e Ven om P h osp h od iester a se (3′-Exo) a n d Ca lf
Sp leen P h osp h od iester a se (5′-Exo) Digestion s. The enzy-
matic digestions of oligomers 26-28 by exonucleases were
followed by MALDI-TOF mass spectrometry according to a
methodology that has been previously described (11, 15).
(5′R)-5′,8-Cyclo-2′-deoxygu an osin e P h osph or am idite De-
r iva tive (25). The procedure adopted was analogous to that
described for the preparation of the phosphoramidite synthon
of (5′R)-CyclodAdo 10 (see above). After purification by silica
gel column chromatography, 25 was obtained as a white foam
(yield of 78%). See above for the FAB-MS data (positive mode).
1H NMR (200 MHz, acetone-d6, two diastereoisomers): δ 7.64-
En zym a tic Digestion s of Oligom er s 27 a n d 28 w ith a
Com bin a tion of Exon u clea ses a n d En d on u clea ses. Puri-
fied oligomers 27 and 28 (0.5 AU260nm) were taken up in 50 µL
of Tris-HCl buffer (10 mM, pH 8.5), containing 10 mM MgCl2.
This solution was incubated with bovine pancreas DNase I (35
units), snake venom phosphodiesterase (3 × 10-3 unit), calf
spleen phosphodiesterase (8 × 10-3 unit), and alkaline phos-
phatase (2 units) for 24 h at 37 °C. The mixture was taken up
in 50 µL of FA (25 mM, pH 6.2) and analyzed by reverse-phase
HPLC with the chromatographic system described above.
6.96 (m, 13H, aromatic H of DMTr), 6.29 and 6.25 (d, J 1′2′′
)
5.0 Hz, J 1′2′′ ) 4.4 Hz, 1H, H-1′), 5.18 and 5.15 (s, 1H, H-4′),
4.84-4.72 (m, 1H, H-3′), 3.91 and 3.90 (s, 6H, CH3O-DMTr),
3.88-3.65 (m, 4H, CH-iPr and CH2OP), 3.15 (s, 1H, H-5′), 3.10-
2.53 (m, 5H, CH2CN, CH-iBu, H-2′ and H-2′′), 1.39-1.05 (m,
18H, CH3-iPr and CH3-iBu). 31P NMR (101 MHz, acetone-d6): δ
152.5 and 150.2 (two diastereoisomers).
Resu lts a n d Discu ssion
Solid -P h a se Syn th esis of Oligon u cleotid es. Oligonucle-
otides containing (5′R)-CyclodAdo 2, (5′S)-CyclodGuo 3, or (5′R)-
CyclodGuo 4 were prepared by means of phosphoramidite solid-
Previous studies have shown the compatibility of 5′,8-
cyclopurine 2′-deoxyribonucleosides with the reagents
used to chemically synthesize and deprotect oligonucle-
otides. In fact, treatments with 32% aqueous ammonia
(room temperature and 55 °C), 80% acetic acid and a
commercial oxidizing solution of iodine for 24 h did not
produce detectable degradation such as epimerization at
C-5′ of 5′,8-cyclopurine 2′-deoxyribonucleosides. The main
consequence of this high stability is the choice of the
standard amino-protecting groups for the modified and
the natural nucleosides: the benzoyl group (Bz) for dAdo
and CyclodAdo and the isobutyryl group (iBu) for dCyd,
dGuo, and CyclodGuo.
i
phase synthesis using the Bz group for dAdo and the Bu group
for dGuo and dCyd. Synthesized phosphoramidites 10, 18, and
25 were found to be insoluble in acetonitrile. Consequently, they
were dissolved in dry dichloromethane and placed in the
additional ports of an ABI model 392 DNA synthesizer. The
standard 1 µmol synthesis scale with retention of the 5′-terminal
DMTr group (trityl-on mode) was used with the modifications
previously described (11).
Dep r otection a n d P u r ifica tion of Oligon u cleotid es.
Following the synthesis, the solid supports were placed in
concentrated aqueous ammonia (32%) in sealed vials and heated
at 55 °C for 15-17 h. The crude 5′-DMTr oligomers were purified
and detritylated on-line by reverse-phase HPLC following the
previously reported method (11).
En zym a tic Digestion s of Oligom er s 26-31 by Nu clea se
P 1 a n d Alk a lin e P h osp h a ta se. Purified oligomers 26-31 (0.5
AU260nm) were taken up in 45 µL of water. Then, 5 µL of a
solution of nuclease P1 (1 unit/µL) in acetate buffer [30 mM
AcONa and 0.1 mM ZnSO4 (pH 5.5)] were added. The resulting
mixture was incubated at 37 °C for 26 h. Subsequently, 5 µL of
Tris-HCl buffer (10×, pH 9) and 2 units of alkaline phosphatase
P r ep a r a tion of th e P h osp h or a m id ite Syn th on of
(5′R)-5′,8-Cyclo-2′-d eoxya d en osin e. Phosphoramidite
synthon 10 of (5′R)-CyclodAdo was synthesized in five
steps from the known (5′S)-5′,8-cyclonucleoside 5 as
described in Scheme 1. The configuration at C-5′ was
inverted using the Corey method (16). This involves
mesylation followed by treatment with potassium super-

Oligonucleotides Containing 5′,8-Cyclopurine Nucleosides
Chem. Res. Toxicol., Vol. 12, No. 5, 1999 417
Sch em e 2. Syn th etic Rea ction s Used for th e P r ep a r a tion of th e P h osp h or a m id ite Syn th on s of
(5′S)-Cyclod Gu o (3) a n d (5′R)-Cyclod Gu o (4)
oxide (KO2) which gives the desired (5′R)-5′,8-cyclo-
nucleoside 7 in a moderate yield (54% from compound
5). Support for the 5′R stereoconfiguration of 7 was
provided by NMR analyses. The 1H NMR spectrum in
CDCl3 exhibited two broad singlets at 4.64 and 4.91 ppm
corresponding to H-4′ and H-5′, respectively. The low
phosphitylation (20) gave phosphoramidite synthon 10
in 80% yield after silica gel chromatography.
P r ep a r a tion of th e P h osp h or a m id ite Syn th on of
(5′S)-5′,8-Cyclo-2′-d eoxygu a n osin e. The synthesis of
the targeted phosphoramidite 18, required for the incor-
poration of (5′S)-CyclodGuo, started with N2-isobutyryl-
2′-deoxyguanosine 11 (13) (Scheme 2). Treatment of 11
with diphenyl disulfide and tributylphosphine (PBu3), in
fact the method of direct substitution of the 5′-OH group
with the phenylthio group reported by Nakagawa et al.
(21, 22), afforded thionucleoside 12 in 70% yield. The far-
UV irradiation of a deaerated acetonitrile solution of 12
in the presence of triethyl phosphite (POEt3), a trapping
agent for the released phenylthiyl radical (23), provided
two main products which were identified as N2-isobu-
tyrylguanine and N2-isobutyryl-5′,8-cyclo-2′,5′-dideox-
yguanosine 13. The 5′,8-cyclonucleoside was isolated by
silica gel chromatography (yield of 46%), and its structure
was confirmed by detailed measurements, including FAB
mass spectrometry in the positive mode (m/z 320.3 ( 0.1
3
magnitude (<1.0 Hz) of J 4′5′ is indicative of a diehedral
angle of ∼90° between these two protons that is compat-
ible with a 5′R stereoconfiguration. No detectable forma-
tion of the 5′S diastereoisomer was found to occur as
inferred from the survey of the reaction by TLC. How-
ever, we have observed a partial desilylation of 6 and 7
that explained the moderate yield of this step. This side
reaction of cleavage of TBDMS ethers by KO2 has been
already reported during the inversion of prostanoid
alcohols (17). Compound 7 was then converted into
DMTr-ether 8 (47% yield) by treatment with an excess
of 4,4′-dimethoxytrityl chloride (DMTr-Cl) in pyridine at
80 °C for 4 h (18). The tert-butyldimethylsilyl (TBDMS)
group was selectively removed by treating 8 with tet-
rabutylammonium fluoride (TBAF) in THF (19), yielding
free 3′-hydroxy derivative 9. A standard method of
1
Da) and NMR analyses. The H NMR spectrum of 13 in
DMSO-d6 showed the lack of the H-8 signal and the

418 Chem. Res. Toxicol., Vol. 12, No. 5, 1999
Ta ble 1. Sequ en ces a n d Molecu la r Ma sses of th e Syn th esized Mod ified ODNs
Romieu et al.
sequence (5′-3′)a
length
mass calcd (Da)
massb found (Da)
26
27
28
29
30
31
ATC GTG XCT GAT CT
ATC GTX ACT GAT CT
ATC GTX ACT GAT CT
CAC TTC GGX TCG TGA CTG ATC T
CAC TTC GXA TCG TGA CTG ATC T
CAC TTC GXA TCG TGA CTG ATC T
14
14
14
22
22
22
4251.80
4251.80
4251.80
6699.40
6699.40
6699.40
4249.26 ( 1.27
4250.50 ( 1.43
4250.63 ( 0.75
6697.51 ( 2.46
6698.15 ( 1.98
6698.84 ( 1.21
a
b
X is (5′R)-CycloA for 26 and 29, (5′S)-CycloG for 27 and 30, and (5′R)-CycloG for 28 and 31. All the oligonucleotide masses were
obtained by ESI-MS in the negative mode.
presence of an ABX system at 3.03, 3.39, and 4.75 ppm
for H-5′′, H-5′, and H-4′, respectively, with the following
3
coupling constants: 2J 5′5′′ ) -17.7 Hz and J 4′5′ ) 6.5 Hz.
3J couplings J 4′5′′, J 3′4′, and J 1′2′ had low magnitudes (<1.0
Hz). The results are consistent with the expected rigid
constrained structure of cyclonucleoside 13 (24). Prior to
the functionalization of C-5′, the 3′-OH residue of 13 was
protected with a TBDMS group. This was accomplished
by treating 13 with TBDMS-Cl and imidazole in DMF
for 24 h at room temperature (19). The expected TBDMS
ether 14 was isolated in 73% yield by silica gel chroma-
tography. Sequential treatment of 14 with selenium oxide
(SeO2) in refluxing 1,4-dioxane and NaBH4 in methanol
(12) gave the (5′S)-5′,8-cyclonucleoside 15 stereoselec-
tively in 42% yield. Thereafter, the (5′S)-5′,8-cyclonucleo-
side 15 was essentially treated as (5′R)-CyclodAdo (vide
supra). Dimethoxytritylation, desilylation, and phosphi-
tylation afforded phosphoramidite synthon 18.
P r ep a r a tion of th e P h osp h or a m id ite Syn th on of
(5′R)-8,5′-Cyclo-2′-d eoxygu a n osin e. The synthesis of
the (5′R)-CyclodGuo phosphoramidite derivative began
with TIPS ether 20 (25) (Scheme 2). Attempts to invert
the C-5′ stereoconfiguration of 20 using the Corey
methodology were unsuccessful. Indeed, the treatment
of dimesyl derivative 21a with KO2 led to the formation
of numerous nonsilylated and unidentified products.
However, alcohol transposition mediated by other oxy-
genated nucleophiles proved to be more successful. Thus,
when 21a was treated with an aqueous solution of NaOH
in ethanol for 14 h (26, 27), the desired inverted alcohol
22 was obtained in good yield. Due to the high stability
of TIPS ethers in alkali media (28), no desilylation side
reactions were observed. Furthermore, the (5′R)-alcohol
can be also obtained by using the Mitsunobu acetylation
method (29) followed by transesterification with sodium
methylate in dry THF (30). Thereafter, (5′R)-5′,8-cyclo-
nucleoside 22 was converted into the 5′-O-DMTr-protect-
ed derivative 23 and finally transformed into the corre-
sponding 3′-O-phosphoramidite building block 25, after
removal of the TIPS group, according to the synthetic
procedures described above for (5′R)-CyclodAdo.
Solid -P h a se Syn t h esis a n d Ch a r a ct er iza t ion of
Oligod eoxyn u cleotid es Th a t Con ta in th e 5′,8-Cy-
clop u r in e 2′-Deoxyr ibon u cleosid es. The incorpora-
tion of 10, 18, and 25 into DNA fragments (Table 1) was
carried out by automated synthesis and proceeded with
a good coupling efficiency (>90%). For deprotection and
isolation of the modified oligomers, we followed the
procedures used for oligonucleotides containing (5′S)-
CyclodAdo residues (11). The determination of the mo-
lecular masses of the modified ODNs was accomplished
by electrospray mass spectrometry (Table 1). These
results confirmed the incorporation of 5′,8-cyclopurine-
2′-deoxyribonucleosides 2-4 and the purity of the oligo-
mers. Further support for the presence of 2-4 in ODNs
F igu r e 1. Reverse-phase HPLC analysis of the enzymatic
digestion mixture of 14-mers 26-28 with nuclease P1 (incuba-
tion for 26 h) and alkaline phosphatase (incubation for 1 h).
was provided by base composition analysis. When modi-
fied 14-mers 26-28 were subjected to enzymatic diges-
tions with nuclease P1 and bacterial alkaline phos-
phatase, the HPLC elution profiles of the resulting
enzymatic digestion mixtures (Figure 1) showed the
presence of 5′,8-cyclonucleosides 2-4 at the expected
retention times and in the correct ratio with respect to
the 13 normal 2′-deoxyribonucleosides. The minor peaks
in the HPLC chromatogramms eluting between dGuo and
Thd and just before (5′S)-CyclodGuo 3 (Figure 1, top and
middle, respectively) correspond to dIno which could
result from the enzymatic hydrolytic deamination of dAdo
and probably to a phosphorylated derivative of 3. These
impurities were not present in synthetic modified ODNs
26 and 27 but were produced during the enzymatic
digestions. The structure and integrity of the released
2-4 from the modified ODNs were further confirmed by
co-injection with standards and by electrospray mass
spectrometric measurements of the collected peaks.
However, the hydrolysis of the phosphodiester bonds
between 2-4 and normal 2′-deoxyribonucleosides is much
slower than that of the corresponding nonmodified ODNs,
requiring 26 h of incubation with nuclease P1. Such
results suggest a partial inhibition of the endonuclease
activity by the lesion in agreement with previously
reported data involving short ODNs containing other free
radical- and singlet oxygen-induced base modifications
(31-33). To obtain more information about the effect of

Oligonucleotides Containing 5′,8-Cyclopurine Nucleosides
Chem. Res. Toxicol., Vol. 12, No. 5, 1999 419
F igu r e 2. MALDI-TOF mass spectra, in the positive mode, of partial 5′-exo enzymatic digestion of 14-mer 27: (a) incubation for 1
min, (b) incubation for 8 min, and (c) incubation for 180 min.
specific tandem DNA lesions 2-4 on the behavior of
nucleases, we have also studied the stability of the
modified ODNs toward exonucleases. The enzymatic
digestions of 27 and 28 by snake venom phosphodi-
esterase (3′-exo) and calf spleen phosphodiesterase (5′-
exo) were followed by MALDI-TOF mass spectrometry
according to the methodology developed by Pieles et al.
(15). Snake venom phosphodiesterase sequentially de-
F igu r e 3. Reverse-phase HPLC analysis of the enzymatic
graded oligonucleotide 27 from the 3′-end until it became
digestion mixture of 14-mer 27 with DNase I, snake venom
phosphodiesterase, calf spleen phosphodiesterase, and alkaline
(5′S)-CyclodGuo 3, which is resistant to further cleavage.
Indeed, the mass spectrum after 3 h of 3′-exo enzymatic
digestion (data not shown) exhibited a single peak at m/z
1805.69 ( 0.1 Da, corresponding to the positive ion [M
+ H]+ of the 6-mer [5′-d(ATC GTX)-3′] 32 (calcd 1805.18
Da). A different behavior was observed in the case of the
enzymatic digestion with calf spleen phosphodiesterase,
which starts from the opposite end (Figure 2). Within 1
min of incubation, the enzyme induced the release of the
first four nucleotides. Thereafter, the presence of modified
nucleoside 3 dramatically reduced the rate of digestion.
The mass spectrum of the enzymatic hydrolysate, after
3 h of incubation, exhibited a single peak at m/z 3014.63
( 0.1 Da corresponding to the positive ion [M + H]+ of
the 10-mer [5′-d(TX ACT GAT CT)-3′] 33 (calcd 3015.94
Da). Thus, calf spleen phosphodiesterase failed to cleave
the phosphodiester linkages between 3 and Thd and
dAdo. Consequently, the modified ODNs could not be
fully digested to the monomer level by a combination of
3′-exo- and 5′-exonucleases. Similar results were obtained
when oligomers 27 and 28 were submitted to the enzyme
mixture (pancreatic deoxyribose DNase I, snake venom
phosphodiesterase, calf spleen phosphodiesterase, and
bacterial alkaline phosphatase) recommended by Diz-
daroglu et al. (6) for the hydrolysis of γ-irradiated DNA.
After incubation for 24 h, the resulting mixture of
phosphatase (incubation for 24 h).
2′-deoxyribonucleosides which was analyzed by reverse-
phase HPLC provided dCyd, dGuo, Thd, dAdo, and dimer
[5′-d(TX)-3′] 34 in a 3/2/4/3/1 ratio (Figure 3). The
structure of 34 was confirmed by electrospray mass
measurement of the collected peak (calcd 569.41 Da,
found 568.98 ( 0.1 Da). The presence of this modified
dinucleoside monophosphate is additional proof of the
high stability of phosphodiester linkages between Cy-
clodGuo and normal 2′-deoxyribonucleosides.
In addition to these enzymatic studies, preliminary
repair experiments (data not shown) showed that 5′,8-
cyclo-2′-deoxyribonucleosides 1-4 were not excised by
formamidopyrimidine DNA N-glycosylase (Fpg) and en-
donuclease III (Endo III), repair enzymes specific for
oxidized purines (34, 35), and hydrated, oxidized, or ring-
fragmentation pyrimidines (36-40), respectively. Further
experiments with other DNA repair enzymatic systems
such Uvr ABC protein complex, which removed bulky
adducts and pyrimidine dimers by a nucleotide excision
repair process (NER) (41), should provide further infor-
mation about the repair mechanisms of these tandem
DNA lesions. To elucidate correlations between the
properties toward enzymatic hydrolysis, of the modified

420 Chem. Res. Toxicol., Vol. 12, No. 5, 1999
Romieu et al.
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Con clu sion a n d P er sp ectives
We have developed a convenient and efficient method
for the synthesis of 5′,8-cyclonucleosides containing oli-
gonucleotides. These modified DNA fragments are suit-
able probes for further investigations of the structural
and biochemical effects of cross-link formation between
the base and 2-deoxyribose moieties. Furthermore, an
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Ack n ow led gm en t. The contributions of Colette Leb-
run (SCIB/LRI/CEA, Grenoble, France), Daniel Ruffieux
(Biochimie C/CHRU, Grenoble, France), and Michel
J aquinod (LSMP/IBS/CEA/CNRS, Grenoble, France) in
obtaining the mass spectrometric measurements are
gratefully acknowledged. This work was supported in
part by grants from the French Ministry of Science and
Research (ACC-SV Grant 8-MESR 1995) and Electricite´
de France (Comite´ de Radioprotection).
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