Site-Specific Introduction of (5'S)-5',8-Cyclo-2'-deoxyadenosine into Oligodeoxyribonucleotides

Full text of DOI:10.1021/jo980083q

J . Org. Chem. 1998, 63, 5245-5249
5245
ization at C-5′ of CyclodAdo was observed after 24 h of
incubation in the three reactional solutions.
Site-Sp ecific In tr od u ction of
(5′S)-5′,8-Cyclo-2′-d eoxya d en osin e in to
Oligod eoxyr ibon u cleotid es
The preparation of the phosphoramidite synthon 12
has required the development of an efficient method for
the synthesis of CyclodAdo. The above-mentioned pho-
tochemical method for the synthesis of CyclodAdo is not
applicable to preparative purpose. In particular, the
yield (roughly 15% for the two diastereomers) was rather
unsatisfactory for the purpose of a large scale prepara-
tion. Due to the unusual structure of cyclonucleosides
with two secondary alcohol functions within the sugar
moiety, the selective introduction of the 4,4′-dimethoxy-
trityl (DMTr) group on the 5′-OH residue represents a
difficult challenge. This has to be taken into consider-
ation in the strategy adopted for the synthesis of Cy-
clodAdo. A survey of the literature revealed a variety of
chemical and photochemical methods for the preparation
of cyclonucleosides, particularly in the RNA series.5 The
methodology developed by Matsuda et al.6,7 for the
synthesis of (5′S)-5′,8-cyclopurine nucleosides was chosen.
In fact, the radical intramolecular cyclization proceeded
in good yields. In addition, the postfunctionalization of
C-5′ by the oxidation-reduction procedure worked out
the issue of the selective introduction of the DMTr group.
The synthesis of the targeted phosphoramidite 12 (Scheme
1) required for the incorporation of compound 2, started
with N6-benzoyl-2′-deoxyadenosine 3.8 The latter nucleo-
side was converted into the 5′-thiophenyl derivative 5 via
the tosyl precursor 4.9 This was accomplished by treating
3 with tosyl chloride in pyridine at -20 °C. Compound
4 was then reacted with thiophenol in the presence of
sodium methylate under refluxing methanol, giving
nucleoside 5 in a moderate yield (42% from compound
3). The key intermediate, N6-benzoyl-5′,8-cyclo-2′,5′-
dideoxyadenosine 6, was synthesized by far-UV irradia-
tion of a deaerated acetonitrile solution of 5 in the
presence of 10 equiv of triethyl phosphite, a trapping
agent for the released phenylthiyl radical.10 After 10 h
of irradiation, which led to the complete degradation of
5, the anhydronucleoside 6 was obtained by crystalliza-
tion from hot methanol in 51% yield. To avoid side
reactions from occurring during the oxidation step and
the reaction with DMTr chloride during the tritylation
step, the 3′-OH residue was protected with a tert-
butyldimethylsilyl (TBDMS) group.11 Thus, 6 was treated
with TBDMS chloride in DMF in the presence of imida-
zole for 20 h at room temperature. The expected TBDMS
ether 7 was isolated in 79% yield by silica gel chroma-
Anthony Romieu, Didier Gasparutto, Didier Molko, and
J ean Cadet*
De´partement de Recherche Fondamentale sur la Matie`re
Condense´e, SCIB/ Laboratoire des le´sions des Acides
Nucle´iques, CEA Grenoble 17, avenue des Martyrs/ F-38054
Grenoble Cedex 9 France
Received J anuary 16, 1998 (Revised Manuscript Received April
24, 1998)
5′,8-Cyclo-2′-deoxyadenosine (CyclodAdo) is one of the
main radiation-induced decomposition products of 2′-
deoxyadenosine (dAdo).1 The formation of CyclodAdo is
explained in terms of intramolecular attack of the C-8,
N-7 double bond of the purine base by the C-5′ carbon
•
radical, produced by the action of OH radicals formed
by the radiolysis of water.2 The reaction is highly
stereoselective since only the (5′R)-diastereomer 1 is
generated, at least, in detectable amount. On the other
hand, the radiation-induced decomposition of 5′-AMP led
to the (5′S)-diastereomer.2,3 CyclodAdo, which may be
considered as a tandem DNA lesion, the 2-deoxyribose
and the adenine residues being both altered, is likely to
have a significant biological impact. To assess the
conformational changes and the biochemical features
associated with the formation of CyclodAdo in DNA, it
is a requisite to prepare oligonucleotides that contain this
double lesion at defined sites.
We report herein the first site-specific incorporation
of (5′S)-CyclodAdo 2 into oligonucleotides using the
phosphoramidite chemistry. Prior to the preparation of
the phosphoramidite synthon 12, the stability of Cy-
clodAdo was checked at room temperature under the
three main experimental conditions used during the solid
support synthesis. These include treatments with 32%
aqueous ammonia, 80% acetic acid, and a commercial
oxidizing solution of iodine. Authentic samples of (5′R)-
CyclodAdo 1 and (5′S)-CyclodAdo 2, obtained by photo-
reaction of a deaerated aqueous solution of 8-bromo-2′-
deoxyadenosine at 254 nm in 12% and 2% yields,
respectively, were used for these stability studies.4 Ali-
quots of the reaction mixtures were taken up at increas-
ing periods of time and analyzed by reverse phase HPLC
(system B). No detectable degradation including epimer-
(5) For examples: (a) Harper, P. J .; Hampton, A. J . Org. Chem.
1972, 37, 795-797. (b) Duong, K. N. V.; Gaudemer, A.; J ohnson, M.
D.; Quillivic, R.; Zybler. J . Tetrahedron Lett. 1975, 34, 2997-3000. (c)
Matsuda, A.; Tezuka, M.; Ueda, T. Tetrahedron 1978, 34, 2449-2452.
(d) Sugawara, T.; Otter, B. A.; Ueda, T. Tetrahedron Lett. 1988, 29,
75-78. (e) Haraguchi, K.; Tanaka, H.; Saito, S.; Kinoshima, S.; Hosoe,
M.; Kanmuri, K.; Yamaguchi, K.; Miyasaka, T. Tetrahedron 1996, 52,
9469-9484.
(6) Matsuda, A.; Muneyama, K.; Nishida, T.; Sato, T.; Ueda, T.
Nucleic Acids Res. 1976, 3, 3349-3357.
* To whom correspondence should be addressed. Phone: (33) 4-76-
88-49-87. Fax: (33) 4-76-88-50-90. E-mail: cadet@drfmc.ceng.cea.fr.
(1) Mariaggi, N.; Cadet, J .; Te´oule, R. Tetrahedron 1976, 32, 2385-
2387.
(2) Cadet, J .; Berger, M. Int. J . Radiat. Biol. 1985, 47, 127-143.
(3) Raleigh, J . A.; Fuciarelli, A. F. Radiat. Res. 1985, 102, 165-
175.
(7) Matsuda, A.; Tezuka, M.; Niizuma, K. Sugiyama, E.; Ueda, T.
Tetrahedron 1978, 34, 2633-2637.
(8) Ti, G. S.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1982,
104, 1316-1319.
(9) Kuhn, R.; J ahn, W. Chem. Ber. 1965, 98, 1699-1704.
(10) Walling, C.; Rabinowitz, R. J . Am. Chem. Soc. 1959, 81, 1243-
1249.
(4) Combat, F.; Berger, M.; Cadet, J . Unpublished results.
(11) Ogilvie, K. K. Can. J . Chem. 1973, 51, 3799-3807.
S0022-3263(98)00083-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/07/1998

5246 J . Org. Chem., Vol. 63, No. 15, 1998
Notes
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′S)-Cyclod Ad oa
ylated on the HPLC column, and again purified by
reverse phase HPLC (system C). The purity and homo-
geneity of the collected fractions were controlled by HPLC
(system D) and gel electrophoresis. Respectively, 16.5
AU260
and 18 AU260 nm of purified oligomers 13 and 14
nm
were obtained. The molecular weight of the oligonucle-
otides 13 and 14 was inferred from electrospray mass
spectrometry analyses in the negative mode (calcd:
4251.80 Da, found: 4250.50 ( 0.62 Da, 13; and calcd:
6699.40 Da, found 6697.61 ( 1.53 Da, 14). These results
confirmed the incorporation of (5′S)-CyclodAdo 2 and the
purity of the oligomers 13, 14. A fraction of 13 was
enzymatically hydrolyzed with nuclease P1, followed by
bacterial alkaline phosphatase. The resulting mixture
of 2′-deoxyribonucleosides was analyzed by reverse phase
HPLC (system E) provided dCyd, dGuo, Thd, dAdo, and
a trimer [5′-d(GXC)-3′] 15 in a 2:2:5:2:1 ratio, confirming
the structure of the modified oligonucleotide (Figure 1a).
The lack of the free modified nucleoside 2 received further
confirmation by means of coinjection of the enzymatic
digestion with an authentic sample of (5′S)-CyclodAdo 2
and HPLC analysis (Figure 1b). The structure of 15 was
confirmed by electrospray mass measurement of the
collected peak (calcd 867.60 Da, found 867.60 ( 0.1 Da).
Further treatment of 15 with nuclease P1 and alkaline
phosphatase did not provide the free 2′-deoxyribonucleo-
sides dCyd, dGuo, and (5′S)-CyclodAdo 2, even after 24
h of incubation. Due to the occurrence of significant
conformational changes associated with the presence of
2, phosphodiester linkages with dCyd and dGuo were not
hydrolyzed by nuclease P1; therefore, the modified nu-
cleoside 2 was not released in the enzymatic hydrolysate.
The high stability of phosphodiester linkages between 2
and normal 2′-deoxyribonucleosides has found further
confirmation with the use of other enzymes. The enzy-
matic digestions of 13 by snake venom phosphodiesterase
(3′-exo) and calf spleen phosphodiesterase (5′-exo) were
followed by MALDI-TOF mass spectrometry.15 To obtain
an optimal distribution of cleavage products for the
oligonucleotide 13 and a given batch of enzyme, aliquots
were removed from the digestion reactions at increasing
time intervals and directly analyzed by MALDI-TOF
mass spectrometry. The mass spectrum after 2 h of
enzymatic digestion with 3′-exo exhibited a single peak
at m/z ) 2119.50 ( 0.1 Da. This peak corresponds to
the 7-mer [5′-d(ATC GTG X)-3′] 16 (calcd 2118.4 Da).
Interestingly, the latter modified 7-mer is not affected
by prolonged enzymatic treatment. The snake venom
phosphodiesterase sequentially degrades the oligonucle-
otide 13 from the 3′-end until it reaches the (5′S)-
CyclodAdo 2 that resists further cleavage. On the other
hand, the treatment of 13 with 5′-exo provided the 9-mer
[5′-(GXC TGA TCT)-3′] 17 (calcd 2711.80 Da). Indeed,
the mass spectrum after 2 h of enzymatic digestion with
5′-exo exhibited a single peak at m/z ) 2710.86 ( 0.1 Da.
The calf spleen phosphodiesterase induced the release
of the first five nucleotides at the 5′-end of the oligo-
nucleotide sequence but failed to cleave phosphodiester
linkages between 2 and dGuo and dCyd, even after
prolonged treatment. With respect to the possible de-
stabilizing structural effect of (5′S)-CyclodAdo 2, the
thermal stability of a (5′S)-CycloA-T base pair has been
studied. The UV melting behavior of oligomer 13 was
a
(a) TsCl (1.2 equiv), pyridine, -20 °C, 24 h, 82%; (b) PhSH
(2.3 equiv), CH3ONa (1.5 equiv), CH3OH, reflux, 2 h, 51%; (c) λ )
254 nm, (EtO)3P (10 equiv), CH3CN, argon, 10 h, 51%; (d)
TBDMSCl (2 equiv), imidazole (4.4 equiv), DMF, 20 h, 79%; (e)
SeO2 (2 equiv), 1,4-dioxane, reflux, 30 min, 87%; (f) NaBH4 (2
equiv), CH3OH, 1 h, 90%; (g) DMTrCl (2 equiv), pyridine, 70 °C,
4 h, 60%; (h) TBAF (2 equiv), THF, 4 h, 72%; (i) P(N-iPr2)2-
O(CH2)2CN (1.5 equiv), N,N′-diisopropylammonium tetrazolate
(0.5 equiv), CH2Cl2, argon, 4 h, 62%.
tography. Sequential treatment of 7 with selenium oxide
in refluxing 1,4-dioxane and NaBH4 in methanol gave
the (5′S)-5′,8-cyclonucleoside 9 stereoselectively and in
high yield (78% from compound 7). Compound 9 was
then converted into the DMTr-ether 10 (60% yield) by
treatment with an excess of DMTr chloride in pyridine
at 70 °C for 4 h.12 Further treatment of compound 10
with a 1 M solution of tetrabutylammonium fluoride
(TBAF) in THF then afforded the free 3′-hydroxyl deriva-
tive 11 in 72% yield after silica gel chromatography.13
Thereafter, the targeted phosphoramidite 12 was ob-
tained from the alcohol 11 in 62% yield using a standard
method of phosphitylation.14
Two oligodeoxynucleotides, namely [5′-d(ATC GTG
XCT GAT CT)-3′)] (14-mer, 13) and [5′-d(CAC TTC GGX
TCG TGA CTG ATC T)-3′] (22-mer, 14), where X ) (5′S)-
CycloA 2, were then prepared following the usual phos-
phoramidite protocol (1 µmol scale). However, the du-
ration of the condensation of the modified monomer 12
was increased by a factor of 2 compared with that of
standard monomers. Furthermore, the duration of the
coupling of the next nucleoside involved in the oligo-
nucleotide sequences (2′-deoxyguanosine, dGuo) was also
increased (330 s instead of 30 s for normal nucleoside
phosphoramidites). This allowed a coupling efficiency of
more than 85%. After standard deprotection with aque-
ous ammonia at 55 °C for 17 h, the crude 5′-DMTr-
oligomers were purified by reverse phase HPLC, detrit-
(12) Sekine, M.; Hata, T. J . Org. Chem. 1983, 48, 3011-3014.
(13) Corey, E. J .; Venkateswarlu, A.J . Am. Chem. Soc. 1972, 94,
6190-6191.
(14) Barone, A. D.; Tang, J .-Y.; Caruthers, M. H. Nucleic Acids Res.
1984, 12, 4051-4061.
(15) Pieles, U.; Zu¨rcher, W.; Scha¨r, M.; Moser, H. E. Nucleic Acids
Res. 1993, 21, 3191-3196.

Notes
J . Org. Chem., Vol. 63, No. 15, 1998 5247
F igu r e 1. (a) HPLC elution profile of the enzymatic digestion mixture (14-mer, 13). (b) HPLC elution profile of (5′S)-CyclodAdo
and the enzymatic digestion mixture (14-mer, 13).
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. Several chromatographic systems were used for the
analytical experiments and the purification steps System A
semipreparative reverse phase HPLC (Macherey-Nagel Nucleosil
C18 column, 7 µm, 21 × 250 mm) with acetonitrile and triethyl-
ammonium acetate buffer (TEAA, 25 mM, pH ) 7) as the eluents
[100% TEAA (4 min), linear gradient from 0 to 30% of aceto-
nitrile (50 min)] at a flow rate of 4 mL/min. The detection was
achieved at 268 nm System B: reverse phase HPLC (Merck
LiChrocart LiChrospher 100RP-18e column, 5 µm, 4 × 125 mm)
with acetonitrile and TEAA (25 mM, pH ) 7) as the eluents
[100% TEAA (10 min), linear gradient from 0 to 30% of
acetonitrile (35 min)] at a flow rate of 1 mL/min. The detection
was achieved at 268 nm. System C: purification of 5′-DMTr-
oligomers by reverse phase HPLC (Hamilton PRP3, polymeric
phase, 10 µm, 7 × 305 mm) with acetonitrile and TEAA (25 mM,
pH ) 7) as the eluents using a nonlinear gradient [100% TEAA
(5 min), then isocratic TEAA/acetonitrile [92/8] v/v (10 min)] to
remove the DMTr group, the oligomers were treated with an
1% aqueous solution of trifluoroacetic acid [isocratic 100% TFA
(1%) (6 min)]. The resulting deprotected oligomers were again
purified using the same eluents [gradient from 2 to 10% of
acetonitrile (24 min)]. The flow rate was 2.5 mL/min and the
detection was achieved at 254 nm. System D: reverse phase
HPLC (Hypersil C18, 5 µm, 4.6 × 250 mm) with acetonitrile and
(TEAA, 25 mM, pH ) 7) as the eluents [100% TEAA (2 min),
linear gradient from 0 to 12% of acetonitrile (25 min)] at a flow
rate of 1 mL/min. The detection was achieved at 260 nm.
System E: system D with a new gradient of acetonitrile and
TEAA (25 mM, pH ) 7) [100% TEAA (10 min), linear gradient
from 0 to 10% of acetonitrile (30 min)].
(5′R)-5′,8-Cyclo-2′-d eoxya d en osin e (1). 8-Bromo-2′-deoxy-
adenosine17 (30 mg, 0.09 mmol) was dissolved in 100 mL of water
and the resulting solution was deaerated by a stream of argon
for 45 min. The solution was irradiated at 254 nm for 12 h,
under an argon atmosphere, in a quartz reactor. Then, the
reaction mixture was concentrated to dryness. The resulting
residue was taken up in 5 mL of water and purified by
semipreparative reverse phase HPLC (system A). The appropri-
ate fraction (38 min < tR < 40 min) was lyophilized, giving 2.7
mg of (5′R)-5′,8-cyclo-2′-deoxyadenosine as a white powder (yield
12%). FAB-MS (positive mode): m/z [M + H]+ ) 250.2 ( 0.1
Da. 1H NMR (200.13 MHz, D2O) δ: 8.28 (s, 1H, H-2′); 6.67 (d,
J 1′2′′ ) 4.9 Hz, H-1′); 4.99 (s, 1H, H-5′); 4.85 (s, 1H, H-4′); 4.56
(q, J 3′2′ ) 7.4 Hz, J 3′2′′ ) 3.9 Hz, 1H, H-3′); 2.69 (q, J 2′2′′ ) -13.9
Hz, 1H, H-2′); 2.31 (m, 1H, H-2′′).
compared with that of the corresponding parent dAdo
containing oligomer. The melting temperature of the
duplex that contained (5′S)-CyclodAdo 2 (Tm ) 48 ( 1
°C) was lower than that of the unmodified duplex (Tm
)
54 ( 1 °C). The noticeable decrease in the melting
temperature (∆Tm ) 6 °C) suggests that the incorporation
of (5′S)-CyclodAdo 2 significantly induces a local desta-
bilization of the duplex DNA structure.
Con clu sion . The synthesis reported herein provides
a facile method for the preparation of oligonucleotides
containing (5′S)-CyclodAdo 2 at specific positions. We
have also demonstrated the high stability of phosphodi-
ester linkages, between 2 and normal 2′-deoxyribonucleo-
sides, toward the enzymatic hydrolysis and that the (5′S)-
CycloA-T base pair is less stable than the A-T base pair.
These modified DNA fragments are suitable for further
studies aimed at determining both the biochemical (mu-
tagenesis, repair) and conformational features of (5′S)-
CyclodAdo 2 into DNA fragments. Furthermore, this
versatile synthetic methodology can be used for the
incorporation into oligonucleotides of other purine cyclo-
nucleosides.16
Exp er im en ta l Section
Gen er a l. The photoreactions at 254 nm were performed with
a Rayonet photochemical reactor (14x15W). 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. Nuclease P1
(Penicilium citrinium) and 0.3 M sodium acetate/ZnCl2 buffer
(pH ) 5.3) were purchased from Sigma (St Louis, MO). Alkaline
phosphatase, snake venom phosphodiesterase, calf spleen phos-
phodiesterase and 1 M Tris-HCl/MgCl2/ZnCl2 buffer (10×, pH
) 9) were obtained from Boehringer Mannheim (Mannheim,
Germany). Anhydrous solvents were purchased from SDS. The
HPLC-grade solvents (acetonitrile and methanol) were obtained
from Carlo Erba (Milan, Italy). Buffers for high performance
liquid chromatography (HPLC) were prepared using water
purified with a Milli-Q system (Milford, MA).
(16) Several oligonucleotides containing (5′R)-CyclodAdo 1 and the
(5′R)- and (5′S)-diastereomers of 5′,8-cyclo-2′-deoxyguanosine are in
preparation.
(17) Wood, M. L.; Esteve, A.; Morningstar, M. L.; Kuziemko, G. M.
Essigmann, J . M. Nucleic Acids Res. 1992, 20, 6023-6032.

5248 J . Org. Chem., Vol. 63, No. 15, 1998
Notes
(5′S)-5′,8-Cyclo-2′-d eoxya d en osin e (2). A second fraction
(44 min < tR < 47 min) was collected and then lyophilized, giving
0.5 mg of (5′S)-5′,8-cyclo-2′-deoxyadenosine as a white powder
(yield 2%). 1H NMR (200.13 MHz, D2O) δ: 8.26 (s,1H, H-2′);
6.53 (d, J 1′2′′ ) 4.8 Hz, H-1′); 5.45 (d, J 5′4′ ) 6.1 Hz, 1H, H-5′);
4.92 (d, 1H, H-4′); 4.84 (q, J 3′2′ ) 7.6 Hz, J 3′2′′ ) 4.2 Hz, 1H, H-3′);
2.77 (q, J 2′2′′ ) -13.9 Hz, 1H, H-2′); 2.35 (m, 1H, H-2′′).
silyl chloride (0.481 g, 3.19 mmol) was added. The mixture was
left at room temperature for 20 h. The reaction was checked
for completion by TLC (CHCl3/CH3OH, 90/10 v/v) and the
mixture was cooled at 5 °C. Then, water (5 mL) was added and
after 10 min, the mixture was evaporated to almost dryness.
The residue was taken up in chloroform (40 mL) and washed
with 5% NaHCO3 (30 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 (0-4%) in chloroform as the
mobile phase. The appropriate fractions were pooled and then
N6-Ben zoyl-5′-O-tosyl-2′-d eoxya d en osin e (4). N6-Benzoyl-
2′-deoxyadenosine8 3 (4 g, 11.25 mmol) was dissolved in dry
pyridine (10 mL) and evaporated to dryness. The resulting white
foam was dissolved in 40 mL of dry pyridine and cooled at -20
°C. Then, tosyl chloride (2.59 g, 13.58 mmol) was added and
the mixture was left at -20 °C for 24 h. The reaction was
checked for completion by TLC (CHCl3/CH3OH, 85/15 v/v).
Water (20 mL) was added, and after 10 min, the mixture was
evaporated to almost dryness. The residue was dissolved in 75
mL of dichloromethane and washed with a saturated solution
of NaCl (80 mL). Then, the organic layer was dried over Na2-
SO4 and evaporated to dryness. The resulting residue was
purified by chromatography on a silica gel (150 g) column with
a step gradient of methanol (0-4%) in chloroform as the mobile
phase. The appropriate fractions were pooled and then concen-
trated to dryness, giving 4.71 g (9.24 mmol) of N6-benzoyl-5′-
tosyl-2′-deoxyadenosine as a white foam (yield 82%). FAB-MS
concentrated to dryness, giving 0.477
g (1.06 mmol) of
N6-benzoyl-3′-O-(tert-butyldimethylsilyl)-5′,8-cyclo-2′,5′-dideoxy-
adenosine as
a yellow foam (yield 79%). EI-MS (positive
mode): m/z [M +•] ) 451.0 ( 0.1 Da. 1H NMR (200.13 MHz,
DMSO-d6) δ: 11.22 (bs, 1H, NH-Bz); 8.77 (s, 1H, H-2); 8.16-
7.62 (m, 5H, aromatic-H of Bz); 6.77 (d, J 1′2′′ ) 4.8 Hz, 1H, H-1′);
4.82 (d, J 4′5′ ) 6.0 Hz, 1H, H-4′); 4.68 (q, J 3′2′ ) 7.0 Hz, J 3′2′′
)
3.2 Hz, 1H, H-3′); 3.60 (dd, J 5′5′′ ) -18.4 Hz, 1H, H-5′); 3.25 (d,
1H, H-5′′); 2.78 (dd, J 2′2′′ ) -13.6 Hz, 1H, H-2′); 2.27 (m, 1H,
H2′′); 0.99 (s, 9H, tBu-TBDMS); 0.21 and 0.18 (s, 6H, CH3-
TBDMS).
N6-Ben zoyl-3′-O-(ter t-b u t yld im et h ylsilyl)-5′,8-cyclo-5′-
oxo-2′-d eoxya d en osin e (8). A mixture of 7 (0.477 g, 1.06
mmol) and selenium oxide (0.254 g, 2.29 mmol) in 140 mL of
anhydrous 1,4-dioxane was stirred for 1 h under reflux. The
reaction was checked for completion by TLC (CHCl3/CH3OH,
95/5 v/v). Then, the mixture was cooled at room temperature
and passed through a Celite column. Eluates were evaporated
to dryness. The resulting residue was purified by chromatog-
raphy on a silica gel (50 g) column with a step gradient of
methanol (0-2%) in chloroform as the mobile phase. The
appropriate fractions were pooled and then concentrated to
dryness, giving 0.430 g (0.92 mmol) of N6-benzoyl-3′-O-(tert-
butyldimethylsilyl)-5′,8-cyclo-5′-oxo-2′-deoxyadenosine as a pink
powder (yield 87%). EI-MS (positive mode): m/z [M +•] ) 465.0
( 0.1 Da. 1H NMR (200.13 MHz, CDCl3) δ: 8.93 (s, 1H, H-2);
8.02-7.48 (m, 5H, aromatic-H of Bz); 6.84 (d, J 1′2′′ ) 5.0 Hz, 1H,
H-1′); 4.92 (s, 1H, H-4′); 4.66 (q, J 3′2′ ) 6.0 Hz, J 3′2′′ ) 2.4 Hz,
1H, H-3′); 2.70 (dd, J 2′2′′ ) -14.1 Hz, 1H, H-2′); 2.52 (m, 1H,
H2′′); 0.91 (s, 9H, tBu-TBDMS); 0.14 and 0.11 (s, 6H, CH3-
TBDMS).
(positive mode): m/z M + H]+ ) 510.2 ( 0.1 Da; [B + 2H]+
)
240.1 ( 0.1 Da 1H NMR (200.13 MHz, CD3OD) δ: 8.53 (s, 1H,
H-8); 8.35 (s, 1H, H-2); 8.03-7.15 (m, 9H, aromatic-H of Bz and
Ts); 6.38 (t, J 1′2′ ) 6.7 Hz, J 1′2′′ ) 6.2 Hz, 1H, H-1′); 4.55 (m, 1H,
H-3′); 4.23 (m, 2H, H-5′ and H-5′′); 4.05 (m, 1H, H-4′); 2.84 and
2.40 (m, 2H, H-2′ and H-2′′); 2.27 (s, 3H, CH3-Ts).
N6-Ben zoyl-5′-th iop h en yl-2′,5′-d id eoxya d en osin e (5). A
mixture of 4 (3.01 g, 5.91 mmol), thiophenol (1.4 mL, 13.72
mmol), and sodium methylate (0.485 g, 8.97 mmol) in 80 mL of
anhydrous methanol was stirred for 2 h under reflux. The
reaction was checked for completion by TLC (CHCl3/CH3OH, 90/
10 v/v) and the mixture was evaporated to dryness. The residue
was taken up in chloroform (100 mL) and washed with water
(100 mL). The organic layer was dried over Na2SO4 and
evaporated to dryness. The resulting residue was then purified
by chromatography on a silica gel (100 g) column with a step
gradient of methanol (0-7.5%) in chloroform as the mobile
phase. The appropriate fractions were pooled and then concen-
trated to dryness, giving 1.35 g (3.02 mmol) of N6-benzoyl-5′-
thiophenyl-2′,5′-dideoxyadenosine as a white foam (yield 51%).
FAB-MS (positive mode): m/z [M + H]+ ) 448.2 ( 0.1 Da; [B +
2H]+ ) 240.1 ( 0.1 Da. 1H NMR (200.13 MHz, CD3OD) δ: 8.62
(s, 1H, H-8); 8.42 (s, 1H, H-2); 8.03-7.01 (m, 10H, aromatic-H
of Bz and PhS); 6.44 (t, J 1′2′ ) J 1′2′′ ) 6.7 Hz, 1H, H-1′); 4.56 (m,
1H, H-3′); 4.09 (m, 1H, H-4′); 3.34 and 3.17 (m, 2H, H-5′ and
H-5′′); 2.98 and 2.43 (m, 2H, H-2′ and H-2′′).
(5′S)-N6-Ben zoyl-3′-O-(ter t-bu tyld im eth ylsilyl)-5′,8-cyclo-
2′-d eoxya d en osin e (9). Compound 8 (0.430 g, 0.92 mmol) was
dissolved in 40 mL of methanol. NaBH4 (67 mg, 1.77 mmol)
was added and the mixture was stirred for 1 h at room
temperature. The reaction was checked for completion by TLC
(CHCl3/CH3OH, 95/5 v/v) and then neutralized with 1 N HCl (3
mL). 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 (0-4%) in
chloroform as the mobile phase. The appropriate fractions were
pooled and then concentrated to dryness, giving 0.387 g (0.83
mmol) of (5′S)-N6-benzoyl-3′-O-(tert-butyldimethylsilyl)-5′,8-cy-
clo-2′-deoxyadenosine as a white foam (yield 90%). FAB-MS
N6-Ben zoyl-5′,8-cyclo-2′,5′-d id eoxya d en osin e (6). Com-
pound 5 (1.35 g, 3.02 mmol) and triethyl phosphite (5 mL, 29.16
mmol) were dissolved in 1 L of anhydrous acetonitrile. Then,
argon was bubbled through the solution for 40 min. The oxygen-
free solution was irradiated at 254 nm for 10 h, under an argon
atmosphere, in a quartz reactor. The reaction was checked for
completion by TLC (CHCl3/CH3OH, 85/15 v/v) and the solution
was evaporated to dryness. The resulting residue was crystal-
lized from hot methanol to give 0.52 g (1.54 mmol) of N6-benzoyl-
5′,8-cyclo-2′,5′-dideoxyadenosine as a yellow powder (yield 51%).
(positive mode): m/z [M + Na]+ ) 490.4 ( 0.1 Da; [M + H]+
)
1
468.2 ( 0.1 Da; H NMR (200.13 MHz, CDCl3) δ: 8.71 (s, 1H,
H-2); 8.03-7.48 (m, 5H, aromatic-H of Bz); 6.51 (d, J 1′2′′ ) 4.4
Hz, 1H, H-1′); 5.47 (d, J 5′4′ ) 5.8 Hz, 1H, H-5′); 5.29 (s, 1H, OH-
5′); 4.82 (q, J 3′2′ ) 7.2 Hz, J 3′2′′ ) 3.8 Hz, 1H, H-3′); 4.51 (d, 1H,
H-4′); 2.54 (dd, J 2′2′′ ) -13.1 Hz, 1H, H-2′); 2.26 (m, 1H, H-2′′);
0.85 (s, 9H, tBu-TBDMS); 0.04 and 0.02 (s, 6H, CH3-TBDMS).
(5′S)-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 (10). Com-
pound 9 (0.193 g, 0.41 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 7 mL of dry pyridine, and 4,4′-dimethoxytrityl chloride
(0.280 g, 0.82 mmol) was added. The mixture was heated at 70
°C and left at this temperature for 4 h. The reaction was checked
for completion by TLC (CHCl3/CH3OH, 97/3 v/v). The solution
was cooled at 5 °C and methanol (1 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 (0-1%) in chloroform/
triethylamine (99/1) as the mobile phase. The appropriate
fractions were pooled and then concentrated to dryness, giving
1
FAB-MS (positive mode): m/z [M + H]+ ) 338.1 ( 0.1 Da; H
NMR (400.13 MHz, DMSO-d6) δ: 9.09 (bs, 1H, NH-Bz); 8.77 (s,
1H, H-2); 8.15-7.64 (m, 5H, aromatic-H of Bz); 6.73 (d, J 1′2′′
)
5.1 Hz, 1H, H-1′); 5.53 (d, J OH-3′ ) 4.15 Hz, 1H, OH-3′); 4.81 (d,
J 4′5′ ) 6.3 Hz, 1H, H-4′); 4.48 (m, J 3′2′ ) 7.2 Hz, J 3′2′′ ) 3.5 Hz,
1H, H-3′); 3.57 (dd, J 5′5′′ ) -18.1 Hz, 1H, H-5′); 3.20 (d, 1H, H-5′′);
2.68 (dd, J 2′2′′ ) -13.5 Hz, 1H, H-2′); 2.26 (m, 1H, H-2′′). 13C
NMR (100.61 MHz, DMSO-d6) δ: 165.6 (1C, C(O) of Bz); 150.9
(1C, C-6); 150.4 (1C, C-4); 148.9 (1C, C-8); 148.2 (1C, C-2); 133.5
(1C, C-1 of Bz); 132.5 (1C, C-4 of Bz); 128.6 (4C, C-2, C-3, C-5,
C-6 of Bz); 125.0 (1C, C-5); 84.2 (1C, C-4′); 82.4 (1C, C-1′); 73.2
(1C, C-3′); 46.2 (1C, C-2′); 30.3 (1C, C-5′).
N6-Ben zoyl-3′-O-(ter t-bu tyld im eth ylsilyl)-5′,8-cyclo-2′,5′-
d id eoxya d en osin e (7). Compound 6 (0.452 g, 1.34 mmol) and
imidazole (0.388 g, 5.70 mmol) were dissolved in dry pyridine
(5 mL) and evaporated to dryness. The resulting residue was
dissolved in 10 mL of anhydrous DMF, and tert-butyldimethyl-

Notes
J . Org. Chem., Vol. 63, No. 15, 1998 5249
0.189 g (0.24 mmol) of (5′S)-N6-benzoyl-3′-O-(tert-butyldimeth-
ylsilyl)-5′-O-(4,4′-dimethoxytrityl)-5′,8-cyclo-2′-deoxyadenosine as
a yellow foam (yield 60%). FAB-MS (positive mode): m/z [M +
H]+ ) 770.3 ( 0.1 Da; [DMTr]+ ) 303.1 ( 0.1 Da. 1H NMR
(200.13 MHz, CDCl3) δ: 8.69 (s, 1H, H-2); 8.14-6.78 (m, 18H,
aromatic-H of Bz and DMTr); 6.34 (d, J 1′2′ ) 4.4 Hz, 1H, H-1′);
5.26 (d, J 4′5′ ) 5.2 Hz, 1H, H-4′); 4.87 (m, 1H, H-3′); 3.76 and
3.74 (s, 6H, CH3O-DMTr); 3.24 (d, 1H, H-5′); 2.54 and 2.10 (m,
2H, H-2′ and H-2′′); 0.82 (s, 9H, tBu-TBDMS); -0.03 (s, 6H, CH3-
TBDMS).
Dep r otection a n d P u r ifica tion of Oligon u cleotid es.
Following synthesis, the solid support was placed in concentrated
aqueous ammonia (32%) in a sealed vial and heated at 55 °C
for 17 h. The crude 5′-DMTr oligomers were purified and
deprotected online by reverse phase HPLC (system C).
En zym a tic Digestion of Oligom er s 13 a n d 14. A 0.5 AU260
nm portion of purified oligomers 13 and 14 was taken up in 45
µL of water. A solution (5 µL) of nuclease P1 in acetate buffer
(1unit/1µL) was added. The resulting mixture was incubated
at 37 °C for 2 h. Subsequently, 5 µL of Tris-HCl buffer (10×)
and 2 units of alkaline phosphatase were added, and the
resulting mixture was incubated for additional 1 h at 37 °C.
Subsequently, the mixture was taken up in 40 µL of TEAA (25
mM, pH ) 7) and the content was analyzed by reverse phase
HPLC (system E).
Sn a k e Ven om P h osp h od iester a se (3′-Exo) Digestion . A
0.2 AU260 nm portion of purified oligomer 13 were taken up in 18
µL of water. Then, 2 µL of ammonium citrate buffer (0.2M, pH
) 9) and 3 × 10-4 unit of 3′-exo were added. The resulting
mixture was incubated at 37 °C and aliquots of 2 µL were taken
up at increasing periods of time. To stop the enzymatic reaction,
each aliquot was diluted with 50 µL of water and frozen in liquid
nitrogen. After lyophilization, the residues obtained were taken
up in 20 µL of 0.1% aqueous solution of trifluoroacetic acid and
analyzed by MALDI-TOF mass spectrometry (matrix: 3-hy-
droxypicolinic acid).
(5′S)-N6-Ben zoyl-5′-O-(4,4′-d im eth oxytr ityl)-5′,8-cyclo-2′-
d eoxya d en osin e (11). Compound 10 (98 mg, 0.12 mmol) was
dissolved in 4 mL of anhydrous THF. A solution of TBAF (0.25
mL, 0.25 mmol) in THF (1 M) was added and the mixture was
stirred for 4 h at room temperature. The reaction was checked
for completion by TLC (CHCl3/CH3OH, 95/5 v/v) and 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 (0-2%) in chloroform/triethylamine (99/
1) as the mobile phase. The appropriate fractions were pooled
and then concentrated to dryness giving 57 mg (0.08 mmol) of
(5′S)-N6-benzoyl-5′-O-(4,4′-dimethoxytrityl)-5′,8-cyclo-2′-deoxy-
adenosine as a white foam (yield 72%). FAB-MS (positive
mode): m/z [M + H]+ ) 656.0 ( 0.1 Da; [DMTr]+ ) 303.0 ( 0.1.
1H NMR (200.13 MHz, CDCl3) δ: 8.75 (s, 1H, H-2); 8.08-6.88
(m, 18H, aromatic-H of Bz and DMTr); 6.36 (d, J 1′2′′ ) 4.7 Hz,
1H, H-1′); 5.30 (d, J 4′5′ ) 5.9 Hz, 1H, H-4′); 4.82 (m, 1H, H-3′);
3.79 and 3.78 (s, 6H, CH3O-DMTr); 2.87 (d, 1H, H-5′); 2.60 (dd,
J 2′2′′ ) -13.8 Hz); 2.13 (m, 1H, H-2′′).
Ca lf Sp leen P h osp h od iester a se (5′-exo) Digestion .
A
similar procedure as described (vide supra) was used with the
following modifications: 1 × 10-3 unit of 5′-exo and ammonium
citrate buffer (0.2M, pH ) 5) were used.
(5′S)-5′,8-Cyclo-2′-d eoxya d en osin e P h osp h or a m id ite De-
r iva t ive (12). Compound 11 (57 mg, 0.08 mmol) and diiso-
propylammonium tetrazolate (7 mg, 0.04 mmol) were dissolved
in dry dichloromethane (3 mL), and the resulting solution was
evaporated to dryness. The operation was repeated twice. The
resulting residue was then dissolved in dry dichloromethane (3
mL) and kept under an argon atmosphere. Subsequently,
(cyanoethyl)bis(diisopropylamino)phosphine (41 µL, 0.13 mmol)
was added with a syringe through a rubber septum. The
reaction mixture was stirred during 4 h. The formation of the
desired product was checked by TLC (AcOEt/Hexane/TEA, 70/
30/1 v/v/v). The mixture was evaporated to dryness. The
resulting residue was taken up in ethyl acetate (20 mL) and
washed successively with 5% NaHCO3 (30 mL) and a saturated
solution of NaCl (25 mL). The organic layer was dried over Na2-
SO4 and evaporated to dryness. The resulting residue was
purified by chromatography on a silica gel (25 g) column with a
step gradient of AcOEt (15-60%) in hexane/triethylamine (98/
2) as the mobile phase. The appropriate fractions were pooled
and then concentrated to dryness, giving 46 mg (0.05 mmol) of
the phosphoramidite synthon 12 as a white foam (yield 62%).
FAB-MS (positive mode): m/z [M + H]+ ) 856.0 ( 0.1 Da;
[DMTr]+ ) 303.0 ( 0.1 Da. 1H NMR (200.13 MHz, acetone-d6)
two diastereomers δ: 8.71 and 8.59 (s, 1H, H-2); 8.38-7.00 (m,
18H, aromatic-H of Bz and DMTr); 6.58 and 6.53 (d, J 1′2′′ ) 4.7
Hz and J 1′2′′ ) 4.4 Hz, 1H, H-1′); 5.41 and 5.35 (d, J 4′5′′ ) 5.6 Hz,
1H, H-4′); 5.17 (m, 1H, H-3′); 3.90 and 3.89 (s, 6H, CH3O-DMTr);
3.83 (s, 6H, CH3O-DMTr); 4.38-3.49 (m, 4H, CH-iPr and CH2-
OP); 3.33 and 3.21 (d, 1H, H-5′); 2.87-2.61 (m, 4H, H-2′, H-2′′
and -CH2CN); 1.40-1.23 (m, 12H, CH3-iPr). 31P NMR (100.21,
acetone-d6) δ: 150.8 and 148.7 (two diastereomers).
Th er m a l Den a tu r a tion Stu d ies. The oligodeoxynucleotides
[5′-d(ATC GTG XCT GAT CT)-3′], where X ) A or (5′S)-CycloA
(0.25 AU260 nm), and its complement [3′-d(TAG CAC TGA CTA
GA)-5′] (0.27 AU260 nm) were mixed in 200 µL of a buffer
containing 0.01 M sodium phosphate, 0.1 M NaCl, 0.001 M
EDTA, pH ) 7. DNA was annealed by heating to 85 °C for 5
min followed by slow cooling to 4 °C (3 h). The hybridization
solutions were diluted with 400 µL of buffer and degassed prior
to each melting experiment. Measurements of UV absorbance
were made in a 0.8 mL quartz cell (0.2 cm path length) with a
UV/vis spectrometer equipped with a Peltier temperature con-
troller. The absorbance of the sample was monitored at 260 nm
from 15 to 85 °C at a heating rate of 1 °C/min. Data reflect the
average of three melting curves per oligodeoxynucleotide duplex.
Ack n ow led gm en t. The contributions of Colette
Lebrun (SCIB/LRI/CEA-Grenoble), Daniel Ruffieux (Bio-
chimie C/CHRU Grenoble) and Michel J aquinod (LSMP/
IBS/CEA/CNRS-Grenoble) to the mass spectrometry
measurements are greatly acknowledged. We thank Dr.
Thierry Douki and Thierry Delatour for helpful discus-
sions concerning the photoreactions of 8-bromo-2′-
deoxyadenosine and the thiophenyl derivative 5. This
work was supported, in part, by the French Ministery
of Science and Research (ACC-SV no. 8-MESR 1995)
and Electricite´ de France (Comite´ de Radioprotection).
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H-, 13C
NMR, and FAB mass spectra of 6, 1H NMR spectrum of 9,
FAB mass spectrum of 12, electrospray ionization mass spectra
of oligomers 13-15, MALDI-TOF mass spectra of the 3′-exo
and 5′-exo enzymatic digestions of 13, and melting curves of
the normal and modified duplexes (11 pages). This material
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
Solid -P h a se Syn th esis of Oligon u cleotid es. Oligonucle-
otides containing (5′S)-CyclodAdo were prepared by phosphora-
midite solid-phase synthesis, using an acetyl protective group
for dCyd, a benzoyl protective group for dAdo, and an isobutyryl
protective group for dGuo. The synthesized amidite 12 (46 mg,
0.05 mmol) was dissolved in 400 µL of dry dichloromethane and
placed in the additional ports of a model 392 DNA synthesizer
(ABI). The standard 1 µmol synthesis scale with retention of
the 5′ terminal DMTr group (trityl-on mode) was used with the
modifications previously mentioned.
J O980083Q