Photolabile N-hydroxypyrid-2(1H)-one derivatives of guanine nucleosides: A new method for independent guanine radical generation

Article abstract of DOI:10.1039/b909138f

One-electron oxidized guanine is an important reactive intermediate in the formation of oxidatively generated damage in DNA and a variety of methods have been utilized for the abstraction of a single electron from the guanine moiety. In this study, an alt

Full text of DOI:10.1039/b909138f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Photolabile N-hydroxypyrid-2(1H)-one derivatives of guanine nucleosides:
a new method for independent guanine radical generation†
Panagiotis Kaloudis,a Cecilia Paris,b Despoina Vrantza,a Susana Encinas,b Raul Pe´rez-Ruiz,a
Miguel A. Miranda*b and Thanasis Gimisis*a
Received 11th May 2009, Accepted 2nd September 2009
First published as an Advance Article on the web 1st October 2009
DOI: 10.1039/b909138f
One-electron oxidized guanine is an important reactive intermediate in the formation of oxidatively
generated damage in DNA and a variety of methods have been utilized for the abstraction of a single
electron from the guanine moiety. In this study, an alternative approach for the site specific,
independent generation of the guanine radical, utilizing N-hydroxypyrid-2(1H)-one as a photolabile
modifier of guanine, is proposed. Novel photolabile 6-[(1-oxido-2-pyridinyl)oxo]-6-deoxy- and
2¢,6-dideoxy-guanosine derivatives capable of generating the neutral guanine radical (G(-H)∑) upon
photolysis were synthesized and characterized. The generation of G(-H)∑ proceeds through homolysis
of the N–O bond and was confirmed through continuous photolysis product analysis and trapping
studies, as well as laser flash photolysis experiments.
the absence of these species, the lifetime can reach ~5 s.22 Recent
Introduction
experiments showed that 3O2 reacts with G(-H)∑ (k < 106 M-1 s-1)
∑
at least 3 orders of magnitude more slowly than O2 -, thereby
Extensive research over the past years on oxidatively produced
DNA damage has uncovered a wealth of chemistry involving
the sugar1 and base2 components of nucleosides, nucleotides,
synthetic oligonucleotides and natural DNA. Of the four DNA
bases, guanine (G) is the most easily oxidized, its reduction
potential being the lowest among the nucleobases, and presents the
richest chemistry.3 Numerous studies on the long-distance hole-
migration process through a DNA double strand have been based
on trapping an electron hole by a guanine base. These studies have
led to a detailed insight into the distance and sequence dependence
of charge movement processes.4-7 One electron oxidation of the G
moiety results in the formation of a wide variety of oxidatively
∑
-
kinetically favoring the reaction with O2
.
21 The reaction of G(-H)∑
∑
with O2 - leads to the formation of imidazolone derivative (dIz), as
the major one-electron oxidation product, which is hydrolytically
unstable and is consequently transformed to oxazolone derivative
(dZ).14 The greater stability and decreased reactivity of G∑+ in
double-stranded DNA leads to hydration at the 8-position and,
after a second one-electron oxidation, to the formation of 8-oxo-
G. The neutral radical G(-H)∑ does not give rise to 8-oxo-G.21
Selective generation of carbon-centered sugar and base radicals
has been accomplished by photo-reactive precursors using nucle-
osides or oligonucleotides (ODNs). Generation of a single radical
species on duplex ODNs provides a powerful tool for elucidating
the role of reactive intermediates in the formation of nucleic acid
lesions. For example, radicals generated at C1¢ and C4¢ and, more
recently, C5¢ positions of the sugar moiety have been studied in
detail by photolysis of the corresponding tert-butyl ketones.23-28
Selective generation of pyrimidine base radicals has been also
accomplished by photolysis of phenylthio-29 or isopropylketones.30
Photolabile precursors for the site selective generation of G(-H)∑
are missing.
generated damage.3,8-10 A variety of oxidants are known to abstract
∑
-
11
a single electron from G. These oxidants include Br2- and SO4
,
∑
-
CO3
,
12,13 light (photoionization) or type I photosensitization.14-16
One electron reduction of 8-bromoguanine has recently been
reported as an efficient method for producing the guanine radical
cation (G∑+).17,18 The produced G∑+ deprotonates at physiological
pH with a pKa of 3.9 at the nucleoside level,11 to produce the
neutral guanine radical [G(-H)∑]. The deprotonation of G∑+ at the
nucleoside level and in DNA occurs with a rate constant in the
order of 107 s-1, at neutral pH.19 A recent report20 suggests that
hydroxyl radicals (OH∑) react with guanine mainly by hydrogen
atom abstraction from the 2-amino group, not by addition to
the C-4 as previously believed,21 resulting in the formation of a
2-aminyl radical which tautomerizes to G(-H)∑. The lifetime of
this radical depends on the presence of reactive species, but in
Towards this effort, we proposed the use of either N-hydroxy-
pyridine-2(1H)-thione (N-HPT) or N-hydroxypyrid-2(1H)-one
(N-HPone) as photolabile modifiers at the 6-position of
2¢-deoxyguanosine or guanosine (Scheme 1). Photolysis of the
aOrganic Chemistry Laboratory, Department of Chemistry, University
of Athens, Panepistimiopolis, 15771 Athens, Greece. E-mail: gimisis@
chem.uoa.gr; Fax: +30 2107274761; Tel: +30 2107274928
bInstituto de Tecnolog´ıa Qu´ımica, Universidad Polite´cnica de Valencia,
Camino de Vera s/n, 46022 Valencia, Spain
† Electronic supplementary information (ESI) available: General experi-
mental procedures, Figures S1-S3 and original NMR spectra. See DOI:
10.1039/b909138f
Scheme 1 Proposed generation of G(-H)∑.
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derivatized guanine moiety was expected to homolytically cleave
the N–O bond and afford the guanine radical. We have recently
evaluated the use of N-hydroxypyridine-2(1H)-thione (N-HPT) as
a photolabile modifier and we did observe products arising from
G(-H)∑ formation.31 However, product studies, as well as laser
flash photolysis experiments indicated that both the chemistry
and photochemistry of this functional group are rather complex.
It has been reported that the photochemistry of N-HPone is
much simpler compared to its sulfur analogue,32,33 and we decided
to utilize N-HPone, with the expectation that homolysis of the
N–O bond will lead to the generation of the guanine radical.
We present herein our results from synthetic and stability studies,
accompanied by continuous photolysis product studies, trapping
experiments and observation of the transient spectra of G(-H)∑ by
laser flash photolysis (LFP).
The synthesis of 8d is presented in Scheme 3. The 2-(4-
nitrophenyl)ethyl group was utilized for the protection of the
O6 position through a Mitsunobu reaction.36 Conversion of 4b
to 5b was accomplished by treatment with tert-butylnitrite in
70% anhydrous HF/pyridine at -20 ◦C using a modification
of the procedure by Robins & Uznanski.37 Unfortunately, the
lability of the TBDMS protecting groups of the sugar moiety
in the presence of the fluoride anion also resulted in partial
deprotection together with the fluorination. Strict adherence to
the experimental protocol is essential to achieve the optimal yield
(71%). Otherwise, reapplication of the reaction mixture to the
silylation conditions was necessary to improve the yield of 5b.
Substitution of the fluoro group by cyclopropylamine, followed by
removal of the 2-(4-nitrophenyl)ethyl protecting group38 afforded
derivative 6b. The photolabile adduct 8d was obtained by following
the previously applied experimental procedure for products 2c and
2d (vide supra) and 8d was obtained in good yield.
Results
Synthesis
The synthesis of O6-[2-oxopyridin-1(2H)-yl]-guanosine and
2¢-deoxyguanosine derivatives 2 is presented in Scheme 2. The
known O6-(2-mesitylsulfonyl) derivatives 1 were synthesized fol-
lowing reported procedures.31,34 Introduction of the 2-oxopyridin-
1(2H)-yl group, through DABCO-induced activation,31 in diox-
ane, led to the formation of compounds 2a and 2b in excellent
yields (94 and 91%, respectively). Products 2a and 2b were readily
deprotected in the presence of TBAF in THF and the water
soluble derivatives 2c and 2d were isolated in satisfactory yields (70
and 75%, respectively). Although the methodology was initially
developed on the ribo-derivative 2d for economy, it was then
extended to 2c and only the 2¢-deoxy analogue 2c was utilized
in the photochemical studies described herein.
Scheme 3 Synthesis of O6-[2-oxopyridin-1(2H)-yl]-N2-cyclopropyl-guan-
osine (8d). (i) TBDMSCl, imidazole, DMF, 87% (ii) NPE, DIAD,
PPh3, dioxane, 84% (iii) HF/pyridine 70/30, t-BuONO, pyridine,
THF, 71% (iv) cyclopropylamine, Et3N, DMF (v) DBU, pyridine,
53% (two steps) (vi) mesitylsulfonyl chloride, DMAP, DCM, 66%
(vii) N-hydroxypyrid-2(1H)-one, DABCO, DBU, dioxane, 94%
(viii) TBAF in THF, 75%.
Protection of the O6 position of the base moiety is compulsory
in order for the fluorination to take place. Utilization of the
2-(4-nitrophenyl)ethyl protecting group was successful, but it nev-
ertheless introduced two additional synthetic steps. An interesting
alternative approach would be to introduce the fluoro group at
derivative 1b, thus eliminating the extra protection–deprotection
steps. Indeed, fluorination of 1b followed by substitution by
N-hydroxypyrid-2(1H)-one were successful, but when the substi-
tution by cyclopropylamine was attempted, only formation of 11b
was observed (Scheme 4).
Scheme
2
Synthesis of O6-[2-oxopyridin-1(2H)-yl]-guanosine and
2¢-deoxyguanosine derivatives. (i) DABCO, dioxane; then N-HPone, DBU.
(ii) TBAF, THF.
Synthesis of a G(-H)∑ chemical trap
In an effort to provide an efficient trap for the guanine radical to be
produced photolytically, a derivative of 2d bearing a cyclopropyl
group at N2 was also synthesized, in accordance with the work of
Saito and coworkers.35 They proposed that the dG radical cation
induces homolytic cyclopropane ring opening, as was evidenced by
the formation of N2-(3-hydroxypropanoyl)dG. Therefore, product
analysis of the photolyzed mixture could provide chemical evi-
dence for G(-H)∑ generation in our system as well.
Scheme 4 Synthesis of 9-(2¢,3¢,5¢-tri-O-TBDMS-b-D-ribofuranosyl)-N-
cyclopropyl-2-fluoro-purin-6-amine (11b). (i) HF/pyridine 70/30,
t-BuONO, pyridine, THF, 44% (ii) N-hydroxypyrid-2(1H)-one, DABCO,
DBU, dioxane, 96% (iii) cyclopropylamine, Et3N, DMF, 73%.
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Photolabile adduct stability
When the photolysis was run in H218O and was monitored by
LC-MS, no isotopic labeling in either 2¢-deoxyguanosine or 2-
hydroxypyridine was detected, indicating that homolysis of the
N–O bond, rather than solvolysis of 2c, had occurred (see also
discussion below).
We did not proceed with a systematic characterization of
the minor products at this stage. In the case of photolysis
under air, however, a new nucleosidic product was identified as
2-amino-5-[(2-deoxy-b-D-ribosyl)amino]-4H-imidazol-4-one (im-
idazolone, dIz), from its characteristic UV (Fig. 1, inset a, lmax at
253 nm) and MS spectra. Upon incubation of the reaction mixture
in the dark, for 24 h, at 37 ◦C, the imidazolone peak gave its place
to the corresponding oxazolone (Fig. S1, see ESI†). This guanine
oxidation product was quantified after 30 min reaction, to ~5%
yield, assuming an e260 of 1.99 ¥ 104 M-1cm-1.41,42
Insertion of the photolabile adduct 2c in synthetic oligonucleotides
would require that suitable protection is applied and that it remains
stable under the standard or modified conditions utilized in au-
tomated oligonucleotide synthesis, on solid support. Compound
2c was indefinitely stable in D2O at natural pD and its solutions
showed no appreciable change after at least 2 months’ storage at
room temperature as indicated by NMR spectroscopy. Moreover,
it was stable for at least 24 h when heated in an aqueous solution, at
80 ◦C, in the dark and exhibited the same stability in 80% aqueous
acetic acid at room temperature as shown by HPLC experiments.
The compound was relatively stable in 0.1 M I2 in THF (1 h, at
r.t., for >90% recovery) and in 25% aq. NH4OH (2.5 h, at r.t., for
>90% recovery).
Therefore, the modification can be inserted in synthetic oligonu-
cleotides, but modified solid phase oligonucleotide synthesis with
more base labile protection (e.g., PAC protection39) would have to
be used.
Photolysis of 8d in aqueous solutions
The analogue 8d was photolyzed in H2O for 25 min, under oxygen,
and the photolyzed mixture was incubated overnight in the dark
at ambient temperature. Analysis by LC-MS revealed that the
major peak (at 17.9 min, Fig. S2, S3, see ESI†) corresponded
to N2-(3-hydroxypropanoyl)guanosine, suggesting that homolytic
cyclopropane ring opening, followed by reaction with molecular
oxygen and subsequent rearrangement to 14d (Scheme 5), has
occurred. Guanosine (3d) is also one of the photolysis products
(peak at 14 min) in accord with the literature report.35
Photolysis of 2c in aqueous solutions
The photolysis of 2c (blue peaks in Fig. 1) with pyrex-filtered
(>320 nm) UVA light in H2O, under argon, at natural pH, was
followed by RP-HPLC. The reaction is practically completed after
30 min (>95% conversion of 2c) yielding two main products
that were characterized by LC/MS and by comparison with
authentic samples. The peak at 16.5 min (Fig. 1) corresponds
to 2¢-deoxyguanosine generated in 38% yield, with respect to
the consumed 2c, whereas the peak at 14 min corresponds to
2-hydroxypyridine (50% yield). Yields were determined by cali-
bration curves, obtained from known concentrations of authentic
samples. The apparent size of the peaks corresponding to 2-
hydroxypyridine (Fig. 1) is a result of the poor absorbance of
this compound at 254 nm40 and detection at 230 nm provides a
more appropriate picture for its formation (Inset b in Fig. 1).
Scheme 5 Major products observed in the photolysis of 8d.
Transient spectroscopy
Direct evidence of G(-H)∑ generation was obtained through time-
resolved experiments. Laser flash photolysis of 2c was performed
by excitation at 308 nm, in acetonitrile:water (4:1) mixture, under
an anaerobic atmosphere. N-Hydroxypyrid-2(1H)-one was also
photolyzed, as a reference. After 2 microseconds, two contribu-
tions were distinguishable in the spectrum of 2c. One of them
corresponded to the 2-pyridyloxyl radical,43,44 as assessed by
comparison with the reference, and the other one displayed the
typical features of the neutral guanine radical. The spectrum of this
radical was obtained by subtraction of the 2-pyridyloxyl radical
from the signal, but it was also obtained directly after longer
delay times (Fig. 2); its assignment to the guanine radical was based
on the characteristic transient absorption bands at ca. 310 nm,
390 nm and 520 nm.11 The quantum yield of this radical was
roughly estimated at 0.2, and its decay was biphasic. A relatively
fast component with t = 25 ms was assigned to the caged G(-H)∑/2-
pyridyloxyl radical pair, whereas the longer lived (>1 ms) residual
absorption was attributed to free G(-H)∑ radical. Quenching of the
obtained guanine radical by oxygen, isopropanol or thiols45 was
not observed; thus the rate constant for these processes must be
lower than 106 M-1 s-1 as has been reported.21
Fig. 1 HPLC profile of the photolysis of 2c in H2O, under argon, detection
at 260 nm. Insets: (a) UV spectra for 2¢-deoxyguanosine (lmax at 251 nm),
2-hydroxypyridine (lmax at 222 & 294 nm) and imidazolone (dIz, lmax at
253 nm). (b) HPLC for the photolysis of 2c (Rt 24 min) after 30 min
reaction; detection at 230 nm.
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cyclopropylamine analogue,46 although a lower estimate for
N-cyclopropylaniline was recently reported.47
The final direct evidence for the independent generation of
G(-H)∑ came from time-resolved experiments, where the char-
acteristic transient absorption bands of G(-H)∑ were observed.
This radical, generated in a significant yield, recombines with
the 2-pyridyloxyl radical within the cage (fast decay component)
or escapes to give a longer lived free radical, which is able
to react further leading to products. The guanine radical is a
well characterized species that has been previously observed by
transient spectroscopy,11,12,48-50 but has not been directly generated
by homolysis of an excited nucleoside derivative.
Conclusions
Fig. 2 Transient absorption spectra obtained 2 ms (squares) and 75 ms
(circles) after excitation with 308 nm excimer laser pulse, generated upon
photolysis of 2c, in a deaerated acetonitrile:water 4:1 solution.
We have synthesized and characterized novel photolabile 6-
[(1-oxido-2-pyridinyl)oxo]-6-deoxy- and 2¢,6-dideoxy-guanosine
derivatives capable of generating the neutral guanine radical
upon photolysis. The generation of G(-H)∑ was confirmed for
2c through continuous photolysis product analysis and trapping
studies, as well as laser flash photolysis experiments, and involves
the homolysis of the N–O bond and the formation of the
neutral guanine radical. In comparison to the previously studied
N-HPT derivative,31 the present system has exhibited, as expected,
simpler photochemistry generating the desired G(-H)∑. It should
be emphasized that this system does not require molecular
oxygen and, therefore, no other reactive oxygen species is
formed. Nevertheless, the possible role of the 2-pyridyloxyl
radical in the fate of G(-H)∑ deserves further study. A systematic
characterization of all minor products in the photolysis of 2c and
utilization of this system for the site-specific formation of G(-H)∑ in
synthetic oligonucleotides under very mild photolytic conditions
is currently under study and will be reported in due course.
Discussion
The main products from the photolysis of 2c, namely 2¢-deoxy-
guanosine and 2-hydroxypyridine could have been produced by
one of two possible routes. The first possible route would involve
simple hydrolysis of either 2c or more likely its corresponding
excited state, since 2c was proven, from the stability studies, to be
stable in natural-pH aqueous solutions. This route was disproven
since the photolysis of 2c in H218O did not lead to any 18O
incorporation in either 2¢deoxyguanosine or 2-hydroxypyridine as
shown by LC-MS studies on the photolysis products. The lack of
18O incorporation in any of the two major products also rules out
the possibility that they are formed from a recombination reaction
between the two radicals followed by hydrolysis of the adducts
formed. The second possible route would involve homolysis of the
N–O bond in 2c, as expected, followed by one electron reduction
of the resulting radical species. This could be effected through
disproportionation reactions, and this possibility would require
further oxidation of one of the two components, resulting in
yields of <50% for the reduced species. The yields observed (38%
for dGuo and 50% for 2-hydroxypyridine) point in this direction,
and this issue deserves further study. Finally, hydrogen abstraction
from the 2-deoxyribose moiety of the nucleosidic products of the
photolysis mixture by a delocalized radical, such as G(-H)∑ or 2-
pyridyloxyl, is expected to be endothermic and therefore could be
ruled out. The presence of dIz, a known dGuo oxidation product,
in the oxygenated photolysis mixture, albeit in low yield, is a first
indication of the intermediacy of G(-H)∑.
Experimental
Synthetic protocols
3¢,5¢-Bis-O-(tert-butyldimethylsilyl)-O6 -(2-oxopyridin-1(2H)-
yl)-2¢-deoxyguanosine (2a). A solution of 1a (dried by heating
over phosphorus pentoxide in vacuo at 56 ◦C for 2 h before
use, 1.87 g, 2.76 mmol) and DABCO (619 mg, 5.52 mmol) in
anhydrous dioxane (25 mL) was stirred for 1 h under Ar in room
temperature. Then, 2-oxopyridine N-oxide (1.53 g, 13.79 mmol)
was added and after 30 min DBU (624 mL, 4.14 mmol) was added
and the reaction mixture was stirred at room temperature for 3 h.
The reaction mixture was diluted with ethyl acetate (20 mL) and
extracted first with water (15 mL), then with a sat. NaHCO3
solution (15 mL) and finally with brine (15 mL). The organic
layer was dried over sodium sulfate and evaporated to dryness.
The residue was purified by column chromatography using 96:4
CH2Cl2–EtOH to give 1.56 g of the product (94%). Rf: 0.35 in
CH2Cl2–EtOH 96:4. 1H-NMR (200 MHz, CDCl3) d 0.07 [s, 12H,
Si(CH3)2] 0.88 [s, 18H, SiC(CH3)3] 2.29–2.59 (m, 2H, H-2b, H-
2a) 3.72 (dd, 1H, JH4¢ = 3.4, JH5¢ = 11.6 Hz, H-5¢¢) 3.80 (dd, 1H,
Nevertheless, more direct and positive means of observing
G(-H)∑ were necessary in order to confirm the suitability of our
system for the independent generation of G(-H)∑. Therefore, we
utilized the radical trap 8d, following the work by Saito and
∑
coworkers.35 According to this work, a G+ containing a cyclo-
propylamino group at the 2-position leads, in the presence of oxy-
gen and through the intermediacy of an N-centered radical cation,
to the ring opened product 14d, thus trapping the electron hole cen-
tered on this specific guanine. Photolysis of the suitably modified
guanosine 8d, in our system, did lead to the same product 14d, thus
confirming the intermediacy of G(-H)∑. If a neutral N-centered
radical is involved in this case, ring opening is expected to proceed
with a rate of around 106 s-1 as has been estimated for a secondary
JH4¢ = 3.4, JH5¢¢ = 11.6 Hz, H-5¢) 3.95 (dd, 1H, JH3¢ = 6.2, JH5¢
=
3.4 Hz, H-4¢) 4.55 (dd, 1H, JH4¢ = 6.2, JH2a = 3.4 Hz, H-3¢) 4.96
(bs, 2H, H-2) 6.18 (dt, 1H, J = 6.9, 6.8, 1.5 Hz, OPyH-5) 6.28
(dd, 1H, JH2b = 6.2, JH2a = 6.2 Hz, H-1¢) 6.71 (dd, 1H, J = 9.2,
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1.5 Hz, OPyH-3) 7.36 (dt, 1H, J = 9.2, 6.8, 2.0 Hz, OPyH-4) 7.51
(dd, 1H, J = 6.8, 2.0 Hz, OPyH-6) 8.02 ppm (s, 1H, H-8). 13C-
NMR (50 MHz, CDCl3) d -5.56, -5.47, -4.89, -4.74 [Si(CH3)2]
17.9, 18.3 [SiC(CH3)3] 25.7, 25.9 [SiC(CH3)3] 41.0 (C-2¢) 62.6
(C-5¢) 71.5 (C-3¢) 83.8 (C-1¢) 87.6 (C-4¢) 104.8 (OPyC-5) 113.4
(C-5) 123.0 (OPyC-3) 136.0 (OPyC-6) 139.0 (C-8) 139.3 (OPyC-4)
25.7, 25.8, 26.1 [SiC(CH3)3] 61.9 (C-5¢) 71.0 (C-3¢) 76.2 (C-2¢) 84.4
(C-4¢) 88.4 (C-1¢) 104.8 (OPyC-5) 113.5 (C-5) 123.1 (OPyC-3)
136.1 (OPyC-6) 139.0 (C-8) 139.6 (OPyC-4) 155.3 (C-6) 157.5
(C-2) 158.6 (C-4) 158.9 ppm (OPyC-O). [a]2D5 = -5.6 (C = 1 M,
-1
=
CHCl3). IR (CHCl3, cm ) 3429, 2959, 2934, 2860, 1674 (C O),
1632, 1600, 1578, 1509, 1202, 836, 716. UV (CHCl3, nm) 246 (e =
9543 M-1cm-1), 289 (e = 13898 M-1cm-1).
155.1 (C-6) 157.4 (C-2) 158.5 (C-4) 158.9 ppm (OPyC-O). [a]D25
=
-7.2 (C = 1 M, CHCl3). IR (CHCl3, cm-1) 3427, 2958, 2934, 2890,
1674(C O), 1632, 1600, 1578, 1463, 835. UV (CHCl3, nm) 288
O6-(2-Oxopyridin-1(2H)-yl)guanosine (2d). To a solution of 2b
(dried by heating over phosphorus pentoxide in vacuo at 56 ◦C for
2 h before use, 380 mg, 0.52 mmol) in anhydrous THF (8 mL) were
added activated molecular sieves (150 mg) and a 1 M solution of
TBAF in THF (1.74 mL, 1.74 mmol). The reaction mixture was
stirred for 1 h at room temperature under Ar, filtrated and washed
with MeOH (15 mL). The filtrate was evaporated to dryness and
the residue was purified by column chromatography using 93:7
CH2Cl2–EtOH to give 149 mg (75%) of the product. Rf: 0.30
in CH2Cl–EtOH 9:1. 1H-NMR (200 MHz, D2O) d 3.75 (dd,
1H, JH4¢ = 3.4, JH5¢ = 13.0 Hz, H-5¢¢) 3.85 (dd, 1H, JH4¢ = 3.4,
JH5¢¢=13.0 Hz, H-5¢) 4.17 (dd, 1H, JH3¢ = 5.4, JH5¢ = 3.4 Hz, H-4¢)
=
(e = 13842 M-1cm-1).
O6-(2-Oxopyridin-1(2H)-yl)-2¢-deoxyguanosine (2c). To a so-
lution of 2a (dried by heating over phosphorus pentoxide in vacuo
at 56 ◦C for 2 h before use, 100 mg, 0.17 mmol) in anhydrous THF
(2 mL) were added activated molecular sieves (50 mg) and a 1 M
solution of TBAF in THF (556 mL, 0.556 mmol). The reaction
mixture was stirred for 1 h at room temperature under Ar, filtrated
and washed with MeOH (5 mL). The filtrate was evaporated to
dryness and the residue was purified by column chromatography
using 95:5 CH2Cl2–EtOH to give 43 mg (70%) of the product. Rf:
1
0.13 in CH2Cl2–EtOH 96:4. H-NMR (400 MHz, DMSO-d6) d
4.36 (dd, 1H, JH4¢ = 5.4, JH2¢ = 5.4 Hz, H-3¢) 4.69 (dd, 1H, JH1¢
=
2.22–2.28 (m, 1H, H-2b) 2.57–2.64 (m, 1H, H-2a) 3.48–3.60 (m,
2H, H-5¢, H-5¢¢) 3.83 (dd, 1H, JH3¢=7.4, JH5¢=4.5 Hz, H-4¢) 4.37
(dd, 1H, JH4¢=7.4, JH2a=3.3 Hz, H-3¢) 6.24 (dd, 1H, JH2b=7.2,
JH2a=7.2 Hz, H-1¢) 6.32 (dt, 1H, J = 6.8, 6.8, 1.6 Hz, OPyH-5)
6.63 (dd, 1H, J = 9.3, 1.6 Hz, OPyH-3) 6.65 (s, 2H, H-2) 7.53
(dt, 1H, J = 9.3, 6.8, 1.9 Hz, OPyH-4) 8.11 (dd, 1H, J = 6.8,
1.9 Hz, OPyH-6) 8.25 ppm (s, 1H, H-8). 13C-NMR (100 MHz,
DMSO-d6) d 40.5 (C-2¢) 61.5 (C-5¢) 70.6 (C-3¢) 82.8 (C-1¢) 87.6
(C-4¢) 104.7 (OPyC-5) 111.5 (C-5) 121.7 (OPyC-3) 137.6 (OPyC-
6) 139.5 (C-8) 140.0 (OPyC-4) 155.4 (C-6) 156.5 (C-2) 158.6 (C-4)
159.2 ppm (OPyC-O). UV (H2O, nm) 214 (e = 19924 M-1cm-1),
246 (e = 7098 M-1cm-1), 291 (e = 10810 M-1cm-1). HR-ESI-MS
5.4, JH3¢ = 5.4 Hz, H-2¢) 5.90 (d, 1H, JH2¢ = 5.4 Hz, H-1¢) 6.56 (dt,
1H, OPyH-5) 6.77 (dd, 1H, OPyH-3) 7.67 (dt, 1H, OPyH-4) 7.89
(dd, 1H, OPyH-6) 8.14 ppm (s, 1H, H-8). 13C-NMR (100 MHz,
DMSO-d6) d 61.3 (C-5¢) 70.4 (C-3¢) 75.7 (C-2¢) 84.5 (C-4¢) 87.9
(C-1¢) 104.8 (OPyC-5) 112.5 (C-5) 122.5 (OPyC-3) 137.1 (OPyC-
6) 139.2 (C-8) 139.8 (OPyC-4) 155.2 (C-6) 156.8 (C-2) 158.6 (C-4)
159.0 ppm (OPyC-O). UV (H2O, nm) 245 (e = 8532 M-1cm-1),
292 (e = 12749 M-1cm-1). HR-ESI-MS for C15H17N6O6+ [M + H]+,
calcd 377.1204; found 377.1188.
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-O6 -[2-(4-nitrophenyl)-
ethyl]-guanosine (4b). To a solution of 2¢,3¢,5¢-tris-O-(tert-
butyldimethylsilyl)-guanosine (3b) (1.00 g, 1.60 mmol) and Ph3P
(462 ◦mg, 1.76 mmol) in anhydrous dioxane (4 mL) were added
at 0 C and under Ar, DIAD (347 mL, 1.76 mmol) and 4-nitro-
phenyl-ethanol (294 mg, 1.76 mmol). The reaction mixture was
stirred at room temperature for 18 h, diluted with CH2Cl2 (15 mL)
and extracted first with water (15 mL), then with a sat. NaHCO3
solution (15 mL) and finally with brine (15 mL). The organic
layer was dried over sodium sulfate and evaporated to dryness.
The residue was purified by column chromatography using 7:3
ethyl acetate–hexane to afford 1.04 g (84%) of the product as a
yellowish powder. Rf: 0.65 in ethyl acetate–hexane 7:3. 1H-NMR
(200 MHz, CDCl3) d -0.22, -0.06 [s, 3H, Si(CH3)2] 0.10, 0.12
[s, 6H, Si(CH3)2] 0.77, 0.91, 0.94 [s, 9H, SiC(CH3)3] 3.27 (t, 2H,
JCH2O = 7.0 Hz, CH2Ph) 3.75 (dd, 1H, JH4¢ = 2.2, JH5¢ = 11.2 Hz,
H-5¢¢) 3.94 (dd, 1H, JH4¢ = 2.2, JH5¢¢ = 11.2 Hz, H-5¢) 4.07 (dd, 1H,
for C15H17N6O5 [M + H]+, calcd 361.1255; found 361.1245.
+
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-O6-(2-oxopyridin-1(2H)-
yl)guanosine (2b). A solution of 1b (dried by heating over
phosphorus pentoxide in vacuo at 56 ◦C for 2 h before use,
100 mg, 0.12 mmol) and DABCO (28 mg, 0.25 mmol) in
anhydrous dioxane (1.5 mL) was stirred for 1 h under Ar at room
temperature. Then, 2-oxopyridine N-oxide (69 mg, 0.62 mmol)
was added and after 30 min DBU (28 mL, 0.19 mmol) was added
and the reaction mixture was stirred in room temperature for
3 h. The reaction mixture was diluted with ethyl acetate (10 mL)
and extracted first with water (5 mL), then with a sat. NaHCO3
solution (5 mL) and finally with brine (5 mL). The organic layer
was dried over sodium sulfate and evaporated to dryness. The
residue was purified by column chromatography using 7:3 ethyl
acetate–hexane to give 89 mg (91%) of the product. Rf: 0.42 in
1
ethyl acetate–hexane 7:3. H-NMR (200 MHz, CDCl3) d -0.05,
JH3¢ = 5.6, JH5¢ = 2.2 Hz, H-4¢) 4.25 (dd, 1H, JH2¢ = 5.6, JH4¢ =
0.00, 0.07, 0.09, 0.12, 0.13 [s, 3H, Si(CH3)2] 0.84, 0.90, 0.94 [s,
9H, SiC(CH3)3] 3.77 (dd, 1H, JH4¢ = 2.2, JH5¢ = 11.4 Hz, H-5¢¢)
4.00 (dd, 1H, JH4¢ = 2.2, JH5¢¢ = 11.4 Hz, H-5¢) 4.05–4.12 (m, 1H,
H-4¢) 4.29 (dd, 1H, JH2¢ = 4.2, JH4¢ = 4.2 Hz, H-3¢) 4.39 (dd, 1H,
JH1¢ = 4.2, JH3¢ = 4.2 Hz, H-2¢) 4.93 (bs, 2H, NH2) 5.89 (d, 1H,
JH2¢ = 4.2 Hz, H-1¢) 6.19 (dt, 1H, J = 7.0, 6.6, 1.5 Hz, OPyH-5)
6.73 (dd, 1H, J = 9.2, 1.5 Hz, OPyH-3) 7.38 (dt, 1H, J = 9.2,
6.6, 2.0 Hz, OPyH-4) 7.53(dd, 1H, J = 7.0, 2.0 Hz, OPyH-6)
8.16 ppm (s, 1H, H-8). 13C-NMR (50 MHz, CDCl3) d -5.43,
-5.38, -4.86, -4.76, -4.26 [Si(CH3)2] 17.9, 18.0, 18.5 [SiC(CH3)3]
5.6 Hz, H-3¢) 4.47 (dd, 1H, JH1¢ = 5.6, JH3¢ = 5.6 Hz H-2¢) 4.71
(t, 2H, JCH2Ph = 7.0 Hz, CH2O) 5.21 (bs, 2H, H-2) 5.91 (d, 1H,
JH2¢ = 5.6 Hz, H-1¢) 7.46 (d, JCH = 8.4 Hz, 2H, Ph) 7.95 (s, 1H,
H-8) 8.14 ppm (d, JCH = 8.4 Hz, 2H, Ph). 13C-NMR (50 MHz,
CDCl3) d -5.45, -5.40, -5.13, -4.76, -4.73, -4.34 [Si(CH3)2] 17.9,
18.0, 18.5 [SiC(CH3)3] 25.6, 25.8, 26.0 [SiC(CH3)3] 35.2 (CH2Ph)
62.7 (C-5¢) 66.0 (CH2O) 72.2 (C-3¢) 76.4 (C-2¢) 85.5 (C-4¢) 87.3
(C-1¢) 115.3 (C-5) 123.6 (2 ¥ Ph) 129.9 (2 ¥ Ph) 137.8 (C-8) 146.0
(C-NO2) 146.8 (Ph) 154.0 (C-4) 159.1 (C-2) 160.6 ppm (C-6). m/z
(ESI) 797.5 (M + Na)+.
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2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-O6 -[2-(4-nitrophenyl)-
ethyl]-2-fluoro-inosine (5b). A solution of 4b (775 mg, 1.00 mmol)
in anhydrous THF (2 mL) was added to a solution of HF/pyridine
70/30 (2.5 mL), pyridine (1 mL) and THF (1 mL) at -25 ◦C under
Ar. Immediately, t-Bu-ONO (714 mL, 6.00 mmol) was added and
the reaction mixture was stirred at -25 ◦C for 7 min exactly. The
reaction mixture was then poured into ice-water (25 mL) and
extracted with CH2Cl2 (5 ¥ 15 mL). The combined organic layers
were extracted with water (25 mL), then with a sat. NaHCO3
solution (25 mL) and finally with brine (25 mL). The organic
layer was dried over sodium sulfate, evaporated to dryness and the
residue was purified by column chromatography using 5:5 ethyl
acetate–hexane to afford 550 mg (71%) of the product. Rf: 0.75 in
JH5¢ = 2.6 Hz, H-4¢) 4.30 (dd, 1H, JH2¢ = 4.6, JH4¢ = 4.6 Hz, H-3¢)
4.62 (dd, 1H, JH1¢ = 4.6, JH3¢ = 4.6 Hz, H-2¢) 5.90 (d, 1H, JH2¢
=
4.6 Hz, H-1¢) 7.97 (s, 1H, H-8) 11.8 ppm (bs, 1H, H-2). 13C-NMR
(50 MHz, CDCl3) d -5.38, -5.32, -4.78, -4.68, -4.42 [Si(CH3)2]
6.89, 7.23 (2 ¥ CH2) 17.9, 18.1, 18.5 [SiC(CH3)3] 23.8 (CH) 25.7,
25.8, 26.0 [SiC(CH3)3] 62.4 (C-5¢) 71.7 (C-3¢) 75.3 (C-2¢) 84.9
(C-4¢) 87.9 (C-1¢) 116.9 (C-5) 135.5 (C-8) 156.7 (C-4) 157.4 (C-2)
158.7 ppm (C-6).
2¢,3¢,5¢-Tris-O -(tert-butyldimethylsilyl)-N2 -cyclopropyl-O6 -
(mesitylsulfonyl)guanosine (7b). To a solution of 6b (dried by
heating over phosphorus pentoxide in vacuo at 56 ◦C for 2 h before
use, 250 mg, 0.38 mmol) and Et3N (210 ml, 1.50 mmol) in 2 mL
of anhydrous CH2Cl2 was added DMAP (12 mg, 0.09 mmol) and
mesitylenechloride (180 mg, 0.83 mmol). The reaction mixture was
stirred at room temperature for 18 h. The solvent was removed
in vacuo and the residue was dissolved in 15 mL of CHCl3. The
CHCl3 solution was extracted first with water (15 mL), then with
a sat. NaHCO3 solution (15 mL) and finally with brine (15 mL).
The organic layer was dried over sodium sulfate and evaporated
to dryness. The residue was purified by column chromatography
using 15:85 ethyl acetate–hexane to give 210 mg of the product
(66%).
1
ethyl acetate–hexane 7:3. H-NMR (200 MHz, CDCl3) d -0.25,
-0.05 [s, 3H, Si(CH3)2] 0.08, 0.12 [s, 6H, Si(CH3)2] 0.78, 0.91, 0.93
[s, 9H, SiC(CH3)3] 3.31 (t, 2H, JCH2O = 6.8 Hz, CH2Ph) 3.77 (dd,
1H, JH4¢ = 2.6, JH5¢ = 11.4 Hz, H-5¢¢) 4.00 (dd, 1H, JH4¢ = 2.6,
JH5¢¢ = 11.4 Hz, H-5¢) 4.11 (dd, 1H, JH3¢ = 5.0, JH5¢ = 2.6 Hz,
H-4¢) 4.28 (dd, 1H, JH2¢ = 5.0, JH4¢ = 5.0 Hz, H-3¢) 4.56 (dd, 1H,
JH1¢ = 5.0, JH3¢ = 5.0 Hz, H-2¢) 4.82 (t, 2H, JCH2Ph = 6.8 Hz, CH2O)
5.96 (d, 1H, JH2¢ = 5.0 Hz, H-1¢) 7.47 (d, JCH = 8.6 Hz, 2H, Ph)
8.14 (d, JCH = 8.6 Hz, 2H, Ph) 8.26 ppm (s, 1H, H-8). 13C-NMR
(50 MHz, CDCl3) d -5.21, -4.46, -4.21 [Si(CH3)2] 18.0, 18.3, 18.7
[SiC(CH3)3] 25.8, 25.0, 26.3 [SiC(CH3)3] 35.2 (CH2Ph) 62.7 (C-5¢)
67.6 (CH2O) 72.1 (C-3¢) 76.2 (C-2¢) 85.9 (C-4¢) 88.7 (C-1¢) 118.3
(C-5) 124.0 (2 ¥ Ph) 130.1 (2 ¥ Ph) 142.0 (C-8) 145.5 (C-NO2)
147.1 (Ph) 155.9 (C-4) 160.1 (C-2) 162.0 ppm (C-6). 19F-NMR
(188 MHz, CDCl3) d -50.23 ppm.
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-N2 -cyclopropyl-O6 -[2-
(4-nitrophenyl)ethyl]guanosine. To a solution of 5b (550 mg,
0.70 mmol) and Et3N (90 mL, 0.85 mmol) in anhydrous DMF
(3.5 mL) was added cyclopropylamine (58 mL, 0.85 mmol) under
Ar and the reaction mixture was stirred at room temperature for
24 h. The reaction mixture was diluted with CHCl3 (35 mL) and
extracted first with water (20 mL), then with a sat. NaHCO3
solution (20 mL) and finally with brine (20 mL). The organic
layer was dried over sodium sulfate and evaporated to dryness.
The residue was used without further purification for the next
step. Rf: 0.72 in ethyl acetate–hexane 7:3. m/z (ESI) 813.5 [100,
(M - H+)-].
1
Rf: 0.29 in ethyl acetate–hexane 15:85. H-NMR (200 MHz,
CDCl3) d -0.22, -0.05 [s, 3H, Si(CH3)2] 0.09 [s, 12H, Si(CH3)2]
0.44–0.47 (m, 2H, CH2) 0.68–0.71 (m, 2H, CH2) 0.77 [s, 9H,
SiC(CH3)3] 0.91 [s, 18H, SiC(CH3)3] 2.28 (s, 3H, CH3) 2.54–2.62
(m, 1H, CH) 2.71 (s, 6H, 2 ¥ CH3) 3.77 (dd, 1H, JH4¢ = 2.4, JH5¢
=
11.0 Hz, H-5¢¢) 3.98 (dd, 1H, JH4¢ = 2.4, JH5¢¢ = 11.0 Hz, H-5¢) 4.07
(dd, 1H, JH3¢ = 5.0, JH5¢ = 2.4 Hz, H-4¢) 4.28 (dd, 1H, JH2¢ = 5.0,
JH4¢=5.0 Hz, H-3¢) 4.60 (dd, 1H, JH1¢ = 5.0, JH3¢ = 5.0 Hz, H-2¢)
5.12 (bs, 1H, H-2) 5.92 (d, 1H, JH2¢ = 5.0 Hz, H-1¢) 6.94 (s, 2H,
Ph) 8.01 ppm (s, 1H, H-8). 13C-NMR (50 MHz, CDCl3) d -5.46,
-4.99, -4.76, -4.50 [Si(CH3)2] 7.17, 7.35 (2 ¥ CH2) 17.7, 18.0, 18.4
[SiC(CH3)3] 21.0 (CH3) 22.7 (2 ¥ CH3) 24.2 (CH) 25.6, 25.7, 26.0
[SiC(CH3)3] 62.4 (C-5¢) 71.8 (C-3¢) 75.2 (C-2¢) 85.0 (C-4¢) 87.8 (C-
1¢) 116.5 (C-5) 129.7 (C-8) 131.5 (2 ¥ m-C) 139.6 (C-S) 140.1, 140.3
(o-C) 143.6 (p-C) 154.6 (C-4) 155.8 (C-2) 159.0 ppm (C-6).
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-N2-cyclopropyl-O6 -(2-
oxopyridin-1(2H)-yl)guanosine (8b). A solution of 7b (dried by
heating over phosphorus pentoxide in vacuo at 56 ◦C for 2 h
before use, 95 mg, 0.11 mmol) and DABCO (25 mg, 0.22 mmol)
in anhydrous dioxane (2 mL) was stirred for 1 h under Ar at room
temperature. Then, 2-oxopyridine N-oxide (62 mg, 0.56 mmol) was
added and after 30 min DBU (25 mL, 0.17 mmol) was added and
the reaction mixture was stirred in room temperature for 3 h. The
reaction mixture was diluted with CHCl3 (15 mL) and extracted
with a sat. NaHCO3 solution (2 ¥ 15 mL) and brine (15 mL).
The organic layer was dried over sodium sulfate and evaporated
to dryness. The residue was purified by column chromatography
using 95:5 CHCl3–MeOH to afford 80 mg of the product (94%)
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-N2 -cyclopropyl-guano-
sine (6b). To a solution of 2¢,3¢,5¢-Tris-O-(tert-butyldimethyl-
silyl)-N2-cyclopropyl-O6-[2-(4-nitrophenyl)ethyl]guanosine (we
consider that the previous reaction was quantitative, 570 mg,
0.70 mmol) in anhydrous pyridine (8 mL) was added DBU
0.5 M (600 mL, 4.00 mmol) under Ar and the reaction mixture
was stirred at room temperature for 3 h. The reaction mixture
was diluted with CHCl3 (20 mL) and extracted first with water
(20 mL), then with a sat. NaHCO3 solution (20 mL) and finally
with brine (20 mL). The organic layer was dried over sodium
sulfate, evaporated to dryness and the residue was purified by
column chromatography using 97:3 CHCl3–MeOH to afford
250 mg of the product as a white solid (overall yield 53%). Rf: 0.29
1
as a yellowish solid. Rf: 0.35 in CHCl3–MeOH 95:5. H-NMR
(200 MHz, CDCl3) d -0.03, 0.01, 0.08, 0.09, 0.11, 0.12, 0.13 [s,
3H, Si(CH3)2] 0.39–0.47 (m, 2H, CH2) 0.61–0.64 (m, 2H, CH2)
0.84, 0.91, 0.93 [s, 9H, SiC(CH3)3] 2.53–2.56 (m, 1H, CH) 3.78
(dd, 1H, JH4¢ = 2.2, JH5¢ = 11.4 Hz, H-5¢¢) 4.04 (dd, 1H, JH4¢ = 2.2,
1
in CHCl3–MeOH 97:3. H-NMR (200 MHz, CDCl3) d -0.12,
-0.01 [s, 3H, Si(CH3)2] 0.11 [s, 12H, Si(CH3)2] 0.76–0.86 (m, 4H,
2 ¥ CH2) 0.83 [s, 9H, SiC(CH3)3] 0.93 [s, 18H, SiC(CH3)3] 2.75
(m, 1H, CH) 3.80 (dd, 1H, JH4¢ = 2.6, JH5¢ = 11.4 Hz, H-5¢¢) 3.98
(dd, 1H, JH4¢ = 2.6, JH5¢¢ = 11.4 Hz, H-5¢) 4.06 (dd, 1H, JH3¢ = 4.6,
JH5¢¢ = 11.4 Hz, H-5¢) 4.08–4.10 (m, 1H, H-4¢) 4.33 (dd, 1H, JH2¢
=
4.4, JH4¢ = 4.4 Hz, H-3¢) 4.48 (dd, 1H, JH1¢ = 4.4, JH3¢ = 4.4 Hz,
H-2¢) 5.14 (bs, 2H, NH2) 5.93 (d, 1H, JH2¢ = 4.4 Hz, H-1¢) 6.14 (dt,
4970 | Org. Biomol. Chem., 2009, 7, 4965–4972
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1H, OPyH-5) 6.73 (dd, 1H, OPyH-3) 7.35 (dt, 1H, OPyH-4) 7.51
(dd, 1H, OPyH-6) 8.14 ppm (s, 1H, H-8). 13C-NMR (50 MHz,
CDCl3) d -5.46, -5.37, -4.81, -4.55, -4.36 [Si(CH3)2] 7.09, 7.24
(2 ¥ CH2) 17.9, 18.0, 18.5 [SiC(CH3)3] 24.1 (CH) 25.7, 25.8, 26.1
[SiC(CH3)3] 61.8 (C-5¢) 70.8 (C-3¢) 75.6 (C-2¢) 84.0 (C-4¢) 88.4
(C-1¢) 104.6 (OPyC-5) 112.1 (C-5) 123.1 (OPyC-3) 136.2 (OPyC-
6) 138.9 (OPyC-4) 139.6 (C-8) 155.3 (C-2) 157.5 (C-6) 158.4
(OPyC-O) 159.1 ppm (C-4).
19F-NMR: (188 MHz, CDCl3) d -49.74 ppm. m/z (ESI) 811.3
[100, (M + H)+].
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-2-fluoro-O6 -(2-oxopy-
ridin-1(2H)-yl)inosine (10b). A solution o◦f 9b (dried by heating
over phosphorus pentoxide in vacuo at 56 C for 2 h before use,
120 mg, 0.15 mmol) and DABCO (34 mg, 0.30 mmol) in anhydrous
dioxane (2 mL) was stirred for 1 h under Ar at room temperature.
Then, 2-oxopyridine N-oxide (83 mg, 0.75 mmol) was added and
after 30 min DBU (34 mL, 0.23 mmol) was added and the reaction
mixture was stirred at room temperature for 3 h. The reaction
mixture was diluted with CHCl3 (15 mL) and extracted with a sat.
NaHCO3 solution (15 mL) and brine (2 ¥ 15 mL). The organic
layer was dried over sodium sulfate and evaporated to dryness.
The residue was purified by column chromatography using 6:4
ethyl acetate–hexane to afford 102 mg of the product (96%). Rf:
N2-Cyclopropyl-O6-(2-oxopyridin-1(2H)-yl)guanosine
(8d).
To a solution of 8b (dried by heating over phosphorus pentoxide
in vacuo at 56 ◦C for 2 h before use, 38 mg, 0.05 mmol) in
anhydrous THF (1 mL) were added activated molecular sieves
(25 mg) and a 1 M solution of TBAF in THF (250 mL, 0.25 mmol).
The reaction mixture was stirred for 1 h at room temperature
under Ar, filtrated and washed with MeOH (15 mL). The filtrate
was evaporated to dryness and the residue was purified by
column chromatography using 9:1 CH2Cl2–EtOH to give 15 mg
1
0.43 in ethyl acetate–hexane 6:4. H-NMR (200 MHz, CDCl3) d
-0.05, 0.03, 0.06, 0.09, 0.14, 0.16 [s, 3H, Si(CH3)2] 0.85, 0.92, 0.96
[s, 9H, SiC(CH3)3] 3.80 (dd, 1H, JH4¢ = 1.8, JH5¢ = 11.6 Hz, H-5¢¢)
4.06 (dd, 1H, JH4¢ = 1.8, JH5¢¢ = 11.6 Hz, H-5¢) 4.13–4.18 (m, 1H,
H-4¢) 4.32 (dd, 1H, JH2¢ = 5.0, JH4¢ = 5.0 Hz, H-3¢) 4.46 (dd, 1H,
JH1¢ = 5.0, JH3¢ = 5.0 Hz, H-2¢) 5.99 (d, 1H, JH2¢ = 5.0 Hz, H-1¢)
6.26 (dt, 1H, J = 8.4, 7.4, 1.6 Hz, OPyH-5) 6.78 (dd, 1H, J =
9.4, 1.6 Hz, OPyH-3) 7.44 (dt, 1H, J = 9.4, 7.4, 1.8 Hz, OPyH-4)
7.58 (dd, 1H, J = 8.4, 1.8 Hz, OPyH-6) 8.54 ppm (s, 1H, H-8).
19F-NMR: (188 MHz, CDCl3) d -49.64 ppm.
1
of the product (75%). Rf: 0.19 in CHCl3–MeOH 93:7. H-NMR
(200 MHz, CDCl3) d 0.34–0.38 (m, 2H, CH2) 0.46–0.52 (m, 2H,
CH2) 2.37–2.39 (m, 1H, CH) 3.65 (dd, 1H, JH4¢ = 2.6, JH5¢
=
11.2 Hz, H-5¢¢) 4.04 (dd, 1H, JH4¢ = 2.6, JH5¢¢ = 11.2 Hz, H-5¢) 4.17
(m, 1H, H-4¢) 4.28–4.30 (m, 1H, H-3¢) 4.73 (m, 1H, H-2¢) 5.71
(d, 1H, JH2¢ = 6.8 Hz, H-1¢) 6.27 (dt, 1H, OPyH-5) 6.70 (dd, 1H,
OPyH-3) 7.42 (dt, 1H, OPyH-4) 7.57 (dd, 1H, OPyH-6) 7.78 ppm
(s, 1H, H-8). 13C-NMR (50 MHz, CDCl3) d 6.92, 7.26 (2 ¥ CH2)
24.2 (CH) 61.5 (C-5¢) 70.9 (C-3¢) 75.9 (C-2¢) 84.6 (C-4¢) 88.0 (C-1¢)
104.6 (OPyC-5) 112.3 (C-5) 122.7 (OPyC-3) 137.4 (OPyC-6)
139.0 (C-8) 139.7 (OPyC-4) 155.5 (C-6) 157.0 (C-2) 158.9 (C-4)
159.3 ppm (OPyC-O). UV (H2O, nm) 247 (e = 7956 M-1cm-1), 292
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-2-fluoro-6-cyclopropyl-
aminopurine riboside (11b). To a solution of 10b (13 mg,
0.018 mmol) and Et3N (38 mL, 0.27 mmol) in anhydrous DMF
(1 mL) was added cyclopropylamine (1.9 mL, 0.027 mmol) under
Ar and the reaction mixture was stirred at room temperature
for 24 h. The reaction mixture is diluted with CHCl3 (5 mL)
and extracted first with water (5 mL), then with a sat. NaHCO3
solution (5 mL) and finally with brine (5 mL). The organic layer
was dried over sodium sulfate and evaporated to dryness. The
residue was purified by column chromatography using 2:8 ethyl
acetate–hexane to afford 10 mg (73%) of the product. Rf: 0.52 in
ethyl acetate–hexane 35:65. 1H-NMR (200 MHz, CDCl3) d -0.16,
-0.01 [s, 3H, Si(CH3)2] 0.09, 0.14 [s, 6H, Si(CH3)2] 0.63–0.71 (m,
2H, CH2) 0.82 [s, 9H, SiC(CH3)3] 0.84–0.87 (m, 2H, CH2) 0.92,
0.95 [s, 9H, SiC(CH3)3] 3.03 (m, 1H, CH) 3.73 (dd, 1H, JH4¢ = 2.8,
JH5¢ = 11.4 Hz, H-5¢¢) 4.02 (dd, 1H, JH4¢ = 2.8, JH5¢¢ = 11.4 Hz,
H-5¢) 4.12 (dd, 1H, JH3¢ = 4.6, JH5¢ = 2.8 Hz, H-4¢) 4.29 (dd, 1H,
(e = 13412 M-1cm-1). HR-ESI-MS for C18H21N6O6 [M + H]+,
+
calcd 417.1517; found 417.1534.
2¢,3¢,5¢-Tris-O-(tert-butyldimethylsilyl)-O6 -(mesitylsulfonyl)-2-
fluoro-inosine (9b). A solution of 1b (400 mg, 0.50 mmol) in
anhydrous THF (2.4 mL) was added to a solution of HF/pyridine
70/30 (4 mL), pyridine (1.2 mL) and THF (1.2 mL) at -25 ◦C
under Ar. Immediately, t-Bu-ONO (354 mL, 3.00 mmol) was added
and the reaction mixture was stirred at -25 ◦C for 7 min exactly.
The reaction mixture was then poured into ice-water (25 mL) and
extracted with CH2Cl2 (5 ¥ 15 mL). The combined organic layers
were extracted with water (25 mL), then with a sat. NaHCO3
solution (25 mL) and finally with brine (25 mL). The organic
layer was dried over sodium sulfate, evaporated to dryness and the
residue was purified by column chromatography using 2% EtOH
in CH2Cl2 to afford 176 mg (44%) of the product. Rf: 0.75 in
CH2Cl2–EtOH 98:2. 1H-NMR (200 MHz, CDCl3) d -0.25, -0.04
[s, 3H, Si(CH3)2] 0.09, 0.13 [s, 6H, Si(CH3)2] 0.78 [s, 9H, SiC(CH3)3]
0.92, 0.94 [s, 18H, SiC(CH3)3] 2.31 (s, 3H, CH3) 2.77 (s, 6H, 2 ¥
CH3) 3.77 (dd, 1H, JH4¢ = 3.0, JH5¢ = 12.0 Hz, H-5¢¢) 3.99 (dd, 1H,
JH2¢ = 4.6, JH4¢ = 4.6 Hz, H-3¢) 4.60 (dd, 1H, JH1¢ = 4.6, JH3¢
=
4.6 Hz, H-2¢) 5.91 (d, 1H, JH2¢ = 4.6 Hz, H-1¢) 8.16 ppm (s, 1H,
H-8). 13C-NMR (50 MHz, CDCl3) d -5.21, -4.84, -4.54, -4.49,
-4.19 [Si(CH3)2] 7.50, 7.64 (2 ¥ CH2) 18.0, 18.3, 18.7 [SiC(CH3)3]
24.0 (CH) 25.9, 26.0, 26.3 [SiC(CH3)3] 62.6 (C-5¢) 72.0 (C-3¢) 75.7
(C-2¢) 85.6 (C-4¢) 88.8 (C-1¢) 131.8 (C-5) 139.5 (C-8) 157.3 (C-
4) 157.7 (C-6) 161.6 ppm (C-2); 19F-NMR: (188 MHz, CDCl3) d
-49.45 ppm. m/z (ESI) 668.3 (M + H)+.
JH4¢ = 3.0, JH5¢¢ = 12.0 Hz, H-5¢) 4.12 (dd, 1H, JH3¢ = 5.0, JH5¢
=
3.0 Hz, H-4¢) 4.28 (dd, 1H, JH2¢ = 5.0, JH4¢ = 5.0 Hz, H-3¢) 4.52
(dd, 1H, JH1¢ = 5.0, JH3¢ = 5.0 Hz, H-2¢) 5.96 (d, 1H, JH2¢ = 5.0 Hz,
H-1¢) 6.99 (s, 2H, Ph) 8.40 ppm (s, 1H, H-8). 13C-NMR (50 MHz,
CDCl3) d -5.56, -5.52, -5.28, -4.88, -4.81, -4.53 [Si(CH3)2] 17.7,
17.9, 18.4 [SiC(CH3)3] 21.0 (CH3) 22.6 (2 ¥ CH3) 25.4, 25.7, 25.9
[SiC(CH3)3] 62.2 (C-5¢) 71.6 (C-3¢) 76.0 (C-2¢) 85.7 (C-4¢) 88.6
(C-1¢) 105.9 (C-5) 131.7 (2 ¥ m-C) 140.8 (C-8) 143.8, 143.9 (o-C)
144.4 (p-C) 151.1 (C-S) 154.4 (C-4) 158.7 (C-6) 160.7 ppm(C-2).
Acknowledgements
Work supported in part by the European Community’s Marie
Curie RTN (6th framework) program under contract MRTN-
CT-2003-505086 (http://clustoxdna.chem.uoa.gr). The project
was co-funded by the European Social Fund and National
Resources – (EPEAEK II) PYTHAGORAS II. FT-HRMS from
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Prof. T. Carell’s laboratory and financial support to P.K. from
the COST action CM0603 entitled “Free Radicals in Chemical
Biology” are highly appreciated.
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