Fluorescent p-substituted-phenyl-imidazolo-cytidine analogues

Article abstract of DOI:10.1016/j.tet.2013.03.005

The synthesis and spectral properties of modified cytidine analogues are described here. To confer fluorescent properties, the cytidine fluorophore was extended by a cinnamyl moiety at the cytidine 5-position, (analogues 1a-c), or extended by a phenyl-imi

Full text of DOI:10.1016/j.tet.2013.03.005

Tetrahedron 69 (2013) 3698e3705
Contents lists available at SciVerse ScienceDirect
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journal homepage: www.elsevier.com/locate/tet
Fluorescent p-substituted-phenyl-imidazolo-cytidine analogues
y
y
*
Marina Kovaliov , Meirav Segal , Bilha Fischer
Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and spectral properties of modified cytidine analogues are described here. To confer
fluorescent properties, the cytidine fluorophore was extended by a cinnamyl moiety at the cytidine
5-position, (analogues 1aec), or extended by a phenyl-imidazolo ring, (analogues 3aed). Those com-
pounds were synthesized via the SuzukieMiyaura coupling reaction and via condensation of 5-amino-
cytidine with p-substituted benzaldehydes, respectively. All analogues were fluorescent in the blue
region (lem: 402e436 nm). Among the above mentioned analogues, p-CF3ephenyl-imidazolo-cytidine,
3d, was found to be a promising fluorescent probe, exhibiting a quantum yield 7000-fold larger than
Received 12 December 2012
Received in revised form 14 February 2013
Accepted 4 March 2013
Available online 13 March 2013
Keywords:
Nucleoside
Cytidine
Fluorescent probe
Imidazolo cytidine
Base-paring
cytidine (
F
¼0.617) and a red-shift of ca. 108 nm of maximum emission (411 nm). Its retained Wat-
soneCrick hydrogen bonding face allows 3d to base-pair specifically with guanosine, similar to cytidine.
In addition, phenyl-imidazolo-cytidine analogues prefer anti conformation and the C30-endo (N) sugar
puckering. These properties make 3d an attractive fluorescent probe for potential use in the various
facets of nucleic acid chemistry.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
were incorporated into an oligonucleotide, which in turn, was
used as a probe for detection of genetic material in cells. The
The fluorescence of natural nucleotide bases is weak, their
fluorescent decay times are short, and the signals are averaged over
all of the bases in the oligonucleotide, resulting in little useful
information.1e5 Cytidine, for example, displays a quantum yield of
0.00008, absorption around 261 nm, and an emission band cen-
tered around 303 nm.3 Over the past decade, fluorescent nucleoside
analogues have gained notable attention due to their important
applications, particularly as pharmaceutical agents and biological
probes or mimetics.6e8 Applications of those analogues include
DNA sequencing for SNP (single nucleotide polymorphism) iden-
tification,9e14 study of nucleic acid dynamics, and nucleic acid-
dligand or protein interactions.7,15e18
photophysical properties of the above mentioned fluorescent
nucleosides were either slightly changed or remained unchanged
upon their incorporation into the oligonucleotides.22e35
We have recently reported that appending various alkenylearyl
moieties at the uridine 5-position, generates a family of responsive
fluorescent nucleoside analogues exhibiting emission in the visible
range andquantumyields upto0.24.36 Here, wereportthe design and
synthesis of a novel series of cytidine-based fluorescent probes, 1e3
(Fig. 1), as well as their spectroscopic and conformational properties.
In addition, we examined the formation of a WatsoneCrick base pair
between guanosine and the most promising fluorescent cytidine
analogue 3d.
Fluorescent nucleoside probes may be divided into two general
classes: those bearing an appended fluorophore, separated elec-
tronically and spatially from the base-paring face,19e21 and those in
which the nucleobase is integral to the fluorophore.22e38 Members
of the latter class are especially attractive because their fluorescent
response can reveal the microenviroment of the nucleobase.
This notion has been applied in fluorescent cytidine nucleosides
analogues, such as pyrrolo-cytidine (pC),22 phenyl-pyrrolo-
cytidine (pCPhe),23 tricyclic 1,3-diaza-2-oxophenothiazinet (tC),24
and 5-furyl deoxycytidine.25,26 These fluorescent nucleosides
* Corresponding author. Tel.: þ972 3 5318303; fax: þ972 3 6354907; e-mail
address: bilha.fischer@biu.ac.il (B. Fischer).
y
These authors contributed equally to this work.
Fig. 1. Novel fluorescent cytidine analogues, 1e3 synthesized in this study.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.03.005

M. Kovaliov et al. / Tetrahedron 69 (2013) 3698e3705
3699
2. Results and discussion
2.1. Probe design
mixtures). All the reactions proceeded reasonably well at 100 ꢀC to
reach full conversion in 3 h. The products were isolated in acceptable
yields of 54e64% after column chromatography.40,41
Our basic design for the development of novel intrinsically
fluorescent cytidine relies on extension of the natural nucleobase
by a conjugated system (e.g., a cinnamyl moiety) or by fusion to
a heterocyclic system. To design fluorescent probes based on suit-
able extensions of the cytosine chromophore, we applied the fol-
lowing strategy: (1) Selection of minimal extensions of the natural
nucleobase at positions not involved in H-bonding with a comple-
mentary base. (2) Selection of probes that may be excited and may
emit at the longest possible wavelengths. (3) Selection of exten-
sions of the cytosine base that can be achieved via a simple and
short synthesis.
2.3. Synthesis of imidazolo-cytidine derivatives 3aed
For the synthesis of imidazolo-cytidine derivatives, cytosine, 6,
was converted to 5-nitro-cytosine, 7, by HNO3/H2SO4 in 64% yield
(Scheme 2).42 Nitration reaction was performed on the base, cy-
tosine, and not on the nucleoside, cytidine, because of the drastic
reaction conditions (pH¼1e2), that would cleave the glycosidic
bond. Next, 5-nitro-cytosine was coupled to protected ribose.
Compound 7, was reacted with 1,1,1,3,3,3-hexamethyldisilazane
(HMDS) to provide 5-nitro-cytosine trimethylsilyl derivative,
which was directly reacted with, 10-acetate-20,30,50-tribenzoate-
b-
In a previous publicationwe reported four types of C5 substituted
uridine analogues where the aromatic systems are: (a) directly
coupled, (b) coupled via an alkenyl linker, (c) coupled via an alkynyl
linker, and (d) coupled via a dienyl linker.36 Analogues in which the
uracil chromophore was extended by a cinnamyl moiety, exhibited
the longest absorption and emission wavelengths and the highest
D
-ribose with stannic tetrachloride (SnCl4) in dichloromethane at
room temperature to give 8 in 70% yield.43 Reduction of 8 by hy-
drogenation with 10% Pd/C/H2 in THF and AcOH provided 9 in 38%
yield. Product 9 is used as a starting material for the preparation of
cytidine derivatives 7, 8 (Scheme 3). Deprotection of 8 with NaOH in
aq EtOH, gave 5-amino cytidine 2 in 70% yield.44
quantum yields, (labs 318 nm, lem 476 nm,
F 0.24), as compared to
uridine.36
Based on our previous findings for uridine fluorescent ana-
logues, we have now designed and synthesized their cytidine
counterparts. We targeted the preparation of pushepull cytidine
containing systems to achieve a maximal enhancement of fluo-
rescent properties.37 Since we did not know whether cytidine plays
the relative role of an electron rich or poor moiety, we have ex-
tended the cytosine scaffold by p-OMe, p-F, and p-CF3 substituted
cinnamyl moieties, to form analogues 1a, 1b, and 1c (Fig. 1). We
selected to couple the cinnamyl moiety at the cytosine C5 position,
since substitution at C6 will drive the nucleoside into the undesired
syn conformation. In addition, we designed bicyclic analogues of
cytidine based on an imidazolo-cytidine scaffold, compounds
3(aed). In the case of phenyl-imidazolo-cytidine analogues, we
prepared compounds bearing various substituents on the para
position of the phenyl group, e.g., H, 3a, p-Me, 3b, p-OMe, 3c, and
p-CF3, 3d.
Scheme 2. Reaction conditions; (a) concd H2SO4, fuming HNO3; (b) i. HMDS, TMSeCl,
reflux, 36 h, ii. 1 M SnCl4 in CH2Cl2, 10-acetate-20,30,50-tribenzoate-
b-D-ribose, CH3CN,
rt, 3 h; (c) 10% Pd/C/H2, THF, AcOH, 6 h; (d) NaOH in aq EtOH, rt, 3 h.
2.2. Synthesis of 5-(cinnamyl)-substituted-cytidine de-
rivatives 1aec
Suzuki coupling reactions are highly useful for the preparation
of biaryl compounds. The Pd-catalyzed SuzukieMiyaura reaction
proved to be effective also for nucleosides cross-coupling38 and was
applied here for the preparation of derivatives 1aec, (Scheme 1).39
Scheme 3. Reaction conditions; (a) appropriate aldehyde 10aed, cat. p-TsOH, 80 ꢀC;
(b) 1 M NaOH in MeOH, rt, 0.5 h.
Scheme 1. Reaction conditions; (a) appropriate trans-vinylboronic acid 5(aec),
0.25 equiv TPPTS, 0.05 equiv Pd(OAc)2, 3 equiv Na2CO3, H2O/CH3CN (2:1), reflux, 3 h.
Thus, coupling of 5-Brecytidine, 4 and the appropriate aryl
boronic acid derivative, 5aec, was performed, using 5 mol % of
Pd(OAc)2, water-soluble tris(3-sulfonatophenyl)phosphane (TPPTS)
ligand, and Na2CO3 in aqueous-phase conditions (water/acetonitrile
Synthesis of bicyclic phenyl-imidazolo-cytidine analogues was
achieved by condensation of protected 5-amino-cytidine 9 with
aryl aldehyde 10aed, in the presence of p-TsOH (20 mol %), in DMF
at 80 ꢀC, to give 11aed in 36e47% yield.45 Addition of 1 M NaOH in

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M. Kovaliov et al. / Tetrahedron 69 (2013) 3698e3705
MeOH, at room temperature for 1 h, yielded 3aed, in 80e85% yield
(Scheme 3).
that the ribose exocyclic group exists predominantly in a gau-
cheegauche (gg) conformation about the C40eC50 bond with the O50
atom projecting over the furanose ring. Here, we employed 1H, and
13C NMR spectroscopy to analyze the solution conformations of
representative nucleoside analogues, 1a and 3b.
2.4. Spectral properties of cytidine derivatives 1e3
We measured the absorption and emission of analogues 1aec
and intermittent compound 2 in H2O, and analogues 3aed in MeOH,
due to solubility consideration. The spectral data for derivatives 1e3
are summarized in Table 1. All compounds were fluorescent in the
blue region (lem: 402e436 nm). Although analogues 1a, 1b, and 1c,
p-substituted by OCH3, F, and CF3, respectively, showed relatively
long emission wavelengths, their quantum yields were low, ranging
from 0.017 to 0.052. However 3aed, in which the chromophore is
extended by fusion to a phenyl-imidazolo-ring system, exhibited
reasonably long absorption and emission wavelengths and up to 36-
fold higher quantum yields as compared to those of 2 and 3aed.
2.5.1. Conformation around the glycosidic bond. Pyrimidine nucle-
osides can adopt two main conformations, syn or anti, in which the
oxygen on C-2 of cytidine is projecting away or toward the sugar
ring. The quantitative determination of the conformation around
the glycosidic bond can be obtained by monitoring the vicinal
3
3
0
0
coupling constants JC6eH1 and JC2eH1 , which are extracted from
13C NMR spectra. A practical rule for the orientation of the base
relative to the ribose was formulated: a value of 3JC2eH1 < JC6eH1
3
0
0
indicates that
indicates that
c
is in the anti conformation, whereas the reverse
c
is in the syn conformation.49
Ippel et al. reparametrized and generalized the Karplus equa-
tions for the glycosidic bond conformation of purine and pyrimi-
dine nucleosides and nucleotides.49 Eqs. 1 and 2 were used in this
Specifically, compounds 3c and 3d, for which
respectively, in MeOH.
F¼0.233 and 0.617,
study to calculate the glycosidic bond angle
c
(O40eC10eN1eC2) in
0
3
nucleoside 1a based on 3JC6eH1 and JC2eH1 values.50,51
Table 1
0
Fluorescent properties of analogues 6e8
3JC6ꢁH10 ¼ 4:5cos2ð
3JC2ꢁH10 ¼ 4:7cos2ð
c
c
ꢁ 60Þ ꢁ 0:6cosð
c
c
ꢁ 60Þ þ 0:1
ꢁ 60Þ þ 0:1
(1)
(2)
Compound
labs (nm)
lem (nm)
F
Solvent
Cytidine
1a
1b
1c
2
3a
3b
3c
3d
261
286
285
297
296
342
344
347
346
303
436
430
424
430
402
403
405
411
0.00008a
0.052a
0.019a
0.017a
0.089b
0.215b
0.198b
0.233b
0.617b
H2O
H2O
H2O
H2O
ꢁ 60Þ þ 2:4cosð
In the case of 3b some of the NMR signals were broaden,
probably because of the tautomeric equilibrium of the imidazole
ring. Hence, small vicinal coupling constants JC6eH1 and JC2eH1
H2O
MeOH
MeOH
MeOH
MeOH
3
3
0
0
,
could not be observed (Fig. 2). NOE experiments are valuable for
examining relative proton positions and deducing the anti or syn
conformation of the nucleoside.52 The conformation of analogue 3b
was determined by cross peaks between H-6 and H-10/20/30 in
NOESY spectrum.53 NOE interactions in compounds 1a, and 3b
were observed between H-6 and H-10/20/30 (Fig. 3). These in-
teractions suggest that the conformation of these compounds is
anti, similar to the conformation of the natural cytidine. Moreover,
a
Data obtained with tryptophan as a reference.
Data obtained with quinine sulfate as a reference.
b
In the bicyclic phenyl-imidazolo nucleosides 3 series, the phenyl
ring substituent caused a significant effect on the fluorescent
properties. Thus, compounds 3b and 3c bearing OMe and Me at the
para position of the phenyl ring, showed very similar fluorescent
properties compared to 3a. However, p-CF3 substitution, in 3d
versus 3a, resulted in enhancement of both lem and quantum yield,
411 nm and 0.617, respectively. Neither analogues 1, nor analogues
3 exhibited characteristics of pushepull systems. In analogues 6
neither electron withdrawing (1b, c), nor electron donating sub-
stituents changed the photophysical behavior of the analogues. No
clear dependence on the electronic character of the substituent was
observed for analogues 3.
calculated
c values for the C5-substitued nucleosides 1a were
within the range defined as anti (Table 2), which concurs with
previously published data.54
In conclusion, all tested cytidine analogues 1e3 showed signif-
icantly improved spectral properties as compared to cytidine.
Analogues in which the cytosine was fused phenyl-imidazolo
moiety exhibited the most promising spectral data, up to 7000-
fold enhancement of the quantum yield of cytidine and red-shift
of ca. 108 nm of maximum emission.
Fig. 2. Tautomers of compound 3b.
2.5. Conformational analysis of cytidine derivatives
2.5.2. Sugar puckering. The ribose moieties of nucleosides most
often adopt either a C30-endo (north, N) or a C20-endo (south, S)
orientation.47 The conformation of the ribose ring of nucleosides 1a
and 3b was analyzed in terms of a dynamic equilibrium between
two favored puckered conformations: North conformer and South
conformer.
Cytosine nucleosides are expected to possess conformational
flexibility, due to possible rotations around the glycosidic bond and
pseudorotation of the ribose ring as well as possible rotations around
the C40eC50 bond angle (
g). However, it is well-established that most
nucleosides adopt a predominant conformation in solution.46 Thus,
it has been shown that the majority of purine and pyrimidine nu-
cleosides favor an anti conformation of the base ring with respect to
the sugar ring.47 Likewise, the ribose ring exhibits a puckered con-
formation in which either the C20 or C30 atom is furthest from the
plane of the other atoms of the ribose ring, named as south (S) and
north (N) conformations, respectively.48 Finally, it has been shown
N and S equilibrium populations were calculated from the
observed J1 2 and J3 4 couplings.51 According to this method, the
observed vicinal couplings are related to the relative proportion
of conformers by Eqs. 3e5:
0
0
0 0
J1020 ¼ 9:3ð1 ꢁ cNÞ ¼ 9:3cS
(3)

M. Kovaliov et al. / Tetrahedron 69 (2013) 3698e3705
3701
classical staggered rotamers, with a preferred gaucheegauche (gg)
conformation. The mole fractions of each staggered rotamer of
C40eC50 were calculated from the following expressions (Eqs. 6e8):
r
r
r
gg ¼ ½ðJt þ JgÞ ꢁ ðJ4050 þ J40500Þꢂ ðJt ꢁ JgÞ
(6)
(7)
(8)
=
tg ¼ ðJ4050 ꢁ JgÞ ðJt ꢁ JgÞ
=
gt ¼ ðJ40500 ꢁ JgÞ=ðJt ꢁ JgÞ
The observed proton signals, labeled H50 and H500, refer to
downfield and upfield signals, respectively. The percentages of gg,
gt, and tg conformers are presented in Table 2. For analogues 1a and
3b there is a clear preference for the gg rotamer around the C40eC50
bond, 88 and 84%, respectively.
2.6. Compound 3d preserves natural H-bonding pattern with
guanosine
Natural base pairing of nucleic acids is essential for
hybridization-based diagnostics.56 Therefore, we examined here
the formation of a WatsoneCrick base pair between guanosine and
p-CF3ephenyl-imidazolo-cytidine, 3d.
Hydrogen bonding results in the formation of planar base pairs
and hence NH2 and NH protons are deshielded due to the presence
of ring currents in the same plane. Since we could not obtain 1H
NMR data on the N3eH of compound 3d, due to tautomerization of
this proton, we studied the interaction of guanosine with 3d by
high-field 1H NMR (700 MHz) and focused on the NH2 and NH
protons of G. Previous NMR studies have shown hydrogen-bonded
association between guanosine and cytidine in DMSO-d6 solu-
tion.57 Here, we applied the same technique with our cytidine an-
alogue 3d. We have obtained data at a range of concentrations of 3d
with constant concentration of guanosine (0.02 M) in DMSO-d6
solution, up to 1.5:1 ratio. The monitoring of spectral changes due
to H-bonding was carried out on the N2eNH2 and N1eNH protons
of guanosine. Upon hydrogen bonding, N2eamino and N1eNH
guanosine protons are deshielded due to the weakening of the NeH
covalent bond. Indeed, downfield shifts of the these protons were
observed with increasing concentrations of analogue 3d with
maximum Dd of 16.61 and 19.53 Hz, respectively, indicating hy-
drogen bonding between guanosine and compound 3d (Fig. 4).
Fig. 3. NOE interactions within compounds (A) 1a, (B) 3b.
3. Conclusions
Table 2
Conformational parameters of analogues 1a and 3b in DMSO-d6 solution
In summary, a novel family of fluorescent cytosine nucleoside
analogues has been prepared and their photophysical and base-
pairing properties have been evaluated. Phenyl-imidazolo-
cytidine compounds 3aed showed high quantum yields. Specifi-
cally, 3d exhibited the highest quantum yield, 0.617 and excitation
and emission maxima of 346 and 411 nm, making it a suitable probe
for study of nucleic acid structure, dynamics, and interactions.
Analogues 3 retain the conformation of cytidine. Futhermore, its
unperturbed WatsoneCrick face allows 3d to base-pair specifically
with guanosine, similar to an unmodified cytidine. Hence, we
suggest 3d as promising cytidine-based fluorescent probe.
Compound
Sugar
Conformer population
Conformation
around glycosidic
puckering %N around C40eC50 bond
bond
c
% gg
% tg
% gt
1a
3b
65
73
88
84
8
8
4
8
anti
anti
J2030 ¼ 4:6cN þ 5:3ð1 ꢁ cN
J3040 ¼ 9:3cN
Þ
(4)
(5)
The mole fraction of conformers S and N can be calculated di-
0
0
0 0
rectly from the observed values of J1 2 and J3 4 . Using the assigned
J-coupling constants and the above equations, the mole fractions of
conformers S and N for nucleoside 1a and 3b were calculated. The
data reveal that analogues 1a and 3b, similar to cytidine55 all prefer
the C30-endo (N) conformation and consist 65 and 73% of the total
population, respectively (Table 2).
4. Experimental section
4.1. General procedure for the synthesis of 5-(cinnamyl)-
substituted-cytidine derivatives (1aec): 4-amino-1-(3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-(4-
methoxystyryl)pyrimidin-2(1H)-one (1a)
2.5.3. Conformations of the exocyclic CH2OH group. The nucleoside
Water/acetonitrile (2:1, 1.6 mL) was added through a septum to
0
0
0 00
coupling constants J4 5 and J4 5 can be interpreted in terms of three
a nitrogen-purged round bottom flask containing 5-Brecytidine,

3702
M. Kovaliov et al. / Tetrahedron 69 (2013) 3698e3705
134.2, 128.7, 128.7, 126.2, 119.7, 115.8, 115.6, 104.2, 90.1, 84.1, 74.8,
68.8, 59.9 ppþm. HRMS (MALDI-TOF) m/z: calculated for
C
17H18FN3NaO5 : 386.112 (MNaþ). Found: 386.109 (MNaþ).
4.3. 4-Amino-1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahy-
drofuran-2-yl)-5-(4-(trifluoromethyl)styryl)pyrimidin-2(1H)-
one (1c)
Product 1c was obtained from 4 (143 mg, 0.48 mmol) and 5c
(130 mg, 0.60 mmol) according to the general procedure. Product
1c was obtained as a white solid in 60% yield (118 mg). Mp
115e117 ꢀC. [
a
]
22 ꢁ25 (c 1.15, DMSO). IR (ZnSe):
n 3334, 2248, 2126,
D
1644, 1485, 1325, 1193, 1109, 1047, 1023, 996, 826, 765, 618 cmꢁ1. 1H
NMR (300 MHz, DMSO-d6):
d 8.62 (s, 1H), 7.76e7.66 (m, 4H), 7.39
(s, 2H), 7.26 (d, J¼15.8 Hz, 1H), 6.89 (d, J¼15.8 Hz, 1H), 5.75
(d, J¼2.3 Hz, 1H), 5.44e5.40 (m, 2H), 4.99e5.97 (m, 1H), 4.06e4.01
(m, 1H), 3.96e3.95 (m, 1H), 3.89e3.86 (m, 1H), 3.82e3.78 (m, 1H),
3.65e3.62 (m, 1H) ppm. 13C NMR (150 MHz, DMSO-d6)
d: 163.3,
154.2, 148.3, 148.2, 141.3, 138.3, 132.1, 131.6, 131.5, 129.2, 128.4,
126.8, 125.2, 124.7, 122.6, 103.2, 89.7, 83.5, 74.5, 68.2, 59.4 ppþm.
HRMS (MALDI-TOF) m/z: calculated for C18H18F3N3NaO5
:
436.109 (MNaþ). Found: 436.109 (MNaþ).
4.4. 4-Amino-5-nitropyrimidin-2(1H)-one (7)
Cytosine, 6 (100 mg, 0.9 mmol) was added after 45 min to
a stirred mixture of fuming HNO3 (0.62 mL) and concd sulfuric acid
(0.124 mL). The resulting mixture was heated at 80 ꢀC for 18 h. A new
spot was seen on TLC (MeOH/CHCl3 6:4) with Rf¼0.64, indicating the
formation of the product. The mixture was poured onto ice (5.25 g)
and neutralized with satd NaOH. The solid was filtered off and
washed with ice-cold water, ice-cold abs EtOH, and ice-cold diethyl
ether, and dried in vacuum, yielding 77 mg (60%). Mp>250 ꢀC
Fig. 4. Changes in the 1H NMR spectra of guanosine upon addition of analogue 3d in
DMSO-d6 solution (700 MHz, 25 ꢀC). A. 1H NMR spectra of N2eNH2 protons of gua-
nosine (ppm). B. 1H NMR spectra of N1eNH protons of guanosine (ppm).
4 (280 mg, 0.87 mmol), trans-2-(4-methoxyphenyl)-vinylboronic
acid 5a (194 mg, 1.09 mmol), Pd(OAc)2 (10 mg, 0.04 mmol), TPPTS
(124 mg, 0.22 mmol), and Na2CO3 (277 mg, 2.61 mmol). The mix-
ture was stirred under reflux for 3 h and monitored by TLC (7:3
CHCl3/MeOH). The solvent was evaporated and water was added.
The resulting solution was freeze dried. The residue was separated
decomp. IR (ZnSe):
1650, 1599, 1567, 1511, 1435, 1343, 1318, 1238, 1137, 1102, 1031, 955,
852, 775, 618 cmꢁ1. 1H NMR (600 MHz, DMSO-d6):
11.88 (br s, 1H),
8.86 (s, 1H), 8.30 (br s, 1H), 8.10 (s, 1H) ppm. 13C NMR (150 MHz,
n 3381, 3168, 3077, 2418, 2050, 1951, 1877, 1681,
d
DMSO-d6) d: 157.5, 154.2, 151.9, 118.5 ppm. HRMS (CI/CH4) m/z:
calculated for C4H4N4O3: 157.036 (MHþ). Found: 157.036 (MHþ).
on a silica gel column (70:30 CHCl3/MeOH). Product 1a was ob-
23
tained as a white solid in a 54% yield (176 mg). Mp 125e127 ꢀC. [
a]
D
ꢁ9.5 (c 1.12, DMSO). IR (ZnSe):
1509, 1482, 1451, 1249, 1176, 1097, 1026, 961, 852, 780, 699 cmꢁ1. 1H
NMR (600 MHz, DMSO-d6):
(s, 2H), 6.92 (d, J¼15.1 Hz, 1 H), 6.90 (d, J¼7.8 Hz, 2H), 6.75
(d, J¼15.1 Hz, 1H), 5.78 (d, J¼3.2 Hz, 1H), 5.36e5.34 (m, 2H),
5.03e5.01(m, 1H), 4.09e4.03 (m, 1H), 3.99e3.97 (m, 1H), 3.87e3.86
(m, 1H), 3.78e3.76 (m, 1H), 3.77 (s, 3H), 3.63e3.60 (m, 1H) ppm. 13C
n 3545, 3401, 2976, 1716, 1636, 1613,
4.5. 2-(4-Amino-5-nitro-2-oxopyrimidin-1(2H)-yl)-5((benzoy-
loxy) methyl)tetrahydrofuran-3,4-diyl dibenzoate (8)
d
8.43 (s,1H), 7.51 (d, J¼7.8 Hz, 2H), 7.32
5-Nitrocytosine, 7 (3 g, 19.23 mmol) was suspended in HMDS
(40 mL, 191.8 mmol) containing TMSeCl (1 mL, 7.82 mmol). The
mixture was heated under reflux for 36 h and then concentrated in
vacuo. To this crude material was added
b-D-ribofuranose-1-
NMR (150 MHz, DMSO-d6) d: 163.5, 158.7, 154.6, 136.9, 129.9, 127.8,
acetate-2,3,5-tribenzoate (9.737 g, 19.30 mmol) and the material
was suspended in MeCN (125 mL). The suspension was degassed
with N2, then SnCl4 (23 mL in 1 M solution in CH2Cl2) was added.
The suspension quickly turned clear and then the solution was
stirred for 3 h at room temperature. Distilled water (100 mL) was
added with vigorous stirring to hydrolyze the remaining SnCl4. This
facilitated the precipitation of the product. The solid was collected,
suspended in EtOAc (500 mL) and washed with aq Na2CO3
(600 mLꢃ4). The organic layer was diluted with EtOAc and washed
with satd NaCl (700 mLꢃ1) and H2O (700 mLꢃ2). The organic layer
was dried over anhydrous Na2SO4 and concentrated in vacuo. The
resulting solid was triturated with CHCl3 and dried in vacuo
yielding 8 g of the product (70%). Mp 218e220 ꢀC decomp.
126.7, 117.1, 113.9, 104.3, 89.6, 83.7, 74.4, 68.6, 59þ.7, 55.2 ppm. HRMS
(MALDI-TOF) m/z: calculated for C18H21N3NaO6 : 398.132 (MNaþ).
Found: 398.134 (MNaþ).
4.2. 4-Amino-1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahy-
drofuran-2-yl)-5-(4-fruorostyryl)pyrimidin-2(1H)-one (1b)
Product 1b was obtained from 4 (103 mg, 0.32 mmol) and 5b
(67 mg, 0.40 mmol) according to the general procedure. Product 1b
was obtained as a white solid in 64% yield (77 mg). Mp 161e163 ꢀC.
23
[
a]
ꢁ22.3 (c 0.98, DMSO). IR (ZnSe):
n 3326, 2855, 1718, 1636,
D
1599, 1507, 1477, 1249, 1160, 1101, 1059, 1023, 992, 962, 827, 763,
700, 671 cmꢁ1 1H NMR (600 MHz, DMSO-d6):
.
d
8.52 (s, 1H),
(Reported mp 211e213 ꢀC decomp.).43
IR (ZnSe):
[
a
]
23 ꢁ101.3 (c 1.02, CHCl3).
D
7.63e7.61 (m, 2H), 7.42 (s, 2H), 7.18e7.15 (m, 2H), 7.40 (d, J¼16.1 Hz,
1H), 6.82 (d, J¼16.1 Hz, 1H), 5.77 (d, J¼3.3 Hz, 1H), 5.37e5.38
(m, 2H), 5.01e5.00 (m, 1H), 4.05e4.04 (m, 1H), 3.99e3.98 (m, 1H),
3.88e3.87 (m, 1H), 3.78e3.76 (m, 1H), 3.63e3.62 (s, 1H) ppm. 13C
n
3480, 3366, 3060, 1716, 1684, 1653, 1601, 1589, 1583,
1491, 1119, 1418, 1363, 1281, 1265, 1130, 1110, 1093, 1039, 1069, 1048,
1024, 927, 891, 851, 819, 761, 736, 703 cmꢁ1 1H NMR (600 MHz,
CDCl3):
9.08 (s, 1H), 8.27 (br s, 1H), 8.07 (d, J¼6.2 Hz, 2H), 7.95
(s, 1H), 7.92e7.89 (m, 4H), 7.55e7.52 (m, 3H) 7.45e7.31 (m, 6H),
.
d
NMR (150 MHz, DMSO-d6) d: 163.6, 162.6, 161.0, 154.6, 137.9, 143.2,

M. Kovaliov et al. / Tetrahedron 69 (2013) 3698e3705
3703
6.33 (d, J¼2.1 Hz, 1H), 5.91e5.91 (m, 2H), 4.83e4.76 (m,
3H) ppm. 13C NMR (150 MHz, CDCl3)
: 166.1, 165.2, 157.7, 151.8,
2928, 1721, 1673, 1583, 1531, 1488, 1451, 1407, 1314, 1262, 1176, 1092,
1068, 1025, 935, 776, 703, 617 cmꢁ1 1H NMR (600 MHz, CDCl3):
d
.
146.4, 133.7, 133.7, 133.5, 129.9, 129.8, 129.7, 128.5, 120.2, 90.5, 81.2,
d
8.56 (s, 1H), 8.38 (m, 2H), 8.12 (d, J¼7.7 Hz, 2H), 8.01 (d, J¼7.52 Hz,
74.9, 70.9, 63.4 ppm. HRMS (MALDI-TOF) m/z: calculated for
2H), 7.94 (d, J¼7.52 Hz, 2H), 7.58e7.53 (m, 4H), 7.48e7.47 (m, 2H),
7.43e7.35 (m, 6H), 6.45e6.44 (m, 1H), 6.07 (br s, 1H), 5.98e5.96 (m,
1H), 4.91e4.89 (m, 1H), 4.86e4.84 (m, 3H) ppm. 13C NMR (175 MHz,
þ
C30H24N4NaO10 : 623.138 (MNaþ). Found: 623.140 (MNaþ).
4.6. 2-((Benzoyloxy)methyl)-5-(4,5-diamino-2-oxopyrimidin-
CDCl3) d: 166.3, 165.2, 165.1, 163.1, 157.62, 154.5, 134.2, 133.6, 133.4,
1(2H)yl)tetrahydrofuran-3,4-diyl dibenzoate (9)
131.8,130.1,130.0,129.9,129.8,129.5,129.1,128.9,128.5,127.9,126.3,
93.1, 81.0, 76.1, 71.2, 64.2 ppm. HRMS (MALDI-TOF) m/z: calculated
þ
To a solution of 8 (1.037 g, 1.72 mmol) in THF (65 mL) and glacial
AcOH (5 mL) was added Pd/C (10%, 377.5 mg). The flask was charged
with H2 (1.5 atm), and stirred for 7 h at room temperature. The
reaction mixture was filtered through a pad of Celite, washed with
excess MeOH, and concentrated in vacuo. The crude material was
purified by automated column chromatography (10% MeOH in
for C37H28N4NaO8 : 679.180 (MNaþ). Found: 679.179 (MNaþ).
4.9. 2-((Benzoyloxy)methyl)-5-(2-oxo-8-(p-tolyl)-2,9-dihydro-
1H-purin-1-yl)tetrahydrofuran-3,4-diyl dibenzoate (11b)
Product 10b was obtained by condensation of 9 (200 mg,
0.35 mmol) with p-tolyl aldehyde 10b (38 mL, 0.35 mmol) with
p-TsOH (13.5 mg, 0.07 mmol) as a catalyst, according to the general
EtOAc) yielding pure 9 (363 mg, 38%). Mp 150e153 ꢀC (Reported mp
23
153e157 ꢀC).43
[
a]
D
ꢁ61 (c 1.10, CHCl3). IR (ZnSe):
n
3064, 1719,
1672,1600,1488,1450,1377,1315,1263,1176,1092,1069,1024,1024,
procedure. Compound 11b was obtained as a white solid in 66%
23
803, 776, 701 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d
8.08 (d, J¼6.2 Hz,
yield (157 mg). Mp 141e144 ꢀC. [
a
]
ꢁ113.2 (c 0.98, CHCl3). IR
D
2H), 7.99 (d, J¼6.2 Hz, 4H), 7.63 (t, J¼5.3 Hz, 3H), 7.51e7.41 (m, 6H),
7.11 (s, 1H), 6.11 (d, J¼5.4 Hz, 1H), 5.90e5.86 (m, 2H), 4.70e4.64 (m,
(ZnSe):
n
2978, 1720, 1675, 1584, 1261, 1091, 1068, 1024, 778, 704,
617 cmꢁ1
.
1H NMR (300 MHz, CDCl3):
d
8.53 (s, 1H), 8.24
3H) ppm. 13C NMR (150 MHz, CDCl3)
d: 165.5, 164.6, 164.6, 161.4,
(d, J¼8.2 Hz, 2H), 8.12e8.11 (m, 2H), 8.02e7.99 (m, 2H), 7.95e7.92
(m, 2H), 7.61e7.50 (m, 3H), 7.42e7.37 (m, 6H), 2.26 (d, J¼8.2 Hz,
2H), 6.43 (d, J¼3.13 Hz, 1H), 6.10e6.08 (m, 1H), 6.01e5.97 (m, 1H),
146.4, 153.7, 133.8, 133.8, 133.5, 129.3, 128.7, 123.2, 116.6, 90.3, 78.3,
78.3, 73.5, 71.1,þ64.1 ppm. HRMS (MALDI-TOF) m/z: calculated for
C30H26N4NaO8 : 593.164 (MNaþ). Found: 593.167 (MNaþ).
4.90e4.83 (m, 3H), 2.32 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3)
d:
166.3, 165.2, 165.2, 163.2, 158.0, 154.6, 142.3, 133.6, 133.4, 129.9,
129.9, 129.8, 129.3, 128.9, 128.7, 128.7, 128.5, 128.5, 127.9, 125.1, 92.6,
80.5, 75.4, 70.7, 63.þ6, 21.5 ppm. HRMS (MALDI-TOF) m/z: calculated
for C38H30N4NaO8 : 693.196 (MNaþ). Found: 693.193 (MNaþ).
4.7. 4,5-Diamino-1-(3,4-dihydroxy-5-(hydroxymethyl)tetra-
hydrofuran-2-yl)pyrimidin-2(1H)-on (2)
Compound 9 (820 mg, 1.45 mmol) was suspended in aq EtOH
(40 mL, 4:1 EtOH/H2O) at room temperature. 1 M NaOH (3.5 mL,
6 mmol) was added at 20 min intervals and the solution was stirred
for 3 h. The reaction was then acidified to pH ca. 2 with 1 M HCl and
EtOH was removed in vacuo. The water layer was extracted with
CHCl3, neutralized to pH ca. 8 with 10% NH4OH, and then concen-
trated in vacuo. The crude material was dissolved in H2O (4 mL)
followed by an addition of EtOH (40 mL), the solution was cooled to
ꢁ20 ꢀC and the resulting solid was collected, and washed with
excess Et2O. The resulting solid was dissolved in H2O and freeze-
4.10. 2-((Benzoyloxy)methyl)-5-(8-(4-methoxyphenyl)-2-oxo-
2,9-dihydro-1H-purin-1-yl)tetrahydrofuran-3,4-diyl di-
benzoate (11c)
Product 11c was obtained by condensation of 9 (200 mg,
0.35 mmol) with anisaldehyde 10c (40 mL, 0.35 mmol) with p-TsOH
(13.5 mg, 0.07 mmol) as a catalyst, according to the general pro-
cedure. Compound 3c was obtained as a white solid in 42% yield
(103 mg). Mp 151e153 ꢀC. [
a
]
D
23 ꢁ110.2 (c 1.05, CHCl3). IR (ZnSe):
n
dried to dryness yielding 2 as a light yellow solid (260 mg, 70%
2993, 1725, 1680, 1615, 1545, 1260, 1094, 1054, 1036, 1022, 778, 734,
23
yield). Mp 190e193 ꢀC decomp. (Reported 191e192 ꢀC).43
[a
]
614 cmꢁ1 1H NMR (300 MHz, CDCl3):
. d 8.48 (s, 1H), 8.31
D
ꢁ14.5 (c 0.88, H2O). IR (ZnSe):
1404, 1312, 1252, 1093, 1046, 935, 856, 741, 617 cmꢁ1
(600 MHz, D2O):
7.51 (s, 1H), 5.88 (d, J¼5.3 Hz, 1H), 4.28e4.26
(m, 1H), 4.19e4.17 (m, 1H), 4.11e4.10 (m, 1H) 3.91e3.78
(m, 2H) ppm. 13C NMR (150 MHz, D2O)
: 159.9, 153.1, 127.7, 115.2,
n
3189, 2921, 1716, 1659, 1574, 1488,
(d, J¼8.7 Hz, 2H), 8.09 (d, J¼7.53 Hz, 2H), 7.98 (d, J¼7.53 Hz, 2H),
7.90 (d, J¼7.53 Hz, 2H), 7.54e7.50 (m, 3H), 7.38e7.25 (m, 6H), 6.96
(d, J¼8.7 Hz, 2H), 6.35 (br s, 1H), 6.14e6.15 (m, 1H), 6.03e6.01
(m, 1H), 4.81e4.45 (m, 3H), 3.78 (s, 3H) ppm. 13C NMR (75 MHz,
.
1H NMR
d
d
CDCl3)
d: 166.3, 165.2, 163.2, 162.7, 154.7, 133.6, 133.4, 129.8, 129.3,
89.7, 84.0, 73.8, 69.2, 60.6 ppm. HRMS (MALDI-TOF) m/z: calculated
128.9, 128.6, 128.5, 128.5, 120.3, 114.5, 114.1, 93.1, 81.0, 76.0, 71.2,
for C9H14N4NaO5 : 281.086 (MNaþ). Found: 281.087 (MNaþ).
64.2 ppm. HRMS (MALDI-TOF) m/z: calculated for C38H30N4NaO9
þ
þ
:
709.191 (MNaþ). Found: 709.194 (MNaþ).
4.8. General procedure for synthesis of protected imidazolo-
cytidine derivatives (11aed): 2-(benzoyloxymethyl)-5-(2-oxo-
8-phenyl-2,9-dihydro-1H-purin-1-yl)tetrahydrofuran-3,4-diyl
dibenzoate (11a)
4.11. 2-((Benzoyloxy)methyl)-5-(2-oxo-8-(4-(trifluoromethyl)
phenyl)-2,9-dihydro-1H-purin-1-yl)tetrahydrofuran-3,4-diyl
dibenzoate (11d)
Benzaldehyde 10a (20
m
L, 0.17 mmol) and 9 (100 mg, 0.17 mmol)
Product 3d was obtained by condensation of 9 (200 mg,
0.35 mmol) with p-trifluoromethylbenzaldehyde 10e (60 L,
0.35 mmol) with p-TsOH (13.5 mg, 0.07 mmol) as a catalyst,
were thoroughly mixed in DMF (2 mL), then p-TsOH (7 mg,
0.034 mmol) was added, and the solution heated and stirred at
80 ꢀC for 2e4 h and monitored by TLC (CHCl3/MeOH 9.5:0.5). When
the reaction was completed, the solution was cooled to room
temperature. The reaction mixture was added dropwise with vig-
orous stirring into a mixture of Na2CO3 (0.034 mmol) and H2O
(10 mL). The crude product was extracted into EtOAc, the organic
phase was washed with H2O, brine and dried (Na2SO4). Evaporation
of solvent gave the crude product, which was purified by column
chromatography over silica gel (9:1 CHCl3/EtOH) yielding 40 mg
m
according to the general procedure. Compound 11d was obtained as
23
a white solid in 47% yield (120 mg). Mp 160e163 ꢀC. [
a
]
D
ꢁ75
(c 1.13, CHCl3). IR (ZnSe):
1175, 1091, 1064, 1025, 778, 705, 616 cmꢁ1
CDCl3): 8.69 (s, 1H), 8.48e8.45 (m, 2H), 8.11e8.09 (m, 2H),
n
2987, 1720, 1644, 1584, 1451, 1316, 1261,
.
1H NMR (300 MHz,
d
8.02e7.99 (m, 2H), 7.93e7.90 (m, 2H), 7.70e7.68 (m, 2H), 7.54e7.52
(m, 3H), 7.40e7.34 (m, 6H), 6.40 (br s, 1H), 6.15 (br s, 1H), 5.94e5.90
(m, 1H), 4.93e4.86 (m, 3H) ppm. 13C NMR (75 MHz, CDCl3)
d
: 170.0,
(36%). Mp 152e155 ꢀC. [
a
]
23 ꢁ81.5 (c 0.97, CHCl3). IR (ZnSe):
n 3181,
166.2, 165.2, 165.1, 162.7, 158.8, 156.1, 154.8, 134.9, 133.8, 133.7, 133.1,
D

3704
M. Kovaliov et al. / Tetrahedron 69 (2013) 3698e3705
129.9, 129.2, 128.7, 128.6, 128.6, 128.5, 128.3, 128.0, 126.0, 125.9,
125.6, 125.0, 92.1, 80.4, 75.2, 70.2, 63.þ2 ppm. HRMS (MALDI-TOF)
m/z: calculated for C38H27F3N4NaO8 : 747.167 (MNaþ). Found:
747.164 (MNaþ).
decomp. [
a
]
23 þ7.9 (c 1.16, DMSO). IR (ZnSe):
n 3298,1664,1619,1584,
D
1499, 1446, 1403, 1373, 1324, 1262, 1170, 1102, 1065, 1046, 1016, 944,
848, 780, 701 cmꢁ1. 1H NMR (600 MHz, DMSO-d6):
d 9.02 (s,1H), 8.02
(d, J¼8.2 Hz, 2H), 7.34 (d, J¼8.2 Hz, 2H), 5.91 (d, J¼2.5 Hz, 1H), 5.53
(br s,1H), 5.39 (br s,1H), 5.04 (br s,1H), 4.07e4.06 (m,1H), 4.04e4.03
(m, 1H), 3.99e3.97 (m, 1H), 3.88e3.84 (m, 1H), 3.70e3.67 (m, 1H),
4.12. General procedure for synthesis of imidazolo-cytidine
derivatives (3aed): 1-(3,4-dihydroxy-5-(hydroxymethyl)tetra-
hydrofuran-2-yl)-8-phenyl-1H-purin-2(9H)-one (3a)
2.38 (s, 3H) ppm. 13C NMR (150 MHz, DMSO-d6)
d: 162.4,157.3, 153.8,
140.5, 133.5, 129.1, 128.8, 126.6, 91.1, 84.1, 74.9, 68.3, 59.6þ, 20.7 ppm.
HRMS (MALDI-TOF) m/z: calculated for C17H15F3N4NaO5 : 435.089
(MNaþ). Found: 435.086 (MNaþ).
Protected nucleoside 11a (100 mg, 0.15 mmol) was added to
a solution of NaOH (9 equiv) in MeOH (2 mL) and the resulting
mixture was stirred at room temperature for 2 h. After evaporation
of the MeOH the product was purified by an automated medium
4.16. NMR base-paring experiments
pressure column chromatography (8:2 CHCl3/MeOH) yielding
Approximate amounts of guanosine and 3d were put into vol-
umetric flasks and stored under vacuum overnight to remove
adsorbed water. All 1H NMR spectra were measured in DMSO-d6 at
700 MHz. The data were collected at 300 K. 1H NMR spectra were
obtained at a range of concentrations of 8d (0.007 M, 0.013 M,
0.020 M, and 0.030 M) with constant concentration of guanosine
(0.02 M) in DMSO-d6 solution.
23
31 mg (60%) of a white solid. Mp 207e210 ꢀC decomp. [
a
]
þ3.2
D
(c 0.94, DMSO). IR (ZnSe):
1476, 1324, 1263, 1168, 1100, 1015, 935, 834, 1091, 778, 704,
702 cmꢁ1 1H NMR (600 MHz, DMSO-d6):
9.09 (s, 1H, H6),
n 3290, 2978, 1701, 1676, 1645, 1619, 1584,
.
d
8.13e8.12 (m, 2H), 7.55e7.54 (m, 3H), 5.91 (d, J¼2.40 Hz, 1H), 5.53
(br s,1H) 5.39 (br s,1H), 5.01 (br s,1H) 4.07e4.06 (m,1H), 4.03e4.02
(m, 1H), 3.97e3.96 (m, 1H), 3.85e3.84 (m, 1H) 3.69e3.68
(m, 1H) ppm. 13C NMR (150 MHz, DMSO-d6)
d: 161.9, 134.66, 131.0,
4.17. Absorbance and fluorescence measurements
129.2, 128.9, 128.9, 126.7, 91.2, 83.9, 75.1, 68.2,þ59.37 ppm. HRMS
(MALDI-TOF) m/z: calculated for C16H16N4NaO5 : 367.101 (MNaþ).
Found: 367.103 (MNaþ).
Absorption spectra were determined in H2O (pH¼7) for com-
pounds 1aec, 2 and MeOH for analogues 3aed. The concentrations
of samples were 2e5 mM. Absorbance was kept less than 0.05 AU in
4.13. 1-(3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-8-(p-tolyl)-1H-purin-2(9H)-one (3b)
order to avoid inner filter distortion. Samples were measured in
a 10 mm quartz cell. For accurate measurements of the fluorescence,
the concentrations of the samples were in the linear range in which
the BeereLambert law applies, the optical density was less than 0.05
to avoid the inner filter effect.58 Emission spectra of compounds 1c,
2 were determined in H2O (pH 7) and in MeOH for compounds 1a,b,
and 3aed. Measurement conditions of analogues 6e8 included
740 V sensitivity and a 4 nm slit. The concentration of the samples
Product 3b was obtained by treating the protected nucleoside
11b (100 mg, 0.15 mmol) with NaOH in MeOH according to the
general procedure, yielding 45 mg (85%) of
a
white solid.
3281,
23
Mp>250 ꢀC decomp. [
a
]
þ5.5 (c 1.12, DMSO). IR (ZnSe):
n
D
2977, 1720, 1674, 1584, 1476, 1414, 1356, 1324, 1261, 1171, 1091, 1068,
1034, 1015, 965, 854, 778, 704, 617 cmꢁ1. 1H NMR (600 MHz, DMSO-
was 3 mM. Samples were measured in a 10 mm quartz cell.
d6):
d
9.02 (s, 1H), 8.02 (d, J¼8.2 Hz, 2H), 7.34 (d, J¼8.2 Hz, 2H), 5.91
(d, J¼2.5 Hz, 1H), 5.53 (br s, 1H), 5.39 (br s, 1H), 5.04 (br s, 1H),
4.06e4.05 (m,1H), 4.02e4.01 (m,1H), 3.96e3.95 (m,1H), 3.83e3.82
(m, 1H), 3.68e3.67 (m, 1H), 2.38 (s, 3H) ppm. 13C NMR (150 MHz,
4.18. Quantum yield measurements
The quantum yield of each compound was calculated from the
observed absorbance and the area of the fluorescence emission
band. For accurate measurements of the fluorescence, the con-
centrations of the samples were in the linear range in which the
BeereLambert law applies, and the optical density was less than
0.05 to avoid the inner filter effect. The fluorescence quantum yield
DMSO-d6) d: 162.4, 157.3, 153.8, 140.5, 133.5, 129.1, 128.8, 126.6, 91.1,
84.1, 74.9, 68.3, 59.þ6, 20.7 ppm. HRMS (MALDI-TOF) m/z: calculated
for C17H18N4NaO5 : 381.117 (MNaþ). Found: 381.118 (MNaþ).
4.14. 1-(3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-8-(4-methoxyphenyl)-1H-purin-2(9H)-one (3c)
(
(
(
FF) was calculated based on
F
F
of a known reference, tryptophan
0.14, labs 280 nm, lem 350 nm in H2O)59 or quinine sulfate
0.54, labs 350 nm, lem 446 nm in 0.1 M H2SO4),60 according to
Product 8c was obtained by treating protected nucleoside 11c
F
(100 mg, 0.15 mmol) with NaOH in MeOH according to the general
Eq. 9. labs and lem values of the selected reference compounds were
in the same range as those of the evaluated compounds.
procedure, yielding 35 mg (65%) of a white solid. Mp 220e223 ꢀC
23
decomp. [
a
]
þ5.6 (c 0.99, DMSO). IR (ZnSe):
n 3324, 2992, 1725,
D
FR I=IRꢄODR=ODꢄh2
=
hR
(9)
1680, 1615, 1547, 1467, 1434, 1389, 1324, 1261, 1167, 1110, 1094, 1055,
FF
¼
1036, 1021, 998, 943, 778, 736, cmꢁ1. 1H NMR (600 MHz, DMSO-d6):
where R¼Reference, I¼Integration of the peak and
h
¼refractive
d
8.91 (s, 1H), 8.08 (d, J¼8.1 Hz, 2H), 7.07 (d, J¼8.1 Hz, 2H), 5.91
index of the solvent.
(d, J¼2.3 Hz, 1H), 5.51 (br s, 1H), 5.36 (br s, 1H), 5.02 (br s, 1H),
4.02e4.01 (m, 1H), 3.96e3.95 (m, 1H), 3.95e3.94 (m, 1H),
3.83e3.82 (m, 4H), 3.68e3.68 (m, 1H) ppm. 13C NMR (150 MHz,
Supplementary data
DMSO-d6)
d
: 161.3,128.5,114.3, 91.2, 83.9, 75.1, 68.3, 59.6,þ55.4 ppm.
Figures of absorbance (UVevis), fluorescence spectra, and
NMR experiments spectra, of 1aec, 2, 3aed (G$8d) are available.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.03.005.
HRMS (MALDI-TOF) m/z: calculated for C17H18N4NaO6 : 397.112
(MNaþ). Found: 397.111 (MNaþ).
4.15. 1-(3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-8-(4-(trifluoromethyl)phenyl)-1H-purin-2(9H)-one (3d)
References and notes
Product 8d was obtained by treating protected nucleoside 11d
(120 mg, 0.17 mmol) with NaOH in MeOH according to the general
procedure, yielding 56 mg (80%) of a yellowish solid. Mp 195e200 ꢀC
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