8-(p-CF3-cinnamyl)-modified purine nucleosides as promising fluorescent probes

Article abstract of DOI:10.1039/c1ob05681f

Natural nucleotides are not useful as fluorescent probes because of their low quantum yields. Therefore, a common methodology for the detection of RNA and DNA is the application of extrinsic fluorescent dyes coupled to bases in oligonucleotides. To overco

Full text of DOI:10.1039/c1ob05681f

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PAPER
8-(p-CF3-cinnamyl)-modified purine nucleosides as promising fluorescent
probes†
Lital Zilbershtein, Alon Silberman and Bilha Fischer*
Received 1st May 2011, Accepted 19th July 2011
DOI: 10.1039/c1ob05681f
Natural nucleotides are not useful as fluorescent probes because of their low quantum yields. Therefore,
a common methodology for the detection of RNA and DNA is the application of extrinsic fluorescent
dyes coupled to bases in oligonucleotides. To overcome the many limitations from which fluorescent
nucleotide–dye conjugates suffer, we have developed novel purine nucleosides with intrinsic fluorescence
to be incorporated into oligonucleotide probes. For this purpose we synthesized adenosine and
guanosine fluorescent analogues 7–25, conjugated at the C8 position with aryl/heteroaryl moieties
either directly, or via alkenyl/alkynyl linkers. Directly conjugated analogues 7–14, exhibited high
quantum yields, f >0.1, and short lem (<385 nm). Alkynyl conjugated analogues 22–25, exhibited low
quantum yields, f <0.075, and lem<385 nm. The alkenyl conjugated analogues 15–21, exhibited lem
408–459 nm. While analogues 15,16, and 20 bearing an EDG on the aryl moiety, exhibited f <0.02,
analogues 17, and 21 with EWG on the aryl moiety, exhibited extremely high quantum yields, f ª 0.8,
suggesting better intramolecular charge transfer. We determined the conformation of selected
adenosine analogues. Directly conjugated analogue 8 and alkynyl conjugated analogue 22, adapted the
syn conformation, whereas alkenyl conjugated analogue 15 adapted the anti conformation. Based on
the long emission wavelengths, high quantum yields, anti conformation and base-paring compatibility,
we suggest analogues 17 and 21 for further development as fluorescent probes for the sensitive detection
of genetic material.
do not interfere with base pairing.7 The latter have been applied to
Introduction
DNA microarray technology.8 This technology involves covalent
Previously, the detection of nucleic acids was based on radioactive
conjugation of nucleotides with large fluorescent dyes followed
by enzymatic incorporation, resulting in a cDNA hybridization-
labeling of nucleic acid probes (e.g. by 32P).1 This labeling allowed
for instance, the detection of specific mRNA sequences.2,3 How-
capable probe. However, a large dye attached to a nucleotide
ever, these radioactive labeled probes suffer from fundamental
alters the efficiency of enzymatic incorporation. The synthesis of
disadvantages: they are hazardous, have short half-lives, and have
long DNA/RNA probes is therefore unreliable and variation of
limited signal emission. In contrast, fluorescent probes do not
fluorescence yield with degree of dye conjugation to the nucleic
suffer from these disadvantages and have largely taken over as
acid probe can significantly reduce the reliability of quantitative
sensitive labels for identification and quantification of nucleic
acids.4
The natural bases of nucleic acids are not useful as fluorescent
measures of hybridization-based assays.
Other currently used conjugated-dye technologies include Flu-
orescent In Situ Hybridization (FISH)9 and Molecular Beacons
probes because of their extremely low quantum yields.5,6 Therefore,
the use of extrinsic probes, namely, fluorescent dyes, is necessary.
Currently marketed dyes for nucleic-acid staining and labeling
include: intercalating dyes that are incorporated non-covalently
to double stranded nucleic acids; minor groove-binding dyes; a
dye-labeled streptavidin to detect a biotinylated probe, and large
fluorescent dyes (e.g., rhodamine, fluorescein, and Cy5/Cy3) that
are incorporated covalently to oligonucleotides at positions that
(MB).10–12 FISH probes are prepared from amino-allyl modified
bases13 used to allow the chemical synthesis of multiply-labeled
fluorescent oligomer hybridization probes14 in a two-step protocol
involving first the incorporation of a slightly modified nucleotide
into nucleic acid, followed by covalent binding of the fluorophore
of interest (e.g. FITC, Cy3, Alexa-488). However, labeling with
these fluorophores requires additional conjugation and purifica-
tion steps following probe synthesis. Furthermore, the detection
of endogenous levels of mRNA is difficult due to fluorescent
background, and typically requires the generation of several probe
sequences to the same mRNA resulting in signal enhancement.
To overcome many of the limitations of the above men-
tioned fluorescent probes, intrinsically fluorescent nucleosides
Department of Chemistry, Gonda-Goldschmied Medical Research Center,
Bar-Ilan University, Ramat-Gan, 52900, Israel. E-mail: bilha.fischer@
biu.ac.il; Fax: +972-3-6354907; Tel: +972-3-5318303
† Electronic supplementary information (ESI) available: 13C and 1H NMR
spectra of reported compounds. See DOI: 10.1039/c1ob05681f
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Fig. 1 Fluorescent purine nucleoside analogues: C8 extended adenosine analogues, 1–4; 2-amino adenosine, 5; N2,N3-etheno-adenosine, 6.
were introduced for numerous applications.15 These nucleosides
are minimally modified, at the nucleobase positions that do
not interfere with base pairing, thus extending the nucleoside’s
fluorophore.
For example, extending the purine p system by C8 modifications,
e.g. 8-vinyladenosine, 1 (Fig. 1) results in a longer emission
wavelength and higher quantum yield as compared to adenosine
(lem 388 nm; f 0.66 vs. lem 319 nm; f 0.00026).16–18
The purine p-system was also extended upon conjugation with
aromatic moieties. Specifically, 8-(p-hydroxy-phenyl) nucleosides,
2–4 emit at ~390 nm with high quantum yields ranging from 0.25
to 0.56 in aqueous solution (pH 7).
2-Amino adenosine, 5, has been employed in studies of nucleic
acid structure and dynamics, due to the significant change of its
quantum yield upon incorporation into single and double-strand
oligonucleotides as compared to that of the monomer in water (f
0.65).19
Here, we report on the design, synthesis, and spectral charac-
terization of a series of C8-aryl/hetero-aryl conjugated adenosine
and guanosine analogues 7–25 (Fig. 2). Moreover, we propose a
mechanism for the analogues’ fluorescence. In addition, we report
on the compatibility of selected analogues for B-DNA formation
based on their conformational analysis. This SAR study resulted
in the identification of highly promising probes 17 (lem 439 nm; f
0.81), and 21 (lem 459 nm; f 0.80) suitable for hybridization assays
upon incorporation in oligonucleotides.
Results
Design of intrinsically fluorescent A and G analogues
We designed novel C8 extended adenine and guanine nucleo-
sides 7–25 as potential intrinsically fluorescent probes that are
hybridization compatible. Extension of a purine nucleoside at C8
position was expected to result in longer excitation and emission
wavelengths, and higher quantum yield, as compared to the parent
compound.21
Here, we targeted a minimal modification of the nucleoside p-
system with small aromatic/hetero-aromatic moieties. We conju-
gated these moieties to the purine ring via an alkenyl or alkynyl
linker, or no linker at all, to study the effect of the linker on
fluorescence.
Fluorescent A or G analogues 7–25 were designed as push–pull
probes to enhance the fluorescent properties of purine nucleosides.
Push–pull molecules are generally composed of three moieties:
an electron donor (D, an electron rich aryl group); an electron
acceptor (A, an electron poor aryl moiety), and an electron rich
linker which is a double/triple bond (analogues 15–25), or a single
bond (analogues 7–14). Since the electron-donating or -accepting
character of A or G in push–pull molecules is relative to the other
moiety, we explored the properties of both electron rich aryl groups
(analogues 8–11, 15–16, 20 and 22–25) and electron poor groups
(analogues 17–19 and 21) attached to either A or G.
Previously, we have extended the adenine chromophore
at C2,N3-positions to give N2,N3-etheno-adenosine, 6, as a
novel probe exhibiting improved fluorescence characteristics (lem
420 nm; f 0.03).20
The limitations of fluorescent dyes used in current
hybridization-based technologies have encouraged us to develop
novel intrinsically fluorescent adenine and guanine nucleosides
that will be later incorporated into oligonucleotides. These probes
include a minimal extension at the C8-position of adenosine (A) or
guanosine (G), rather than attachment of large hydrophobic dyes
to the nucleobase. In addition, A- and G- fluorescent nucleosides
proposed here were designed to satisfy the following requirements:
relatively long absorption and emission wavelengths, satisfactory
quantum yield, and base-pairing compatibility. Specifically, long
absorption and emission wavelengths (>450 nm), as well as
satisfactory quantum yield (>0.1) are required to overcome auto-
fluorescence of many cellular components excited by UV–vis
radiation of suitable wavelengths (from 300 nm for aromatic amino
acids to 550 nm for riboflavin). In addition, we extended A and
G at the C8 position, which is not involved in base pairing, to
obtain hybridization-compatible probes upon incorporation into
oligonucleotides.
Likewise, we investigated the effect of the number (1–3) and
positions (p, m) of EDGs on the probe’s fluorescence. The effect
of the nucleobase (i.e., adenine vs. guanine) on fluorescence was
evaluated as well.
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Fig. 2 Adenosine and guanosine analogues synthesized and studied here.
Synthesis of fluorescent nucleoside probes 7–25
basic conditions,26 the crude reaction residue was washed with
boiling water. All salts and any unreacted 8-Br-G were removed by
this procedure. The solid residue was then washed with EtOAc and
Et2O to remove any palladium complexes and traces of boronic
acid, thus providing a highly pure product 11 in 21% yield.
Analogues 15, 16 and 20 were prepared from (E)-4-
methoxystyrylboronic acid and 8-Br-A, 8-Br-dA, and 8-Br-G
under Suzuki coupling conditions. The pure products were
typically obtained from the reaction mixture as follows: ana-
logue 15 was precipitated upon neutralization of the reaction
mixture by 10% aqueous HCl, vacuum filtered and purified
by excessive washing with H2O and ACN resulting in pure
15 in 42% yield. However, analogues 16 and 20 were purified
on a silica gel column and obtained in 83% and 10% yield,
respectively. The low yield obtained for products 11 and 20
We have applied the Suzuki–Miyuara coupling22,23 for the prepa-
ration of analogues 7–21, and the Sonogashira coupling24,25 for
the preparation of analogues 22–25. The great advantage of these
procedures is that no protecting groups are needed, thus allowing
a single step reaction towards the desired conjugated nucleoside
analogues.
Specifically,
A and G were directly conjugated with
aryl/heterocyclic boronic acids in the presence of Na2CO3 and
a water-soluble catalytic system consisting of Pd(OAc)2 : P(m-
C6H4SO3Na)3, in water–acetonitrile (2 : 1) at ~100 ◦C.22,23 Products
7–10 and 12–14 were obtained in 13%–89% yield upon silica gel
chromatography. To separate 8-(4-methoxyphenyl)-guanosine, 11,
from guanosine palladium complexes formed under the reaction’s
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Table 1 Spectral properties of analogues 7–25 measured in MeOH
Analogue
lab (nm)
lem (nm)
f
Standard
Adenine17a
361
275
284
284
288
292
290
309
306
285
345
340
340
338
333
350
357
323
323
316
299
319
303
0.00026
0.0003
0.11
0.105
0.46
0.113
0.23
0.054
0.46
—
—
Guanine17a
7
365, 387
361, 382
362, 381
365, 385
362, 383
354
360, 381
358, 382
394, 419
408
439
438
436
430
tryptophan
tryptophan
tryptophan
tryptophan
tryptophan
quinine sulfate
quinine sulfate
tryptophan
quinine sulfate
quinine sulfate
quinine sulfate
quinine sulfate
quinine sulfate
quinine sulfate
quinine sulfate
quinine sulfate
tryptophan
quinine sulfate
tryptophan
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
0.21
0.005
0.0025
0.81
Fig. 3 A representative emission spectrum of analogue 13.
cases, quantum yield calculations are based on the area under all
emission bands. The energy difference between the spectral lines,
of all above mentioned analogues, was 1000–2000 cm-1, and was
therefore considered to be due to a vibrational progression rather
than an additional fluorescence band due to contamination.
We evaluated the dependence of probe fluorescence on the
following parameters: type of linker, presence of an ED or an
EW substituent on the aryl moiety, number of ED substituents,
aryl vs. heteroaryl substituents, and type of nucleobase.
0.74
0.025
0.018
0.80
459
355, 367, 382
355, 368, 384
365, 385
316, 330, 344
0.05
0.062
0.075
0.054
a Fluorescence data were obtained at room temperature in neutral aqueous
solutions.
Type of linker. The effect of the linker between the nucleobase
and the aryl group on fluorescence of A and G analogues was
evaluated. Three types of linkers were compared: analogues 8
and 11, 8-(4-methoxy-phenyl)-A and -G, analogues in which the
aromatic moiety is directly coupled to the A/G C8-position,
analogues 15 and 20, 8-(2-(4-methoxyphenyl)-ethenyl)-A and -G,
analogues in which the aromatic moiety is coupled to the A/G
C8-position via an alkenyl linker, and analogues 22 and 25, 8-
(2-(4-methoxyphenyl)-ethynyl)-A and -G, analogues in which the
aromatic moiety is conjugated to the A/G C8-position via an
alkynyl linker. The analogues in which the aromatic moiety was
directly coupled to the nucleobase 8 and 11, exhibited relatively
high quantum yields, f 0.105 and 0.23, respectively, but short
emission wavelengths ~360 and 380 nm. The analogues with the
alkenyl linker, 15 and 20, exhibited the lowest quantum yields f
0.005 and 0.018, respectively, but, lem >400 nm. The analogues
with the alkynyl linker, 22 and 25, exhibited low quantum yields, f
0.05 and 0.054 respectively, and emission wavelengths of 355, 367,
382 nm for analogue 22, and 316, 330, 344 nm for analogue 25,
which were the shortest emission wavelengths.
is due to the reduced reactivity of 8-bromoguanosine in the
Suzuki–Miyaura reaction.26 Analogues 17, 18 and 21, prepared
from (E)-[4-(trifluoromethyl)phenyl]vinylboronic acid and 8-Br-
A, 8-Br-dA, and 8-Br-G, and analogue 19 prepared from (E)-
(4-fluorophenyl)vinylboronic acid and 8-Br-A, were purified on a
silica gel column and obtained in 40–73% yield.
The analogues bearing an alkynyl linker, 22–25, were pre-
pared by conjugation of 8-Br-A, 8-Br-dA, and 8-Br-G with 4-
methoxyphenyl-ethynyl or 3,5-dimethoxyphenyl-ethynyl (with 8-
Br-A). Specifically, the reactions were carried out in DMF at room
temperature for 18 h (products 22–24), or at 50 ◦C for 24 h (product
25), in the presence of Et3N and a catalytic system consisting of
Pd(Ph3P)4/CuI. Purification of analogues 22–24 was achieved by
chromatography on a silica gel column, resulting in products 22–24
in 26–95% yield. However, purification of the corresponding 8-((4-
methoxyphenyl)ethynyl)-guanosine, 25, was achieved as described
above for 11, resulting in only 15% yield. Here again, the reduced
reactivity of 8-bromoguanosine in palladium catalyzed cross-
coupling reactions resulted in a low yield of analogue 25.
Spectral properties of analogues 7–25
Type of substituent on the aryl moiety (EDG vs. EWG). We
have studied the dependence of the probe’s fluorescence on the
type of group at the para-phenyl position, for example, EWG
i.e., CF3 in analogue 17, vs. EDG, i.e., OCH3, in analogue 15.
Analogue 17, 8-(4-trifluoromethyl-phenyl)-A, exhibited a longer
emission wavelength as compared to the corresponding p-EDG
substituted analogue 15, (lem 439 vs. 394, 419 nm). Furthermore,
the quantum yield of 17 was dramatically increased 160-fold as
compared to that of 15 (f 0.81 vs. 0.005).
The absorption and emission spectra of compounds 7–25 were
measured in methanol, and the fluorescence quantum yields were
determined compared to known standards such as tryptophan in
H2O (lex 286 nm, lem 355 nm, f 0.13) or quinine sulfate in 0.1 M
H2SO4, (lex 350 nm, lem 446 nm, f 0.54)27,28 (Table 1).
We have observed more than one emission band in the emission
spectra of analogues 7–11, 13–15, and 22–25 (a representative
spectrum of analogue 13 is shown in Fig. 3). This phenomenon
occurs due to vibrational progressions, explained by the Franck–
Condon principle. The electron radiates back from different
vibrational states in the first excited singlet state S1 to different
vibrational states on the ground state S0, resulting in more than
one emission wavelength, where the energy difference between
the emission wavelengths is between 1000–2000 cm-1.29–31 In these
Number of substituents on the aryl moiety. The dependence
of fluorescence on the number of methoxy groups on the phenyl
moiety was evaluated by comparing analogue 9, 8-(4-methoxy-
phenyl)-dA, to 10, 8-(3,4,5-methoxy-phenyl)-dA. Addition of an
EDG at the meta positions of analogue 10, did not change the
emission wavelength of analogue 9 (365, 385 nm, as compared to
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Fig. 4 Nucleoside analogues selected for conformational analysis.
362, 381 nm for analogue 9). Furthermore, the quantum yield of
10 has decreased (0.46 vs. 0.113 in analogue 10).
equilibrium to favor the syn conformation, due to steric hindrance.
When a nucleos(t)ide possessing a predominant syn conformation
is incorporated into an oligonucleotide, which in turn undergoes
hybridization to form a duplex, it might cause considerable
destabilization of the B-DNA duplex structure and possibly
enhance Z-DNA formation. To learn the possible consequences of
incorporation of nucleoside analogues 7–25 in DNA duplexes, we
studied by NMR the dependence of the syn–anti equilibrium and
the ribose sugar puckering on the linkers of three representative
analogues, 8, 15 and 22 (Fig. 4).
The representative nucleoside analogues 8, 15, and 22, bear
at the purine C8 position a p-methoxyphenyl moiety linked
directly or indirectly via an alkenyl or an alkynyl linker (Fig. 4).
These analogues are ribonucleosides, but we assume that the 2¢-
deoxyribose analogues will exhibit similar characteristics.
Purine nucleosides in solution undergo rapid syn/anti
interchange.32 However, the ratio of syn/anti conformers is
controlled by chemical modifications of the base. Specifically,
8-substituted purines may favor syn conformation due to steric
hindrance between the 8-substituent and the ribose ring.33,34
The furanose ring in nucleosides does not possess a rigid struc-
ture in solution, but interconverts between South (S) and North
(N) conformers.35 In solution, both states are in equilibrium,
however, in helical polynucleotides, the conformation is fixed in
either S or N form.36 In the common B-DNA structure the ribose
conformation is South.36
The conformation around the glycosidic bond was qualitatively
determined for analogues 8, 15, and 22 by NOESY experiments,
indicating typical H–H interactions between the ribose ring
protons and the 8-substituent or the nucleobase. Specifically, the
1H–1H-NOESY spectrum of analogue 22, indicated clear cross
peaks between H1¢ and Ha, and between H1¢ and Hb (Fig. 5).
In addition, a clear cross peak was observed between H2 and
H(5¢-OH), indicating a predominant syn conformation around
the glycosidic bond. 1H–1H-NOESY spectrum of analogue 15
indicated clear cross peaks between H1¢/H2¢ and H-vinyl, in
addition to a cross peak between H5¢ and H-Ar, indicating a
predominant anti conformation around the glycosidic bond (Fig.
5). In the same way, the predominant conformation of analogue 8
was determined and found to be syn (Table 2).
The conformation of the D-ribose ring of nucleosides 8, 15,
and 22 was analyzed in terms of a dynamic equilibrium in
solution between the two favored puckered conformations: N and
S conformers.37–39 We calculated N and S equilibrium populations
from observed J1¢2¢ and J3¢4¢ couplings for compounds 8, 15, and
22, as previously reported.37 The observed vicinal couplings are
Aryl vs. heteroaryl substituent. The effect of the type of the
aryl moiety on the fluorescence of adenosine was evaluated
by comparing analogue 7 (aryl = Ph) to 12–14 (aryl = furyl
or thiophenyl). We noticed that the nature of the aryl group
directly coupled to the adenosine C8 position, whether phenyl
or heteroaryl (furyl or thiophenyl) does not make a significant
difference in maximum emission, i.e., lem values of 7, 13 and 14
were 387, 381, 382 nm, f values were 0.11, 0.46, 0.21, respectively.
Unlike the latter compounds, analogue 12 (aryl = 5-methyl-
2-thiophenyl), exhibited a relatively short emission wavelength
(354 nm) and a low quantum yield (0.054).
Type of nucleobase. We also compared the properties of 8-
(4-methoxy-phenyl)-A, 8, 8-(2-(4-methoxyphenyl)-ethenyl)-A, 15,
and 8-((4-methoxyphenyl)-ethynyl)-A, 22, to the corresponding G
analogues 11, 20, and 25, respectively, to evaluate the contribution
of the nucleobase to fluorescence. Analogues 8 and 11 exhibited
the same emission wavelengths. Similar observations were made
for analogues 15 and 20. However, analogue 22 exhibited emission
wavelength longer by ~40 nm than 25.
In summary, analogues bearing an alkenyl linker at the
purine C8 position (15–21), exhibited the longest emission wave-
lengths 408–439 nm, compared to the analogues in which the
aryl/heteroaryl groups were linked directly to the nucleobase
(~380 nm), analogues 7–14, or via an alkynyl linker (~380 nm),
analogues 22–25. Extremely large quantum yields (~0.8) were
found for analogues 17 and 21 bearing a p-EWG-Ph on an alkenyl
linker. Substitutions of EDG at the phenyl meta position do
not shift the emission wavelength of the probe. Conjugation of
either aryl or heteroaryl to A or G resulted in similar emission
wavelengths. Likewise, the purine nucleobase (A vs. G) does
not affect the nucleoside probe fluorescence. High quantum
yields were determined for analogues 9, 13, and 14, bearing
aryl/heteroaryl groups directly linked to the nucleobase (f 0.46,
0.46 and 0.21). Yet, substitution of an EWG instead of an
EDG at the phenyl para position, as in analogue 17 vs. 15,
respectively, causes a dramatic 160-fold enhancement of the quan-
tum yield (~0.8), and a 57 nm bathochromic shift of maximum
emission.
Conformational analysis of analogues 8, 15, and 22
Extension of the natural fluorophore of purines by a large C8
substitution is expected to shift the nucleoside conformational
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Fig. 5 Determination of the conformation of compounds 15 and 22. A) 1H,1H-NOESY spectrum of compound 22; B) NOE interactions in 15 and 22.
Table 2 Conformational data of analogues 8, 15, and 22 as determined
these nucleoside analogues, the purine C8-position was conjugated
either directly, or through an alkenyl or alkynyl linker, to an
aryl/hetero aryl moiety. These extensions of the natural purine
nucleobases significantly enhanced the spectral properties of the
latter. For instance, the quantum yields of analogues 9 and 21
are 1770 and 3100-fold higher than adenosine and guanosine,
respectively, and emission is red shifted by 60 and 156 nm,
respectively.
by NMR
Sugar puckering
Conformation around
Analogue
the glycosidic bond
XS (%)
XN (%)
8
15
22
syn
anti
syn
81
76
68
19
24
32
Moreover, some of our new analogues exhibited better spectral
characteristics than currently used purine fluorescent probes. For
example, 2-amino adenosine, 5,19 and 8-vinyladenosine, 1,17 have
high quantum yields (f 0.65 and 0.66, respectively) and therefore,
are useful probes for monitoring the structure and dynamics of
DNA and for detecting base stacking in a duplex. These studies
are based on the significant change in quantum yield of the
probe upon incorporation into single-strand and double-strand
oligonucleotide.17,19,40,41 However, both 2-amino adenosine and
8-vinyladenosine emit at relatively short wavelengths, ~370 and
360 nm, respectively, while some of our purine derivatives emit at
longer wavelengths with higher quantum yield (e.g. compound 17
– lem 439 nm; f 0.81).
The enhanced fluorescence of some of our analogues may be
attributed to the fact that they undergo an intramolecular charge
transfer (ICT) from the donor to the acceptor moiety in the
molecule.42,43 In analogue 9, the adenine moiety is the acceptor
and the aryl moiety is the donor, while in analogue 17 the adenine
is the donor and the aryl group is the acceptor.
related to the relative proportion of conformers, given by eqn (1)–
(3):
J1¢2¢ = 9.3(1 - XN ) + 9.3XS
J2¢3¢ = 4.6XN + 5.3(1 - XN )
J3¢4¢ = 9.3XN
(1)
(2)
(3)
Using the assigned J-coupling constants and the above equa-
tions, the mole fractions of conformers S and N for nucleotides 8,
15, and 22 were calculated (Table 2). For all three analogues the
predominant ribose puckering is S, ranging from 68 up to 81% of
the conformers’ population.
Discussion
Internal charge transfer process from the donor to the acceptor
via the linker results in a large dipole moment in the excited state
compared to that of the ground state, therefore the excited state is
We have designed and synthesized analogues 7–25 as novel
fluorescent nucleoside probes based on a minimal extension of
the non-fluorescent adenosine or guanosine chromophore. In
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more stabilized by the polar solvents and the excited-state energy
is lowered, resulting in longer emission wavelengths.44–46
the 2¢-deoxyribose analogue 16 had lower quantum yield (2-fold)
than the ribose analogue 15, and the emission wavelength was
slightly shorter (408 vs. 419 nm).
All directly conjugated adenosine and guanosine analogues, 7–
14 (except analogue 12), exhibited high quantum yields (>0.1),
especially analogues 9 and 13 with a quantum yield of 0.46. How-
ever, as shown for analogue 8, directly conjugated adenosine and
guanosine analogues adopt preferentially the syn conformation
due to steric hindrance between the aryl/heteroaromatic moiety
at the C-8 position and the ribose ring. Adenosine and guanosine
analogues 7–14 may not be attractive as fluorescent probes for
incorporation in oligonucleotides, because their syn conformation
might enhance the formation of the thermodynamically less stable
Z-DNA duplex.
The alkenyl conjugated nucleoside analogues 15–21 displayed
longer excitation and emission wavelengths as compared to di-
rectly conjugated analogues 7–14 (lem 408–459 nm) as anticipated
according to the ICT theory. In addition to long lem values, a major
advantage of analogues 15–21 is their preferred anti conformation.
The phenyl moiety is shifted away from the ribose ring by the
(E)-configured vinyl linker, thus minimizing steric hindrance and
enabling the nucleoside standard anti conformation. Furthermore,
a population of ca. 80% Southern sugar puckering indicates
that upon incorporation into an oligonucleotide, followed by
hybridization with target nucleic acid, analogues 15–21 are
expected to contribute to the formation of stable B-DNA duplex.
Analogues 15, 16, and 20, where the purine moiety is an acceptor,
exhibited the lowest quantum yields of all analogues (£0.018),
a fact that may reduce their usefulness as fluorescent probes.
However, analogues 17 and 21, where the purine moiety is a donor,
have the highest quantum yields (f 0.81 and 0.8, respectively)
and the longest emission wavelengths (lem 439 nm and 459 nm,
respectively). Namely, purine nucleosides play either the acceptor
or the donor role in push–pull systems 15–21. Yet, dramatically
enhanced fluorescence is obtained when the purine moiety plays
the donor role in those push–pull molecules.47
For the alkynyl conjugated analogues 22–25, we also antici-
pated long lem values, as for the alkenyl conjugated nucleosides;
however, they exhibited relatively short emission wavelengths
(£385 nm). This phenomenon has been reported for p-amino
styryl-naphthalene derivatives with different linkers between the
aryl moieties,48 and was explained by the delocalization of charge
along the p system which includes more resonance structures for
the alkenyl containing compound than for the alkynyl containing
compound (Scheme 1). In addition, as shown for analogue 22,
alkynyl conjugated compounds adopt the undesired syn confor-
mation due to steric hindrance between the alkynylaryl moiety and
the ribose ring. Therefore, analogues 22–25 may not be suitable
for incorporation in oligonucleotides to make fluorescent probes.
The changes in the lem between A and G analogues bearing the
same donor moieties were not significant, e.g. the adenine analogue
8 and the guanine analogue 11 exhibited the same emission
wavelength (~360 and 380 nm). Therefore, we conclude that the
nucleobase does not have a significant effect on fluorescence.
To our surprise, there was a difference in the fluorescent prop-
erties of the ribose analogues compared to their corresponding
2¢-deoxyribose analogues, yet no clear dependence of fluorescence
on the presence of 2¢-OH could be found. For instance, the 2¢-
deoxyribose analogue 9 had higher quantum yield (~4-fold) than
the ribose analogue 8, but the same emission wavelength. However,
Probes 7–25, upon incorporation in oligonucleotides, were
designed to be used in cells, but, fluorescence microscopy analysis
requires probes with long absorption and emission wavelengths,
due to auto-fluorescence of cells.49 Therefore, out of analogues
7–25, the alkenyl conjugated nucleoside analogues, 15–21, are our
most promising candidates as fluorescent probes, in particular,
analogues 17 and 21 with the longest emission wavelength and the
extremely high quantum yield.
The promising nucleoside probes will be eventually incorpo-
rated in oligonucleotides to be used for identification of target
nucleic acids by hybridization assays. In those hybridization assays
a B-DNA, rather than Z-DNA, structure is desired for the
duplexes between probes and target nucleic acids, due its higher
thermodynamic stability. Yet, the DNA conformation depends on
various factors including chemical modifications of the bases,50
which may affect syn/anti 51,52 or S/N conformers.
To ensure that our planned duplexes will adopt B-DNA
structure, the conformation around the glycosidic bond and sugar
puckering were determined for selected nucleoside probes. As
was shown for analogue 15, the alkenyl conjugated nucleoside
analogues adopt preferentially the anti and S conformation, which
may induce the formation of the favored B-DNA.
The potential of compounds 17 and 21 (and the corresponding
2¢-deoxy-analogues) for the detection and quantification of genetic
material upon incorporation in oligonucleotide probes will be
reported in due course.
Experimental section
General
1H-NMR and 13C-NMR spectra were obtained on Bruker AC-
200 and DPX-300 and DMX-600 spectrometers. For conforma-
1
tional analysis H-NMR and NOESY spectra were obtained on
Bruker DMX-600 with DMSO-d6 as solvent. Chemical shifts are
expressed in ppm downfield from Me4Si (TMS) used as internal
standard. The values are given in d scale. All moisture sensitive
reactions were carried out in flame-dried reactions flasks with
rubber septa, and the reagents were introduced with a syringe.
Progress of the reaction was monitored by TLC on precoated
Merck silica gel plates (60F-254). Visualization was accomplished
by UV light. Flash chromatography was carried out on silica gel
(Davisil Art. 1000101501). Medium pressure chromatography was
carried out using automated flash purification system (Biotage
SP1 separation system, Uppsala, Sweden). The purity of the
analogues was evaluated on HPLC (Merck-Hitachi) system, using
an analytical reverse-phase column (Gemini 5u, C-18, 110A, 150 ¥
4.60 mm, 5 micron, Phenomenex, Torrance, CA, USA), in two
solvent systems as described below. The purity of the nucleosides
was generally ≥95%. New compounds were analyzed under
ESI (electron spray ionization) conditions on a Q-TOF micro-
instrument (Waters, UK). High resolution mass spectra were
recorded on an AutoSpec Premier (Waters, UK) spectrometer by
chemical ionization. Absorption spectra were measured on a UV
instrument (UV-2401PC UV–VIS recording spectrophotometer,
Shimadzu, Kyoto, Japan). Emission spectra were measured using
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Scheme 1 Resonance structures of analogues 15 and 22.
Aminco-Bowman series 2 (AB2) Luminescence Spectrometer General procedure for the preparation of analogues 22–25
(Thermo electron corporation, Markham, Ontario, Canada). As
a standard we used tryptophan in H2O (lex 286 nm, lem 355 nm, f
0.13), or quinine sulfate in H2O (lex 350 nm, lem 446 nm, f 0.54).27
Analogues 7,53 8,53 9,5411,22 14,55 22,56 and 2524 were synthesized
according to the literature.
To a solution of 8-bromoadenosine (0.43 mmol, 1 eq) or 8-
bromoguanosine (0.41 mmol, 1 eq) in dry DMF (12.7 mL),
and Et3N (1.95 mL) under an atmosphere of Ar were added
tetrakis(triphenylphosphine)palladium (0.043 mmol, 0.1 eq) and
CuI (0.003 mmol). The appropriate terminal alkyne (1.29 mmol,
3 eq) was added and the reaction mixture was stirred at room
temperature for overnight. The solvent was removed in vacuo
and the residue was purified on a silica gel column using
CHCl3 : MeOH, 8 : 2, as the eluent.
General procedure for the preparation of analogues 7–21
Water–acetonitrile (2 : 1, 3 mL) solvent mixture was added through
a septum to an argon-purged round bottom flask containing 8-
bromoadenosine (0.29 mmol, 1 eq) or 8-bromoguanosine (0.27
mmol, 1 eq), Pd(OAc)2 (0.014 mmol, 0.05 eq), sodium carbonate
(0.86 mmol, 3 eq), TPPTS (0.072 mmol, 0.25 eq), and the
appropriate boronic acid derivative (0.36 mmol, 1.25 eq). The
mixture was stirred at 90 ◦C under argon atmosphere for ~2 h.
The reaction was cooled to room temperature and few drops of
37% HCl were added (up to pH~7). The product was purified on
a silica gel column using CHCl3 : MeOH, 8 : 2, as the eluent.
5-(6-Amino-8-(3,4,5-trimethoxyphenyl)-9H-purin-9-yl)-2-
(hydroxymethyl)tetrahydrofuran-3-ol (10)
Product 10 was obtained as an orange solid (110 mg, 87%),
starting from 8-Br-2¢-deoxy-adenosine (100 mg, 0.303 mmol) and
◦
1
3,4,5-trimethoxyphenylboronic acid. Mp 118–119 C; H-NMR
(300 MHz, DMSO-d6): d 8.14 (s, 1H, 2-H), 7.43 (s, 2H, NH2),
7.00 (s, 2H, Ph-H), 6.20 (dd, J = 8.3 and 5.5 Hz, 1H, 1¢-H), 5.59
(dd, J = 8.5 and 3.9 Hz, 1H, OH), 5.26 (d, J = 4.6 Hz,1H, OH),
7770 | Org. Biomol. Chem., 2011, 9, 7763–7773
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◦
1
4.45 (m, 1H, 3¢-H), 3.86 (m, 1H, 4¢-H), 3.84 (s, 6H, m-OCH3), 3.76
(s, 3H, p-OCH3), 3.68 (m, 1H, 5¢-H), 3.52 (m, 1H, 5¢¢-H), 3.20 (m,
1H, 2¢-H), 2.20 (m, 1H, 2¢¢-H) ppm; 13C-NMR (75 MHz, DMSO-
d6): d 156.1, 152.9, 151.9, 150.4, 149.9, 138.9, 124.8, 119.0, 107.0,
88.3, 85.8, 71.5, 62.4, 60.2, 56.1, 37.4 ppm. Purity data obtained
on an analytical column: tR 6.4 min, using solvent system I (85 : 15
to 50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 95% purity; tR
5.46 min, solvent system II (60 : 40 to 20 : 80 H2O : MeOH over
10 min, 1 mL min-1) 97% purity. UV–vis (CH3OH) lmax 292 nm.
MS ES+ m/z: 418 (MH+); HR MALDI calcd for C19H24N5O6:
418.2376, found 418.1721.
(4-methoxyphenyl)vinylboronic acid. Mp 228–229 C (dec); H
NMR (600 MHz, DMSO-d6): d 8.26 (s, 1H, H-2), 7.75 (d, J =
14 Hz, 1H, CH), 7.73 (d, J = 8.7 Hz, 2H, Ph-H), 7.47 (d, J = 14 Hz,
1H, CH), 7.00 (d, J = 8.7 Hz, 2H, Ph-H), 6.14 (d, J = 6.9 Hz, 1H,
H-1¢), 4.68 (t, J = 6.9 Hz, 1H, H-2¢), 4.22 (dd, J = 4.9 and 1.9 Hz,
1H, H-3¢), 4.02 (m, 1H, H-4¢), 3.81 (s, 3H, CH3), 3.73 (m, 1H, H-
5¢), 3.64 (m, 1H, H-5¢¢) ppm; 13C-NMR (150 MHz, DMSO-d6): d
160.3, 158.8, 149.9, 149.7, 137.0, 129.4, 128.1, 127.4, 118.8, 114.4,
112.0, 87.5, 86.4, 72.7, 70.1, 61.5, 55.3 ppm. Purity data obtained
on an analytical column: tR 4.59 min, using solvent system I (75 : 25
to 50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 95% purity; tR 6.5
min, solvent system II (60 : 40 to 20 : 80 H2O : MeOH over 10 min,
1 mL min-1) 98% purity. UV–vis (CH3OH) lmax 345 nm. MS ES+
m/z: 400 (MH+); HR MALDI calcd for C19H22N5O5: 400.1615,
found 400.1621.
2-(6-Amino-8-(5-methylthiophen-2-yl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (12)
Product 12 was obtained as a white solid (25 mg, 24%),
starting from 8-Br-adenosine (100 mg, 0.29 mmol) and 5-
methyl-2-thiopheneboronic acid. Mp 210–211 ◦C (dec); 1H-NMR
(300 MHz, DMSO-d6): d 8.12 (s, 1H, 2-H), 7.48 (s, 2H, NH2), 7.45
(d, J = 3.4 Hz, 1H, CH), 6.99 (d, J = 3.4 Hz, 1H, CH), 5.99 (d,
J = 7.1 Hz, 1H, 1¢-H), 5.79 (dd, J = 8.7 and 3.2 Hz, 1H, OH),
5.50 (d, J = 6.3 Hz, 1H, OH), 5.19 (m, 1H, OH), 5.18 (m, 1H,
2¢-H), 4.20 (m, 1H, 3¢-H), 3.97 (m, 1H, 4¢-H), 3.68 (ddd, J = 12,
6.7, and 3 Hz, 1H, 5¢-H), 3.56 (ddd, J = 12, 9.6, and 3.70 Hz,
1H, 5¢¢-H), 2.54 (s, 3H, CH3) ppm; 13C-NMR (75 MHz, DMSO-
d6): d 155.9, 152.0, 149.8, 145.1, 143.7, 130.1, 128.1, 126.6, 119.1,
89.1, 86.7, 71.3, 71.0, 62.2, 14.9 ppm. Purity data obtained on
an analytical column: tR 6.67 min, using solvent system I (85 : 15
to 50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 96% purity; tR
6.14 min, solvent system II (60 : 40 to 20 : 80 H2O : MeOH over 10
min, 1 mL min-1) 95% purity. UV–vis (CH3OH) lmax 309 nm. MS
ES+ m/z: 364 (MH+); HR MALDI calcd for C15H17N5NaO4S:
386.2363, found 386.0893.
5-(6-Amino-8-((E)-4-methoxystyryl)-9H-purin-9-yl)-2-
(hydroxymethyl)tetrahydrofuran-3-ol (16)
Product 16 was obtained as a yellow solid (96 mg, 83%), starting
from 8-Br-2¢-deoxy-adenosine (100 mg, 0.303 mmol◦) and trans-2-
1
(4-methoxyphenyl)vinylboronic acid. Mp 230–231 C (dec); H-
NMR (300 MHz, DMSO-d6): d 8.09 (s, 1H, 2-H), 7.71 (d, J =
8.6 Hz, 2H, Ph-H), 7.68 (d, J = 15.7 Hz, 2H, CH), 7.45 (d, J =
15.7 Hz, 2H, CH), 7.28 (s, 2H, NH2), 6.98 (d, J = 8.6 Hz, 2H, Ph-
H), 6.59 (dd, J = 5.7 and 1.4 Hz, 1H, 1¢-H), 5.49 (dd, J = 6.9 and
4.1 Hz, 1H, OH), 5.31 (d, J = 4.1 Hz, 1H, OH), 4.51 (m, 1H, 3¢-H),
3.89 (m, 1H, 4¢-H), 3.80 (s, 3H, O-CH3), 3.71 (m, 1H, 5¢-H), 3.59
(m, 1H, 5¢¢-H), 2.93 (m, 1H, 2¢-H), 2.19 (m, 1H, 2¢¢-H) ppm; 13C-
NMR (75 MHz, DMSO-d6): d 160.1, 155.5, 151.7, 149.9, 148.3,
135.9, 129.1, 128.4, 119.1, 114.3, 112.6, 87.8, 83.4, 70.7, 61.7, 55.3
ppm. Purity data obtained on an analytical column: tR 8.35 min,
using solvent system I (85 : 15 to 50 : 50 H2O : CH3CN over 10 min,
1 mL min-1) 99% purity; tR 5.93 min, solvent system II (50 : 50 to
20 : 80 H2O : MeOH over 10 min, 1 mL min-1) 95% purity. UV–vis
(CH3OH) lmax 340 nm. MS ES+ m/z: 384 (MH+); HR MALDI
calcd for C19H21N5O4: 384.2238, found 384.1666.
5-(6-Amino-8-(furan-2-yl)-9H-purin-9-yl)-2-
(hydroxymethyl)tetrahydrofuran-3-ol (13)
Product 13 was obtained as a white solid (85 mg, 89%), starting
from 8-Br-2¢-deoxy-adeno◦sine (100 mg, 0.303 mmol) and 2-
furanboronic acid. Mp 186 C (dec); 1H-NMR (300 MHz, DMSO-
d6): d 8.13 (s, 1H, 2-H), 8.01 (dd, J = 1.8 and 0.8 Hz, 1H,CH), 7.49
(s, 2H, NH2), 7.12 (dd, J = 3 and 0.8 Hz, 1H, CH), 6.78 (dd, J = 3
and 1.8 Hz, 1H, CH), 6.54 (dd, J = 8 and 6.3 Hz, 1H, 1¢-H), 5.50
(dd, J = 8.2 and 4 Hz, 1H, OH), 5.30 (d, J = 3.2 Hz, 1H, OH), 4.50
(m, 1H, 3¢-H), 3.90 (m, 1H, 4¢-H), 3.67 (ddd, J = 11.8, 7.8, and
4 Hz, 1H, 5¢-H), 3.52 (ddd, J = 11.8, 7.8, and 4.5 Hz, 1H, 5¢¢-H),
3.29 (m, 1H, 2¢-H), 2.19 (ddd, J = 13.2, 6.3, and 2.6 Hz, 1H, 2¢¢-
H) ppm; 13C-NMR (150 MHz, DMSO-d6): d 156.1, 152.2, 149.6,
145.4, 143.5, 141.0, 119.4, 113.6, 112.1, 88.3, 85.5, 71.3, 62.2, 37.5
ppm. Purity data obtained on an analytical column: tR 4.6 min,
using solvent system I (75 : 25 to 50 : 50 H2O : CH3CN over 10 min,
1 mL min-1) 95% purity; tR 5.93 min, solvent system II (60 : 40 to
20 : 80 H2O : MeOH over 10 min, 1 mL min-1) 95% purity. UV–vis
(CH3OH) lmax 306 nm. MS ES+ m/z: 318 (MH+); HR MALDI
calcd for C14H15N5O4: 318.1714, found 318.1197.
2-(6-Amino-8-((E)-4-(trifluoromethyl)styryl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (17)
Product 17 was obtained as a white solid (70 mg, 55%), start-
ing from 8-Br-adenosine (100 mg, 0.29 mmol) and trans-2-[4-
(trifluoromethyl)phenyl]vinylboronic acid. Mp 212–213 ◦C (dec);
1H-NMR (200 MHz, DMSO-d6): d 8.12 (s, 1H, 2-H), 7.98 (d, J =
7.6 Hz, 2H, Ph-H), 7.78 (s, 2H, CH), 7.76 (d, J = 7.6 Hz, 2H,
Ph-H), 7.46 (s, 2H, NH2), 6.16 (d, J = 7 Hz, 1H, 1¢-H), 5.82 (dd,
J = 7.5 and 3.5 Hz, 1H, OH), 5.37 (d, J = 6.5 Hz, 1H, OH), 5.26
(d, J = 4.5 Hz, 1H, OH), 4.71 (m, 1H, 2¢-H), 4.20 (m, 1H, 3¢-H),
4.03 (m, 1H, 4¢-H), 3.71- 3.65 (m, 2H, 5¢-H and 5¢¢-H) ppm; 13C-
NMR (50 MHz, DMSO-d6): d 155.8, 152.2, 150.1, 147.5, 139.7,
134.4, 128.4, 128.1, 125.6, 117.8, 87.5, 86.5, 72.8, 70.4, 61.8 ppm.
Purity data obtained on an analytical column: tR 9.67 min, using
solvent system I (75 : 25 to 50 : 50 H2O : CH3CN over 10 min, 1 mL
min-1) 99% purity; tR 6.57 min, solvent system II (90 : 10 to 70 : 30
MeOH : CH3CN over 10 min, 1 mL min-1) 98% purity. UV–vis
(CH3OH) lmax 340 nm. MS ES+ m/z: 438 (MH+); HR MALDI
calcd for C19H18F3N5O4: 437.3762, found 438.1380.
2-(6-Amino-8-(4-methoxystyryl)-9H-purin-9-yl)-5-
hydroxymethyl)tetrahydrofuran-3,4-diol (15)
Product 15 was obtained as a yellow solid (47 mg, 42%),
starting from 8-Br-adenosine (100 mg, 0.29 mmol) and trans-2-
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5-(6-Amino-8-(4-(trifluoromethyl)styryl)-9H-purin-9-yl)-2-
ES+ m/z: 438 (MNa+); HR MALDI calcd for C19H21N5NaO6:
(hydroxymethyl)tetrahydrofuran-3-ol (18)
438.1384, found 438.1371.
Product 18 was obtained as a yellow solid (82 mg, 65%), starting
from 8-Br-2¢-deoxy-adenosine (100 mg, 0.303 mmol) and trans-
2-[4-(trifluoromethyl)phenyl] vinylboronic acid. Mp 219–220 ◦C
(dec); 1H-NMR (200 MHz, DMSO-d6): d 8.12 (s, 1H, 2-H), 7.99
(d, J = 7.78 Hz, 2H, Ph-H), 7.86 (d, J = 12.46 Hz, 1H, CH), 7.77
(d, J = 12.46 Hz, 1H, CH), 7.76 (d, J = 7.78 Hz, 2H, Ph-H), 7.41
(s, 2H, NH2), 6.64 (t, J = 7.78 Hz, 1H, 1¢-H), 5.46 (t, J = 4.67 Hz,
1H, OH), 5.34 (d, J = 4.67 Hz, 1H, OH), 4.52 (m, 1H, 3¢-H), 3.92
(m, 1H, 4¢-H), 3.70–3.60 (m, 2H, 5¢-H and 5¢¢-H), 2.91 (m, 1H, 2¢-
H), 2.22 (m, 1H, 2¢¢-H) ppm; 13C-NMR (150 MHz, DMSO-d6): d
155.7, 152.8, 149.9, 147.3, 139.7, 134.2, 128.5, 127.6, 125.7, 120.3,
119.2, 118.1, 87.9, 83.5, 70.1, 61.6, 39.2 ppm. Purity data obtained
on an analytical column: tR 6.84 min, using solvent system I (65 : 35
to 50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 95% purity; tR
7.47 min, solvent system II (90 : 10 to 70 : 30 MeOH : CH3CN over
10 min, 1 mL min-1) 98% purity. UV–vis (CH3OH) lmax 338 nm.
MS ES+ m/z: 423 (MH+); HR MALDI calcd for C19H19F3N5O3:
422.3836, found 422.1435.
2-Amino-9-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-8-(4-(trifluoromethyl)styryl)-1H-purin-6(9H)-one (21)
Product 21 was obtained as a yellow–green solid (89 mg, 73%),
starting from 8-Br-guanosine (100 mg, 0.27 mmol) and trans-2-[4-
(trifluoromethyl)phenyl]vinylboronic acid. Mp 188–190 ◦C (dec);
1H NMR (300 MHz, DMSO-d6): 7.92 (d, J = 8.44 Hz, 2H, Ph-H),
7.70 (d, J = 8.44 Hz, 2H, Ph-H), 7.69 (d, J = 19 Hz, 1H, CH),
7.64 (d, J = 19 Hz, 1H, CH), 6.58 (s, 2H, NH2), 5.96 (d, J =
7.39 Hz, 1H, H-1¢), 5.38 (t, J = 5.23 Hz, 1H, OH), 5.34 (d, J =
5.23 Hz, 1H, OH), 5.10 (d, J = 3.49 Hz, 1H, OH), 4.45 (m, 1H,
H-2¢), 4.14 (m, 1H, H-3¢), 3.92 (m, 1H, H-4¢), 3.70 (m, 2H, H-5¢
and H-5¢¢) ppm; 13C-NMR (150 MHz, DMSO-d6): d 156.3, 153.8,
152.3, 143.8, 140.3, 131.6, 131.1, 127.7, 125.6, 125.5, 123.4, 118.7,
116.8, 86.4, 85.6, 72.9, 69.8, 61.2 ppm. Purity data obtained on an
analytical column: tR 8.27 min, using solvent system I (75 : 25 to
50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 99% purity; tR 4.85
min, solvent system II (90 : 10 to 70 : 30 MeOH : CH3CN over 10
min, 1 mL min-1) 99% purity. UV–vis (CH3OH) lmax 357 nm. MS
ES+ m/z: 476 (MNa+); HR MALDI calcd for C19H18F3N5NaO5:
476.1752, found 476.1152.
2-(6-Amino-8-(4-fluorostyryl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (19)
Product 19 was obtained as a yellow solid (45 mg, 40%),
starting from 8-Br-adenosine (100 mg, 0.29 mmol) and trans-2-(4-
5-(6-Amino-8-((4-methoxyphenyl)ethynyl)-9H-purin-9-yl)-2-
(hydroxymethyl)tetrahydrofuran-3-ol (23)
◦
1
fluorophenyl)vinylboronic acid. Mp 215–216 C (dec); H-NMR
(200 MHz, DMSO-d6): d 8.10 (s, 1H, 2-H), 7.82 (m, 2H, Ph-H),
7.74 (d, J = 15.88 Hz, 1H, CH), 7.53 (d, J = 15.88 Hz, 1H, CH),
7.40 (s, 2H, NH2), 7.26 (m, 2H, Ph-H), 6.12 (d, J = 7.4 Hz, 1H,
1¢-H), 5.82 (t, J = 3.7 Hz, 1H, OH), 5.35 (m,1H, OH), 5.27 (m,
1H, OH), 4.7 (m, 1H, 2¢-H), 4.20 (m, 1H, 3¢-H), 4.01 (m, 1H, 4¢-
H), 3.70–3.62 (m, 2H, 5¢-H and 5¢¢-H) ppm; 13C-NMR (50 MHz,
DMSO-d6): d 155.7, 151.9, 150.0, 148.1, 135.1, 132.4, 129.8, 129.6,
116.1, 115.6, 114.8, 87.6, 86.5, 72.7, 70.4, 61.8 ppm; 19F-NMR
(188 MHz, DMSO-d6): d -112.4 ppm. Purity data obtained on
an analytical column: tR 9.23 min, using solvent system I (85 : 15
to 50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 98% purity; tR
3.47 min, solvent system II (90 : 10 to 70 : 30 MeOH : CH3CN over
10 min, 1 mL min-1) 95% purity. UV–vis (CH3OH) lmax 333 nm.
MS ES+ m/z: 388 (MH+); HR MALDI calcd for C18H19FN5O4:
388.3771, found 388.1416.
Product 23 was obtained as a brown solid (30 mg, 26%),
starting from 8-Br-2¢-deoxy-adenosine (100 mg, 0.303 mmol) and
4-ethynylanisole. Mp 167–168 ◦C (dec); 1H-NMR (200 MHz,
DMSO-d6): d 8.16 (s, 1H, 2-H), 7.63 (d, J = 8.3 Hz, 2H, Ph-
H), 7.59 (s, 2H, NH2), 7.06 (d, J = 8.3 Hz, 2H, Ph-H), 6.52 (dd,
J = 7.1 and 6 Hz, 1H, 1¢-H), 5.36 (d, J = 4.4 Hz, 1H, OH), 5.34
(dd, J = 7.4 and 4.4 Hz, 1H, OH), 4.50 (m, 1H, 3¢-H), 3.92 (m,
1H, 4¢-H), 3.83 (s, 3H, O-CH3), 3.65 (m, 1H, 5¢-H), 3.51 (m, 1H,
5¢¢-H), 3.12 (m, 1H, 2¢-H), 2.26 (m, 1H, 2¢¢-H) ppm; 13C-NMR
(50 MHz, DMSO-d6): d 160.8, 155.9, 153.2, 148.5, 133.7, 133.3,
114.8, 111.7, 94.9, 88.3, 85.0, 77.6, 77.4, 71.3, 62.2, 55.5, 37.8
ppm. Purity data obtained on an analytical column: tR 8.74 min,
using solvent system I (85 : 15 to 50 : 50 H2O : CH3CN over 10 min,
1 mL min-1) 98% purity; tR 7.99 min, solvent system II (60 : 40 to
20 : 80 H2O : MeOH over 10 min, 1 mL min-1) 99% purity. UV–vis
(CH3OH) lmax 323 nm. MS ES+ m/z: 381 (MH+); HR MALDI
calcd for C19H19N5O4: 382.2080, found 382.1510.
2-Amino-9-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-
yl)-8-(4-methoxystyryl)-1H-purin-6(9H)-one (20)
2-(6-Amino-8-((3,5-dimethoxyphenyl)ethynyl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (24)
Product 20 was obtained as a yellow solid (10.8 mg, 10%),
starting from 8-Br-guanosine (100 mg, 0.27 mmol)◦and trans-2-
1
(4-methoxyphenyl)vinylboronic acid. Mp 205–206 C (dec); H
Product 24 was obtained as a pale yellow solid (117 mg, 95%),
NMR (600 MHz, DMSO-d6): d 7.64 (d, J = 8.2 Hz, 2H, Ph-H),
7.49 (d, J = 15.6 Hz, 1H, CH), 7.28 (d, J = 15.6 Hz, 1H, CH), 6.94
(d, J = 8.2 Hz, 2H, Ph-H), 6.47 (s, 2H, NH2), 5.92 (d, J = 6.8 Hz,
1H, H-1¢), 5.31 (m, 2H, OH), 5.07 (m, 1H, OH), 4.50 (m, 1H, H-
2¢), 4.16 (m, 1H, H-3¢), 3.90 (m, 1H, H-4¢), 3.79 (s, 3H, CH3), 3.72
(m, 1H, H-5¢), 3.65 (m, 1H, H-5¢¢) ppm. Purity data obtained on
an analytical column: tR 5.57 min, using solvent system I (85 : 15
to 50 : 50 H2O : CH3CN over 10 min, 1 mL min-1) 99% purity; tR
6.96 min, solvent system II (70 : 30 to 30 : 70 H2O : MeOH over 10
min, 1 mL min-1) 99% purity. UV–vis (CH3OH) lmax 350 nm. MS
starting from 8-Br-adenosine (100 mg, 0.29 mmol) and 1-ethynyl-
◦
1
3,5-dimethoxybenzene. Mp 208 C (dec); H-NMR (300 MHz,
DMSO-d6): d 8.18 (s, 1H, 2-H), 7.67 (s, 2H, NH2), 6.82 (d, J =
2.3 Hz, 2H, Ph-H), 6.70 (t, J = 2.3 Hz, 1H, Ph-H), 6.04 (d, J =
6.6 Hz, 1H, 1¢-H), 5.53 (dd, J = 8.6 and 3.9 Hz, 1H, OH), 5.48
(d, J = 6.5 Hz, 1H, OH), 5.25 (d, J = 4.7 Hz, 1H, OH), 5.00 (m,
1H, 2¢-H), 4.20 (m, 1H, 3¢-H), 4.00 (m, 1H, 4¢-H), 3.81 (s, 6H,
O-CH3), 3.70 (m, 1H, 5¢-H), 3.55 (m, 1H, 5¢¢-H) ppm; 13C-NMR
(75 MHz, DMSO-d6): d 160.6, 156.2, 153.5, 148.6, 133.2, 121.3,
119.6, 109.7, 103.2, 94.3, 89.4, 86.7, 78.1, 71.8, 71.0, 62.2, 55.6,
7772 | Org. Biomol. Chem., 2011, 9, 7763–7773
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45.8 ppm. Purity data obtained on an analytical column: tR 6.61
min, using solvent system I (85 : 15 to 50 : 50 H2O : CH3CN over 10
min, 1 mL min-1) 99% purity; tR 6.32 min, solvent system II (50 : 50
to 20 : 80 H2O : MeOH over 10 min, 1 mL min-1) 99% purity. UV–
vis (CH3OH) lmax 316 nm. MS ES+ m/z: 428 (MH+); HR MALDI
calcd for C20H21N5O6: 428.4228, found 428.1565.
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Abbreviations
27 J. Yvon,
A Guide to recording fluorescence quantum yields,
A
adenosine
http://www.jobinyvon.com.
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G
guanosine
NOESY
TPPTS
EDG
EWG
ICT
Nuclear Overhauser Enhancement spectroscopy
tris(3-sulfonatophenyl) phosphine trisodium salt
electron donating group
electron withdrawing group
intramolecular charge transfer
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