Rules for the design of highly fluorescent nucleoside probes: 8-(substituted cinnamyl)-adenosine analogues

Article abstract of DOI:10.1021/jo402050x

Currently, there are no tools that can help the design of useful fluorescent analogues. Hence, we synthesized a series of 8-(substituted cinnamyl)-adenosine analogues, 5-17, and established a relationship between their structure and fluorescence properties. We attempted to find a correlation between maximum emission wavelengths (λ_em) of 5-17 or their quantum yields (φ), and Hammett constants (σ_p and σ_m) of the substituent on the cinnamyl moiety. A linear correlation was observed at low-medium σ values, but not at high σ values (≥0.7). Next, we explored correlation between λ_em and φ of 5-17 and computed HOMO and LUMO energy levels of fragments of 5-17, i.e., 8-vinyl 9-Me-adenine (fluorescent molecule), 18, and substituted toluene rings (fluoresence modulators), 19-30. High φ correlated with relatively close LUMO levels of 19-30 and 18 (-0.076 to -0.003 eV). The electron density of LUMO of nitro analogues 9 and 15 is localized on the aryl ring only, which explains their low φ. Calculation of HOMO-LUMO gap of 5-17 enables accurate prediction of the λ_abs for a planned analogue, and LUMO levels of an aryl moiety vs 8-vinyl 9-Me-adenine, allows the prediction of high or low φ. These findings lay the ground for prediction of fluorescence properties of additional analogues having a similar structure.

Full text of DOI:10.1021/jo402050x

Article
pubs.acs.org/joc
Rules for the Design of Highly Fluorescent Nucleoside Probes:
8‑(Substituted Cinnamyl)-Adenosine Analogues
Lital Zilbershtein-Shklanovsky, Michal Weitman, Dan Thomas Major, and Bilha Fischer*
Department of Chemistry, Gonda-Goldschmied Medical Research Center and the Lise-Meitner-Minerva Center of Computational
Quantum Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
S
* Supporting Information
ABSTRACT: Currently, there are no tools that can help the
design of useful fluorescent analogues. Hence, we synthesized
a series of 8-(substituted cinnamyl)-adenosine analogues, 5−
17, and established a relationship between their structure and
fluorescence properties. We attempted to find a correlation
between maximum emission wavelengths (λem) of 5−17 or
their quantum yields (φ), and Hammett constants (σp and σm)
of the substituent on the cinnamyl moiety. A linear correlation
was observed at low-medium σ values, but not at high σ values
(≥0.7). Next, we explored correlation between λem and φ of
5−17 and computed HOMO and LUMO energy levels of fragments of 5−17, i.e., 8-vinyl 9-Me-adenine (fluorescent molecule),
18, and substituted toluene rings (fluoresence modulators), 19−30. High φ correlated with relatively close LUMO levels of 19−
30 and 18 (−0.076 to −0.003 eV). The electron density of LUMO of nitro analogues 9 and 15 is localized on the aryl ring only,
which explains their low φ. Calculation of HOMO−LUMO gap of 5−17 enables accurate prediction of the λabs for a planned
analogue, and LUMO levels of an aryl moiety vs 8-vinyl 9-Me-adenine, allows the prediction of high or low φ. These findings lay
the ground for prediction of fluorescence properties of additional analogues having a similar structure.
significantly improved by extending the purine/pyrimidine π-
system.19−22
INTRODUCTION
■
Fluorescent organic molecules constitute an important class of
compounds in many fields of science and are widely used as
biological imaging probes, sensors, and light emitting devices,
as well as for identification and quantification of nucleic
acids.1−4 For the latter application, natural nucleotides are not
useful as fluorescent probes because of their extremely low
quantum yields.5−7 Hence, the use of extrinsic probes, namely,
fluorescent dyes, is necessary.8−11 These dyes are used for
detecting genetic material in DNA microarrays,12 fluorescent in
situ hybridization (FISH),13 gels,14 and cells, by fluorescence
microscopy or electrophoresed gels.15
However, commonly used fluorescent dyes suffer from
various limitations. For instance, most dye molecules are
large and hydrophobic and have limited water solubility. In
addition the large dye molecule attached to a nucleotide alters
the efficiency of enzymatic incorporation into DNA/RNA,
resulting in different levels of labeling and prohibits
quantification of nucleic acids.
Because of limitations and complications of the current
methodologies applying extrinsic dyes, the development of
useful fluorescent nucleosides has been a subject of intensive
research. Over the past decade a number of nucleoside
analogues with intrinsic fluorescence, replacing natural DNA
bases in oligonucleotide probes, have been reported as an
alternative to dye-substituted nucleosides.16−18 Thus, the
fluorescent properties of the natural nucleosides were
For example, extending the purine π-system by C8
modifications, e.g., 8-vinyl-adenosine, 1, results in a longer
emission wavelength and higher quantum yield as compared to
adenosine (λem 388 nm; φ 0.66 vs λem 319 nm; φ 0.00026).23,24
Another example is the extension of adenine chromophore at
C2,N3-positions to give N2,N3-etheno-adenosine, 2, exhibiting
improved fluorescence characteristics (λem 420 nm; φ
0.03).25,26
Fluorescent pyrimidine nucleosides include pyrrolo-C, 3, φ
0.04, and the pyrrolo-dC, 4, φ 0.05, analogues, which maintain
a proper Watson−Crick H-bonding. These analogues were
incorporated into an oligonucleotide; however, their quantum
yields decreased upon incorporation and were quenched even
further upon duplex formation (Figure 1).27
Previously, we have designed and synthesized fluorescent
purine nucleoside probes based on a minimal extension of the
nonfluorescent adenosine or guanosine chromophore.28 In
these nucleoside analogues, the purine C8-position was
conjugated either directly, or through an alkenyl or alkynyl
linker, to an aryl/hetero aryl moiety to determine the effect of
the type of linker on fluorescence. Moreover, the influence of
the type of the substituent on the aryl moiety, i.e., electron-
donating-group (EDG) vs electron-withdrawing-group (EWG)
Received: September 15, 2013
© XXXX American Chemical Society
A
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Figure 1. Fluorescent purine and pyrimidine analogues: 8-vinyladenosine, 1; C2,N3-etheno-adenosine, 2; Pyrrolo-C, 3; Pyrrolo-dC, 4.
on fluorescence and the effect of number of substituents were
studied as well. These extensions of the natural purine
nucleobases at the C-8 position significantly enhanced their
spectral properties. For instance, the emission wavelength of
the natural adenosine was red-shifted by 36−140 nm, and the
quantum yield increased 10−3100-fold. In particular, alkenyl
conjugated nucleoside analogues (e.g., 5) displayed the longest
shift of emission wavelengths (75−140 nm) vs parent
nucleosides. However, the quantum yields of alkenyl
conjugated analogues were either very high for several
analogues (e.g., φ 0.81, for analogue 5), or very low for others
(e.g., φ 0.005, for analogue 6), Figure 2. These contradictory
Specifically, we report on the synthesis and spectral
characterization of a series of 8-(cinnamyl)-modified adenosine
analogues 5−17 (data for analogues 5, 6, and 8 were previously
reported),28 Figure 3. We explored a possible correlation
between maximum emission wavelengths or quantum yields
and Hammett constants (σp and σm) of the substituent on the
cinnamyl moiety.
Figure 2. Example of previously studied analogues, 5 and 6.
Figure 3. 8-(Cinnamyl)-adenosine analogues synthesized and studied
here.
data led us to extend our research and to establish a relationship
between the probes’ structure and their fluorescence properties,
with a view to setting rules for prediction of improved
fluorescent nucleoside probes.
Although there are numerous reports on the structural
dependence of fluorescence properties,29−31 i.e., λem and φ
values, there are no clear rules that can help predict
fluorescence characteristics of a molecule based on its chemical
structure.
Previous attempts at finding a relationship between the
fluorescent characteristics of several organic compounds such as
N-hexyl-1,8-naphthalimides,32 7-hydroxycumarins,33 and ben-
zofurazan34 and sigma Hammett constants revealed an excellent
correlation, which enabled the prediction of the fluorescent
characteristics of additional analogues. However, these
predictions are specific to the studied structure and cannot be
used for predicting fluorescence of nucleoside analogues.
We targeted the establishment of the relationship between
the chemical nature of the substituents on the cinnamyl moiety
and fluorescence characteristics of the resulting C-8-cinnamyl
nucleoside analogues 5−17.
In addition, we explored the correlation between the
fluorescence properties of 5−17 and calculated HOMO and
LUMO energy levels of 8-(substituted cinnamyl)-9-Me-adenine
analogues, or their fragments: 8-vinyl 9-Me-adenine, 18, and
variously substituted toluene analogues, 19−31 (Figure 4).
RESULTS
■
Selection of a Set of Adenosine Analogues for
Establishing Rules for Prediction of Fluorescent Nucleo-
side Analogues. Previously, we have reported the large
enhancement (up to 162-fold) of the quantum yield of 8-
cinnamyl-adenosine analogues triggered by the p-substituent on
the aryl group (p-CF3, F, or OMe, instead of H).28
Here, we explored the fluorescence of a series of 8-cinnamyl-
adenosine analogues representing a wider chemical space
(analogues 5−17).
We have designed and synthesized a series of 8-cinnamyl
adenosine analogues bearing various substituents at p- or m-
position of the aryl moiety. Selection of the substituents was
done on the basis of their Hammett substituent constants, σp
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state energy is lowered, resulting in longer emission wave-
lengths.37−39
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 or a single
bond. Since the electron-donating or -accepting character of
one moiety in push−pull molecules is relative to the other
moiety, the purine moiety can play either the acceptor or the
donor role. In our previous results, we observed a dramatic
enhancement of fluorescence when the purine moiety and the
aryl group played the donor and acceptor role, respectively, by
adding EWG substituents on the aryl moiety.28
We have applied the Suzuki−Miyuara coupling40,41 for the
preparation of analogues 5−8 and 12−13 and the Heck
coupling42 for the preparation of analogues 9−11 and 14−17.
The great advantage of these procedures is that no protecting
groups are needed, thus allowing a single step reaction toward
the desired conjugated nucleoside analogues. Specifically, for
the Suzuki−Miyuara coupling 8-Br-adenosine43 was directly
conjugated with the appropriate boronic acid 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. Products 5−8 and 12−13 were obtained in 30−35%
yield upon silica gel chromatography.
Figure 4. 8-(Substituted cinnamyl)-9-Me-adenine analogues for which
spectral properties and HOMO−LUMO energies were calculated. (A)
8-(Substituted cinnamyl)-9-Me-adenine analogues studied here. (B) 8-
(Substituted cinnamyl)-9-Me-adenine constituents studied here: 8-
vinyl 9-Me-adenine, 18, and substituted toluene rings, 19−30.
For Heck coupling, 8-vinyl adenosine44 was directly
conjugated with the appropriate aryl iodide/bromide in the
presence of Et3N, Pd(OAc)2, and P(o-tol)3, in DMF at 100−
105 °C. Products 9−11 and 14−17 were obtained in 10−47%
yield upon silica gel chromatography.
and σm, to achieve the widest possible range of σ values.
Hammett constants are based on dissociation of benzoic acid
and p/m-substituted benzoic acids. The sigma values are
derived from the equation below:
Fluorescence Characteristics of Analogues 5−17.
Maximum excitation and maximum emission wavelengths of
compounds 5−17 were measured in a methanolic solution, and
fluorescence quantum yields were determined compared to
quinine sulfate in 0.1 M H2SO4 (λex 350 nm, λem 446 nm, φ
0.54).45 The concentrations of the samples were within the
linear range in which the Beer−Lambert law applies, and the
optical density (OD) was less than 0.05 to avoid the inner filter
effect.46 The fluorescence properties of the analogues are
summarized in Table 1, and their fluorescence intensity and
normalized fluorescence are presented in Figure 5.
σ = log(Kx/K0)
x
where σx is the sigma constant for substituent x, K0 is the acid
dissociation constant for the ionization of benzoic acid and Kx
is the acid dissociation constant for the ionization of a
substituted benzoic acid with a given substituent (x) at a given
position on the aromatic ring.
The fluorescent analogues were designed as push−pull
molecules that undergo an intramolecular charge transfer
(ICT).35,36 ICT process from the donor to the acceptor via a
linker results in a large dipole moment in the excited state
compared to that of the ground state; therefore, the excited
state is more stabilized by the polar solvents, and the excited-
The highest quantum yields were obtained for analogues 5/
10 and 14/16 bearing a CF3 or CN group at either para or
meta position (φ: 0.81, 0.61, 0.55, and 0.66, respectively) and
Table 1. Fluorescent Properties of Analogues 5−17 in MeOH Using Quinine Sulfate As a Standard
Hamett constant
47
47
analogue
Y
X
σp
σm
λex (nm)
ε (M−1 cm−1
)
λem (nm)
φF
5
CF3
H
0.54
−0.27
0
0
0
0
0
0
0
0
0
340
345
336
333
367
350
342
338
336
334
335
337
356
25300
26100
20610
21550
26450
21180
24560
23920
21350
23180
26170
23600
22680
439
0.81
6
OCH3
H
H
394, 419
422
0.005
0.026
0.025
0.002
0.61
7
H
8
F
H
0.06
0.78
0.66
0.4348
−0.17
0
436
9
NO2
CN
COOMe
CH3
H
H
445
10
11
12
13
14
15
16
17
H
463
H
469
0.56
H
437
0.004
0.28
F
0.34
0.43
0.71
0.56
0.43
428
H
CF3
NO2
CN
CF3
0
432
0.55
H
0
420
0.002
0.66
H
0
435
CN
0.66
494
0.45
C
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Figure 5. Fluorescence spectra (A) and normalized fluorescence spectra (B) of analogues 5−17 in MeOH. The concentrations of all the samples
were 2−2.5 μM, and the OD was adjusted to 0.05.
for analogue 11 bearing a COOMe substituent at the para
position (φ: 0.56). On the contrary, the lowest quantum yields
were obtained for analogues with a NO2 substituent at either
the para or the meta position, with quantum yield of 0.002 for
each analogue. Likewise, analogues 6 and 12 with an electron-
donating group, OMe or Me, respectively, at the para position
exhibited quantum yields of 0.005 and 0.004, respectively.
Surprisingly, no additive effect was observed for analogue 17
bearing CN and CF3 at the para and meta positions,
respectively, showing a lower quantum yield, 0.45, than
analogues with only CN or CF3 (φ 0.61 and 0.55, respectively).
We have observed more than one emission band in the
emission spectrum of analogue 6. This phenomenon probably
occurs due to vibrational progressions.28
We attempted to analyze the substituent-dependent
fluorescence of analogues 5−17, by seeking first correlation
with σ values of the substituents.
A plot of φ values vs σp or σm values, and a plot of λem values
vs σp or σm values for analogues 5−16, are presented in Figure
6A and 6B, respectively. As observed from Figure 6A there is a
parabolic dependence between the φ values and σp values, with
the exception of analogues 6 and 12, having negative σp values.
In addition, a linear dependence is observed between the φ
values and σm values. The nitro substituted derivatives (9 and
15) are outliers to the general behavior (see Discussion below).
Figure 6B shows that there is no clear dependence of λem on
σp; however, λem of analogues substituted at the meta position
exhibited a linear dependence on σm, with the exception of m-
nitro analogue 15.
The longest λem was observed for analogue 17 (494 nm)
bearing EWGs at both para and meta positions.
Theoretical Modeling. Since no clear correlation was
found between φ or λem and σp/σm values of substituents of the
nucleosides’ aryl moiety, we concluded that parameters
Figure 6. Dependence of φ values of adenosine analogues 5−16 on σp
affecting electronically only part of the molecule are not useful
○
▲
( ) values (A). Dependence of λem of adenosine
( ) and σm
for the prediction of novel highly fluorescent analogues based
on 8-cinnamyl adenosine scaffold. Alternatively, we considered
molecular properties of analogues 5−17 and explored a
possible correlation between φ or λem and HOMO−LUMO
energy gap.49 HOMO and LUMO orbital energies were
calculated for the ground state. Since the ribose group is not
○
▲
) values (B).
analogues 5−16 on σp ( ) and σm
(
part of the fluorophore excitation and emission process, we
simplified the structure and used a methyl group instead of a
ribose group.
D
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Table 2. Absorption Wavelengths (λ), Oscillator Strengths (f), Molecular Orbital Energies, and Molecular Orbital Energy Gaps
of Compounds 5−17, Obtained from Calculations Run at the M062X/6-311++G(d,p) Level
analogue
substituent
λabs (nm) exp
λabs (nm) calc
f
HOMO (eV)
LUMO (eV)
Δ (eV)
φ
5
p-CF3
p-OMe
H
340
345
336
333
367
350
342
338
336
334
335
337
356
332
334
328
328
365
346
344
330
330
329
341
333
358
1.08
1.24
1.07
1.06
1.12
1.21
1.23
1.18
1.05
1.04
0.70
1.01
1.15
−0.266
−0.254
−0.261
−0.261
−0.268
−0.266
−0.265
−0.258
−0.264
−0.265
−0.266
−0.266
−0.269
−0.058
−0.045
−0.050
−0.049
−0.083
−0.067
−0.064
−0.048
−0.054
−0.056
−0.074
−0.059
−0.077
0.208
0.209
0.211
0.212
0.185
0.199
0.201
0.210
0.210
0.209
0.192
0.207
0.192
0.81
6
0.005
0.26
7
8
p-F
0.025
0.002
0.61
9
p-NO2
p-CN
10
11
12
13
14
15
16
17
p-COOMe
p-Me
0.56
0.004
0.28
m-F
m-CF3
m-NO2
m-CN
p-CN, m-CF3
0.55
0.002
0.66
0.45
The experimental and computed absorption wavelengths are
presented in Table 2 along with the energy of HOMO and
LUMO orbitals for compounds 5−17. In general, absorbance is
related to the gap between HOMO and LUMO levels, and
indeed the electronic transition obtained from the TD DFT
calculations are mainly composed of HOMO → LUMO
transition (Table S27, Supporting Information), but additional
excitations also contribute to the first excited state. The
oscillator strengths suggest that these are allowed transitions.
The energies of HOMO and LUMO of most substituted 8-
cinnamyl-adenosine compounds are lower than that of the
parent compound, 7, with the exception of the p-OMe and the
p-Me substituted compounds (analogues 6 and 12), which have
slightly higher frontier orbital energies, and F-substituted
compounds (analogues 8 and 13), which are similar to the
parent compound.
The HOMO−LUMO energy gap of the parent compound,
7, is 0.211 eV. In all compounds with electron-withdrawing
groups the HOMO−LUMO gap is similar or lower than that of
the parent compound. The p-nitro-compound, 9, is at the
extreme low end of energy gap with a value of 0.185 eV. The
excellent agreement found between the calculated and the
experimental absorption wavelengths validated the approach
used for further calculations.
To gain further insight into the photophysical excitation and
emission process of analogues 5−17, we decided to divide the
analogues into two moieties. The first moiety is 8-vinyl-
adenosine, a known fluorescent nucleoside,23,24 and the second
moiety is a substituted toluene ring, which functions as a
fluorescence modulator. We computed the HOMO−LUMO
energies of model molecules representing 8-(substituted
cinnamyl)-9-Me-adenine moieties: 8-vinyl 9-Me-adenine, 18,
and substituted toluene rings, 19−30 (Figure 4). By calculating
each part separately, we hoped to achieve a better under-
standing of the correlation between the toluene ring substituent
and the fluorescence of the fluorophore. Moreover, since
analogues 5−17, likely undergo an ICT process from the donor
moiety to the acceptor moiety, we examined cases in which the
8-vinyl 9-Me-adenine likely plays the donor role and the
substituted toluene plays the acceptor role (analogues 5, 8−11,
and 13−17, with the electron-withdrawing group on the
toluene ring), and cases in which the 8-vinyl 9-Me-adenine
likely plays the acceptor role and the substituted toluene plays
the donor role (analogues 6 and 12, with the electron-donating
group on the toluene ring).
Here, HOMO levels of all substituted toluene derivatives,
26−29, had lower energy (−0.266 to −0.334 eV) than that of
8-vinyl-9-Me-adenine, 18, (−0.253 eV). Moreover, the
HOMO−LUMO energy gap of all the analogues was larger
(ΔE ≥ 0.234 eV) than the energy gap for 8-vinyl-9-Me-adenine,
18, (0.207 eV), with the exception of analogue 25 with energy
gap of 0.191 eV, Table 3. The most interesting results were
Table 3. Molecular Orbital Energies of Compounds 18−30,
Obtained from Calculations Run at the M062X/6-311++G
(d,p) Level
HOMO
(eV)
LUMO
(eV)
analogue
substituent
f
Δ (eV)
18
8-vinyl-9-Me-
adenine
1.08
−0.253
−0.046
0.207
19
20
21
22
23
24
25
26
27
28
29
30
p-CF3
0.81
0.005
0.03
0.00
0.61
0.56
0.00
0.28
0.55
0.02
0.66
0.45
−0.314
−0.266
−0.291
−0.334
−0.312
−0.317
−0.272
−0.299
−0.310
−0.330
−0.310
−0.314
−0.013
−0.002
−0.003
−0.094
−0.032
−0.058
−0.081
−0.003
−0.018
−0.096
−0.038
−0.076
0.301
0.264
0.288
0.24
p-OMe
p-F
p-NO2
p-CN
0.28
p-COOMe
p-Me
0.259
0.191
0.296
0.292
0.234
0.272
0.238
m-F
m-CF3
m-NO2
m-CN
p-CN, m-CF3
obtained for the LUMO levels of the analogues. The LUMO
levels of all meta substituted toluene derivatives, 26−29, were
found to have higher energies (from −0.038 to −0.003 eV)
than 8-vinyl-9-Me-adenine, 18, (−0.046 eV) with the exception
of nitro-toluene, 28, which exhibited significantly lower LUMO
energy level (−0.096 eV). The para-substituted toluene series
demonstrates a wide range of LUMO energies (from −0.094 to
−0.002 eV) as compared to 8-vinyl-9-Me-adenine’s LUMO
energy (Table 3). When all LUMO energy levels of all
analogues were plotted, we noticed that meta or para
substituted derivatives with LUMO energy level relatively
close to LUMO energy of 8-vinyl-9-Me-adenine (−0.076 to
−0.003 eV) exhibited high φ values (0.28−0.81), Figure 7.
Figure 8 depicts the HOMO and LUMO orbitals of the
unsubstituted compound, 7, as well as those of p- and m-NO2
derivatives 9 and 15. Inspection of the frontier orbitals of all
compounds reveals that the molecular orbitals are delocalized
E
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be of great use for the prediction of absorption wavelengths of
future analogues.
To further our understanding of the influence of the aryl
substitution on the fluorescence of analogues 5−17, we studied
the electronic effects of the fragments of the 8-cinnamyl-
substituted analogues: 8-vinyl-9-Me-adenine, 18, and substi-
tuted toluene rings, 19−30.
A similar computational approach was adopted before for a
fluorescein derivative that consists of a donor−acceptor
system.50 Fluorescein was divided into two parts: the xanthene
moiety as a fluorophore and the benzene moiety as a
fluorescence-controlling moiety, even though there is no
obvious structured linker between them. By the division of
the molecule and taking advantage of the intramolecular
donor−acceptor system, a relationship between the fluorescent
quantum yield and the reduction potential of the substituted-
benzene moiety was determined.
We observed that the ΔELUMO between LUMO level of 8-
vinyl-9-Me-adenine and LUMO levels of substituted toluene
rings is the main factor that determines the quantum yield. This
rule applied to analogues in which 8-vinyl-9-Me-adenine played
either a donor or acceptor role. Thus, we divide nucleoside
analogues, 5−17, into three groups (Table 3, Figure 7): (1)
Analogues exhibiting low quantum yields (0.002−0.004) are
characterized by low LUMO energy levels (m-nitro, 15, p-nitro,
9, and p-Me, 12, E ≤ −0.081 eV). (2) Analogues exhibiting
high quantum yield (0.28−0.81) are characterized by medium
LUMO energy levels (p-CN and m-CF3, 17, p-COOMe, 11, m-
CN, 16, p-CN, 10, m-CF3, 14, p-CF3, 5, m-F, 13, E −0.076 to
−0.003 eV). (3) Analogues exhibiting low quantum yield
(0.005 and 0.025) showing high LUMO energy levels (p-OMe,
6, and p-F, 8, E ≥ −0.002 eV).
Figure 7. Relationship between LUMO levels of substituted toluene
rings, 19−30 and 8-vinyl-9-Me-adenine, 18, and the fluorescent
properties of analogues 5−17.
In this paper, we examined the influence of the addition of an
aryl moiety as a fluorescence modulator, on the fluorescent
properties of the fluorophore 8-vinyl-adenosine. The data
showed that the emission wavelength of 8-vinyl-adenosine was
red-shifted by 31−106 nm, in analogues 5−17, as expected
from the elongation of the parent π system. However, for most
analogues there was a reduction of the quantum yield upon
conjugation of the aryl moiety vs that of the parent compound
(0.002−0.61 vs 0.66). The exceptions to this observation were
analogue 16, with m-CN substituent, having the same φ value
(0.66), and analogue 5, with p-CF3 substituent, which exhibited
a higher φ value (0.81).
In earlier studies, it was found that fluorescence decreases or
even disappears upon introduction of a nitro group to
fluorescent compounds.51−53 The mechanism of quenching
was related to greatly lowered LUMO energy levels of the
aromatic compounds due to the strong electron-withdrawing
effect of nitro groups,54 and this finding is echoed in our results.
Indeed, we found that the electron density distribution of the
LUMO levels of the nitro analogues, 9 and 15, are different
than that of analogues, 5−8, 10−11, and 16−17. While the
other compounds have highly delocalized LUMOs, where the
electron density is spread over the entire ring system, in the
nitro analogues LUMOs, the electrons are localized mostly on
the aryl ring (Figure 8). The localized electron density on the
aryl moiety upon excitation causes an electron deficiency in the
fluorophore moiety, and a resulting nonradiative pathway with
a concomitant fluorescence quenching. This suggests a nearly
complete charge-transfer to the aryl group, followed by a very
fast recombination pathway, which outcompetes the radiative
decay.
over both rings in most cases, similar to the parent compound
(Supporting Information). However, the p- and m- nitro
compounds 9 and 15 are exceptions to this rule. Electron
density of the LUMO orbitals of nitro-compounds 9 and 15 is
largely localized on the benzene ring, probably due to the
strong electron-withdrawing effect of the substituent.
DISCUSSION
■
We attempted to establish general guidelines that will enable
the prediction of fluorescent properties of new nucleoside
analogues. For this purpose, we have designed and synthesized
C8-extended adenosine analogues 5−17. These 8-(substituted
cinnamyl)-adenosine analogues cover a wide chemical space.
Indeed, the variously substituted cinnamyl moiety significantly
changed the spectral properties (e.g, λabs, λem, and φ) of
adenosine.
Next, we attempted to correlate quantum yields and emission
data with the electronic properties of the cinnamyl moiety in
analogues 5−17 as reflected by sigma Hammett constants (σm
or σp) of the cinnamyl substituents. Previous reports indicated a
linear correlation between sigma values and fluorescent
properties of several organic compounds.32−34 Indeed, here,
at sigma values of 0 up to 0.54 for σp, or up to 0.56 for σm, a
linear correlation with the quantum yield was observed.
However, at higher or lower σ values no correlation was
observed.
Additionally, we sought for a correlation between molecular
descriptors (e.g., HOMO−LUMO energies) and fluorescence
parameters. The calculated λabs were in excellent agreement
with experimental λabs indicating that these calculations might
F
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Figure 8. HOMO and LUMO orbitals of analogues 7, 9, and 15. (A) Analogue 7; (B) Analogue 15; (C) Analogue 9.
In conclusion, we reported here on the synthesis and
calculations made for a series of 8-(substituted cinnamyl)-
adenosine analogues, 5−17. We established a relationship
between the ΔELUMO energy levels of model substituted
toluene rings and 8-vinyl-9-Me-adenine and the quantum yields
of the corresponding nucleoside analogues. Identification of
these relationships between the chemical structure and the
fluorescence characteristics of analogues 5−17 allows us to
design useful fluorescent nucleoside probes. For further
development of nucleoside probes based on the scaffold of 8-
vinyladenosine moiety, we can expect that only analogues in
which the fluorophore modulator moiety exhibits LUMO
energy levels within an energy window similar to that of the
parent fluorophore will exhibit high φ value.
In addition, we suggested that the quenching character of the
nitro group in these analogues is based on their aryl-localized
electron density observed in the LUMO orbitals, and hence
reduced electron density on the fluorophore, resulting
eventually in a nonradiative decay. The correlation identified
here constitutes an important tool for the further development
of new fluorescent nucleosides to be used in solid phase
synthesis for the preparation of hybridization probes,
potentially useful for the detection of genetic material.
EXPERIMENTAL SECTION
General Methods. H NMR spectra were obtained using a 200,
■
1
300, 400, or 600 MHz spectrometer. 13C NMR spectra were obtained
at 75, 100, or 150 MHz spectrometer. Chemical shifts are expressed in
ppm downfield from Me4Si (TMS) used as internal standard. The
values are given in δ scale. All air and moisture sensitive reactions were
carried out in flame-dried, nitrogen flushed flasks sealed with rubber
septa, and the reagents were introduced with a syringe. The reactants
were dried overnight in a vacuum oven. Progress of the reactions was
monitored by TLC on precoated silica gel plates. Visualization was
accomplished by UV light. Flash chromatography was carried out on
silica gel. Medium pressure chromatography was carried out using
automated flash purification system. New compounds were analyzed
under ESI (electron spray ionization) conditions on a Q-TOF
microinstrument, and high resolution MS-MALDI-TOF spectra were
recorded with an autoflex TOF/TOF instrument. Absorption spectra
were measured on a UV instrument using a 1 cm path length quartz
cell. Emission spectra were measured using luminescence spectrom-
eter, and the emission was corrected for the monochromator
sensitivity according to the standard procedure. All commercial
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Article
reagents were used without further purification. The fluorescence
quantum yield (φ) was determined relative to quinine sulfate in 0.1 M
H2SO4 (λex 350 nm, λem 446 nm, φ 0.54).45 Starting materials 8-vinyl
adenosine44 and 8-Br-Adenosine,43 and analogues 5,28 6,28 7,55 and 828
were synthesized according to literature.
(74 mg, 0.34 mmol): mp 218 °C (dec); 1H NMR (300 MHz, DMSO-
d6) δ 8.12 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H),
7.81 (d, J = 15.3 Hz, 1H), 7.73 (d, J = 15.3 Hz, 1H), 7.44 (s, 2H), 6.16
(d, J = 7.2 Hz, 1H), 5.82 (dd, J = 7.8 and 3.6 Hz, 1H), 5.36 (m, 1H),
5.27 (m, 1H), 4.73 (m, 1H), 4.21 (m, 1H), 4.03 (m, 1H), 3.87 (s, 3H),
3.77−3.60 (m, 2H) ppm; 13C NMR (75 MHz, DMSO-d6) δ165.9,
155.8, 152.1, 150.1, 147.6, 140.2, 134.7, 129.6, 129.5, 127.7, 119.3,
117.6, 87.5, 86.5, 72.8, 70.4, 61.8, 52.2 ppm; UV−vis (CH3OH) λmax
342 nm; HRMS (MALDI) m/z calcd for C20H22N5O6 [M + H]+
428.1565, found 428.1560.
DFT Calculations. To elucidate the absorption spectra of the 8-
(substituted cinnamyl)-adenosine analogues (SPEC), we performed
time-dependent density functional theory (TD-DFT) calculations.56
The M06-2X functional57 was employed, as it has been shown to
provide excellent agreement with experimental spectra.58 We note that
this global functional is most appropriate for valence and Rydberg
excitations, while somewhat less so for charge transfer processes.59 As
a comparison, the absorption spectra computed with the M06HF
functional is provided in Table S28 (Supporting Information). All
calculations were performed using the polarizable continuum model
solvent description modeling MeOH.60 The TD-DFT calculations
were performed using a 6-31+G (d,p) basis set or geometry
optimizations of the ground states, while single point calculations
used the 6-311++G(3df,2p) basis set.61 Electronic excitation spectra
calculated at the ground state optimized geometry correspond to the
absorption spectra. In addition to the absorption wavelengths,
oscillator strengths were computed, to provide information regarding
the intensity of the electronic transitions. All calculations used the
Gaussian 09 program.62
General Procedure for the Preparation of Analogues 9−11
and 14−17.42 A mixture of Pd(OAc)2 (4 mg, 0.02 mmol), P(o-tol)3
(10.2 mg, 0.03 mmol), Et3N (111 μL, 0.8 mmol), 8-vinyladenosine
(50 mg, 0.17 mmol), and aryl bromide/aryl iodide (0.34 mmol) in
DMF (2 mL) was prepared in an oven-dried vial, equipped with a
stirring bar, and was heated to 100−105 °C. Although the general
procedure described is for reactions on the 50 mg scale of 8-
vinyladenosine, some reactions were conducted at different scales, and
in these cases the proportions of reagents and solvents were
appropriately adjusted. Reactions were monitored by TLC, and
upon consumption of the starting material, the reaction mixtures were
diluted with EtOAc (40 mL) and washed with brine (20 mL) and
water (40 mL). The organic layer was separated, dried over Na2SO4,
and concentrated. The crude reaction products were purified by silica
gel column chromatography using DCM:MeOH (98:2−95:5) or
EtOAc:MeOH (98:2) as the eluent.
2-(6-Amino-8-(3-(trifluoromethyl)styryl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (14). Product 14 was
obtained as a light brown solid (56 mg, 47%), starting from 8-
vinyladenosine (80 mg, 0.27 mmol) and 3-bromobenzotrifluoride (123
mg, 0.54 mmol): mp 211 °C (dec); 1H NMR (600 MHz, DMSO-d6) δ
8.17 (s, 1H), 8.12 (s, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 15 Hz,
1H), 7.75 (d, J = 15 Hz, 1H), 7.71 (d,J = 7.5 Hz, 1H), 7.66 (t, J = 7.5
Hz, 1H), 7.41 (s, 2H), 6.17 (d, J = 7.2 Hz, 1H), 5.78 (dd, J = 8.4 and
4.2 Hz, 1H), 5.34 (d, J = 7.2 Hz, 1H), 5.22 (d, J = 4.8 Hz, 1H), 4.76
(q, J = 7.2 Hz, 1H), 4.22 (m, 1H), 4.03 (m, 1H), 3.73 (m, 1H), 3.63
(m, 1H) ppm; 13C NMR (150 MHz, DMSO-d6) δ 155.8, 152.0, 150.0,
147.7, 136.8, 134.6, 131.6, 130.1, 129.9, 129.7, 129.5, 126.8, 125.3,
125.3, 125.0, 123.8, 123.7, 123.2, 121.4, 119.3, 116.9, 87.7, 86.6, 72.7,
70.5, 61.9 ppm; UV−vis (CH3OH) λmax 334 nm; HRMS (MALDI)
m/z calcd for C19H18F3N5NaO4 [M + H]+ 460.1203, found 460.1230.
2-(6-Amino-8-(3-nitrostyryl)-9H-purin-9-yl)-5-(hydroxymethyl)-
tetrahydrofuran-3,4-diol (15). Product 15 was obtained as a brown
solid (20 mg, 18%), starting from 8-vinyladenosine (80 mg, 0.273
mmol) and 1-bromo-3-nitrobenzene (111 mg, 0.546 mmol): mp 188
°C (dec); 1H NMR (600 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.20 (d, J =
8.4 Hz, 2H), 8.12 (s, 1H), 7.85 (d, J = 16.2 Hz, 1H), 7.81 (d, J = 16.2
Hz, 1H), 7.71 (t, J = 8.4 Hz, 1H), 7.43 (s, 2H), 6.18 (d, J = 7.2 Hz,
1H), 5.79 (dd, J = 7.8 and 3.6 Hz, 1H), 5.35 (m, 1H), 5.25 (m, 1H),
4.76 (m, 1H), 4.21 (m, 1H), 4.04 (m, 1H), 3.74 (m, 1H), 3.63(m, 1H)
ppm; 13C NMR (150 MHz, DMSO-d6) δ 155.8, 152.1, 150.0, 148.5,
147.5, 137.5, 133.8, 130.3, 123.3, 121.7, 121.6, 119.3, 117.9, 87.7, 86.6,
72.7, 70.5, 61.9 ppm; UV−vis (CH3OH) λmax 335 nm; HRMS
(MALDI) m/z calcd for C18H18N6O6Na [M + Na]+ 437.1186, found
437.1184.
3-((E)-2-(6-Amino-9-(3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-9H-prin-8-yl)vinyl)benzonitrile (16). Product
16 was obtained as a brown solid (11 mg, 10%), starting from 8-
vinyladenosine (80 mg, 0.273 mmol) and 3-bromobenzonitrile (100
mg, 0.546 mmol): mp 177 °C; 1H NMR (600 MHz, DMSO-d6) δ 8.31
(s, 1H), 8.11 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 7.8 Hz,
1H), 7.74 (s, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.42 (s, 2H), 6.16 (d, J =
7.2 Hz, 1H), 5.81 (dd, J = 8.4 and 4.2 Hz, 1H), 5.32 (d, J = 6.6 Hz,
1H), 5.23 (d, J = 4.2 Hz, 1H), 4.75 (m, 1H), 4.22 (m, 1H), 4.03 (m,
1H), 3.73 (m, 1H), 3.63 (m, 1H) ppm; 13C NMR (150 MHz, DMSO-
d6) δ 155.8, 152.1, 150.0, 147.5, 136.9, 133.9, 132.2, 132.1, 130.7,
130.0, 119.3, 118.6, 117.3, 112.1, 87.7, 86.6, 72.7, 86.6, 72.7, 70.4, 61.9
ppm; UV−vis (CH3OH) λmax 337 nm; HRMS (MALDI) m/z calcd
for C19H19N6O4 [M + H]+ 395.1468, found 395.1475.
2-(6-Amino-8-(4-nitrostyryl)-9H-purin-9-yl)-5-(hydroxymethyl)-
tetrahydrofuran-3,4-diol (9). Product 9 was obtained as a dark brown
solid (20 mg, 28%), starting from 8-vinyladenosine (50 mg, 0.1706
mmol) and 4-bromonitrobenzene (69 mg, 0.34 mmol): mp 198 °C
(dec); 1H NMR (600 MHz, DMSO-d6) δ 8.25 (d, J = 9 Hz, 2H), 8.13
(s, 1H), 8.03 (d, J = 9 Hz, 2H), 7.84 (s, 2H), 7.46 (s, 2H), 6.17 (d, J =
7.2 Hz, 1H), 5.82 (dd, J = 7.8 and 3.6 Hz, 1H), 5.35 (d, J = 7.2 Hz,
1H), 5.26 (d, J = 4.8 Hz, 1H), 4.71 (q, J = 7.2 Hz, 1H), 4.21 (m, 1H),
4.04 (m, 1H), 3.74 (m, 1H), 3.64 (m, 1H) ppm; 13C NMR (150 MHz,
DMSO-d6) δ 155.9, 152.4, 150.2, 147.3, 147.1, 142.3, 133.6, 128.5,
124.1, 119.43, 119.40, 87.5, 86.5, 73.0, 70.4, 61.8 ppm; UV−vis
(CH3OH) λmax 367 nm; HRMS (MALDI) m/z calcd for C18H19N6O6
[M + H]+ 415.1361, found 415.1360.
4-((E)-2-(6-Amino-9-(3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-9H-purin-8-yl)vinyl)benzonitrile (10). Product
10 was obtained as a yellow solid (20 mg, 30%), starting from 8-
vinyladenosine (50 mg, 0.1706 mmol) and 4-iodobenzonitrile (78 mg,
4-((E)-2-(6-Amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-8-yl)vinyl)-2-
(trifluoromethyl)benzonitrile (17). Product 17 was obtained as a
yellow solid (55 mg, 35%), starting from 8-vinyladenosine (100 mg,
0.341 mmol) and 4-bromo-2-(trifluoromethyl)benzonitrile (171 mg,
1
0.34 mmol): mp 219 °C (dec); H NMR (200 MHz, DMSO-d6) δ
8.12 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.78
(s, 2H), 7.46 (s, 2H), 6.16 (d, J = 7.4 Hz, 1H), 5.82 (dd, J = 7.2 and
3.2 Hz, 1H), 5.35 (d, J = 7.2 Hz,1H), 5.25 (d, J = 4.4 Hz, 1H), 4.71
(m, 1H), 4.20 (m, 1H), 4.03 (m, 1H), 3.74−3.62 (m, 2H) ppm; 13C
NMR (75 MHz, DMSO-d6) δ 156.3, 152.7, 150.6, 147.9, 140.7, 134.7,
133.2, 128.7,119.8, 119.3, 119.0, 111.3, 88.1, 87.0, 73.3, 70.9, 62.3
ppm; UV−vis (CH3OH) λmax 350 nm; HRMS (MALDI) m/z calcd
for C19H19N6O4 [M + H]+ 395.1462, found 395.1470.
Methyl 4-((E)-2-(6-amino-9-(3,4-dihydroxy-5-(hydroxymethyl)-
tetrahydrofuran-2-yl)-9H-purin-8-yl)vinyl)benzoate (11). Product
11 was obtained as a brown solid (25 mg, 35%), starting from 8-
vinyladenosine (50 mg, 0.1706 mmol) and methyl 4-bromobenzoate
1
0.682 mmol): mp 236 °C (dec); H NMR (600 MHz, DMSO-d6) δ
8.42 (s, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.13
(s, 1H), 7.95 (d, J = 15.6 Hz, 1H), 7.85 (d, J = 15.6 Hz, 1H), 7.46 (s,
2H), 6.19 (d, J = 7.2 Hz, 1H), 5.77 (dd, J = 7.5 and 3 Hz, 1H), 5.34 (d,
J = 6.6 Hz, 1H), 5.24 (d, J = 4.2 Hz, 1H), 4.74 (m, 1H), 4.22 (m, 1H),
4.04 (m, 1H), 3.73(m, 1H), 3.64 (m, 1H) ppm; 13C NMR (150 MHz,
DMSO-d6) δ 156.0, 152.4, 150.1, 147.1, 141.1, 135.8, 132.9, 131.8,
131.5, 131.3, 131.1, 125.42, 125.39, 125.3, 123.5, 121.7, 120.4, 119.8,
119.5, 115.7, 87.7, 86.6, 72.9, 70.4, 61.9 ppm; UV−vis (CH3OH) λmax
356 nm; HRMS (MALDI) m/z calcd for C20H18N6O4F3 [M + H]+
463.1342, found 463.1345.
H
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General Procedure for the Preparation of Analogues 5−8
and 12−13.40,41 Water−acetonitrile (2:1, 3 mL) mixture was added
through a septum to an argon-purged round-bottom flask containing
8-bromo-adenosine (0.29 mmol, 1 equiv), Pd(OAc)2 (0.011 mmol,
0.05 equiv), sodium carbonate (0.87 mmol, 3 equiv), tris(3-
sulfonatophenyl) phosphine trisodium salt (Tppts) (0.072 mmol,
0.25 equiv), and the appropriate boronic acid derivative (0.36 mmol,
1.25 equiv). 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 (98:2−94:6) as the eluent.
2-(6-Amino-8-((E)-4-methylstyryl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (12). Product 12 was
obtained as a light yellow solid (30 mg, 27%), starting from 8-
bromo-adenosine (100 mg, 0.289 mmol) and trans-2-(4-
methylphenyl)vinylboronic acid (59 mg, 0.36 mmol): mp 213 °C
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1
(dec); H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.72 (d, J =
15.4 Hz, 1H), 7.65 (d, J = 7.6 Hz, 2H), 7.48 (d, J = 15.4 Hz, 1H), 7.36
(s, 2H), 7.24 (d, J = 7.6 Hz, 2H), 6.11 (d, J = 6.8 Hz, 1H), 5.80 (m,
1H), 5.33 (d, J = 6.8 Hz, 1H), 5.22 (d, J = 4.2 Hz, 1H), 4.76 (m, 1H),
4.20 (m, 1H), 4.02 (m, 1H), 3.73 (m, 1H), 3.61 (m, 1H), 2.34 (s, 3H)
ppm; 13C NMR (150 MHz, DMSO-d6) δ 155.6, 151.7, 149.9, 148.3,
138.8, 136.3, 132.9, 129.5, 127.5, 119.2, 113.6, 87.6, 86.5, 72.6, 70.5,
61.8, 21.0 ppm; UV−vis (CH3OH) λmax338 nm; HRMS (MALDI) m/
z calcd for C19H22N5O4 [M + H]+ 384.1672, found 384.1669.
2-(6-Amino-8-(3-fluorostyryl)-9H-purin-9-yl)-5-(hydroxymethyl)-
tetrahydrofuran-3,4-diol (13). Product 13 was obtained as a white
solid (39 mg, 35%), starting from 8-bromo-adenosine (100 mg, 0.289
mmol) and trans-2-(3-fluorophenyl)vinylboronic acid (60 mg, 0.36
mmol): mp 265 °C (dec); 1H NMR (300 MHz, DMSO-d6) δ 8.11 (s,
1H), 7.74 (d, J = 15.9 Hz, 1H), 7.64 (d, J = 15.9 Hz, 1H), 7.69 (m,
1H), 7.56 (m, 1H), 7.46 (m, 1H), 7.43 (s, 2H), 7.2 (m, 1H), 6.15 (d, J
= 7.2 Hz, 1H), 5.84 (dd, J = 8.1 and 3.9 Hz, 1H), 5.34 (d, J = 6.5 Hz,
1H), 5.26 (d, J = 3.9 Hz, 1H), 4.73 (m, 1H), 4.21 (m, 1H), 4.03 (m,
1H), 3.76−3.71 (m, 1H), 3.66−3.64 (m, 1H) ppm; 13C NMR (100
MHz, DMSO-d6) δ 162.5, 155.7, 151.9, 150.4, 149.9, 147.7, 138.1,
134.8, 130.7, 124.1, 119.2, 116.3, 115.7, 113.3, 87.6, 86.5, 72.7, 70.3,
61.8 ppm; UV−vis (CH3OH) λmax 336 nm; HRMS (MALDI) m/z
calcd for C18H19FN5O4 [M + H]+ 388.1416, found 388.1420.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
13C and H NMR spectra of reported analogues, fluorescence
spectra for selected analogues, and theoretical data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Fax: 972-3-6354907. Tel.: 972-3-5318303. E-mail: bilha.
fischer@biu.ac.il.
Notes
The authors declare no competing financial interest.
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