Synthesis of two 6-aza-uridines modified by benzoheterocycle as environmentally sensitive fluorescent nucleosides

Article abstract of DOI:10.1016/j.tetlet.2017.05.087

Two fluorescent nucleosides were synthesized via incorporating benzothiophene and benzofuran into position 5 of 6-aza-uridines. With more intensive push-pull interactions between electron-deficient uridine core and electron-rich extended aromatic moieties

Full text of DOI:10.1016/j.tetlet.2017.05.087

Accepted Manuscript
Synthesis of Two 6-Aza-Uridines Modified by Benzoheterocycle as Environ-
mentally Sensitive Fluorescent Nucleosides
Changge Zheng, Wenji Zhai, Jianquan Hong, Xiaoxiao Zhang, Zhenhua Zhu,
Ling Wang
PII:
S0040-4039(17)30689-5
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.05.087
TETL 48980
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
24 March 2017
16 May 2017
26 May 2017
Please cite this article as: Zheng, C., Zhai, W., Hong, J., Zhang, X., Zhu, Z., Wang, L., Synthesis of Two 6-Aza-
Uridines Modified by Benzoheterocycle as Environmentally Sensitive Fluorescent Nucleosides, Tetrahedron
Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.05.087
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Tetrahedron Letters
Synthesis of Two 6-Aza-Uridines Modified by Benzoheterocycle as Environmentally
Sensitive Fluorescent Nucleosides
Changge Zheng,a,,b Wenji Zhai, Jianquan Hong, Xiaoxiao Zhang, Zhenhua Zhu, Ling Wang
a
a
a
b
b
a
Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122,
†
Jiangsu Province, P. R. China
b
School of Chemical Engineering, Xinjiang Agricultural University, Urumqi 830052, Xinjiang Uygur Autonomous Region, P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Two fluorescent nucleosides were synthesized via incorporating benzothiophene and benzofuran
into position 5 of 6-aza-uridines. With more intensive push-pull interactions between electron-
deficient uridine core and electron-rich extended aromatic moieties, both extended 6-aza-
uridines show enhanced bathochromic shifts with remarkable sensitivity to polarity, pH or
viscosity changes, suitable as a useful nucleobase probe to be sensitive to changes of
microenvironment. The extended nucleoside modified by benzofuran display much high
emission quantum yields and emission intensity in apolar solvents.
Available online
Keywords:
Nucleoside
2
009 Elsevier Ltd. All rights reserved.
6
-Aza-Uridines
Fluorescent
Environmentally Sensitive
Benzoheterocycle
Fluorescence spectroscopy has been widely utilized in
investigating the structure and binding properties of nucleic acids
with many advantages, such as high sensitivity, low cost, easy
deficient 6-aza-uridines, a donor motif of an extended aromatic
system can promote further emission shift into the red region of
the spectrum.
1
detection, etc. With sensitivity to their local environment,
synthetic fluorescent nucleoside analogs have become powerful
tools for investigating nucleic acids structure, dynamics,
recognition and damage, beyond essentially nonemissive
2
property of natural nucleobases. The photophysical properties of
designed nucleoside analogs can be tuned by introducing
substituents at conjugated position with distinct electronic
3
characteristics. Some extended fluorescent nucleoside analogues
can not only show emission in the visible region and maintain a
reasonable quantum yield, but also importantly keep sensitivity
4
to environmental polarity, viscosity and acidity. By extending
the π conjugation of the aromatic pyrimidine core at the 5-
position, these extended fluorescent nucleoside can add a new
5
level of interplay between structure and properties, based on
impact of electronic nature and their conformation around the
6
aryl-aryl bond between two aromatic entities. Some 5-modified
uridine display emission in the visible range (390-443 nm) with
Scheme 1. Synthesis of modified 6-aza-uridines 5a-b. See the
Supporting Information for experimental details. Reagents: (1) SeO2,
pyridine, 110 °C, ~4 h, 70%, 54%; (2) (i) semicarbazide
−1
very large Stokes shifts (8400-9700 cm ) and poor low quantum
7
efficiency (Φ
=
0.01-0.035). Through introducing the
electronegative nitrogen at the pyrimidine’s 6-position, further
enhancing the polarization of the conjugated electron-
poor/electron-rich biaryl system will result in red-shifted
absorption, emission maxima and substantially augmented
hydrochloride, H O, 60 °C, 17 h; (ii) NaOH aq, 60 °C, 4 h, 65%,
2
5
2
5%; (3) N,O-bis(trimethylsilyl)acetamide, TMSOTf, 1-O-acety-
,3,5-tri-O-benzoyl-β-D-ribofuranose, CH CN, 87%, 53%; (4)
3
CH OH/ NH , 65 °C, 24 h, 75%, 73%.
3
3
8
quantum yields (Φ = 0.2-0.8). Conjugated to the electron-
—
——

†
Corresponding author. Tel.: +86-510-85917763; Fax: +86-510-85917763; E-mail: cgzheng@jiangnan.edu.cn (C.-G. Zheng)
Permanent address.

2
Tetrahedron Letters
The 5-modified uridines with thienyl and furanyl moieties
lifetime of 1.6 ns than in dioxane (Fig. 1, Table 1). Compared
with 5a, nucleoside 5b exhibits the higher fluorescence intensity
in water and acetonitrile, or lower in methanol and dioxane.
Nucleoside 5b in water had the largest red-shift of emission
maxima in 471 nm among various solvents, along with the lowest
emission intensity. The fluorescence intensity of both 5a and 5b
was enhanced with the decreasing of the solvent polarity.
possess strong emissive property and relatively low quantum
yields. While benzothienyl and benzofuranyl are employed, the
9
5
-modified uridine analogues have shown higher quantum yields
10
with a larger conjugation. With stronger push-pull effect added
1
1
to the native nucleobases, the 5-modified 6-aza-uridine with the
substituent of benzothiophene and benzofuran will be expected
reasonable emission and susceptivity to local environment. To
know more about the fluorescence-structure relationship of the
conjugated electron-poor/electron-rich biaryl system, two
fluorescent nucleosides were designed and synthesized by
extending the π conjugation of benzoheterocycle to the position 5
1
.0
350
a)
water
3
2
2
1
1
5
0
00
50
00
50
00
0
methanol
acetonityile
dioxane
0.8
0.6
1
2
0.4
of electron-deficient-6-aza-uridines.
0.2
Modified 6-aza-uridines have been synthesized according to
the procedure illustrated in Scheme 1. Firstly, benzothiophene
glyoxylic acid 2a were obtained via oxidation of the
corresponding 2-acetylbenzothiophene 1a using selenium
dioxide. Upon heating under basic conditions, 3a was
synthesized in good yields through in situ cyclization of imine
from condensation of 2a with semicarbazide hydrochloride.
Glycosylation of 3a was performed in a one-pot, two-step
0.0
250 300 350 400 450 500 550 600 650

(nm)
1.0
1000
800
600
400
200
0
1
3
b)
water
9.0 c)
8.8
water
0
.8
.6
methanol
acetonityile
dioxane
methanol
acetonitrile
dioxane
8.6
0
8.4
8.2
8.0
0.4
8
b
procedure as the reported procedure.
The glycosylated
0
.2
.0
7.8
7.6
intermediate 4a was got from reaction of the nucleobase with β-
D-ribofuranose 1-acetate 2,4,5-tribenzoate about 2 h, in good
yields by purification with column chromatography.
Deprotection with methanolic ammonia was carried at 65 °C. The
desired 5a was purified by column chromatograph. From starting
material 1b, the desired 5b was synthesized, using the same
process as described above. Both modified nucleosides were
0
2
50 300 350 400 450 500 550 600
(nm)
35
40
45
50
55
60
65

E
(30) (kcal/mol)
T
8
8
7
7
6
6
5
.5 d)
water
methanol
acetonitrile
dioxane
.0
.5
.0
.5
.0
.5
1
13
thoroughly characterized by H and C NMR spectroscopy as
well as by MS.
3
5
40
45
50
55
60
65
To assess their sensitivity to the microenvironment, the
photophysical features of ribonucleoside 5a and 5b were
examined in solvents with distinct polarity, viscosity and acidity.
The photophysical properties of 5a and 5b are summarized in
Table 1. Polarity-sensitive fluorescent nucleosides can be used as
a probe in investigating nucleic acid dynamics and the
recognition process by monitoring changes in fluorescence
properties. To evaluate the responsiveness of the modified
nucleosides to polarity changes, UV absorption and steady-state
fluorescence spectroscopic measurements were performed in
solvents of different polarity. The absorption spectrum of 5a in
water revealed distinct absorption bands at 262 and 335 nm,
attributed to 6-aza-uridine core and modified nucleoside,
respectively (Fig. 1). While the absorption maxima of 5a and 5b
were marginally affected by solvent polarity, significant changes
have been shown in both emission wavelengths and intensity.
With gradually increasing of the solvent polarity from dioxane to
water, 5a displays a remarkable bathochromic shift from 455 to
E
(30) (kcal/mol)
T
Figure 1. Absorption (50 μM, solid) and emission (5.0 μM, dash)
spectra of ribonucleoside (a) 5a and (b) 5b in various solvents,
respectively. Linear relationship between stokes shift and solvent
polarity E (30) of (c) 5a and (d) 5b in various solvents, respectively.
T
8
b
In comparison with reported 5-(thiophen-2-yl)-6-aza-uridine,
the emission of nucleoside 5a shows remarkable bathochromic
shift effect (24-44 nm) along with larger Stokes shift (7521-8974
cm ). For introducing the electronegative nitrogen at the 6-
position of reported 5-benzo[b]thien-2-yl-uridine, further
enhancing “push-pull” interaction of 5a promote significant
bathochromic shift in the absorption (16-17 nm) and emission
2
b
-
1
1
0c
maxima (20-31 nm). Compared with reported 5-(furan-2-yl)-6-
8
b
aza-uridine, the absorption and emission maxima of 5b further
shift into the red region of the spectrum about 13-17 nm and 12-
28 nm respectively. Nucleoside 5b shows significantly red-
shifted absorption (15-20 nm) and emission maxima (22-37 nm)
1
4
4
79 nm and nearly 2.6-fold weaker emission intensity. A
in various solvents, in contrast with 5-(2-benzofuranyl)-uridine.
fluorescence emission of 5a was observed at 479 nm in the
visible region under excitation at 335 nm in an aqueous solution,
with a lower quantum yield of 0.01 and a longer fluorescence
Table 1 Selected spectroscopic properties of 5a and 5b.
a
a
a
b
-1
c
Compound
Solvent
water
λ
max (nm)
λ
em (nm)
479
477
465
455
511
480
479
479
429
471
457
447
426
483
465
I
rel
Stocks shift (cm )
Φ
τ[ns]
1.60
-
335
337
338
339
339
332
330
339
342
337
339
341
342
341
332
1
8974
8709
8080
7521
9929
9287
9426
8622
5930
8442
7617
6954
5766
8622
8615
0.01
0.01
0.02
methanol
acetonitrile
dioxane
pH 2.19
pH 6.96
pH 11.26
MeOH
0.9
1.6
2.6
< 0.1
0.7
1.2
-
0.03
0.53
d
5
a
< 0.01
< 0.01
0.01
0.01
0.03
0.04
0.06
0.453
0.78
< 0.01
0.06
0.43
-
1.48
d
0.9
1.7
0.616
0.56
1.89
-
Glycerol
water
1
methanol
acetonitrile
dioxane
pH 2.19
pH 6.96
1.9
7.9
26.4
< 0.1
1.6
-
5
b
3.98
d
0.51
-

d
pH 11.26
MeOH
Glycerol
330
339
344
468
463
458
2.1
1.9
5.3
8936
7900
7236
0.08
0.06
0.23
1.98
3.06
0.94
a
b
Absorption and steady-state emission spectroscopy studies were performed using samples prepared from a concentrated DMSO stock solution. Emission
c
d
intensity relative to intensity in water. Fluorescence standards: Coumarin 1 was used for 5a-b. Values for pH 2.91 and pH 10.23 represent the lower- and upper
plateau values of the sigmoidal fit.
8
b
Strikingly different from the 5-(thiophen-2-yl)-6-aza-uridine
in different pH values, the influence of pH to the emission
properties is not necessarily accompanied by significant shifts of
1
5
and 5-(thiophen-2-yl)-uridine, especially noteworthy is that the
emission intensity of 5b was amazingly 26.4-fold higher in
dioxane than in water, with a respectable quantum yield (Φ =
2
2
the emission maxima. Nucleoside 5a showed a significant
increase in fluorescence intensity with accompany of a red shift
from 479 nm (Φ = 0.01, τ = 1.48 ns) to 511 nm (Φ < 0.01, τ =
0.43 ns), going from pH 11.26 to 2.19, respectively (Fig. 3, Table
1). The emission intensity of 5b increased dramatically in pH =
6-7 then to decay gradually, until the intensity remained constant.
The emission maximum shifted from 468 nm (Φ = 0.08) to 483
nm (Φ < 0.01), going from pH 11.26 to 2.19, respectively. With
gradually change from acidic to basic conditions, an increase of
the quantum yield was also observed along with increase of
fluorescence lifetimes from 1.98 to 0.51 ns.
0
.78) and a fluorescence lifetime of 3.98 ns. (Table 1).
Furthermore, the quantum yields of both 5a and 5b increase with
fluorescence intensity, corresponding to the decreasing of the
solvent polarity. Plotting the Stokes shift in various solvents as a
1
6
function of Reichardt’s microscopic solvent polarity parameter,
E (30), a steady increase of E (30) values for 5a and 5b was
T
T
observed with increase of the solvent polarity. Additionally, a
good linear correlation is shown for related emissive nucleosides,
quantifying the sensitivity to polarity (Fig. 1). It is also necessary
for 5a and 5b as a useful nucleobase probe to be sensitive to
changes in solvent-polarity environment. Such fluorescent
nucleosides would be also valuable for monitoring the polarity of
Generally, the quantum yield of benzofuran-modified 6-aza-
uridine 5b is higher than benzothiophene-modified 6-aza-uridine
5a in distinct polarity, viscosity and acidity. While the quantum
yield is low for 5b in water (Φ = 0.04) and methanol (Φ = 0.06),
the quantum yield of 5b in dioxane (Φ = 0.78) and acetonitrile (Φ
= 0.453) is reasonably high for most 5-substituted uracil
9b
major grooves in nucleic acids.
The emission intensity of molecular rotors depends on the
solvent’s viscosity through fluorescence emission and non-
radiative deexcitation. If intramolecular rotation is inhibited by
high viscosity of the microenvironment, fluorescence intensity
1
4,22,23
nucleotide and nucleoside fluorophores (Table 1).
Interestingly, the quantum yield of benzofuran-5-modified
uridine nucleolipids is pretty higher than the corresponding
1
7
increases with increased viscosity of the solvent. Both modified
nucleosides of 5a and 5b are endowed with molecular rotor
behavior, through introduction of a rotatable aryl-aryl bond
2
4
benzothiophene-5-modified, similar as the higher quantum
yield of 5-benzofuran-modified uracil PNA monomer than 5-
1
8
25
separating a 1,2,4-triazine motif and conjugated aromatic ring.
benzothiophene-modified uracil PNA monomers. While lower
Upon increasing the viscosity of solution from methanol (η
=
than 5-thiophene-modified 6-aza-uridine, the quantum yield of
thiophene-modified uridines is higher to furan-modified
2
0°C
1
1a,19
0
.583 cP) to glycerol (η20°C = 1317 cP),
nucleoside 5a shows
2
6
enhancing emission intensity 2.4-fold higher in glycerol than in
methanol, with a discernible blue shift of 43 nm in the emission
maximum (Fig. 2, Table 1). Similar as 5a, the fluorescence
intensity of 5b also increased gradually with the solvent viscosity
upto 3.4-fold higher in glycerol than in methanol, but without
apparent change of shift in emission maximum. It can be
attributed to restriction of the rotation of the C-C bond between
uridines. The unexpected low fluorescence quantum yield of 5a
(Φ = 0.01-0.03) shows that photophysical properties are difficult
to predict from the molecular structure alone. However, the
fluorescence quantum yield of 5b is substantially augmented (Φ
= 0.04-0.78), compared with the quantum yield of 5-(2-
1
4
benzofuranyl)-uridine (Φ = 0.063-0.212).
1
60
400
350
300
250
200
150
100
50
2
0
the benzoheterocycle and uracil rings. Fluorescence properties
have been shown to be sensitive to molecular crowding and
viscosity. The result indicates that 5a and 5b can be affected by
viscosity and potentially suitable as a probe to immediate
environment. When incorporated into RNA oligonucleotides, 5a
and 5b could also be employed to explore the structure and
recognition properties of DNA oligonucleotides, through the
responsiveness of these ribonucleoside analogues to changes in
140 a)
pH=2.91
pH=6.96
pH=11.26
intermediate pH
b)
pH=2.19
pH=6.96
pH=11.26
intermediate pH
1
1
20
00
8
6
0
0
40
2
0
0
0
350
4
00
450
500
550
600
650
400
450
500
550
600
650

(nm)
(nm)
2
1
polarity and the neighbouring base environment.
1.0
.8
c)
1
0
0
0
.0
.8
.6
.4
d)
0
2
2
1
1
40
00
60
20
1000
800
0.6
0.4
0.2
0.0
a)
b)
methanol
glycerol
methanol
glycerol
glycerol/methanol
100:0
glycerol/methanol
pK : 6.78
pK : 6.56
a
2
a
1
00:0
600
0.2
.0
2
R
: 0.99739
R :0.99225
0:100
0:100
0
4
2
00
00
0
8
4
0
0
2
4
6
8
10
12
2
4
6
8
10
12
pH
pH
Figure 3. The sensitivity of the fluorescent modified 6-Aza uredines
to pH. Emission of (a) 5a and (b) 5b in phosphate buffers of different
pH values; Plot of normalized PL intensity of (c) 5a and (d) 5b vs pH
3
50
400
450
500
550
600
400
450
500
550
600
650

(nm)
(nm)
Figure 2. Emission spectra of (a) 5a and (b) 5b in in media of
different viscosity.
(solid squares ) and a Boltzmann fit (red line).
Plotting the normalized emission intensity versus pH gives a
To investigate the sensitivity of the fluorescent modified 6-
aza-uridines to pH, both 5a and 5b were performed in aqueous
phosphate buffers with pH values ranging from 1 to 12. Although
an impact were observed in the absorption spectra of 5a and 5b
characteristic titration S-curve yielding a pKa value for a two
state (de)protonation process of 6.78 and 6.56 for 5a and 5b
respectively (Fig. 3), in agreement with the reported value of 6-

4
Tetrahedron Letters
aza-uridine and 7its analogs (pKa
=
6.7-6.8) close to
and the Fundamental Research Funds for the Central Universities
(JUSRP115 A08 and JUSRP51513).
2
physiological pHs. With the same downward trend of the
fluorescent intensity of both 5a and 5b at very low pH, it further
shows that aromatic benzoheterocyclic substituent in the 5-
position of 6-aza-uridine has little impact on the pKa of the NH
in its electronic influence. In view of significantly deviation from
the reported values for the parent uridine (pKa = 9.3-9.5), the
additional nitrogen in 6-aza-uridine was assumed to increase
greatly the acidity of the NH of modified fluorescent nucleosides,
Supplementary Material
Supplementary material for this article is available online at
http://dx.doi.org/10.1016/j.tetlet.
2
8
References and notes
8
in accordance with the reported pKa value. With the
fluorescence intensity and emission maximum sensitivity, a pKa
1
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value close to physiological pH make 5a and 5b an attractive
2
9
probe for studies involving (de)protonation events on RNA.
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fluorescent nucleosides with pH-dependent emission
characteristics can be exploited as a DNA oligonucleotides
1
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AMP, GMP, CMP, and TMP) as quenchers in aqueous
(
(f) Rist MJ, Marino JP. Curr Org Chem. 2002;6:775–793;
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quenching rate constants K , for evaluating the degree of
sv
3
0
quenching dependence on the neighboring bases. Stern-Volmer
plots illustrate that all four nucleoside monophosphates display a
linear quenching behavior to fluorescent nucleoside analog 5a
and 5b (Fig. 4). Monophosphate nucleotides AMP to 5a exhibits
greater quenching than CMP, GMP and TMP with quenching
2
013;54:3374–3377.
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.
Segal M, Fischer, B. Org Biomol Chem. 2012;10:1571–1580.
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378.
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sv
5
.
(a) Pesnot T, Wagner GK. Org Biomol Chem. 2008;6:2884–2891;
(
Similar as 5a, quenching constants of 5b is 0.13, 0.10, 0.10 and
b) ꢀoꢁ MS, Sinkeldam RW, Tor Y. J Org Chem. 2013;78:8123−8128.
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Hopkins PA, Sinkeldam RW, Tor Y. Org Lett. 2014;16:5290−5293.
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1
0
.06 mM to AMP, CMP, GMP and TMP, respectively.
(
7
.
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
1.8
a)
a)
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1.6
1.5
1.4
1.3
1.2
A
C
G
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2
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2
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[
NMP](mM)
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(
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Figure 4. Steady-state Stern–Volmer plot for the titration of
nucleoside (a) 5a and (b) 5b with AMP, GMP, CMP and TMP.
(
1
In conclusion, sensitive toward polarity, pH and viscosity
changes, two fluorescent 6-aza-uridine analogs with desirable
photophysical properties have been synthesized, tagged at the 5-
position of 6-aza-uridines with benzothiophene or benzofuran.
With increase of viscosity, the higher emission intensification of
2
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6
Tetrahedron Letters
Highlights
•
One-pot desilylation/Sonogashira coupling with
trimethylsilyl alkynes was developed.
Two new nucleosides are highly fluorescent and
potential antivirals.
A reasonable quantum yield for Nucleoside 5.
Enhanced bathochromic shifts.
Remarkable sensitivity to polarity, pH or viscosity
changes.

7
nucleosides enhanced bathochromic shifts with remarkable
sensitivity to polarity, pH or viscosity changes.
Graphical Abstract
A stronger push-pull effect between the conjugated electron-
poor/electron-rich biaryl system endow two fluorescent
Synthesis of Two 6-Aza-Uridines Modified
by Benzoheterocycle as Environmentally
Sensitive Fluorescent Nucleosides
Leave this area blank for abstract info.
Changge Zheng, Wenji Zhai, Jianquan Hong, Xiaoxiao Zhang, Zhenhua Zhu, Ling Wang
Emission spectra of two 6-aza-uridines modified by benzoheterocycle in various solvents