Synthesis and spectroscopic properties of fluorescent 5-benzimidazolyl- 2′-deoxyuridines 5-fdU probes obtained from o-phenylenediamine derivatives

Article abstract of DOI:10.1039/c3ob27519a

Fluorescent nucleosides (dU^bmz) with desirable fluorescence quantum yield (Φ) are synthesized from almost non-fluorescent 5-fdU and o-phenylenediamine derivatives. The fluorescence of these nucleosides is quite sensitive to pH and organic solve

Full text of DOI:10.1039/c3ob27519a

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Synthesis and spectroscopic properties of fluorescent
5-benzimidazolyl-2’-deoxyuridines 5-fdU probes
obtained from o-phenylenediamine derivatives†
Pu Guo,‡a Xiaowei Xu,‡a Xiaoyu Qiu,a Yimin Zhou,a Shengyong Yan,a
Changcheng Wang,a Chunjiang Lu,a Wen Ma,a Xiaocheng Weng,a
Xianzheng Zhanga and Xiang Zhou*a,b
Cite this: Org. Biomol. Chem., 2013, 11,
1610
Received 31st December 2012,
Accepted 22nd January 2013
DOI: 10.1039/c3ob27519a
www.rsc.org/obc
Fluorescent nucleosides (dUbmz
)
with desirable fluorescence reported.10 In that work, both the microwaves and the pro-
quantum yield (Φ) are synthesized from almost non-fluorescent tected hydroxyl hamper the detection of 5-fdU. Therefore, we
5-fdU and o-phenylenediamine derivatives. The fluorescence of synthesized series of fluorescent 2′-deoxyuridine derivatives
these nucleosides is quite sensitive to pH and organic solvents. without protecting hydroxyl groups in one step, and we used
4-Methoxybenzene-1,2-diamine was used for the detection of this reaction to distinguish 5-fdU from natural bases. Herein,
5-fdU among natural nucleosides.
we report the synthesis of a series of highly fluorescent 5-benz-
imidazolyl-2′-deoxyuridines (dUbmz, Table 1) that were readily
synthesized based on o-phenylenediamine derivatives from a
commercial source. The o-phenylenediamine derivatives (2a–j)
exhibit no fluorescence before the reaction with the target
nucleoside, 5-fdU. After the reaction of 2a–j with 5-fdU, the
formyl group at the 5-position of 5-fdU is converted into a benz-
imidazole ring, which is directly conjugated to the uracil group
to greatly improve the fluorescence (Scheme 1a).
High-fidelity DNA replication is essential to maintain the
genetic integrity of living cells. The mutation of bases tends to
be associated with serious diseases, such as cancer.1 In the
past few years, numerous unnatural bases with diverse chemi-
cal biology functions have been reported. These unnatural
bases minimally perturb DNA replication and transcription.2
Naturally, identifying and detecting new bases with novel bio-
logical functions are becoming great challenges. Among these
new bases is 5-formyl-2′-deoxyuridine (5-fdU), an oxidized thy-
midine lesion formed by reactive oxygen species resulting
from UV light,3 and the formation of this lesion may cause
aging and/or carcinogenesis.4 Thus, the convenient detection
of 5-fdU is imperative. Several approaches for the detection of
5-fdU have been developed.5 Until now, only one fluorescence-
based approach for detecting 5-fdU has been reported,6 but
the reagent used for 5-fdU detection in this method is difficult
to synthesize and is unstable. In addition, fluorescent nucleo-
sides are playing an increasingly important role in the study of
the biological functions of nucleic acids, including the moni-
toring of real-time biochemical events such as drug binding,7
RNA folding and cleavage,8 and RNA–protein interactions.9
Recently, a complicated microwave-assisted synthesis of 5-benz-
imidazolyl-2′-deoxyuridines with antibacterial activity was
First, we synthesized dUbmz in one step (Scheme 1).6
5-Formyl-2′-deoxyuridine (5-fdU, 1) was mixed with o-phenylene-
diamine derivatives (2) in DMF, and hydrogen peroxide was
added as an oxidant to oxidize the C–N bond to a CvN double
bond. The reaction occurred in the presence of Sc(OTf)3 and
gave 5-benzimidazolyl-2′-deoxyuridines (dUbmz, 3) with satis-
factory yields, as shown in Table 1.
To explore the electronic effects of substituents on the
o-phenylenediamine ring on the spectroscopic properties of
the products (3a–j), the UV absorbance and fluorescence of
dUbmz were investigated. The UV absorbance and fluorescence
spectra show that compounds 3a–f, which were synthesized
with o-phenylenediamine derivatives containing electron-
donating groups, exhibit significantly higher fluorescence
levels than compounds 3g–i, which have much weaker fluo-
rescence (see ESI†). The reason for this difference is likely that
the structures of compounds 3a–j include conjugated systems
that undergo an intramolecular charge transfer process. The
uracil moiety of compounds 3a–j is an electron-withdrawing
structure. When there are electron-donating groups on the
o-phenylenediamine moiety, dUbmz exhibits high fluorescence
emission. In contrast, 3g–i, which contain electron-withdraw-
ing groups on the o-phenylenediamine moiety, exhibit no fluo-
rescence emission.
aCollege of Chemistry and Molecular Sciences, Key Laboratory of Biomedical
Polymers of Ministry of Education, State Key Laboratory of Virology,
Wuhan University, Hubei, Wuhan, 430072, P. R. China. E-mail: xzhou@whu.edu.cn;
Fax: +86-27-87336380; Tel: +86-27-61056559
bState Key Laboratory of Natural and Biomimetic Drugs, Peking University, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob27519a
‡These two authors contributed equally to this work.
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Table 1 Structure of o-phenylenediamine derivatives and dUbmz with yield
Table 1 (Contd.)
Substrate
Product
Yield
77%
Substrate
Product
Yield
83%
77%
93%
87%
84%
Scheme 1 (a) The diagram for production of fluorescent dUbmz. (b) Synthesis
of dUbmz
.
83%
92%
Table 2 Photoproperties of 3a–3f and 1 in 100 mM HCl (aq)
abs
max
flu
max
R
λ
[nm]
λ
[nm]
Stokes shift [nm]
Φ
3a
3b
3c
3d
3e
3f
1
H
320
335
324
330
325
329
260
405
475
426
448
423
405
308
85
0.039
0.023
0.065
0.088
0.070
0.016
0.00003
OMe
Me
2Me
t-Bu
Br
140
102
118
98
76
48
Table 2 shows that the Stokes shift of the fluorescent
80%
79%
nucleosides becomes longer with the greater electron-donating
ability of R. This result is consistent with the character of the
intramolecular charge transfer effect. Among dUbmz, the struc-
ture with the methoxy group (R = MeO) has the longest Stokes
shift. In Table 2, we present the main optical properties of
compounds 3a–3f, including the maximum excitation wave-
length (λambasx), the maximum fluorescence emission wavelength
flu
max
(λ ), the Stokes shift, and the fluorescence quantum yield
(Φ) in 100 mM aqueous hydrogen chloride (HCl) solution.
Compared with that of 5-fdU (Φ = 0.00003), the fluorescence
quantum yields (Φ) of these fluorescent nucleosides (3a–3f)
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are excellent. All of the fluorescence quantum yields were dUbmz in organic solvents is lower than that in 100 mM HCl (aq),
determined using quinine sulfate (0.56) in 100 mM H2SO4 as a most likely because the formation of the imidazole dication
standard.
under acid conditions could hold the benzimidazole ring and
We decided to analyze the optical properties of dUbmz in the uracil in the same plane via hydrogen bonding. Therefore,
100 mM aqueous hydrogen chloride (HCl) solution because the fluorescence of dUbmz in 100 mM HCl (aq) is higher than
the fluorescence of these compounds is sensitive to pH (see that in organic solvents, in which there is only one proton on
Fig. S45–S49†). When the pH of the buffer is lower than 7, the the benzimidazole ring. Somewhat surprisingly, all com-
fluorescence intensity of dUbmz would increase quickly with pounds exhibit almost identical fluorescence quantum yields
decreasing pH. However, if the pH is neutral or basic, the fluo- (Φ) in 1,4-dioxane. Due to the distinct solubilities of these
rescence intensity would remain relatively low. Under acidic compounds in organic solvents with diverse polarities, the
conditions, abundant hydrogen ions may facilitate the for- fluorescence quantum yields (Φ) of dUbmz in acetone,
mation of the imidazole dication due to the protonation of methanol, ethanol, and acetonitrile are quite low. Although it
both nitrogen atoms. A hydrogen bond is formed spon- was very low, the fluorescence quantum yield (Φ) of dUbmz con-
taneously between the hydrogen atom of the secondary amine taining bromide (R = Br) is greater in DMSO than in aqueous
in the benzimidazole and the oxygen atom on C4 of deoxyuri- solution and is significantly greater than that of compounds
dine. This hydrogen bond keeps the benzimidazole ring and with other substituent groups in organic solvents (acetone,
the uracil base in the same plane.11 This behavior is the pro- methanol, ethanol, and acetonitrile, see ESI†). The assay
posed reason for the change in fluorescence at an acidic pH.
results indicate that the fluorescence quantum yields (Φ) of
Compound 2j has been used as a fluorescent probe for 3a–3f display moderate sensitivity to different solvents. Further
nitric oxide (NO).12 This compound is almost non-fluorescent experiments are required to elucidate the influence of the
before combination with NO because of its sufficiently high solvent.
HOMO energy level. However, the triazolobenzene produced
Because of the excellent fluorescence properties of dUbmz
,
after the reaction with NO has a lower HOMO energy level, and this series of compounds such as 3a–3e (pKa = 4.4, 4.9, 4.8,
this compound is highly fluorescent. Analogously, 3j, which 4.3, 4.5, see Fig. S56†) can be used in fluorescence detection,
contains a benzimidazolyl structure synthesized from 5-fdU fluorescence labeling, cell imaging, and other applications.13
and 2j, should be highly fluorescent. As shown in Fig. S50,† at The water solubility of these nucleoside analogs is significant
approximately 503 nm there is a peak at which there is a much for research in the biology field. Therefore, dUbmz, which have
higher fluorescence intensity for 3j than for 2j. 3j and 2j were fluorescence intensities in H2O, especially under the acidic
even different colors in water exposed to UV light, and these conditions that are crucial to organisms, could be used to
differences were visible with the naked eye. Thus, probe 2j can exploit this advantage in biological detection. Above all, we
also be used to detect 5-fdU using fluorescence.
have demonstrated that it is possible to use o-phenylenedi-
In addition to water or buffer as a solvent, we also tested amine derivatives to detect 5-fdU (Fig. 2).
the fluorescence and UV absorbance of dUbmz in different
Finally, 10 μM 4-methoxybenzene-1,2-diamine was added to
types of organic solvents. The solvent-related properties of aqueous solutions (10 mM CH3COONH4, pH = 4.5) containing
nucleosides 3a–3f are summarized in Fig. 1. As expected, the 5-formyl-2′-deoxyuridine (5-fdU), 2′-deoxyguanosine (dG),
unique structure had an impact on the photophysics of 3a–3f 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), or 2′-deoxythymi-
in organic solvents. 3a–3f in 100 mM HCl (aq) have high fluo- dine (dT) at a concentration of 100 μM, followed by the
rescence quantum yields (Φ). The behavior in water is similar addition of dithiothreitol (DTT). Then, the fluorescence emis-
to that in dimethylsulfoxide (DMSO). The fluorescence of sion of the reaction mixture was tested at 400 nm–560 nm with
an excitation wavelength of 370 nm after a 12 h incubation at
Fig. 2 (a) Fluorescence spectra of 10 μM 4-methoxybenzene-1,2-diamine
toward 100 μM 5-fdU (green), dC (black), dT (red), dA (blue), dG (purple) in
10 mM CH3COONH4 buffer, pH = 4.5, with DTT 10 μM. (b) Fluorescence spectra
of 10 μM 4-methoxybenzene-1,2-diamine toward total concentration of
a
Fig. 1 Fluorescence quantum yield (Φ) of compounds 3a–3f in several solvents
(100 mM HCl (aq), H2O, DMSO, 1,4-dioxane, acetone, MeOH, EtOH, and
CH3CN). Concentrations of compounds are 1 μM.
100 μM mixture of dA, dG, dT, dC, 5-fdU in 10 mM CH3COONH4 buffer, pH =
4.5, with 10 μM DTT, including 5% 5-fdU (black), control with only natural
nucleosides (red).
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37 °C (Fig. 2a). The results indicate that 4-methoxybenzene- “the Fundamental Research Funds for the central Universities”
1,2-diamine can selectively react with 5-fdU but not with the (2012203020212).
natural nucleosides. Additionally, we mixed 5-fdU into
aqueous buffer (10 mM CH3COONH4, pH = 4.5) containing the
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This work was supported by the National Basic Research
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Shanghai Institute of Organic Chemistry, the Chinese
Academy of Sciences; by the Program for Changjiang Scholars
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