Analytical Chemistry
triple domino reaction initiated by Wittig olefination.
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and the efficient photocatalytic strategy make it the fastest
method for 5fC selective-labelling so far. What’s more,
our study provides a new idea for distinguishing 5fC and
5fU from the perspective of reaction mechanism and also
broadened the application of Wittig reaction in the field of
chemical biology.
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Subsequently, fluorescence titration experiments were
conducted. As depicted in Figure 2c, the emission peak
centred at 410 nm belonging to 5fC-CN-Close gradually
increased and the fluorescence intensity exhibited an excellent
linear relationship with the concentration range of 0-10 μM
(Figure 2d). Additionally, the corresponding detection limit
(3σ/slope) was calculated to be 35.7 nM, which indicates the
high sensitivity of YC-CN toward 5fC.
ASSOCIATED CONTENT
Supporting Information
Recently, Wagner et al28 have reported that 5mC
exposed to ionizing radiation would produce a variety of
oxidation analogs, including 5fC. Encouraged by the
above findings, we continue to investigate whether YC-
CN is capable of detecting 5fC mutations caused by γ-
rays. Typically, an aerated aqueous solution of 5mC (10
mM), prior to being bubbled with oxygen for 1 h, was
exposed to a 60Co γ-source at a dose rate of 16.7 Gy/min.
Aliquots were taken from this solution at different time
points to obtain testing samples with a series of irradiation
doses. We next performed our domino reaction derivation
with YC-CN, scanned their fluorescence spectra, and
calculated the 5fC lesions using the emission intensity at
410 nm. As can be seen from Figure 3b, the yield of 5fC
was proportional to the dose of γ-rays with a formation
rate of (0.20 5fC/106 nucleosides)/Gy. This data is in
reasonable agreement with (0.45 5fC/106 nucleosides)/Gy
reported by Wagner et al., who delivered the γ-ray at a
completely different dose rate of 1.2 Gy/min. Taken
together, the above results demonstrate that our
independently developed fluorescence detection method
based on the Wittig-initiated domino reactions is reliable
for quantifying 5fC.
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The Supporting Information is available free of charge on the
ACS Publications website.
Experimental details, NMR, ESI-MS and additional figures (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: kli@scu.edu.cn.
*E-mail: xqyu@scu.edu.cn.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was financially supported by National Natural Science
Foundation of China (No.: 21572147 and 21877082). We also
thank the Comprehensive Training Platform of Specialized
Laboratory, College of Chemistry, Sichuan University for sample
analysis.
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Figure 3. (a) Fluorescence spectra of γ-irradiated 5mC after
treatment with YC-CN; (b) Quantification of 5fC mutations in γ-
irradiated 5mC at different irradiation doses (0–3000 Gy). λex
/λem=345/410 nm.
In summary, after screening from a wide variety of P-
ylides, for the first time, we developed an effectively
derivatization method for 5fC using a commercially
available Wittig reagent YC-CN. Then, through detailed
analysis and identification of the obtained products, we
propose a photo-assisted triple domino reaction strategy
to achieve rapid qualitative and quantitative detection of
5fC. Specifically, upon UV irradiation, the resulting
Wittig olefin, nonfluorescent trans-5fC-CN, further
undergoes trans-to-cis isomerization and intramolecular
cycloaddition of exocyclic 4-NH2 to -CN to afford highly
fluorescent nucleoside 5fC-CN-Close, making it feasible
for us to distinguish 5fC from 5fU and accurately quantify
5fC lesions caused by γ-irradiation with significant
selectivity and sensitivity. The high reactivity of YC-CN
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