Scheme 1. Synthesis of the Probe RHF and RHP
aqueous solution based on an intramolecular charge trans-
fer (ICT) mechanism.10
The p-nitrobenzyl moiety is used frequently in the devel-
opment of prodrugs for hypoxia.11 Therefore, we designed
our hypoxia probe RHP by connecting a p-nitrobenzyl
moietyand a signaling moietyRHF via a carbamate group.
The electron-withdrawing carbamate group weakens the
ICT effect and results in a fluorescent emission wavelength
blue shift. Upon reduction, the amino group of RHF will
be released and the fluorescence emission will be restored12
(Figure 1). Both probe RHP and bioreduction product
RHF were efficiently synthesized (Scheme 1) and well
characterized.
Next, we studied their spectral characteristics in chemi-
cal, enzymatic, and cell media. Spectroscopic evaluation of
RHP and RHF was carried out under physiological con-
ditions at 37 °C in PBS buffer (pH=7.0, 0.01 M) with 1%
DMSO (Figure S1). The absorption spectra of RHP and
RHF have an intersection at a wavelength of 410 nm
implying that they could be equally excited at this wave-
length. Thus, wechose 410 nm asthe excitationwavelength
for ratiometric studies. The maximum emission wave-
length of the RHP locates at 475 nm. And that of RHF is
longer by ca.75 nm and falls at 550 nm (Figures S2, S3).
Thus, the probe exhibited a favorable ratiometric signal
change13 (Table S1).
Figure 1. Proposed detection mechanism of RHP.
Various methods for hypoxia detection are available.5
However, their practical applications are restricted due to
the invasiveness. Fluorescence based techniques havesome
obvious advantages since they are noninvasive, display
high sensitivity, and provide high spatiotemporal resolu-
tion. They have also been viewed as a powerful and
versatile toolbox in the field of life sciences, environmental
monitoring, and disease diagnosis.6 Based on nitro groups,
which can be reduced to amino groups under hypoxic
conditions, several fluorescent probes for hypoxia were
reported.7 Fraser et al. reported an elegant sensing plat-
form that emits both fluorescence and phosphorescence
for hypoxia imaging.8 Probes with dual emission are
particularly viable because they enable a built-in calibra-
tion for environmental effects.9 Herein, we describe a novel
prodrug-derived ratiometric fluorescent probe RHP in
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expressed in Escherichia coli. The fluorescence spectra of
RHP were collected in the presence of NTR (3.2 unit of
NTR), and a time dependent fluorescence change originated
from RHP was observed (Figure 2). Within 10 min, the
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of RHF to RHP emission upon excitation at 410 nm exhibited
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