A concise colorimetric and fluorimetric probe for sarin related threats designed via the covalent-assembly approach

Article abstract of DOI:10.1021/ja502945q

A turn-on signal from zero background allows sensitive detection of a weak signal and is highly desired. The covalent-assembly probe design principle is powerful in this regard. Herein, we report an embodiment of this principle (NA570) for detection of Sa

Full text of DOI:10.1021/ja502945q

Communication
pubs.acs.org/JACS
A Concise Colorimetric and Fluorimetric Probe for Sarin Related
Threats Designed via the “Covalent-Assembly” Approach
Zuhai Lei‡ and Youjun Yang*,†,‡
‡
†State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China
University of Science and Technology, Meilong Road 130, Shanghai 200237, China
S
* Supporting Information
not available to us, diethylphosphinic chloride (6) and
ABSTRACT: A turn-on signal from zero background
allows sensitive detection of a weak signal and is highly
desired. The “covalent-assembly” probe design principle is
powerful in this regard. Herein, we report an embodiment
of this principle (NA570) for detection of Sarin related
threats, based on a phenylogous Vilsmeier−Haack
reaction. NA570 bears a concise molecular construct,
exhibits a colorimetric and a fluorimetric signal, and has
potential for real applications.
chlorodiethylphosphate (7) were used as mimics for Sarin,
and 3 as a mimic for 2. The fact that 3, 6, 7, SOCl2, and
C2O2Cl2 are all strongly electrophilic makes it chemically
possible for a single probe to detect them all, and yet such a
probe has not been reported to our knowledge. Methylphos-
phonic acid (4) and isopropyl methylphosphonic acid (5) were
excluded from our list of chemical species to detect because
they are not toxic and present minimal threat. Also the
phosphorus atom present in reagents 4 or 5 is not prone to
nucleophilic attack and requires different chemistry for
detection.
arin, a weapon of mass destruction developed during
While fluorescence based probes are highly sensitive and
fluorimeters can be very portable for field use as demonstrated
by FIDO,6 a color change facilitating naked-eye detection could
be necessary in situations where a fluorimeter is unavailable or
fails unexpectedly. Therefore, a probe generating both a
colorimetric and a fluorimetric turn-on signal in response to
the relevant chemical species is attractive. Further, optimal
detection sensitivity is achieved when a probe gives zero
background signal, a feat not readily obtained through the
classic dye-linker-receptor principle.7 These considerations
promote us to explore a novel design principle, which we
term “covalent-assembly” (Figure 2a). The fundamental feature
of the “covalent-assembly” principle is that the color-enabling
push−pull conjugative backbone of a dye is split into two
fragments in the probe. These two fragments are covalently
assembled to restore the dye through a chemical cascade, which
is triggered selectively by the analyte of interest. This feature
warrants both a turn-on colorimetric and a turn-on fluorimetric
signal from a zero background, hence an optimal detection
sensitivity. A second important feature of the “covalent-
assembly” approach is that the probes are simple in terms of
structural complexity and synthesis. Synthesis of a dye with
proper chemical handles is not always easy, and a multistep
cascade carrying a dye scaffold is even more challenging. A
“covalent-assembly” type probe employs the analyte of interest
to carry out the dye synthesis in situ, thereby alleviates synthetic
efforts.
S
WWII, acts by inhibition of acetylcholinesterase and
thereby overstimulation of the central nervous system.1 Though
the possibility of military use is low with the Chemical Weapon
Convention2 in force, terroristic use of Sarin (1) is a real threat3
and demands countermeasures including robust and convenient
detection.4
Though Sarin is the active toxin, high specificity of an assay
toward Sarin itself could ironically be a disadvantage, because
Sarin is not stockpiled in the arsenal owing to its limited shelf
life and high toxicity, but rather its two synthetic precursors, i.e.,
methylphosphonic difluoride (2) and 2-propanol are (Figure
1).5 For this reason, Sarin and 2 are both an immediate threat.
Figure 1. Chemical species related to Sarin threat.
Herein, we report our progress toward the development of a
practical probe (NA570) fighting against Sarin related threats.
Existing probes for nerve agents generally rely on
nucleophilicity of an aliphatic hydroxyl,8 hydroxyl oxime,9 or
amine10 toward electrophilic phosphorus atoms. However, the
In addition, synthesis of difluoride 2 from commercially
available methylphosphonic acid (4) is also straightforward,
i.e., through a chlorination with thionyl chloride (SOCl2) or
oxalyl chloride (C2O2Cl2) followed by a halide exchange with a
fluoride source like NaF or HF.5 Thus, the presence of SOCl2,
C2O2Cl2, and methylphosphonic dichloride (3) away from their
authorized storage sites should also trigger an alarm against
potential Sarin related terroristic activities. As Sarin and 2 are
Received: March 24, 2014
Published: April 25, 2014
© 2014 American Chemical Society
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Figure 2. (a) The general working principle of molecular probes
designed via the “covalent-assembly” approach. (b) The structure of
NA570 and its proposed detection mechanism. (c) Chemical synthesis
of NA570.
existing chemistry for nerve agent recognition could not be
readily coupled for use with the proposed covalent-assembly
principle. Therefore, novel chemistry, i.e., a phenylogous
Vilsmeier−Haack reaction, was employed in our work and
lead to the design of NA570 (Figure 2b). Highly electron
deficient phosphorus centers of the nerve agents, their mimics,
or precursors (vide infra) are expected to be able to activate the
4-diethylaminobenzaldehyde in NA570 toward nucleophilic
attack by the nearby diethylaniline and ultimately furnish
Pyronine B (8), a highly absorbing and fluorescing xanthene
dye (ε = 83 000 cm−1·M−1 and ϕ = 0.64 in chlorobenzene).
NA570 was conveniently synthesized by a simple condensation
of commercial 3-diethylaminophenol (9) and 2-bromo-4-
diethylaminobenzaldehyde (10) in a 78% yield (Figure 2c).
NA570 is indefinitely stable if protected from light and acid. No
decomposition of NA570 was observed during the course of
the project.
The viability of this design was validated by reactions
between NA570 and PO(OEt)2Cl (7) in a list of nonprotic
solvents. Upon addition of 7 (10 equiv) into a solution of
NA570 (50 μM), the kinetics of pyronine B (8) formation was
monitored with a fluorimeter. In chlorobenzene, dichloro-
methane, dichloroethane, or nitrobenzene, the fluorescence
signal rose instantaneously from the baseline upon addition of
7. The signal intensities increased steadily over the course of
monitoring for 2 h (Figure 3a). In comparison, signal intensities
in tetrahydrofuran, ethyl acetate, and dimethyl sulfoxide were
much lower, suggesting that solvents bearing a nucleophilic
oxygen atom are not an optimal working medium. On the basis
of these results, chlorobenzene was chosen for further studies.
Figure 3. (a) Fluorescence enhancement upon addition of 10 equiv of
PO(OEt)2Cl (7) into a solution of NA570 (50 uM) in various
solvents. λex = 563 nm and λem = 573 nm. (b) The absorption and
fluorescence emission spectra of NA570 solution before and 30 min
after addition of 7. (c) The picture of the NA570 solution 30 min after
addition of 7, taken under white light (left) and 532 laser excitation
(right).
The absorption and emission spectra of the solution before and
30 min after addition of 7 were recorded in chlorobenzene
(Figure 3b). Prior to the addition of 7, the NA570 solution
displays no absorption beyond 400 nm, is colorless, and
exhibits no background fluorescence with excitation at 563 nm.
After 30 min, pyronine B (8) generated in situ is easily captured
by its distinctive pink color with a maximum absorption at 563
nm, or by its intense yellow-orange fluorescence with a
maximum emission at 573 nm (Figure 3c).
We next examined the responses of the NA570 solution in
chlorobenzene (50 μM) toward the model set of compounds
indicative of immediate or potential Sarin threat: 3, 6, 7, SOCl2,
and C2O2Cl2. All these species (at 1 equiv) induced a
fluorescence turn-on (Figure 4a). Within the first ca. 150 s,
signal intensity was in the order of 7 > 6 ≈ C2O2Cl2 > SOCl2 ≈
3. Then, the signal enhancement from C2O2Cl2 and 3 gradually
slowed down, while the rest continued increasing steadily
during the entire course of monitoring for 1800 s. In
comparison, nontoxic methylphosphonic acid (4) did not
induce any signal enhancement even at a concentration as high
as 100-fold excess. Additionally, titrations showed that the
signal intensities caused by these Sarin related species were
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Communication
should yield stronger signals and display higher sensitivity than
all of the species tested in this work.
In conclusion, we have developed an optical probe (NA570)
for detection of Sarin related threats, which we have shown to
have potential for practical applications. NA570 was designed
via a novel “covalent-assembly” principle, advantages of which
include compact probe structure, convenient probe synthesis,
and a highly sensitive colorimetric and fluorimetric turn-on
signal from zero background. We expect the covalent-assembly
principle to find additional applications for a broader scope of
substrates.
ASSOCIATED CONTENT
■
S
* Supporting Information
General methods, NA570 synthesis, H and 13C NMR, HRMS
1
and supplementary spectral results. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
youjunyang@ecust.edu.cn
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work is supported by Sponsored by Shanghai Rising-Star
Program (No. 13QA1401200), Innovation Program of
Shanghai Municipal Education Commission (No. 12ZZ047),
Doctoral Fund of Ministry of Education of China (No.
20110074120008) and the National Natural Science Founda-
tion of China (Nos. 21106043 and 21372080). We thank Prof.
Amanda E. Hargrove and Dr. Michelle A. Ivy for help with
manuscript preparation.
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