A rhodamine-deoxylactam based sensor for chromo-fluorogenic detection of nerve agent simulant

Article abstract of DOI:10.1016/j.bmcl.2012.08.077

N-(rhodamine B)-deoxylactam-5-amino-1-pentanol (dRB-APOH) was designed and prepared as the chromo-fluorogenic sensor for detection of a nerve agent simulant via analyte triggered tandem phosphorylation and opening of the intramolecular deoxylactam. The su

Full text of DOI:10.1016/j.bmcl.2012.08.077

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6358–6361
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Bioorganic & Medicinal Chemistry Letters
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A rhodamine-deoxylactam based sensor for chromo-fluorogenic detection
of nerve agent simulant
⇑
Zhisheng Wu, Xuanjun Wu, Yuhui Yang, Ting-bin Wen, Shoufa Han
Department of Chemical Biology, College of Chemistry and Chemical Engineering and the Key Laboratory for Chemical Biology of Fujian Province Xiamen University, 361005
Xiamen, China
a r t i c l e i n f o
a b s t r a c t
Article history:
N-(rhodamine B)-deoxylactam-5-amino-1-pentanol (dRB-APOH) was designed and prepared as the
chromo-fluorogenic sensor for detection of a nerve agent simulant via analyte triggered tandem phos-
phorylation and opening of the intramolecular deoxylactam. The successful detection of diethyl chloro-
phosphate suggests the utility of rhodamine-deoxylactams as the chromo-fluorogenic signal reporting
platform for design of sensors targeting reactive chemical species via various chemistries.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 30 March 2012
Revised 14 June 2012
Accepted 21 August 2012
Available online 27 August 2012
Keywords:
Rhodamine-deoxylactam
Nerve agent
Chromo-fluorogenic
Tandem reaction
Visual detection
Nerve agents, e.g. Sarin, are a group of lethal organo-phosphates
that can inactivate acetylcholinesterase of the human nerve sys-
tem.1 The inhibition of acetylcholinesterase leads to accumulation
of acetylcholine, resulting in neuromuscular paralysis and eventu-
ally death.2 Nerve agents are of extraordinary toxicity and are an
increasing homeland security concern due to the horrifying effects
that could be administered from a terrorist attack. For example, the
purposeful release of Sarin gas in Tokyo subway in 1995 leaded to
12 deaths.3 As such, methods allowing facile detection of nerve
agents will be highly desirable. Conventional assay methods for
nerve agents, example liquid chromatography/mass spectrometry,
enzyme based biosensors,4 usually show limitations such as lack of
portability or storage/stability issues that limits their success in
some conditions. Chemosensors are advantageous in the afore-
mentioned aspects as they require widely used instruments and of-
fer the possibility to detect nerve agents with naked eyes.
A number of chemical probes have been designed to afford
altered fluorescence emission or color change in the presence of
nerve agent mimics via various chemistries.5–7 One notable
strategy, as shown in Scheme 1, proceeds via analyte mediated
phosphorylation of the judiciously positioned hydroxyl moiety of
the probes followed by intramolecular SN2 reaction to liberate
the phosphate group. The cyclization is concomitant with the
formation of chromophores or fluorophores for signal detection
(Scheme 1).5,7j–l
Rhodamines, a group of fluorone dyes with visible colors, are
widely utilized in cellular imaging and biotechnology applications
owing to the exceptional photophysical properties. Historically,
rhodamine-lactam have been extensively utilized to detect metal
ions by virtue of reversible ring-opening of the spirolactam, which
gives rise to highly fluorescent and colored rhodamine fluoro-
phore.8 In contrast, the rhodamine-deoxylactams have largely
remained unexplored for analyte detection.9 To complement the
methodology described in Scheme 1 further, we report the chro-
mo-fluorogenic detection of a nerve agent simulant via analyte
triggered irreversible ring opening of a rhodamine-deoxylactam
based chemosensor (Scheme 2).
Aminolysis of the methyl ester of rhodamine B with 5-amino-
1-pentanol affords N-(rhodamine B)-lactam-5-amino-1-pentanol
(RB-APOH). Reduction of RB-APOH with lithium aluminium hy-
dride in anhydrous THF gave N-(rhodamine B)-deoxylactam-5-
amino-1-pentanol (dRB-APOH) as the desired product in 50% yield
(Supplementary data, Scheme S1).dRB-APOH, which is colorless
and nonfluorescent, quickly turned into red color in dimethylform-
amide (DMF) upon addition of diethyl chlorophosphate which was
widely used as the nerve agent simulant.5,6,7c To optimize the as-
say, the color formation rates of dRB-APOH with diethyl chloro-
phosphate in selected solvents were monitored by fluorescence
emission. Time course studies showed that DMF was the preferred
assay medium relative to other less polar solvents. The assay was
also efficient in aqueous DMF containing 2% (v/v) of water
(Fig. 1A). Kinetic analysis of the reaction between dRB-APOH and
diethyl chlorophosphate showed that the fast phase of the signal
⇑
Corresponding author. Tel.: +86 0592 2181728.
E-mail address: shoufa@xmu.edu.cn (S. Han).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.077

Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6358–6361
6359
O
P
OEt
Cl
OEt
Cyclization
O
P
OEt
N
N
O
OEt
N
OH
Fluorescent
weakly fluorescent
Scheme 1. Reported fluorogenic sensor (B)6 for nerve agent mimics.
O
Cl P OEt
N
N
O
N
O
N
OEt
O
O
P OEt
OH
Phosphorylation
N
N
OEt
1
Intermediate
dRB-APOH
nonfluorescent and colorless
cyclization
N
O
N
N
2
highly fluorescent and colored
Scheme 2. Fluoro-chromogenic detection of diethyl chlorophosphate with dRB-APOH.
production (50% of the maximal fluorescence intensity) is com-
plete at about 5 min (Fig. 1B).
on-spot detection of nerve agent with the aid of instruments or
possibly with ‘‘naked eyes’’.
The sensitivity of dRB-APOH based assay was probed by fluores-
cence emission and UV–vis spectrometry of dRB-APOH (1 mg
mlÀ1) in DMF in the presence of various amount of diethyl chloro-
phosphate. The solutions were incubated at room temperature for
20 min and then recorded for fluorescence emission spectra. Fig-
ure 2 showed the maximum fluorescence emission intensity at
590 nm increased as a function of diethyl chlorophosphate concen-
trations. As low as 25 ppm can be clearly identified under the assay
condition. The UV–vis absorption spectra of the reaction solutions
revealed a new strong absorption band at 560 nm with dose
dependant color intensities (Fig. 3). Relative to the colorless sub-
strate, the deep red colored species generated in the assay system
is highly visible to naked eyes, suggesting the potential utility for
To access the identity of the colored species generated in the assay
solution, which is presumably compound 2 (Scheme 1), the assay
solution was analyzed by high resolution mass spectrometry (HRMS).
A major peak located at 496.3320 was identified (Fig. 4), which is in
consistence with the theoretical molecular weight of compound 2
(C33H42N3O+; MW: 496.3322), confirming formation of compound
2 in the assay system. The fluorescence spectra of the resultant solu-
tion were almost identical to that of rhodamine B (Supplementary
data, Fig. S1 and S2). Taken together, the experimental data
supported the formation of rhodamines from opening of the deoxy-
lactam of dRB-APOH under the assay condition (Scheme 2).
To verify the critical role of the deoxylactam in the assay mech-
anism, dRB-APOH and RB-APOH were simultaneously tested for
2500
500 ppm
DMF
A
2500
2000
1500
1000
500
B
250 ppm
170 ppm
75 ppm
2000
1500
1000
500
0
DMF+2% H2O
CH3CN
0 ppm
Blank
0
0
200
400
600
800 1000 1200
0
200
400
600
800
1000 1200
Time (seconds)
Time (seconds)
Figure 1. Effects of solvents on the reaction rates between dRB-APOH (1 mg mlÀ1) and diethyl chlorophosphate (170 ppm) (A); kinetic profiles of the reaction of dRB-APOH
(1 mg mlÀ1) with various amounts of diethyl chlorophosphate in DMF. The fluorescence emission intensity at 590 nm (kex@560 nm) was recorded as a function of time (B).

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Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6358–6361
3000
2500
2000
1500
1000
500
3000
2500
2000
1500
1000
500
496.3320
N
O
N
N
Exact mass: 496.3322
0
0
100 200 300 400 500
ppm
0
560 580 600
620 640 660 680
700
Wavelength (nm)
Figure 2. Fluorescence emission spectra of dRB-APOH in the presence of diethyl
chlorophosphate (500, 170, 100, 50, 25, and 0 ppm, from top to bottom)
(kex@560 nm).
480
485
490
495
500
505
m/z
Figure 4. HRMS confirmation of formation of compound 2 in reaction of dRB-APOH
with diethyl chlorophosphate.
their differential responses to diethyl chlorophosphate. RB-APOH,
the structural ananlog of dRB-APOH, differs from dRB-APOH in that
it contains an amide moiety (lactam, in red, Fig. 5A). Analysis
showed that no fluorescent or colored species was produced in
reaction of RB-APOH with diethyl chlorophosphate (Fig. 5B), indi-
cating that the intramolecular lactam of RB-APOH remained closed
under the assay conditions. We envision that the non-nucleophilic
nature of the lactam impedes the intramolecular cyclization and
thus prevents the formation of colored and fluorescent species in
the assay solution. The differential responses of dRB-APOH relative
to RB-APOH highlighted the crucial role of the deoxylactam of dRB-
APOH in sensing of diethyl chlorophosphate.dRB-APOH efficiently
sensed diethyl chlorophosphate vis O-phosphorylation mediated
intra-molecular N-alkylation. In contrast, N-(rhodamine B)-deoxy-
lactam-2-aminoethanol (dRB-AE) responded to diethyl chloro-
phosphate by O-phosphorylation mediated intra-molecular
N-phosphorylation,10 suggesting the versatile chemistry of
rhodamine deoxylactams with structural alterations.
A
N
N
O
N
O
N
OH
OH
N
N
O
dRB-APOH
RB-APOH
B
3000
2500
2000
1500
1000
500
Rhodamine-hydroxamate11 have been reported for fluorogenic
detection of diethyl chlorophosphate. Hence, the assay efficiency
of dRB-APOH towards diethyl chlorophosphate was compared with
rhodamine-hydroxamate. The new assay exhibited improved assay
kinetics and gave species with highly stable fluorescence proper-
ties relative to rhodamine-hydroxamate (Supplementary data,
Scheme S3, S4, and Fig. S4).
0
560 580 600 620 640 660 680 700
Wavelength (nm)
In summary, a chromo-fluorogenic detection of diethyl chloro-
phosphate was developed based on analyte triggered O-phosphor-
ylation mediated intramolecular N-alkylation, yielding visible
Figure 5. Chemical structures RB-APOH and dRB-APOH (A) and the differential
fluorogenic responses of dRB-APOH (B, in black) and RB-APOH (B, in red) in the
presence of diethyl chlorophosphate in DMF.
A
B
0.3
0.3
0.2
0.1
0.2
0.1
0.0
400
450
500
550
600
650
700
0
100
200
300
ppm
400
500
Wavelength (nm)
Figure 3. UV–vis absorbance spectra of the dRB-APOH based assay solution. (A) Uv–vis absorption spectra of dRB-APOH in the presence of diethyl chlorophosphate (500, 170,
100, 50, 25, and 0 ppm, from top to bottom). The inset shows the color of dRB-APOH (1 mg mlÀ1) in DMF with (left) or without (right) diethyl chlorophosphate (50 ppm); (B)
The titration curve was plotted by absorbance at 560 nm vs the analyte concentrations.

Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6358–6361
3. Vale, A. Przegl Lek 2005, 62, 528–532.
6361
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instruments or ‘‘naked eyes’’. The successful detection of diethyl
chlorophosphate with dRB-APOH suggests the compatibility of
rhodamine-deoxylactams as the chromo-fluorogenic signal report-
ing platform for design of sensors targeting reactive chemical spe-
cies via various chemistries.
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Acknowledgments
Dr. S. Han was supported by grants from NSF China (21272196),
Natural Science Foundation of Fujian Province of China
(2011J06004) and the Fundamental Research Funds for the Central
Universities (2011121020); Dr. T. Wen was supported by NSF Chi-
na (21072161) and 973 National Basic Research Program of China
(2012CB821600).
Supplementary data
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