Aggregation induced emission enhancement behavior of conformationally rigid pyreneamide-based probe for ultra-trace detection of picric acid (PA)

Article abstract of DOI:10.1016/j.dyepig.2018.04.014

In this study, we designed and prepared pyrene-based dipodal probes with a range of molecular flexibility for picric acid (PA) detection. These probes commonly contain a central rigid core, but probe 1 contains relatively short linkers/spacers compared to probes 2 and 3. The dipodal probes produced dominant pyrene excimer fluorescence in an aqueous solution, and aggregation-induced emission enhancement (AIEE) was observed. The fluorescence emission of probe 1 was significantly quenched only by the presence of PA, while probes 2 and 3 were responsive to 4-nitrophenol or 2,4-dinitrophenol as well as PA. Of the three dipodal probes, the highest binding strength was observed for probe 1 with PA, which gave an association constant of K_a = 3.96 × 10⁷ M^?1.¹H NMR measurements and DFT calculations indicate that, upon addition of PA, these dipodal probes formed individual probe-PA complexes via π–π interactions between the pyrene ring and PA in an aqueous solution. This complex formation enabled energy transfer from electron-rich pyrene to electron-poor PA upon excitation, leading to fluorescence quenching. Probe 1 provided ultra-trace detection of PA in water, with observed and calculated limit of detections (LOD) of 1 nM and 0.13 pM, respectively, one of the lowest values reported. Effective fluorescence quenching was also obtained using probe 1-coated filter paper or TLC plate with PA solution/vapor, suggesting the significant potential of this sensing system in real-time applications. These studies were supported by FE-SEM, TEM, and UV-visible, fluorescence, NMR and DFT calculations.

Full text of DOI:10.1016/j.dyepig.2018.04.014

Accepted Manuscript
Aggregation induced emission enhancement behavior of conformationally rigid
pyreneamide-based probe for ultra-trace detection of picric acid (PA)
Ashwani Kumar, Pil Seok Chae
PII:
S0143-7208(18)30390-5
10.1016/j.dyepig.2018.04.014
DYPI 6675
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 16 February 2018
Revised Date: 10 April 2018
Accepted Date: 10 April 2018
Please cite this article as: Kumar A, Chae PS, Aggregation induced emission enhancement behavior of
conformationally rigid pyreneamide-based probe for ultra-trace detection of picric acid (PA), Dyes and
Pigments (2018), doi: 10.1016/j.dyepig.2018.04.014.
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ACCEPTED MANUSCRIPT
Aggregation induced emission enhancement behavior of
conformationally rigid pyreneamide-based probe for ultra-trace
detection of picric acid (PA)
Ashwani Kumar*, and Pil Seok Chae*
Department of Bionanotechnology, Hanyang University, Ansan, 15588, Republic of Korea
Corresponding authors: Tel.: +82-31-400-5205.
E-mail address: ashwanirubal@gmail.com, pchae@hanyang.ac.kr
Pyrene-appended probe 1 exhibited selective PA detection with LOD of 0.13 pM in water.

ACCEPTED MANUSCRIPT
Aggregation induced emission enhancement behavior of
conformationally rigid pyreneamide-based probe for ultra-trace
detection of picric acid (PA)
Ashwani Kumar* and Pil Seok Chae*
Department of Bionanotechnology, Hanyang University, Ansan, 155-88, Republic of Korea
Corresponding authors: Tel.: +82-31-400-5205.
E-mail address: ashwanirubal@gmail.com, pchae@hanyang.ac.kr
Abstract
In this study, we designed and prepared pyrene-based dipodal probes with a range of
molecular flexibility for picric acid (PA) detection. These probes commonly contain a central
rigid core, but probe 1 contains relatively short linkers/spacers compared to probes 2 and 3.
The dipodal probes produced dominant pyrene excimer fluorescence in an aqueous solution,
and aggregation-induced emission enhancement (AIEE) was observed. The fluorescence
emission of probe 1 was significantly quenched only by the presence of PA, while probes 2 and
3
were responsive to 4-nitrophenol or 2,4-nitrophenol as well as PA. Of the three dipodal
probes, the highest binding strength was observed for probe 1 with PA, which gave an
7
−1
1
a
association constant of K = 3.96 × 10 M . H NMR measurements and DFT calculations
indicate that, upon addition of PA, these dipodal probes formed individual probe-PA complexes
via π–π interactions between the pyrene ring and PA in an aqueous solution. This complex
formation enabled energy transfer from electron-rich pyrene to electron-poor PA upon
1

ACCEPTED MANUSCRIPT
excitation, leading to fluorescence quenching. Probe 1 provided ultra-trace detection of PA in
water, with observed and calculated limit of detections (LOD) of 1 nM and 0.13 pM,
respectively, one of the lowest values reported. Effective fluorescence quenching was also
obtained using probe 1-coated filter paper or TLC plate with PA solution/vapor, suggesting the
significant potential of this sensing system in real-time applications. These studies were
supported by FE-SEM, TEM, and UV-visible, fluorescence, NMR and DFT calculations.
Keywords: AIEE, Pyreneamide, excimer emission, PA, vapor sensing
1. Introduction
Due to the continued wars, battles and army conflicts in various regions, there is an
increased possibility for chemical and biological attacks by terrorsits or criminals. For the global
safety and security, thus, we need some real time monitoring systems for the detection of
hazardous materials such as viruses [1-2], bacteria [3-4], toxic ions or gases [5-9], and
explosives [10-11]. Some hazardous pollutants include many organic molecules (e.g.,
nitrophenols, picric acid (PA), 2,4-dinitrophenol (2,4-DNP), 4-nitrophenol (4-NP), trinitrotoluene
(TNT), and dinitrotoluene (DNT)) [10-11]. Among these, PA that is highly explosive and toxic has
a good water solubility and thus can easily pollute ground water [12-13] and irrigation land [14].
In addition, PA ingestion by humans can cause severe poisoning, including skin and eye
irritations, headache, nausea, vomiting, liver malfunction and chronic diseases such as
destruction of erythrocytes, cyanosis, and cancer [15-17]. The lowest limit of detection (LOD)
2

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-1
for PA in ground water is 0.001 mgL allowed by WHO (World health Organization) [18-19]
while its lethal dose (oral) in rabbits is 120 mg/Kg [20]. Additionally, a metabolite of PA (i.e., 2-
amino-4,6-dinitrophenol) was found to be 10 times more mutagenic than PA itself [17]. Despite
its potential enviromental and health issues, PA has widely used in dye industries, rocket fuels,
fireworks, analytical chemistry of metals and minerals, chemistry laboratories and
pharmaceutical industries as an antiseptic component [21-22]. Protecting a clean and safe
environment from hazardous pollutants is the main goal of every society for the well-being of
our present and future generations. Therefore, it is of high interest and demand to develop
sensors for the selective PA determination.
There are several ways to detect PA at a trace level including electrochemical analysis, HPLC,
chromatographic, mass, Raman and fluorescence-spectroscopies [23-26]. Among these
analytical methods, fluorescence technique has become the most convenient tool for PA
detection due to its simplicity of operation, high sensitivity, real-time monitoring with rapid
response and portability [27-29]. A number of probes including COFs (covalent organic
frameworks) [30-31], MOFs (metal-organic frameworks) [32-34], coordination polymers [35],
gels [36-37], supramolecular self-assemblies [38], organometallic compounds, cages [39-40],
and nanoparticles/nano-composites, nano-fibers [41-42], and small fluorescent probes have
been invented for the detection of a trace amount of PA and TNT [43-44]. For the sensitive
detection of these explosives, several research groups developed new molecular probes that
exhibited aggregation-induced emission enhancement and investigated their aggregates in
terms of morphology and luminescence behavior [44-49]. The physical and chemical properties
3

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of these small fluorescent probes such as their emission efficiency, water-solubility, and analyte
selectivity can be conveniently optimized via chemical modification.
Tweezers or clips were effectively able to recognize aromatic guests using multivalent
interactions (H-bonding, π-π interactions, etc.) [50-52]. Recently, imidazolium-based tweezers
or dipodes were used for PA detection in aqueous solution [25,28,44,53-54]. However, the
selectivity for PA detection, along with sensitive fluorescence quenching is still lacking. Thus, in
order for sensitive and selective detection of PA, we have synthesized neutral tweezer or clip-
based probes (probe 1, 2 and 3). We have chosen pyrene and a rigid 4,4'-
(Hexafluoroisopropylene)bis(p-penyleneoxy)dianiline (HFIBPA) as fluorophore and linker units,
respectively, to provide the excimer emission of two pyrene units through intramolecular π-π
interactions. These two units (pyrene and HFIBPA) were conjugated through different amino
acids varying the spacer length and studied their effect on selectivity and sensitivity for PA
detection in water. These probes exhibited aggregation-induced emission enhancement (AIEE)
phenomena in a DMSO-water mixed solvent system and were evaluated for PA detection in an
aqueous solution. Of these probes, probe 1 displayed the highest efficacy toward PA sensing in
terms of selectivity and sensitivity, with a limit of detection (LOD) observed and calculated at 1
nM and 0.13 pM, respectively. The ultra-sensitive and selective PA detection observed for
probe 1 was achieved by the formation of strong intermolecular π-π interactions between the
pyrene units and PA, allowing energy transfer from the probe to the guest molecule. For
comparison of the dipodal probes 1-3, we synthesized monopodal probe 4. The probe
behaviors with PA were investigated in detail using various analytical methods such as FE-SEM,
4

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TEM, and UV-visible, fluorescence, and nuclear magnetic resonance (NMR) spectroscopies, in
addition to density functional theory (DFT) calculations.
Fig. 1. DFT-optimized (B3LYP-631G*) chemical structures of probes 1-4.
2
2
2
2
. Experimental Section
.1 Details for general procedure and theoretical calculations were given in ESI
.2 Synthesis of probes 1 – 4
.2.1.
(Compound
7)
Di-tert-butyl
((((((perfluoropropane-2,2-diyl)bis(4,1-
phenylene))bis(oxy))bis(4,1-phenylene))bis(azanediyl))bis(2-oxoethane-2,1-diyl))dicarbamate
5

ACCEPTED MANUSCRIPT
A mixture of (tert-butoxycarbonyl)glycine (351 mg, 2 mmol), hydroxybenzotriazole (HOBt,
05 mg, 3 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 575 mg, 3 mmol)
4
was dissolved in dichloromethane-dimethylformamide (CH Cl : DMF (1:1), 20 ml) and stirred
2
2
o
vigorously at 0 C for 20 minutes under an argon atmosphere. Then, 4,4'-
Hexafluoroisopropylene)bis(p-penyleneoxy)dianiline (HFIBPA) (519 mg, mmol) and
(
1
diisopropylethylamine (DIPEA) (388 mg, 3 mmol) were added to the reaction mixture and
stirred at room temperature for another 3 h. After completion of the reaction (detected by TLC
analysis), the solvent of the reaction mixture was reduced under vacuum. The crude solid was
dissolved in H O and extracted with CH Cl , and the organic layer was washed with H O (2 × 50
2
2
2
2
mL) and a 5% aqueous NaCl solution (100 mL). The collected organic layer was then dried over
anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure,
2 2
and the residue was purified by column chromatography using CH Cl as the eluent to afford
1
compound 7 as a white solid (750 mg, 90% yield). H NMR (400 MHz, CDCl ): δ 1.48 (s, 18H, 6 x
3
3 2
CH ), 3.96 (d, J = 6.0 Hz, 4H, 2 x CH ), 5.44 (bt, 2H, 2 x aliphatic NH), 6.90 (d, J = 9.0 Hz, 4H, 4 x
ArH), 7.00 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.31 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.51 (d, J = 8.9 Hz, 4H, 4 x
1
3
ArH), 8.46 (bs, 2H, 2 x CONH); C NMR (100 MHz, CDCl
3
): δ 28.52, 45.72, 81.07, 117.21, 120.76,
121.92, 127.56, 131.88, 134.03, 152.38, 158.53, 168.10.
2.2.2. (Compound 8) 2,2'-(((((Perfluoropropane-2,2-diyl)bis(4,1-phenylene))bis(oxy))bis(4,1-
phenylene))bis(azanediyl))bis(2-oxoethan-1-aminium)
To a CH Cl (30 ml) solution of compound 7 (417 mg, 0.5 mmol), trifluoroacetic acid (TFA, 4
2
2
mmol) was added drop-wise, and the mixture was stirred overnight at room temperature under
6

ACCEPTED MANUSCRIPT
an argon atmosphere. After completion of the reaction (analyzed by TLC), the reaction mixture
was evaporated under reduced pressure and precipitated by the addition of diethyl ether. The
suspension was filtered, and the solid was washed with diethyl ether to afford compound 8 as a
1
white solid (300 mg, 94% yield). H NMR (400 MHz, DMSO-d ): δ 3.79 (bs, 4H, 2 x CH ), 7.04 (d,
6
2
J = 7.2 Hz, 4H, 4 x ArH), 7.14 (d, J = 7.2 Hz, 4H, 4 x ArH), 7.33 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.64 (d, J
+
13
=
7.2 Hz, 4H, 4 x ArH), 8.12 (bs, 6H, 2 x NH ), 10.51 (s, 2H , 2 x NHCO); C NMR (100 MHz,
3
DMSO-d ): δ 40.97, 117.14, 120.69, 120.95, 126.17, 131.48, 134.88, 150.83, 158.22, 164.70.
6
2.2.3.
(Compound
9)
Di-tert-butyl
((((((perfluoropropane-2,2-diyl)bis(4,1-
phenylene))bis(oxy))bis(4,1-phenylene))bis(azanediyl))bis(3-oxopropane-3,1-diyl))dicarbamate
The synthetic procedure for this preparation is similar to that used for compound 7 using 3-
((tert-butoxycarbonyl)amino)propanoic acid (378 mg,
2
mmol), instead of (tert-
butoxycarbonyl)glycine (351 mg, 2 mmol). Compound 9 was obtained as a white solid (820 mg,
1
9
5% yield). H NMR (400 MHz, CDCl ): δ 1.44 (s, 18H, 6 x CH ), 2.63 (d, J = 6.0 Hz, 4H, 2 x OCH ),
3
3
2
3
.51 (q, J = 6.0 Hz, 4H, 2 x NCH ), 5.17 (bs, 2H, 2 x aliphatic NHCOO), 6.93 (d, J = 9.2 Hz, 4H, 4 x
2
ArH), 7.04 (d, J = 9.2 Hz, 4H, 4 x ArH), 7.32 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.55 (d, J = 8.9 Hz, 4H, 4 x
1
3
ArH), 7.89 (bs, 2H, 2 x ArCONH); C NMR (100 MHz, CDCl ): δ 28.60, 36.70, 37.93, 80.08,
3
117.18, 120.79, 121.82, 127.52, 131.87, 134.41, 152.25, 158.59, 168.82.
2.2.4. (Compound 10) 3,3'-(((((Perfluoropropane-2,2-diyl)bis(4,1-phenylene))bis(oxy))bis(4,1-
phenylene))bis(azanediyl))bis(3-oxopropan-1-aminium)
7

ACCEPTED MANUSCRIPT
The synthetic procedure for this preparation is similar to that used for compound 8 using
compound 9 (430 mg, 0.5 mmol) instead of compound 7. Compound 10 was obtained as a
1
white solid (300 mg, 90% yield). H NMR (400 MHz, DMSO-d ): δ 2.70 (t, J = 6.8 Hz, 4H, 2 x
6
2
CH ), 3.07-3.12 (m, 4H, 2 x CH ), 7.02 (d, J = 9.2 Hz, 4H, 4 x ArH), 7.11 (dd, J = 8.4 Hz, J = 2.0
2 2 1 2
Hz, 4H, 4 x ArH), 7.32 (d, J = 8.4 Hz, 4H, 4 x ArH), 7.66 (dd, J = 7.2 Hz, J = 2.0 Hz, 4H, 4 x ArH),
1
2
+
7
.77 (bs, 6H, 2 x NH ), 10.25 (s, 2H , 2 x NHCO).
3
2.2.5. (Probe 1) N,N'-((((((perfluoropropane-2,2-diyl)bis(4,1-phenylene))bis(oxy))bis(4,1-
phenylene))bis(azanediyl))bis(2-oxoethane-2,1-diyl))bis(pyrene-1-carboxamide)
The synthetic procedure for this preparation is similar to that used for compound 7 using
pyrene-1-carboxylic acid (247 mg, 1 mmol), instead of (tert-butoxycarbonyl)glycine and
1
compound 8 (317 mg, 0.5 mmol). Probe 1 was obtained as a white solid (500 mg, 91% yield). H
NMR (400 MHz, DMSO-d ): δ 4.24 (d, J = 6.0 Hz, 4H, 2 x 2CH ), 7.07 (dd, J = 7.6 Hz, J = 2.0 Hz,
6
2
1
2
4
H, 4 x ArH), 7.16 (dd, J = 7.2 Hz, J = 2.0 Hz, 4H, 4 x ArH), 7.35 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.76
1 2
(d, J = 8.8 Hz, 4H, 4 x ArH), 8.12 (t, J = 7.6 Hz, 2H, 2 x ArH), 8.21-8.30 (m, 8H, 8 x ArH), 8.35 (d, J =
8.0 Hz, 6H, 6 x ArH), 8.71 (d, J = 9.2 Hz, 2H, 2 x ArH), 9.05 (t, J = 6.0 Hz, 2H, 2 x CONHC), 10.30 (s,
1
3
2
H, 2 x ArNHCO); C NMR (100 MHz, DMSO-d ): δ 43.32, 116.97, 120.68, 120.90, 123.63,
6
123.78, 124.45, 124.94, 125.35, 125.65, 125.82, 126.02, 126.60, 127.24, 127.94, 128.11, 128.36,
130.25, 130.73, 131.47, 131.58, 131.69, 135.86, 150.27, 158.47, 167.76, 169.49; HRMS (EI):
+
+
42 6 4 6
calcd. for C65H F N O + H (M+H ): 1089.3081; found: m/z 1089.3090.
2.2.6. (Probe 2) N,N'-((((((perfluoropropane-2,2-diyl)bis(4,1-phenylene))bis(oxy))bis(4,1-
phenylene))bis(azanediyl))bis(3-oxopropane-3,1-diyl))bis(pyrene-1-carboxamide)
8

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The synthetic procedure for this preparation is similar to that used for compound 7 using
pyrene-1-carboxylic acid (247 mg, 1 mmol), instead of (tert-butoxycarbonyl)glycine and
compound 10 (331 mg, 0.5 mmol). Probe 2 was obtained as a white solid (500 mg, 90% yield).
1
H NMR (400 MHz, DMSO-d ): δ 2.78 (t, J = 6.8 Hz, 4H, 2 x 2CH ), 3.72 (q, J = 6.4 Hz, 4H, 2 x
6
2
2
CH ), 7.03 (d, J = 9.2 Hz, 4H, 4 x ArH), 7.12 (d, J = 9.2 Hz, 4H, 4 x ArH), 7.32 (d, J = 8.4 Hz, 4H, 4 x
2
ArH), 7.74 (d, J = 8.8 Hz, 4H, 4 x ArH), 8.07-8.11 (m, 4H, 4 x ArH), 8.16-8.35 (m, 12H, 12 x ArH),
1
3
8.50 (d, J = 9.2 Hz, 2H, 2 x ArH), 8.87 (t, J = 5.6 Hz, 2H, 2 x CONH), 10.21 (s, 2H, 2 x ArNHCO);
C
NMR (100 MHz, DMSO-d ): δ 36.23, 36.44, 116.85, 120.59, 120.89, 123.64, 123.75, 124.42,
6
124.77, 125.21, 125.54, 125.79, 125.94, 126.55, 127.23, 127.70, 128.00, 128.25, 130.19, 130.72,
131.44, 131.51, 132.10, 136.14, 150.09, 158.50, 169.00, 169.45; HRMS (EI): calcd. for
+
+
C H F N O +H (M+H ): 1117.3394; found: m/z 1117.3403.
6
7 46 6 4 6
2.2.7. (Probe 3) N,N'-((((((perfluoropropane-2,2-diyl)bis(4,1-phenylene))bis(oxy))bis(4,1-
phenylene))bis(azanediyl))bis(2-oxoethane-2,1-diyl))bis(4-(pyren-1-yl)butanamide)
The synthetic procedure for this preparation is similar to that used for compound 7 using
pyrene-1-butyric acid (289 mg, 1 mmol), instead of (tert-butoxycarbonyl)glycine and compound
1
8
(317 mg, 0.5 mmol). Probe 3 was obtained as a white solid (550 mg, 93% yield). H NMR (400
MHz, CDCl ): δ 2.14-2.19 (m, 4H, 2 x 2CH ), 2.35 (t, J = 7.2 Hz, 4H, 2 x 2CH ), 3.28 (t, J = 7.2 Hz,
3
2
2
4
H, 2 x 2CH ), 3.95 (d, J = 5.2 Hz, 4H, 2 x 2CH ), 6.79 (d, J = 9.0 Hz, 4H, 4 x ArH), 6.82 (t, J = 5.2
2
2
Hz, 2H, 2 x CONH), 6.88 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.23 (d, J = 8.8 Hz, 4H, 4 x ArH), 7.49 (d, J =
8.8 Hz, 4H, 4 x ArH), 7.72 (d, J = 7.8 Hz, 2H, 2 x ArH), 7.87- 7.98 (m, 10H, 10 x ArH), 8.05 (t, J =
1
3
9
.2 Hz, 4H, 4 x ArH), 8.14 (d, J = 9.2 Hz, 2H, 2 x ArH), 9.13 (s, 2H, 2 x ArNHCO); C NMR (100
9

ACCEPTED MANUSCRIPT
MHz, CDCl ): δ 27.40, 32.90, 35.78, 44.63, 117.14, 120.74, 121.64, 123.25, 124.92, 124.99,
3
125.17, 126.07, 126.95, 127.44, 127.57, 127.61, 128.83, 130.14, 130.91, 131.49, 131.82, 134.33,
+
1
35.48, 152.22, 158.65, 167.37, 174.13; HRMS (EI): calcd. for C H F N O (M ): 1172.3948;
7
1 54 6 4 6
found: m/z 1172.3950.
2.2.8. (Compound 12) Tert-butyl (2-((4-methoxyphenyl)amino)-2-oxoethyl)carbamate
The synthetic procedure for this preparation is similar to that used for compound 7 using p-
anisidine (370 mg, 3 mmol) instead of 4,4'-methylenedianiline. The procedure afforded
1
compound 12 as a thick liquid (800 mg, 95% yield). H NMR (400 MHz, CDCl ): δ 1.47 (s, 9H, 3 x
3
t
CH ), 3.79 (s, 3H, OCH ), 3.92 (d, J = 6.0 Hz, 2H, CH ), 5.28 (br s, 1H, NHCOOBu ), 6.86 (d, J = 10.4
3
3
2
1
3
Hz, 2H, 2 x ArH), 7.40 (d, J = 10.0 Hz, 2H, 2 x ArH), 8.04 (br s, 1H, NHCO); C NMR (100 MHz,
CDCl ): δ 28.51, 55.68, 77.55, 144.38, 122.04, 130.67, 156.79, 167.77.
3
2.2.9. (Compound 13) 2-((4-methoxyphenyl)amino)-2-oxoethan-1-aminium
The synthetic procedure for this preparation is similar to that used for compound 8. The
1
procedure afforded compound 13 as a brownish-white solid (500 mg, 91% yield). H NMR (400
MHz, DMSO-d ): δ 3.73 (s, 3H, OCH ), 3.74 (d, J = 5.6 Hz, 2H, CH ), 6.94 (d, J = 6.8 Hz, 2H, 2 x
6
3
2
+
3
ArH), 7.50 (d, J = 6.8 Hz, 2H, 2 x ArH), 8.10 (br s, 3H, NH ), 10.31 (s, 1H, NHCO).
2.2.10. (Probe 4) N-(2-((4-methoxyphenyl)amino)-2-oxoethyl)-4-(pyren-1-yl)butanamide
The synthetic procedure for this preparation is similar to that used for probe 1 using
compound 13 (181 mg, 1.0 mmol) instead of compound 8 and pyrene-1-butyric acid (289 mg, 1
1
mmol). The procedure afforded probe 4 as a white solid (405 mg, 90% yield). H NMR (400
10

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MHz, DMSO-d ): δ 2.01-2.09 (m, 2H, CH ), 2.36 (t, J = 7.2 Hz, 2H, CH ), 3.30-3.39 (m, 2H, CH ),
6
2
2
2
3.72 (s, 3H, CH ), 3.89 (d, J = 5.6 Hz, 2H, CH ), 6.88 (d, J = 7.2 Hz, 2H, 2 x ArH), 7.50 (d, J = 9.2 Hz,
3 2
2H, 2 x ArH), 7.98 (d, J = 7.6 Hz, 1H, ArH), 8.07 (t, J = 7.6 Hz, 1H, ArH), 8.12–8.17 (m, 2H, 2 x
ArH), 8.20–8.29 (m, 5H, 4 x ArH, + 1H, PyCONH ), 8.43 (d, J = 9.2 Hz, 1H, ArH), 9.83 (s, 1H,
1
3
ArNH); C NMR (100 MHz, DMSO-d ): δ 27.56, 32.19, 34.83, 42.57, 55.12, 113.84, 120.63,
6
123.59, 124.14, 124.22, 124.76, 124.89, 124.93, 126.11, 126.47, 127.18, 127.45, 127.55, 128.16,
129.28, 130.42, 130.88, 132.07, 136.66, 155.14, 167.41, 172.50; HRMS (EI): calcd. for
+
C H N O (M+H ): 451.2016; found: m/z 451.2025.
2
9 26 2 3
3. Results and discussion
Scheme 1. Synthetic scheme for the preparation of individual probes (1-4); (a) DCM-DMF (1:1),
o
(
tert-butoxycarbonyl)glycine, HOBt, EDC, diisopropylethylamine, 0 C, RT, 8h, 90-95%; (b) DCM,
11

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TFA, RT, 3h, 90-94%; (c) 3-((tert-butoxycarbonyl)amino)propanoic acid, DCM-DMF (1:1), HOBt,
o
EDC, diisopropylethylamine, 0 C, RT, 8h, 95%; (d) pyrene-1-carboxylic acid, DCM-DMF (1:1),
o
HOBt, EDC, diisopropylethylamine, 0 C, RT, 8h, 90-91%; (e) pyrene-3-butyric acid, DCM-DMF
o
(
1:1), HOBt, EDC, diisopropylethylamine, 0 C, RT, 8h, 90-93%.
3.1. Synthesis and structural characterization
A common architecutre of 4,4'-(hexafluoroisopropylene)bis(p-penyleneoxy)dianiline (HFIBPA)
are present in the central core region of the three dipodal probes (1-3) connected into two
pyrene units using amino acid linker. But these probes structurally differ from each other in
terms of the amino acid linker. Glycine and β-alanine were used for the preparations of probe
1/3 and probe 2, respectively. Another structural difference between these dipodal probes is in
the length of the alkyl spacer between the pyrene ring and the amino acid linker. Pyrene-1-
carboxylic acid was utilized to prepare probes 1 and 2 while pyrene-1-butanoic acid was utilized
to prepare probe 3 (Fig. 1). Probe 4 with a single pyrene unit, correponding to the monopod
analogues of probe 3, was also prepared for comparison. Probes 1-4 were synthesized by an
EDC-based coupling of an amine-functionalized compound 8/10/13 with a respective acid
component (Scheme 1). The chemical structures of probes 1-4 (Fig. 1) were well supported by
1
13
H and C NMR spectra as well as by FAB mass data (see ESI).
3.2. UV-vis. absorption and fluorescence studies of probes 1- 4
The UV-visible absorption spectra of the individual probes (probes 1-4) in DMSO produced a
typical pyrene signature with three absorption maxima (λmax) at 330, 345, and 372 nm. Due to
12

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the presence of two pyrene units, absorbance values of the dipodal probes (1-3) were roughly
two-fold higher than that of monopod probe 4 (Fig. S1). The optimal excitation wavelength for
maximum fluorescence intensity was obtained by measuring fluorescence intensity of probe 1
or 2 in a range of excitation wavelength from 300 to 360 nm. Both probes dissolved in ethanol
gave maximum fluorescence intensity upon excitation at 340 nm (Fig. S2 & S3). Under the
conditions tested, these dipodal probes (1-2) gave rise to a large pyrene monomer peak in the
region of 380-390 nm, along with a small pyrene excimer peak at 480-490 nm, indicating that
each pyrene unit of these probes exists primarily as a monomeric form. To investigate the
solvent effect on the fluorescence emission of these probes, sample solutions were prepared by
dissolving the individual probes in various solvent systems (see ESI). Under illumination at 365
nm, the solutions of probe 1 showed different colors depending on solvent polarity (Fig. S4).
Among the various solvents, the green color was dominant when water was used as a solvent,
indicating that the probe tends to form a pyrene-pyrene stacking conformation (i.e., pyrene
excimer) in the aqueous system. The dominant presence of pyrene excimer over monomer in
water was further supported by the green and blue emission intensities measured at 488 and
388 nm, represented by I488 and I388, respectively (Figs. S4b-c).
The excimer emission intensity (I485) of probe 2 was comparable to that of probe 1 (I488) in all
tested solvent systems (Fig. S5). However, the tendency to form pyrene excimer dramatically
was increased with probe 3 (Fig. S6). The excimer emission intensity (I470) of this probe was
substantially higher than those of probes 1 and 2, presumably due to the high conformational
flexibility of this probe relative to the others. This probe contains a flexible propylene spacer
13

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between pyrene and the amino acid linker instead of the direct connection of pyrene used for
probes 1 and 2. The dominant presence of pyrene excimer over monomer in water could be
clearly seen by plotting emission intensity ratio (Iexcimer/Imonomer) of the individual probes (1-3)
with a variable of solvent system (Fig. S7). For all of these probes, use of 99% water resulted in
the highest values of the emission intensity ratio, responsible for the prominent green color of
the probe solutions detected under illumination at 365 nm (Figs. S4-6). This result implies that
the solvent properties are important in determining the relative tendency to form
intramolecular π-π stacking of the two pyrene rings in these dipodal probes. In case of
monopod probe 4, in contrast, no excimer emission was observed in water (Fig. S13) as readily
expected from the presence of monopod pyrene unit. Next, we measured fluorescence spectra
with increasing probe concentration in DMSO to investigate the aggregation behavior of these
probes. The peak intensities corresponding to both pyrene monomer and excimer tended to
increase with increasing probe concentration in all probe cases, but reached individual maxima
at a certain probe concentration in a range of 5 to 10 µM. When the solvent was changed from
DMSO to water, all dipodal probes (1-3) mainly produced the pyrene excimer peak, which is
consistent with the preferable formation of pyrene-pyrene excimer in this polar solvent system
(Figs. S8-9). The large increases in the excimer peak intensity observed here suggest the AIEE
behaviors of these probes in water.
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Fig. 2. (A) Changes in solution color of probe 1 (1 µM) with various water concentrations in a
mixed water-DMSO solvent system, under 365 nm illumination and (B) corresponding changes
in the fluorescence spectra at λex = 340 nm. D and W denote DMSO and water, respectively, and
the numbers 0, 10, 20, ...., 100 indicate water percentage. (C) Bar graph of the pyrene
monomer and excimer emission at 388 and 488 nm, respectively, and (D) FE-SEM and TEM
images of the aggregates formed by probe 1. Probe 1 solutions were prepared from different
water content in DMSO.
We next investigated solvent composition effect on the photo-physical properties of the
individual probes in the binary system of water and DMSO. The UV-visible absorption spectra of
the dipodal probes (1-3) became abruptly broader at a water concentration greater than 40%,
15

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along with the appearance of leveling-off spectral tails for wavelengths higher than 380 nm (Fig.
S1). This result suggests the formation of large probe aggregates at this range of water
concentration (> 40%) in DMSO. A similar behavior was observed for monopod probe 4, but the
spectral features indicating the formation of large probe aggregates started to appear at a
water concentration much higher than those detected for the dipodal systems (40% vs. 80%).
The fluorescence spectra of the dipodal probes varied significantly with increasing water
content in DMSO (Fig. 2 & Figs. S10-11). For probe 1, the blue emission peak intensity (I388
)
corresponding to monomeric pyrene remained almost constant up to 30% water content, but
rapidly decreased with a further increase of water content, along with a concomitant increase
in the green emission peak intensity (I488) arising from pyrene excimer (Figs. 2A-C). This result
further supports formation of intramolecular pyrene dimer at high water content in DMSO, as
well as AIEE behavior suggested by the solvent composition-dependent absorbance study (Fig.
S1). The AIEE behavior of this probe was further supported by the analysis of probe aggregates
prepared using a set of water concentrations (0, 20, 50, and 80%) in DMSO via FE-SEM and TEM
studies (Fig. 2D). A similar dependency of aggregate size depending on water content was
found in the DLS profiles of this probe (Fig. S10). Thus, the intense pyrene excimer emission
(green color) of this probe 1 observed in water is likely ascribed to both the enhanced tendency
to form intramolecular pyrene-pyrene stacking and AIEE behavior of the probe in this solvent
system. A similar behavior was observed for probe 2/3 under the same conditions tested (Figs.
S11-12). As for probe 3, the green emission peak intensity (I470) was rather invariant with
increasing water content while the blue emission peak intensity (I380) sharply dropped starting
from 40% water in DMSO (Fig. S12). The FE-SEM image obtained from a solution of probe 3
16

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prepared using 80% water in DMSO showed the presence of large probe aggregates with 1-10
µm in size (Fig. S12D). A difference in the trend between probe 1/2 and probe 3 was also
observed when the emission intensity ratio (Iexcimer/Imonomer) was plotted with water percentage
in DMSO (Fig. S13). The emission intensity ratios of probes 1 and 2 reached individual maxima
at 60% water in DMSO in both cases, while 99% water content was necessary for probe 3 to
produce a maximum value of the intensity ratio. The monopod probe 4 produced only the
monomer emission peak, which increased substantially with increasing water percentage,
suggesting AIEE phenomenon of this probe as well (Fig. S14). The decreased fluorescence
intensity of probe 4 observed in 80% or 100% water content in DMSO is probably due to
aggregation-caused quenching (ACQ) behavior. These results are reasonably consistent with
previously reported pyrene sulfonamide-based probes containing one or two pyrene units [55-
56].
The interesting photo-physical properties prompted us to evaluate probe 1 for selective PA
detection in an aqueous medium. For this purpose, a variety of phenol derivatives (Ps), along
with trinitrotoluene (TNT) were individually added to a probe 1 solution using the probe and
phenol derivatives at 1 and 20 µM, respectively. Among TNT and various Ps, only PA was highly
effective at quenching the fluorescence emission of probe 1. The fluorescence emission of the
probe decreased more than 95% by the addition of PA, while TNT and other Ps did not show
any significant changes (Fig. 3a & Fig. S15). Because of the high selectivity toward PA, probe 1
could detect this guest molecule without interference even in the presence of individual Ps or
TNT at 20 µM (Fig. S16). In order to investigate the binding characteristics of probe 1 with PA, a
17

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fluorescence titration experiment was carried out. The stepwise addition of PA into probe 1
sharply quenched the emission of the probe, with more than 90% emission quenched upon the
addition of 2 equivalents of PA (Fig. 3b and Fig. S17). The fitting of this titration data using a 1:2
7
binding model gave a strong association constant between the probe and PA (K = 3.96 × 10
a
−1
M ) (Table 1). Further, the 1:2 binding ratio deduced from the titration data was confirmed by
a Job’s plot showing an inflection point at 0.7 mole fraction of PA (Fig. S18). The limit of
detection (LOD) of probe 1 for PA was observed to be less than 1 nM, which was obtained from
a linear relationship between the fluorescence intensity (I488) of probe 1 at 488 nm and [PA]
(
inset of Fig. 3B). The calculated LOD from the slope of this line was determined to be 0.13 pM
Fig. S19). A similar binding strength and fluorescence quenching effect was observed for probe
(
2
or 3 with the same guest molecule (i.e., PA), but the additions of 2,4-DNP and 4-NP were also
somewhat effective at quenching the fluorescence emission of the respective probe (Figs. S20-
3). This result indicates that probe 2 or 3 does not possess a high selectivity toward PA
2
detection, which was observed for probe 1. All three dipodal probes (probe 1-3) showed the
same binding stoichiometry (1:2), as expected from the presence of the two pyrene units.
However, the binding strength of probe 1 toward PA was highest of the three probes (Table 1).
2,4-DNP and 4-NP were found to bind to probe 2, but their binding strengths with this probe
were roughly 10 and 100 times weaker than PA, respectively.
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Fig. 3. (a) Fluorescence quenching effect of TNT and various phenol derivatives (Ps) used at 20
µM for probe 1 (1 µM) in water. I and I represent fluorescence intensities of probe 1 before
o
and after addition of individual Ps or TNT, respectively. The inset shows the color change of
probe 1 upon addition of PA, under 365 nm illumination. (b) Fluorescence titration of probe 1 (1
µM, water) with PA. The inset shows a linear relationship between the fluorescence intensity of
probe 1 at 488 nm (I488) and [PA], λex = 340 nm. (c) FE-SEM and TEM images of probe 1 and its
PA complex, prepared from 80% water in DMSO.
Table 1. Association constants (K ) of probes 1-4 (1 µM, water) with three nitro-functionalized
a
phenols (PA, 2,4-DNP, and 4-NP).
−
1
Association constant, K (M )
a
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1
2
3
4
7
7
6
5
5
4
PA
3
.96 × 10 3.08 × 10 9.44 × 10 1.61 × 10
6
5
2,4-DNP
-----
-----
1
.50 × 10 3.01 × 10 1.59 × 10
5
4
4-NP
1
.25 × 10 9.56 x 10 1.89 × 10
The binding strengths of these nitro-phenol derivatives (2,4-DNP and 4-NP) were further
decreased when tested with probe 3 (Table 1), probably due to an increased flexibility in probe
conformation. These results indicate that probes 2 and 3 are inferior to probe 1 in terms of
selective binding to PA. The association strength was further decreased for monopod probe 4
(Table 1), without significant differentiation between PA and 2,4-DNP/4-NP (Figs. S24-25). We
performed FE-SEM/TEM analysis on [probe 1 + PA (2 equiv.)] prepared using 80% water in
DMSO (Fig. 3C). FE-SEM/TEM images showed a decrease in aggregate size from 10 µm to 500
nm upon complexation of the probe with PA, probably due to the increased polarity of the
[
probe 1 + PA] complex relative to that of the probe alone. Such a large polarity increase
induced partial de-aggregation of the aggregates in an aqueous solution. A similar behavior was
also observed for probe 3 complexed with PA (Fig. S22).
The quenching effect of PA on the fluorescence emission of each probe was further
investigated by plotting the fluorescence intensity ratio (I /I) as a function of PA concentration
o
(Stern-Volmer plot) (Fig. S26). Stern-Volmer constants (Ksv) were calculated using the Stern-
Volmer equation (I /I = 1 + K [Q]). With PA, Stern-Volmer constants of the dipodal probes (1-3)
o
sv
6
6
6
-1
were higher (3.46 x 10 , 2.94 x 10 , and 1.86 x 10 M , respectively) than that of the monopod
20

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5
-1
probe 4 (6.54 x 10 M ) (Fig. S26B), indicating that the fluorescence emissions of the dipodal
probes are more effectively quenched by PA than is that of monopod probe, probably due to
the strong binding of individual dipodal probes with this guest molecule.
3.3. Binding studies of probes with PA
1
The molecular interactions between probe 1 and PA were further investigated by the H NMR
spectra of probe 1 collected in the absence or presence of PA. First, the individual NMR peaks
were assigned to the protons of the probe to determine which region of the probe interacts
with PA. For this peak assignment, we used the coupling constants and proton-proton
1
1
1
correlations obtained from the 1D and 2D H NMR spectra ( H- H COSY & NOESY) of probe 1 in
DMSO-d , respectively (Fig. 4A, Figs. S27-28). In the NOESY spectrum, the highly downfield
6
shifted NH proton of ArNHCO, designated the z-proton, was correlated with the d-proton of the
phenyl ring (Fig. S28). Both COSY and NOESY spectra also showed a strong correlation between
c- and d-protons and between a- and b-protons, indicating that these pairs of protons are
neighbors. This result was further supported by the same coupling constants estimated for the
1
corresponding H NMR peaks. We also found that the coupling constants for x- and y-proton
peaks were the same, and these two protons were correlated with each other in the 2D NMR
spectra (Figs. S27-28).
21

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1
1
1
Fig. 4. (A) H- H COSY (2D NMR) spectra of probe 1 in DMSO-d . (B) H NMR spectra of probe 1
6
(i) before addition of PA, (ii) upon addition of 1 equiv., (iii) 2 equiv., (iv) 3 equiv. of PA, and (v)
PA alone in DMSO-d :D O (4:1).
6
2
With this NMR peak assignment, we conducted an NMR titration of probe 1 with PA. No
substantial changes in chemical shifts were detected for the individual signals of the probe or
PA when the sample solution was made from DMSO-d
observed for probe 2/3 (Figs. S30-31). When the same experiment was conducted with probe 1
dissolved in (DMSO-d :D O) (4:1) instead of DMSO-d , however, noticeable upfield shifts for the
6
(Fig. S29). A similar behavior was
6
2
6
proton signals were observed by PA addition (Fig. 4B & Fig. S32). The changes in chemical shift
were increased with increasing amount of PA and were maximized by the addition of 2
equivalents of PA. A further addition of PA did not induce additional changes in chemical shift,
further supporting the 1:2 binding stoichiometry between the probe and PA. This result
22

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indicates that, upon addition of PA, probe 1 undergoes a large conformational change due to
the strong association between the pyrene ring of the probe and PA in the (DMSO-d :D O) (4:1)
6
2
system, which is responsible for the sensitive quenching effect of the guest molecule on the
fluorescence emission of the probe.
3.4. Theoretical calculation of probes 1- 4 with PA
Energy-minimized structures of the individual probes (1-4) complexed with PA were obtained
by DFT calculations at the B3LYP/6-31G* level. As can be seen in Fig. 5, the optimized structure
of each dipodal probe (1-3) alone showed intramolecular pyrene-pyrene interaction at a
distance of 3.86 - 4.19 Å, comparable to a previous report [57-58]. In the presence of PA, these
probes were calculated to show the strong interactions between the pyrene ring and PA at a
distance of 3.53 - 3.67 Å (Fig. 5). These new π–π interactions make it possible for efficient
energy transfer from the electron-rich pyrene ring to an electron-deficient PA upon excitation
at 340 nm, leading to the quenching of fluorescence emission. The energy-minimized structure
of the [probe 4 + PA] complex indicates the formation of pyrene- PA complex at a distance of
3.67 Å, resulting in fluorescence quenching of the probe (Fig. 5).
23

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Fig. 5. Energy-minimized structures of individual probes (probe 1-4) in the absence or presence
of PA were obtained from DFT calculations at the B3LYP/6-31G* level. Each pair of parallel
dotted lines in red indicates π–π interaction between two aromatic rings (pyrene-pyrene or
pyrene- PA). The dotted lines in blue indicate the presence of intramolecular hydrogen bonding
between two amide groups within the probe or between the amide group of the probe and the
nitro group of PA.
DFT calculations on frontier molecular orbitals (FMOs) showed that the HOMOs and LUMOs of
these probes (probe 1, 2, 3, and 4) are mainly localized on one of the pyrene rings of the probes
(Figs. S33-36). Upon complexation with PA, their HOMO locations did not change, while their
LOMO locations were changed from the pyrene ring to PA. This implies energy transfer
between these two π electron systems upon excitation, resulting in fluorescence emission
quenching for the probe- PA complexes. The energy gaps (−ΔE) between HOMOs and LUMOs of
the respective probes were significantly reduced upon complexation with PA compared to
those of the individual probes alone (Table S1); this gap reduction was mainly due to the
24

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decrease in LUMO energy upon complexation, resulting from changes in their locations from
the pyrene ring to PA in all probe cases (Figs. S33-36).
3.5. Mechanism of probe- PA interactions
The fluorescence quenching of individual probes in the presence of PA was further investigated
by finding the spectral overlaps between the absorption spectrum of PA and the fluorescence
emission spectra of probes 1-4. The presence of significant spectral overlap suggests that the
fluorescence emission of the respective probe can be efficiently absorbed by nearby PA
molecules (Fig. S37). This is similar to Förster resonance energy transfer (FRET) between two
fluorophores. Fluorescence emission can be quenched via one of three processes: a static,
dynamic, or combined process. The Stern-Volmer plots obtained from the dipodal probe- PA
systems, particularly for probe 1, tend to show a non-linear relationship between intensity ratio
o
(I /I) and [PA], suggesting that both static and dynamic quenching processes occur
simultaneously (Fig. S26A). Excited state lifetime (τ) measured for [probe 1/3 + PA] system
further supported the involvement of a dynamic process in fluorescence quenching of the
system. This lifetime significantly decreased from τ int = 40.85 to 28.69 ns for probe 1 and from τ
int = 54.10 to 33.04 ns for probe 3, after the addition of 2.0 equivalents of PA (Fig. 6).
25

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Fig. 6. Fluorescence lifetime decay profile of (A) probe 1 and (B) probe 3 alone or in the
presence of PA in water. The insets show changes in the ratios of excited state lifetime (τ /τ) of
0
0
both probes with increasing PA concentration. τ and τ represent excited state lifetimes of the
probe in the absence and presence of the guest molecule, respectively.
The non-linearity of the Stern-Volmer plot and the substantial decrease in excited state lifetime
observed for probe 1/3 with PA suggest that both dynamic and static processes occur together,
yet dynamic process is likely dominant in the fluorescence quenching. The dominant role of
dynamic process in fluorescence quenching was further supported by temperature-dependent
fluorescence studies of these probes. In the presence of 2 equivalent of PA, fluorescence
intensity of probe 1 or 3 was steadily decreased with increasing temperature from 20 °C to 40
°C (Fig. S38).
3.6. Application of probe 1 as a PA sensor
The utility of probe 1 was further demonstrated even in real samples such as HEPES buffer (pH
7.4), lake water and sea water for PA detection. By utilizing the linear relationship between
=
26

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I488 and [PA], the LOD values were observed to be 1 nM (Fig. S39) in all different solvent
samples systems including water. The use of water or HEPES buffer gave a little more sensitive
decrease in fluorescence intensity than that of lake or sea water with increasing PA
concentration.
Fig. 7. (a) Changes in the fluorescence spectrum of a probe 1-coated filter paper strip (10 µM)
with time after exposure to saturated PA vapor, and (b) respecitve point diagram using
fluorescence eintensity at 476 nm (I476). (c-f) Fluorescence quenching behaviors of PA for
written letters ‘HYU’ (c,d) or dropped spots (e,f) on a TLC plate. The letters and spots were
prepared using a probe 1 solution (10 µM, EtOH:H O (1:9)). (g) Changes in fluorescence
2
intensity of the letters ‘HYU’ monitored at regular intervals during a 30-min exposure to
saturated PA vapor, under 365 nm irradiation.
To develop an inexpensive and convenient tool for PA detection, dry test strips of filter paper
were prepared using probe 1 solution (10 μM, EtOH:H O (1:9)). The probe 1-coated filter paper
2
strip showed a sharp decrease in fluorescence intensity with time after exposure to saturated
27

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PA vapor (Figs. 7a,b). A silica-coated test strip with the letters ‘HYU’ was prepared using probe 1
solution (10 μM, EtOH:H O (1:9)). The clear green emission of these letters was significantly
2
reduced after dipping the plate into a 1 µM PA solution prepared using tap water (Figs. 7c,d).
The detection limit of this system was estimated by preparing a TLC plate with four probe spots
and having these individual spots contacting with water or a PA solution at 1, 10, or 100 nM (Fig.
7e,f). A notable quenching of the green emission was observed by a naked-eye even with a PA
concentration as low as 1 nM. To further explore the utility of probe 1 for PA vapor detection,
another silica-coated test strip with the letters ‘HYU’ was prepared and exposed to the
saturated PA vapor for 30 min. The fluorescence intensity of the letters was gradually
decreased with time, reaching significant fluorescence quenching after 30 min (Fig. 7g). This
result indicates that probe 1 can be effectively used to detect PA even in a vapor state. This
demonstrates the potential utility of this probe in real time-sensing applications. In addition,
the repeatability of probe 1 towards PA sensing was supported by multiple fluorescence
titration experiments conducted at two and five months after the first experiment with almost
7
−1
similar association constant (K = 3.91 × 10 M ) (Fig. S40).
a
Finally, probe 1 was compared with previously reported probes for the PA detection in terms of
LOD (Table S2). The calculated LOD of probe 1 for PA detection (i.e., 0.13 pM) is significantly
lower than those of most of the reported probes. Therefore, probe 1 is one of the best probes
for PA detection in an aqueous solution..
4. Conclusions
28