Colorimetric analysis of malononitrile via the formation of a novel NBD-based CH-acidic dye

Article abstract of DOI:10.1016/j.dyepig.2019.108034

Malononitrile is a key starting material used in synthetic organic, medical, and industrial chemistry, but it is also a notorious cyanogenic toxicant. However, selective and sensitive analytical methods for this key chemical are rare. In this research, a

Full text of DOI:10.1016/j.dyepig.2019.108034

Journal Pre-proof
Colorimetric analysis of malononitrile via the formation of a novel NBD-based CH-
acidic dye
Na Yeong Kim, Myung Gil Choi, Kang Min Lee, Sungguan Hong, Suk-Kyu Chang
PII:
S0143-7208(19)31407-X
DOI:
https://doi.org/10.1016/j.dyepig.2019.108034
DYPI 108034
Reference:
To appear in: Dyes and Pigments
Received Date: 17 June 2019
Revised Date: 6 August 2019
Accepted Date: 7 November 2019
Please cite this article as: Kim NY, Choi MG, Lee KM, Hong S, Chang S-K, Colorimetric analysis of
malononitrile via the formation of a novel NBD-based CH-acidic dye, Dyes and Pigments (2019), doi:
https://doi.org/10.1016/j.dyepig.2019.108034.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.

Graphical Abstract

Colorimetric analysis of malononitrile via the formation of a novel
NBD-based CH-acidic dye
1
2
3
4
Na Yeong Kim, Myung Gil Choi, Kang Min Lee, Sungguan Hong,* and Suk-Kyu Chang*
5
Department of Chemistry, Chung-Ang University, Seoul 06974, Republic of Korea
6
7
*Corresponding author. Tel.: +82 2 820 5199; Fax: +82 2 825 4736.
8
E-mail address: sungguanhong@cau.ac.kr (S. Hong); skchang@cau.ac.kr (S.-K. Chang)
9
10
11
12
13
14
15
16
17
18
Abstract:
Malononitrile is a key starting material used in synthetic organic, medical, and
industrial chemistry, but it is also a notorious cyanogenic toxicant. However,
selective and sensitive analytical methods for this key chemical are rare. In this
research, a novel reaction-based colorimetric signaling probe for the selective
analysis of malononitrile via the SNAr-type nucleophilic substitution reaction of an
NBD-based phenyl selenoether was investigated. The probe exhibited a pronounced
colorimetric response, with a color change from yellow to violet due to its CH-
acidity. The interference of sulfide ions and several transition metal ions was
1

19
20
21
22
23
24
25
26
27
effectively circumvented with the use of copper foil as a sulfide scavenger and
EDTA as a masking agent for the metal ions. The detection limit of the probe for
malononitrile was found to be 264 nM. It was also demonstrated that this convenient
colorimetric analysis of malononitrile in chemical and industrial applications using
the probe can be conducted with an office scanner.
Keywords: Malononitrile sensing; 4-Nitrobenzoxadiazole; Colorimetric signaling;
CH-acidic dye; Masking agent; Office scanner.
28
1. Introduction
29
30
31
32
33
34
35
Malononitrile is a versatile compound with exceptional reactivity that acts as a key
starting material for a number of synthetic organic, medical, and industrial chemistry
applications [1]. For example, it is frequently used in the synthesis of various
heterocyclics, pharmaceuticals, pesticides, fungicides, and charge-transfer salts [2].
It is also used in the manufacture of lubricating oil additives [3], dyes and acrylic
fibers [4], thiamine and anti-cancer agents [5], 2-chlorobenzalmalononitrile (CS-gas,
also known as tear gas), which is used for self-defense purposes [6], and as a
2

36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
leaching agent for gold [7]. In analytical chemistry, malononitrile has been used in
the detection of 1,2-quinones in color tests via paper chromatography and spot plates
[8,9], the spectrophotometric determination of vitamin K3 [10], and the analysis of
sialic acids using postcolumn fluorescence labeling in high-performance liquid
chromatography [11].
Despite these practical uses, malononitrile is a notorious cyanogenic toxicant [3]
that is characterized by a number of serious symptoms, such as irritation of the eyes
and skin, headaches, dizziness, convulsions, dyspnea, nausea, and vomiting [12].
Therefore, particular care should be taken in handling malononitrile because it
exhibits extensive chemical reactivity and has a low LD50 (66 mg/kg) [13]. As such,
the National Institute for Occupational Safety and Health (NIOSH) has established a
recommended exposure limit (REL) of 3 ppm (8 mg/m3) as an 8-hour time-weighted
average [14]. Therefore, the development of a selective and sensitive analytical
method for the detection of malononitrile is very important not only for the health of
workers in various industrial activities but also for analytical assays involving this
key reagent.
Despite this need, only a few studies have analyzed malononitrile and its derivatives
from this perspective (Table S1, Supplementary Data). Malononitrile has primarily
3

54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
been analyzed using gas chromatography [15] and gas chromatography−mass
spectrometry [16]. Polarographic analysis based on its electroactivity compared to
the closely related compounds cyanoacetic acid, malonic acid, and succinic acid
dinitrile has also been reported [17]. In addition, due to their greater convenience,
several colorimetric detection methods have been reported. For instance,
malononitrile and its derivatives have been detected and quantified according to their
reaction with benzofuran oxide in an alkaline medium, which produces an intense
violet color [18]. In addition, the reaction of malononitrile with 4-(4′-
nitrobenzyl)pyridine leads to the generation of a yellow color [19], while it also
reacts with conventional coloring reagents (e.g., diazotized p-nitroaniline) to produce
a blue color [20]. Colorimetric spot tests using nitroprusside (Fe(CN)5NO2) [21] and
the 2,4-dinitrobenzenediazonium coupling reaction [22] have also been reported.
However, a more selective and sensitive analytical method is required because most
of these colorimetric methods, except the benzofuran oxide method, essentially use a
qualitative approach involving TLC visualization or spot testing and thus cannot
provide practical and analytically useful quantitative information.
For this reason, we designed a new colorimetric reaction-based probe for the
selective and sensitive signaling of malononitrile using the nucleophilic aromatic
4

72
73
74
75
76
77
78
79
80
81
82
substitution (SNAr) reaction with a selenoether derivative of 7-nitrobenz-2-oxa-1,3-
diazole (NBD) dye. Recently, selective colorimetric probes for hydrogen sulfide
based on the SNAr-type reaction of NBD thioethers through the thiol extrusion and
formation of a uniquely colored nitrobenzofurazan-thiol (λmax = 534 nm) have been
reported [23–26]. Following these reports, a variety of NBD derivatives based on
amines [27], ethers [28], and thioethers [29] with several dye subunits, such as
rhodamine and fluorescein, have been designed through the thiolysis-type cleavage
of C–N, C–O, and C–S bonds to detect hydrogen sulfide and various biothiols,
including cysteine (Cys) and reduced glutathione (GSH); these have been
summarized in a recent review [30].
83
2. Materials and methods
84
85
86
87
88
2.1 General. Malononitrile, 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl),
phenol, thiophenol, benzeneselenol, and diphenyl diselenide were purchased from
Merck KGaA. Other chemicals and solvents were obtained from commercial sources
1
at spectroscopic grade. H (300 MHz and 600 MHz) and 13C (150 MHz) NMR
spectra were measured on Varian Gemini 2000 and VNS NMR spectrometers, using
5

89
90
residual solvent signals as a reference. UV–vis spectra were recorded using a Scinco
S-3100 spectrophotometer. Fluorescence spectra were measured with a Scinco
FluoroMate FS-2 fluorescence spectrophotometer. Mass spectra were acquired using
a Micromass Autospec mass spectrometer.
91
92
93
2.2 Preparation of malononitrile-selective NP-O, NP-S, and NP-Se probes. NBD-
based malononitrile-selective probes (NP-O, NP-S, and NP-Se) were prepared
according to a previously reported procedure [31]. Phenol (0.28 g, 3.0 mmol, for NP-
O), thiophenol (0.33 g, 3.0 mmol, for NP-S), or benzeneselenol (0.47 g, 3.0 mmol,
for NP-Se) was dissolved in ethanol (20 mL), and triethylamine (0.42 mL, 3.0 mmol)
was added to the solution. A solution of NBD-Cl (0.20 g, 1.0 mmol) in 5 mL of
ethanol was slowly added to the reaction mixture and stirred at room temperature
overnight. The reaction mixture was diluted with distilled water and extracted with
dichloromethane. The organic phase was separated, washed with water several times,
and then evaporated. The residue was purified using short-column chromatography
94
95
96
97
98
99
100
101
102
103
104
105
106
1
(silica gel, dichloromethane). NP-O: 65%. Yellow crystal. H NMR (600 MHz,
DMSO-d6) δ 8.64 (d, J = 8.4 Hz, 1H), 7.61 (dd, J = 8.5, 7.3 Hz, 2H), 7.48–7.40 (m,
3H), 6.69 (d, J = 8.4 Hz, 1H); 13C (150 MHz, DMSO-d6) δ 153.7, 153.3, 145.8, 144.8,
136.0, 131.3, 130.6, 127.4, 121.3, 109.9; LRMS (EI+); m/z calcd for C12H8N3O4
6

1
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
[M+H]+: 258.0, found 258.1. NP-S: 69%. Yellow crystal. H NMR (600 MHz,
DMSO-d6) δ 8.51 (d, J = 8.0 Hz, 1H), 7.79–7.74 (m, 2H), 7.71–7.63 (m, 3H), 6.83 (d,
J = 8.0 Hz, 1H); 13C (150 MHz, DMSO-d6) δ 149.0, 143.3, 140.4, 135.7, 133.3,
133.0, 131.6, 131.2, 126.4, 123.8; LRMS (EI+); m/z calcd for C12H8N3O3S [M+H]+:
1
274.1, found 274.0. NP-Se: 72%. Amber crystal. H NMR (600 MHz, DMSO-d6) δ
8.45 (d, J = 7.8 Hz, 1H), 7.83–7.78 (m, 2H), 7.67–7.56 (m, 3H), 7.03 (d, J = 7.8 Hz,
13
1H); C (150 MHz, DMSO-d6) δ 150.2, 143.0, 136.9, 134.2, 132.5, 131.05, 131.04,
127.7, 124.2; LRMS (EI+); m/z calcd for C12H8N3O3Se [M+H]+: 321.8, found 321.9.
2.3 Preparation of NBD malononitrile derivative 1. Signaling product 1 was
independently prepared via the reaction of NBC-Cl with malononitrile in ethanol.
NBD-Cl (0.20 g, 1.0 mmol) was dissolved in ethanol (10 mL), and sodium carbonate
(0.11 g, 1.0 mmol) was added to the solution. Malononitrile (0.07 g, 1.0 mmol) was
added to the reaction mixture, and the solution was stirred at 50 °C. After stirring for
3 h, the solvent was removed under reduced pressure. Acetone (10 mL) was added to
the residue, and the precipitate was filtered and dried in a vacuum oven. 1: 95%.
1
Dark violet crystal. H NMR (600 MHz, DMSO-d6) δ 8.19 (d, J = 9.0 Hz, 1H), 6.49
13
(d, J = 9.0 Hz, 1H); C (150 MHz, DMSO-d6) δ 146.8, 144.7, 142.6, 134.3, 119.9,
7

–
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
118.8, 118.6, 108.6, 51.1; HRMS (ESI–); m/z calcd for C9H2N5O3 [M–H]–: 228.0163,
found 228.0159.
2.4 Preparation of stock solutions. Stock solutions (5.0 × 10–4 M) of NP-O, NP-S,
and NP-Se were prepared in spectroscopic-grade DMSO, and a stock solution of
malononitrile (1.0 × 10–2 M) was prepared in distilled water. Other stock solutions of
metal ions and anions (1.0 × 10–2 M) were prepared in distilled water using the
perchlorate and sodium salts of metal ions and anions, respectively. A solution of
ethylenediaminetetraacetic acid (EDTA, 1.0 × 10–1 M) was also prepared in
deionized water.
2.5 Measurement of signaling behavior. All measurements were carried out under
optimal conditions with a 10% aqueous DMSO solution buffered with tris buffer (pH
9.0). The solutions used to measure malononitrile signaling were prepared by mixing
a stock solution of the probe (NP-O, NP-S, or NP-Se; 60 µL, 5.0 × 10–4 M),
malononitrile or the tested metal ions and anions (30 µL, 1.0 × 10–2 M), and tris
buffer solution (150 µL, pH 9.0, 0.20 M) in a vial. The sample solution was diluted
using deionized water and DMSO to give a final volume of 3.0 mL with a 1:9 (v/v)
ratio of water to DMSO. The final concentrations of the probe, analyte (malononitrile
8

141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
or metal ions/anions), and buffer in the solution were 1.0 × 10–5 M, 1.0 × 10–4 M, and
1.0 × 10–2 M, respectively.
2.6 Suppression of sulfide and transition metal ion interference.
A. Suppression of sulfide interference. To prevent sulfide interference, copper foil
(battery electrode grade) was used. A square piece of copper foil (10.0 mm × 10.0
mm, 18 mg) was fully soaked in the prepared sample solution for 1 min and was then
removed. The NP-Se probe was added to the solution treated with copper foil, and
changes in the UV–vis spectrum or green channel of the sample solution were
measured.
B. Suppression of transition metal ion interference. The interference of transition
metal ions in malononitrile signaling was successfully prevented using an EDTA as a
masking agent. After the signaling was complete, 15.0 µL of EDTA solution (1.0 ×
10–1 M) was added to the solution, and changes in the UV–vis spectrum or green
channel of the sample solution were measured.
2.7 Determination of the detection limit. The detection limit for malononitrile was
calculated according to IUPAC recommendations using the equation 3sbl/m, where sbl
is the standard deviation of the blank responses (number of measurements = 15), and
m is the slope of the obtained titration curve [32].
9

159
160
161
162
163
2.8 Calculation of pKa for CH-acidic dye 1. To calculate the acid dissociation
constant (Ka) for CH-acidic dye 1, changes in the absorbance at 577 nm of CH-acid 1
as a function of the solution pH in water were measured. The pH of the sample
solution was adjusted using HCl solution. From the obtained pH-dependent plot, the
pKa of CH-acid 1 was calculated using the Henderson-Hasselbalch equation [33]:
–
ꢃ ꢄ
A
pꢀꢁ = pH – log ꢂ
ꢅ
ꢃ
ꢄ
HA
164
165
166
167
168
169
170
171
172
173
174
175
2.9 Evidence for the signaling process. A deuterated phosphate buffer solution
(0.10 mL, pH 7.0) containing malononitrile (6.6 mg, 0.10 mmol) was added to a
solution of NP-Se (3.2 mg, 0.010 mmol) in 0.90 mL of DMSO-d6. After mixing, the
1H NMR spectrum of the mixture (NP-Se + malononitrile) was obtained. As a
1
reference, the H NMR spectra of independently prepared compound 1 and diphenyl
diselenide were acquired in the same deuterated phosphate-buffered DMSO-d6 (1:9,
v/v).
2.10 Colorimetric analysis using an office scanner [34]. The red, green, and blue
channel values of the signaling solutions were recorded using an office scanner
(Perfection V550, Epson Inc.). To obtain images of the samples, solutions with
varying concentrations of malononitrile were treated with the probe, and 0.30 mL of
the solution was placed in a 96-well plate. Images of the plate were captured using a
10

176
177
178
179
180
181
transmittance-type scanner (resolution: 1200; image type: 24-bit color). The color
channels of the scanned images were estimated using a color analysis program
(Adobe, Photoshop CS6). Because the change in the green channel levels was more
pronounced than the red or blue channels, the ∆Green value (255 – green channel
level) was used when plotting the results.
182
3. Results and discussion
183
184
185
186
187
188
189
190
191
192
A malononitrile-selective probe was designed based on the nucleophilic substitution
capability of malononitrile on NBD phenyl ether derivatives. We expected that the
reaction of a reactive malononitrile anion at the ether-substituted C-4 atom of the
malononitrile moiety of the probe would result in the formation of a novel CH-acidic
dye. The produced compound exhibited pronounced chromogenic behavior owing to
the deprotonation of the methine proton of the malononitrile moiety and to the
extended cross-conjugation of the anionic species through the nitro group of the
NBD moiety. To achieve this, three candidate NBD-based compounds containing
ether, thioether, and selenoether functions (NP-O, NP-S, and NP-Se) were prepared
via the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with phenol, thiophenol,
11

193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
and benzeneselenol, respectively (Scheme 1). Preliminary research demonstrated that
all three compounds readily reacted with malononitrile to generate intensely violet-
o
colored solutions within 1 h at 20 C. However, the reaction speed of the probes with
malononitrile differed, decreasing in the order of NP-O > NP-Se > NP-S (Fig. S1,
Supplementary Data). The ether derivative NP-O exhibited the most favorable
signaling speed; however, the stability of the probe in the measurement solution was
not satisfactory. The nearly colorless NP-O solution progressively became more
yellow due to the formation of a hydroxylated NBD derivative (7-nitro-2,1,3-
benzoxadiazol-4-ol) [35] via a hydrolysis reaction (Fig. S2, Supplementary Data).
The thioether NP-S also showed a similar undesirable response but to a lesser extent.
On the other hand, the selenoether NP-Se showed relatively good stability, remaining
stable for up to 12 h after preparation. After further optimization, NP-Se was
selected as the probe for the sensing of malononitrile because it demonstrated
satisfactory selectivity, reaction speed, and probe stability under the target
measurement conditions.
12

209
210
Scheme 1. Preparation of NBD-based malononitrile signaling probes (NP-O, NP-S,
and NP-Se).
211
212
213
214
215
216
217
218
219
220
221
222
223
224
The NP-Se probe solution was lightly yellow-colored and exhibited a UV–vis
absorption maximum at 434 nm in tris-buffered DMSO (pH = 9.0, 1:9, v/v). The
mixing of the NP-Se solution with malononitrile resulted in the development of a
violet-colored solution. In addition, a 250-fold increase in absorbance (A/A0) at 577
nm was measured, while there were no discernible changes in the absorption spectra
in the presence of other tested common metal ions (Fig. 1). However, of the tested
anions, significant responses to sulfide ions were observed (Fig. S3, Supplementary
Data). Sulfide-induced interference was not unexpected because the NBD phenyl
selenoether derivative has previously been used as a colorimetric sulfide-signaling
probe [23–26]. We attempted to suppress sulfide interference to allow exclusive
malononitrile-selective signaling using several transition metal ions, metal powders,
and metal foils. Subsequently, it was found that, in the presence of copper foil
13

225
226
227
228
229
230
231
232
233
234
(battery electrode grade), sulfide ion interference was completely suppressed without
significantly affecting malononitrile signaling (Fig. S4, Supplementary Data). The
sulfide ions were found to be removed from the solution via deposition on the
surface of the copper foil, which was confirmed by energy-dispersive X-ray analysis
(Fig. S5, Supplementary Data). In fact, copper or copper naphthenate has been used
as a sulfide scavenger for corrosion prevention via the deactivation of corrosive
sulfur [36]. In contrast to this prominent colorimetric response, no practically useful
changes in the fluorescence of the NP-Se probe were observed for any of the tested
species (Fig. S6, Supplementary Data).
300
0.6
250
+ Malononitrile
NP-Se
0.4
0.2
0
200
150
100
50
only
+ Mn+
NP-Se
NP-Se
350
400
450 500
550
600
Wavelength (nm)
0
235
14

236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
Fig. 1. Selective signaling of malononitrile by NP-Se compared to common metal
ions as expressed by the absorbance ratio (A/A0) at 577 nm. Inset: Changes in the
absorption spectra of the probe in the presence of malononitrile or the metal ions
shown in the main graph. [NP-Se] = 1.0 × 10–5 M, and [Malononitrile] = [Mn+] = 1.0
× 10–4 M in a mixture of tris buffer (pH = 9.0, 0.10 M) and DMSO (1:9, v/v). MAL =
malononitrile.
The prominent colorimetric response of the NP-Se probe is due to the SNAr-type
reaction with malononitrile to form the strongly colored CH-acidic dye 1 (Scheme 2).
The postulated conversion of NP-Se to dye 1 via the well-established nucleophilicity
of malononitrile anions was supported by NMR and mass spectroscopy. As shown in
Fig. 2, in the presence of 1 equiv of malononitrile, the 1H NMR resonance ascribable
to NP-Se disappeared completely, and only new resonances for compound 1 and
diphenyl diselenide were observed. The nucleophilic substitution of the NP-Se probe
with malononitrile produced dye 1 and benzeneselenol, which was subsequently
oxidized by the DMSO in the signaling solution to diphenyl diselenide as shown in
Scheme 2. It is known that DMSO works well as a mild oxidant in many organic
synthetic applications [37,38]. The postulated conversion was corroborated by the
15

1
254
255
256
257
258
259
260
observation that the H NMR spectra of benzeneselenol and diphenyl diselenide in
deuterated phosphate-buffered DMSO-d6, as well as the mass spectra of these
compounds in DMSO solution, were nearly identical (Figs. S7 and S8,
Supplementary Data). In addition, the mass spectrum of the purified reaction product
also confirmed the postulated structure of compound 1 by revealing a peak at m/z =
228.0159 (calcd. for C9H2N5O3 [M–H]– = 228.0163; Fig. S9, Supplementary Data).
–
261
262
Scheme 2. Colorimetric signaling of malononitrile using the NP-Se probe.
263
16

264
265
Fig 2. Partial 1H NMR spectra of NP-Se in the presence and absence of malononitrile,
reference 1, and diphenyl diselenide in a mixture of deuterated phosphate buffer (pH
7.0) and DMSO-d6 (1:9, v/v). [NP-Se] = [1] = [Diphenyl diselenide] = 1.0 × 10–2 M.
The 1H NMR spectrum of NP-Se + malononitrile was obtained by mixing NP-Se (10
mM) and malononitrile (10 mM) without further purification.
266
267
268
269
270
271
272
273
274
275
276
The violet color of the signaling product is due to the strong CH acidity of the
methine proton in signaling product 1, which is ascribable to the presence of efficient
electron-withdrawing substituents of an NBD moiety and two cyano groups.
Malononitrile itself is an inherently CH-acidic compound with a pKa of 11.2 [6]. The
presence of the NBD moiety, which has strong electron-withdrawing and resonance-
stabilizing properties, on the malononitrile subunit would further enhance the acidity
17

277
278
279
280
281
of the resulting compound [39]. The effective stabilization of anionic structure 1 can
be demonstrated using resonance structures (Scheme 2). The acid dissociation
constant (pKa) of the resulting CH-acid 1 was estimated using UV–vis spectroscopy
with the Henderson–Hasselbalch equation and found to be –0.83 (Fig. 3).
0.5
pKa = – 0.83
0.4
0.3
0.2
0.1
0
-1.1
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
pH
282
283
Fig 3. Changes in the absorbance at 577 nm of CH-acid dye 1 as a function of
solution pH in water. [1] = 1.0 × 10–5 M in distilled water. The pH of the solution
was adjusted with HCl solution.
284
285
286
287
288
Based on these promising results, we carried out further experiments to
characterize the colorimetric signaling properties of NP-Se with regards to
18

289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
malononitrile in detail. To confirm possible interference from other commonly
encountered ionic species, the effect of common metal ions and anions on
malononitrile signaling was investigated. We found that some metal ions,
particularly the transition metal ions Co2+, Ni2+, Cu2+, Zn2+, and Pb2+, led to an
undesirable negative deviation in absorbance, possibly due to the interaction with the
phenolate anion and the adjacent nitrogen ligand of the oxadiazole moiety of
signaling product 1 (Fig. S10, Supplementary Data). Therefore, to expand the
applicability of the probe for use in practical applications where these interfering
ions might be present, we attempted to suppress unwanted changes using a chelating
agent, which could sequester the interfering transition metal ions. To achieve this,
after the signaling had been completed, EDTA solution (5 equiv for the analyte) was
added to the signaling mixture. As a result, most of the interference from the
transition metal ions was found to be effectively suppressed; the absorbance ratio
AMetal+MAL/AMAL observed at 577 nm varied between 0.96 and 1.12 (Fig. 4). On the
other hand, for anions, no measurable interference was observed, with a nearly
constant AAnion+MAL/AMAL ratio (Fig. S11, Supplementary Data).
19

with EDTA treatment
1
0.8
0.6
0.4
0.2
0
+ Malononitrile
306
307
Fig. 4. Competitive signaling of malononitrile by NP-Se in the presence of common
metal ions in the background expressed by the absorbance ratio (AMetal+MAL/AMAL) at
577 nm. [NP-Se] = 1.0 × 10–5 M, and [Malononitrile] = [Mn+] = 1.0 × 10–4 M,
[EDTA] = 5.0 × 10–4 M in a mixture of tris buffer (pH = 9.0, 0.10 M) and DMSO
(1:9, v/v). All measurements were carried out after treatment of the signaling solution
with EDTA solution. MAL = malononitrile.
308
309
310
311
312
313
314
315
316
317
The signaling speed of NP-Se for malononitrile was relatively slow, with complete
signaling requiring about 40 min (Fig. S12, Supplementary Data). However, the
signaling process could be sped up by increasing the temperature of the solution. As
o
the temperature rose, the signaling speed progressively increased; at 20 C, a
20

318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
saturated signal was attained after about 40 min, which fell to about 20 min and 10
min at 30 oC and 40 oC, respectively.
Next, to determine the detection limit of NP-Se for malononitrile, UV–vis titration
was carried out (Fig. S13, Supplementary Data). The absorbance of NP-Se at 577 nm
steadily increased with an increase in the concentration of malononitrile, whereas the
absorbance at 434 nm simultaneously decreased. The malononitrile concentration-
dependent change in absorbance for NP-Se exhibited a linear calibration trend up to
1.0 × 10–5 M. From this plot, the detection limit of NP-Se for malononitrile was
calculated to be 3.66 × 10–7 M according to IUPAC recommendations (3sbl/m) [33].
The observed sensitivity of the probe for malononitrile detection was thus found to
be superior to other reported analytical methods (Table S1, Supplementary Data).
Finally, the rapid and convenient detection of malononitrile with a daily-use IT
device was investigated. Recently, colorimetric analysis using smartphones and
scanners that do not rely on complex and/or heavy instruments has attracted
significant research interest [40]. The proposed probe is particularly suitable for this
purpose because the colorimetric response to malononitrile is clearly evident to the
naked eye. We tested the use of an office scanner as a signal-capturing device for the
rapid and convenient analysis of malononitrile. Using the red, green, and blue
21

336
337
338
339
340
341
342
channel levels of images of the signaling solution attained using a color analysis
program (Adobe, Photoshop CS6), linear calibration curves were plotted as a
function of the concentration of malononitrile up to 2.0 × 10–5 M, with satisfactory
R2 values (0.9814 – 0.9954; Fig. S14, Supplementary Data). Based on the most
sensitive plot using the green channel, the detection limit of malononitrile measured
by the scanner was calculated to be 2.64 × 10–7 M (Fig. 5).
100
80
60
y = 3.0978x + 11.468
R2 = 0.9954
40
∆
∆
∆
∆
Detection limit:
3 x Blank STDEV (0.272517) / slope (3.0978)
= 264 nM
20
0
0
2
4
6
8
10
12
14
16
18
20
[Malononitrile] ( M)
µ
343
344
Fig. 5. Malononitrile concentration-dependent changes in ∆Green values (∆Green
value = 255 – green channel level) of NP-Se. Inset: photographic images of the
signaling solutions in the presence of varying concentrations of malononitrile. [NP-
Se] = 1.0 × 10–5 M, and [Malononitrile] = 0 – 1.0 × 10–4 M in a mixture of tris buffer
345
346
347
22

348
349
350
351
352
353
354
(pH = 9.0, 0.10 M) and DMSO (1:9, v/v). The inset images were obtained using a
transmittance-type office scanner, and the error bars were estimated from three
independent experiments.
355
4. Conclusions
356
357
358
359
360
361
362
363
In this study, we developed a novel reaction-based colorimetric probe for the
synthetically important chemical malononitrile. The selenoether-based probe
demonstrated a marked change in color from yellow to violet via an SNAr-type
reaction with malononitrile to form a novel CH-acidic dye. Interference from sulfide
ions and some transition metal ions was readily suppressed using copper foil and
post-treatment with the chelating agent EDTA, respectively. By elevating the
temperature of the signaling solution, the signaling speed was noticeably enhanced.
Based on colorimetric behavior of the probe, which can be readily detected by the
23

364
365
366
naked eye, malononitrile monitoring and analysis can be successfully conducted
using an office scanner with a detection limit of 2.64 × 10–7 M.
367
368
Acknowledgments
This research was supported by a Chung-Ang University research grants in 2017.
369
Appendix. Supplementary data
370
371
Supplementary data associated with this article can be found in the online version at
doi:**.****.
372
373
References
374
375
376
377
378
379
[1]
[2]
Freeman F. Chemistry of malononitrile. Chem Rev 1969;69:591–624.
Fatiadi AJ, New applications of malononitrile in organic chemistry - Part I.
Synthesis 1978;1978:165–204.
[3]
Lin Z, Zhang W, Wang L, Yu H, Wu C. Mechanism of the synergistic
toxicity of malononitrile and p-nitrobenzaldehyde with photobacterium
phosphoreum. Toxicol Mech Methods 2003;13:241–5.
24

380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
[4]
[5]
Clayton GD, Clayton FE. Patty's industrial hygiene and toxicology. In: Vol.
2A, 2B, 2C, 2D, 2E, 2F: Toxicology. 4th ed. New York: John Wiley & Sons
Inc.; 1993-1994, p. 3157.
Jain AK, Sharma S, Vaidya A, Ravichandran V, Agrawal RK. 1,3,4-
Thiadiazole and its derivatives: a review on recent progress in biological
activities. Chem Biol Drug Des 2013;81:557–76.
[6]
[7]
[8]
[9]
Kirk-Othmer Encyclopedia of Chemical Technology. In: Vo. 1, 4th ed. New
York: John Wiley & Sons Inc.; 1995, p. 942.
Lewis RJ. Hawley's condensed chemical dictionary. 13th ed. New York: John
Wiley & Sons, Inc.; 1997, p. 696.
Junek H, Hamböck H. Eine farbreaktion zum nachweis von o-chinonen der
naphthalin- und phenanthrenreihe. Mikrochim Acta 1966;54, 522–5.
Junek H, Hamböck H, Sterk H. Synthesen mit nitrilen, 18. mitt.:
untersuchungen über den farbstoff aus phenanthrenchinon und malonitril.
Mon Chem 1968;99:1028–33.
[10] Hassan SSM. Manual and semiautomated spectrophotometric determination
of vitamin K3 by reactions with piperidine and malononitrile. J Assoc Off
Anal Chem 1981;64:611–5.
25

398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
[11] Honda S, Iwase S, Suzuki S, Kakehi K. Fluorometric determination of sialic
acids using malononitrile in weakly alkaline media and its application to
postcolumn labeling in high-performance liquid chromatography. Anal
Biochem 1987;160:455–61.
[12] Karalliedde L, Wheeler H, Maclehose R, Murray V. Possible immediate and
long-term health effects following exposure to chemical warfare agents.
Public Health 2000;114:238–48.
[13] Rao P, Singh P, Yadav SK, Gujar NL, Bhattacharya R. Acute toxicity of
some synthetic cyanogens in rats: Time-dependent cyanide generation and
cytochrome oxidase inhibition in soft tissues after sub-lethal oral intoxication.
Food Chem Toxicol 2013;59:595–609.
[14] NIOSH Pocket Guide to Chemical Hazards. "#0378". National Institute for
Occupational Safety and Health (NIOSH). Last reviewed: November 29, 2018.
[15] Sass S, Fisher TL, Jascot MJ, Herban J. Gas-liquid chromatography of some
irritants at various concentrations. Anal Chem 1971;43:462–4.
[16] USEPA; EMMI. EPA's Environmental Monitoring Methods Index. Version
1.1. PC# 4082. Rockville, MD: Government Institutes, 1997.
26

415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
[17] Zollinger DP, Bos M, Van Veen-Blaauw AMW, Van Der Linden WE.
Polarographic determination of malononitrile. Talanta 1984;31:723–5.
[18] Haddadin M, Khalidi U, Turjuman N, Ghougassian R. Sensitive test for
detection and determination of malononitrile and its derivatives. Anal Chem
1974;46:2072.
[19] Ludemann WD, Stutz MH, Sass S. Thin-layer chromatography of some
irritants. Anal Chem 1969;41:679–81.
[20] Eulenhöfer HG. Dünnschichtchromatographische trennung von dinitrilen
unter besonderer berücksichtigung tautomerer formen des malonsäuredinitrils
und einfache nachweisreaktion. J Chromatogr A 1968;36:198–204.
[21] Johar GS, Singh JP. New spot tests for nitroprusside, malononitrile, and
dithiophthalimide. Mikrochim Acta 1974;62:437–41.
[22] Juvvik
P,
Solheim
E.
Thin-layer
chromatography
of
2,4-
dinitrobenzenediazonium coupling products with malononitrile and some
analogues. J Chromatogr A 1973;84:414–6.
[23] Montoya LA, Pearce TF, Hansen RJ, Zakharov LN, Pluth MD. Development
of selective colorimetric probes for hydrogen sulfide based on nucleophilic
aromatic substitution. J Org Chem 2013;78:6550–7.
27

433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
[24] Wei C, Wei L, Xi Z, Yi L. A FRET-based fluorescent probe for imaging H2S
in living cells. Tetrahedron Lett 2013;54:6937–9.
[25] Montoya LA, Pluth MD. Hydrogen sulfide deactivates common
nitrobenzofurazan-based fluorescent thiol labeling reagents. Anal Chem
2014;86:6032–9.
[26] Hammers MD, Pluth MD. Ratiometric measurement of hydrogen sulfide and
cysteine/homocysteine ratios using a dual-fluorophore fragmentation strategy.
Anal Chem 2014;86:7135–40.
[27] Song F, Li Z, Li J, Wu S, Qiu X, Xi Z, Yi L. Investigation of thiolysis of
NBD amines for the development of H2S probes and evaluating the stability
of NBD dyes. Org Biomol Chem 2016;14:11117–24.
[28] Wei C, Zhu Q, Liu W, Chen W, Xi Z, Yi L. NBD-based colorimetric and
fluorescent turn-on probes for hydrogen sulfide. Org Biomol Chem
2014;12:479–85.
[29] Sun L, Jiang Y, Zhang C, Ji X, Lv D, Xi Z, Yi L. A NBD-S-rhodamine dyad
for dual-color discriminative imaging of biothiols and Cys/Hcy. New J Chem
2018;42:15277–83.
28