Tuning the sensitivity towards mercury: Via cooperative binding to d-fructose: Dual fluorescent chemosensor based on 1,8-naphthyridine-boronic acid derivative

Article abstract of DOI:10.1039/d1ra02122b

A novel fluorescent chemosensor naphthyridine-boronic acid derivative (1.1) was synthesized and its ability to act as a selective chemosensor was examined for various metal ions. Compound 1.1 displayed highly selective fluorescence quenching upon interaction with Hg2+, possibly by means of photo induced electron transfer (PET) mechanism. The binding stoichiometry of the naphthyridine-boronic acid-Hg2+ complex and the association constant was determined. It was found that in the presence of d-fructose at physiological concentration, the sensitivity of chemosensor 1.1 towards Hg2+ improved by at least 7 times, perhaps as a result of the cooperative binding of both d-fructose and mercury ion to the sensor. Till now, the presented dual d-fructose-mercury chemosensor is the first example of utilizing boronic acid-diol complexation for enhancement of the sensor's sensitivity towards a toxic metal ion. The utility of compound 1.1 lays in applications in the food industry, e.g. for detection of mercury contamination of high fructose corn syrup, or in estimation of mercury in polluted biological samples and underground water. This journal is

Full text of DOI:10.1039/d1ra02122b

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Tuning the sensitivity towards mercury via
cooperative binding to D-fructose: dual fluorescent
chemosensor based on 1,8-naphthyridine-boronic
acid derivative†
Cite this: RSC Adv., 2021, 11, 14862
Marina Rajadurai * and E. Ramanjaneya Reddy
A novel fluorescent chemosensor naphthyridine-boronic acid derivative (1.1) was synthesized and its ability
to act as a selective chemosensor was examined for various metal ions. Compound 1.1 displayed highly
2+
selective fluorescence quenching upon interaction with Hg , possibly by means of photo induced
2+
electron transfer (PET) mechanism. The binding stoichiometry of the naphthyridine-boronic acid–Hg
complex and the association constant was determined. It was found that in the presence of D-fructose
2+
at physiological concentration, the sensitivity of chemosensor 1.1 towards Hg improved by at least 7
times, perhaps as a result of the cooperative binding of both D-fructose and mercury ion to the sensor.
Till now, the presented dual D-fructose–mercury chemosensor is the first example of utilizing boronic
acid–diol complexation for enhancement of the sensor's sensitivity towards a toxic metal ion. The utility
of compound 1.1 lays in applications in the food industry, e.g. for detection of mercury contamination of
high fructose corn syrup, or in estimation of mercury in polluted biological samples and underground water.
Received 17th March 2021
Accepted 6th April 2021
DOI: 10.1039/d1ra02122b
rsc.li/rsc-advances
trained professionals in order to perform the analysis. At the
same time, mercury detection based on uorescence changes
1
. Introduction
Mercury and mercuric salts are extensively circulated in atmo- allows rapid, convenient and inexpensive detection. Fluores-
spheric air, water and soil, and considered to be highly toxic and cence chemosensors offer denite advantages, such as high
1
hazardous to humans and the environment. For humans, selectivity, high sensitivity, accuracy and possibility to investi-
mercury contamination may cause a wide variety of symptoms, gate molecule–molecule recognition in both environmental and
7
including neuro-disorders, neuromuscular changes, memory biological samples. Till date, numerous uorescent chemo-
2
+
2
loss and carcinogenic diseases. Despite the danger mercury sensors for Hg are developed based on rhodamine and BOD-
8
9
possesses, especially to unborn children, it is still used in IPY, various nanoparticles, for example silver, modied
10
industry. Its contaminants are oen detected in various prod- naphthalimide derivatives and other sensors. However, these
ucts, including food industry products, for example in high sensors oen suffer from such drawbacks, as laborious,
fructose corn syrup, which is cheaper and sweeter than regular complex or expensive synthesis, low aqueous solubility or
sugar. Traces of mercury were found in high fructose corn syrup limited selectivity as result of interference with other metal
containing beverages/confectioneries, and the alarming ions.
amounts of up to 0.570 micrograms of mercury per gram of high
1,8-Naphthyridine and its derivatives are extensively studied
fructose corn syrup were determined. Various methods have for molecular recognitions events, including nucleosides
3
2
+
11
been employed for the detection of Hg , including cold vapor sensing (e.g. for guanine, cytosine and thymidine), mono-
13
4
12
atomic absorption spectroscopy, high performance liquid saccharide sensing and heavy transition metal ions sensing
chromatography and inductively coupled plasma atomic (e.g. for Zn , Hg , Cd ). In order to improve uorescent
emission spectrometry. The above methods are extremely properties, affinity, aqueous solubility and stability of 1,8-
sensitive and able to detect Hg in the nanomolar range. naphthyridine based chemosensors, we report design and
However, their drawbacks are: costly equipment, time- synthesis of novel uorescent chemosensor 1.1 [(2-((((5-(7-
consuming and laborious procedures, and need for the acetamido-1,8-naphthyridin-2-yl)thiophen-2-yl)methyl)(methyl)
amino)methyl)phenyl)boronic acid]. In order to strengthen an
2
+
2+
2+
5
6
2
+
interaction of mercury ion with the chemosensor, 1,8-naph-
Center for Innovation in Molecular & Pharmaceuticals Sciences (CIMPS), Dr Reddy's
Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad thyridine was conjugated to thiophene moiety, keeping in mind
5
†
1
00046, Telangana, India. E-mail: marinasraj@gmail.com
Electronic supplementary information (ESI) available. See DOI:
0.1039/d1ra02122b
high affinity of mercury to sulfur. At the same time, boronic acid
group, which is known to easily form cyclic ester with
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monosaccharides, was additionally introduced in the same Spectroscopic data matches with the one from reported proce-
1
scaffold aiming cooperative action of boronic acid and metal dure. H NMR (CDCl , 400 MHz) d: 7.22–7.20 (1H, m), 6.94–6.92
3
chelate, which could possibly enhance sensitivity and selectivity (2H, m), 4.87 (1H, brs), 4.47 (2H, d, J ¼ 5.2 Hz), 1.46 (9H, s).
either to metal ion or sugar. There are very few examples are
2.2.2. tert-Butyl((5-(tributylstannyl)thiophen-2-yl)methyl)
15
known for using boronic acid containing compounds for direct carbamate (2.2). To a solution of compound 2.1 (0.4 g, 2.62
14
detection of metal ions.
mmol) in dry THF (30 mL) was added n-butyl lithium (1.6 M in
ꢀ
ꢀ
Thus, we report the synthesis of naphthyridine-boronic acid hexane, 3.58 mL, 5.73 mmol) at ꢁ78 C. Aer 1 h at ꢁ78 C,
based uorescent chemosensor 1.1, its uorescence properties tributyl tin chloride (1.57 mL, 5.81 mmol) was added drop wise
and investigation of interaction with various metal ions and to the above reaction mixture. Aer stirring over 6 h at room
monosaccharides (D-glucose, D-fructose, D-mannose and D- temperature, the reaction mixture was quenched with 20 mL of
galactose). The ability of chemosensor 1.1 to form reversible saturated aqueous ammonium chloride solution and extracted
covalent bonds with D-fructose was employed for the rst time with THF. The organic phase was dried with anhydrous sodium
to signicantly enhance selectivity towards metal ion, namely sulphate, concentrated under reduced pressure, and the
2
+
Hg .
resulting residue 2.2 (0.87 g, 93%) was used without further
purication. Spectroscopic data matches with the one from re-
1
ported procedure. H NMR (CDCl
3
3
, 400 MHz) d: 7.05 (1H, d, J ¼
2. Materials and methods
.2 Hz), 6.99 (1H, d, J ¼ 2.8 Hz), 4.87 (1H, brs), 4.51 (2H, s), 1.59–
1.51 (6H, m), 1.46 (9H, s), 1.37–1.28 (6H, m), 1.07–1.04 (6H, m),
.91–0.87 (9H, m).
.2.3. tert-Butylmethyl((5-(tributylstannyl)thiophen-2-yl)
methyl)carbamate (1.3). To a solution of compound 2.2 (5.54 g,
2.1. General
0
The materials 2-thiophene carboxaldehyde, 2,6-diaminopyridine,
n-butyl lithium, tributyltin chloride, bis(triphenylphosphine)
palladium(II) dichloride, acetic anhydride, methyl amine (40%
aqueous solution), 2-thiophenemethylamine, potassium bis(-
trimethylsilyl)amide, 2-(bromomethyl)phenylboronic acid and
paraformaldehyde were purchased from Sigma Aldrich. N-Bro-
mosuccinimide, phosphorus trichloride oxide, azobisisobutyr-
onitrile, di-tert-butyl dicarbonate, triuoroacetic acid, methyl
iodide were purchased from Spectro Chem. Commercial reagents
2
1
2
ꢁ
1.0 mmol) in 30 mL of dry THF was added 1 M KHMDS (22 mL,
ꢀ
2 mmol) dropwise at ꢁ10 C. The reaction was maintained at
ꢀ
10 C for 1.5 h, and methyl iodide (2.05 mL, 33.0 mmol) was
added drop wise. The ice bath was removed and reaction
mixture was stirred at room temperature for 4 h. The reaction
mixture was washed with brine (4 ꢂ 100 mL) and extracted with
hexane. The organic layer was collected, dried over anhydrous
sodium sulphate, concentrated under reduced pressure to
provide compound 1.3 (5.45 g, 96% yield) as a dark brown oil,
(DL-malic acid, ethane-1,2-diol, sodium borohydride, ammonium
bicarbonate, mercury(I) chloride and HCl) were purchased from
Merck and Rankem and were used as received. The solvents THF,
DMF, toluene, 1,4-dioxane and DCM were distilled and dried
before reactions and for extracting purposes. All reactions were
carried out under an inert atmosphere with dry solvents, unless
which was used without further purication. TLC (10% EtOAc in
hexane): R
3
1
1.10–1.06 (6H, m), 0.98–0.93 (9H, m). C NMR (CDCl
MHz) d: 155.3, 146.1, 136.7, 134.8, 127.1, 79.9, 47.3, 33.7, 28.9,
2
H
1
f
¼ 0.39; H NMR (CDCl
3
, 400 MHz) d: 7.03 (1H, d, J ¼
.2 Hz), 7.01 (1H, d, J ¼ 3.2 Hz), 4.56 (2H, s), 2.86 (3H, s), 1.67–
.62 (2H, m), 1.59–1.54 (4H, m), 1.49 (9H, s), 1.39–1.31 (6H, m),
otherwise stated. Syringes and needles for the transfer of reagents
1
3
ꢀ
, 100
3
were dried at 100 C and allowed to cool in a desiccator over P
O
2 5
before use. Reactions were monitored by thin layer chromatog-
raphy (TLC) on silica gel plates (60 F254), using UV light detec-
tion. Merck silica gel (particle size 100–200 mesh) was used for
column chromatography. For UV-vis and uorescence measure-
ments spectroscopic grade solvents were used.
+
8.4, 27.2, 13.6, 10.7. MS (EI, 70 eV): m/z [M] calcd for C23
-
-
+
2 43
43NO SSn: 517.2, found 517.2 [M] . Anal. calcd for C23H
2
NO SSn: C, 53.50; H, 8.39; N, 2.71; O, 6.20; S, 6.21; Sn, 22.99,
found: C, 53.46; H, 8.36; N, 2.75.
.2.4. 7-Amino-1,8-naphthyridin-2-ol
minopyridine 2.3 (1.1 g, 10.07 mmol) and malic acid (1.48 g,
1.07 mmol) were placed in a three necked round bottom ask
16
2
(2.4).
2,6-Dia-
Buffers and samples preparations for uorescence studies
and synthesis of the compounds of routes 2–4 are given in ESI.†
1
outtted with an additional funnel, reux condenser and
mechanical stirrer. The mixture was cooled to 0 C and con.
2
(
.2. Synthesis of target compound 1.1 using route I
Schemes 1 and 2)
.2.1. tert-Butyl(thiophen-2-ylmethyl)carbamate
ꢀ
H
H
2
SO
4
4
(5 mL) was added dropwise. Aer addition of conc.
was completed, the reaction mixture was slowly heated to
15
2
(2.1).
2
SO
ꢀ
Thiophene-2-methyl amine (0.5 g, 4.42 mmol) was dissolved in 110 C and stirred for 3 h. The reaction mixture was cooled to
ꢀ
dry DCM (20 mL), and triethyl amine (0.95 mL, 6.84 mmol) was 0 C and the solution was made alkaline (pH ¼ 8) by careful
added to the solution. Boc-anhydride (1.22 mL, 5.31 mmol) was addition of aqueous ammonium hydroxide. The crude solid was
added portion wise to the above reaction mixture. Aer collected by vacuum ltration and washed thoroughly with
complete addition of Boc-anhydride the reaction mixture was water and methanol to give compound 2.4 as a yellowish solid
stirred at room temperature for 4 h. Aer 4 h, the reaction (1.38 g, 86%). Spectroscopic data matches with the one from
1
mixture was washed with water and extracted with DCM. The reported procedure. H NMR (DMSO-d
6
, 400 MHz) d: 11.91 (1H,
organic phase was dried with anhydrous sodium sulphate, s), 7.65 (2H, d, J ¼ 9.2 Hz), 7.03 (2H, s), 6.34 (1H, d, J ¼ 8.4 Hz),
concentrated under reduced pressure, and the resulting residue 6.12 (1H, d, J ¼ 9.2 Hz).
2.1 (0.8 g, 86%) was used without further purication.
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.2.5. N-(7-Hydroxy-1,8-naphthyridin-2-yl)acetamide
Paper
2
8.01 (1H, d, J ¼ 8.4 Hz), 7.86 (1H, d, J ¼ 4.4 Hz), 7.08 (1H, d, J ¼
17
13
(
2.5). A suspension of compound 2.4 (5.0 g, 15.5 mmol) in 3.6 Hz), 3.93 (2H, s), 2.35 (3H, s), 2.14 (3H, s). C NMR (DMSO-
ꢀ
50 mL of acetic anhydride was stirred at 110 C for 3 h. Aer 3 h,
d , 100 MHz) d: 170.5, 155.2, 154.9, 154.8, 143.8, 139.6, 138.0,
6
reaction mixture was cooled to room temperature and the 127.9, 127.5, 119.2, 116.8, 114.2, 49.7, 35.2, 24.5. MS (EI, 70 eV):
+
+
precipitate was collected by vacuum ltration, washed with m/z [M] calcd for C16
hexane and dried at reduced pressure to give compound 2.5 as calcd for C16 OS: C, 61.52; H, 5.16; N, 17.93; O, 5.12; S,
a yellow solid (5.33 g, 89%). Spectroscopic data matches with 10.26, found: C, 61.42; H, 5.23; N, 17.85.
16 4
H N OS: 312.1, found 313.1 [M + 1] . Anal.
16 4
H N
1
the one from reported procedure. H NMR (DMSO-d , 400 MHz)
2.2.9. (2-((((5-(7-Acetamido-1,8-naphthyridin-2-yl)
6
d: 11.90 (1H, s), 10.50 (1H, s), 8.02 (1H, d, J ¼ 8.4 Hz), 7.90 (1H, d, thiophen-2-yl)methyl)(methyl)amino)methyl)phenyl)boronic
J ¼ 8.8 Hz), 7.82 (1H, d, J ¼ 9.6 Hz), 6.40 (1H, d, J ¼ 9.6 Hz), 2.12 acid (1.1). Anhydrous K
CO (0.176 g, 1.32 mmol) was added to
2 3
a stirred suspension of compound 1.2 (0.14 g, 0.44 mmol) in dry
DMF (2 mL). Aer 30 min, a solution of 2-(bromomethyl)phe-
(3H, s).
18
2.2.6. N-(7-Chloro-1,8-naphthyridin-2-yl)acetamide (1.4).
A mixture of compound 2.5 (10.0 g, 49.25 mmol) and POCl
3
(175 nylboronic acid (0.096 g, 0.44 mmol) in DMF (2 mL) was added,
mL) was heated at 100 C for 2 h. The reaction mixture was and the reaction mixture was stirred at room temperature for
ꢀ
cooled to room temperature, and excess of POCl was removed 12 h. DMF was removed under reduced pressure, and the crude
3
by distillation. The crude residue was dissolved in ice water, and residue was washed with water, hexane and toluene to yield
ꢀ
1
the solution was made alkaline (pH ¼ 8) by careful addition of compound 1.1 as a white solid (0.14 g, 70%). Mp: 210–212 C. H
concentrated ammonium hydroxide. The crude solid was NMR (DMSO-d , 400 MHz) d: 11.12 (1H, s), 9.03 (2H, brs), 8.34–
6
collected by vacuum ltration, air-dried, and continuously 8.27 (3H, m), 8.0 (1H, d, J ¼ 8.4 Hz), 7.87 (1H, d, J ¼ 3.6 Hz),
extracted (Soxhlet extraction) with chloroform for 12 h. Chlo- 7.72–7.69 (1H, m), 7.35–7.25 (3H, m), 7.10 (1H, d, J ¼ 3.6 Hz),
1
1
roform was removed at reduced pressure, and the crude product 3.75 (2H, s), 3.70 (2H, s), 2.14 (3H, s), 2.13 (3H, s). B NMR
1
3
was puried using column chromatography on silica gel using (CD
3 3
OD, 130 MHz) d: 26.5. C NMR (CD OD, 100 MHz) d: 170.5,
MeOH/DCM (1 : 9) as an eluent to yield compound 1.4 in form 155.2, 154.9, 154.8, 143.8, 139.6, 138.0, 127.9, 127.5, 119.2,
1
+
of a golden needles (6.47 g, 60%). H NMR (DMSO-d , 400 MHz) 116.8, 114.2, 49.7, 35.2, 24.5. MS (EI, 70 eV): m/z [M] calcd for
6
+
d: 8.70 (1H, s), 8.56 (1H, d, J ¼ 8.8 Hz), 8.20 (1H, d, J ¼ 8.8 Hz),
C
C
23
H
H
23BN
23BN
4
O
O
3
S: 446.1, found 447.1 [M + 1] . Anal. calcd for
S: C, 60.38; H, 4.73; N, 18.78; O, 5.36; S, 10.75,
8
.18 (1H, d, J ¼ 8.4 Hz), 7.40 (1H, d, J ¼ 8.0 Hz), 2.30 (3H, S).
.2.7. tert-Butyl((5-methyl(7-acetamido-1,8-naphthyridin-
-yl)thiophen-2-yl)methyl)carbamate (2.6). To a solution of
23
4
3
2
found: C, 61.76; H, 5.12; N, 12.65.
2
compound 1.3 (6.2 g, 12.0 mmol) in dry 1,4-dioxane (50 mL)
3
. Results and discussion
were added compound 1.4 (2.0 g, 9.0 mmol) and PdCl (PPh3)2
2
3.1. Synthesis of naphthyridine-boronic acid (1.1)
(0.315 g, 0.45 mmol) under nitrogen atmosphere. Aer 12 h at
reux, the solvent was removed under reduced pressure, and The key intermediate in synthesis of the chemosensor 1.1 is
the crude residue was dissolved in EtOAc, passed through celite compound 1.2, which in its turn could be synthesized through
and solvent was evaporated. Crude product was puried by four different routes (Scheme 1). We selected the route I which
column chromatography using 50% EtOAc in hexane to give 2.6 allowed to successfully synthesizing key intermediate 1.2 and
ꢀ
as yellowish solid (2.5 g, 67%). Mp: 115–118 C; TLC (50% EtOAc target compound 1.1 as described in Scheme 2. Thiophene
1
in hexane): R
f
¼ 0.26; H NMR (DMSO-d
6
, 400 MHz) d: 11.07 (1H, methyl amine, which was protected with Boc-anhydride to give
bs), 8.33 (3H, m), 8.01 (1H, d, J ¼ 8.8 Hz), 7.87 (1H, d, J ¼ 4.4 Hz), compound 2.1 served as the starting material for this synthetic
15
7
1
1
1
.08 (1H, d, J ¼ 3.6 Hz), 4.53 (2H, s), 2.80 (3H, s), 2.14 (3H, s), route. Compound 2.1 was subjected to lithiation using n-BuLi
13
.43 (9H, s). C NMR (CDCl , 100 MHz) d: 169.5, 155.6, 154.6, followed by treatment with tributyl tin chloride resulting in
3
15
53.9, 145.7, 143.5, 139.2, 139.0, 137.0, 126.8, 119.3, 116.9, compound 2.2 in excellent yield. Further, compound 2.2 was
+
14.3, 114.0, 80.2, 48.0, 33.8, 28.4, 24.9. MS (EI, 70 eV): m/z [M]
methylated using methyl iodide and potassium hexamethyldi-
S: 412.16, found 413.2 [M + 1] . Anal. calcd silazane as a base, to give compound 1.3 in 96% yield. In order
S: C, 61.14; H, 5.86; N, 13.58; O, 11.64; S, 7.77, to build naphthyridine moiety, 2,6-diamino pyridine (2.3), was
found: C, 61.26; H, 5.89; N, 13.45.
cyclised using DL-malic acid in the presence of conc. H SO
.2.8. N-(7-(5-((Methylamino)methyl)thiophen-2-yl)-1,8-
to give 7-amino-1,8-
+
calcd for C21
24 4 3
H N O
for C21H N O
24 4 3
2
4
,
16
2
according to standard procedure,
naphthyridin-2-yl)acetamide (1.2). Compound 2.6 (0.293 g, 0.71 naphthyridin-2-ol (2.4) in 86% yield. Amino group of 7-amino-
mmol) was dissolved in TFA : DCM (3 mL : 3 mL) and stirred at 1,8-naphthyridin-2-ol was protected using acetic anhydride to
17
RT for 2 h. Aer 2 h solvents were removed and the reaction obtain acetamide protected compound 2.5 in 89% yield. This
mixture was treated with sodium bicarbonate solution and step allowed subsequent selective conversion of hydroxyl group
18
extracted with DCM. The organic solvent was removed under to the halogen via chlorination with phosphorus oxychloride
reduced pressure and the crude residue was then puried using to obtain 1.4 in 60% yield. In the next step, coupling of
column chromatography on silica gel using MeOH/DCM (2 : 8) compound 1.3 with 1.4 in Stille coupling conditions in 1,4-
as an eluent to yield compound 1.2 as a yellowish solid (0.189 g, dioxane using palladium(II) catalyst resulted in compound 2.6
ꢀ
1
8
5%). Mp: 260–262 C; TLC (20% MeOH in DCM): R ¼ 0.21. H in 67% yield. In order to introduce phenyl-boronic acid moiety
f
NMR (DMSO-d , 400 MHz) d: 11.08 (1H, brs), 8.35–8.28 (3H, m), in this scaffold, Boc-protection of the compound 2.6 was
6
removed using triuoroacetic acid to give key intermediate 1.2
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Scheme 1 Plausible retrosynthesis of key intermediate 1.2 and target molecule 1.1.
in 82% yield, followed by N-alkylation with commercially
(a) Imine formation followed by reduction using interme-
available 2-bromomethylphenylboronic acid, using potassium diate 1.5, as presented in route II.
carbonate as a base. Final compound was obtained in 70% yield
(b) Conversion of bromine to amine, via azide formation
and its structure was conrmed by different methods, including followed by N-alkylation using intermediate 1.6, as presented in
1
NMR spectroscopy and mass-spectrometry. Particularly,
H
route III.
(c) N-Alkylation using intermediate 1.7, as presented in
peak at 9.03 ppm, additionally route IV.
EI-MS spectrum showed predominant peak with m/z value of
NMR spectrum showed an appearance of characteristic N–CH2
peak at 3.72 ppm and B–(OH)
2
4
47.1 corresponding to target molecule (ESI Fig. S1 and S2†).
Apart from the successful route described above, other
3.2. Photophysical properties of compound 1.1
attempts to synthesize target compound 1.1 were made as
sketched in Scheme 1 (see experimental procedures and
discussion in ESI, Schemes S1–S3†), including:
To explore utility of the nal uorophore 1.1, we investigated
the photophysical properties of 1.1 in MeOH and MeOH/H
2
O
O
solutions. In both solutions, MeOH and MeOH/H
2
Scheme 2 Route I towards key intermediate 1.2 and target compound 1.1.
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1+
2+
methanolic buffer at pH 7.5, and metal ions such as Ag , Zn ,
1
+
2+
2+
2+
2+
1+
3+
Cu , Mg , Pb , Cd , Hg , Li and Fe were tested. Among
2+
them, addition of Hg to 1.1 showed a signicant decrease of
uorescence intensity by 75% (Fig. 2A). This can be explained by

the fact that aer coordination to metal ion uorophore becomes
electron decient, and as a result the PET from proximal tertiary
nitrogen to adjacent chromophore increases and the uorescence
intensity of the uorophore decreases. Apart from that, there could
be possible contribution of strong spin–orbit coupling associated
2+
with Hg to the luminescence quenching of 1.1. Fluorescence
ꢁ
5
properties of compound 1.1 (10 M) were also studied upon
2+
increasing the concentration of Hg (1–10 eq.), and it was
2+
observed that as the concentrations of Hg increases, the uo-
Fig. 1 UV-vis absorption spectra of compound 1.1 in MeOH and rescent intensity of 1.1 gradually decreases as shown in Fig. 2B and
MeOH/H
2
O, and its emission spectra in MeOH and MeOH/H
2
O, lex
¼
C. In order to estimate the binding stoichiometry of
3
56 nm (insert).
2+
naphthyridine-boronic acid–Hg complex, Job plot experiments
19
2+
were employed. In Job method, equal concentrations of Hg and
.1 were prepared, and mixed in different proportions maintaining
1

uorophore 1.1 displayed identical absorption maxima at
a total concentration of 10 mM. Using the obtained data, a graph
was plotted between the emission intensity (xed at 408 nm) and
a mole fraction of compound 1.1 (Fig. 2D). The minimum emis-
sion intensity was reached when the mole fraction was 0.5. This
356 nm and a shoulder at 371 nm (Fig. 1 and Table S1 in ESI†).
Fluorescent emission spectra (lex ¼ 356 nm) in both solvents
exhibited similar uorescence bands, with a maximum at
401 nm for 1.1 in MeOH and 403 nm in MeOH/H
2
O. The solu-
2+
result indicates the formation of naphthyridine-boronic acid–Hg
tion state quantum yield of 1.1 in MeOH/H O (F
slightly higher than that in MeOH (F
new uorophore 1.1 was 31 nm.
2
f
¼ 0.26) was
a
complex in a 1 : 1 ratio. The association constant (K ) of
¼ 0.20), Stokes shi of the
2+
f
naphthyridine-boronic acid–Hg was calculated from the varia-
tion of uorescence intensity as a function of the concentration of
2+
20
Hg by using Benesi–Hildebrand equation (eqn (1)) and tting
the emission wavelength at 408 nm. The calculated K for
a
3
.3. Examination of binding ability of 1.1 to metal ions
2+
5
ꢁ1
a complex of 1.1 with Hg was 2.842 ꢂ 10 M
.
Fluorescence response of compound 1.1 in presence of various
metal ions was investigated. The study was carried out in aqueous
2
Fig. 2 (A) Fluorescence spectra of compound 1.1 in MeOH/H O in presence of various metal ions; (B) fluorescence spectra of compound 1.1 in
2
+
2+
MeOH/H
complex in MeOH/H
2
O in presence of Hg at various concentrations (0–10 eq.); (C) Benesi–Hildebrand plot of the naphthyridine-boronic acid–Hg
2+
2
2
O solutions; and (D) Job plot of naphthyridine-boronic acid–Hg complex in MeOH/H O.
14866 | RSC Adv., 2021, 11, 14862–14870
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/(F ꢁ F ) ¼ 1/(K (F
RSC Advances
2
+
1
ꢁ F )[Hg ]) + 1/(Fmax ꢁ F0),
(1) Lewis acidity of the central boron atom. As result B–N (proximal
0
a
max
0
23
tertiary nitrogen) interactions are strengthening and PET from
where F is the uorescence intensity at 408 nm at any given proximal tertiary nitrogen to adjacent uorophore is sup-
2
+
concentration of Hg , F is the uorescence intensity at 408 nm pressed and the uorescence of the uorophore is
0
+
2
22,24
in the absence of Hg , Fmax is the uorescence intensity at enhancing.
0
The uorescence enhancement I/I obtained for
is association D-glucose, D-galactose, D-mannose and D-fructose is in the order
2
+
408 nm in the presence of Hg in solution and K
a
constant.
D-fructose > D-galactose > D-mannose > D-glucose as shown in
In order to understand the structure of naphthyridine- Fig. 4.
2
+
boronic acid–Hg complex, proton NMR spectroscopy was
The binding selectivity of 1.1 towards D-fructose is around
employed. Addition of Hg to the DMSO solution of 1.1 two and half fold higher compared to other monosaccharides
resulted in a downeld chemical shi of the H proton from (D-galactose, D-mannose, D-glucose) at lower concentrations (<10
.10 ppm to 7.55 ppm, additionally a moderate down eld mM). The association constant (K ) of naphthyridine-boronic
21
2+
a
7
a
chemical shis observed for H and Hh,i,j (Fig. 3). These changes acid based uorescence chemo sensor 1.1 with D-fructose, D-
e
2
+
indicate that Hg interacts mainly with the sulphur atom, glucose, D-galactose and D-mannose were calculated from the
which was expected as high affinity of mercury for sulphur variation of uorescence intensity (Fig. 4 and S21–S23 in ESI†)
atoms is well known, and partly binds to the N and N of the as a function of the concentration of D-monosaccharides, using
1
8
20
naphthyridine moiety; at the same time there are no interaction Benesi–Hildebrand equation (eqn (2)) and tting the emission
with N-methyl nitrogen (no shi observed in NMR spectrum, wavelength at 403, 409, 410 and 408 respectively. The highest
not shown in Fig. 3). The decrease of uorescence intensity or affinity of 1.1 was observed towards fructose (the association
ꢁ
1
down eld chemical shis of H
a
, H
e
and Hh,i,j upon addition of constants (K ) ¼ 104.91 M ), while to other sugars it was
a
2
+
ꢁ1
Hg are due to decrease of electron density aer coordination signicantly lower (K ¼ 14.42–39.46 M ). The affinity for 1.1
a
2
+
of Hg to the respective binding site as shown in Fig. 3. were in the following order: D-fructose > D-galactose > D-
Therefore, these both observations support the theory of PET mannose > D-glucose, and the calculated association constant
mechanism for presented here chemosensor.
are listed in Tables S2–S5 in ESI.†
1
/(F ꢁ F ) ¼ 1/(K (F ꢁ F )[sugar]) + 1/(F
ꢁ F0),
(2)
0
a
max
0
max
3.4. Examination of binding ability of 1.1 to
monosaccharides
where F is the uorescence intensity at 403/409/410/408 nm at
ꢁ5
any given concentration of D-fructose/D-galactose/D-mannose/D-
Fluorescence titration studies of compound 1.1 (10 M) in
presence of various monosaccharides was carried out in
aqueous methanolic phosphate buffer at pH 8.20. As expected,
the uorescence intensity of compound 1.1 increased upon
increasing of saccharides concentration (D-glucose, D-mannose,
glucose respectively, F
0
is the uorescence intensity at 403/408/
409/410 nm in the absence of D-saccharides, Fmax is the uo-
rescence intensity at 403/409/410/408 nm in the presence of D-
fructose/D-galactose/D-mannose/D-glucose respectively in solu-
tion, and K is association constant.
a
Fluorescence response of the boronic acid based uorescent
chemosensors is usually pH dependent. Therefore, uorescence
22
D-fructose and D-galactose). As reported by Shinkai et al. such
uorescence response explained by the fact that boronic acids
such as compound 1.1) form cyclic esters with cis 1,2-diol or
,3-diol of mono saccharides, which leads to increase of the

(
1
response
of
naphthyridine-boronic
acid
uorescent
2+
1
Fig. 3 (A) Proposed coordination complex structure of Hg ions with 1.1, and (B) H NMR spectra of compound 1.1 along and in the presence of
6
Hg salt (1 : 3 eq.) in DMSO-d .
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Fig. 4 (A) Fluorescence intensity of compound 1.1 in MeOH/H
2
O (v/v ¼ 1 : 1, 5 mM PBS, pH 8.20) in the presence of D-glucose, D-galactose, D-
ꢁ5
mannose and D-fructose; (B) fluorescence spectra of compound 1.1 (10 M) in presence of D-fructose at various concentrations (0–10 mM); (C)
ꢁ5
pH vs. fluorescent intensity of 1.1 (10 M) in presence and absence of D-fructose in MeOH/H
2
O (v/v ¼ 1 : 1, 5 mM), at various pH solutions and (D)
Benesi–Hildebrand plot of the naphthyridine-boronic acid–fructose complex in MeOH/H
emission wavelength was monitored at 403 nm.
2
O (v/v ¼ 1 : 1, 5 mM PBS, pH 8.20) solutions, the
2
+
chemosensor 1.1 was measured at different pH, both in the response was examined in presence of Hg and various D-
presence and absence of D-fructose. As Fig. 4C displays, in both monosaccharides. It was expected, that cooperative binding of
cases the uorescent response was similar. Fluorescent inten- both sugar and metal ion will result in increased sensitivity of
sity was increasing upon pH change from 2 to 5.5 due to the chemosensor to either of analytes, decreasing/eliminating
protonation of tertiary amine, and was decreasing as pH raised at the same time interference by various factors, which are
ꢁ
from 5.5 to 12 due to deprotonation of tertiary amine and OH
adduct formation with boronic acid group.
common for chemosensors acting through “turn-off” mecha-
25
25a
nism. The study was carried out in aqueous methanolic buffer
2
+
at pH 7.5. Addition of Hg to a solution containing both che-
mosensor 1.1 and D-fructose complex resulted in dramatic
decrease of uorescence intensity by seven folds (Fig. 5A). This
2
+
3
.5. Competitive sensing of Hg and D-monosaccharide
As compound 1.1 has incorporated phenyl boronic acid moiety
which is expected to bind to mono-saccharides, its uorescence

uorescent response was ꢃ2.8 times higher compared to one,
ꢁ
5
2+
2+
Fig. 5 Fluorescence spectra of 1.1 alone (10 M), 1.1 with Hg and 1.1 with Hg and D-monosaccharides in MeOH/H
2
O (v/v ¼ 1 : 1, 5 mM, at
various pH) solutions (A) and comparative bar graph of the emission intensity change for the same solutions (B).
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when compound 1.1 was interacting with mercury without
addition of D-fructose (Fig. 5B). At the same time, no any
Author contributions
signicant change was observed in the emission intensity of the E. Ramanjaneya Reddy: investigation, writing – original dra.
probe 1.1 in the presence of other hexose sugars.
Marina Rajadurai: conceptualization, supervision, writing –
Till date, there are no reports, where the sensitivity of the review & editing.
chemosensor for metal ions was enhanced as result of interac-
tion with fructose, although there are few investigations
Conflicts of interest
utilizing metal–sensor coordination in order to improve sensi-
25a
tivity towards sugar. For example, Seiji Shinkai et al. reported
There are no conicts to declare.
new saccharide receptor for uronic acids, based on two-point
interactions of boronic acid and coordination with zinc(II), or
another work employing cooperative binding of fructose and
Acknowledgements
14
metal ion performed by group of Tony D. James. In contrary, we ERR acknowledges CSIR-New Delhi for a Senior Research
obtained an interesting result indicating that the sensitivity of Fellowship. This research was supported by grants from DST
the chemosensor 1.1 towards mercury ions increases signi- (SR/SI/CS-131/2009), CSIR (01(2411)/10/EMR-II) and DBT (BT/
cantly in presence of D-fructose moiety. Thus, presented here PR32386/MED/32/681/2019), New Delhi to MR.
work is the rst example of employing boronic acid–diol
complexation to achieve signicant enhancement of the
sensor's sensitivity towards toxic metal ion.
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