Rational design of a highly selective fluorescent sensor for l-histidine detection in aqueous solution

Article abstract of DOI:10.1039/c5dt04214c

Computational studies in combination with experimental research were used to design a new rapid, selective and sensitive "turn-on" fluorescent sensor (H3) for l-histidine, which can be first quenched by Ni²⁺ and then recovered upon addition of His.

Full text of DOI:10.1039/c5dt04214c

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Rational design of a highly selective fluorescent
sensor for L-histidine detection in aqueous
solution†
Cite this: DOI: 10.1039/c5dt04214c
Received 27th October 2015,
Accepted 24th November 2015
a,b
a
a
a
a
a
DOI: 10.1039/c5dt04214c
Yan Qiao, Bin Chen, Yangyang Yang, Xin Wang, Yufang Xu* and Honglin Li*
www.rsc.org/dalton
Computational studies in combination with experimental research development of some novel fluorescent probes for biologically
8
were used to design a new rapid, selective and sensitive “turn-on” or environmentally relevant analytes. From a theoretical point
fluorescent sensor (H3) for L-histidine, which can be first quenched of view, if the analyte can snatch the chelated metal ion, the
2
+
by Ni and then recovered upon addition of His.
binding energy for the ion must be lower than that of the
chemosensor. This inspires us to theoretically predict and
then experimentally confirm rationally designed highly selec-
tive chemosensors for amino acids such as His. We propose
that a chemosensor that can bind a metal ion more weakly
than His but more strongly than all other amino acids can be
used for selective His detection. If true, this hypothesis would
certainly provide valuable insights for the rational design of
highly selective chemosensors for other analytes.
Among the twenty proteinogenic amino acids, histidine plays a
pivotal role in biochemistry due to its aromatic imidazole
moiety, which is a common coordinating ligand in metallo-
proteins and a part of the catalytic site in certain enzymes. Histi-
dine is active in maintaining healthy tissues and protecting
nerve cells that transport messages from the brain to various
other parts of the body. An abnormal histidine level is an
indicator of many diseases such as chronic kidney disease,
acute liver failure, and rheumatoid arthritis. Therefore, the
development of high-quality methods for histidine detection is
extremely necessary and important. Numerous studies have
dealt with the detection of histidine using techniques such as
1
2
3
3a
Previous studies have revealed that His is always a good
3
b
3c
2+
ligand in Ni containing metal enzymes; furthermore, the
2+
octahedral complex of Ni with free His molecules has been
9
crystallized previously. Examination of the crystal structure
shown in Fig. 1 shows that all three His functional groups, i.e.,
4
5
voltammetry, UV/vis spectroscopy, luminescence spectro-
NH , COOH, and the imidazole group, can coordinate with
2
6
7
scopy methods, and fluorescence spectroscopy. However,
most of the available probes exhibit poor selectivity or require
sophisticated detection systems such as the use of organic sol-
vents. The development of reliable, rapid and accurate
methods for the determination of histidine is still a highly
challenging area of research.
The addition of transition metal ions to the chemosensor
ensemble can usually change fluorescence signals due to the
chelation of metal ions by the chemosensors. The subsequent
snatching of the chelated metal ions from the ensemble by
some analytes can again change the fluorescence signal. The
two-step signal change phenomenon has been used for the
2+
2+
Ni , indicating that the binding interaction of His with Ni
should be sufficiently strong. Moreover, as a paramagnetic
2+
metal ion, Ni has a pronounced quenching effect on fluoro-
phores, with this characteristic leading to its wide use as a
10
2+
chelated metal ion in probe design. Therefore, Ni is an
ideal choice for use as the chelated metal ion in the present
work. Due to the excellent intrinsic fluorescence characteristics
and the fluorescence quenching effect triggered by coordi-
nation with paramagnetic metal ions, 7-hydroxycoumarin and
a
Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor
Engineering, School of Pharmacy, East China University of Science & Technology,
Shanghai 200237, China
b
Pathophysiology Department, Basic Medical College of Zhengzhou University,
Zhengzhou 450001, China
†
Electronic supplementary information (ESI) available: Computational details,
2+
General experimental procedure, experimental procedures, spectroscopic pro-
Fig. 1 Complex of Ni with 2His. (a) Schematic illustration of the Ni-
2His complex. (b) Crystal structure of the Ni-2His complex. (c) Opti-
mized structure for the Ni-2His complex. All distances are in Å.
1
13
perties, H NMR, C NMR and HRMS of compounds. See DOI: 10.1039/
c5dt04214c
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replace the other His molecule with H3 or other amino acids
such as Asp and Glu. Therefore, four models were considered:
Ni-2His, Ni-His-H3, Ni-His-Asp, and Ni-His-Glu. The optimized
Scheme 1 Illustration of the designed chemosensor H3.
Scheme 2 Schematic illustration of a speculated fluorescent signal
change.
Fig. 2 (a and b) Binding mode and optimized structures of the Ni-His-
H3 complex. (c and d) Binding mode and optimized structures of the Ni-
His-Asp complex. All the distances are in Å.
its fluorescent derivatives have been widely used as indicators
1
1
of various metal ions. Thus, 7-hydroxycoumarin is con-
sidered a good candidate fluorophore. Herein, considering
these two factors, we devised a chemosensor (H3) (Scheme 1)
incorporating His into 7-hydroxycoumarin via a methylene
group. First, to preserve the inherent sensitivity of the fluoro-
phore, we reserve the whole 7-hydroxycoumarin group in the
devised chemosensor and assume that the 7-hydroxyl group
2
+
can coordinate with Ni . Second, to improve the selectivity,
2
+
the ability of the devised chemosensor to bind Ni should be
between that of His and all other amino acids. Because the
7
-hydroxyl group has been assumed as a coordinating group,
2
+
we preserve the imidazole and NH groups of His as Ni
ligands but protect the third ligand, i.e., the carboxyl group,
2
+
from coordinating with Ni . Therefore, in the devised chemo-
sensor H3, the carboxyl group of His was protected by methyl-
ation, and the methylene group is used as a linker to connect
His with 7-hydroxycoumarin in order to set the 7-hydroxyl
2
+
group at an appropriate position for coordination with Ni .
We assume that the intrinsic fluorescence emitted by chemo-
2
+
sensor H3 can be first quenched by Ni , and then recovered
by addition of histidine (as shown in Scheme 2). To the best of
our knowledge, this is the first report of a computationally
designed chemosensor for selective detection of His. The suc-
cessful strategy outlined in the present work can be used in
rational design of highly selective chemosensors for other
analytes.
Prior to the synthesis of H3, we performed quantum mech-
anical calculations to compare the Ni binding abilities of H3, Fig. 3 Fluorescence emission spectra of H3 upon addition of Ni . (a)
2
+
2+
2
+
H3 concentration in MOPS buffer (50 mM, pH 7.2) was 5 μM. Ni con-
centration was 1–10 μM. Excitation and emission wavelengths were 336
and 455 nm, respectively. Slit: 2.5 nm, 5 nm. (b) Graph of the fluor-
His and other amino acids. Here, we do not need to consider
all the amino acids, but only His, Asp and Glu, which are most
2
+
commonly involved in coordinating with Ni . To compare the
binding abilities more directly, we can keep one molecule of
2+
escence responses to the Ni ion (1 μM–10 μM). Inset shows fluor-
escence intensity of H3 (5 μM) at 455 nm as a function of Ni
2+
2
+
His coordinating with Ni in all the systems considered and concentration (0–6 μM).
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structures of Ni-His-H3 and Ni-His-Asp complexes are shown 2His, Ni-His-H3, Ni-His-Asp, and Ni-His-Glu models are −71.4,
−
1
in Fig. 2. In all calculations, we assumed that the coordination −70.4, −67.8, and −65.1 kcal mol , respectively, indicating
2
+
2+
mode of Ni with the ligands is similar to that found in the that the binding of Ni with H3 should be weaker than that of
Ni-2His crystal structure, i.e., hexacoordinated. All calculations His but stronger than that of other amino acids such as Asp
were performed in the framework of density functional theory and Glu.
1
2
13
2+
(
DFT) using Gaussian 09 software. The M06-L functional
with a def2-TZVP basis set for the metal ion and a 6-31+G* addition of amino acids were then investigated (Fig. 3). We
basis set for light atoms was employed in structural optimi- first prepared a solution of the H3-Ni complex by mixing the
4 2
zations, while a combination of 6-311++G** for light atoms H3 solution and a Ni(ClO ) solution (H3/Ni , 1/1 equiv.). The
and def2-TZVP for the metal ion was used in energy refine- H3-Ni complex was relatively insensitive to pH in the 6.5–8.0
ment. Solvation effects were taken into consideration by using range (Fig. S4†). Fluorescence emission spectra of histidine
the SMD solvation model with water as the solvent. The titration were measured under physiological conditions
details of the interaction energy calculation method are sum- (50 mM MOPS) with λex: 350 nm. It was found that the fluo-
marized in the ESI (Tables S1, S2, and Fig. S1†). According to rescence intensity increased with the addition of histidine
The fluorescence changes in the H3-Ni complex upon the
1
4
2
+
2
+
2
+
1
5
2
+
the calculation results, the interaction energies for the Ni- (Fig. 4). The H3-Ni complex exhibited a maximum absorp-
tion at 360 nm, which gradually shifted to a shorter wavelength
2
+
2+
Fig. 4 Emission spectra of the H3-Ni complex in the presence of his-
tidine (0–100 µM) in MOPS (50 mM, pH 7.2) solution. Excitation and
emission wavelengths were 350 and 454 nm, respectively. Slit: 2.5 nm,
Fig. 6 Job’s plot of the [H3 + Ni ] complex in MOPS (50 mM, pH 7.2)
2+
buffer solution. The total concentration of [H3 + Ni ] and histidine was
10 μM. Excitation and emission wavelengths were 350 and 454 nm,
respectively, slit: 2.5 nm–5 nm.
2
+
5
nm. The H3-Ni complex was prepared in situ by mixing H3 and
Ni(ClO (H3: 5 µM, Ni(ClO : 5 µM).
4
)
2
4 2
)
2+
Fig. 7 Fluorescence emission spectra of the H3-Ni complex (5 μM)
upon addition of different amino acids (100 μM) in MOPS (50 mM, pH
7.2) buffer solution. Concentration of histidine was 50 μM. Excitation
wavelength was 350 nm. Black bars represent addition of different
amino acids to H3-Ni buffer solution. Red bars represent subsequent
addition of 50 μM histidine to the above solution.
2+
Fig. 5 Absorption spectra of [H3 + Ni ] (5 μM) for different concen-
trations of histidine (0–80 µM) in MOPS (50 mM, pH 7.2) buffer solution.
Excitation and emission wavelengths were 350 and 454 nm, respectively.
Slit: 2.5 nm, 5 nm.
2+
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at 330 nm close to the H3 absorption with the sequential identical conditions (red column); the observed fluorescence
addition of histidine. Fig. 5 illustrates that histidine displaced increased immediately upon the addition of histidine into
2
+
H3 to bind with Ni , liberating H3 from the quenching solutions that already contained the different amino acids and
2
+
environment and turning the fluorescence “on”. Two isosbes- the H3-Ni complex.
tic points at 344 nm and 384 nm were also observed (Fig. 5). Further evidence for H3 chelating the Ni ion with 1 : 1
A Job’s plot analysis was also performed indicating that the stoichiometry is provided directly by ESI Mass (Fig. S6†), and it
2
+
2
+
ratio of H3-Ni and L-histidine was 1 : 2 (Fig. 6).
can also be seen that the 7-hydroxyl of coumarin in the result-
2
+
To confirm the specificity of the fluorescence response of ing H3-Ni complex is deprotonated. To prove that the
2
+
the H3-Ni complex to histidine, we next measured the fluore- 7-hydroxyl is indeed involved in coordination, we synthesized
scence spectra. As shown in Fig. 7 (black column), while no the reference compound H3-1 (Scheme 3), where 7-hydroxyl is
fluorescence intensity changes were observed in the emission replaced by 7-methoxy, and tested its ability to bind different
spectra of the other 19 amino acids, a significant fluorescence metal ions by UV-absorbance and fluorescence spectroscopy
enhancement was observed in the presence of histidine under (Fig. S7†). We found that the Ni ion had nearly no or low
response to H3-1 (Fig. 8). The unchanged absorbance and fluo-
2
+
rescence indicated that H3-1 cannot be effectively combined
with the above metal ions. These results proved that the
7-hydroxyl is one of the chelating sites in H3 in the process of
coordination with these metal ions.
In summary, based on the computational predictions, a
2
+
new highly selective “turn-on” fluorescent sensor (H3-Ni ) for
L-histidine was designed and synthesized. Experimental data
2
+
proved that the H3-Ni complex exhibits excellent selectivity
for L-histidine over the other 19 amino acids in a neutral
buffer solution. The recognition mechanism was also
deduced; this consists of two L-histidine molecules binding
with Ni and releasing H3 to enable fluorescence recovery.
This methodology shows great potential for practical appli-
cation, such as the detection of other disease-associated bio-
markers and pharmaceuticals and in clinical analysis.
Scheme 3 The structure of H3-1.
2
+
This work was supported by the National Natural Science
Foundation of China (grants 81222046, 81230076 and
21403199), the Shanghai Committee of Science and Techno-
logy (grant 14431902100) and China Postdoctoral Science
Foundation (No. 2014M552010).
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