U.S. patent application number 16/610085 was filed with the patent office on 2021-02-18 for fluorescent probe for detecting nitroreductase and preparation method and use thereof in enzymatic reaction.
This patent application is currently assigned to SOUTH CHINA UNIVERSITY OF TECHNOLOGY. The applicant listed for this patent is SOUTH CHINA UNIVERSITY OF TECHNOLOGY. Invention is credited to Ling NI, Shuizhu WU, Lingfeng XU, Fang ZENG.
Application Number | 20210048393 16/610085 |
Document ID | / |
Family ID | 1000005235996 |
Filed Date | 2021-02-18 |
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United States Patent
Application |
20210048393 |
Kind Code |
A1 |
WU; Shuizhu ; et
al. |
February 18, 2021 |
FLUORESCENT PROBE FOR DETECTING NITROREDUCTASE AND PREPARATION
METHOD AND USE THEREOF IN ENZYMATIC REACTION
Abstract
The present invention relates to a fluorescent probe for
detecting nitroreductase and a preparation method and use thereof
in enzymatic reactions, belonging to the field of industrial
analysis and detection. The fluorescent probe is
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-yl)v-
inyl)quinolin-1-ium-1-yl)propane-1-sulfonate. The fluorescent probe
of the present invention, with the introduction of hydrophilic
groups, sulfonate and quinolinium, the probe's hydrophilicity is
enhanced, under the enzymatic catalysis of nitroreductase (NTR),
1,6-rearrangement and elimination reaction occurs, and hydroxyl
group is generated. Detection and analysis of the NTR in the
industrial enzymatic reactions can be realized due to the change of
fluorescence which is induced by the intramolecular charge transfer
(ICT) effect. This method has such advantages as easy preparation,
high yield and being suitable for detecting high concentration of
enzyme in the enzymatic reactions, and it shows an extensive
application prospect in the field of enzyme-detection in the
industrial enzymatic reaction systems.
Inventors: |
WU; Shuizhu; (Guangdong,
CN) ; XU; Lingfeng; (Guangdong, CN) ; NI;
Ling; (Guangdong, CN) ; ZENG; Fang;
(Guangdong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SOUTH CHINA UNIVERSITY OF TECHNOLOGY |
Guangdong |
|
CN |
|
|
Assignee: |
SOUTH CHINA UNIVERSITY OF
TECHNOLOGY
Guangdong
CN
|
Family ID: |
1000005235996 |
Appl. No.: |
16/610085 |
Filed: |
March 26, 2019 |
PCT Filed: |
March 26, 2019 |
PCT NO: |
PCT/CN2019/079616 |
371 Date: |
November 1, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09K 2211/1018 20130101;
G01N 21/6428 20130101; G01N 21/76 20130101; G01N 2021/6432
20130101; C09K 11/06 20130101; C07D 215/14 20130101; C12Q 1/26
20130101 |
International
Class: |
G01N 21/76 20060101
G01N021/76; C12Q 1/26 20060101 C12Q001/26; C07D 215/14 20060101
C07D215/14; C09K 11/06 20060101 C09K011/06; G01N 21/64 20060101
G01N021/64 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 2018 |
CN |
201811454833.X |
Claims
1. A fluorescent probe for detecting nitroreductase, wherein the
fluorescent probe is
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate, having a structural
formula as follows: ##STR00003##
2. A preparation method of the fluorescent probe for detecting
nitroreductase according to claim 1, wherein comprising the
following steps: (1) dissolving
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde into
dimethyl sulfoxide and dissolving 1-(bromomethyl)-4-nitrobenzene
into tetrahydrofuran, followed by ultrasonic treatment respectively
and then mixing together, adding cesium carbonate, controlling a
reaction temperature in the range of 50.degree. C.-150.degree. C.,
separating and purifying a reaction product to obtain
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in yellow solid powder; (2) dissolving
3-(4-methylquinoline-1-bromine)propane-1-sulfonate into pyridine,
then adding acetic acid, followed by sufficient mixing, then adding
the
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
obtained in step (1), heating to 25.degree. C.-80.degree. C. with
stirring to perform reaction, separating and purifying a reaction
product to obtain
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphe-
nyl]-4-yl)vinyl)quinoline-1-bromine)propane-1-sulfonate in
purplish-red solid powder.
3. The preparation method according to claim 2, wherein a molar
ratio of dosages of
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde to
1-(bromomethyl)-4-nitrobenzene in step (1) is 1:1.5-2.
4. The preparation method according to claim 2, wherein a molar
ratio of dosages of cesium carbonate to
1-(bromomethyl)-4-nitrobenzene in step (1) is 4 5:1.
5. The preparation method according to claim 2, wherein a molar
ratio of dosages of
3-(4-methylquinoline-1-bromine)propane-1-sulfonate to
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in step (2) is 1:1-2.
6. The preparation method according to claim 2, wherein a molar
ratio of dosages of acetic acid to
3-(4-methylquinoline-1-bromine)propane-1-sulfonate in step (2) is 2
4:1.
7. The preparation method according to claim 2, wherein the
reaction in step (1) lasts for 5 hours to 48 hours.
8. The preparation method according to claim 2, wherein the
reaction in step (2) lasts for 3 hours to 24 hours.
9. The preparation method according to claim 2, wherein the
separating and purifying in step (1) are as follows: cooling a
reaction mixture to room temperature, extracting with
dichloromethane/deionized water, collecting an organic phase
followed by drying and filtering, removing a solvent by rotary
evaporation, and purifying the obtained solid via a silica gel
chromatographic column; and wherein the separating and purifying in
step (2) are as follows: cooling a reaction mixture to room
temperature, removing a solvent by rotary evaporation, then adding
ethyl acetate and washing with hydrochloric acid and saturated salt
solution respectively, followed by drying and filtering, removing a
solvent by rotary evaporation, and purifying the obtained solid via
a silica gel chromatographic column.
10. (canceled)
Description
BACKGROUND
Technical Field
[0001] The present invention relates to the technical field of
industrial analysis and detection, and specifically relates to a
fluorescent probe for detecting nitroreductase and a preparation
method and use thereof in enzymatic reaction.
Description of Related Art
[0002] Nitro compounds are widely used in the fields of medicine,
dyes, pesticides, explosives, and etc. However, owing to their
carcinogenesis to human, most of the nitro compounds may cause many
diseases and thus are harmful to human health. Amine compounds are
essential to the synthesis of various fine chemical products and
intermediates such as pesticides, medicine, dyes, synthetic resins,
surfactants, and etc., for the introduction of amino group makes
the change of function of the fine chemicals possible. For example,
the introduction of amino group causes a red shift in the
absorption and emission spectra of the compounds, the introduction
of amino group to the ortho-position of a dye chromophore may
result in a color change of the dye, and the introduction of amino
group may alter the printing and dyeing property of the dye. More
importantly, the amine compounds show less toxicity compared with
the nitro compounds. At present, most of the aromatic amine
compounds in the industry are prepared from the reduction of
aromatic nitro compounds. Therefore, the reaction of reducing nitro
group into amino group plays an important role in the industrial
production.
[0003] Generally, the major reduction methods in the industry are
as follows: reduction with iron powder, reduction with alkali
sulfide, catalytic hydrogenation reduction and etc. However, these
methods still have such drawbacks as complicated technological
process, complex post-treatment, numerous wastes generated during
the process, and high preparation cost. In recent years, biological
method which can reduce the nitro compound into the amino compound
is developing rapidly and it has become one of the methods that are
environmentally-friendly and green-chemistry approaches. Enzyme,
also called as ferment, being a kind of biocatalyst, is a
biomacromolecule having biocatalytic function. Most of the enzymes
are proteins, having relatively good biocompatibility and
environmental friendliness. Enzymatic catalysis is regarded as a
catalytic reaction between homogeneous phase catalytic reaction and
heterogeneous phase catalytic reaction, which possesses not only
characteristics of the general catalysts but also uniquenesses that
differ from those of the general catalysts. Compared with the
general catalysts, the enzymatic catalyst shows several advantages
as follows: 1) an enzymatic reaction has high efficiency; 2) the
enzymatic reaction has high specificity; 3) the enzymatic reaction
is rather mild; 4) the diversity of the enzymes results in the
diversity of the enzymatic reactions; 5) the performance of the
enzymatic reaction can be adjusted by modulating the activity of
the enzyme. However, since most of the enzymes are proteins, the
activity of the enzyme may be affected by temperature, acidity or
alkalinity, and concentration of the substrate, and even more the
enzyme may be inactivated. Therefore, performing the enzymatic
reaction in the aqueous media is preferable and conducive to
reducing the environmental pollution caused by organic solvents and
to facilitating the enzymatic reaction.
[0004] In recent years, with the improvement of the separation and
purification technologies for enzymes, using a free enzyme to
directly act on the reduction of nitro compounds has become a new
field of the bio-organic chemistry. In particular, using
oxidoreductases to reduce the nitro compounds has become a hot
topic of research. The oxidoreductases that are mainly used for
such kind of enzymatic reactions at present include nitroreductase
and nitrate reductase. Particularly, nitroreductase is a kind of
enzyme having a wide range of application and the source thereof is
widely available. The conditions of the enzymatic reaction are
mild, and the effect thereof is better. The research of the
enzymatic reaction has been conducted more in-depth, and the
reaction mechanism is relatively mature. In the meantime, there are
two types of enzymes including the one sensitive to oxygen and the
other one insensitive to oxygen, with wide range of applications.
Thus, in order to guarantee the efficacy and stability of the
enzymatic reactions that convert the nitro compounds into amine
compounds in the industrial applications, it is of great
significance to study and develop a fluorescent probe which is
capable of measuring such kind of nitroreductase.
[0005] Fluorescence method has several excellent characteristics in
analytic detection such as good selectivity, high sensitivity,
quick response speed and ease of operation. Also, fluorescent
compounds can fulfill the different needs of detecting various
analytes, for they are easy to be designed, modified and improved
in chemical structure. Therefore, the fluorescence method is
particularly suitable for the analysis and detection of
nitroreductase in the enzymatic reactions in industry. Chinese
patent CN201610050741.X prepares a two-photon fluorescent probe for
detecting nitroreductase in hypoxic region. The aromatic nitro in
the compound can be reduced to an aromatic amino group by the
nitroreductase, and the 1,6-rearrangement and elimination reaction
occurs, releasing a fluorophore and resulting in a change of
fluorescence. However, such fluorescent probe has poor
water-solubility and exhibits aggregation-caused quenching of
fluorescence. So, it is difficult to realize the detection and
analysis of enzyme of high concentration and in an aqueous media.
In the meantime, the two-photon detection instruments are rather
complicated and expensive, the probe's application field mainly
focuses on hypoxia in cells. Chinese patent CN201610471060.0
discloses a two-photon fluorescent probe for detecting
nitroreductase, wherein the nitro group is directly coupled to the
fluorophore, and with the increasing concentration of
nitroreductase, the fluorescence intensity increases gradually with
an emission wavelength ranging from 425 nm to 475 nm and from 500
nm to 550 nm. The probe is not suitable for use in the aqueous
media, and fluorescence quenching would easily occur when a high
concentration of nitroreductase is present. This probe is mainly
used in the biological field such as cell imaging, without
application potential for industrial enzymatic reaction in large
scale.
[0006] When a fluorescent material with aggregation-induced
emission (AIE) feature exists in the form of monomolecularly
dissolved state in solution, electrons in the excited state return
to the ground state through the intramolecular motions; when the
molecules are in the aggregation state, the intramolecular motions
are restricted and the electrons in the excited state may return to
the ground state only through the radiative pathway, and thus
enhanced fluorescence can be observed which has extensive
applications in many fields. Chinese patent CN201710009923.7
discloses a fluorescent probe based on AIE feature for detecting
nitroreductase, wherein a nitro group is directly coupled to
tetraphenylethylene. Before response to nitroreductase, it shows
strong fluorescence due to the D-.pi.-A electronic effect; and
after the response, the fluorescence becomes faint and blue shift
occurs due to the D-.pi.-D structure. The detection of
nitroreductase is realized by using such change of fluorescence.
However, the probe is mainly used in the cells and unable to be
applied in the detection and analysis of the concentration of
enzyme in the industrial enzymatic reaction systems.
[0007] Although there's already some progress of the fluorescent
probes for detecting nitroreductase in the field of biological
detection and imaging, it is still rare to apply the fluorescent
probes to detection and analysis of the enzymatic activity in the
industrial enzymatic reactions. It is clear that, there's urgent
need of developing a probe with specific catalysis effect for
detecting and analyzing enzymatic activity for the field of
industrial enzymatic reactions.
SUMMARY
[0008] In order to solve the drawbacks and deficiencies in the
prior art, the primary objective of the present invention is to
provide a fluorescent probe compound. The fluorescent probe has
aggregation-induced emission feature. With the introduction of
hydrophilic groups, sulfonate and quinolinium, the hydrophilicity
of the probe is enhanced, a 1,6-rearrangement and elimination
reaction occurs under the catalysis of a nitroreductase (NTR), and
a hydroxyl group is generated. Detection and analysis of NTR in the
industrial enzymatic reactions can be realized due to the change of
fluorescence which is induced by the intramolecular charge transfer
(ICT) effect.
[0009] Another objective of the present invention is to provide a
preparation method of the fluorescent compound.
[0010] Another objective of the present invention is to provide use
of the fluorescent compound for detecting activity of the
nitroreductase in the industrial enzymatic reactions for converting
aromatic nitro into aromatic amino.
[0011] The objectives of the present invention are realized by the
following technical solutions.
[0012] A fluorescent probe for detecting nitroreductase, wherein
the fluorescent probe is
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-yl)v-
inyl)quinoline-1-bromine)propane-1-sulfonate, having a structural
formula as follows:
##STR00001##
[0013] A preparation method of the above fluorescent probe for
detecting nitroreductase, includes the following steps:
[0014] (1) dissolving
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde into
dimethyl sulfoxide to obtain a solution 1, dissolving
1-(bromomethyl)-4-nitrobenzene into tetrahydrofuran to obtain a
solution 2, subjecting the solution 1 and the solution 2 to
ultrasonic treatment respectively and then mixing together, adding
cesium carbonate to perform a reaction, controlling a reaction
temperature in the range of 50.degree. C.-150.degree. C.,
separating and purifying a reaction product to obtain
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in yellow solid powder;
[0015] (2) dissolving
3-(4-methylquinoline-1-bromine)propane-1-sulfonate into pyridine,
then adding acetic acid, followed by sufficient mixing, then adding
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
obtained in step (1), heating to 25.degree. C.-80.degree. C. with
stirring to perform reaction, separating and purifying a reaction
product to obtain
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphe-
nyl]-4-yl)vinyl)quinoline-1-bromine)propane-1-sulfonate in
purplish-red solid powder.
[0016] Preferably, a molar ratio of dosages of
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde to
1-(bromomethyl)-4-nitrobenzene in step (1) is 1:(1.5-2).
[0017] Preferably, a molar ratio of dosages of cesium carbonate to
1-(bromomethyl)-4-nitrobenzene in step (1) is (4-5):1.
[0018] Preferably, a molar ratio of dosages of
3-(4-methylquinoline-1-bromine)propane-1-sulfonate to
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in step (2) is 1:(1-2).
[0019] Preferably, a molar ratio of dosages of acetic acid to
3-(4-methylquinoline-1-bromine)propane-1-sulfonate in step (2) is
(2-4):1.
[0020] Preferably, the reaction in step (1) lasts for 5 hours to 48
hours.
[0021] Preferably, the reaction in step (2) lasts for 3 hours to 24
hours.
[0022] Preferably, the separating and purifying in step (1) are as
follows: cooling a reaction mixture to room temperature, extracting
the reaction mixture with dichloromethane/deionized water,
collecting an organic phase followed by drying and filtering,
removing a solvent by rotary evaporation, and purifying the
obtained solid via a silica gel chromatographic column.
[0023] Preferably, the separating and purifying in step (2) are as
follows: cooling a reaction mixture to room temperature, removing a
solvent by rotary evaporation, then adding ethyl acetate and
washing with hydrochloric acid and saturated salt solution
respectively, followed by drying and filtering, removing a solvent
by rotary evaporation, and purifying the obtained solid via a
silica gel chromatographic column.
[0024] Use of the fluorescent probe for detecting nitroreductase in
detecting and analyzing the nitroreductase in an enzymatic reaction
of converting aromatic nitro into aromatic amino in the
industry.
[0025] The fluorescent compound, the product obtained in the
present invention, is
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate (TAE-NQS), with a
molecular formula of C.sub.45H.sub.37N.sub.3O.sub.6S and a relative
molecular weight of 747.24. Being purplish-red and odourless solid
powder, the TAE-NQS is slightly soluble in water and easily soluble
in solvents such as DMSO and DMF. Having good photostability and
being non-toxic, the compound is suitable for being used in
enzymatic reactions in aqueous media. Since the TAE-NQS has a
triphenylamine group, and the fluorescence is significantly
quenched due to the nitro group on the recognition moiety, there's
hardly fluorescence emission near 750 nm under the excitation of
500 nm. When the TAE-NQS reacts with the nitroreductase, a
1,6-rearrangement and elimination reaction occurs, and hydroxyl
group is generated through cleavage reaction (the product is
3-(4-(2-(4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-yl)vinyl)quinolin-
e-1-bromine)propane-1-sulfonate, TAE-NQS-OH). Meanwhile, the probe
product after response also has the aggregation-induced emission
feature due to the existence of the AIE-active triphenylamine
group. The fluorescent probe of the present invention can be used
for detecting the activity of nitroreductase in the industrial
enzymatic reactions for converting aromatic nitro into aromatic
amino. The recognition mechanism is shown as follows:
##STR00002##
[0026] The present invention provides a fluorescent probe for
detecting the nitroreductase in the reactions of converting phenyl
nitro into phenyl amino in the industrial enzymatic reactions. The
probe has merely weak fluorescence, but with the enzymatic reaction
by nitroreductase, the phenyl nitro is reduced into phenyl amino,
and the 1,6-rearrangement and elimination reaction occurs to
generate the hydroxyl group through cleavage reaction resulting in
strong fluorescence.
[0027] Compared with the prior art, the present invention has the
following advantages and beneficial effects:
[0028] (1) The fluorescent compound, TAE-NQS, of the present
invention has aggregation-induced emission feature. In most cases,
in order to improve the reaction efficiency of the enzymatic
reaction in the field of chemical industry, high concentration of
enzyme would usually be added to the reaction system. Whereas with
the existence of high concentration of substrate, the present probe
will not be quenched, and the detecting effect with good
sensitivity and accuracy can be obtained.
[0029] (2) After the enzymatic catalysis reaction by
nitroreductase, 1,6-rearrangement and elimination reaction occurs
in the probe TAE-NQS of the present invention. After the cleavage
reaction, the intramolecular "mechanical rotations" and also the
non-radiative energy dissipation pathway from the excited state to
the ground state are restricted because of the existence of
triphenylamine, hence the probe still has aggregation-induced
emission feature. Meanwhile, hydroxyl group is generated so that
the fluorescence changes. Therefore, the probe TAE-NQS can be used
in the detection and analysis of nitroreductase in the industrial
enzymatic reactions, especially the reactions of converting
aromatic nitro into aromatic amino.
[0030] (3) The fluorescent probe of the present invention has
relatively long emission wavelength reaching up to 750 nm, and has
significant fluorescence-enhancement effect.
[0031] (4) The fluorescent probe of the present invention is
suitable for relatively harsh and complicated environment in the
industrial enzymatic reactions, having good structural stability
and being easy to be promoted and applied in the enzymatic
reactions in chemical industry.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows a synthetic route of a fluorescent probe
compound of the present invention.
[0033] FIG. 2 shows a .sup.1H-NMR spectrum of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in Example 1.
[0034] FIG. 3 shows a .sup.1H-NMR spectrum of
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate in Example 1.
[0035] FIG. 4 shows absorption spectra of the fluorescent probe of
the present invention before and after response.
[0036] FIG. 5 shows fluorescence spectra of the fluorescent probe
of the present invention before and after response.
[0037] FIG. 6 shows fluorescence spectra displaying the
aggregation-induced emission feature of the response product
TAE-NQS-OH.
[0038] FIG. 7 shows fluorescence spectra of the fluorescent probe
TAE-NQS in response to nitroreductase for different time.
[0039] FIG. 8 shows fluorescence spectra of the fluorescent probe
TAE-NQS in response to different concentrations of
nitroreductase.
DESCRIPTION OF THE EMBODIMENTS
[0040] The present invention is further described in detail with
the examples and accompanied drawings, and implementation of the
present invention is not limited to these.
[0041] A synthetic route of a fluorescent probe compound of the
present invention is shown as FIG. 1.
EXAMPLE 1
[0042] (1) 365 mg of
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde was
dissolved in 10 mL of dimethyl sulfoxide, 324 mg of
1-(bromomethyl)-4-nitrobenzene was dissolved in 10 mL of
tetrahydrofuran, followed by ultrasonic treatment respectively, and
then they were mixed together. 1.96 g of cesium carbonate was added
to perform a reaction of which a reaction temperature was
maintained at 50.degree. C. and which lasted for 5 hours. An
obtained reaction mixture was cooled to room temperature and
extracted with dichloromethane/deionized water, an organic phase
was collected, dried and filtered, the solvent was removed by
rotary evaporation, and an obtained solid was purified via a silica
gel chromatographic column (an eluent used is
dichloromethane/petroleum ether, V/V=2:1). A product, 405 mg of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbald-
ehyde in yellow solid powder, was obtained (with a yield of 81%).
The product was characterized by .sup.1H-NMR, wherein .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. (TMS, ppm): 10.58 (s, 1H), 8.27 (d,
J=8.6 Hz, 2H), 8.07 (d, J=2.4 Hz, 1H), 7.74 (dd, J=8.6, 2.4 Hz,
1H), 7.65 (d, J=8.5 Hz, 2H), 7.43 (d, J=8.6 Hz, 2H), 7.28 (s, 1H),
7.24 (d, J=10.8, 4.8 Hz, 2H), 7.12 (d, J=8.3 Hz, 6H), 7.04 (dd,
J=12.8, 5.6 Hz, 4H), 5.33 (s, 2H). Specifically, the proton peak at
10.58 ppm is the proton peak of the aldehyde group in the structure
of salicylaldehyde, the proton peaks at 8.02 ppm, 7.75 ppm and 7.28
ppm are proton peaks of three hydrogen atoms in the aromatic ring
of salicylaldehyde, the proton peaks at 8.26 ppm and 7.66 ppm are
proton peaks of four hydrogen atoms in
1-(bromomethyl)-4-nitrobenzene, the characteristic peaks of four
protons on one of the aromatic rings of triphenylamine are near
7.40 ppm and 7.27 ppm, the characteristic peaks of the rest 10
protons on the aromatic rings of triphenylamine lie at 7.0 ppm-7.24
ppm, and the proton peaks at 5.33 ppm are the characteristic peaks
of methylene in 1-(bromomethyl)-4-nitrobenzene. It can be
determined through the analysis of .sup.1H-NMR spectrum that the
product synthesized is the target intermediate. The .sup.1H-NMR
spectrum of the obtained product is shown as FIG. 2.
[0043] (2) 265 mg of
3-(4-methylquinoline-1-bromine)propane-1-sulfonate was dissolved in
10 mL of pyridine, and 114 .mu.L of acetic acid was added, followed
by sufficient mixing. Then, 500 mg of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
was added, heated to 25.degree. C. with stirring to perform a
reaction which lasted for 3 hours. An obtained reaction mixture was
cooled to room temperature and subjected to rotary evaporation to
remove the solvent, and then excessive ethyl acetate was added. The
mixture was washed with hydrochloric acid for 3 times and salt
solution for 1 time respectively, dried with anhydrous sodium
sulfate, and subjected to suction filtration and rotary evaporation
to remove solvent. An obtained solid was purified via a silica gel
chromatographic column (an eluent used is dichloromethane/methanol,
V/V=5:1). 448 mg of
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate in purplish-red solid
powder was obtained (with a yield of 60%). The product was
characterized by .sup.1H-NMR, wherein .sup.1H NMR (600 MHz, DMSO)
.delta. (TMS, ppm): 9.41 (d, J=6.5 Hz, 1H), 8.81 (d, J=8.2 Hz, 1H),
8.64 (d, J=9.0 Hz, 1H), 8.46 (t, J=11.2 Hz, 2H), 8.37-8.29 (m, 4H),
8.27-8.22 (m, 1H), 7.98-7.94 (m, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.72
(dd, J=20.4, 9.8 Hz, 3H), 7.37-7.31 (m, 4H), 7.30 (d, J=8.8 Hz,
1H), 7.11-7.04 (m, 8H), 5.53 (s, 2H), 5.02 (t, J=7.5 Hz, 2H),
2.12-2.05 (m, 2H), 1.76-1.69 (m, 2H). Specifically, the proton
peaks at positions a, b and c are the characteristic peaks of three
methylene protons on the alkylsulfonate respectively, the proton
peaks at position d are the characteristic peaks of the methylene
protons in 1-(bromomethyl)-4-nitrobenzene, the proton peaks at
position e are the characteristic peaks of the protons on the
conjugated double bond structure, the protons at position g are the
characteristic peaks of 16 hydrogen protons on the triphenylamine
and the aromatic ring of salicylaldehyde coupled thereto, the
proton peaks of the quinolinium and the aromatic ring of
1-(bromomethyl)-4-nitrobenzene and those near the double bond of
salicylaldehyde lie at 7.78 ppm-9.5 ppm, and there are 11
characteristic peaks of hydrogen protons in total. It can be
determined through the analysis on .sup.1H-NMR spectrum that the
product synthesized is the target intermediate. The .sup.1H-NMR
spectrum of the obtained product is shown as FIG. 3.
EXAMPLE 2
[0044] (1) 365 mg of
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde was
dissolved in 10 mL of dimethyl sulfoxide, 389 mg of was dissolved
in 10 mL of tetrahydrofuran, followed by ultrasonic treatment
respectively, and then they were mixed together. 2.64 g of cesium
carbonate was added to perform a reaction of which a reaction
temperature was maintained at 100.degree. C. and which lasted for
24 hours. An obtained reaction mixture was cooled to room
temperature and extracted with dichloromethane/deionized water, an
organic phase was collected, dried and filtered, the solvent was
removed by rotary evaporation, and an obtained solid was purified
via a silica gel chromatographic column (an eluent used is
dichloromethane/petroleum ether, V/V=2:1). A product, 415 mg of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in yellow solid powder, was obtained (with a yield of 83%).
[0045] (2) 265 mg of
3-(4-methylquinoline-1-bromine)propane-1-sulfonate was dissolved in
10 mL of pyridine, and 171 .mu.L of acetic acid was added, followed
by sufficient mixing. Then, 750 mg of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
was added, heated to 50.degree. C. with stirring to perform a
reaction which lasted for 12 hours. An obtained reaction mixture
was cooled to room temperature and subjected to rotary evaporation
to remove the solvent, and then excessive ethyl acetate was added.
The mixture was washed with hydrochloric acid for 3 times and salt
solution for 1 time respectively, dried with anhydrous sodium
sulfate, and subjected to suction filtration and rotary evaporation
to remove the solvent. An obtained solid was purified via a silica
gel chromatographic column (an eluent used is
dichloromethane/methanol, V/V=5:1). 485 mg of
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate in purplish-red solid
powder was obtained (with a yield of 65%).
[0046] The characterization results for the obtained intermediate
compound and the fluorescent probe compound TAE-NQS in the present
example are the same as those in Example 1.
EXAMPLE 3
[0047] (1) 365 mg of
4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde was
dissolved in 10 mL of dimethyl sulfoxide, 432 mg of
1-(bromomethyl)-4-nitrobenzene was dissolved in 10 mL of
tetrahydrofuran, followed by ultrasonic treatment respectively, and
then they were mixed together. 3.26 g of cesium carbonate was added
to perform a reaction of which a reaction temperature was
maintained at 150.degree. C. and which lasted for 48 hours. An
obtained reaction mixture was cooled to room temperature and
extracted with dichloromethane/deionized water, an organic phase
was collected, dried and filtered, solvent was removed by rotary
evaporation, and an obtained solid was purified via a silica gel
chromatographic column (an eluent used is dichloromethane/petroleum
ether, V/V=2:1). A product, 430 mg of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
in yellow solid powder, was obtained (with a yield of 86%).
[0048] (2) 265 mg of
3-(4-methylquinoline-1-bromine)propane-1-sulfonate was dissolved in
10 mL of pyridine, and 228 .mu.L of acetic acid was added, followed
by sufficient mixing. Then, 1000 mg of
4'-(diphenylamino)-3-((4-nitrobenzyl)oxy)-[1,1'-biphenyl]-4-carbaldehyde
was added, heated to 80.degree. C. with stirring to perform a
reaction which lasted for 24 hours. An obtained reaction mixture
was cooled to room temperature and subjected to rotary evaporation
to remove solvent, and then excessive ethyl acetate was added. The
mixture was washed with hydrochloric acid for 3 times and salt
solution for 1 time respectively, dried with anhydrous sodium
sulfate, and subjected to suction filtration and rotary evaporation
to remove solvent. An obtained solid was purified via a silica gel
chromatographic column (an eluent used is dichloromethane/methanol,
V/V=5:1). 470 mg of
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate in purplish-red solid
powder was obtained (with a yield of 63%).
[0049] The characterization results for the obtained intermediate
compound and the fluorescent probe compound TAE-NQS in the present
example are the same as those in Example 1.
[0050] Tests of the obtained fluorescent probe compound of the
present invention used to detect activity of nitroreductase in an
enzymatic reaction system:
[0051] 1.5 mg of the solid fluorescent compound,
3-(4-(2-(4'-(diphenylamino)-3-((4-nitrobenzyl)oxyl)-[1,1'-biphenyl]-4-yl)-
vinyl)quinoline-1-bromine)propane-1-sulfonate (TAE-NQS, prepared in
Example 1), was dissolved in 2 mL of DMSO, and prepared into a 1 mM
stock solution of the fluorescent compound. Before the test,
concentration of the fluorescent compound was diluted to 10 .mu.M
with a phosphate buffer (10 mM, pH 7.4), and a solution system to
be tested containing 1% DMSO was obtained.
[0052] (1) Fluorescence property of the probe compound TAE-NQS:
[0053] 3 .mu.L of the above-mentioned stock solution of the
fluorescent compound was drawn, and a blank control sample and test
samples were prepared with PBS buffer solution (10 mM, pH=7.4). The
concentration of the probe compound in the blank sample was 10
.mu.M, without adding nitroreductase and coenzyme substance
(reduced form of nicotinamide adenine dinucleotide (NADH)), as the
control sample. The concentration of the probe compound in the test
samples was controlled to 10 .mu.M, and the final concentration of
nitroreductase was controlled to 2 .mu.g/mL, and the concentration
of the coenzyme substance NADH was controlled to 100 .mu.M. The
samples were incubated at 37.degree. C. for 15 minutes, then the
absorption spectra ranging from 350 nm to 700 nm were recorded, and
the fluorescence spectra were measured under the excitation light
of 500 nm. The results are shown as FIG. 4 and FIG. 5. Compared
with the blank sample, red shift in the absorption of the test
samples occured and the fluorescence intensity changed
significantly. This is because when the NTR was present,
1,6-rearrangement and elimination reaction occurred in the probe
molecules in the test samples, after the cleavage reaction, the
generated hydroxyl group was an electron-donating group, and then
3-(4-(2-(4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-yl)vinyl)quinolin-
e-1-bromine)propane-1-sulfonate (TAE-NQS-OH) was formed, resulting
in the intramolecular charge transfer effect (ICT effect) and thus
a red shift in the fluorescence. Meanwhile, the fluorescent
molecule has a relatively good AIE effect due to the existence of
triphenylamine--the AIE group. The test results of the AIE effect
of TAE-NQS-OH are shown as FIG. 6 (by adjusting a ratio of water to
N,N-dimethylformamide to be 0%-99% and controlling the
concentration in each test solution to be 10 .mu.M, the test
solutions for the aggregation-induced emission feature were
prepared).
[0054] (2) Fluorescent response test of the probe compound TAE-NQS
to different concentrations of NTR in PBS buffer, and response time
test:
[0055] When the concentration of NTR was 2 .mu.g/mL and the
concentration of the probe was 10 .mu.M, the fluorescence intensity
varied over time, shown as FIG. 7. Additionally, a series of PBS
buffer solutions (pH=7.4) with the concentration of the probe being
10 .mu.M and the concentration of NTR being 0, 0.25, 0.5, 0.75, 1,
1.5, 2, 3, 5 .mu.g/mL respectively, were prepared. By controlling
the temperature to be 37.degree. C. and the incubation time to be 5
minutes, the fluorescence spectra under the excitation wavelength
of 500 nm for each test sample were recorded. The test results were
shown as FIG. 8. It can be seen from FIG. 7 and FIG. 8 that the
fluorescent probe prepared by the present invention has relatively
good detecting effect on the NTR in the enzymatic reaction system.
With the increasing concentration of NTR (0 .mu.g/mL to 5
.mu.g/mL), there was sufficient response within 30 minutes, and the
fluorescence changed significantly after the response. It
demonstrates that the probe is suitable for being used in detecting
the nitroreductase in reactions of converting aromatic nitro into
aromatic amino.
[0056] This method has advantages including easy preparation, high
yield and being suitable for detecting high concentration of enzyme
in the enzymatic reactions, and it shows an extensive application
prospect in the field of enzyme-detection in the industrial
enzymatic reaction systems.
[0057] The above examples are preferable implementations of the
present invention, and the implementations of the present invention
are not limited to the above examples. Any other variation,
modification, substitution, combination and simplification that are
made without departing from the spirit and scope of the present
invention are intended to be equivalents, and should be included in
the scope of protection of the present invention.
* * * * *