CN108676024A - The near-infrared squaraine dye and its preparation method and application of phenyl boric acid modification - Google Patents
The near-infrared squaraine dye and its preparation method and application of phenyl boric acid modification Download PDFInfo
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- CN108676024A CN108676024A CN201810575198.4A CN201810575198A CN108676024A CN 108676024 A CN108676024 A CN 108676024A CN 201810575198 A CN201810575198 A CN 201810575198A CN 108676024 A CN108676024 A CN 108676024A
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- solvent
- atp
- silica gel
- column chromatography
- gel column
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- IHXWECHPYNPJRR-UHFFFAOYSA-N 3-hydroxycyclobut-2-en-1-one Chemical compound OC1=CC(=O)C1 IHXWECHPYNPJRR-UHFFFAOYSA-N 0.000 title claims abstract description 36
- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical compound OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 238000012986 modification Methods 0.000 title claims abstract description 22
- 230000004048 modification Effects 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000002904 solvent Substances 0.000 claims abstract description 33
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 16
- -1 dicyanoethenyl squaric acid Chemical compound 0.000 claims abstract description 13
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- 238000006243 chemical reaction Methods 0.000 claims description 25
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- 238000010992 reflux Methods 0.000 claims description 15
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- FLHJIAFUWHPJRT-UHFFFAOYSA-N 2,3,3-trimethylindole Chemical class C1=CC=C2C(C)(C)C(C)=NC2=C1 FLHJIAFUWHPJRT-UHFFFAOYSA-N 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
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- 238000010189 synthetic method Methods 0.000 claims description 8
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- 238000000034 method Methods 0.000 claims description 7
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- 238000010438 heat treatment Methods 0.000 claims description 3
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- 239000000047 product Substances 0.000 claims description 3
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- SVENKGKXRLHCTC-UHFFFAOYSA-N B(O)(O)O.BrCC=1C=CC=CC1 Chemical compound B(O)(O)O.BrCC=1C=CC=CC1 SVENKGKXRLHCTC-UHFFFAOYSA-N 0.000 claims description 2
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 claims description 2
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 47
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 24
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- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
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- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
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- 235000014304 histidine Nutrition 0.000 description 3
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention discloses a kind of near-infrared squaraine dyes and its preparation method and application of phenyl boric acid modification, it is the phenyl boronic acid derivative for modifying indoles and the mixing of dicyanoethenyl squaric acid derivertives, through dissolving, flow back, remove under reduced pressure solvent and silica gel column chromatography purifying, be made the near-infrared squaraine dye.The squaraine dye has preferable stability and excellent optical property, especially align substituted phenyl boric acid group, it can be combined with the c/s-diol part in ATP molecules, therefore it can be acted on by the multiple electrostatic between dyestuff, ATP and CTAB molecules, cause the change of dyestuff Absorption and fluorescence spectrum, so as to the fluorescence probe detected as ATP, fluorescence imaging for intracellular ATP detects, and the probe has preferable biocompatibility and cell permeability, lower bio-toxicity, has preferable detection sensitivity to ATP.
Description
Technical field
The invention belongs to analytical chemistry fields, and in particular to a kind of near-infrared squaraine dye of phenyl boric acid modification and its preparation
Method and its application in fluorescence imaging detects intracellular ATP.
Background technology
Adenosine 5'-triphosphate（ATP）It is made of adenine, ribose and three phosphate groups.ATP is not only to lead in living cells
Energy currency, and participate in a variety of biologies such as triphosphoric acid cycle, ion channel, neurotransmission, cell division, DNA synthesis
The signal transduction medium of process（Xu, Z.; Singh, N. J.; Lim, J. et al. Unique sandwich
stacking of pyrene-adenine-pyrene for selective and ratiometric fluorescent
sensing of ATP at physiological pH. Journal of the American Chemical Society,
2009, 131(42), 15528-15533. Ashcroft, F. M.; Gribble, F. M. ATP-sensitive K+
channels and insulin secretion: their role in health and disease.Diabetologia, 1999, 42, 903-919. Burnstock, G. Historical review: ATP as a
neurotransmitter. Trends in Pharmacological Sciences, 2006, 27, 166-176.
Shen, X.; Mizuguchi, G.; Hamiche, A. et al. A chromatin remodelling complex
involved in transcription and DNA processing. Nature, 2000, 406(6795), 541.）.
In addition, several pathological states such as the abnormal level of ATP and ischemic, hypoglycemia Parkinson's disease and angiocardiopathy are closely related
(Bush, K. T.; Keller, S. H.; Nigam, S. K. Genesis and reversal of the
ischemic phenotype in epithelial cells. The Journal of Clinical Investigation, 2000, 106(5), 621-626. Przedborski, S.; Vila, M. MPTP: a
review of its mechanisms of neurotoxicity. Clinical Neuroscience Research,
2001, 1(6), 407-418. Mäkelä, A.; Kuusi, T.; Schröder, T. Inhibition of serum
phospholipase-A2 in acute pancreatitis by pharmacological agents in vitro.Scandinavian Journal of Clinical and Laboratory Investigation, 1997, 57(5),
401-407.).Therefore, simple, quick, sensitive and accurate ATP analysis methods are established for medical clinic applications and body generation
It thanks to horizontal research to be of great significance, while in the screening of tumour medicine sensibility, Apoptosis detection, a variety of important cells activity
And content of microorganisms analysis etc. has important value.
There are many detection methods of ATP, including electrophoresis (Yangyuoru, P. M.; Dhakal, S.; Yu, Z.;
et al. Single-molecule measurements of the binding between small molecules
and DNA aptamers. Analytical Chemistry, 2012,84 (12), 5298-5303.), chemoluminescence method
(Zhang, S.; Yan, Y.; Bi, S. Design of molecular beacons as signaling probes
for adenosine triphosphate detection in cancer cells based on
chemiluminescence resonance energy transfer. Analytical Chemistry, 2009, 81
(21), 8695-8701.), biloluminescence method (Liu, B. F.; Ozaki, M.; Hisamoto, H.; et al.
Microfluidic chip toward cellular ATP and ATP-conjugated metabolic analysis
with bioluminescence detection. Analytical Chemistry, 2005, 77(2), 573-578.)
And electrochemical methods (Goyal, R. N.; Oyama, M.; Singh, S. P. Simultaneous
determination of adenosine and adenosine-5′-triphosphate at nanogold modified
indium tin oxide electrode by osteryoung square-wave voltammetry.Electroanalysis, 2007,19 (5), 575-581.) etc., the context of detection of these methods ATP in the cell faces not
Same challenge.In addition some developed are used to measure the analysis method of intracellular ATP concentration, mostly use offline mode, pass through
Smudge cells obtain cell extract and are analyzed, and can not achieve real-time, in situ detection (Manfredi, G.; Yang, L.;
Gajewski, C. D.; et al. Measurements of ATP in mammalian cells. Methods,
2002, 26(4), 317-326. Li, M.; Zhang, J.; Suri, S.; et al. Detection of
adenosine triphosphate with an aptamer biosensor based on surface-enhanced
Raman scattering. Analytical Chemistry, 2012, 84, 2837-2842.).In contrast, fluorescence at
As method due to easy to operate, cost economy, to analyte high selectivity, while it can realize the real-time in-situ detection to object
And show specific advantage.
Squaraine dye is that the substitutions of 1,3- bis- generated with electron rich aryl compound or aminated compounds condensation by side's acid are spread out
Biology is the dyestuff of a kind of amphoteric ion type structure with resonance stabilized（Ajayaghosh, A.Chemistry of
squaraine-derived materials: near-IR dyes, low band gap systems, and cation
sensors. Acc. Chem. Res. 2005, 38(6), 449-459.）.Such compound is noteworthy characterized by visible
Light area to near-infrared region（620-670 nm）There are narrow and strong absorption band and higher quantum yield, molar absorption coefficient to be more than
105 L•mol-1•cm-1.This photoelectric characteristic is mainly derived from the strong Donor-Acceptor-Donor of intramolecular（donor-
acceptor-donor）Between charge transfer interaction.Since it has excellent fluorescent emission performance and chemical property, good concurrently
Optical stability, easily modification the features such as, have broad application prospects in the design of chemical small molecule probe of new generation.
The present invention is optimized by the structure to squaraine dye, the near-infrared squaraine dye of synthesis phenyl boric acid modification, and
Its application in ATP detections is realized, there is good development prospect.
Invention content
The purpose of the present invention is to provide the near-infrared squaraine dyes and its ATP fluorescence in the cell of a kind of modification of phenyl boric acid
Application in image checking.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of near-infrared squaraine dye of phenyl boric acid modification, structural formula are as follows：
。
The preparation method of the near-infrared squaraine dye of the phenyl boric acid modification includes the following steps：
（1）The phenyl boronic acid derivative that indoles is modifiedWith dicyanoethenyl squaric acid derivertives
Mixing, is dissolved in solvent, N2The lower reflux of protection；
（2）It is cooled to room temperature, solvent is removed under reduced pressure, obtain crude product；
（3）It is purified through silica gel column chromatography, obtains the near-infrared squaraine dye.
Wherein, step（1）Described in solvent by n-butanol and toluene by volume 1:1 composition；The time of the reflux is 5
Hour；Step（3）Described in silica gel column chromatography use volume ratio for 15:1 dichloromethane and the mixed solution of methanol, which are used as, to be washed
De- agent.
The phenyl boronic acid derivative of the indoles modificationSynthetic method include the following steps：
a）By 2,3,3- tri-methyl indolesIt is mixed with bromomethyl benzene boric acid, is dissolved in solvent, N2The lower reflux of protection
Divide water；
b）It is cooled to room temperature, solvent is removed under reduced pressure, obtain crude product；
c）Crude product is purified through silica gel column chromatography, obtains the phenyl boronic acid derivative of the indoles modification；
Wherein, step a）Solvent for use is acetonitrile；The time of the reflux is 24 hours；Step c）The silica gel column chromatography uses
Volume ratio is 20:1 dichloromethane and the mixed solution of methanol are eluant, eluent.
The 2,3,3- tri-methyl indolesSynthetic method include the following steps：
i）Phenylhydrazine and methyl isopropyl Ketone are dissolved in solvent, then instills the concentrated sulfuric acid and is heated to reflux；
ii）It is cooled to room temperature, after dropwise addition NaOH solution is adjusted to alkalinity, is extracted with dichloromethane, solvent is removed under reduced pressure；
iii）It is purified through silica gel column chromatography, obtains described 2,3,3- tri-methyl indoles；
Wherein, step i）Middle solvent for use is absolute ethyl alcohol；The temperature of the reflux is 80 DEG C, and the time is 3 hours；Step ii）
In plus NaOH solution be adjusted to pH be 8；Step iii）Described in silica gel column chromatography use volume ratio for 3:1 petroleum ether and acetic acid second
The mixed solution of ester is as eluant, eluent.
The dicyanoethenyl squaric acid derivertivesSynthetic method include the following steps：
a）By malononitrile,It is dissolved in solvent with triethylamine, reaction is stirred at room temperature；
b）After the completion of reaction, reaction bulb is placed in refrigerator cooling, is then extracted with dichloromethane, solvent is removed under reduced pressure, is obtained described
Dicyanoethenyl squaric acid derivertives；
Wherein, step a）Middle solvent for use is benzene；It is 6 hours to be stirred to react the time；Step b）The time of middle cooling is 24 hours.
It is describedSynthetic method include the following steps：
i）Square acid is dissolved in solvent, revolving removes solvent after heating reflux reaction；
ii）Repeat step i）Back flow reaction is primary when long again after three times；
iii）Products therefrom is purified through silica gel column chromatography, is obtained described；
Wherein, step i）Solvent for use is ethyl alcohol, and reflux temperature is 80 DEG C, and the reaction time is 3 hours；Step ii）It flows back when long
The temperature of reaction is 80 DEG C, and the time is 24 hours；Step iii）Described in silica gel column chromatography use volume ratio for 2:1 petroleum ether
Mixed solution with ethyl acetate is as eluant, eluent.
The near-infrared squaraine dye of gained phenyl boric acid modification especially aligns near-infrared side's acid of substituted phenyl boric acid modification
Dyestuff can be made into the ATP probes of fluorescence response, and the fluorescence imaging for intracellular ATP detects.
Ortho position, meta position and contraposition phenyl boric acid are connected on dicyanovinyl side's acid cyanines skeleton by the present invention, have been obtained close red
Outer squaraine dye SQ-PBA 1-3.Cationic surfactant cetyl trimethyl is added in the PBS buffer solution of pH=7.4
Ammonium bromide（CTAB）, using the electrostatic repulsion between dyestuff and CTAB, the coherent condition between dye molecule is made to occur
Change；Be added ATP after, phenyl boric acid group can be combined with the c/s-diol part in ATP molecules, so as to by dyestuff, ATP and
Between CTAB molecules multiple electrostatic effect, make dye molecule from State Transition be disaggregation state, fluorescence release, to
Realize the detection to ATP.
Gained squaraine dye fluorescence probe stability of the invention is good, excellent in optical properties, and has preferable bio-compatible
Property and cell permeability, lower bio-toxicity, use it for intracellular ATP fluorescence imaging detection can show preferably to examine
Survey sensitivity.After testing, which has higher sensitivity in the PBS solution containing 0.25 mM CTAB to ATP,
Its detection is limited to 28 nM（3σ/k）, and due to the similitude of the nucleotide structures such as ADP, UTP, GTP and CTP, therefore the probe dye
Have different degrees of response to these substances etc., this can with side light its be to the identification of ATP response main cause because
Phenyl boric acid leads to the disaggregation of dye molecule in the solution, to which Fluorescence Increasing occur, and its is right to the specific recognition of glycol
Some amino acid（Proline, histidine, cysteine, arginine, serine, lysine, glutamic acid, asparatate, junket ammonia
Acid, glutathione）And some metal ions（Zn2+、Na+、K+、Mg2+、Ca2+）Without response.Therefore, the probe can be used for monitor by
Intracellular nucleoside triphosphate total content variation caused by drug or disease.
By fluorescence co-focusing microscopic image analysis, which has preferable cell permeability, in the cell together
Sample shows preferable ATP detection sensitivities, and without apparent deleterious cellular effects, can be used for the fluorescence imaging of intracellular ATP
Detection.
Description of the drawings
Fig. 1 is the ultraviolet spectrogram after squaraine dye SQ-PBA3 is combined with different surfaces activating agent, wherein（A）Not add
Enter surfactant,（B）For nonionic surfactant TW80 is added,（C）For cationicsurfactants are added.
Fig. 2 is the ultraviolet spectrogram after squaraine dye SQ-PBA3 is combined with various concentration CTAB.
Fig. 3 is ortho position, meta position, fluorescence spectrum response conditions of the SQ-PBA to ADP, AMP and ATP for aligning three kinds of structures
Figure.
Fig. 4 is fluorescence spectrum response condition figures of the squaraine dye SQ-PBA3 to different bio-related substances, wherein 1- dried meat
Propylhomoserin, 2- histidines, 3- cysteines, 4- arginine, 5- glutathione, 6- lysines, 7- glutamic acid, 8- asparatates,
9- tyrosine, 10 serines, 11-Zn2+, 12-Na+, 13-K+, 14-Mg2+, 15-Ca2+, 16-CDP, 17-CTP, 18-UTP, 19-
GTP, 20-UMP, 21-AMP, 22-ADP, 23-ATP, 24-GDP, 25-UDP.
Fig. 5 is fluorescence when various concentration ATP (0-50 μM) being added dropwise in the PBS buffer solution of squaraine dye SQ-PBA3
Spectrogram.
Fig. 6 is the linear relationship of fluorescence intensities of the squaraine dye SQ-PBA3 at 700 nm and ATP concentration (2-22 μM)
Scheme (λexThe nm/5 of=670 nm, slit=5 nm, PMT=650 V).
Fig. 7 is the confocal fluorescent image of MCF-7 cells, wherein（A）To use squaraine dye SQ-PBA3 (4.0 μ
M it) is incubated 2 hours,（B）First (4.0 μM) of squaraine dye SQ-PBA3 to be used to be incubated 2 hours, then with taxol (10 nM) incubation
24 hours.
Specific implementation mode
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
Preparation：
20 mL ethyl alcohol are first added in 50 mL round-bottomed flasks, add 2.00 sides g acid（17.6 mmol）, flow back at 80 DEG C anti-
It answers 3 hours, vacuum rotary steam removes ethyl alcohol after the side's of waiting for acid all dissolves, and back flow reaction 24 is small again after operating repeatedly three times
When, after reaction terminates, using petroleum ether:Ethyl acetate is 2:1 (v/v) is that eluant, eluent carries out silica gel column chromatography separation, is obtained
2.09 g of pale yellowish oil product liquid, yield 70%.1H NMR (400 MHz, CDCl3) δ 4.74 (q, J = 7.1
Hz, 4H), 1.48 (t, J = 7.1 Hz, 6H)。
Embodiment 2
Dicyanoethenyl squaric acid derivertivesPreparation：
By 1.77 g malononitrile（26.8 mmol）It is dissolved in 35 mL dry benzenes, is placed in 100 mL round-bottomed flasks, and drip dropwise
Enter the preparation of 5g embodiments 1（29.4 mmol）, then by 2.98 g triethylamines（29.4 mmol）It is slowly added into
In above-mentioned reaction solution, 6 h are stirred at room temperature.After the completion of reaction, reaction bulb is placed in cooling 24 hours in refrigerator, with appropriate CH2Cl2
Extraction, takes organic layer in vacuo to concentrate, obtains 5.28 g of yellow solid product, yield 62%.1H NMR (400 MHz, CDCl3)
δ 9.19 (brs, 1H), 4.76 (q, J = 7.0 Hz, 2H), 3.32-3.25 (m, 6H), 1.99 (s, 1H),
1.49 (t, J = 7.1 Hz, 3H), 1.38 (t, J = 7.3 Hz, 9H)。
Embodiment 3
2,3,3- tri-methyl indolesPreparation
20 mL are added in 50 mL round-bottomed flasks and contain 500 mg phenylhydrazines（4.62 mmol）Absolute ethyl alcohol, then by 438 mg first
Base nezukone（5.09 mmol）It is slowly dropped in above-mentioned solution, solution becomes light yellow, and 0.25 is instilled in 0.5 h
The mL concentrated sulfuric acids, at this time reaction system become yellow turbid solution, 80 DEG C of 3 h of back flow reaction again.With the raising of reaction temperature, reaction
System color slowly becomes orange-yellow from yellow, becomes orange-red turbid solution in turn.TLC is detected to the reaction was complete, reaction
After liquid cooling to room temperature, it is 8 that NaOH solution, which is added dropwise, and is adjusted to pH.Use CH2Cl2Three times, solvent is removed under reduced pressure, using petroleum ether in extraction:Second
Acetoacetic ester is 3:1（v/v）Silica gel column chromatography purifying is carried out for eluant, eluent, obtains 614 mg of bronzing oily liquids, yield 83%.1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 7.6 Hz, 1H), 7.35-7.24 (m, 2H), 7.18
(t, J = 7.3 Hz, 1H), 2.27 (s, 3H), 1.28 (s, 6H); 13C NMR (100 MHz, CDCl3): δ
187.84, 153.47, 145.46, 127.41, 124.94, 121.13, 119.69, 53.41, 22.91, 15.20。
Embodiment 4
The phenyl boronic acid derivative of indoles modificationPreparation：
The 2,3,3- tri-methyl indoles of 120 mg embodiments 3 preparation are added in 25 mL round-bottomed flasks（0.75 mmol）, with 4
ML acetonitriles dissolve, and 4 mL are then added and contain 150 mg bromomethyl benzene boric acids（0.70 mmol）Acetonitrile solution, 80 DEG C of return stirrings
Solution is in peony after reaction 24 hours, with dichloromethane:Methanol is 20:1（v/v）It is pure that silica gel column chromatography is carried out for eluant, eluent
Change, obtains khaki solid.
Ortho position is modified：170 mg, yield 65%.1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 7.2
Hz, 1H), 7.80 (t, J = 4.3 Hz, 1H), 7.58 (d, J = 10.2 Hz, 2H), 7.51 (d, J =
7.6 Hz, 1H),7.37 (t, J = 4.6 Hz, 2H) 6.93 (t, J = 4.9 Hz 1H), 6.03 (s, 2H),
2.89 (s, 3H), 1.63 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ 198.72, 142.43,
141.91, 136.43, 135.81, 130.89, 129.89, 129.40, 128.00, 125.89, 124.06,
116.32, 54.91, 51.74, 22.60, 15.01; ESI-MS m/z: [M]+ Calcd for C18H21NBO2
294.1665; found 294.1656。
Meta position is modified：153 mg, yield 58%.1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 6.4
Hz, 1H), 7.80 (d, J = 6.9 Hz, 1H), 7.67–7.57 (m, 3H), 7.46 (d, J = 6.7 Hz,
1H), 7.25 (d, J = 6.7 Hz, 1H), 5.84 (s, 2H), 2.99 (s, 3H), 1.62 (s, 6H); 13C
NMR (100 MHz, DMSO-d6): δ 172.44, 161.24, 146.14, 137.36, 136.75, 134.84
133.12, 128.51, 127.99, 124.08, 122.37, 118.92, 116.40, 105.95, 54.95, 51.33,
21.52, 15.01; ESI-MS m/z: [M]+ Calcd for C18H21NBO2 294.1665; found 294.1657。
Contraposition modification：140 mg, yield 53%.1H NMR (400 MHz, DMSO-d6) δ 7.87 (d, J = 7.0
Hz, 1H), 7.81 (d, J = 7.8 Hz, 2H), 7.78 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.2
Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.37 (d, J = 7.6 Hz, 2H), 5.82 (s,2H),
2.98 (s, 3H), 1.60 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ 198.85, 142.43,
141.91, 136.45, 135.81, 130.89, 129.89, 129.40, 128.00, 125.89, 124.06,
116.32, 54.91, 51.74, 22.60, 15.01; ESI-MS m/z: [M]+ Calcd for C18H21NBO2
294.1665; found 294.1657。
Embodiment 5
Near-infrared squaraine dyePreparation
The phenyl boronic acid derivative of the indoles modification of 99 mg embodiments 4 preparation is added in 50 mL twoport flasks（0.265 mmol）
The dicyanoethenyl squaric acid derivertives prepared with 38 mg embodiments 2（0.132 mmol）, it is dissolved in 20 mL n-butanols-toluene
（1:1, v/v）In, connect water knockout drum, N2The lower heating of protection divides water to flow back 5 h, and TLC is monitored to the reaction was complete postcooling to room temperature,
Washing three times, is dried with anhydrous sodium sulfate, is filtered, solvent is removed under reduced pressure, using dichloromethane:Methanol is 15:1（v/v）To wash
De- agent carries out silica gel column chromatography purification, obtains dark green solid, yield 56%.
SQ-PBA1：53 mg, yield 56%.1H NMR (400 MHz, DMSO-d6): δ 7.75 (d, J = 7.2
Hz, 2H), 7.62 (d, J = 7.3 Hz, 2H), 7.33–7.21 (m, 8H), 7.15 (d, J = 6.9 Hz,
2H), 6.73 (d, J = 7.2Hz, 2H), 6.35 (s, 2H), 5.57 (s, 4H), 1.82 (s, 12H); 13C
NMR (100 MHz, DMSO-d6): δ 172.88, 172.53, 166.65, 165.48, 142.38, 142.27,
133.98, 132.49, 128.70, 128.40, 125.42, 122.85, 118.54, 111.92, 89.06, 79.04,
66.75, 49.81, 47.37, 33.46, 31.99, 31.62, 29.95, 29.47, 26.54, 20.95; ESI-MS
m/z: [M+H]+ Calcd for C43H39B2N4O5713.3107; found 713.3104。
SQ-PBA2：33 mg, yield 35%.1H NMR (400 MHz, DMSO-d6): δ7.72–7.63 (m, 8H),
7.37–7.32 (m, 8H), 6.37 (s, 2H), 5.34 (s, 4H), 1.76 (s, 12H); 13C NMR (100
MHz, DMSO-d6): δ 172.88, 172.53, 166.65, 165.48, 142.38, 142.27, 133.98,
132.49, 128.70, 128.42, 125.42, 122.85, 118.54, 111.91, 89.16, 78.98, 66.79,
49.81, 47.38, 33.42, 32.02, 29.92, 26.54, 20.95; ESI-MS m/z: [M+H]+ Calcd for
C43H39B2N4O5 713.3107; found 713.3101。
SQ-PBA3：31 mg, yield 33%.1H NMR (400 MHz, DMSO-d6): δ 7.74 (d, J = 7.6
Hz, 4H), 7.62 (d, J = 7.2 Hz, 2H), 7.44–7.37 (m, 4H), 7.28 (d, J = 7.3 Hz,
2H), 7.22 (d, J = 7.0 Hz, 4H), 6.34 (s, 2H), 5.33 (s, 4H), 1.74 (s, 12H); 13C
NMR (100 MHz, DMSO-d6): δ 172.76, 172.40, 172.39, 165.45, 142.44, 142.25,
137.00, 135.10, 128.76, 126.06, 125.47, 122.90, 118.56, 111.86, 89.20, 49.78,
47.30, 33.42, 32.07, 29.47, 26.52, 20.95; ESI-MS m/z: [M+H]+ Calcd for
C43H39B2N4O5713.3107; found 713.3140。
Application Example
1. the absorption maximum of the near-infrared squaraine dye of gained phenyl boric acid modification of the invention is located near 700 nm, have very high
Molar absorption coefficient and fluorescence quantum yield.Self aggregation easily occurs in aqueous solution for squaraine dye, exists to improve compound
Responding ability under physiological condition, the binding ability of enhancing and ATP, respectively to the PBS buffer solution of the squaraine dye containing SQ-PBA3
（PH=7.4,10 mM）Middle addition cationicsurfactants and non-ionic surfactant Tween 80（TW80）And equivalent
ATP, then carry out ultraviolet spectroscopy, the results are shown in Figure 1.As seen from Figure 1, it is not added with surfactant or nonionic is added
When surfactant, SQ-PBA3 to ATP without response, and when cationic surfactant is added, the monomer absorption of 700 nm or so
The absorption intensity at peak is remarkably reinforced, and shows that the addition of Surfactant CTAB effectively alleviates the coherent condition of dyestuff.
2. in the PBS solution containing 4.0 μM of SQ-PBA3 and various concentration CTAB（PH=7.4,10 mM）Middle addition etc.
Ultraviolet spectroscopy is carried out after amount ATP, the results are shown in Figure 2.From Figure 2 it can be seen that as a concentration of 0.25 mM of CTAB solution suction
Light varience value is maximum, it is thus determined that 0.25 mM is the optimum detection concentration of CTAB.
3. the substituent group of different location often influences the acid-base property of organic matter due to the difference of electronic effect, to shadow
Ring reacting between phenyl boric acid and vicinal diamines.Fig. 3 is ortho position, meta position, aligns the SQ-PBA of three kinds of structures to ADP, AMP and ATP
Fluorescence spectrum response condition figure.SQ-PBA 1-3 fluorescence in the PBS buffer solution of pH=7.4 is in cancellation state, and when difference
The change of different fluorescence intensities is presented in three kinds of dye solutions when ADP, AMP and ATP is added.As seen from Figure 3, meta position SQ-PBA2
To ADP, AMP and ATP without response, fluorescence intensity does not change；And the SQ-PBA of ortho position and contraposition substitution is to ADP, AMP
There is response with ATP, and the response effect for aligning SQ-PBA3 pairs of three kinds of substances is best, the fluorescence intensity of system, which has, relatively to increase
By force.
4. squaraine dye SQ-PBA3 is to some metal ions present in different biosystems, amino acid and nucleotide
Fluorescence spectrum response condition is shown in Fig. 4.From fig. 4, it can be seen that dyestuff SQ-PBA3 is to some amino acid（Proline, histidine, half Guang ammonia
Acid, arginine, serine, lysine, glutamic acid, asparatate, tyrosine, glutathione）And some metal ions (Zn2+,
Na+, K+, Mg2+, Ca2+) without response, and since the nucleotide structures such as ATP and ADP, UTP, GTP and CTP have a similitude, therefore the dye
Material has these substances different degrees of response.ATP plays main effect in cellular energy metabolism in human body, internal
It is mainly to be embodied with generating and consuming ATP that energy, which is discharged and absorbed,.And UTP, CTP and GTP are also some substances synthesis generation
Energy source in thanking.Therefore the probe can be used for monitoring the intracellular nucleoside triphosphate total content caused by drug or disease
Variation.
5. in the PBS solution of the SQ-PBA3 containing 0.25 mM CTAB（10 mM, pH=7.4）The middle fluorescence drop for carrying out ATP
Fixed experiment, is as a result shown in Fig. 5 and Fig. 6 respectively.As seen from Figure 5, SQ-PBA3 is in fluorescent quenching in PBS solution, when into system
When ATP is added, the fluorescence intensity at 700nm gradually increases, and when a concentration of 40 μM of ATP are added, fluorescence reaches saturation, glimmering
Luminous intensity about enhances 6 times.As shown in fig. 6, ATP concentration is within the scope of 2.0-22 μM, fluorescence intensity and the ATP concentration of solution
Good linear relationship is presented（R2=0.9918, k=3.12 × 108）.According to 3 σ of formula/k, calculate its detection is limited to 28 nM.
6. by Laser scanning confocal microscopy technology, further research institute obtains the reality of squaraine dye in vivo
Using.Experimental selection breast cancer cell MCF-7 is cell model, fragmentation effect of the examination taxol to MCF-7 cells.First,
MCF-7 cells are incubated 2 h with 4 μM of squaraine dye SQ-PBA3, are then proceeded to the cell culture containing 10 nM taxols
Liquid is incubated 24 h, and confocal fluorescent image is shown in Fig. 7.As seen from Figure 7, the intracellular hair being only incubated with dyestuff SQ-PBA3
Bright red fluorescence, and good dispersion in the cell are projected, illustrates that probe has preferable Cell permeable；Continue to use
The intensity of cellular fluorescence that taxol is incubated obviously weakens, and illustrates the intracellular ATP content being incubated through taxol reduction.Thus it demonstrate,proves
Bright, which can realize detection response well to ATP in the cell, obtain good cell imaging figure, and have
Good Premeabilisation of cells rate, without apparent cytotoxicity.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (8)
1. a kind of near-infrared squaraine dye of phenyl boric acid modification, it is characterised in that：Its structural formula is as follows：
。
2. a kind of method preparing near-infrared squaraine dye as described in claim 1, it is characterised in that：Include the following steps：
（1）The phenyl boronic acid derivative that indoles is modifiedWith dicyanoethenyl squaric acid derivertives
Mixing, is dissolved in solvent, N2The lower reflux of protection；
（2）It is cooled to room temperature, solvent is removed under reduced pressure, obtain crude product；
（3）It is purified through silica gel column chromatography, obtains the near-infrared squaraine dye.
3. preparation method according to claim 2, it is characterised in that：The solvent is by n-butanol and toluene by volume 1:
1 composition；
The time of the reflux is 5 hours；
The silica gel column chromatography uses volume ratio for 15:1 dichloromethane and the mixed solution of methanol are as eluant, eluent.
4. preparation method according to claim 2, it is characterised in that：The phenyl boronic acid derivative of the indoles modificationSynthetic method include the following steps：
a）By 2,3,3- tri-methyl indolesIt is mixed with bromomethyl benzene boric acid, is dissolved in solvent, N2Protect flow point next time
Water；
b）It is cooled to room temperature, solvent is removed under reduced pressure, obtain crude product；
c）Crude product is purified through silica gel column chromatography, obtains the phenyl boronic acid derivative of the indoles modification；
Wherein, step a）Solvent for use is acetonitrile；The time of the reflux is 24 hours；
Step c）The silica gel column chromatography uses volume ratio for 20:1 dichloromethane and the mixed solution of methanol are eluant, eluent.
5. preparation method according to claim 4, it is characterised in that：The 2,3,3- tri-methyl indoles
Synthetic method include the following steps：
i）Phenylhydrazine and methyl isopropyl Ketone are dissolved in solvent, then instills the concentrated sulfuric acid and is heated to reflux；
ii）It is cooled to room temperature, after dropwise addition NaOH solution is adjusted to alkalinity, is extracted with dichloromethane, solvent is removed under reduced pressure；
iii）It is purified through silica gel column chromatography, obtains described 2,3,3- tri-methyl indoles；
Wherein, step i）Middle solvent for use is absolute ethyl alcohol；The temperature of the reflux is 80 DEG C, and the time is 3 hours；
Step ii）In plus NaOH solution be adjusted to pH be 8；
Step iii）Described in silica gel column chromatography use volume ratio for 3:1 petroleum ether and the mixed solution of ethyl acetate, which are used as, to be washed
De- agent.
6. preparation method according to claim 2, it is characterised in that：The dicyanoethenyl squaric acid derivertivesSynthetic method include the following steps：
a）By malononitrile,It is dissolved in solvent with triethylamine, reaction is stirred at room temperature；
b）After the completion of reaction, reaction bulb is placed in refrigerator cooling, is then extracted with dichloromethane, solvent is removed under reduced pressure, is obtained described
Dicyanoethenyl squaric acid derivertives；
Wherein, step a）Middle solvent for use is benzene；It is 6 hours to be stirred to react the time；
Step b）The time of middle cooling is 24 hours.
7. preparation method according to claim 6, it is characterised in that：It is describedSynthetic method include such as
Lower step：
i）Square acid is dissolved in solvent, revolving removes solvent after heating reflux reaction；
ii）Repeat step i）Back flow reaction is primary when long again after three times；
iii）Products therefrom is purified through silica gel column chromatography, is obtained described；
Wherein, step i）Solvent for use is ethyl alcohol, and reflux temperature is 80 DEG C, and the reaction time is 3 hours；
Step ii）The temperature of back flow reaction is 80 DEG C when long, and the time is 24 hours；
Step iii）Described in silica gel column chromatography use volume ratio for 2:1 petroleum ether and the mixed solution of ethyl acetate, which are used as, to be washed
De- agent.
8. a kind of application of near-infrared squaraine dye as described in claim 1 in ATP detections, it is characterised in that：It will be described
The ATP probes of fluorescence response are made in the near-infrared squaraine dye of phenyl boric acid modification, and the fluorescence imaging for intracellular ATP detects.
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| CN109456349A (en) * | 2018-12-13 | 2019-03-12 | 中南民族大学 | A kind of ATP multidigit point combination fluorescence enhancement type probe molecule and its preparation method and application |
| CN114573621A (en) * | 2022-01-19 | 2022-06-03 | 南京邮电大学 | Phenylboronic acid modified water-soluble near-infrared two-region fluorescent contrast agent and application thereof |
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| US20100143960A1 (en) * | 2007-03-09 | 2010-06-10 | Cis Bio International | Cyanine derivatives, fluorescent conjugates containing same and use thereof |
| WO2010083471A1 (en) * | 2009-01-15 | 2010-07-22 | Seta Biomedicals, Llc | Luminescent compounds |
| CN103923481A (en) * | 2014-04-14 | 2014-07-16 | 福州大学 | Adamantyl-modified near-infrared squaraine dye as well as preparation method and application thereof |
| US20140216283A1 (en) * | 2010-11-15 | 2014-08-07 | Deepak Shukla | Method of flexographic patterning using photocurable composition |
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| US20100143960A1 (en) * | 2007-03-09 | 2010-06-10 | Cis Bio International | Cyanine derivatives, fluorescent conjugates containing same and use thereof |
| WO2010083471A1 (en) * | 2009-01-15 | 2010-07-22 | Seta Biomedicals, Llc | Luminescent compounds |
| US20140216283A1 (en) * | 2010-11-15 | 2014-08-07 | Deepak Shukla | Method of flexographic patterning using photocurable composition |
| CN103923481A (en) * | 2014-04-14 | 2014-07-16 | 福州大学 | Adamantyl-modified near-infrared squaraine dye as well as preparation method and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109456349A (en) * | 2018-12-13 | 2019-03-12 | 中南民族大学 | A kind of ATP multidigit point combination fluorescence enhancement type probe molecule and its preparation method and application |
| CN109456349B (en) * | 2018-12-13 | 2021-01-29 | 中南民族大学 | ATP multi-site combined fluorescence enhanced probe molecule and preparation method and application thereof |
| CN114573621A (en) * | 2022-01-19 | 2022-06-03 | 南京邮电大学 | Phenylboronic acid modified water-soluble near-infrared two-region fluorescent contrast agent and application thereof |
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