CN117820256A - Preparation method of a near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe and its imaging application in cells or in vivo - Google Patents

Abstract

The invention discloses a preparation method of a near infrared ratio type lysosome targeted carbon monoxide fluorescent probe and imaging application of the near infrared ratio type lysosome targeted carbon monoxide fluorescent probe in cells or living bodies. The technical scheme of the invention is as follows: a near infrared ratio type lysosome targeted carbon monoxide detection fluorescent probe has the structural formula:the invention also specifically discloses a preparation method of the fluorescent probe and an imaging application of the fluorescent probe in cells or living bodies. In the selectivity experiment, the fluorescence enhancement of the fluorescent probe can only be induced by carbon monoxide,but not by other metals and biological thiols. Thus, the fluorescent probe shows good selectivity for carbon monoxide. Based on the method, a rapid and simple fluorescence detection method for detecting carbon monoxide is established.

Description

Preparation method of a near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe and its imaging application in cells or in vivo

Technical Field

The invention belongs to the technical field of carbon monoxide detection fluorescent probes, and specifically relates to a preparation method of a near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe and its imaging application in cells or living bodies.

Background technique

Carbon monoxide (CO) is a colorless, odorless gas that has traditionally been considered toxic or hazardous due to its strong affinity for hemoglobin. Long-term exposure to CO can cause headaches and nausea, and severe acute exposure can lead to anemia or neurological damage, or even death. Clinical studies have shown that exposure to CO in patients with heart disease can lead to worsening of the disease and increased risk of arrhythmias and ischemia. Animal models have provided further evidence that CO can induce compensatory hemodynamic changes, cardiac hypertrophy, and atherosclerosis. However, CO is also considered an endogenous gasotransmitter (signaling molecule) that is continuously produced in the human body through the endogenous degradation of heme by heme oxygenase (HO) with the help of _O2 and NADPH. Similar to other signaling molecules (NO and _H2S ), CO has shown important functions in regulating immune responses, modulating inflammation and controlling tissue damage in a manner tailored to tissue needs. For example, there are many literatures demonstrating the cardioprotective effects of CO administration. The possible reason is that endogenous CO can improve the blood supply to the heart by dilating coronary blood vessels and reduce Ca ²⁺ influx through L-type Ca ²⁺ channels, thereby reducing the risk of Ca ²⁺ overload in cardiomyocytes. Similarly, CO administration has also been shown to have neuroprotective effects, and many studies have shown that exposure to CO or induction of HO-1 can have neuroprotective effects on cerebral ischemia, traumatic brain injury (TBI), and neurodegenerative diseases. These results indicate that CO has important physiological functions and is of great significance for the treatment of a variety of diseases. Therefore, real-time tracking of CO molecules in vivo has been a hot topic in recent years.

Traditional methods such as gas chromatography, colorimetric detection, and electrochemical analysis have been used to detect CO, but these methods cannot selectively detect CO in living systems in a non-invasive manner. In contrast, fluorescence technology is very attractive as a non-destructive detection method due to its high sensitivity and real-time detection capability. Recent reports have shown that different fluorophores such as coumarin, BODIPY, fluorescein, naphthylimide, and nitrobenzoxadiazole have been proposed for CO detection. However, these probes have more or less some defects, such as a part of their emission in the blue-green region, and the penetration of the blue-green region is not as strong as that of near-infrared fluorescent probes. Some fluorescent "on" type probes cannot self-calibrate to improve accuracy like ratiometric probes, and non-targeted probes cannot detect targets in specific areas. Therefore, these unfavorable characteristics mentioned above limit their application in biological systems.

Recently, near-infrared (NIR) fluorescent probes have been widely used to detect CO in living organisms because of their deep tissue penetration, minimal damage to biological samples, and low background interference. However, most of them are single-channel signal-modulated or non-organelle-targeted fluorescent probes. Ratiometric fluorescent probes have attracted extensive attention due to their ability to overcome the interference of instrument parameters, probe concentration, excitation intensity, and temperature fluctuations by simultaneously modulating the emission signals in two channels. So far, only a few ratiometric fluorescent probes have been designed for the detection of CO. In addition, organelles are important sites for many biochemical reactions in cells, and abnormalities in their composition or function are associated with many physiological or pathological processes. Recently, several CO probes targeting organelles such as mitochondria, lysosomes, or endoplasmic reticulum have been developed, but to the best of our knowledge, a probe that simultaneously possesses the three characteristics of near-infrared emission, ratiometric fluorescence, and organelle targeting has not been reported. Its successful design will provide an important tool for accurately studying the concentration or function of CO at the subcellular level.

Based on the above research and development background, the present invention designs and prepares a lysosome-targeted near-infrared ratiometric fluorescent probe (TBM-CO) for detecting the concentration of CO in cells or in vivo. The fluorescent probe TBM-CO uses TBM-NH ₂ as a ratiometric fluorescent signal unit because its near-infrared fluorescence characteristics have a significant large Stokes shift (~200nm), efficient intramolecular charge transfer (ICT) characteristics and excellent biocompatibility. Traditionally, the allyl carbamate part is introduced as a Tsuji-Trost reaction site to react with CO. At the same time, its electron-withdrawing properties effectively regulate the electron push-pull system and change the photophysical properties of TBM-NH _2. The probe TBM-CO itself shows weak ICT characteristics with a maximum emission peak of 616nm. However, after adding CO in the presence of Pd ²⁺ , the allyl carbamate is cleaved to generate TBM-NH ₂ , which has a stronger ICT effect and an emission red shift of 686nm. Therefore, through self-calibration in two emission bands, TBM-CO can provide an effective ratiometric fluorescent probe for CO, which has obvious fluorescence enhancement in the NIR region. In addition, lysosomes are digestive organelles, and staining lysosomes can minimize potential effects on cell activities, avoid interference with cell migration caused by membrane staining, or changes in membrane potential caused by mitochondrial staining. Therefore, as a physical marker for traditional lysosome targeting, the morpholino fragment was transplanted into the probe, which enabled the probe to effectively detect CO in lysosomes in living cells. In summary, it is expected that TBM-CO can become a useful molecular tool for exploring CO in cellular lysosomes or in vivo under physiological conditions.

Summary of the invention

The technical problem solved by the present invention is to provide a method for preparing a near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe and its imaging application in cells or living bodies. The fluorescent probe prepared by the method can be used for the detection of endogenous or exogenous carbon monoxide in Hela living cells, and can be used for the detection of carbon monoxide in living bodies, and can also be used for imaging in cells or living bodies.

The present invention adopts the following technical solution to solve the above technical problems, a near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe, characterized in that the structural formula of the fluorescent probe is:

The method for preparing the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe of the present invention is characterized by the following specific steps:

Step S1, adding compound a and malononitrile into anhydrous ethanol, then adding piperidine and subjecting the mixed solution to reflux reaction to obtain a white solid compound b;

Step S2, adding compound b and 3-hydroxy-4-nitrobenzaldehyde into dry CH ₃ CN, then adding piperidine and subjecting the obtained mixed solution to reflux reaction under N ₂ to obtain compound TB-OH;

Step S3, dissolving the compound TB-OH and 2-morpholineethanol in dry THF solvent, adding triphenylphosphine, cooling the mixed solution to 0°C, adding DIAD, and then heating to room temperature and stirring to react to obtain the compound TBM-NO ₂ ;

Step S4, dissolving the compound TBM-NO ₂ in anhydrous ethanol, adding SnCl ₂ ·2H ₂ O and stirring the mixture under N ₂ to obtain the compound TBM-NH ₂ ;

Step S5, dissolving the compound TBM- _NH2 and allyl chloroformate in DCM, adding NEt3 and stirring at room temperature to react to obtain the target compound TBM-CO;

The corresponding synthetic route in the preparation process is:

The invention discloses an application of the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe in the preparation of a selective detection preparation for endogenous or exogenous carbon monoxide in Hela living cells.

The invention discloses an application of the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe in the preparation of a preparation for selective detection of carbon monoxide in living bodies.

Furthermore, the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe has fluorescence at 616 nm before the reaction. As a reactive fluorescent probe, its fluorescence intensity at 616 nm decreases in the presence of carbon monoxide and Pd2 ⁺ , while the near-infrared fluorescence intensity at 686 nm is enhanced. Based on this process, a lysosome-targeted near-infrared probe for carbon monoxide ratio detection is designed. The fluorophore has lysosome targeting due to its morpholine group, and the fluorescent probe has good water solubility and pH adaptability, and can be used as a biocompatible fluorescent probe.

The invention discloses an application of the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe in the preparation of cell or in vivo imaging preparations.

The present invention prepares a novel near-infrared fluorescent probe for detecting CO based on a dicyanoisophorone skeleton and a Tsuji-Trost reaction mechanism. The fluorescent probe endowed with a morpholine fragment can be used to detect CO in lysosomal subcellular organelles. The ICT effect can be adjusted by the cleavage of an allylcarbamate (AF) unit to achieve ratio fluorescence changes in two emission channels. In vitro pH dependence and selectivity experiments show that it can detect CO under physiological conditions. There is a good linear relationship between the concentration and the ratio fluorescence intensity signal, indicating that the method can be used for the quantitative detection of CO with a low detection limit. Cell fluorescence imaging and co-localization experiments further prove that the near-infrared fluorescent probe can be used to detect endogenous or exogenous CO in lysosomal subcellular organelles, and can also be used to monitor changes in CO concentration in living mice.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the principle of detecting CO using the fluorescent probe TBM-CO.

Figure 2 (A) is the UV-Vis absorption spectrum of the fluorescent probe TBM-CO (10.0 μM) after the reaction of Pd ²⁺ (20.0 μM) and CROM-3 (100.0 μM). Inset: Color change of TBM-CO solution before and after adding CO; (B) Changes in fluorescence spectra of the probe system (10μM) TBM-CO and (20μM) PdCl ₂ after adding different concentrations of CORM-3, each spectrum was obtained 45 minutes after mixing; (C) Linear relationship between fluorescence intensity ratio (I ₆₈₆ \/I ₆₁₆ ) and CO concentration from 0 to 50μM; (D) Fluorescence intensity ratio (I ₆₈₆ \/I ₆₁₆ ) response of the probe system (10mΜ) probe and 20μM PdCl ₂ to various analytes (100μM each), including: (1) probe, (2-11) K ⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Co ²⁺ , Cr ³⁺ , Al ³⁺ , Cd ²⁺ , Ni ⁺ , Cu ²⁺ , (12-20) F ^- , Br ^- , HCO ₃ ^- , CO ₃ ^2- , SO ₄ ^2- , SO ₃ ^2- , NO ₃ ^- , HS ^- , S ^2- , (21-24)Hcy (100μM), Cys (100μM), GSH (1mM), CO (CORM-3, 100μM); (E) Time-dependent fluorescence intensity ratio (I ₆₈₆ \/I 616 ) enhancement of the probe system (10μM TBM-CO and 20μM PdCl ₂ ) at different CO concentrations; (F) Effect of pH on the fluorescence intensity ratio (I ₆₈₆ \/I ₆₁₆ ) of the probe system (10μM TBM-CO and 20μM PdCl ₂ ) and the amount of CO added (CORM-3, _100μM ). Unless otherwise stated, all spectra were acquired at 37°C, _λex = 488 nm, slit width: _dex = _dem = 10 nm after mixing for 45 min in PBS buffer (10 mM, pH 7.4, 40% DMSO, v/v).

Figure 3 shows the reaction mechanism of the interaction between fluorescent probe TBM-CO and CO.

Figure 4 shows the energy-optimized geometric structures and frontier molecular orbitals of compounds TBM-NH ₂ and TBM-CO.

Figure 5 Confocal fluorescence imaging of exogenous CO in HeLa cells by the probe system (10uM probe and 20uM PdCl ₂ ). a1-a4: incubated with fluorescent probe (TBM-CO) for 30min; b1-b4: incubated with TBM-CO and PdCl ₂ for 30min, c1-c4: pre-incubated with 100mM CORM-3 for 30min, then added TBM-CO and PdCl ₂ and incubated together for 30min. (λex＝488nm, yellow channel: λem: 570～620nm. Red channel: λem: 663～738nm, scale bar＝100μm) The histogram on the right shows the quantitative information of the fluorescence ratio (Fred/Fyellow) of the red channel and the yellow channel during CO incubation.

Figure 6 is a confocal image of endogenous CO stimulated by heme in HeLa cells. (Left) Pretreatment with 100 μM heme for different time periods (0, 2, 4, 8 h), followed by incubation with the fluorescent probe system (TBM-CO + PdCl ₂ , 10 μM and 20 μM) for 30 min. (Right) Fluorescence ratio (red/yellow) of the corresponding fluorescence images in the left panel. λex = 488 nm, yellow channel: λem: 570-620 nm. Red channel: λem: 663-738 nm, scale bar = 20 μm.

Figure 7 Colocalization imaging of HeLa cells stained with LysoTracker Green and TBM-CO with PdCl ₂ and CORM-3. (a) Cells were stained with Lyso Tracker Green (60 nM) in the green channel; (b) cells were stained with TBM-CO (10 μM), PdCl ₂ (2 equivalents) and CORM-3 (10 equivalents) in the red channel; (c) merged image; (d) bright field image; (e) intensity scatter plot; (f) fluorescence intensity distribution of the region of interest (white line in a and b); scale bar is 20 μm.

Figure 8 is a fluorescent image of mice. (a) Mice were intraperitoneally injected with TBM-CO (200 μM, 50 μL) only; (b) Mice were intraperitoneally injected with the probe (200 μM) and PdCl ₂ (2 equivalents); (cf) Mice were intraperitoneally injected with the probe (200 μM), PdCl ₂ (2 equivalents) and CORM-3 (10 equivalents) (c) 1 min, (d) 4 min, (e) 7 min, (f) 12 min.

FIG9 is the ¹ H NMR of compound b in DMSO-d6 solution (400 MHz, 298 K).

FIG10 is an ESI-MS spectrum of compound b.

FIG. 11 is the 1H NMR of compound TB-OH in DMSO-d6 solution (400 MHz, 298 K).

FIG. 12 is the ¹³ C NMR of compound TB-OH in DMSO-d6 solution (400 MHz, 298 K).

FIG13 is an ESI-MS spectrum of compound TB-OH.

FIG. 14 is the 1H NMR of compound TBM-NO ₂ in DMSO-d6 solution (400 MHz, 298 K).

FIG. 15 is the ¹³ C NMR of compound TBM-NO ₂ in DMSO-d6 solution (400 MHz, 298 K).

Figure 16 is the ESI-MS spectrum of compound TBM-NO ₂ .

FIG. 17 is the 1H NMR of compound TBM-NH2 in DMSO-d6 solution (400 MHz, 298 K).

FIG. 18 is ¹³ C NMR of compound TBM-NH2 in DMSO-d6 solution (400 MHz, 298 K).

Figure 19 is the ESI-MS spectrum of compound TBM-NH2.

FIG20 is the 1H NMR of compound TBM-CO in DMSO-d6 solution (400 MHz, 298 K).

FIG. 21 is the ¹³ C NMR of compound TBM-CO in DMSO-d6 solution (400 MHz, 298 K).

FIG. 22 is the ESI-MS spectrum of compound TBM-CO.

FIG. 23 is a comparison of the UV-Vis spectra of TBM-CO after addition of CO and other analytes in PBS buffer (10 mM, pH 7.4, 40% DMSO, v/v).

FIG24 is a comparison of HPLC results of compounds TBM-NH ₂ (10 mM), TBM-CO (10 mM), and Pd ²⁺ (2.0 equivalents) and CO (10.0 equivalents), eluent: CH ₃ OH:H ₂ O=9:1, v/v.

FIG25 is the positive ion mode ESI-MS spectrum of the reaction of compound TBM-CO (10.0 mM) with Pd ²⁺ (2 equivalents) and CO (10 equivalents).

Figure 26 shows the cell viability of Hela cells after incubation with different concentrations of TBM-CO (left) and TBM-CO and Pd ²⁺ ions (1:2 ratio) for 24 hours.

Detailed ways

The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

Example

Unless otherwise specified, the instruments and reagents used in the present invention were obtained from commercial sources.

1 Experimental part

1.1 Materials and reagents

All chemicals were purchased from Anhui Energy Chemical Co., Ltd., China. All other reagents were of analytical grade and used without further purification unless otherwise specified.

1.2 Main instruments

In addition, NMR spectra of organic compounds were obtained on a Bruker Ascend ^™ Electron Spray Ionization (ESI) mass spectra were recorded on a Bruker Microtof QII spectrometer. UV-visible absorption spectral data were obtained by a Shimadzu UV2600 spectrophotometer. Fluorescence spectra were recorded using an Edinburgh Instruments FS-5 fluorescence spectrophotometer. Cell imaging was recorded using a NIKONA1R confocal fluorescence microscope.

1.3 Methods

1.3.1 Preparation of fluorescent probes

synthetic route

The fluorescent probe is synthesized according to the synthesis route shown above. The specific synthesis process is:

The specific preparation method of compound b is as follows: In a 100mL round-bottom flask, compound a (1.11g, 8.0mmol) and malononitrile (0.95g, 14.4mmol) are added to 20mL of anhydrous ethanol, and piperidine (0.19g, 2.2mmol) is added, and the resulting solution is refluxed for 8h. The reaction progress is monitored by TLC, and after the reaction is completed, the resulting mixed solution is treated with column chromatography (EA/PE=1:10, v/v) to obtain a white solid compound b (0.95g, yield 64%).

Structural characterization of compound b: ¹ H NMR (400 MHz, DMSO) δ=6.55 (s, J=1.1, 1H), 2.53 (s, 2H), 2.23 (s, 2H), 2.04 (s, 3H), 0.95 (s, 6H). ESI-MS, (m/z), Calcd for [C ₁₂ H ₁₅ N ₂ ] ⁺ , 187.1229; found, 187.1219.

The specific preparation method of compound TB-OH is as follows: In a 100 mL round-bottom flask, compound b (0.4 g, 2.2 mmol) and 3-hydroxy-4-nitrobenzaldehyde (0.36 g, 2.2 mol) are added to 50 mL of dry CH ₃ CN, and then piperidine (51 mg, 0.6 mmol) is added. The resulting solution is refluxed under N ₂ for 8 h. The progress of the reaction is monitored by thin layer chromatography (EA/PE=1:1, v/v). After the reaction is completed, the resulting mixed solution is treated with column chromatography (EA/PE=1:4, v/v) to obtain a yellow solid compound TB-OH (0.31 g, yield 42%).

Structural characterization of compound TB-OH: ¹ H NMR (400 MHz, DMSO) δ = 11.02 (s, 1H), 7.94 (d, J = 8.7, 1H), 7.54 (d, J = 16.2, 1H), 7.37 (dd, J = 8.7, 1.6, 1H), 7.33 (d, J = 1.4, 1H), 7.25 (d, J = 16.2, 1H), 6.99 (s, 1H), 2.62 (s, 2H), 2.54 (s, 2H), 1.01 (s, 6H). ¹³ C NMR (101 MHz, DMSO) δ＝170.17, 154.65, 152.73, 143.06, 135.95, 134.76, 133.59, 125.82, 124.68, 118.45, 117.96, 113.62, 112.79, 78.03, 42.24, 38.05, 31.70, 27.43. ESI-MS (m/z), Calcd for [C ₁₉ H ₁₆ N ₃ O ₃ ] ^- : 334.1197; found: 334.1222.

Compound TB-OH (100 mg, 0.3 mmol) and 2-morpholineethanol (58 mg, 0.44 mmol) were dissolved in dry THF (5 mL) solvent, and triphenylphosphine (116 mg, 0.44 mmol) was added, cooled to 0°C, and DIAD (88 mg, 0.44 mmol) was added, and then the reaction was warmed to room temperature and stirred overnight. The reaction was monitored by TLC (EA/PE=1:1, v/v) to completion, and the resulting solution was treated by column chromatography (EA/PE=1:2, v/v) to finally obtain yellow oil compound TBM-NO ₂ (74 mg, yield 55%).

Structural characterization of compound TBM-NO ₂ : ¹ H NMR (400MHz, DMSO) δ＝7.91(d, J＝8.4, 1H), 7.69(d, J＝1.1, 1H), 7.62(d, J＝16.5, 1H), 7.40(dd, J＝8.5, 1.2, 1H), 7.31(d, J＝16.2, 1H), 6.97(s, 1H), 4.34(t, J＝5.7, 2H), 3.61-3.48(m, 4H), 2.74(t, J＝5.7, 2H), 2.64(s, 2H), 2.54(s, 2H), 2.48(d, J＝4.2, 4H), 1.03(s, 6H). ¹³ C NMR (101 MHz, CDCl3) δ＝169.05,152.83,152.47,141.75,134.16,132.90,132.23,126.60,125.57,119.80,113.16,112.85,112.51,80.55,68.51,67.05,57.26,54.26,42.99,39.20,32.18,28.11 _. ESI-MS, (m _/ z), Calcd for _{[C25H29N4O4 ]} ⁺ : 449.2183; found: 449.2187.

Compound TBM- _NO2 (140 mg, 0.3 mmol) was dissolved in 50 mL of anhydrous ethanol, and _SnCl2 · _2H2O (309 mg, 1.49 mmol) was added to the solution, and stirred at RT for 6 h under _N2 conditions. The resulting solution was pre-treated with TLC (MeOH/ _CH2Cl2 = _1:50 , v/v) to obtain dark red solid compound TBM- _NH2 (80 mg, yield 64%).

Structural characterization of compound TBM-NH ₂ : ¹ H NMR (400 MHz, DMSO) δ = 7.27 (s, 1H), 7.16 (q, J = 15.9, 2H), 7.04 (dd, J = 8.1, 1.2, 1H), 6.73 (s, 1H), 6.62 (d, J = 8.1, 1H), 5.56 (s, 2H), 4.12 (t, J = 5.8, 2H), 3.63-3.54 (m, 4H), 2.73 (t, J = 5.7, 2H), 2.57 (s, 2H), 2.51 (d, J = 1.4, 6H), 1.00 (s, 6H). ¹³ C NMR (101 MHz, DMSO) δ＝169.89, 157.55, 145.33, 141.57, 140.10, 124.79, 124.12, 123.97, 120.00, 114.55, 113.79, 113.10, 110.83, 72.68, 66.21, 66.13, 57.10, 53.56, 42.32, 38.25, 31.64, 27.45. ESI-MS, (m/z), Calcd for [C ₂₅ H ₃₁ N ₄ O ₂ ] ⁺ : 419.2442; found: 419.2453.

Compound TBM- _NH2 (40 mg, 0.095 mmol) and allyl chloroformate (58 mg, 0.47 mmol) were dissolved in DCM (10 mL), and two drops of _NEt3 were added and stirred at room temperature overnight. After the reaction was completed, the orange solid compound TBM-CO (31 mg, yield 64%) was obtained using the pre-TLC method (MeOH:DCM: _NEt3 ＝1:1:0.02, v/v/v).

Structural characterization of compound TBM-CO: ¹ H NMR (400MHz, DMSO) δ＝8.89(s, 1H), 7.81(d, J＝8.4, 1H), 7.53(d, J＝1.6, 1H), 7.37(d, J＝16.0, 1H), 7.30-7.19(m, 2H), 6.85(s, 1H), 6.07-5.91(m, 1H), 5.37(dd, J＝17.2, 1.6, 1H), 5.25 (dd, J = 10.4, 1.5, 1H), 4.63 (dt, J = 5.5, 1.3, 3H), 4.22 (t, J = 5.7, 2H), 3.65-3.56 (m, 5H), 2.67 (t, J = 5.7, 2H), 2.62 (s, 2H), 2.54 (s, 2H), 1.23 (s, 3H), 1.02 (s, 6H). ¹³ C NMR (101 MHz, DMSO) δ＝170.34,156.19,153.08,148.42,137.57,136.91,133.06,131.67,128.44,122.95,122.81,122.26,120.04,117.87,113.98,113.23,76.93,65.91,65.13,65.07,56.55,53.31,42.31,38.18,31.71,27.45. ESI-MS, (m/z), Calcd for [C ₂₉ H ₃₅ N ₄ O ₄ ] ⁺ :503.2653; found:503.2657.

1.3.2 Cell culture

Culture: Hela cells were cultured in DMEM medium, and 10wt% fetal bovine serum (FBS) and 1wt% penicillin-streptomycin were added. The cells were incubated at 37°C with a volume fraction of 5% CO ₂ , and the cells were passaged when the cell saturation exceeded 70%.

Inoculation: Add DMEM complete medium containing 10wt% FBS to a 24-well plate and culture in an incubator at 37°C and 5% _CO2 for 12 hours.

Counting and photography: The number and status of cells in the 24-well plate were observed using an inverted biological microscope.

2 Results and discussion

2.1 Design and synthesis of TBM-CO

The dicyanoisophorone skeleton (electron acceptor) is combined with the benzene ring through a double bond to form a large conjugated system. The amino group of aniline (strong electron donor) enhances the ICT effect, and a near-infrared emission spectrum (686 nm) is emitted from the solution of TBM- _NH2 . When allyl formate is introduced as a CO trigger to form the probe TBM-CO, the ICT effect is weakened, and the maximum emission peak moves from 686 nm to 616 nm. Therefore, after removing the allyl formate group, obvious near-infrared ratio fluorescence can be observed. In addition, in order to detect CO in lysosomal subcellular organelles, a morpholine fragment is introduced into the structure of TBM-CO as a targeting group.

2.2 Spectral response of TBM-CO to CO

After obtaining the probe, the corresponding spectral experiments were carried out. First, the UV-vis absorption spectrum of the fluorescent probe TBM-CO in PBS buffer (10mM, pH 7.4, 40% DMSO, v/v). For the fluorescent probe TBM-CO, its own absorption is centered at 428nm. After reacting with CO (CROM-3, CO releaser) and Pd ²⁺ ions, the maximum absorption peak red-shifted to 460nm, and a new absorption peak appeared at 525nm. It can be observed in the inserted picture that the color of the test solution changed from light yellow to dark orange, which is conducive to the detection of CO with the naked eye. These significant changes in absorption spectra indicate the generation of new CO-mediated Tsuji-Trost reaction products. The addition of other analytes, such as metal cations, anions and thiols (GSH, Cys, Hcy) does not result in obvious absorption changes. This color change shows that the fluorescent probe TBM-CO can be used as a colorimetric indicator for CO molecules.

Fluorescence experiments of the reaction of fluorescent probe TBM-CO with CO were also performed. When excited at 460 nm, the free fluorescent probe TBM-CO showed medium-intensity orange fluorescence at 616 nm. With the addition of Pd ²⁺ (2.0 equivalents) ions and increasing the concentration of CROM-3 (CO releaser), the fluorescence intensity at 616 nm decreased, and the new emission peak at 686 nm gradually increased. These results indicate that the D--A structure of the fluorescent probe TBM-CO is interrupted by CO attack and the intramolecular charge transfer effect (ICT) is enhanced, which leads to an obvious ratio change from orange to red in the fluorescence emission spectrum. The ratio signal (I ₆₈₆ \/I ₆₁₆ ) shows a change of more than 10 times (from 0.58 to 5.96) and reaches a plateau at 80 μM. In the range of 0 to 50 μM, there is a good linear relationship between the ratio fluorescence signal (I ₆₈₆ \/I ₆₁₆ ) and the CO concentration (R ² = 0.996). Based on 3σ/slope (σ＝0.00883), the corresponding detection limit of the fluorescent probe TBM-CO for CO was 0.38 μM. These results indicate that TBM-CO can be used for the quantitative analysis of CO with high sensitivity.

In addition, by detecting various potential interfering substances, such as K ⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Co ²⁺ , Cr ³⁺ , Al ³⁺ , Cd ²⁺ , Ni ⁺ , Cu ²⁺ , F ^- , Br ^- , HCO ₃ ^- , CO ₃ ^2- , SO ₄ ^2- , SO ₃ ^2- , NO ₃ ^- , HS ^- , S ^2- , Hcy, Cys and GSH, it can be seen that the above analytes have no obvious interference with the detection of CO. Time-dependent fluorescence experiments were performed to explore the kinetic response ability of the fluorescent probe TBM-CO to different concentrations (25.0 mM, 50 mM, 100.0 mM) of CO in PBS buffer (10 mM, pH 7.4, 40% DMSO, v/v). The fluorescence ratio signal (I ₆₈₆ \/I ₆₁₆ ) of the TBM-CO solution increased and reached a plateau within 40 min. In addition, pH-dependent fluorescence experiments were performed, and the ratio intensity (I ₆₈₆ \/I ₆₁₆ ) of the fluorescent probe TBM-CO changed minimally in a wide pH range of 3.0 to 10.0, indicating that the fluorescent probe TBM-CO has good stability. The addition of CO produced significant sensing capabilities in the pH range of 4.0 to 8.0. These results indicate that the fluorescent probe TBM-CO can detect CO with excellent selectivity under physiological conditions, as shown in Figure 2.

2.3 Response mechanism of TBM-CO to CO

According to the above spectral changes, when CO is added to the solution of the fluorescent probe TBM-CO with PdCl ₂ , Pd ²⁺ is first reduced by CO to produce Pd(0), which then mediates the Tsuji-Trost reaction and finally releases TBM-NH _2. The TBM-CO fluorescent probe itself emits orange fluorescence at 616nm. Since the amino group of the fluorophore has a medium intensity, it is protected by the formate (AF) unit, which reduces the electron-donating ability of the amino group and weakens the intramolecular charge transfer (ICT) effect. After adding Pd ²⁺ and CO, the AF unit is cleaved and the amino group is deprotected, which leads to the recovery of the ICT process and the emission spectrum moves to 686nm to emit red fluorescence. Therefore, the ratio fluorescence signal (I ₆₈₆ \/I ₆₁₆ ) of the fluorescent probe in the solution can be clearly observed. In order to better understand its mechanism, density functional theory (DFT) calculations were performed using the B3LYP/6-311G method. The optimized structures of TBM-CO and TBM-NH ₂ are shown in the figure. The electron density on both HOMO and LUMO is mainly located on the TBM unit, so obvious fluorescence can be observed for both. When the allyl formate (AF) group is introduced to form a carbamate group with the amino group of TBM- _NH2 , the electron-withdrawing effect of the carbamate causes the electron to transfer from the amino group to the carbamate group, which reduces the electron-donating ability of the amino group and weakens the intramolecular charge transfer (ICT) process. Therefore, in theory, it will lead to obvious spectral changes, which is consistent with the experimental results, that is, the emission spectrum moves from 686nm to 616nm. In addition, the energy gaps (HOMO-LUMO) of TBM-CO and TBM- _NH2 are calculated to be 2.85eV and 2.70eV, respectively. Theoretical calculations well support the experimental results and rationalize the changes in the ratio signal. At the same time, ESI-MS experiments were performed to confirm the structural transformation (Figure 4). For the fluorescent probe TBM-CO, the only peaks were at 503.2652 (m/z, [M+H] ⁺ , calculated) and 525.2643 (m/z, [M+Na] ⁺ ), and after the addition of Pd ²⁺ and CO, a peak at 419.2439 (m\/z, [M+H] ⁺ ) corresponding to TBM-NH ₂ appeared. This also indicates that CO induced the conversion of TBM-CO to TBM-NH ₂ .

2.4 Imaging of exogenous and endogenous CO in HeLa cells

Based on the above spectral response to carbon monoxide, the effectiveness of the fluorescent probe TBM-CO for detecting CO in living cells was further examined. First, the cytotoxicity of the fluorescent probe TBM-CO to HeLa cells in the absence and presence of Pd ²⁺ ions was studied by conventional MTT method. Under the experimental conditions, more than 80% of the cells were viable when exposed to the fluorescent probe or TBM-CO with Pd ²⁺ ions in the concentration range of 0 to 20 μM (1:2 ratio). Therefore, the results show that the fluorescent probe TBM-CO has low cytotoxicity to cultured cells and is suitable for biological applications.

After obtaining the results of the fluorescent probe with low cytotoxicity, the fluorescent TBM-CO was used for imaging of exogenous CO in HeLa cells, as shown in Figure 5, and the yellow channel and red channel (yellow: 570-620 nm, red: 663-738 nm) were monitored. In the control group, the fluorescent probe TBM-CO and TBM-CO containing PdCl ₂ induced bright fluorescence in the yellow channel and very weak fluorescence in the red channel. When treated with CO, the red fluorescence in the red channel increased significantly, and the fluorescence intensity ratio (red/yellow) was as high as 2.2 times. Then, the feasibility of the fluorescent probe to detect endogenous CO release in Hela cells was further examined, as shown in Figure 6. It is well known that heme can induce cells to produce endogenous CO through heme oxygenase (HO). In the experiment, the cells were pre-incubated with heme (100 μM) for 0 h, 2 h, 4 h, and 8 h, and then incubated with the fluorescent probe and PdCl ₂ (10 μM, 1:2) for 0.5 h. As shown in Figure 6, enhanced red fluorescence can also be observed in the red channel, and the fluorescence ratio intensity (red/yellow) shows a time-dependent characteristic. These results indicate that the fluorescent probe TBM-CO has the ability to image exogenous and endogenous CO levels in living cells.

Morpholine fragments are often introduced into probe structures as traditional lysosomal targeting entities. In order to further verify the targeting ability of the fluorescent probe TBM-CO, as shown in Figure 7, HeLa cells were stained with LysoTracker Green (a commercial lysosomal tracker), and the fluorescent probe was co-localized with _PdCl2 and CORM-3. Cells stained with LysoTracker Green showed green fluorescence in the green channel (a in Figure 7). Cells stained with TBM-CO containing _PdCl2 and CORM-3 showed red fluorescence in the red channel. The merged image showed that the red image and the green image fit well, and the Pearson's correlation coefficient was as high as 0.83 (c in Figure 7). Bright field showed that the cells were feasible throughout the imaging experiment (d in Figure 7). In addition, the intensity scatter plot in Figure 7 e showed a high correlation and indicated that the probe was mainly in the lysosome. In addition, the changes in the fluorescence intensity distribution of the region of interest (white lines in a and b) in HeLa cells tended to be synchronized (f in Figure 7). These results confirmed that the fluorescent probe TBM-CO can specifically target the lysosomes of living HeLa cells.

2.7 In vivo fluorescence imaging

The near-infrared ratiometric fluorescence properties of the fluorescent probe TBM-CO may penetrate deep into tissues with low background autofluorescence interference and little damage to biological samples. To prove this concept, in vivo mouse imaging experiments were performed. As shown in Figure 8, live mice treated with TBM-CO alone (a in Figure 8) or with a mixture of TBM-CO and PdCl ₂ (b in Figure 8) showed negligible fluorescence. However, when TBM-CO, PdCl ₂ , and CORM-3 were injected into live mice, a significant enhancement over time was observed (cf in Figure 8). The above results indicate that the fluorescent probe TBM-CO is an effective in vivo CO probe.

3.0 Conclusion

In general, a novel near-infrared fluorescent probe for the detection of CO was prepared based on the dicyanoisophorone skeleton and the Tsuji-Trost reaction mechanism. The morpholine fragment-endowed probe can be used to detect CO in lysosomal subcellular organelles. The ICT effect can be regulated by the cleavage of the ethyl formate (AF) unit to achieve ratiometric fluorescence changes in two emission channels. In vitro pH-dependency and selectivity experiments showed that it can detect CO under physiological conditions. There is a good linear relationship between the concentration and the ratiometric fluorescence intensity signal, indicating that this method can be used for the quantitative detection of CO with a low detection limit. Cell fluorescence imaging and colocalization experiments further demonstrated that the probe can be used to detect endogenous and exogenous CO in lysosomal subcellular organelles. The successful monitoring of CO concentration changes in living mice also showed that the near-infrared fluorescence emission characteristics endowed the probe with good tissue penetration ability.

The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims (6)

1. A near-infrared ratiometric lysosomal targeted carbon monoxide fluorescent probe, characterized in that the structural formula of the fluorescent probe is: 2. A method for preparing the near-infrared ratiometric lysosomal targeted carbon monoxide fluorescent probe according to claim 1, characterized in that the specific steps are: Step S1, adding compound a and malononitrile into anhydrous ethanol, then adding piperidine and subjecting the mixed solution to reflux reaction to obtain a white solid compound b; Step S2, adding compound b and 3-hydroxy-4-nitrobenzaldehyde into dry CH ₃ CN, then adding piperidine and subjecting the obtained mixed solution to reflux reaction under N ₂ to obtain compound TB-OH; Step S3, dissolving the compound TB-OH and 2-morpholineethanol in dry THF solvent, adding triphenylphosphine, cooling the mixed solution to 0°C, adding DIAD, and then heating to room temperature and stirring to react to obtain the compound TBM-NO ₂ ; Step S4, dissolving the compound TBM-NO ₂ in anhydrous ethanol, adding SnCl ₂ ·2H ₂ O and stirring the mixture under N ₂ to obtain the compound TBM-NH ₂ ; Step S5, dissolving the compound TBM- _NH2 and allyl chloroformate in DCM, adding NEt3 and stirring at room temperature to react to obtain the target compound TBM-CO; The corresponding synthetic route in the preparation process is: 3. Use of the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe according to claim 1 in the preparation of a preparation for selective detection of endogenous or exogenous carbon monoxide in living Hela cells. 4. Use of the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe according to claim 1 in the preparation of a preparation for selective detection of carbon monoxide in vivo. 5. The use according to claim 3 or 4, characterized in that: the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe has fluorescence at 616nm before the reaction. As a reactive fluorescent probe, its fluorescence intensity at 616nm decreases in the presence of carbon monoxide and Pd2 ⁺ , while the near-infrared fluorescence intensity at 686nm increases. Based on this process, a lysosome-targeted near-infrared probe for carbon monoxide ratio detection is designed. Because its morpholine group makes the fluorophore have lysosomal targeting, and the fluorescent probe has good water solubility and pH adaptability, and can be used as a biocompatible fluorescent probe. 6. Use of the near-infrared ratiometric lysosome-targeted carbon monoxide fluorescent probe according to claim 1 in the preparation of cell or in vivo imaging preparations.