Carbon monoxide probe molecules

Abstract

The present disclosure relates generally to fluorescent amide functionalized palladium coordination complexes and their use for the detection of carbon monoxide. A method for detecting carbon monoxide, as described herein, comprises the steps of combining a test sample with a fluorescent amide-functionalized palladium coordination complex, thereby reacting carbon monoxide in the test sample with the fluorescent amide-functionalized palladium coordination complex, thereby forming a fluorescent imide, and detecting fluorescence emitted by the fluorescent imide so as to detect carbon monoxide in the test sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 395,286, filed on August 4, 2022, which is hereby incorporated by reference in its entirety.

[0003] STATEMENT AS TO RIGHTS TO INVENTS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under DK119202 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Since the signaling function of carbon monoxide (CO) was first reported in the 1990s, a large number of laboratories have conducted extensive research on the physiological and pharmacological effects of CO. Although the public generally believes that CO is toxic at high concentrations, under normal physiological conditions, CO actually exists in the general circulation in the high micromolar range, mainly endogenously produced by heme degradation mediated by heme oxygenase. In addition, whether endogenously produced or exogenously delivered, CO provides anti-inflammatory, cell and organ protective activities against various stress conditions, such as lipopolysaccharide attack, ischemia-reperfusion injury and chemically induced organ damage. The prospect of developing CO as a therapeutic agent for the treatment of various diseases (such as colitis, sickle cell disease, acute kidney injury, etc.) is imminent. Therefore, in recent years, people have made extensive efforts to study CO gas inhalation in clinical trials and develop non-gaseous CO delivery methods, including liquid preparations, metal-based CO release molecules (CORMs) and organic CO prodrugs and their preparations.

[0006] As a gas molecule, the unique challenge faced by CO research and development is to determine its concentration and related pharmacokinetic work. Under physiological and near-equilibrium conditions, CO in the circulatory system exists mainly in the form of carboxyhemoglobin (COHb) because it has a high binding affinity to hemoglobin. In addition, the high concentration of Hb (about 8mM in the human body) also enables it to play a role in storing CO. Therefore, COHb concentration is often used as a surrogate indicator for assessing systemic CO exposure levels because it is easier to assess by testing peripheral blood. On the other hand, CO in tissues is the actual executor that determines biological activity and thus therapeutic effects and / or toxicity. CO binds to heme proteins in tissues, including enzymes, proteins for transporting and storing oxygen, iron regulatory proteins, sensors, transcription factors, ion channels, etc., to exert its biological functions. Due to the thermodynamic equilibrium between COHb and heme proteins according to their relative binding affinities, the tissue CO concentration is proportional to the blood COHb concentration. In addition to COHb levels and heme protein concentrations, tissue CO saturation levels also depend on other factors such as local CO and oxygen partial pressures. We can draw an analogy with traditional small molecule assessments, where COHb levels are equivalent to albumin-bound drugs and CO in tissues is equivalent to tissue drug concentrations. In fact, in practice, overall CO bioavailability is often evaluated by calculating the area under the curve (AUC) of COHb levels after CO is delivered to the body. As a best practice, many studies also evaluate tissue CO concentrations to show the correspondence between observed biological effects and increased CO levels. Therefore, to understand the pharmacology of CO in order to develop CO-based therapeutics, evaluating CO concentrations in blood and tissues is an important and necessary component of in vivo and ex vivo studies. For in vitro cellular studies, intracellular CO levels can also be evaluated to witness the correlation of CO with observed biological effects.

[0007] Studying the relationship between CO dose response and observed biological effects requires measuring CO concentrations in tissues and other biological samples, but the analytical methods required to achieve this can be challenging for many laboratories. Existing methods for quantifying CO in cell culture samples and tissue samples include myoglobin-coupled chemiluminescence; 14 C radioactive method, which requires [ 14 C]glycine pre-labeling; and UV spectroscopy using synthetic heme-containing CO sensors. However, these approaches have not been widely implemented for a number of reasons.

[0008] There are two mainstream methods for determining CO in biological samples reported in the literature. One is direct quantification using gas chromatography (GC). Specifically, CO is released by treating tissue with a denaturant or oxidant in a headspace vial. To detect trace CO in the headspace vial, a methanator-coupled flame ionization detector (FID), mercury reduction gas detector (RGD), or semiconductor sensor gas chromatograph (SGC) is required. According to the manufacturer's specifications, the detection limits for a 100 μL gas injection are 0.5 ppm for the methanator FID, 50 ppb for the RGD, and 50 ppb for the SGC. The thermal conductivity detector (TCD), which is a general-purpose gas detector, cannot detect such low CO concentrations due to its low sensitivity (about 1000 ppm for a 100 μL injection). However, these sophisticated gas chromatography instruments are not commonly used in biological laboratories and require chemical knowledge to design experiments and interpret data. For example, a methanator can also detect CO in the air. 2 to methane; therefore, CO must be reduced to methane using a GC column. 2 The RGD method requires sufficient separation of CO from the methanogen or requires flushing the sample with nitrogen or helium before CO is released. For RGD methods, due to the extremely sensitive detector, the carrier gas must be pre-purified with a CO trap, which is rarely available, to eliminate trace amounts of CO. The consumability of the methanogen and RGD due to poisoning by sulfide species in biological samples, the cumbersome sample handling procedures (for RGD), and the limited access to GC in most biological laboratories can be obstacles to such methods.

[0009] The second approach to sensing CO in biological systems is to use fluorescent or chromogenic probes, including genetically encoded CO sensor proteins and small molecule-based CO probes ( Figure 1 ). Since CO confers binding ability to the heme prosthetic group in heme proteins, the bacterial CO sensing heme protein CooA was fused to a modified yellow fluorescent protein (YFP). Binding of CO to the CooA domain restored the fluorescent conformation of YFP, increasing its fluorescence by approximately twofold. A comparable approach along this line is the cyclodextrin-encapsulated heme analog HemoCD1 ( Figure 1), which imparts a high binding affinity for CO and is used to quantify CO in cell culture and tissues by UV-Vis spectroscopy. The reversibility of these metal binding methods enables real-time assessment of CO in cells under near-equilibrium conditions. For reaction-based fluorescent sensors, there are two main strategies for designing such CO probes. One strategy is to form a transition metal (in most cases Pd) complex with the fluorophore, thereby quenching the fluorescence through heavy atom electronic effects. After reaction with CO, the Pd is removed by palladium-mediated carbonylation or proton decomposition. In either case, the fluorescence quenching effect of Pd is relieved, thereby restoring the fluorescence of the fluorophore. This "dequenching" strategy has been implemented in the design of CO probes such as COP-1, its analogs, and CC-CO. Another strategy is to cap the fluorophore with an allylic group. Although the pro-fluorescent compounds do not contain metals, PdCl 2 Must be applied simultaneously with the probe to sense CO. Reduction of Pd with CO 2+ Afterwards, the allyl capping group is removed by the formed Pd(0) via the Tsuji-Trost reaction to restore fluorescence. These CO probes, especially COP-1, have been widely used in cell imaging-based studies and have greatly helped in understanding the biology of CO. However, depending on the sensing mechanism, the reported probes have their limitations, such as low signal-to-noise ratio (SNR) and limited specificity and sensitivity to CO. For probes based on dequenching, nucleophiles (such as thiols) that are ubiquitous in the biological environment can react and remove the palladium quenching group, which can be seen by the slight turn-on effect of thiol species reported in the literature. For probes based on the Tsuji-Trost reaction, the capping group may be removed by enzyme metabolism, and it has been reported that Pd 2+ Can also be reduced by ascorbic acid, which can lead to false positives or compatibility issues.

[0010] There are also several nitro-reduction based probes that were initially reported to sense CO, but the sensing mechanism was later found to depend on the reactivity of the ruthenium complex. Therefore, they should not be considered as general CO probes, but rather as ruthenium-based CORM probes. In addition to these nitro-reduction sensing mechanism probes, the recently reported CORM-3 fluorescent probe utilizes the CORM-3-mediated isomerization-hydrolysis reaction of the allylic end group to turn on fluorescence. One would not expect that CO alone could undergo such nitro-reduction or isomerization reactions under physiological conditions. Therefore, the necessity of using CO gas to define the true analyte of CO probes and to understand the reactivity of CORM is emphasized.

[0011] There are no reported applications of using fluorescent probes to quantify CO in cell culture, blood, or tissue samples. Although CO oximeters can be applied to test blood COHb levels in humans and animals, not all laboratories have such access to CO oximeters, and alternative feasible methods are needed. Therefore, there is a need for highly reliable fluorescent CO probes with ultra-low background fluorescence that not only detect carbon monoxide with high specificity and ultra-high sensitivity, but also enable semi-quantitative and quantitative determination of CO concentrations in biological samples, as well as imaging of intracellular CO accumulation in living cells. Summary of the invention

[0012] In one aspect, a method for detecting carbon monoxide is described herein, the method comprising: combining a test sample with a palladium coordination complex functionalized with a fluorescent amide, thereby reacting carbon monoxide in the test sample with the palladium coordination complex functionalized with a fluorescent amide, thereby forming a fluorescent imide, and detecting fluorescence emitted by the fluorescent imide, thereby detecting carbon monoxide in the test sample. In some embodiments, the amide in the palladium coordination complex functionalized with a fluorescent amide used in the method for detecting carbon monoxide described herein is benzamide or naphthamide.

[0013] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula I:

[0014]

[0015] in:

[0016] Each R 1 Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 alkyl;

[0017] R 2 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 2a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties;

[0018] Each R 2a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NRa C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O) 2 R c 、-S(O) 2 OR a 、-S(O) 2 NR a R b and-NR a S(O) 2 R c ;

[0019] Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl;

[0020] Each R c It is C 1-6 alkyl;

[0021] Part L 1 -L 2 It is a bidentate ligand;

[0022] X is an anionic ligand;

[0023] subscript m is 0 or 1; and

[0024] Subscript n is 0, 1, 2 or 3.

[0025] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula Ia:

[0026]

[0027] Among them A - It is a non-coordinating anion.

[0028] In some embodiments, in the methods described herein, the fluorescent imide formed by the reaction between carbon monoxide in the test sample and the fluorescent amide functionalized palladium coordination complex of Formula I or Formula Ia is a compound according to Formula II:

[0029]

[0030] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula III:

[0031]

[0032] in:

[0033] Each R 3a and R 3b Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 Alkyl, or R 3a and R 3b Merge to form C 3 -C 7 Cyclic amine ring,

[0034] R 4 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 4a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties;

[0035] Each R 4a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O) 2 R c 、-S(O) 2 OR a 、-S(O) 2 NR a R b and-NR a S(O) 2 R c ;

[0036] Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl;

[0037] Each R c It is C 1-6 alkyl;

[0038] Part L 3 -L 4 It is a bidentate ligand;

[0039] X is an anionic ligand;

[0040] subscript p is 0 or 1; and

[0041] The subscripts q and t are independently 0, 1, 2 or 3.

[0042] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula IIIa:

[0043]

[0044] Among them A - It is a non-coordinating anion.

[0045] In some embodiments, in the methods described herein, the fluorescent imide formed by the reaction between carbon monoxide in the test sample and the fluorescent amide functionalized palladium coordination complex of Formula III or Formula IIIa is a compound according to Formula IV:

[0046]

[0047] In some embodiments, the test sample used in any of the methods described herein is a biological fluid sample. In some embodiments, the test sample used in any of the methods described herein is a tissue sample. In some embodiments, the test sample used in any of the methods described herein is a cell sample.

[0048] In another aspect, the present disclosure provides a compound according to Formula I:

[0049]

[0050] in

[0051] Each R 1 Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 alkyl;

[0052] R 2 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 2a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties;

[0053] Each R 2a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -ORa 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O) 2 R c 、-S(O) 2 OR a 、-S(O) 2 NR a R b and-NR a S(O) 2 R c ;

[0054] Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl;

[0055] Each R c It is C 1-6 alkyl;

[0056] Part L 1 -L 2 selected from the group consisting of: alkylenediamines, bipyridines, and phenanthrolines;

[0057] X is an anionic ligand;

[0058] subscript m is 0 or 1; and

[0059] Subscript n is 1, 2, or 3.

[0060] In some embodiments, the compound of Formula I has a structure according to Formula Ia:

[0061]

[0062] Among them A - It is a non-coordinating anion.

[0063] In another aspect, the present disclosure provides a compound according to Formula III:

[0064]

[0065] in:

[0066] Each R 3a and R3b Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 Alkyl, or R 3a and R 3b Merge to form C 3 -C 7 Cyclic amine ring,

[0067] R 4 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 4a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties,

[0068] Each R 4a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O) 2 R c 、-S(O) 2 OR a 、-S(O) 2 NR a R b and-NR a S(O) 2 R c ;

[0069] Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl;

[0070] Each R c It is C 1-6 alkyl;

[0071] Part L 3 -L 4 It is a bidentate ligand;

[0072] X is an anionic ligand;

[0073] subscript p is 0 or 1; and

[0074] The subscripts q and t are independently 0, 1, 2 or 3.

[0075] In some embodiments, the compound of Formula III has a structure according to Formula IIIa:

[0076]

[0077] Among them A - It is a non-coordinating anion.

[0078] In another aspect, the present disclosure provides a kit comprising a compound of Formula I, Formula Ia, Formula III or Formula IIIa as described herein, and instructions for use of the compound in carbon monoxide detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Shown are (a) a CO probe reported in the literature, and (b) a CO probe as described herein.

[0080] Figure 2 The design and fluorescence properties of the benzamide-based CO probe molecule are shown. a. CO sensing chemistry and structure of the benzamide-based CO probe molecule; b. 500 μM 5a was incubated with various amounts of CO gas (0-100 nmol) in a headspace vial for 1 hour, and the fluorescence spectra (λ Ex =394 nm, bandwidth = 5 nm); inset: linear regression of fluorescence intensity on molar concentration of CO gas; c. SNR of 5a at concentrations of 1, 10, and 100 μM in PBS with or without incubation with CO gas for 1 h (SNR was calculated as the ratio between the AUC of the emission spectra with and without incubation with CO gas); inset: regression of SNR on probe concentration in PBS; d. Fluorescence spectra of 22 and 17 (bandwidth = 5 nm); e. Selectivity of 20 μM 5a in pH 7.4 PBS (1% CO gas in air at 1 atm, concentration of other substances was 100 μM, incubation for 1 h, λ Ex =395 nm, bandwidth = 5 nm); inset: images of 50 μM 5a in PBS with or without incubation with pure CO gas for 30 min, the images of both cuvettes were taken under the same 365 nm UV light (5 W); f. 500 μM 14 in PBS was incubated with 0-100 nmol CO gas in a headspace vial for 1 h, and the fluorescence spectra (λ Ex=385 nm, bandwidth = 5 nm); inset: linear regression of fluorescence intensity on molar concentration of CO gas; g. SNR of 14 at concentrations of 1, 10, and 100 μM in PBS with or without CO gas incubation for 30 min; inset: regression of SNR on probe concentration in PBS (bandwidth = 3 nm); h. Selectivity of 10 μM 14 in pH 7.4 PBS (1% CO gas in air at 1 atm, concentration of other CO probe substances was 100 μM, incubation for 30 min, λ Ex =385 nm, bandwidth = 5 nm); i. Fluorescence spectra of 20 μM 25 and 10 μM 19 in PBS (bandwidth = 5 nm); j. CO sensing kinetics of CO probe molecules (800 μL 12.5 μM CO probe and 200 μL 1 mM CO saturated PBS at T 0 The mixture was mixed at 4 °C and the fluorescence at 509 nm or 499 nm was recorded every second at 25 °C. The progress of the reaction at each time point was calculated as the percentage of the fluorescence intensity of the maximum fluorescence. Inset: extended range 0-360 s).

[0081] Figure 3 The stability of 5 and 5a in PBS as tested using fluorescence recovery is shown. 10 μM 5 and 5a were incubated in PBS in headspace vials for the indicated times, and CO was injected to turn on the probes followed by fluorescence measurements. (λ Ex =395nm,λ Em =511nm, bandwidth =5nm).

[0082] Figure 4 The fluorescence spectra of the CO probe molecule 5a are shown. The selectivity of 10 μM 5a in pH 7.4 PBS (1% CO gas in air at 1 atmosphere; the concentration of other analytes is 100 μM; incubation for 1 h; λ Ex =395nm, bandwidth =3nm).

[0083] Figure 5 NMR mechanism studies of CO sensing using 5a are shown. (I) NMR mechanism of 5a 1 H NMR spectrum; (II) 5a was incubated with CO gas (0.3 equivalent) for 1 h; (III) 22. Solvent: 10 mM PBS in D 2 O:DMSO-d 6 =1:1(v / v).

[0084] Figure 6 HPLC mechanism study of CO sensing using 5a in DI water (a) and EtOH (b) solutions. Conditions: (1) 100 μM 5a in H 2O or EtOH solution; (2) 100 μM 5a was incubated with excess CO gas (>10 equivalents) for 5 min; (3) 100 mM PBS (10×) was added to the sample in (2), and the final PBS concentration was 10 mM (1×); (4) 200 μM 22 in PBS (1×). (UV detector monitored at 214 nm).

[0085] Figure 7 LC-MS spectra for identifying intermediate substances and fluorescent products generated from the reaction between CO gas and 5a are shown: IM-1 (a), 22 (b), and IM-2 (c). For a and b, 100 μM 5a was dissolved in 0.01 M HCl (pH = 2), and 0.5 ml of CO gas was injected, followed by incubation at room temperature. After injection for identification of IM-1, the reaction was neutralized by adding 100 μL of 10×PBS, followed by injection into LCMS for identification of 22. For c, 100 μM 5a was dissolved in ethanol, and 0.5 ml of CO gas was injected, followed by incubation at room temperature, followed by injection into LCMS for identification of IM-2.

[0086] Figure 8 The pH dependence of the fluorescent products 22 (a) and 25 (b) is shown. 30 μM 22 and 10 μM 25 were used at λ EX Fluorescence was measured at =395 nm and 385 nm, bandwidth = 5 nm, low sensitivity setting.

[0087] Fig. 9 The fluorescence spectrum of the CO probe molecule 14 is shown. The selectivity of 10 μM 14 in pH 7.4 PBS (1% CO gas in air at 1 atmosphere; the concentration of other analytes is 100 μM; incubation for 30 min; λ Ex =385nm, bandwidth =3nm).

[0088] Fig.10 It is shown that 14 quantitatively forms 25 and produces the same fluorescence intensity after incubation with CO gas. 200 μM 14 was dissolved in DMA and incubated with 1 ml CO gas for 15 min in a 2 ml headspace vial, then diluted 16-fold to 12.5 μM with PBS and compared with 12.5 μM 25 in PBS (mean ± SD, n = 3, ns: not significant).

[0089] Fig.11The design and fluorescence properties of naphthamide-based CO probe molecules are shown. a. CO sensing chemistry and structure of naphthamide-based CO probe molecules; b. Fluorescence spectra of 10 μM 34 and 29 (bandwidth = 5 nm); c. SNR of 31a at concentrations of 1, 10, 25, and 50 μM in PBS with or without CO gas incubation for 1 hour (SNR is calculated as the ratio between the AUC of the emission spectra with and without CO gas incubation); inset: linear regression of SNR against probe concentration in PBS (bandwidth: ex = 3 nm, em = 5 nm); d. CO sensing kinetics of 31a (800 μL of 12.5 μM 31a was incubated with 200 μL of 1 mM CO-saturated PBS at T 0 The mixture was mixed at 457 nm or 499 nm, and the fluorescence at 457 nm or 499 nm was recorded every second at 25°C. The progress of the reaction at each time point was calculated as the percentage of the fluorescence intensity of the maximum fluorescence); e. and f. Selectivity of 10 μM 31a in pH 7.4 PBS (1% CO gas in air at 1 atmosphere, the concentration of other substances was 100 μM; incubation for 1 h; λ Ex =377 nm; bandwidth = 5 nm; image taken under 385 nm UV light).

[0090] Fig.12 Shown is the stability study of 31 and 31a in PBS solution incubated at 37° C. Monitoring was performed by injecting the solution into HPLC every 15 min.

[0091] Fig.13 LC-MS spectra identifying the fluorescent products generated from the reaction between CO gas and 31a and the CO sensing mechanism are shown. 31a was dissolved in PBS at a concentration of 50 μM. After 20 μL was injected into the LCMS system, 1 ml of CO gas was bubbled into the vial and incubated at room temperature for 20 min before injection into the LC-MS.

[0092] Fig.14 Determination of CO concentration in biological samples is shown. a. Blood of mice with various COHb levels pre-determined by CO-oximetry was incubated with 10 mM 5a for 30 min and then denatured; EX= Fluorescence spectra were collected at 395 nm; inset: calibration curve of COHb versus fluorescence intensity. b. COHb levels in blood from five individual mice were saturated with CO gas and then diluted 2-fold with fresh blood because the maximum limit of the CO oximeter is 80%. c. Blood COHb levels of untreated mice and mice treated with BW-AC-306 (100 mg / kg) were determined using 5a or CO oximeter; inset: calibration curve of COHb versus fluorescence intensity (counts per second (CPS)). d. Blood COHb levels of untreated mice and mice treated with BW-CO-306 (200 mg / kg) were determined using 11a or CO oximeter; inset: calibration curve of COHb versus fluorescence intensity (CPS). e. Relative CO levels of HeLa cells treated with 0.3 μM CDDO-Me (6h) or 250 ppm CO gas (2h), as represented by fluorescence intensity (CPS) tested with a multiplate reader. f. Western blot analysis of mouse heme oxygenase 1 (HO-1) antibody in HeLa cells treated with 0.3 μM CDDO-Me (6 h), with β-actin probed as a loading control. g. Fluorescence spectra of 1 mM 14 in DMA incubated with various CO calibration gases and subsequently diluted with PBS; inset: CO concentration (ppm) relative to the peak at 499 nm (λ EX =385 nm). h. CO concentration in liver tissue of untreated mice and mice treated with BW-CO-306 (200 mg / kg) measured using 14 or methanator-FID-GC. i. CO concentration in kidney tissue of untreated mice and mice treated with BW-CO-306 (200 mg / kg) measured using 14 or methanator-FID-GC. j. Summary data of Figures h and i. k. HeLa cells were treated with CO gas and CO prodrug BW-CO-111 (50 μM) for 2 hours, and CO concentrations tested with 14. For all experiments, n≥3, ****P<0.0001, ns: not significant (P>0.05).

[0093] Fig.15 The cytotoxicity of 31 and 31a in HeLa cells is shown. After 24 h of incubation, cell viability was determined by CCK-8 assay.

[0094] Fig.16Fluorescence microscopy imaging of CO in living cells using naphthamide-based CO probe molecule 31 is shown. HeLa cells were treated with vehicle control (DMSO), 250ppm CO gas, 50μM BW-CO-201, 50μM CORM-401 for 1 hour, followed by the addition of 20μM 31 and incubation for 1 hour. Living cells were washed once with PBS and imaged in Fluorobrite DMEM medium. Exposure time: 0.7s, scale bar: 20μm (BW-CO-201 was reported in Ji, X. et al. Chem Commun (Camb) 2017, 53 (69), 9628-9631; CORM-401 was reported in Crook, SH et al. Dalton Trans 2011, 40, 4230-4235).

[0095] Fig.17 Fluorescence microscopy imaging of CO in living cells using naphthamide-based CO probe molecule 31a is shown. HeLa cells were treated with (a) DMSO vehicle control for 1 h, (b) 250 ppm CO gas for 1 h, or (c) 50 μM BW-CO-201 for 1 h, followed by the addition of 20 μM 31a and incubation for 1 h (scale bar: 20 μm); HeLa cells were treated with (d) DMSO vehicle for 6 h, or (e) 0.3 μM CDDO-Me for 6 h, followed by incubation with 20 μM 31a for 1 h (scale bar: 50 μm); (f) Background normalized maximum signal intensity of cells in the image (*P<0.05, n=3). Living cells were washed twice with PBS and imaged in Fluorobrite DMEM medium; (g) and (h) illustrate the line profile ROI of (f), line profiles 1-3 are used to calculate signal intensity, and line profile 4 is used for background subtraction.

[0096] Fig.18 Determination of the second-order reaction constants of CO probe molecules 6a and 14 for CO is shown.

[0097] Fig.19 Determination of the detection limits of benzamide-based CO probe compounds 5a, 11a, and 14 are shown.

[0098] Fig. 20 Determination of the detection limits of naphthamide-based CO probe compounds 31 and 31a is shown.

[0099] Fig.21 X-ray crystallographic structures of naphthamide-based CO probe compounds 5 and 5a are shown.

[0100] Fig. 22X-ray crystallographic structures of naphthamide-based CO probe compounds 31 and 31a are shown.

[0101] Fig.23 The depalladium species formed by thiol are shown. (a) LC-MS (XIC, extracted ion chromatography) analysis of the reaction between 5 mM 5a and 5 mM GSH in PBS. (b) LC-MS (XIC) analysis of the reaction between 100 μM 5a and 100 μM NaHS in PBS. (c) 1H-NMR and MS of 17 recovered from the reaction of 5a and NaHS (1:2) in DMA.

[0102] Fig.24 The effect of thiol species on 5a is shown. (a) 5a was pre-incubated with or without 5 mM GSH at 5 mM (1:1), 10 mM (2:1), or 5 mM (probe only) in PBS at 37°C for 15 min, followed by incubation with 600 ppm CO gas (0.1 equivalent) for 30 min to partially turn on fluorescence. (b) 5a at a concentration of 100 μM was pre-incubated with NaHS at a concentration of 100 μM (1:1), 50 μM (2:1), 25 μM (4:1), 10 μM (10:1), or 0 (probe only) in PBS at 37°C for 15 min, followed by incubation with 120 ppm CO gas (0.5 equivalent) for 30 min to partially turn on fluorescence. (c) 200 μM 5a was preincubated with 200 μM GSH or 20 μM NaHS in 500 μL PBS for 15 min, then mixed with 500 μL CO2-saturated PBS (about 1 mM), and the fluorescence intensity was recorded every second. (λ Ex =395nm,λ Em =511nm, bandwidth =5nm). DETAILED DESCRIPTION

[0103] I. Overview

[0104] Described herein are fluorescent amide-functionalized palladium coordination complexes that selectively detect carbon monoxide by constructing fluorescent products via sequential CO insertion-carbonylation-amidation reactions with high specificity, high sensitivity, rapid response, and little or no background fluorescence. Also described herein are methods for detecting and quantifying carbon monoxide in biological samples, and methods for imaging intracellular CO accumulation in living cells using fluorescent CO probe compounds.

[0105] II. Definitions

[0106] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are defined.

[0107] As used herein, the terms "a," "an," or "the" include not only elements having one member, but also elements having more than one member. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds, etc.

[0108] As used herein, the terms "about" and "about" etc. are used herein to modify numerical values ​​and indicate a range limited around the value. If "X" is a value, "about X" or "about X" generally indicates a value of 0.90X to 1.10X. Any reference to "about X" indicates at least the following values: X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Therefore, "about X" is intended to be disclosed, for example, "0.98X". When "about" is applied to the beginning of a numerical range, it is applied to both ends of the range. Thus, "about 6 to 8.5" is equivalent to "about 6 to about 8.5". When "about" is applied to the first value of a group of values, it applies to all values ​​in that group. Thus, "about 7, 9 or 11%" is equivalent to "about 7%, about 9% or about 11%".

[0109] As used herein, the terms "comprising" and "comprises" are intended to mean that methods, compounds, compositions, and their respective components include the listed elements, but do not exclude other elements. "Consisting essentially of" refers to those elements required for a given embodiment. The phrase allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of a given embodiment (e.g., method, compound, or composition). "Consisting of" refers to methods, compounds, compositions, and their respective components as described herein, excluding any elements not listed in the description of the embodiment. Embodiments defined by each of these transition terms are within the scope of the invention.

[0110] As used herein, the terms "detect," "detecting," or "detection" refer to the general act of finding or discerning or the specific observation of carbon monoxide.

[0111] As used herein, the term "carbon monoxide" or "CO" refers to and and other forms of carbon monoxide formed under physiological conditions.

[0112] As used herein, the term "fluorescent" refers to the property of a substance that is initially non-fluorescent, and subsequently becomes fluorescent by way of a chemical reaction.

[0113] As used herein, the term "fluorescent amide functionalized palladium coordination complex" refers to a non-fluorescent amide functionalized palladium coordination complex that forms a fluorescent imide after a sequential CO insertion-carbonylation-amidation-cyclization reaction between CO and the fluorescent amide functionalized palladium coordination complex. "Fluorescent amide functionalized palladium coordination complex" refers to a compound having a structure according to Formula I, Formula Ia, Formula III or Formula IIIa as described herein, wherein the palladium metal center contains a coordinated amide-containing ligand.

[0114] As used herein, the term "fluorescent imide" refers to a compound having a structure according to Formula II or Formula IV as described herein, which is formed by a sequential CO insertion-carbonylation-amidation-cyclization reaction between CO and a palladium coordination complex functionalized with a fluorescent amide. Fluorescent imides absorb light energy at a specific wavelength and re-emit light at a longer wavelength for detecting the presence of carbon monoxide in a test sample.

[0115] As used herein, the term "alkyl", alone or as part of another substituent, refers to a straight or branched chain, saturated, aliphatic group having the indicated number of carbon atoms. Alkyl groups may include any number of carbon atoms, such as C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 1-11 , C 1-12 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 3-4 , C 3-5 , C 3-6 , C 4-5 , C 4-6 and C 5-6 For example, C 1-6Alkyl includes but is not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isopentyl, hexyl etc.Alkyl can also refer to an alkyl group with up to 20 carbon atoms, such as but not limited to heptyl, octyl, nonyl, decyl etc.Unless otherwise indicated, an alkyl group can be substituted or unsubstituted.For example, a "substituted alkyl" group can be an alkyl group substituted by one or more groups selected from halo, hydroxyl, amino, aminoalkyl, amide and alkoxy.

[0116] As used herein, the term "alkoxy", alone or as part of another substituent, refers to a group having the formula -OR, wherein R is alkyl as described above.

[0117] As used herein, the term "cycloalkyl", alone or as part of another substituent, refers to a saturated or partially unsaturated monocyclic, fused bicyclic or bridged polycyclic combination containing 3 to 12 ring atoms or the indicated number of atoms. Cycloalkyl may include any number of carbon atoms, such as C 3-6 , C 4-6 , C 5-6 , C 3-8 , C 4-8 , C 5-8 , C 6-8 , C 3-9 , C 3-10 , C 3-11 and C 3-12 . Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2]bicyclooctane, decalin, and adamantane. Cycloalkyl groups may also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3-isomers and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-isomers, 1,4-isomers, and 1,5-isomers), norbornene, and norbornadiene. When the cycloalkyl group is a saturated monocyclic C 3-8 When cycloalkyl is a saturated monocyclic C 3-6 When cycloalkyl, exemplary groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The cycloalkyl group may be substituted or unsubstituted. Unless otherwise indicated, the "substituted cycloalkyl" group may be substituted by one or more groups selected from halo, hydroxyl, amino, alkylamino, amide, acyl, nitro, cyano and alkoxy.

[0118] As used herein, the term "alkylene" refers to an alkyl group as defined above, which is linked to at least two other groups (i.e., a divalent alkyl group). The two moieties linked to the alkylene group may be linked to the same carbon atom or different carbon atoms of the alkylene group.

[0119]

[0046] As used herein, the term "halo" or "halogen," by itself or as part of another substituent, refers to a fluorine, chlorine, bromine, or iodine atom.

[0120] As used herein, the term "haloalkyl", alone or as part of another substituent, refers to an alkyl group in which some or all of the hydrogen atoms are replaced by halogen atoms. As with alkyl groups, haloalkyl groups can have any suitable number of carbon atoms, such as C 1-6 For example, haloalkyl includes trifluoromethyl, fluoromethyl, and the like. In some cases, the term "perfluoro" may be used to define a compound or group in which all hydrogens are replaced by fluorine. For example, perfluoromethyl refers to 1,1,1-trifluoromethyl.

[0121] As used herein, the term "aryl", alone or as part of another substituent, refers to an aromatic ring system having any suitable number of carbon ring atoms and any suitable number of rings. Aryl groups may include any suitable number of carbon ring atoms, such as C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 or C 16 , and C 6-10 , C 6-12 or C 6-14 . The aryl group may be monocyclic, fused to form a bicyclic (e.g., benzocyclohexyl) or tricyclic group or connected by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl with a methylene linker. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl and benzyl. The aryl group may be substituted or unsubstituted. Unless otherwise indicated, a "substituted aryl" group (such as a substituted phenyl or substituted benzyl group) may be substituted by one or more groups selected from halo, hydroxy, amino, alkylamino, amide, acyl, nitro, cyano and alkoxy.

[0122] As used herein, the term "amino" refers to the moiety -NR 2, wherein each R group is H or alkyl. The amino moiety can be ionized to form the corresponding ammonium cation. "Dialkylamino" refers to an amino moiety wherein each R group is alkyl.

[0123] As used herein, the term "sulfonyl" refers to the moiety -SO 2 R, wherein the R group is alkyl, haloalkyl, or aryl. The amino moiety can be ionized to form the corresponding ammonium cation. "Alkylsulfonyl" refers to an amino moiety wherein the R group is alkyl.

[0124] As used herein, the term "hydroxy" refers to the moiety -OH.

[0125] As used herein, the term "cyano" refers to a carbon atom triple-bonded to a nitrogen atom (ie, the moiety -C≡N).

[0126] As used herein, the term "carboxyl" refers to the moiety -C(O)OH. The carboxyl moiety can be ionized to form the corresponding carboxylate anion.

[0127] As used herein, the term "amido" refers to the moiety -NRC(O)R or -C(O)NR 2 , wherein each R group is H or an alkyl group.

[0128] As used herein, the term "nitro" refers to the moiety -NO 2 .

[0129] As used herein, the term "oxo" refers to an oxygen atom double-bonded to a compound (ie, O=).

[0130] As used herein, the term "bidentate ligand" refers to a ligand having two binding groups (e.g., oxygen groups, sulfur groups, nitrogen groups, phosphorus groups) that can attach to palladium metal ions. Non-limiting examples of bidentate ligands include alkylenediamines (e.g., ethylenediamine), bipyridines, substituted bipyridines, phenanthrolines, and ethylenebis(dimethylphosphine). In some embodiments, the bidentate ligand may be separated by two monodentate ligands, such as trialkylphosphines or triarylphosphines (e.g., P(CH 3 ) 3 PPh 3 The bidentate ligand portion of the palladium metal ion attached to the fluorescent amide functionalized palladium coordination complex of the present invention is represented by "L 1 -L 2 ” or “L 3 -L 4 ". The two monodentate ligands attached to the palladium metal ion of the fluoroamide functionalized palladium coordination complex of the present invention are represented by "L 1 and L 2 ” or “L 3 and L 4”It should be understood by those skilled in the art that in the fluorescent amide functionalized palladium coordination complex of the present invention, the bidentate ligand "L 1 -L 2 ” or “L 3 -L 4 A bidentate ligand such as that shown in Formula I, Ia, III and IIIa may be bound by two monodentate ligands "L 1 and L 2 ” or “L 3 and L 4 "Replacement, that is, L 1 is a monodentate ligand and L 2 is a monodentate ligand, which may be the same or different (ie, independently selected), or L 3 is a monodentate ligand and L 4 are monodentate ligands, and the two may be the same or different (ie, independently selected).

[0131] As used herein, the term "test sample" refers to a gas sample (e.g., an ambient air sample, a respiratory air sample, a gas sample collected from a denatured biological sample (such as a cell or tissue lysate), or an industrial gas sample) or various biological sample types obtained or isolated from a subject, which can be used in any of the methods described herein. Biological samples include, but are not limited to, cell cultures or extracts thereof; biopsy materials or extracts thereof obtained from animals (e.g., mammals); and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. For example, the term "biological sample" refers to any solid or fluid sample obtained from a subject, excreted or secreted by a subject, wherein "subject" includes any living organism, including unicellular microorganisms (such as bacteria and yeast) and multicellular organisms (such as plants and animals, e.g., vertebrates or mammals, and especially human subjects). Biological samples can be in any form, including solid materials, such as whole organs, tissues, cells, cell pellets, cell extracts, cell homogenates, or cell fractions; or biopsies or biological fluids. Examples of sources of such samples include muscle, eye, skin, gonad, lymph node, heart, brain, lung, liver, kidney, spleen, thymus, pancreas, solid tumor, macrophage, breast, mesothelium, etc. Biological fluids can be obtained from any part (e.g., whole blood, saliva or mouthwash containing oral cells, tears, plasma, serum, urine, bile, cerebrospinal fluid, amniotic fluid, peritoneal fluid and pleural fluid or cells therefrom, aqueous humor or vitreous humor or any body secretion), transudate, exudate (e.g., fluid obtained from an abscess or any other infection or inflammation site) or fluid obtained from a joint. Biological samples can be obtained from any organ or tissue (including biopsy or autopsy samples), or can include cells (whether primary cells or cultured cells) or culture medium conditioned by any cell, tissue or organ. Biological samples can also include tissue sections, such as frozen sections collected for histological purposes. Biological samples also include mixtures of biomolecules produced by partial or complete fractionation of cell or tissue homogenates, including proteins, lipids, carbohydrates and nucleic acids. Although the sample is preferably taken from a human subject, the biological sample can be from any animal, plant, bacterium, virus, yeast, etc. As used herein, the term animal refers to humans and non-human animals at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms, and unicellular. Cell culture and biopsy samples are considered to be the plural form of animals. In certain exemplary embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, or pig). The animal can be a transgenic animal or a human clone. If desired, the biological sample can be preliminarily processed, including a preliminary separation technique.

[0132] As used herein, the term "non-coordinating anion" refers to any negatively charged ion that acts as a counterion to the positively charged fluoramide-functionalized palladium coordination complex of Formula Ia and Formula IIIa. The non-coordinating anion balances the charge of the positively charged fluoramide-functionalized palladium coordination complex and does not formally bond or share electrons with the metal center of the fluoramide-functionalized palladium coordination complex in a covalent bond. Examples of suitable non-coordinating anions include, but are not limited to, the following: OTf-(CF 3 SO 3 - ), NO 3 - CF 3 COO - , CH 3 COO - , 4-CF 3 C 6 H 4 SO 3 - 、4-CH 3 C 6 H 4 SO 3 - 、F - , Cl - Br - ,I - 、SO 4 2- , CO 3 2- wait.

[0133] III. Fluorochrome-functionalized palladium coordination complexes

[0134] In certain embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula I:

[0135]

[0136] in:

[0137] Each R 1 Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 alkyl;

[0138] R 2 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 2a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties;

[0139] Each R 2a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O) 2 R c 、-S(O) 2 OR a 、-S(O) 2 NR a R b and-NR a S(O) 2 R c ;

[0140] Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl;

[0141] Each R c It is C 1-6 alkyl;

[0142] Part L 1 -L 2 It is a bidentate ligand;

[0143] X is an anionic ligand;

[0144] subscript m is 0 or 1; and

[0145] Subscript n is 0, 1, 2 or 3.

[0146] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula Ia:

[0147]

[0148] Among them A - It is a non-coordinating anion.

[0149] In some embodiments, R 2is a functional group containing at least two sulfonic acid or sulfonate moieties. For example, in some cases, R 2 Selected from the group consisting of:

[0150]

[0151] where v is 0, 1, 2, or 3.

[0152] In some embodiments, in the methods described herein, the fluorescent imide formed by the reaction between carbon monoxide in the test sample and the fluorescent amide functionalized palladium coordination complex of Formula I or Formula Ia is a compound according to Formula II:

[0153]

[0154] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula III:

[0155]

[0156] in:

[0157] Each R 3a and R 3b Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 Alkyl, or R 3a and R 3b Merge to form C 3 -C 7 Cyclic amine ring,

[0158] R 4 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 4a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties,

[0159] Each R 4a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b、-S(O)R c 、-S(O) 2 R c 、-S(O) 2 OR a 、-S(O) 2 NR a R b and-NR a S(O) 2 R c ;

[0160] Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl;

[0161] Each R c It is C 1-6 alkyl;

[0162] Part L 3 -L 4 It is a bidentate ligand;

[0163] X is an anionic ligand;

[0164] subscript p is 0 or 1; and

[0165] The subscripts q and t are independently 0, 1, 2 or 3.

[0166] In some embodiments, the fluoroamide-functionalized palladium coordination complex used in the methods described herein for detecting carbon monoxide is a compound according to Formula IIIa:

[0167]

[0168] Among them A - It is a non-coordinating anion.

[0169] In some embodiments, R 4 is a functional group containing at least two sulfonic acid or sulfonate moieties. For example, in some cases, R 4 Selected from the group consisting of:

[0170]

[0171] where v is 0, 1, 2, or 3.

[0172] In some embodiments, in the methods described herein, the fluorescent imide formed by the reaction between carbon monoxide in the test sample and the fluorescent amide functionalized palladium coordination complex of Formula III or Formula IIIa is a compound according to Formula IV:

[0173]

[0174] In the embodiments disclosed herein, wherein X is an anionic ligand, the anionic ligand may be independently selected from the group consisting of: a halogen ion and -SO 3 R, wherein R is selected from the group consisting of: H, C 1 -C 8 In some embodiments, the anionic ligand (ie, X) is a halide ion such as F. - , Cl - Br - and I - .

[0175] In the embodiments disclosed herein, wherein LL is a bidentate ligand, such as, for example, alkylenediamines, bipyridines, and phenanthroline, the bidentate ligand may be replaced by two monodentate ligands. Examples of bidentate ligands and monodentate ligands suitable for use in the present disclosure include, but are not limited to, substituted or unsubstituted pyridines, substituted or unsubstituted triphenylphosphine, tri-tert-butylphosphine, bipyridines, substituted bipyridines, 1,10-phenanthroline, 2-aminomethylpyridines, 4-aminomethylimidazoles, and analogs thereof (such as, for example, histidine).

[0176] IV. Examples

[0177] In general, unless otherwise indicated, all chemical reagents and solvents used to synthesize the compounds described herein were obtained from commercial suppliers (Sigma-Aldrich, Oakwood, and Fisher Scientific) and used without further purification. Certified carbon monoxide calibration gas was obtained from GASCO. When necessary, silica obtained from Sigma-Aldrich was used and flash column chromatography was performed on a Biotage SP1 system. HPLC analysis was performed on an Agilent 1100 HPLC system (column: Kromasil C18 5 μm, 4.6×150 mm. Mobile phase A: 0.1% trifluoroacetic acid (TFA) in H 2O solution. Mobile phase B: 0.1% TFA in acetonitrile (ACN). Flow rate: 1 mL / min. Gradient: 5% to 95% B, 0 to 10 min; 95% B, 10 to 12 min; 95% to 5% B, 12 to 12.1 min; 5% B, 12.1 to 15 min. Detector: DAD monitored at 220 nm and 254 nm). LCMS analysis was performed on an AB Sciex API 3200 LC-MS / MS (ESI) system with an Agilent 1200 HPLC as the LC module (column: Waters SunFire C18 3.5 μm, 3×150 mm. Mobile phase A: 0.1% formic acid (FA) in H 2 O solution. Mobile phase B: 0.1% FA in ACN. Flow rate: 0.5 ml / min. Gradient: 5% to 95% B, 0 to 10 min; 95% B, 10 to 12 min; 95% to 5% B, 12 to 12.1 min; 5% B, 12.1 to 15 min. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV-400 MHz Ultra Shield NMR instrument. 1 H was recorded at 400 MHz and 13 C was recorded at 101 or 151 MHz. Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively, using the corresponding solvents ( 1 H NMR, 13 C NMR) was used as an internal reference. Fluorescence spectra were recorded on a Shimadzu RF-5301PC fluorescence spectrophotometer.

[0178] Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin-EDTA (0.05%) were purchased from Gibco BRL. The 250 ppm carbon monoxide gas used in the cell culture experiment was customized by Nexair LLC (250 ppm CO, 5% CO 2, balanced air). Cell culture incubator: VWR symphony. To determine cell viability in cytotoxicity assays, a cell counting kit-8 (CCK-8) was purchased from Dojindo and used according to the manufacturer's manual. Optical density (OD) and microplate fluorescence assays were measured using a PerkinElmer Victor3 multi-wavelength plate reader. For CCK assays, OD was measured at 450nm. For fluorescence readings, fluorescence intensity (counts per second, CPS) was measured at 405nm (excitation) and 535nm (emission), with a bandwidth of 10nm, normal pore size and a signal collection time of 2 seconds per well. Cell imaging was performed on an Olympus IX73 inverted fluorescence microscope. Carboxyhemoglobin (COHb) was tested with an AVoximeter 4000 according to the manufacturer's manual.

[0179] All data are expressed as mean ± standard deviation (n ≥ 3). Statistical analysis was performed by Student's t-test using GraphPad Prism 9 to compare the two groups. A p value of less than 0.05 was considered statistically significant.

[0180] Example 1. Synthesis of CO probe molecules (compounds 5, 5a, 6 and 6a).

[0181] Fluoroamide-functionalized palladium coordination complexes 5, 5a, 6 and 6a were synthesized as shown in Scheme 1 below.

[0182] Solution 1

[0183]

[0184] Reagents and conditions: a) HCl, NaNO 2 , KI, 0°C then 90°C, 5 hours; b) i. Oxalyl chloride, DMF (catalytic), DCM, 0°C, 5 minutes then 40°C, 1 hour; ii. n-Butylamine, TEA, 0°C then rt, 2 hours; c) BBr 3 , DCM, -78°C, 1 hour; d) Pd(0)(dba) 2 , TMEDA or 2,2'-bipyridine, DCM, rt, 1.5-2 hours; e) AgOTf, acetone, rt, 30 minutes.

[0185] Preparation of 2-iodo-6-methoxybenzoic acid (2). Commercially available 2-amino-6-methoxybenzoic acid (1,300 mg, 1.8 mmol) was dissolved in a mixture of HCl (3M, 4 mL) and acetone (1 mL), and then cooled to 0 ° C. A mixture of sodium nitrite (250 mg, 3.6 mmol) in 1.5 mL of water was slowly added to this solution, and the reaction was stirred at 0 ° C for 30 minutes, and then a solution of KI (30 mg, 2.0 mmol) in 1 mL of water was added with a pipette. The resulting brown-purple mixture was then heated to 90 ° C for 5 hours. The reaction was monitored to completion using thin layer chromatography (TLC, hexane: ethyl acetate = 3: 1, v: v). After cooling to room temperature, the reaction was heated to 40 ° C with saturated NH 4 Cl solution (20 mL), extracted with dichloromethane (DCM, 50 mL), and washed successively with brine (3×50 mL). 2 SO 4 Dried and concentrated under vacuum to give a brown residue.The residue was purified by flash column chromatography to give Compound 2 (376 mg, 75% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ9.40(s-br,1H),7.44(d,J=8.0Hz,1H),7.08(t,J=8.0Hz,1H),6.94(d,J=8.0Hz,1H),3.87(s,3H). 13 C NMR (101 MHz, CDCl 3 )δ172.32,156.76,131.94,131.22,129.10,110.89,92.18,77.36,77.04,76.72,56.25. The obtained NMR spectrum of compound 2 is comparable to that reported in the literature. See Whyte, A. et al. Org Lett. 2018, 20, 345-348.

[0186] Preparation of N-butyl-2-iodo-6-methoxybenzamide (3). Under argon protection, compound 2 (376mg, 1.352mmol) is dissolved in 5mL anhydrous DCM, and cooled to 0 DEG C. Then catalytic amount (two drops) of anhydrous dimethylformamide (DMF) is added to the mixture, followed by addition of oxalyl chloride (0.18mL, 2.03mmol). The reaction is heated to 40 DEG C for 1 hour, and then the light yellow reaction mixture is concentrated to dryness under vacuum. After the obtained residue is dissolved in 2mL anhydrous DCM, a mixture of n-butylamine (0.16mL, 1.62mmol) and triethylamine (0.223mL, 1.62mmol) in 1mL DCM is slowly added via a syringe at 0 DEG C, and allowed to stir at room temperature for 2 hours. The reaction progress is monitored by TLC (DCM: methanol = 20: 1, v: v). After completion, the reaction was quenched with brine (10 mL), extracted with DCM (50 mL), and then washed with saturated NaHCO 3 solution (3×20 mL) and brine (3×20 mL), and 2 SO 4 The mixture was then concentrated under vacuum to give compound 3 (406 mg, 90% yield) as a white solid without further purification. 1 H NMR (400 MHz, CDCl 3 )δ7.38(dd,J=8.0,0.8Hz,1H),7.00(t,J=8.1Hz,1H),6.86(d,J=8.3Hz,1H),5.66(s,1H),3.79( s,3H),3.45(td,J=7.1,5.8Hz,2H),1.65–1.54(m,2H),1.50–1.36(m,2H),0.95(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ167.74,156.72,132.84,131.30,131.22,110.89,94.21,56.09,39.78,31.45,20.23,13.88. HRMS (ESI, m / z): C 12 H 17 O 2 NI[M+H] + The calculated value is 334.0298 and the measured value is 334.0292.

[0187] Preparation of N-butyl-2-hydroxy-6-iodobenzamide (4). A dry 20 mL reaction vial containing a stirring bar and a solution of compound 3 (0.377 mg, 1.131 mmol) in 1 mL of anhydrous DCM was cooled to -78 °C using an acetone / dry ice bath. BBr 3 The mixture was stirred for 30 minutes at -78 °C and then quenched with methanol (MeOH, 2 mL). The mixture was then diluted with 50 mL DCM and washed with brine (3 × 50 mL). The organic layer was stirred for 10 minutes at -78 °C for 30 minutes at -78 °C. 2 SO 4 Dried and concentrated in vacuo.The residue was purified by flash column chromatography (DCM:MeOH=50:1) to give compound 4 (342 mg, 90% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ10.91(s,1H),7.42(dd,J=5.5,3.5Hz,1H),6.99–6.87(m,2H),6.84–6.63(m,1H),3.5 0 (q, J = 6.8 Hz, 2H), 1.65 (p, J = 7.3 Hz, 2H), 1.46 (h, J = 7.4 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H). 13 CNMR (101MHz, CDCl 3 )δ168.68,160.53,133.46,132.49,121.65,118.52,91.70,39.98,31.15,20.47,13.85. HRMS (ESI, m / z): C 11 H 15 O 2 NI[M+H] + The calculated value is 320.0142 and the measured value is 320.0132.

[0188] (2-(Butylcarbamoyl)-3-hydroxyphenyl)(N 1 ,N 1 ,N 2 ,N 2 Preparation of bis(dibenzylideneacetone)palladium(II)iodide (5). Under argon protection, compound 4 (100 mg, 0.313 mmol) and bis(dibenzylideneacetone)palladium (Pd(dba) 2, 179 mg, 0.313 mmol) was dissolved in 5 mL of DCM. Then, deoxygenated N 1 ,N 1 ,N 2 ,N 2 -Tetramethylethylenediamine (TMEDA, 57 μL, 0.375 mmol) was injected into a sealed reaction vial and the reaction was stirred at room temperature. After stirring for 30 minutes, the color of the reaction mixture was observed to change from red to yellow-black. The reaction was allowed to stir for an additional 60 minutes until completion, as indicated by TLC. The reaction mixture was passed through a dry MgSO4 column and precipitated. 2 SO 4 The filtrate was concentrated to dryness and washed with hexane and anhydrous ether (Et 2 O) to form an orange solid precipitate, which was isolated as Compound 5 (151 mg, 89% yield) without further purification. 1 H NMR (400 MHz, CDCl 3 )δ12.29(s,1H),9.39(s,1H),7.12(d,J=7.6Hz,1H),6.86(t,J=7.8Hz,1H),6.44(d,J=8.0Hz,1H),3.57(m,1H),3.47(m,1H),2.74(s,3H),2. 70(s,3H),2.66–2.64(m,2H),2.58(m,2H),2.26(s,3H),2.11(s,3H),1.75(qu,J=7.4Hz,2H),1.52(sx,J=7.3Hz,2H),0.97(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ171.48,161.28,145.10,129.79,128.41,121.04,112.75,95.27,62.37,58.67,50.28,50.17,49.95,39.72,31.84,20.76,13.94. HRMS (ESI, m / z): C 17 H 30 N 3 O 2 Pd[MI] + The calculated value is 414.1380 and the measured value is 414.1396.

[0189] (2-(Butylcarbamoyl)-3-hydroxyphenyl)(N 1 ,N 1 ,N 2 ,N 2-Tetramethylethylenediamine) palladium ring trifluoromethanesulfonate (5a) preparation. In a dry 20mL reaction vial with a sealable septum and a stirring bar, compound 5 (120mg, 0.221mmol) was dissolved in anhydrous acetone (8mL). To this solution, silver trifluoromethanesulfonate (AgOTf, 57mg, 0.222mmol) was added as a solid, and a precipitate was formed immediately. The resulting mixture was stirred for 30 minutes and filtered through diatomaceous earth. The yellow filtrate was then concentrated, passed through a short silica gel column (DCM: MeOH = 20: 1), and the main fraction was concentrated to dryness under vacuum to obtain compound 5a (108mg, 87% yield) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.91(s,1H),8.86(t,J=5.7Hz,1H),7.08(t,J=7.9Hz,1H),6.99(d,J=8.2Hz,1H),6.50(d,J=7.5Hz,1H),3.37(q,6. 8Hz,2H),2.99–2.83(m,8H),2.83–2.64(m,8H),1.57(quint,7.2Hz,2H),1.37(sx,7.3Hz,2H),0.93(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ178.89,155.25,131.99,126.71,122.01,118.86,114.74,65.28,57.58,52.10,47.98,39.98,31.07,20.05,13.69. HRMS (ESI, m / z): C 17 H 30 O 2 N 3 Pd[M] + The calculated value is 414.1367 and the measured value is 414.1367.

[0190] Preparation of (2-(butylcarbamoyl)-3-hydroxyphenyl)(bipyridine)palladium(II) iodide (6) and (2-(butylcarbamoyl)-3-hydroxyphenyl)(bipyridine)palladium cyclotriflate (6a). Under argon protection, compound 4 (133 mg, 0.417 mmol), Pd(dba) 24-(2-(2-(2-pyridyl)-1-yl)-2-nitropropene (262mg, 0.458mmol) and 2,2'-bipyridine (BIPY, 65mg, 0.417mmol) were dissolved in 10mL DCM. The reaction mixture was stirred at room temperature and changed from red to dark green-yellow within 30 minutes. The reaction was allowed to stir for an additional 30 minutes until completion, as indicated by TLC. The reaction mixture was diluted with DCM (10mL) and dried over MgSO4 on diatomaceous earth. 2 SO 4 The mixture was stirred for 30 minutes and filtered through diatomite. The yellow filtrate was concentrated to dryness and purified by flash column chromatography to obtain compound 6 (146 mg, 60% yield) as a yellow solid intermediate, which was then dissolved in anhydrous acetone (12 mL). AgOTf (65 mg, 0.251 mmol) was added to this solution as a solid, and a precipitate was formed immediately. The resulting mixture was stirred for 30 minutes and filtered through diatomite. The yellow filtrate was concentrated, passed through a short silica gel column (DCM: MeOH = 20: 1), and the main fraction was collected and concentrated to dryness under vacuum to obtain compound 6a (117 mg, 78% yield) as a bright yellow solid. 1 H NMR (400MHz, CD 3 OD-CDCl 3 Mixture) δ8.89–8.48(m,2H),8.36(d,J=7.9Hz,2H),8.21(t,J=7.6Hz,2H),7.71–7.64(m,2H),7.09(t,J=7.9Hz,1H),6.71 (br-s,1H),6.62(d,J=8.1Hz,1H),3.47(t,J=6.9Hz,2H),1.71–1.58(m,2H),1.46(dd,J=7.3Hz,2H),0.97(br-s,3H). 13 C NMR (151 MHz, CD 3 OD-CDCl 3 Mixture) δ 156.33, 152.77, 151.13, 150.78, 147.50, 139.83, 139.69, 130.90, 126.47, 126.21, 123.08, 121.96, 112.50, 39.03, 30.41, 19.31, 12.35. HRMS (ESI, m / z): C 21 H 22 O 2 N 3 Pd[M+H] + The calculated value is 454.0741 and the measured value is 454.0759.

[0191] Example 2. Synthesis of CO probe molecules (compounds 11, 11a and 12-14).

[0192] Fluoroamide-functionalized palladium coordination complexes 11, 11a, and 12-14 were synthesized as shown in Scheme 2 below.

[0193] Solution 2

[0194]

[0195] Reagents and conditions: a) HCl, NaNO 2 , KI, 0℃ then 90℃, 3-5 hours; b) Na 2 S 2 O 4 , H 2 O / THF, 50°C, 3 hours; c) i.SOCl 2 , toluene, reflux, 3 hours; ii. n-butylamine or n-propylamine, TEA, DCM, 0°C, 2 hours; d) Pd(0)(dba) 2 , TMEDA or 2,2'-bipyridine, DCM, rt, 1.5-2 hours; e) AgOTf, THF, rt, 30 min.

[0196] Preparation of 2-iodo-6-nitrobenzoic acid (8). In a 50 mL round-bottom flask, 2-amino-6-nitrobenzoic acid (7, 500 mg, 2.74 mmol) was dissolved in a mixture of HCl (37%, 5 mL) and acetone (5 mL), and then cooled to 0 ° C. To this solution, a solution of sodium nitrite (250 mg, 3.6 mmol) in 1.5 mL of water was slowly added, and the reaction was stirred at 0 ° C for 30 minutes, and then a solution of KI (462 mg, 2.8 mmol) in 1 mL of water was added with a pipette. The resulting brown-purple mixture was then heated to 90 ° C for 3 hours. The reaction was monitored by TLC (dichloromethane: methanol = 10: 1, v: v) until completion. After cooling to room temperature, the reaction was washed with saturated NH 4 The mixture was quenched with Cl solution (30 mL), extracted with DCM (50 mL), and then washed with brine (3×50 mL). 2 SO 4 Dried and concentrated under vacuum to give a brown residue.The residue was purified by flash column chromatography (DCM:MeOH=2-10%, with 0.5% acetic acid) to give compound 8 (473 mg, 57% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ8.26 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H). It is worth noting that the obtained 1 The H NMR spectrum is comparable to that reported in the literature. See Fu, Z. et al. Org. Lett. 2019, 21(9), 3003-3007.

[0197] Preparation of 2-iodo-6-aminobenzoic acid (9). In a 100 mL round-bottom flask, compound 8 (473 mg, 1.55 mmol) was dissolved in tetrahydrofuran (THF, 20 mL), and sodium dithionite (Na 2 S 2 O 4 , 2.697 g, 15.5 mmol) in 15 mL of deionized (DI) water while stirring. The mixture was vigorously stirred at 50 ° C for 3 hours until completion, as indicated by TLC. The reaction mixture was diluted with HCl (1 M, 30 mL) and then extracted with ethyl acetate (EtOAc, 80 mL). The organic layer was washed with brine (3×80 mL) and purified by Na 2 SO 4 Drying and then concentration in vacuo gave an off-white residue.The residue was purified by flash column chromatography to give compound 9 (186 mg, 46% yield) as an off-white solid. 1 HNMR (400MHz, DMSO-d6): δ7.03(d,J=7.5Hz,1H), 6.78(t,J=7.9Hz,1H), 6.70(d,J=8.2Hz,1H); 13 C NMR (101 MHz, CD 3 OD)δ171.86,147.18,132.10,129.52,128.81,116.55,94.34,40.62,32.14,21.41,14.11. HRMS (ESI, m / z): C 7 H 7 N 2 OI[M+H] + The calculated value is 263.9516 and the measured value is 263.9510.

[0198] Preparation of 2-amino-N-butyl-6-iodobenzamide (10a). To a solution of compound 9 (97 mg, 0.36 mmol) in toluene (10 mL) was added thionyl chloride (SOCl) at room temperature. 2, 130 μL, 1.8 mmol), and the mixture was heated at 110°C for 3 hours. Afterwards, the solvent was removed under vacuum to obtain crude 2-amino-6-iodobenzoyl chloride as a yellow oil, which was immediately used in the next step without purification or characterization. Triethylamine (Et 3 N, 50 μ L, 0.36 mmol) was added to a solution of n-butylamine (35 μ L, 0.36 mmol) in DCM (10 mL), and stirred for 10 minutes, then a solution of crude 2-amino-6-iodobenzoyl chloride in DCM (2 mL) was added dropwise at 0 ° C. The reaction was allowed to stir for 2 hours until completion, as confirmed by TLC. The solvent was then removed under reduced pressure, and the residue was purified by flash column chromatography (hexane: EtOAc = 1: 1, v / v) to obtain compound 10a (85 mg, 75% yield) as a white solid. 1 H NMR (400MHz, CD 3 OD)δ7.12(d,J=7.7Hz,1H),6.80(t,J=7.9Hz,1H),6.73(d,J=8.2Hz,1H),3.35 (t,J=7.1Hz,2H),1.67–1.60(m,2H),1.52–1.43(m,2H),0.97(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CD 3 OD)δ171.86,147.18,132.10,129.52,128.81,116.55,94.34,40.62,32.14,21.41,14.11. HRMS (ESI, m / z): C 11 H 16 N 2 OI([M+H] + ) is calculated to be 319.0302 and the measured value is 319.0295.

[0199] Preparation of 2-amino-N-propyl-6-iodobenzamide (10b). Compound 10b (95 mg, 87% yield) was prepared according to a procedure similar to that used to prepare compound 10a, using n-propylamine (30 μL, 0.36 mmol) instead of n-butylamine. 1 H NMR (400 MHz, CDCl 3 )δ7.15(d,J=7.8Hz,1H),6.77(t,J=7.9Hz,1H),6.61(d,J=8.2Hz,1H),5.96(s,1H),3.38(q,7.2Hz,2H),1.72–1.58(m,2H),0.99(t,J=7.5Hz,3H). 13C NMR (101 MHz, CDCl 3 )δ169.02,145.84,131.47,128.99,127.64,115.97,93.37,41.76,22.59,11.77. HRMS (ESI, m / z): C 10 H 14 N 2 OI[M+H] + The calculated value is 305.0151 and the measured value is 305.0148.

[0200] (2-(Butylcarbamoyl)-3-aminophenyl)(N 1 ,N 1 ,N 2 ,N 2 Preparation of -tetramethylethylenediamine)palladium(II) iodide (11). Under argon protection, compound 10a (60 mg, 0.188 mmol) and Pd(dba) 2 4-(4-(4-(4-(4-(4-nitro-1-yl)-2-nitropropene))-1-nitropropene)-2-nitropropene ... 2 SO 4 The mixture was filtered through a pad of 5% paraffin wax and the filtrate was concentrated to dryness and washed with hexane and anhydrous Et 2 O to obtain a yellow solid which was isolated as Compound 11 (151 mg, 89% yield) without further purification. 1 H NMR (400 MHz, CDCl 3 )δ8.17(s,1H),6.88(d,J=7.3Hz,1H),6.69(t,J=7.7Hz,1H),6.18(d,J=7.5Hz,1H),3.72–3.31(m,2H),2.8 1–2.47(m,10H),2.36(s,3H),2.21(s,3H),1.84–1.66(m,2H),1.49(q,J=7.5Hz,2H),0.95(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3)δ170.50,146.80,143.53,128.00,127.41,124.81,112.09,62.43,58.54,50.84,50.13,50.07,49.92,39.64,31.98,20.71,13.99. HRMS (ESI, m / z): C 17 H 32 N 4 OIPd[M+H] + The calculated value is 541.0656 and the measured value is 541.0640.

[0201] (2-(Butylcarbamoyl)-3-aminophenyl)(N 1 ,N 1 ,N 2 ,N 2 -Tetramethylethylenediamine) palladium ring trifluoromethanesulfonate (11a) preparation. Compound 11 (151mg, 0.279mmol) was dissolved in degassed THF (10mL) and cooled in an ice / water bath, then AgOTf (75mg, 0.293mmol) was added in a solid state under argon protection. Precipitation was immediately formed and the reaction was stirred for 30 minutes. The reaction mixture was then filtered through diatomaceous earth and the filtrate was concentrated under vacuum. Compound 11a (112mg, 72% yield) was obtained as a light yellow solid by flash column chromatography (DCM: MeOH = 10: 1). 1 H NMR (400 MHz, CDCl 3 )δ9.32(s,1H),7.06(t,J=7.7Hz,1H),6.70(d,J=7.8Hz,1H),6.65(d,J=7.6Hz,1H),3.99(s,2H),3.43-3.36(m,2H),2 .93(s,6H),2.74(d,J=5.2Hz,2H),2.70–2.25(m,8H),1.63-1.56(m,3H),1.37(q,J=7.3Hz,2H),0.93(t,J=7.2Hz,3H). 13CNMR (101MHz, CDCl3) δ179.43,151.97,144.36,131.36,130.88,124.82,120.25,65.43,57.67,52.00,48.02,40.24,31.11,20.28,13.84. HRMS (ESI, m / z): C 17 H 31 N 4 OPd[M+H] + The calculated value is 413.1533 and the measured value is 413.1551.

[0202] (2-(Propylcarbamoyl)-3-aminophenyl)(N 1 ,N 1 ,N 2 ,N 2 Preparation of -tetramethylethylenediamine)palladium(II) iodide (12). Under argon protection, compound 10b (50 mg, 0.164 mmol) and Pd(dba) were mixed in a dry 20 mL reaction vial with a sealable septum. 2 4-(4-(4-(4-(4-(4-(4-nitro-1-yl)-2-yl)-2-nitropropene)-1-yl)-4-nitropropene-1-yl)-2 ... 2 SO 4 The dried filtrate was then washed with hexane and anhydrous Et 2 O to obtain a yellow solid, which was further purified by flash column chromatography (DCM:MeOH=20:1) to give compound 12 (73 mg, 85% yield). 1 H NMR (400MHz, CD 3 OD) δ7.10 (t, J = 7.8 Hz, 1H), 6.76-6.73 (doublet, J = 7.8, 2H), 3.46 (t, J = 7.1 Hz, 2H), 2.96 (s, 6H), 2.81-2.76 (m, 2H), 2.74 (s, 6H), 2.60 (s, 2H), 1.70 (sex, J = 7.4 Hz, 2H), 1.02 (t, J = 7.4 Hz, 3H). 13 C NMR (CD 3 OD)δ181.0,153.4,147.2,132.3,130.8,125.1,119.8,66.3,58.4,55.0,52.1,48.0,44.5,42.9,23.5,11.8.

[0203] Preparation of (2-(butylcarbamoyl)-3-aminophenyl)(bipyridyl)palladium(II) iodide (13). Compound 10a (41 mg, 0.13 mmol), Pd(dba) 2(82mg, 0.142mmol) and 2,2'-bipyridine (BIPY, 22mg, 0.130mmol) are weighed together and put into a dry 20mL vial with a sealable diaphragm, and rinsed with argon. Dry degassed DCM (2mL) is added to the reaction vial via a syringe to fully dissolve the reactant. The reaction mixture is stirred at room temperature and changes from dark red to yellow-brown within 30 minutes of stirring. The reaction is allowed to stir for 1 hour until compound 10a is completely consumed, as indicated by TLC. The reaction mixture is then purified by flash column chromatography (DCM / MeOH, 0%-5% MeOH gradient) to obtain compound 13 (60mg, 73% yield) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.62(d,J=3.5Hz,1H),8.07–8.01(m,3H),8.00–7.91(m,2H),7.60(d,J=5.5H z,1H),7.54(t,J=6.4Hz,1H),7.39(t,J=5.7Hz,1H),7.01(d,J=7.6Hz,1H),6.7 7(t,J=7.7Hz,1H),6.32(d,J=7.8Hz,1H),4.68(br-s,2H),3.54–3.45(m,1H),3 .17-3.13(m,1H),1.45-1.34(m,2H),1.29-1.18(m,2H),0.51(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ170.03,155.27,153.93,152.83,150.54,147.29,144.49,138.79,138.75,128.12, 127.57,127.05,126.75,124.97,121.89,121.57,112.47,39.17,31.90,20.38,13.57. HRMS (ESI, m / z): C 21 H 25 N 4 OIPd[M+H] + The calculated value is 581.0030 and the measured value is 581.0032.

[0204] Preparation of (2-(propylcarbamoyl)-3-aminophenyl)(bipyridyl)palladium(II) iodide (14). Compound 10b (69 mg, 0.206 mmol), Pd(dba) 210b) was added to the mixture of 4-nitropropene (118mg, 0.206mmol) and BIPY (32mg, 0.206mmol) weighed together and put into a dry 20mL vial with a sealable diaphragm, and rinsed with argon. Dry degassed DCM (2mL) is added to the reaction vial via a syringe to fully dissolve the reactant. The reaction mixture is stirred at room temperature, and changes from crimson to yellow-brown within 30 minutes of stirring. The reaction is allowed to stir for 1 hour until compound 10b is completely consumed, as indicated by TLC. The reaction mixture is then purified by flash column chromatography (DCM / MeOH, 0%-5% MeOH gradient) to obtain compound 14 (68mg, 58% yield) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.62(d,J=5.1Hz,1H),8.06–8.02(m,3H),7.99–7.95(m,2H),7.61(dd,J=5.5,2.1Hz,1H),7.56–7.55(m,1H),7.43–7.40(m,1H),7.02(d,J= 7.6Hz,1H),6.79(t,J=7.7Hz,1H),6.35(d,J=7.9Hz,1H),3.40(p,J=6.7Hz,1H),3.25–3.10(m,1H),1.53–1.37(m,2H),0.74(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ170.21,155.47,154.10,153.06,150.74,147.09,144.61,138.87,138.85,128. 30,127.93,127.20,126.91,125.30,121.98,121.64,112.76,41.33,23.07,11.95. HRMS (ESI, m / z): C 20 H 22 N 4 OIPd[M+H] + The calculated value is 566.9868 and the measured value is 566.9886.

[0205] Example 3. Synthesis of original depalladium products (Compounds 17 and 19).

[0206] The ortho-depalladiumated benzamide materials 17 (Scheme 3) and 19 (Scheme 4) were synthesized as shown below.

[0207] Solution 3

[0208]

[0209] Reagents and conditions: a) i. Oxalyl chloride, DMF (catalytic), DCM, 0°C, 5 minutes then 40°C, 1 hour; ii. n-Butylamine, TEA, 0°C then rt, overnight; b) BBr 3 , DCM, -78°C, 1 hour.

[0210] Preparation of N-butyl-2-methoxybenzamide (16). Under argon protection, in a 20mL reaction vial, 2-methoxybenzoic acid (15, 200mg, 1.314mmol) was dissolved in 10mL anhydrous DCM, and anhydrous DMF of a catalytic amount (two drops) was added. The mixture was cooled to 0°C, and oxalyl chloride (167μL, 1.971mmol) was added to the reaction vial via a syringe. The reaction was allowed to stir for 5 minutes at 0°C, heated to 40°C and stirred for 1 hour, and then the reaction mixture was concentrated to dryness under vacuum. After the obtained residue was dissolved in 5mL anhydrous DCM, a mixture of n-butylamine (156μL, 1.577mmol) and triethylamine (271.5μL, 1.971mmol) was added via a syringe at 0°C, and it was allowed to stir at room temperature overnight. The reaction mixture was diluted with DCM (30mL), saturated NaHCO 3 solution (3×50 mL) and brine (3×50 mL), and 2 SO 4 The mixture was then concentrated under vacuum to give compound 16 (223 mg, 83% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ8.20(dd,J=7.8,1.8Hz,1H),7.85(s,1H),7.47–7.37(m,1H),7.07(t,J=7.5Hz,1H),6.96(d,J=8.3 Hz, 1H), 3.46 (q, J = 7.0Hz, 2H), 1.60 (p, J = 7.3Hz, 2H), 1.41 (sx, J = 7.3Hz, 2H), 0.96 (t, J = 7.3Hz, 3H). 13 C NMR (101 MHz, CDCl 3 )δ165.03,157.26,132.39,131.92,121.58,121.00,111.17,55.76,39.29,31.52,20.11,13.67. HRMS (ESI) calculated value: 230.1141 (C 12 H 17 NO 2 Na, [M+Na] + ); Measured value: 230.1148.

[0211] Preparation of N-butyl-2-hydroxybenzamide (17). A dry 20 mL reaction vial containing a stirring bar and a solution of compound 16 (218 mg, 1.063 mmol) in 8 mL of anhydrous DCM was cooled to -78 °C using an acetone / dry ice bath. BBr 3 The mixture was stirred for 30 minutes at -78 °C and then quenched with MeOH (5 mL). The mixture was then diluted with 50 mL DCM and washed with brine (3 × 50 mL). The organic layer was stirred for 10 minutes at -78 °C for 30 minutes at -78 °C. 2 SO 4 Dried and concentrated in vacuo.The residue was purified by flash column chromatography (DCM:MeOH=50:1) to give compound 17 as a light yellow oil which turned into a waxy solid at -15°C (186 mg, 92% yield). 1 H NMR (400 MHz, CDCl 3 )δ12.49(s,1H),7.42(d,J=8.0Hz,1H),7.35(t,J=8.6Hz,1H),6.94(d,J=9.3Hz,1H),6.80(t,J=8.2Hz,1H) ,6.73(s,1H),3.41(q,J=7.2Hz,2H),1.57(p,J=7.3Hz,2H),1.36(sx,J=7.3Hz,2H),0.91(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ170.05,161.29,134.09,125.64,118.78,118.39,114.51,39.52,31.50,20.14,13.75. HRMS (ESI): calculated value: 216.1000 (C 11 H 15 NO 2 Na, [M+Na] + ); Measured value: 216.0992.

[0212] Solution 4

[0213]

[0214] Reagents and conditions: a) n-propylamine, H 2 O, rt, 2 hours.

[0215] The preparation of N-propyl-2-aminobenzamide (19).In a 50mL round-bottomed flask, isatoic anhydride (18,200mg, 1.227mmol) is suspended in 10mL DI water, stirred simultaneously, and n-propylamine (108.6mg, 1.84mmol) is slowly added, so as to cause the suspension to become clear, and form a white precipitate.Allow the reaction to be stirred for another 2 hours, then dilute with DI water and filter.The collected solid is washed with water successively and dried under vacuum, to obtain compound 19 (216mg, 99% yield) in white solid shape, it does not need to be further purified. 1 H NMR (400 MHz, CDCl 3 )δ7.30(d,J=7.9Hz,1H),7.20(t,J=8.4Hz,1H),6.68(d,J=8.2Hz,1H),6.65(t,J=7.5Hz ,1H),6.10(s,1H),3.37(q,J=7.2Hz,2H),1.63(sx,J=7.3Hz,2H),0.98(t,J=7.4Hz,3H); 13 C NMR (101 MHz, CDCl 3 )δ169.45,148.53,132.26,127.13,117.48,116.83,116.68,41.50,23.05,11.60. HRMS(ESI):C 10 H 15 N 2 O[M+H] + Calculated value: 179.1184, measured value: 179.1187.

[0216] Example 4. Synthesis of fluorescent products (Compounds 22-25).

[0217] Fluorescent imide species 22-23 (Scheme 5) and 24-25 (Scheme 6) were synthesized as shown below.

[0218] Solution 5

[0219]

[0220] Reagents and conditions: a) AcOH, reflux, 3 hours.

[0221] Preparation of 3-hydroxy-N-butylphthalimide (22). To a solution of 3-hydroxyphthalic anhydride (20a, 250mg, 1.52mmol) in acetic acid (AcOH, 3mL), n-butylamine (21a, 133mg, 1.82mmol) was added dropwise. The resulting mixture was refluxed for 3 hours, cooled to room temperature, and then poured into 10mL of purified water. The formed precipitate was filtered and washed with DI water to produce pure compound 22 (235mg, 70% yield) as a white solid. 1 H NMR (400MHz, DMSO) δ7.60–7.49(m,1H),7.22(d,J=7.1Hz,1H),7.13(d,J=8.4Hz,1H) ,3.46(t,J=7.0Hz,2H),1.52–1.39(m,2H),1.24–1.10(m,2H),0.81(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ170.74,168.17,154.74,136.41,132.30,122.65,116.00,114.78,37.78,30.77,20.19,13.76. HRMS (ESI, m / z): C 12 H 14 O 3 N([M+H] + ) calculated value: 220.0968; measured value: 220.0970.

[0222] Preparation of 3-hydroxy-N-propylphthalimide (23). To a solution of 3-hydroxyphthalic anhydride (20a, 150mg, 0.91mmol) in AcOH (2mL), n-propylamine (21b, 69mg, 1.18mmol) was added dropwise. The resulting mixture was refluxed for 3 hours, cooled to room temperature, and then poured into 10mL DI water. The precipitate was filtered and washed with pure water to obtain pure compound 23 (148mg, 79% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ11.13(s,1H),7.59(dd,J=8.3,7.2Hz,1H),7.25(d,J=7.1Hz,1H), 7.17(d,J=8.3Hz,1H), 3.45(t,J=7.1Hz,2H), 1.54(h,J=7.4Hz,2H), 0.82(t,J=7.4Hz,3H). 13C NMR (101MHz, DMSO) δ167.87,166.81,155.13,135.99,133.58,123.24,114.66,114.01,38.73,21.41,11.26. HRMS (ESI, m / z): C 11 H 12 O 3 N([M+H] + ) calculated value: 206.0817; measured value: 206.0813.

[0223] Solution 6

[0224]

[0225] Reagents and conditions: a) AcOH, reflux, 3 hours; b) Na 2 S 2 O 4 , THF / H 2 O, rt, 4 hours.

[0226] Preparation of 3-nitro-N-butylphthalimide (24.1) and 3-amino-N-butylphthalimide (24). To a solution of 3-nitrophthalic anhydride (20b, 250 mg, 1.29 mmol) in AcOH (3 mL) was added n-butylamine (21a, 122 mg, 1.68 mmol) dropwise. The resulting mixture was refluxed for 3 hours, cooled to room temperature, and then poured into 10 mL DI water. The precipitate was filtered and washed with purified water to produce an intermediate compound 24.1 in the form of a white solid, which was directly used in the next synthesis step. Sodium dithionite (224 mg, 1.29 mmol) was then added to the intermediate compound 24.1 in THF (2 mL) and H 2 O (2mL) mixture. The reaction was allowed to stir at room temperature for 4 hours, then the reaction mixture was diluted with water (40mL) and extracted with EtOAc (40mL). The organic layers were combined and washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure to give a crude product which was then purified by flash column chromatography to obtain pure compound 24 (187 mg, 67% yield). 1 H NMR (400 MHz, CDCl 3)δ7.38(dd,J=8.1,7.3Hz,1H),7.12(d,J=7.1Hz,1H),6.83(d,J=8.3Hz,1H),5.20(s,2 H), 3.61 (t, J = 7.2Hz, 2H), 1.67–1.55 (m, 2H), 1.40–1.28 (m, 2H), 0.93 (t, J = 7.4Hz, 3H). 13 C NMR (101 MHz, CDCl 3 )δ170.49,168.85,145.23,135.12,133.00,121.04,112.73,111.56,37.48,30.86,20.20,13.79. HRMS (ESI, m / z): C 12 H 15 O 2 N 2 ([M+H] + ) calculated value: 219.1128; measured value: 219.1130.

[0227] Preparation of 3-nitro-N-propylphthalimide (25.1) and 3-amino-N-propylphthalimide (25). To a solution of 3-nitrophthalic anhydride (20b, 250 mg, 1.29 mmol) in AcOH (3 mL) was added n-propylamine (21b, 98 mg, 1.68 mmol) dropwise. The resulting mixture was refluxed for 3 hours, cooled to room temperature, and then poured into 10 mL of purified water. The precipitate was filtered and washed with purified water to produce an intermediate compound 25.1 in the form of a white solid, which was directly used in the next synthetic step. Sodium dithionite (224 mg, 1.29 mmol) was then added to the intermediate compound 25.1 in THF (2 mL) and H 2 O (2mL) mixture. The reaction mixture was allowed to stir at room temperature for 4 hours, then quenched by water (40mL) and extracted with EtOAc (40mL). The organic layers were combined and washed with anhydrous Na 2 SO 4 Dried and concentrated under reduced pressure to give a crude product which was then purified by flash column chromatography to obtain pure compound 25 (181 mg, 69% yield). 1 H NMR (400 MHz, DMSO) δ7.44–7.40 (m, 1H), 6.97–6.94 (m, 2H), 6.43 (s, 2H), 3.45 (t, J = 7.2 Hz, 2H; with H 2 O peak collision), 1.56 (p, J = 7.6 Hz, 2H), 0.84 (t, J = 7.4 Hz, 3H). 13C NMR (101MHz, DMSO) δ170.01,168.63,146.85,135.56,132.77,121.83,111.13,109.36,38.94,21.89,11.67. HRMS (ESI, m / z): C 11 H 13 O 2 N 2 ([M+H] + ) calculated value: 205.0972; measured value: 205.0973.

[0228] Example 5. Synthesis of CO probe molecules (compounds 31 and 31a).

[0229] Fluoroamide-functionalized palladium coordination complexes 31 and 31a were synthesized as shown in Scheme 7 below.

[0230] Solution 7

[0231]

[0232] Reagents and conditions: a) i. SOCl 2 , 60°C, 2 hours; ii. MeOH, DCM, 0°C, 1 hour; b) i. CuBr, EtAOc, CH 3 ONa / MeOH, reflux, 3 hours; ii. NaOH, MeOH / H 2 O, reflux, 2 hours; c) i.SOCl 2 , toluene, reflux, 2 hours; ii. n-butylamine, TEA, DCM, 0°C, 1 hour; d) NBS, H 2 SO 4 , AcOH / TFA, 0°C, 30 minutes; e) Pd(dba) 2 , 2,2'-bipyridine, DCM / toluene, 80°C, 1.5 hours; f) AgOTf, acetone, rt, 30 minutes.

[0233] Preparation of methyl 5-bromo-1-naphthoate (27). To a dried 100 mL flask containing 5-bromo-1-naphthoic acid (26, 300 mg, 1.195 mmol) was added SOCl 2(30mL, 413mmol). The turbid reaction mixture was stirred at 60°C for 2 hours. Afterwards, the now clear reaction mixture was thoroughly dried under reduced pressure to obtain a 5-bromo-1-naphthoyl chloride intermediate in the form of an off-white solid, which can be used for the next step of the reaction without purification or characterization. The 5-bromo-1-naphthoyl chloride intermediate was then dissolved in anhydrous DCM (10mL); the flask was placed in an ice / water bath, cooled to 0°C, and anhydrous MeOH (5mL) was added to the reaction mixture via a syringe. After stirring at room temperature for 60 minutes, the reaction mixture was concentrated in vacuo, and the residue was then dissolved in EtOAc (80mL), successively with saturated NaHCO 3 solution (3×80 mL) and brine (3×50 mL), and the organic layer was washed with Na 2 SO 4 The mixture was then concentrated under vacuum to give compound 27 (316 mg, 100% yield) as a white solid without further purification. 1 H NMR (400 MHz, CDCl 3 )δ8.89(d,J=8.7Hz,1H),8.50(d,J=8.6Hz,1H),8.21(d,J=7.3Hz,1H),7.84(d,J=7.4Hz,1H),7.60(t,J=8.4Hz,1H),7.44(t,J=8.4Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ167.79,132.76,132.43,132.36,130.91,130.68,128.03,127.84,126.04,125.87,123.43,52.54. HRMS (ESI, m / z): C 12 H 9 O 2 BrNa[M+Na] + The calculated value is 286.9684 and the measured value is 286.9673.

[0234] Preparation of 5-methoxy-1-naphthoic acid (28). Compound 28 was synthesized according to the method reported in the literature. See Lukeman, M. et al. Can. J. Chem. 2004, 82, 240-253. Briefly, compound 27 (0.31 g, 1.17 mmol) was reacted with CuBr (50.3 mg, 0.351 mmol), EtOAc (115 μL, 1.17 mmol) and CH 3Ona was mixed with a solution of MeOH (25%, 2.2 mL, 10 mmol). The mixture was refluxed for 3 hours until completion, as confirmed by TLC. The reaction mixture was then filtered through celite, the filtrate was concentrated under vacuum, and a small sample of the concentrated filtrate was passed through a flash silica gel column to isolate the intermediate compound methyl-5-methoxy-1-naphthoate for NMR characterization: 1 H NMR (400MHz, CDCl3) δ8.52(d,J=8.4Hz,1H),8.45(d,J=8.8Hz,1H),8.18(q,J=7 .2,Hz,1H),7.58–7.42(m,2H),6.88(d,J=7.7Hz,1H),4.01(s,3H),4.00(s,3H). 13 C NMR (101 MHz, CDCl 3 )δ168.30,155.49,132.35,130.59,127.85,127.19,126.81,126.16,123.78,117.92,104.15,55.62,52.18. To the remaining filtered reaction mixture was added NaOH (160 mg, 4 mmol) in MeOH (5 mL) and H 2 O (0.5 mL), the mixture was refluxed for 2 hours until completion, as confirmed by TLC. When the hydrolysis was complete, the reaction mixture was concentrated, acidified to pH 1-2 with HCl (1 M, 10 mL), extracted with EtOAc (100 mL), and washed with brine (3×100 mL). The organic layer was washed with Na 2 SO 4 The mixture was dried and then concentrated under vacuum to obtain compound 28 (0.22 g, 93% yield after two-step synthesis) as an off-white solid. TLC confirmed the presence of one product, namely compound 28 (R f =0.2; DCM:MeOH=20:1). 1 H NMR (400MHz, CD 3 OD) δ8.48 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 8.8 Hz, 1H), 8.17 (d, J = 6.4 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 7.7 Hz, 1H), 4.02 (s, 3H). 1 H NMR spectrum is consistent with NMR data reported in the literature. See Meyers, AI et al. J. Org. Chem. 1987, 52(20), 4592-4597. HRMS (ESI, m / z): C 12 H 9O 3 [MH] - The calculated value is 201.0552 and the measured value is 201.0559.

[0235] Preparation of N-butyl-5-methoxy-1-naphthamide (29, also used as the original depalladium naphthamide material). In an oven-dried 20 mL scintillation vial, compound 28 (200 mg, 0.99 mmol) was mixed with SOCl 2 (10mL, 138mmol) and toluene (2mL) were combined. The reaction was refluxed at 80°C for 2 hours, and the color changed from turbid yellow to dark green. Afterwards, the solvent was removed under vacuum to obtain the intermediate compound 5-methoxy-1-naphthoyl chloride, which can be used for the next step of the reaction without characterization or further purification. The 5-methoxy-1-naphthoyl chloride intermediate was dissolved in anhydrous DCM (8mL) under argon protection. After cooling to 0°C in an ice / water bath, n-butylamine (101μL, 1mmol) and Et 3 N (137 μ L, 1mmol) mixture is slowly added to the reaction mixture, and the color of the reaction mixture changes from dark green to yellow. Allow the reaction to be stirred at room temperature for 1.5 hours until completion, as confirmed by TLC. The reaction mixture is then diluted with EtOAc (50mL), and with saturated NaHCO 3 (3×50 mL) and brine (3×50 mL). The organic layer was washed with Na 2 SO 4 The crude mixture was purified by HPLC and then concentrated under vacuum to afford crude compound 29 as a light yellow solid. 2 O was recrystallized to give compound 29 (206 mg, 81% yield) as an off-white solid. 1 H NMR (400MHz, CD 3 OD)δ8.54(s,1H),8.34(d,J=8.3Hz,1H),7.70(d,J=8.4Hz,1H),7.54(d,J=8.6Hz,1H),7.45(t,J=8.2Hz,2H),6.94( d,J=9.3Hz,1H),3.99(s,3H),3.51–3.38(m,2H),1.73–1.55(m,2H),1.46(sx,J=7.6Hz,2H),1.00(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CD 3OD)δ172.78,156.80,135.77,132.37,128.11,127.07,126.47,125.09,125.03,118.31,105.32,56.09,40.78,32.61,21.24,14.17. HRMS (ESI, m / z): C 16 H 20 NO 2 [M+H] + The calculated value is 258.1494 and the measured value is 258.1500.

[0236] Preparation of 8-bromo-N-butyl-5-methoxy-1-naphthamide (30). In a 20 mL scintillation vial, compound 29 (87 mg, 0.283 mmol) was combined with 0.2 ml AcOH (0.2 mL) and TFA (0.2 mL). After cooling to 0 °C in an ice / water bath, N-bromosuccinimide (NBS, 61 mg, 0.340 mmol) was added as a solid to the stirred reaction mixture, followed by concentrated H 2 SO 4 (0.7 mM, 38 μL). The reaction was allowed to stir at 0 ° C for 30 minutes until completion, as indicated by TLC. After quenching with sodium acetate (NaOAc, 890 mg, 10 mmol), the reaction mixture was diluted with brine (50 mL) and then extracted with EtOAc (50 mL). The organic layer was washed with saturated NaHCO 3 solution (3×50 mL) and brine (3×50 mL), and 2 SO 4 The mixture was then concentrated under vacuum and purified by flash column chromatography to give compound 30 (84.2 mg, 77% yield) as a dark blue resinous solid. 1 H NMR (400 MHz, CDCl 3 )δ8.29(d,J=8.3Hz,1H),7.65(d,J=8.3Hz,1H),7.46(d,J=5.5Hz,1H),7.36(t,J=8.3Hz,1H),6.63(d,J=8.4Hz,1H),6.0 3(t,J=8.0Hz,1H),3.94(s,3H),3.43-3.33(m,2H),1.55(p,J=7.3Hz,2H),1.36(sx,J=7.3Hz,2H),0.91(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3)δ170.88,155.21,135.34,132.89,128.60,128.52,127.66,124.73,124.23,109.59,105.07,55.82,40.36,30.97,30.93,20.30,13.82. HRMS(ESI):C 16 H 19 N 2 Obr[M+H] + The calculated value is 336.0594 and the measured value is 336.0600.

[0237] Preparation of (8-(butylcarbamoyl)-4-methoxynaphthyl)(bipyridyl)palladium(II) bromide (31). Under argon protection, compound 30 (230 mg, 0.682 mmol), Pd(dba) 2 4-[4-(414mg, 0.72mmol) and BIPY (112mg, 0.72mmol) were dissolved in DCM (5mL) and toluene (5mL). The reaction was slowly heated to 80°C in about 1 hour, while stirring, and the color of the reaction mixture changed from red to dark yellow. The reaction was allowed to stir for an additional 30 minutes until completion, as indicated by TLC. The reaction mixture was diluted with DCM (15mL), filtered through diatomaceous earth, and the filtrate was concentrated to dryness. The residue was washed with anhydrous Et 2 The mixture was triturated with 0, the supernatant was decanted to remove dibenzylideneacetone, and the solid was further purified by flash column chromatography to give Compound 31 (146 mg, 60% yield) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 )δ9.44(d,J=4.9Hz,1H),8.30(dd,J=8.3,1.5Hz,1H),8.05-7.98(m,3H),7.87(td,J =7.9,1.6Hz,1H),7.73(d,J=7.9Hz,1H),7.62–7.48(m,2H),7.42-7.37(m,2H),7.13 –7.04(m,1H),6.75(d,J=7.9Hz,1H),6.46(t,J=4.6Hz,1H),3.98(s,3H),3.35(br-s ,1H),2.95(br-s,1H),1.56–1.45(m,2H),1.26-1.20(m,3H),0.82(t,J=7.4Hz,3H). HRMS(ESI):C 26 H 27 N 3 O 2BrPd[M+H] + The calculated value is 598.0321 and the measured value is 598.0347.

[0238] Preparation of (8-(butylcarbamoyl)-4-methoxynaphthyl)(bipyridine)palladium ring trifluoromethanesulfonate (31a). In a dry 100mL round-bottom flask, compound 31 (176mg, 0.294mmol) was dissolved in anhydrous acetone (50mL), and AgOTf (76mg, 0.294mmol) was added as a solid while stirring. A white precipitate was formed immediately, and the reaction was stirred at room temperature for 30 minutes until completion, as confirmed by TLC (DCM: MeOH = 10: 1). The reaction mixture was then filtered through a diatomaceous earth pad and the filtrate was concentrated under vacuum. The residue was purified by flash column chromatography (DCM: MeOH = 20: 1), and the fraction containing the target product was recrystallized from dried and degassed chloroform to obtain pure compound 31a (118mg, 60% yield) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ9.13–9.07(m,2H),8.72–8.68(m,2H),8.40–8.30( m,3H),7.94(s,1H),7.77(d,J=7.1Hz,1H),7.69(s,1H),7.59–7.55(m,2H), 7.51(d,J=8.0Hz,1H),6.96(d,J=7.2Hz,1H),3.98(s,3H),2.97(q,J=6.7Hz ,2H), 1.21(p,J=7.1Hz,2H), 1.10(sx,J=7.4Hz,2H), 0.70(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ171.85,155.43,153.66,153.45,151.62,149.27,141.02,134.50,133.28,132.99,127.66,1 27.30,127.20,125.88,125.49,124.23,123.71,123.42,122.28,119.08,105.07,55.63,30.31,19.63,13.56(CH 2 -NH peak collides with DMSO-d6 peak). HRMS (ESI): C 26 H 26 N 3 O 2 Pd 104 [M+H] + The calculated value is 516.1065 and the measured value is 516.1076.

[0239] Example 6. Synthesis of fluorescent product (Compound 34).

[0240] The fluorescent imide substance 34 was synthesized as shown in Scheme 8 below.

[0241] Solution 8

[0242]

[0243] Reagents and conditions: a) n-butylamine, EtOH, reflux, 8 hours; b) CH 3 Ona、CuSO 4 ·5H 2 O, MeOH, 80°C, 12 hours.

[0244] Preparation of 4-bromo-N-butyl-1,8-naphthalimide (33). Compound 33 was synthesized from 4-bromo-1,8-naphthalene dicarboxylic anhydride (32) using a method reported in the literature. 1 H NMR (400 MHz, CDCl 3 )δ8.64 (dd, J=7.3,1.2 Hz,1H),8.55 (dd, J=8.5,1.2 Hz,1H),8.40 (d, J=7.9 Hz,1H),8.03 (d, J=7.9 Hz,1H),7.83 (dd, J=8.5,7.3 Hz,1H),4.22–4.13 (m,2H),1.77–1.65 (m,2H),1.44 (h, J=7.4 Hz,2H),0.98 (t, J=7.3 Hz,3H). 1 The H NMR spectrum is consistent with the NMR data reported in the literature. See Feng, L. et al. Anal. Chem. 2018, 90(22), 13341–13347.

[0245] Preparation of 4-methoxy-N-butyl-1,8-naphthalimide (34). Compound 33 (50 mg, 0.151 mmol), CH 3 ONa (66 mg, 1.21 mmol) and copper sulfate pentahydrate (5 mg, 0.019 mmol) were weighed into a 10 mL sealed tube containing a stirring bar. Anhydrous MeOH (2 mL) was added and the tube was sealed with an aluminum crimp cap with a PTFE / silicone rubber septum. The reaction mixture was stirred at 80 ° C for 12 hours until completion, as confirmed by TLC. The reaction was quenched with HCl (1 M, 30 mL), extracted with EtOAc (50 mL), and washed with brine (3 × 50 mL). The organic layer was then filtered through Na 2 SO 4Dried and concentrated under vacuum.The concentrated residue was then purified by flash column chromatography (25% EtOAc in hexanes) to give compound 34 (44 mg, 100% yield) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ8.51(dd,J=7.3,1.1Hz,1H),8.48–8.43(m,2H),7.62(dd,J=8.2,7.4Hz,1H),6.96(d,J=8.3Hz,1H),4. 18–4.10(m,2H),4.08(s,3H),1.69(tt,J=7.7,6.6Hz,2H),1.43(sx,J=7.6Hz,2H),0.96(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ164.53,163.96,160.75,133.38,131.48,129.32,128.55,125.93,123.45,122.45,115.15,105.19,56.25,40.16,30.36,20.51,13.98. The obtained NMR spectrum of compound 34 is consistent with the spectrum reported in the literature. See Feng, L. et al. Anal. Chem. 2018, 90(22), 13341–13347.

[0246] Example 7. Sensing chemistry and spectroscopic properties of CO probe molecules.

[0247] For most reaction-based CO sensing methods, such as probes based on the Tsuji-Trost reaction and palladium ring-based probes, the sensing mechanism is based on the removal of the quenching group of the fluorescent molecule with CO. Therefore, background fluorescence is inevitable due to insufficient quenching of the fluorescent molecule and the removal of the quenching group via interference, metabolism, and simple chemical degradation of other functional groups. According to some comprehensive reviews in the field, the signal-to-noise ratio (SNR) of most reported probes is less than 200:1, which is insufficient for quantitative analysis. Quantitative measurements require sensors with true fluorescence onset and low background noise. Selectivity is another problem of the reported probes because they are sensitive to various nucleophiles commonly present in biological samples, including amines and thiol species that can release fluorophores and induce CO-independent fluorescence increases.

[0248] Our strategy for sensing CO is not to utilize CO to remove the quencher group (dequenching), but to achieve sensing by constructing the fluorophore via two sequential reactions, which essentially eliminates background fluorescence and provides a very high level of selectivity ( Figure 1The design utilizes palladium-mediated carbonylation of CO followed by spontaneous amidation to “build” the fluorophore from scratch ( Figure 2 a). Therefore, fluorescence can only be turned on after reaction with CO. To achieve this goal, O-hydroxyphthalimide (22) was selected as the fluorescent product, which has a well-defined excited state intramolecular proton transfer (ESIPT) fluorescence mechanism, a large Stokes shift (114 nm), and a small overlap between the excitation and emission spectra ( Figure 2 d). The quantum yield (Φ) of 22 in phosphate buffered saline (PBS) at pH 7.4 was determined using quinine sulfate as a reference. F ) is 0.23.

[0249] The first probe series was synthesized from 2-amino-6-methoxybenzoic acid (Scheme 1). The palladium ring was not constructed via an ortho group, as reported for other probes such as COP-1 or CC-CO, since this would lead to the formation of dimers with higher molecular weights and could sometimes lead to concerns about the formation of regioisomers. The introduction of an iodine group at the ortho position of the amide group by diazotization-iodination allowed the oxidative insertion of palladium to be directed using tetramethylethylenediamine (TMEDA) as a ligand. Thus, palladium complex 5 was synthesized in good yield under mild conditions. TMEDA was chosen as a ligand due to the stability and water solubility of the resulting complex. In fact, this molecule can react with CO and can also serve as a CO fluorescent probe (COFP). The iodine group was then removed by treatment with AgOTf in acetone, yielding the palladium ring 5a. The structure of 5a was characterized by NMR and X-ray crystallography (crystallographic data Fig.21 ). It was found that the reaction kinetics of 5 towards CO was the same as that of 5a ( Figure 2 j). Both CO probe molecules 5 and 5a are soluble and stable in PBS solution at pH 7.4 ( Figure 3 ).

[0250] Incubation of 5a with CO gas in the headspace vial resulted in a CO concentration-dependent fluorescence turn-on ( Figure 2 b and Figure 2 e inset). The main advantages of this strategy compared to other “dequenched” CO probes include: the CO probe molecules 5 and 5a have higher SNR and lower background due to the completely dark nature of the probes, the Stokes shift of the fluorescent product 22 is large (114 nm), and the maximum excitation wavelength of the fluorescent product 22 is similar to that of the original depalladium species 17 (λ Ex =329nm, λ Em=420nm) do not overlap. In addition, compared with other "dequenched" CO probes, CO probe molecules 5 and 5a show enhanced selectivity and tolerance to the presence of multiple molecules. Under the excitation wavelength range of 385-405nm of 22, the depalladium product 17 does not show any fluorescence signal ( Figure 2 d).

[0251] Similar palladium complexes are known to be reactive toward thiol species. Such reactivity is believed to be caused by the original demetallation of COP-1 to thiol, because the depalladium species of the probe can have similar fluorescence characteristics as the CO sensing product. Due to the presence of thiol species (such as H 2 S and GSH), and the exclusion of such CO-independent reactions can significantly enhance the reliability of CO detection. LC-MS studies have shown that 5a reacts with glutathione (GSH) or H2S (generated from NaHS) in PBS to form a depalladium species (17) ( Fig.23 a, 23b). 5a was directly reacted with two equivalents of NaHS in dimethylacetamide (DMA) to afford 17 as the major product ( Fig.23 c). However, 17 does not show any fluorescence at the excitation wavelength used for CO detection, thus preventing false positive reactions due to depalladium ( Fig.24 ac).

[0252] The SNR of our probe strategy is proportional to the concentration of the probe. For example, when the concentration of the probe is 100 μM, the SNR of 5a exceeds 1200:1 ( Figure 2 c). At a concentration of 10 μM, the SNR is 320:1, which is still higher than most existing CO probes. Again, the reason we can use high concentrations of the probe is due to its completely dark nature. Our design enables "fail-safe" detection of CO in the presence of other substances, which can lead to demetallation (original depalladium) and fluorescence onset of the probe in literature cases. In terms of selectivity, 5a can only be turned on by CO gas ( Figure 2 e and Figure 4 ). When 5a was exposed to thiols, persulfides, peroxides, NO 2 - , glutathione, glutathione disulfide, cysteine, CN - No fluorescence changes were detected when , hydrogen sulfide, or HClO were present. Therefore, CO probe 5a showed higher selectivity compared with other palladium ring-based probes such as COP-1, which responds to the presence of H in biological samples to some extent. 2 S or other thiols.

[0253] Regarding the sensing mechanism, the probe was designed in such a way that after CO insertion between the palladium and the phenyl carbon, followed by hydrolysis, a carboxylic acid group was formed (see Scheme 9). Due to the proximity of the amide nitrogen and the kinetic favor of the formation of the five-membered phthalimide ring, the fluorescent o-hydroxyphthalimide was formed as the final product.

[0254] Solution 9

[0255]

[0256] To verify the mechanism shown in Scheme 9, the reaction between 5a and CO gas was studied using proton NMR, HPLC, and LC-MS. In the NMR study, we monitored the 2 The transition in O-DMSO solution showed the formation of 22 ( Figure 5 ).

[0257] HPLC studies showed that after CO gas injection in PBS solution of 5a and incubation at 37 °C for 30 min, the phthalimide fluorescent product 22 was formed as the major product ( Figure 6 a). At the same time, a black precipitate (presumably palladium) was observed in the reaction mixture. A small peak at 6.2 minutes was shown in the CO sensing reaction and the pure 22 sample. LC-MS confirmed that it was the ring-opening product IM-1 ( Figure 7 a), according to previous studies on the hydrolysis of phthalimide, the ring-opening product is an intermediate of the CO sensing reaction that exists in the hydrolysis equilibrium. In order to capture the intermediate of the CO sensing reaction, an ethanol solution of 5a was used because ethanol is not a good leaving group for the lactamization reaction. HPLC and LC-MS showed that the ethyl ester intermediate IM-2 and the cyclized product 22 ( Figure 6 b and Figure 7 b-7c). After adding PBS to this reaction mixture, IM-2 was almost immediately and completely converted to 22, indicating a rapid intramolecular lactamization reaction in aqueous solution. Such results also indicate that the insertion of CO into the palladium complex is the rate-determining step. In addition, after 5a reacts with CO gas, the fluorescence intensity is the same as 22 at the same concentration. For this reason, the sensing mechanism is confirmed by quantitatively converting the probe into a fluorescent product in PBS solution.

[0258] Example 8. Structural optimization of CO probe molecules.

[0259] The CO probe was optimized to achieve better sensitivity, stability, and quantum yield for biological applications. Sensitivity is very important for the probe to detect low concentrations of CO. To achieve this goal, improvements in quantum yield and reactivity toward CO were investigated. The pKa of the phenol group is close enough to physiological pH, which makes the fluorescence sensitive to pH under near physiological conditions ( Figure 8 a). To increase the quantum yield and minimize the pH dependence of the fluorescent product, the amino analogue 25 was designed as a new reporter product, and its quantum yield was significantly increased to 0.33. The maximum excitation and emission wavelengths of 25 were blue-shifted by about 10 nm compared with those of 22 ( Figure 2 d, 2i). Due to the low pKa of the conjugate acid and the good ESIPT effect of aniline, the pH dependence around physiological pH 25 also largely disappears ( Figure 8 b), indicating improved signal stability. Based on 25, several analogs were designed and synthesized by changing the ligand moiety and amide chain, and their sensing reaction kinetics were tested under normalized conditions.

[0260] First, 11a was synthesized, which is a complex with TMEDA as a ligand and a butyl chain on the benzamide nitrogen. Compared with the hydroxy derivative 5a, compound 11a showed a faster reaction toward CO ( Figure 2 j) Next, compound 12 was prepared using TMEDA as a ligand and a propyl chain on the benzamide nitrogen. Figure 2 As shown in Figure 1, the amide alkyl chain in compound 12 was shortened to a propyl group, resulting in a slower reaction of compound 12 to CO gas compared to 11a and the hydroxy derivative 5a (both of which have a butyl group on the benzamide). When the amide chain was butyl and the ligand was changed to the more electron-withdrawing 2,2'-bipyridine (BIPY), the resulting complex 13 showed a significant increase in reaction kinetics compared to 12, 11a, and 5a. Still using the BIPY ligand, but shortening the length of the alkylamide chain from butyl to propyl (compound 14), the reaction kinetics were further increased, with a second-order reaction rate constant of 220.6 ± 27.2 M -1 s -1 The reaction kinetics of 14 enables near real-time sensing of CO gas. The CO probe molecule 14 also exhibits excellent concentration-dependent SNR ( Figure 2 g) and selectivity for CO ( Figure 2 h and Fig. 9 ). In a mixture of dimethylacetamide (DMA) and PBS solution, 14 was quantitatively converted to the fluorescent product 25, which was also confirmed by fluorescence recovery ( Fig.10 Interestingly, when 5a was modified by replacing the TMEDA ligand with BIPY, the resulting palladacycle 6a showed a much slower response to CO than 5a ( Figure 2 j). The second-order rate constant of compound 6a was determined to be 13.8 ± 1.4 M -1 s -1 The CO sensing kinetic parameters generally fall within the region defined by 6a and 14 ( Figure 2j) The results indicate that changing the bidentate ligand and changing the substituents on the benzamide of the Pd complex achieves the CO insertion reaction.

[0261] The detection sensitivity of 5a, 11a and 14 was tested by incubating the CO probe molecules with 1.6-8ppm CO in the air in a headspace vial. Due to their high SNR, it should be feasible to use these probes at high concentrations to increase sensitivity. To test this hypothesis, the detection sensitivity of 5a and 11a was tested at low (100μM) and high (1mM) concentrations in DMA, and the detection sensitivity of 14 was tested at 1mM. The detection limits of 5a, 11a and 14 were determined to be approximately 0.1-0.2ppm, which is equivalent to 0.45-0.9nM in solution according to Henry's law. As expected, higher probe concentrations result in higher sensitivity, as generally shown by lower detection limits. High sensitivity coupled with high SNR enables stable determination of CO concentrations in biological samples, as discussed herein.

[0262] Since phthalimide fluorophores exhibit relatively low intracellular accumulation and photostability in live cell imaging studies, we developed an additional series of probe molecules that are capable of generating photostable and cell-permeable fluorophores in the presence of CO. Since naphthalimide fluorophores are known to be photostable, cell-permeable, and have tunable quantum yields, two naphthalimide-based CO probes 31 and 31a were designed to increase the photostability and cellular retention of the fluorescent products in intracellular CO accumulation imaging applications. Based on a strategy similar to that used for benzamide-based CO probes, naphthamide-based CO probes ( Schemes 7 and ) were synthesized. Fig.11 ). While the preparation of the naphthamide Pd complex with TMEDA as the bidentate ligand resulted in spontaneous decomposition during the synthesis, the preparation of the palladium complex 31 using BIPY did not present any decomposition issues. As shown in Scheme 7, the bromide in 31 was extracted with AgOTf to form the six-membered palladium ring 31a. The structures of both 31 and 31a were characterized by NMR and X-ray crystallography (X-ray crystallography data are shown in Fig. 22 middle).

[0263] like Fig.12 As shown, it was found that both 31 and 31a were stable in PBS solution at 37°C for at least 1 hour. The apparent reaction kinetics of compound 31a towards CO were determined to be as fast as that of 14 ( Fig.11 d). LC-MS studies confirmed that the reaction of 31a with CO in PBS resulted in the formation of the fluorescent naphthylimide product 34 ( Fig.13 ). The quantum yield of 34 in water was determined to be 0.82. Fig.11As shown in b, the original depalladium-dephosphoryl naphthamide species 29 is characterized by its excitation spectrum being different from that of the naphthimide CO sensing product 34, which enables the realization of a concentration-dependent SNR ( Fig.11 c) and high selectivity ( Fig.11 e, 11d). The CO detection limits of 31 and 31a were determined using 20 μM 31 in PBS or 20 μM 31a in PBS. Assuming a CO solubility of 1 mM in PBS at 760 mmHg CO partial pressure, the CO detection limits of 31 and 31a were calculated to be 2.74 nM and 2.06 nM, respectively, which should enable detection of endogenous CO in cells at micromolar concentrations.

[0264] Example 9. Application of benzamide-based CO probe and naphthamide-based CO probe.

[0265] CO probe molecules 5a, 11a, 14, and 31a were selected for further study to determine the feasibility of COFP in research applications. In general, there are two applications for CO probes: 1) measuring CO levels in blood, tissue, and cell culture samples; and 2) imaging intracellular CO accumulation in living cells. Each CO probe molecule has its own physical, chemical, and biological characteristics suitable for a variety of applications.

[0266] Semi-quantitative determination of CO in blood and cell culture samples. Since CO sensing uses an on-state mechanism, the probe confers a "fail-proof" feature in three aspects: 1) the probe is completely dark; 2) the original depalladium does not produce fluorescence; and 3) only CO insertion enables the construction of a fluorophore. Therefore, when biological samples contain high CO concentrations, the probe can provide a large linear range, and the presence of nucleophilic / enzymatic substances in the biological sample does not present interference. Due to the coordinated TMEDA ligands and triflate forms of 5a and 11a, compounds 5a and 11a are more soluble in water than compounds 14 and 31a. Specifically, the solubility of 5a and 11a in PBS was determined to be about 5mM, and 14 was 100μM, and 31a was 50μM. In addition, the fluorescent products of 5a and 11a (i.e., phthalimide compounds 22 and 24, respectively) are very stable in serum. Although the fluorescence of 22 was determined to be pH-dependent, blood pH is not expected to fluctuate significantly; in addition, PBS (1×) was used to buffer the test fluid, which helps prevent pH fluctuations in biological samples. Therefore, 5a and 11a were selected to measure carboxyhemoglobin (COHb) levels in mouse blood samples.

[0267] Based on the fact that the blood hemoglobin tetramer concentration is about 2 mM, it was determined that 100% COHb would produce a CO concentration of about 8 mM. Therefore, testing COHb levels up to 50% requires a CO probe concentration of at least 4 mM. Due to the turn-on sensing mechanism constructed via a CO-intercalated fluorophore, the background signal is negligible even at this high concentration. By incubating 5a at a concentration of 10 mM directly in blood samples with different COHb levels pre-determined by CO-oximetry, a correlation between fluorescence intensity and COHb levels determined by CO-oximetry was established ( Fig.14 a). By using this standard curve, the COHb level of the unknown portion of the CO-saturated blood sample with a fluorescence intensity of 117.48 au was calculated to be 7.9%. CO oximeter readings of the same sample showed a COHb level of 8.2 ± 0.45%, showing good agreement. In order to make the determination of COHb levels more feasible without the support of a CO oximeter, a clear COHb calibration level of blood is required. It has been reported that blood pre-saturated with pure CO gas results in a COHb level of approximately 90%. We personally measured CO-saturated blood with a CO oximeter and consistently verified a COHb level of 90% in blood samples collected from five different mice ( Fig.14 b) By saturating CO with N 2 The COHb level of the washout blood was assigned to 90% and serial dilutions were made with normal blood, allowing a calibration curve to be established using COSM. Thus, COHb levels of unknown samples can be determined by the probe using a fluorescent microplate reader without the need for a CO oximeter, GC, or even a fluorometer. This approach was successfully validated in two ex vivo experiments. Following oral administration of the CO prodrug BW-CO-306 and its activated carbon formulation BW-AC-306 to mice, the blood COHb levels detected by the probe were in good agreement with those determined using a CO oximeter. Specifically, blood samples from mice dosed with 100 mg / kg BW-AC-306 were determined to have COHb of approximately 3% when measured using either a 5a or CO oximeter, while COHb in control mice was determined to be approximately 1.5% ( Fig.14 c). Blood COHb levels were approximately 0.9% and 6.1% in untreated mice and mice treated with 200 mg / kg BW-CO-306, respectively, as measured with 11a and confirmed by CO-oximeter readings ( Fig.14 d) There was no statistically significant difference between the results of the CO probe method and CO oximetry.

[0268] The same approach can also be used for in vitro cell experiments to determine the relative CO saturation changes induced by endogenous CO induced by external CO sources (such as CO gas) or HO-1 activators. To demonstrate this, bardoxolone methyl (CDDO-Me) was chosen as the HO-1 inducer because there is no spectral interference caused by CDDO-Me compared to chromogenic heme. HeLa cells were incubated with 250ppm CO gas for 2 hours, or with 0.3μM CDDO-Me for 6 hours and then collected with a cell scraper. The washed cell pellets were incubated with 100μM 11a, followed by fluorescence measurements using a microplate reader, showing that the fluorescence signal increased by about 28% after CO gas treatment and by 7.5% after CDDO-Me activation compared to the DMSO vehicle control (Figure 2A). Fig.14 e). Western blot analysis confirmed that CDDO-Me treatment significantly increased HO-1 expression ( Fig.14 f), which probably explains the elevated CO production.

[0269] Determination of the absolute amount of CO in tissue and cell culture samples. Vreman et al. previously used the RGD-GC method to determine that the endogenous CO concentration of various organs in mouse tissues was approximately 2-10 pmol / mg. Theoretically, if approximately 100 mg of tissue releases all bound CO into the 1 ml headspace in the headspace vial, at least 4.5 ppm of CO should be produced, which is much higher than the detection limit of probe 14. Since the detection limit of the CO probe molecule 14 is 0.1 ppm and the reaction kinetics are fast, it was used in CO concentration quantification experiments. In order to develop the quantification method, 14 was dissolved in 50 μL of degassed dimethylacetamide (DMA) at a concentration of 1 mM and sealed in a 0.5 ml vacuum headspace vial with a PTFE / silicone crimp septum. DMA was chosen for the following reasons: 1) DMA has a relatively high solubility for CO (approximately 4.5 mM at 760 mmHg), allowing higher CO concentrations to react with the probe in solution; 2) Compound 14 is soluble in DMA, which allows a high ratio between probe and analyte for rapid response and sensitive detection; and 3) DMA is miscible with PBS, which helps normalize pH and increase accuracy. This probe-charged headspace vial (CO detection vial) is used as a CO "detector" by injecting a CO-containing gas, followed by incubation, and then fluorescence measurement using a fluorescence spectrometer or plate reader. The CO concentration is determined by the external standard curve method. Specifically, 250 μL of standard CO air calibration gas (CO concentration of 10-100 ppm) is injected into the detection vial to establish a standard curve for 14 ( Fig.14g). The excellent reproducibility and goodness of fit indicate that the experimental setup was reasonable. To verify its utility in ex vivo studies of CO donor administration, we used organ tissue from the same mouse dosed with 200 mg / g BW-CO-306 in the above COHb blood analysis study. Aliquots of liver and kidney homogenates were tested simultaneously using compound 14 and a GC equipped with a methanator-FID detector. To release protein-bound CO in the headspace vials, the tissues were treated and denatured using 3% 5-sulfosalicylic acid (SSA) according to established procedures. The liver tissue CO concentration of the control was determined to be approximately 5 pmol / mg, and the liver tissue CO concentration of the BW-CO-306-treated group was 31 pmol / mg ( Fig.14 h). The renal tissue CO concentration of the control group was 11 pmol / mg, and the renal tissue CO concentration of the CO-306 treated group was 26 pmol / mg ( Fig.14 i). There was no statistical difference between the results obtained using the GC-methanizer and the CO probe molecule 14 ( Fig.14 h, 14i, 14j), confirming that our CO detection protocol using 14 can quantify CO in tissue samples with accuracy and reproducibility comparable to that of a methanogen-FID-GC system, which is expensive to set up and is considered the “gold standard” for measuring tissue CO concentrations. Tissue CO concentrations in control mice were also in a similar range to those tested by Vreman et al. using the RGD-GC method, demonstrating the reproducibility of our method.

[0270] Using a method similar to that used for CO detection in tissue samples, the CO gas detection tube containing compound 14 can also be used to determine CO concentration in cell culture samples. Fig.14 k results showed that the CO concentration in HeLa cells was increased from 25 pmol / 10 6 The number of cells increased significantly to 92 pmol / 10 6 cells and 203pmol / 10 6 Since the cells were washed twice with PBS before denaturation and CO determination, the significantly higher CO concentration in the BW-CO-111 treated group can be attributed to the total amount of CO bound by heme proteins and residual intracellular CO prodrug. To this end, we have demonstrated that 5a, 11a, and 14 can be used to semi-quantitatively and quantitatively determine CO concentrations in blood, tissue, and cell culture samples with excellent reproducibility and accuracy.

[0271] Fluorescence imaging of CO in cell culture. Fluorescence imaging, as a key visualization method in biological research, has significantly promoted the understanding of CO function and the development of various useful CO donors that can deliver CO to the intracellular space. The naphthamide-based CO probe molecule series 31 and 31a have high selectivity, high sensitivity, low background fluorescence, and can produce photostable naphthamide products with pH-independent fluorescence. In addition, both 31 and 31a showed no cytotoxicity to HeLa cells at less than 50 μM within 24 hours ( Fig.15 ), and as a triflate salt, compound 31a has improved water solubility. Both 31 and 31a were used for fluorescence imaging of intracellular CO accumulation from various sources, including CO gas and the CO prodrug BW-CO-201, which was chosen in part because its CO release products do not produce fluorescent interference ( Fig.16 ).like Fig.16 and Fig.17 As shown, live HeLa cells treated with CO followed by the addition of 20 μM 31 or 31a showed strong blue fluorescence under the DAPI channel, while the control group not treated with CO did not show any fluorescence under the same imaging conditions. The low background fluorescence in the vehicle control group again demonstrated the advantage of the insertion-on strategy. In addition, 31a was also able to sense the increase in endogenous CO production induced by 0.3 μM CDDO-Me using a longer exposure time of 6 seconds ( Fig.17 d, 17e, 17f). Fig.17 The line profile region of interest (ROI) of f was used to calculate signal intensity, and line profile 4 was used for background subtraction ( Fig.17 g, 17h).

[0272] Summary. In order to improve the reliability and reproducibility of CO detection and quantification in biological samples, we have developed a new CO sensing strategy involving a sequential CO insertion-carbonylation-amidation reaction. The CO probe molecules described herein produce fluorescent products in the presence of CO with high specificity, high sensitivity, rapid response and high SNR (low background fluorescence). In this strategy, CO is the key reactant for the de novo synthesis of analytes and fluorophores. By utilizing the unprecedented detection characteristics of CO probes derived from this strategy, benzamide-based CO probe molecules (such as 5a, 11a and 14) are used to achieve detection and quantification of CO in blood, tissue and cell culture samples with high sensitivity, selectivity, accuracy and reproducibility. The same design strategy has also been used to develop naphthamide-based CO probe molecules 31 and 31a, which have been shown to be very useful for detecting and imaging exogenous CO from CO gas or CO prodrugs in living cells and endogenous CO production. In summary, the present disclosure shows the advantages of insertion and fluorescence methods in the development of CO fluorescent probes.

[0273] Example 10. Kinetic determination of CO probe molecules.

[0274] General procedure for determining the second-order rate constants of 6a and 14. Saturated CO PBS solution (1 mM) was freshly prepared by bubbling pure CO gas into 4 mL of 10 mM PBS (pH = 7.4) solution in a 6 mL headspace vial (silicone oil seal) at room temperature for more than 10 minutes (see, Almeida, AS et al. J Biol Chem 2012, 287, 10761-10770). The stock solution of the probe was prepared as a 1 mM solution in DMSO. The observed pseudo-first-order reaction constant (k obs ) versus CO concentration to estimate the second-order kinetic constants of 6a and 14 ( Fig.18 ). To adapt to the pseudo-first-order reaction conditions, the CO concentration was kept 5 times greater than the probe concentration.

[0275] Determination of the secondary rate constant of 6a. 890 μL, 790 μL, or 690 μL PBS was added to a 1 mL cuvette, followed by 10 μL 6a probe stock solution. The final concentration of 6a was 10 μM. The cuvette was sealed with a rubber stopper. At an excitation wavelength of 395 nm, 100 μL, 200 μL, or 300 μL of saturated CO PBS solution was injected into the cuvette via a 1 mL syringe. Therefore, the final CO concentration was 100 μM, 200 μM, or 300 μM, respectively. The emitted fluorescence signal was recorded until a steady level was reached. The half-life (t) was then estimated by plotting a graph of the time course of the emission intensity using Graphpad Prism 9. 1 / 2 ), and k is calculated based on pseudo-first-order reaction kinetics obs :k obs =ln2 / t 1 / 2 The experiment was repeated three times.

[0276] Determination of the secondary rate constant of 14. 940 μL, 890 μL or 790 μL PBS was added to a 1 mL cuvette, followed by 10 μL of 14 probe stock solution. The final concentration of 14 was 10 μM. The cuvette was sealed with a rubber stopper. At an excitation wavelength of 385 nm, 50 μL, 100 μL or 200 μL of saturated CO PBS solution was injected into the cuvette via a 1 mL syringe. Therefore, the final CO concentration was 62.5 μM, 125 μM or 250 μM, respectively. The emitted fluorescence signal was recorded until a steady level was reached. The half-life (t) was then estimated by plotting a time course graph of the emission intensity using Graphpad Prism 9. 1 / 2 ), and k is calculated based on pseudo-first-order reaction kinetics obs :k obs=ln2 / t 1 / 2 The experiment was repeated three times.

[0277] Kinetic determination of other CO probe molecules (5, 5a, 11a, 12, 13, 31 and 31a). Other CO probe molecules (5, 5a, 11a, 12, 13, 31 and 31a) were tested under uniform conditions: CO probe molecule concentration = 10 μM; CO concentration = 200 μM. The maximum signal intensity (stable) percentage versus time was plotted. Therefore, the reaction kinetics can be qualitatively compared under normalized conditions. Specifically, 790 μL PBS was added to a 1 mL cuvette, followed by 10 μL CO probe stock solution. The cuvette was sealed with a rubber stopper. At a specific excitation wavelength, 200 μL of saturated CO PBS solution was injected into the cuvette through a 1 mL syringe. The emitted fluorescence signal was recorded until it reached a stable level.

[0278] Example 11. Evaluation of the quantum yield (ΦF) of the fluorescent product.

[0279] By using a well-defined quinine sulfate as a reference standard (Φ f =0.55, at 0.5MH 2 SO 4 In, η = 1.346, I r =7040.262: bandwidth = 5 / 5nm, ex / em, I r =1933.334: bandwidth = 3 / 5nm, ex / em, A r =0.0086) and according to the following formula reported in the literature (Heller, CA et al. Journal of Chemical & Engineering Data 1974, 19, 214-219), the quantum yields of the fluorescent products 22, 23, 24, 25 and 34 were compared and determined:

[0280]

[0281] wherein the subscripts r and x represent the reference compound and the test compound, respectively; Φ is the quantum yield; I is the integrated area of ​​the fluorescence spectrum; A is the UV / visible absorbance at the excitation wavelength; λ is the excitation wavelength; and η is the refractive index of the solvent.

[0282] Each fluorescent imide substance 22, 23, 24, 25 and 34 was dissolved in the culture medium indicated in the following Table 1 at a concentration of 10-20 μM. The UV absorbance at its maximum excitation wavelength was recorded. The fluorescence spectrum was recorded and the area under the emission spectrum was integrated.

[0283] Table 1. Quantum yield determination of fluorescent products 22, 23, 24, 25 and 34.

[0284]

[0285] [a] Bandwidth 5 / 5nm (Ex / Em); [b] Bandwidth 3 / 5nm (Ex / Em); [c] η values ​​of PBS are reported in Hoang, VT et al. Applied Sciences 2019, 9, 1145.

[0286] Example 12. Determination of the detection limit of CO probe molecules.

[0287] The detection limits of CO probe molecules 5a, 11a, 14, 31 and 31a were determined by the following formula reported in the literature (Mei, Q. et al. RSC Advances 2015, 5, 74924-74931):

[0288] Detection limit = 3σ / K

[0289] where σ represents the standard deviation of blank measurements, and K represents the slope of CO concentration versus the corresponding fluorescence intensity.

[0290] For benzamide-based CO probe compounds 5a, 11a, and 14, 1 mM or 100 μM of 5a and 11a and 1 mM of 14 were freshly prepared in 50 μL of degassed DMA in a 300 μL headspace vial. Various volumes of 20 ppm CO gas (in air) were injected by a gas-tight syringe, and 0, 20, 40, 60, 80, and 100 μL of CO gas were added. The resulting mixture was incubated on an oscillator at room temperature for 1 hour. Then, fluorescence was measured. The background signal was measured using a freshly prepared CO probe solution of the same concentration for detection. The detection limits of 5a, 11a, and 14 were evaluated by plotting the relationship between CO concentration and the corresponding fluorescence intensity (Tables 2 and Fig.19 ).

[0291] Table 2. Detection limits of CO probe molecules 5a, 11a, and 14.

[0292]

[0293]

[0294] [a] Standard deviation of blank measurements; [b] Fluorescence intensity change at each CO concentration.

[0295] For naphthamide-based CO probe compounds 31 and 31a, 20 μM of 31 and 31a were freshly prepared in 4 mL of degassed PBS buffer (pH = 7.4) in a 6 mL headspace vial. Various volumes of 100 ppm CO gas (in air) were injected via a gas-tight syringe, and 0, 22, 44, 88, and 176 μL of CO gas were added. The resulting mixture was incubated on an oscillator at room temperature for 1 hour. Then, the fluorescence was measured. The background signal was measured using a freshly prepared CO probe solution (20 μM). Based on the solubility of CO in PBS (1 mM at a partial pressure of 760 mmHg), the CO concentration was calculated to be 1 ppm CO in the air to produce a 1 nM CO concentration in PBS. The detection limits of 31 and 31a were evaluated by plotting the relationship between CO concentration and the corresponding fluorescence intensity (Tables 3 and Fig. 20 ).

[0296] Table 3. Detection limits of CO probe molecules 31 and 31a.

[0297]

[0298] Example 13. Determination of the signal-to-noise ratio of the CO probe molecule.

[0299] Compounds 5a, 14 and 31a were dissolved in DMSO to prepare 10mM stock solutions and diluted with PBS in 6ml headspace vials (actual volume 8.8ml) and sealed with PTFE-silicone rubber septa. The final concentrations of 5a and 14 were 1, 10 and 100 μM. Since the solubility of 31a in PBS is low, its final concentration is 1, 10, 25 and 50 μM. At room temperature, for the CO reaction group, 1ml pure CO gas (45 μmol) was injected into the headspace vial and incubated for 15 minutes. For the blank control group, the probe was incubated in PBS without CO gas injection. The fluorescence spectra of 5a, 14 and 31a were recorded at excitation wavelengths of 395nm, 385nm and 377nm, respectively. The area under the fluorescence spectrum curve (AUC) was calculated by Graphpad Prism 9. The signal-to-noise ratio (SNR) of each concentration level was calculated using the following formula:

[0300]

[0301] Example 14. Administration of CO prodrugs to mice and blood / tissue sample collection protocol.

[0302] CD-1 mice (25-30 g) were purchased from Envigo (Indianapolis, USA) and fed and watered ad libitum. Animals were housed according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi (IACUC protocol: 19-012). Mice were randomly divided into two groups (n=3 per group), untreated controls and CO prodrug-treated groups. The CO prodrug-treated group was administered a 200 mg / kg dose of BW-CO-306 by oral gavage. See De La Cruz, LK et al. Chemical Science 2021, 12, 10649-10654. BW-CO-306 was prepared by dissolving 13.4 mg BW-CO-306 in 120 μl DMA, then mixed with 430 μl PEG400, and then gavage to minimize the CO yield loss caused by the hydrolysis of BW-CO-306 in PEG400. The control group was not subjected to any treatment. After the CO prodrug was administered, mice were monitored with a CO oximeter, and mice were killed after blood samples were collected (blood COHb levels were measured 10 minutes before and after oral administration). Blood (0.6-0.8 ml) was collected by retroorbital bleeding and transferred to a test tube containing 30 units of heparin. After killing, mice were then perfused with HBSS buffer throughout the body. Liver and kidney were collected and quickly frozen by immersing them in liquid nitrogen for 30 seconds and stored at -80 ° C until CO determination experiments were performed.

[0303] Example 15. Relative quantification of COHb levels in blood samples using 5a and 11a.

[0304] Blood collection. CD-1 mice (25-30 g) were purchased from Charles River Laboratories (Wilmington, MA, USA) and fed and watered ad libitum. Animals were raised according to the National Institutes of Health's Guide to Laboratory Animal Care and Use. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Georgia State University (IACUC protocol: A21055). Mice (male) were anesthetized with 2% isoflurane / oxygen, and blood (approximately 1 ml) was collected via cardiac puncture and transferred to a test tube containing 40 units of heparin.

[0305] Standard curve. 50-100 μL of blood was saturated with pure CO gas in a headspace vial at room temperature under normal atmospheric pressure for 20 minutes. The headspace was then flushed with ultrapure nitrogen in an ice bath for 5 minutes to remove unbound CO. Because the CO saturation level was tested to be about 90% COHb. Therefore, CO saturated blood was serially diluted in Eppendorf tubes with fresh blood from a no CO control group and equilibrated for 20 minutes to form 45%, 22.5%, 11.25%, 5.63%, 2.81%, 1.41% COHb calibrants.

[0306] COHb assay. 20 μL of blood was mixed with 2 μL of 100 mM 5a or 11a DMSO stock solution and incubated at room temperature for 30 minutes. The mixture was then diluted with 80 μL of cold PBS and subsequently diluted with 100 μL of cold ACN to precipitate protein. After centrifugation at 20 000 × g for 5 minutes at 4 ° C, 100 μL of clarified supernatant was transferred to a black 96-well plate (Corning, New York, USA) for fluorescence determination, and the fluorescence signal was read with a plate reader (filter: Ex: 405 nm / Em: 535 nm, 10 nm bandwidth). COHb levels were calculated based on linear regression of the calibrator standard curve.

[0307] Example 16. Relative quantification of CO in cell culture samples using 11a.

[0308] HeLa cells were seeded in 6-well plates in DMEM cell culture medium (without phenol red and sodium pyruvate, supplemented with 10% FBS and 100 units of penicillin and 100 μg / ml streptomycin) and incubated at 37°C, 5% CO 2 When the cells reached about 90% confluence, the medium was replaced with the same fresh DMEM full medium containing 0.5% DMSO as a solvent control, 0.3% CDDO-Me (DMSO concentration was 0.5%), and 0.5% DMSO (for the CO gas incubation group). For the control and CDDO-Me treatment groups, the cells were incubated at 37°C, 5% CO 2 For the CO gas treatment group, the cell culture plate was placed in a humidified atmosphere filled with 250 ppm CO (balanced with 5% CO in the air). 2) in an airtight chamber and incubated at 37°C for 2 hours. After incubation, all culture media were removed, and the cells were washed 3 times with cold PBS and scraped in PBS. After being transferred to a 1.5 ml Eppendorf tube and centrifuged at 250 × g for 4 minutes, the cell pellet was lysed with 80 μL ACN containing 100 μM 11a and incubated at room temperature for 1 hour. Then 40 μL PBS was added and incubated for another 15 minutes, followed by centrifugation at 20000 × g for 5 minutes. The supernatant was transferred to a black 96-well plate (Corning, New York, USA), and the fluorescence signal was read with a plate reader (filter: Ex: 405nm / Em: 535, 10nm bandwidth).

[0309] Example 17. Absolute quantification of CO in tissue samples using 14.

[0310] Preparation of tissue homogenate and release of CO. Depending on the number of repetitions required for the test, approximately 150-300 mg of tissue was weighed into an Eppendorf tube. Ultrapure water was then added to the tissue (water volume: tissue weight = 4 μL: 1 mg). The test tube was placed on ice, the tissue was cut into small pieces with iris scissors, and then homogenized with a Tissue-Tearor homogenizer (Biospec, Bartleville, Oklahoma, USA). 300 μL of tissue homogenate was transferred to a 2 ml headspace vial (vial capacity: 2 mL), and then 1200 μL of 3.75% 5-sulfosalicylic acid in ultrapure water was added at one time, and the vial was quickly sealed with a crimped seal cap with a PTFE-silicone rubber septum. The headspace vial was incubated at 37° C. for 2 hours.

[0311] Preparation of gas detection tube containing 14. Compound 14 was dissolved in DMA without CO at a concentration of 1 mM. DMA without CO was prepared by freeze-thaw method and purged three times with ultrapure argon. 300 μL headspace vials were filled with 50 μL DMA solution of 14, and the test tube was sealed by crimping cap (8 mm) PTFE-silicone rubber septum. The pressure in the vial was reduced by sealing the test tube under vacuum or by extracting 200 μL headspace gas through a syringe with a 28-gauge needle.

[0312] Preparation of calibration curve and testing of CO in tissue homogenate. Fill the gas sampling bag (Supel TM, inert multilayer foil, Sigma-Aldrich, Saint Louis, Missouri, USA). 100 μL of headspace gas was injected from the sampling bag or tissue homogenate vial into the gas detection tube through a gastight syringe with a sample lock valve (Hamilton, Reno, Nevada, USA). The gas detection tube was placed in the tray of the orbital oscillator and oscillated for 1 hour at room temperature. 25 μL of solution in the detection vial was transferred to a cuvette and diluted with PBS purged with 975 μL ultrapure nitrogen. The fluorescence signal at 499 nm was measured with an excitation wavelength at 385 nm. A calibration curve was obtained by calibrating the relationship between the CO concentration of the gas and the fluorescence intensity. The CO concentration of the tissue sample was determined by using a standard curve and fluorescence intensity.

[0313] Example 18. Absolute quantification of CO in cell culture samples using 14.

[0314] Cell treatment. HeLa cells were cultured at 37°C and 5% CO 2 The cells were cultured in a 15 cm dish in a DMEM-based complete medium (supplemented with 10% FBS and 100 units of penicillin and 100 μg / ml streptomycin) under a humidified atmosphere to about 90% confluence. The cell culture medium was replaced with fresh medium containing or without CO prodrug. The DMSO concentration of all groups was 0.5%. For CO prodrug, BW-CO-111 (100 μM) with a half-life of 24 minutes was used to match the incubation time of the CO gas treatment group. For CO gas treatment, the cells were incubated at 37°C in a humidified atmosphere containing 250 ppm CO, 5% CO 2 And incubate in an airtight chamber with balanced air. All groups were incubated for 2 hours and then rinsed twice with PBS. Cells were extracted from the culture dish by scraping a minimum amount of PBS (about 1 ml). After cell counting by a hemocytometer, the cells were transferred to an Eppendorf tube and then centrifuged at 250 × g for 4 minutes to obtain a cell pellet. After removing the supernatant and rinsing once with ultrapure water (without disturbing the pellet), the cell pellet was transferred to a 2mL headspace vial by resuspending in 300 μL ultrapure water. Then, 1200 μL of an ultrapure aqueous solution of 3.75% 5-sulfosalicylic acid was added once, and the vial was quickly sealed by a crimping seal cap with a PTFE-silicone rubber diaphragm. The headspace vial was incubated at 37 ° C for 2 hours.

[0315] Measurement of CO in Cell Lysates The CO concentration in the headspace was measured as described in Example 17 and a calibration curve was prepared using CO calibration gas.

[0316] Example 19. Absolute quantification of CO in tissue samples using gas chromatography.

[0317] Gas chromatography setup. The GC was tested on an Agilent 7820a GC system. The packed inlet was purged at 150°C. Column: Restek Molesieve 5A, 80 / 100 mesh, 0.53 mm x 2 m (Centre County, Pennsylvania, US). Carrier gas: Helium. Column flow rate: 4.5 ml / min. Oven temperature: 100°C isocratic for 5 min. Detector: Restek methanator (CH4izer, Centre County, Pennsylvania, US) was coupled between the column and the FID detector; it uses a nickel catalytic tube and hydrogen to convert CO and CO 2 Converted into methane, which can be detected by FID detector; Catalytic tube temperature: 380℃; Catalyst H 2 Flow rate: 25 ml / min. FID detector: temperature 300°C; H 2 Flow rate: 15ml / min; air flow rate: 400ml / min.

[0318] Preparation of calibration curve and testing of CO in tissue homogenate. Fill the gas sampling bag (Supel TM , inert multilayer foil, Sigma-Aldrich, Saint Louis, Missouri, USA). 100 μL of headspace gas was injected into the GC from a sampling bag or a tissue homogenate vial (prepared according to Example 17). The CO peak area was plotted against the CO concentration of the calibration gas to obtain a standard curve that can be used to determine the CO concentration in tissue samples.

[0319] Example 20. Cell imaging using 31 and 31a.

[0320] HeLa cells were cultured at 5×10 4 Cells were seeded at a density of 100 μg / dish in 3.5 cm Cellvis glass bottom culture dishes (Mountain View, California, USA) in FluoroBrite DMEM medium (ThermoFisher, Carlsbad, California, USA) supplemented with 10% FBS, 100 units of penicillin and 100 μg / ml streptomycin. After overnight culture, cells were treated with the indicated conditions and incubated for 1 hour: vehicle control: 0.5% DMSO in culture medium; CO donor: 50 μM BW-CO-201 or 50 μM CORM-401; CO gas: 250 ppm CO, 5% CO 2and balance air in the chamber. Then 31 or 31a was added to the culture medium at a concentration of 20 μM, and the cells were incubated continuously for 1 hour under the previous conditions. The cells were then washed twice with PBS and incubated in fresh FluoroBrite DMEM medium. Live cells were imaged using an Olympus IX-73 inverted fluorescence microscope in the DAPI channel and phase contrast transmission settings.

[0321] Example 21. Western blot analysis of HO-1.

[0322] HeLa cells were cultured at 75 cm 2 The cells were cultured in DMEM medium (without sodium pyruvate) supplemented with 10% fetal bovine serum, 100 units / mL penicillin, and 100 μg / mL streptomycin in a cell culture flask. After the confluence reached 80%, the cells were trypsinized and cultured at 1×10 6 Cells were seeded at a density of 100 cells / well in 6-well plates with 3 mL of culture medium containing 0.5% DMSO (vehicle control group) or 0.3 μM CDDO-Me in DMSO (treatment group, DMSO concentration: 0.5%). The cells were incubated at 37°C in a humidified cell culture incubator with 5% CO 2 The cells were incubated in an air atmosphere for 6 hours at 4 ° C. The cells were washed 3 times with cold 1×PBS and lysed with 200 μL 1×Laemmili loading buffer (containing 2.5% mercaptoethanol). The cell lysate was denatured at 95 ° C for 5 minutes. 5 μL of denatured cell lysate was separated by 4-15% SDS-PAGE gel and transferred to a PTFE membrane. According to the manufacturer's manual, the target protein was blotted using the iBind Flex protein blotting system (Invitrogen, USA). Primary antibody (mouse): HO-1 (1:200, Santa Cruz, USA), β-actin (1:400, Bio-rad, USA); HRP-conjugated goat anti-mouse secondary antibody (1:2000, Bio-rad, USA). The blot was detected with Pierce ECL plus protein blotting substrate (Thermo-Fisher, USA) and imaged with a GE LAS4000 micro chemiluminescent imager.

[0323] Example 22. Cytotoxicity evaluation of 31 and 31a.

[0324] HeLa cells were cultured at 1×10 5Cell / 100 μ L / hole density is seeded in 96 well plates.Cells are incubated overnight in 37 ℃ of cell culture incubators under wet conditions in DMEM complete medium (supplemented with 10% FBS and 100 units / ml penicillin and 100 μ g / mL streptomycin).After adhesion, the cell culture medium is replaced with the culture medium of the compound load of 2 × serial dilutions containing 31 or 31a of 100 μ L, and the concentration is 1.56-50 μ M.DMSO is used to prepare stock solution, and the final concentration of DMSO in all wells (including control group wells) is 0.5%.After incubation for 24 hours, 10 μ L CCK-8 is added in each well, and then further incubated for 2 hours.The optical density at 450nm is read using a plate reader, and cell viability is calculated according to the following formula:

[0325]

[0326] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, those skilled in the art will appreciate that certain changes and modifications may be made within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were incorporated by reference individually.

[0327] Example 23. X-ray crystallographic structure determination of palladium complex

[0328] 5a, 14, 31, and 31a were recrystallized by vapor diffusion (solvent system: dichloromethane-ether). X-ray crystallography was performed at Emory University. Crystals of appropriate size were selected and mounted on a Rigaku XtaLAB Synergy-S diffractometer (Tokyo, Japan) on a paratone ring and chromatographically analyzed using a Cu K α radiation Or Mo K α radiation During data collection, the crystal remained stable at T = 100 (1) K. The structure was solved with the ShelXT solver using an iterative approach and by using Olex2 1.3-alpha as a graphical interface. The model was refined by olex2.refine 1.3-alpha using full matrix least squares minimization on F2.

[0329] The crystallographic data of 5a, 14, 31 and 31a have been deposited in the Cambridge Crystallographic Data Center: CCDC 2181545-2181548.

[0330] Table 4. Overall comparison of representative Pd-based CO probes designed in this paper and compounds.

[0331]

[0332]

[0333] *LOD: limit of detection. # SNR: signal-to-noise ratio (>20 equivalent excess CO)

Claims

1. A method for detecting carbon monoxide, the method comprising: The test sample is combined with a fluoroamide-functionalized palladium coordination complex. thereby causing the carbon monoxide in the test sample to react with the fluoroamide-functionalized palladium coordination complex to form a fluoroimide, and The fluorescence emitted by the fluorescent imide is detected, thereby detecting carbon monoxide in the test sample.

2. The method of claim 1, wherein the amide in the fluorescent amide-functionalized palladium coordination complex is benzamide or naphthamide.

3. The method of claim 1 or 2, wherein the fluoroamide-functionalized palladium coordination complex is a compound according to formula I: in: Each R 1 Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 alkyl; R 2 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 2a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties; Each R 2a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O)2R c 、-S(O)2OR a 、-S(O)2NR a R b and-NR a S(O)2R c ; Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl; Each R c It is C 1-6 alkyl; The part L 1 -L 2 It is a bidentate ligand; X is an anionic ligand; subscript m is 0 or 1; and Subscript n is 0, 1, 2 or 3.

4. The method of claim 3, wherein each R 1 Independently selected from the group consisting of: -OR a and-NR a R b .

5. The method according to claim 3 or 4, wherein each R 1 Yes-OR a ; and R a Selected from the group consisting of: H, methyl, ethyl, propyl and butyl.

6. The method of claim 3 or 4, wherein each R 1 Yes - NR a R b ; and R a and R b Independently selected from the group consisting of H, methyl, ethyl, propyl and butyl.

7. The method of claim 6, wherein R a is H; and R b Selected from the group consisting of: H, methyl, ethyl, propyl and butyl.

8. The method of any one of claims 3-7, wherein subscript m is 1.

9. The method of any one of claims 3-7, wherein subscript m is 0 and the carbonyl oxygen is coordinated to the palladium in the compound of formula I.

10. The method of claim 9, wherein the fluoroamide-functionalized palladium coordination complex is a compound according to formula Ia: Among them A - It is a non-coordinating anion.

11. The method of any one of claims 3 to 10, wherein R 2 Selected from the group consisting of: where v is 0, 1, 2, or 3.

12. The method of any one of claims 3 to 11, wherein subscript n is 1.

13. The method of claim 12, wherein R 1 It is -OH or -NH2.

14. The method of any one of claims 3 to 13, wherein the fluorescent imide is a compound according to formula II:

15. The method of claim 1 or 2, wherein the fluoroamide-functionalized palladium coordination complex is a compound according to formula III: in: Each R 3a and R 3b Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 Alkyl, or R 3a and R 3b Combine to form a C3-C7 cyclic amine ring, R 4 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 4a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties, Each R 4a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O)2R c 、-S(O)2OR a 、-S(O)2NR a R b and-NR a S(O)2R c ; Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl; Each R c It is C 1-6 alkyl; The part L 3 -L 4 It is a bidentate ligand; X is an anionic ligand; subscript p is 0 or 1; and The subscripts q and t are independently 0, 1, 2 or 3.

16. The method of claim 15, wherein each R 3a and R 3b Independently selected from the group consisting of: -OR a and-NR a R b .

17. The method of claim 15 or 16, wherein each R 3a and R 3b Yes-OR a ; and R a Selected from the group consisting of: H, methyl, ethyl, propyl and butyl.

18. The method of claim 15 or 16, wherein each R 3a and R 3b Yes - NR a R b ; and R a and R b Independently selected from the group consisting of H, methyl, ethyl, propyl and butyl.

19. The method of claim 18, wherein R a is H; and R b Selected from the group consisting of: H, methyl, ethyl, propyl and butyl.

20. The method of any one of claims 15-19, wherein subscript p is 1.

21. The method of any one of claims 15-19, wherein subscript p is 0 and the carbonyl oxygen is coordinated to the palladium in the compound of formula III.

22. The method of claim 21, wherein the fluoroamide-functionalized palladium coordination complex is a compound according to formula IIIa: Among them A - It is a non-coordinating anion.

23. The method of any one of claims 15-22, wherein R 4 Selected from the group consisting of: where v is 0, 1, 2, or 3.

24. The method of any one of claims 15-23, wherein subscript q is 0 and subscript t is 1.

25. The method of claim 24, wherein R 3b It is -OCH3.

26. The method of any one of claims 15-25, wherein the fluorescent imide is a compound according to formula IV:

27. The method of any one of claims 3-26, wherein the anionic ligand is selected from the group consisting of halide ions and -SO3R, wherein R is selected from the group consisting of H, -C1-C8 alkyl, benzyl, and substituted benzyl.

28. The method of any one of claims 3 to 26, wherein the non-coordinating anion is selected from the group consisting of: OTf-(CF3SO3 - )、NO3 - CF3COO - 、CH3COO - 、4-CF3C6H4SO3 - 、4-CH3C6H4SO3 - 、F - , Cl - Br - ,I - 、SO4 2- and CO3 2- .

29. The method of any one of claims 3-27, wherein the bidentate ligand is selected from the group consisting of alkylenediamines, bipyridines, and phenanthrolines.

30. The method of any one of claims 1-29, wherein the test sample is a biological fluid sample.

31. The method of claim 30, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, and serum.

32. The method of any one of claims 1-29, wherein the test sample is a tissue sample.

33. The method of claim 32, wherein the tissue sample is selected from the group consisting of liver, kidney, heart, brain and lung.

34. The method of any one of claims 1-29, wherein the test sample is a cell sample.

35. The method of claim 34, wherein the cell sample is a cell culture.

36. A compound according to formula I: in Each R 1 Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 alkyl; R 2 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 2a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties; Each R 2a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O)2R c 、-S(O)2OR a 、-S(O)2NR a R b and-NR a S(O)2R c ; Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl; Each R c It is C 1-6 alkyl; The part L 1 -L 2 selected from the group consisting of: alkylenediamines, bipyridines, and phenanthrolines; X is an anionic ligand; subscript m is 0 or 1; and Subscript n is 1, 2, or 3.

37. The compound of claim 36, having a structure according to Formula Ia: Among them A - It is a non-coordinating anion.

38. The method of claim 36 or 37, wherein R 2 Selected from the group consisting of: where v is 0, 1, 2, or 3.

39. A compound according to formula III: in: Each R 3a and R 3b Independently selected from the group consisting of: -OR a 、-NR a R b and C 1-6 Alkyl, or R 3a and R 3b Combine to form a C3-C7 cyclic amine ring, R 4 Selected from the group consisting of: C 1-6 Alkyl, which is optionally substituted by one or more R 4a substituted, and a functional group containing at least two sulfonic acid or sulfonate moieties, Each R 4a Independently selected from the group consisting of: C 1-6 Alkyl, halogen, -CN, -OR a 、-C(O)R c 、-C(O)OR a 、-OC(O)R c 、-NR a R b 、-NR a C(O)R c 、-C(O)NR a R b 、-S(O)R c 、-S(O)2R c 、-S(O)2OR a 、-S(O)2NR a R b and-NR a S(O)2R c ; Each R a and R b Independently selected from the group consisting of: H and C 1-6 alkyl; Each R c It is C 1-6 alkyl; The part L 3 -L 4 It is a bidentate ligand; X is an anionic ligand; subscript p is 0 or 1; and The subscripts q and t are independently 0, 1, 2 or 3.

40. The compound of claim 39, having a structure according to Formula IIIa: Among them A - It is a non-coordinating anion.

41. The method of claim 39 or 40, wherein R 4 Selected from the group consisting of: where v is 0, 1, 2, or 3.

42. A kit comprising the compound according to any one of claims 36 to 41 and instructions for use of the compound in the detection of carbon monoxide.

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