CROSS REFERENCE TO RELATED APPLICATION

[0001] 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.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under DK119202 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

[0003] Since the first report of the signaling functions of carbon monoxide (CO) in the 1990s, there have been extensive studies of the physiological and pharmacological roles of CO by a large number of labs. Despite its commonly held public perception of being poisonous at high concentrations, CO is actually present in the general circulation in the high micromolar range under normal physiological conditions, largely through the endogenous production by heme oxygenase-mediated degradation of heme. Further, whether endogenously produced or delivered exogenously, CO offers anti-inflammatory, cellular and organ-protective activity against various stress conditions such as lipopolysaccharide challenge, ischemia-reperfusion injury, and chemical induced organ damage. The prospect of developing CO into a therapeutic agent for treating various diseases such as colitis, sickle cell disease, acute kidney injury, among others is on the horizon. Therefore, extensive efforts have been made in recent years in investigating CO gas inhalation in clinical trials, and developing the non-gaseous CO delivery approaches, including liquid formulations, metal-based CO- releasing molecules (CORMs), and organic CO prodrugs and its formulations.

[0004] As a gas molecule, a unique challenge posing to the research and development of CO is the determination of its concentration and associated pharmacokinetic work. Under physiological and near-equilibrium conditions, CO in the circulation system predominantly exists in the form of carboxyhemoglobin (COHb), owing to its high binding affinity to hemoglobin. Further, the high concentration of Hb (about 8 mM in human) also allows its reservoir roles for CO. Therefore, due to the easier assessment through testing peripheral blood, COHb concentration is commonly used as a surrogate for assessing systemic CO exposure levels. On the other hand, CO in the tissue is the actual executor that dictates the biological activity thus therapeutic effect and/or toxicity. CO binds to hemoproteins in tissues, including enzymes, proteins for oxygen transport and storage, iron-regulatory proteins, sensors, transcription factors, ion channels, and others to exert its biological functions. Due to the thermal dynamic equilibrium between COHb and the hemoproteins according to their relative binding affinities, tissue CO concentration is proportional to the blood COHb concentration. In addition to COHb level and hemoprotein concentration, tissues CO saturation levels are also dependent on other factors, such as local partial pressure of CO and oxygen. One may draw an analogy with traditional small molecule assessments, whereas the level of COHb is the equivalent of albumin-bound drug, while CO in the tissue is the equivalent of tissue drug concentration. Indeed in practice, the overall CO bioavailability is often evaluated by calculating the area under the curve (AUC) of COHb levels after delivering CO to the body. As the best practice, tissue CO concentration is also assessed to show the correspondence between the observed biological effect and elevated CO level in many studies. As such, to understand the pharmacology of CO for the development of CO- based therapeutics, assessing CO concentration in both blood and tissue is an important and essential component of in-vivo an ex-vivo study. For in-vitro cell study, it would also be amenable to evaluate intracellular CO levels to witness the relevance of CO and observed biological effects.

[0005] Studying the relationship between CO dose-response and the observed biological effects requires determining the CO concentration in tissues and other biological samples, but the analytical methods necessary to do this can present challenges for many labs. Existing methods for quantifying CO in cell culture samples and tissue samples include a myoglobin- coupled chemiluminescence method; a ¹⁴ C radioactivity method, which requires pre-labeling with [ ¹⁴ C] glycine; and a UV spectroscopic method using a synthetic heme-containing CO sensor. However, these methods have not been widely implemented for many reasons.

[0006] There are two mainstream ways of determining CO in biological samples in the reported literature. One is the direct quantification using gas chromatography (GC). Specifically, CO is liberated by treating the tissue with a denaturing or oxidizing agent in the headspace vial. To detect the trace amounts of CO in the headspace vial, it requires using a methanizer coupled flame ionization detector (FID), a mercury reduction gas detector (RGD), or a semiconductor sensor gas chromatograph (SGC). The detection limits with 100 pL gas injection are 0.5 ppm, 50 ppb, and 50 ppb for methanizer FID, RGD, and SGC, respectively, according to the manufacturer’s specification. Thermal conductivity detector (TCD) as the universal gas detector is not able to detect such low CO concentration due to its lower sensitivity (about 1000 ppm with 100 pL injection). However, these delicate gas chromatograph instruments are not commonly available in biology labs and require chemistry knowledge in designing experiments and interpreting data. For example, methanizer can also reduce CO2 in the air to methane; therefore, CO2 and CO must be well separated with a GC column, or the sample needs to be flushed with nitrogen or helium before CO liberation. For the RGD method, since the detector is very sensitive, the carrier gas must be pre-purified with a scarcely available CO trap to eliminate trace CO. The consumable nature of the methanizer and RGD due to the poisoning by sulfide species in the biospecimen, the tedious sample processing procedure (for RGD), and the limited access to the GC for most biology labs may pose hurdles to such methods.

[0007] The second approach to sense CO in the biological system is to use fluorescent or chromogenic probes, including a genetically encoded CO sensing protein and small molecule reaction-based CO probes (FIG. 1). As CO endows binding capability to the heme prosthetic group in hemoproteins, a bacterial CO sensing hemoprotein C00A was fused to a modified yellow fluorescent protein (YFP). Binding CO to C00A domain restored the fluorescent conformation of YFP, thus increasing its fluorescence by about two-fold. A comparable approach along this line is the cyclodextrin encapsuled heme analog HemoCDl (FIG. 1), which endows high binding affinity to CO and was used to quantify CO in the cell culture and tissue by UV-Vis spectroscopy. The reversibility of these metal binding approaches allows for assessing CO in the cell under a near-equilibrium condition in a real-time fashion. As for the reaction-based fluorescent sensors, there are two major strategies in designing such CO probes. One strategy is to form a transition metal (Pd in most cases) complex with a fluorophore therefore quenching the fluorescence by the heavy atom electronic effect. Upon reaction with CO, Pd is removed by either palladium-mediated carbonylation or protonolysis. In either case, the fluorescence quenching effect of Pd is lifted, hence recovering the fluorescence of the fluorophore. This “dequenching” strategy has been implemented in designing CO probes such as COP-1, its analogs, and CC-CO, among others. Another strategy is to use an allyl group to cap the fluorophore. Although the pro-fluorescent compound is metal-free, PdCb has to be administered concomitantly with the probe to sense CO. Upon reduction of Pd ²⁺ with CO, allyl capping group is removed by the formed Pd(0) via Tsuji-Trost reaction to recover the fluorescence. These CO probes, especially COP-1, have been extensively used in cellular imaging-based studies and have tremendously helped the understanding of CO’s biology. 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 towards CO. For dequenching-based probes, ubiquitous nucleophiles such as thiols in the biological milieu can react and remove the palladium quenching group which could be seen with the slightly turn-on effect by the thiol species reported in the literature. For Tsuji-Trost reach on -based probes, the capping group could be potentially removed by enzymatic metabolism, and Pd ²⁺ was also reported to be able to get reduced by ascorbic acid, which may result in false positive or compatibility issues.

[0008] There are also several nitro-reduction-based probes that initially were reported to sense CO, but the sensing mechanism was later found to be dependent on the reactivity of the ruthenium complex. Thus, they should not be considered as general CO probes, but rather probes for ruthenium-based CORMs. Aside from these nitro reduction sensing mechanism probes, a recently reported CORM-3 fluorescent probe utilized CORM-3 mediated isomerization-hydrolysis reactions of an allyl capping group to turn-on fluorescence. One would not expect CO alone is able to carry out such nitro-reduction or isomerization reaction under physiological conditions. Hence, it underscores the necessity of using CO gas to define the true analyte of CO probe and understanding the reactivity of CORMs.

[0009] There have not been any reported applications of using a fluorescence probe to quantify CO in cell culture, blood, or tissue samples. Though CO-oximetry can be applied to test blood COHb levels in human and animal, not all laboratories have such access to the CO- oximeter, demanding for alternative feasible approaches. Therefore, there is a need for highly reliable fluorescent CO probes with extra-low background fluorescence that not only detect carbon monoxide with high specificity and ultrahigh sensitivity, but are also capable of determining CO concentrations in biological samples, both semi-quantitatively and quantitatively, as well as imaging intracellular CO accumulation in live cells. BRIEF SUMMARY OF THE DISCLOSURE

[0010] In one aspect, described herein is a method for detecting carbon monoxide, the method comprising: combining a test sample and a fluorogenic amide-functionalized palladium coordination complex, allowing carbon monoxide in the test sample to react with the fluorogenic amide-functionalized palladium coordination complex, 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 fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a benzamide or a naphthamide.

[0011] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula I: (I), wherein: each R ¹ is independently selected from the group consisting of-OR ^a , -NR ^a R ^b , and Ci-6 alkyl;

R ² is selected from the group consisting of Ci-6 alkyl, which is optionally substituted with one or more R ^2a , and a functional group containing at least two sulfonic acid or sulfonate moieties; each R ^2a is independently selected from the group consisting of Ci-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 ; each R ^a and R ^b are independently selected from the group consisting of H and

Ci-6 alkyl; each R ^c is Ci-6 alkyl; the moiety L ¹ — L ² is a bidentate ligand;

X is an anionic ligand; subscript m is 0 or 1; and subscript n is 0, 1, 2, or 3.

[0012] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula la: (la), wherein A is a non-coordinating anion.

[0013] In some embodiments, the fluorescent imide formed from the reaction between carbon monoxide in the test sample and the fluorogenic amide-functionalized palladium coordination complex of Formula I or Formula la in the methods described herein is a compound according to Formula II:

[0014] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula III: wherein: each R ^3a and R ^3b is independently selected from the group consisting of-OR ^a , -NR ^a R ^b , and Ci-6 alkyl, or R ^3a and R ^3b are combined to form a C3-C7 cycloamine ring, R ⁴ is selected from the group consisting of Ci-6 alkyl, which is optionally substituted with one or more R ^4a , and a functional group containing at least two sulfonic acid or sulfonate moieties; each R ^4a is independently selected from the group consisting of Ci-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 ; each R ^a and R ^b are independently selected from the group consisting of H and Ci-6 alkyl; each R ^c is Ci-6 alkyl; the moiety L ³ — L ⁴ is a bidentate ligand;

X is an anionic ligand; subscript p is 0 or 1; and subscripts q and t are independently 0, 1, 2, or 3.

[0015] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula Illa: wherein A is a non-coordinating anion.

[0016] In some embodiments, the fluorescent imide formed from the reaction between carbon monoxide in the test sample and the fluorogenic amide-functionalized palladium coordination complex of Formula III or Formula Illa in the methods described herein is a compound according to Formula IV:

[0017] 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.

[0018] In another aspect, the disclosure provides a compound according to Formula I: wherein each R ¹ is independently selected from the group consisting of-OR ^a , -NR ^a R ^b , and Ci-6 alkyl;

R ² is selected from the group consisting of Ci-6 alkyl, which is optionally substituted with one or more R ^2a , and a functional group containing at least two sulfonic acid or sulfonate moieties; each R ^2a is independently selected from the group consisting of Ci-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 ; each R ^a and R ^b are independently selected from the group consisting of H and Ci-6 alkyl; each R ^c is Ci-6 alkyl; the moiety L ¹ — L ² is selected from the group consisting of alkylenediamine, bipyridine, and phenanthroline;

X is an anionic ligand; subscript m is 0 or 1; and subscript n is 1, 2, or 3.

[0019] In some embodiments, the compound of Formula I has a structure according to

Formula la: (la), wherein A“ is a non-coordinating anion.

[0020] In another aspect, the disclosure provides a compound according to Formula III: wherein: each R ^3a and R ^3b is independently selected from the group consisting of-OR ^a , -NR ^a R ^b , and Ci-6 alkyl, or R ^3a and R ^3b are combined to form a C3-C7 cycloamine ring,

R ⁴ is selected from the group consisting of Ci-6 alkyl, which is optionally substituted with one or more R ^4a , and a functional group containing at least two sulfonic acid or sulfonate moieties, each R ^4a is independently selected from the group consisting of Ci-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 ; each R ^a and R ^b are independently selected from the group consisting of H and Ci-6 alkyl; each R ^c is Ci-6 alkyl; the moiety L ³ — L ⁴ is a bidentate ligand;

X is an anionic ligand; subscript p is 0 or 1; and subscripts q and t are independently 0, 1, 2, or 3.

[0021] In some embodiments, the compound of Formula III has a structure according to Formula Illa: wherein A is a non-coordinating anion.

[0022] In another aspect, the disclosure provides a kit comprising a compound of Formula I, Formula la, Formula III, or Formula Illa, as described herein, and instructions for use of the compound in the detection of carbon monoxide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows (a) CO probes reported in the literature, and (b) CO probes as described herein.

[0024] FIG. 2 shows the design and fluorescence properties of benzamide-based CO probe molecules, a. CO sensing chemistry and structure of the benzamide-based CO probe molecules; b. 500 pM 5a was incubated with various amounts of CO gas (0-100 nmol) in the headspace vial for 1 hour and the fluorescence spectra were recorded (Z _X =394 nm, bandwidth=5 nm); insert: linear regression of fluorescence intensity on CO gas molarity; c. SNR of 5a at concentrations of 1, 10, and 100 pM in PBS incubated with or without CO gas for 1 hour (SNR was calculated as the ratio between the AUC of emission spectra with and without CO gas incubation); insert: the regression of SNR on probe concentration in PBS; d. Fluorescence spectra of 22 and 17 (bandwidth=5 nm); e. Selectivity of 20 pM 5a in pH 7.4 PBS (CO gas 1% in air at 1 atm, concentration of other species is 100 pM, incubated for Ih, ,EX=395 nm, bandwidth=5 nm); insert: image of 50 pM 5a in PBS with or without incubation with pure CO gas for 30 min, image of the two cuvettes was taken under the same 365 nm UV light (5W); f. 500 pM 14 in PBS were incubated with 0-100 nmol of CO gas in the headspace vial for 1 hour and the fluorescence spectra were recorded ( E _X =385 nm, bandwidth=5 nm); insert: linear regression of fluorescence intensity on CO gas molarity; g. SNR of 14 at a concentration of 1, 10, and 100 pM in PBS incubated with or without incubating with CO gas for 30 min; insert: the regression of SNR on probe concentration in PBS (bandwidth=3 nm); h. Selectivity of 10 pM 14 in pH 7.4 PBS (CO gas 1% in air at 1 atm, concentration of other CO probe species is 100 pM, incubated for 30 min, XE _X =385 nm, bandwidth=5 nm); i. Fluorescence spectra of 20 pM 25 and 10 pM 19 in PBS (bandwidth=5 nm); j. CO sensing kinetics of CO probe molecules (800 pL 12.5 pM CO probe was mixed with 200 pL 1 mM CO saturated PBS at To and the fluorescence at 509 nm or 499 nm was recorded every second at 25 °C. Reaction progress of each time point is calculated as the percentage of the fluorescence intensity of the maximum fluorescence. Insert: expanded range of 0-360 s).

[0025] FIG. 3 shows the stability of 5 and 5a in PBS as tested using fluorescence recovery ratio. 10 pM 5 and 5a were incubated in PBS in a head-space vial for the designated time and CO was injected to turn on the probe, followed by fluorescence measurements. (A,EX=395 nm, ,Em=511 nm, bandwidth=5 nm).

[0026] FIG. 4 shows the sensing selectivity fluorescence spectra of CO probe molecule 5a. Selectivity of 10 pM 5a in pH 7.4 PBS (CO gas 1% in air at 1 atm; concentration of other analyte species was 100 pM; incubated for Ih; XEX=395 nm, bandwidth=3 nm).

[0027] FIG. 5 shows NMR mechanistic studies of CO sensing using 5a. ¹ H NMR spectra of (I) 5a; (II) 5a 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).

[0028] FIG. 6 shows HPLC mechanistic studies of sensing CO using 5a in D.I. water (a) and in EtOH (b). Conditions: (1) 100 pM 5a in H ₂ O or EtOH; (2) 100 pM 5a was incubated with excessive CO gas (>10 equivalent) for 5 min; (3) 100 mM PBS (10x) was added to the sample of (2), final PBS concentration is 10 mM (l ^x ); (4) 200 pM 22 in PBS (l ^x ). (UV detector monitored at 214 nm).

[0029] FIG. 7 shows LC-MS spectra identifying the intermediate species and fluorescent products generated from the reaction between CO gas and 5a: IM-1 (a), 22 (b), and IM-2 (c). For a and b, 100 pM 5a was dissolved in 0.01 M HC1 (pH=2) and 0.5 ml CO gas was injected followed by incubation at room temperature. After injection for identification of IM- 1, the reaction was neutralized by adding 100 pL lOx PBS followed by injection to LCMS for 22 identification. For c, 100 pM 5a was dissolved in ethanol and 0.5 ml CO gas was injected followed by incubation at room temperature followed by injection to LCMS for IM-2 identification.

[0030] FIG. 8 shows the pH-dependence of fluorescent products 22 (a) and 25 (b). 30 pM 22 and 10 pM 25 were used, fluorescence measured at XEX= 395 nm and 385 nm, respectively, bandwidth=5 nm, low sensitivity setting.

[0031] FIG. 9 shows the sensing selectivity fluorescence spectra of CO probe molecule 14. Selectivity of 10 pM 14 in pH 7.4 PBS (CO gas 1% in air at 1 atm; concentration of other analyte species was 100 pM; incubated for 30 min, XEX=385 nm, bandwidth=3 nm).

[0032] FIG. 10 shows 14 quantitatively forms 25 and produces the same fluorescence intensity after incubation with CO gas. 200 pM 14 was dissolved in DMA and incubated in a 2 ml headspace vial with 1 ml CO gas for 15 min, then diluted by 16-fold with PBS to 12.5 pM and compared with 12.5 pM 25 in PBS (mean ± SD, n=3, ns: not significant).

[0033] FIG. 11 shows the design and fluorescence properties of naphthamide-based CO probe molecules, a. CO sensing chemistry and structure of the naphthamide-based CO probe molecules; b. Fluorescence spectra of 10 pM 34 and 29 (bandwidth = 5 nm); c. SNR of 31a at concentrations of 1, 10, 25, and 50 pM in PBS incubated with or without CO gas for 1 hour (SNR was calculated as the ratio between the AUC of emission specturm with and without CO gas incubation); insert: linear regression of SNR on probe concentraion in PBS (bandwidth: ex=3 nm, em=5 nm); d. CO sensing kinetics of 31a (800 pL 12.5 pM 31a was mixed with 200 pL 1 mM CO saturated PBS at To and the fluorescence at 457 nm or 499 nm was recorded every second at 25 °C. Reaction progress of each time point is calculated as the percentage of the fluorescence intensity of the maximum fluorescence); e. and f. Selectivity of 10 pM 31a in pH 7.4 PBS (CO gas 1% in air at 1 atm, concentration of other species is 100 pM; incubated for Ih; XEX=377 nm; bandwidth=5 nm; image taken under 385 nm UV light).

[0034] FIG. 12 shows stability studies of 31 and 31a in PBS solution incubated at 37 °C. Monitored by injecting the solution to HPLC every 15 min.

[0035] FIG. 13 shows LC-MS spectra identifying the fluorescent products generated from the reaction between CO gas and 31a and the CO sensing mechanism. 31a was dissolved in PBS at a concentration of 50 pM. After injection 20 pL to the LCMS system, 1 ml CO gas was bubbled into the vial and incubated at room temperature for 20 min, followed by injection to LC-MS.

[0036] FIG. 14 shows the determination of CO concentration in biological samples, a. mouse blood with various COHb levels pre-determined by CO-oximeter was incubated with 10 mM 5a for 30 min, followed by denaturation; the fluorescence spectra were collected under XEX=395 nm; insert: calibration curve of COHb vs fluorescence intensity, b. COHb levels of the blood from five individual mice was saturated with CO gas, followed by 2-fold dilution by fresh blood due to the 80% maximum limit of the CO-oximeter. c. blood COHb levels of undosed mice and mice dosed with BW-AC-306 (100 mg/kg), determined using either 5a or a CO-oximeter; insert: calibration curve of COHb vs fluorescence intensity (counts per sec. CPS), d. blood COHb levels of undosed mice and mice dosed with BW-CO- 306 (200 mg/kg), determined using either Ila or a CO-oximeter; insert: calibration curve of COHb vs fluorescence intensity (CPS), e. relative CO levels of the HeLa cells treated with 0.3 pM CDDO-Me (6 h) or 250 ppm CO gas (2 h), as represented by the fluorescence intensity (CPS) tested with multi-plate reader, f. Western-blot of mouse heme oxygenase 1 (HO-1) antibody in HeLa cells treated with 0.3 pM CDDO-Me (6 h), P-actin was probed as the loading control, g. Fluorescence spectra of 1 mM 14 in DMA incubated with various CO calibration gas followed by PBS dilution; insert: calibration curve of CO concentration (ppm) vs fluorescence intensity at 499 nm (XEX=385 nm). h. CO concentration in liver tissue of undosed mice and mice dosed with BW-CO-306 (200 mg/kg), determined using either 14 or a methanizer-FID-GC. i. CO concentration in kidney tissue of undosed mice and mice dosed with BW-CO-306 (200 mg/kg), determined using either 14 or a methanizer-FID-GC. j. Collective data of h and i panels, k. CO concentration in the HeLa cells treated with CO gas and CO prodrug BW-CO-111 (50 pM) for 2 hours and tested with 14. For all experiments, n>3, ****P<0.0001, ns: not significant ( >0.05).

[0037] FIG. 15 shows the cytotoxicity of 31 and 31a in HeLa cells. Cell viability was determined by the CCK-8 assay after 24-h incubation.

[0038] FIG. 16 shows fluorescence microscopic imaging of CO in live cells using naphthamide-based CO probe molecule 31. HeLa cells were treated with vehicle control (DMSO), 250 ppm CO gas, 50 pM BW-CO-201, 50 pM CORM-401 for 1 hour followed by addition of 20 pM 31 and incubated for 1 hr. Live cells were washed with PBS once and imaged in Fluorobrite DMEM culture medium. Exposure time: 0.7 s, scale bar: 20 pm (BW- CO-201 is reported in Ji, X. et al. Chem Commun (Camb) 2017, 53(69), 9628-9631; CORM- 401 is reported in Crook, S.H., et al. Dalton Trans 2011, 40, 4230-4235).

[0039] FIG. 17 shows fluorescence microscopy imaging of CO in live cells using naphthamide-based CO probe molecule 31a. HeLa cells were treated with (a) DMSO vehicle control for 1 h, (b) 250 ppm CO gas for 1 h, or (c) 50 pM BW-CO-201 for 1 h, followed by addition of 20 pM 31a and incubation for 1 hour (scale bar: 20 pm); HeLa cells were treated with (d) DMSO vehicle for 6 hrs, or (e) 0.3 pM CDDO-Me for 6 hrs, followed by incubation with 20 pM 31a for Ih (scale bar: 50 pm); (f) Background-normalized maximum signal intensity of the cells in the image (*P<0.05, n=3). Live cells were washed with PBS twice and imaged in Fluorobrite DMEM culture medium; (g) and (h) illustrate the line profile ROI of (f), line profile 1-3 was used for the calculation of signal intensity, line profile 4 is used for background subtraction.

[0040] FIG. 18 shows the determination of the second-order reaction constants of CO probe molecules 6a and 14 towards CO.

[0041] FIG. 19 shows the determination of the detection limits for benzamide-based CO probe compounds 5a, Ila, and 14.

[0042] FIG. 20 shows the determination of the detection limits for naphthamide-based CO probe compounds 31 and 31a.

[0043] FIG. 21 shows the X-ray crystallography structure for naphthamide-based CO probe compounds 5 and 5a.

[0044] FIG. 22 shows the X-ray crystallography structure for naphthamide-based CO probe compounds 31 and 31a.

[0045] FIG. 23 shows the formation of depalladation species by thiols, (a) LC-MS (XIC, extract 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 pM 5a and 100 pM NaHS in PBS. (c) 1H-NMR and MS of 17 recovered from the reaction of 5a and NaHS (1 :2) in DMA.

[0046] FIG. 24 shows the effect of thiol species on 5a. (a) Preincubation of 5a at 5 mM (1 : 1), 10 mM (2: 1), or 5 mM (probe only) with or without 5 mM GSH, in PBS at 37 °C for 15 minutes, followed by incubation with 600 ppm CO gas (0.1 eq. in quantity) for 30 minutes to partially turn on the fluorescence, (b) Preincubation of 5a at a concentration of 100 pM with NaHS at concentrations of 100 pM (1 : 1), 50 pM (2: 1), 25 pM (4: 1), 10 pM (10: 1), or 0 (probe only) in PBS at 37 °C for 15 minutes, followed by incubation with 120 ppm CO gas (0.5 eq. in quantity) for 30 minutes to partially turn on the fluorescence, (c) 200 pM 5a was preincubated with 200 pM GSH or 20 pM NaHS for 15 min in 500 pL PBS followed by mixing with 500 pL CO saturated PBS (about 1 mM) and fluorescence intensity was recorded every second. (XE _X =395 nm, Z,Em=511 nm, bandwidth=5 nm).

DETAILED DESCRIPTION OF THE DISCLOSURE

I. General

[0047] Described herein are fluorogenic amide-functionalized palladium coordination complexes that selectively detect carbon monoxide through the construction of a fluorescent product via a sequential CO insertion-carbonylation-amidation reaction, with high specificity, high sensitivity, fast response, and little or no background fluorescence. Methods for detecting and quantifying carbon monoxide in biological samples and methods for imaging intracellular CO accumulation in live cells using the fluorogenic CO probe compounds, are also described.

II. Definitions

[0048] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined.

[0049] The terms “a,” “an,” or “the” as used herein not only include elements with one member, but also include elements with more than one member. For instance, 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, and so forth.

[0050] As used herein, the terms “about” and “around,” and the like, are used herein to modify a numerical value and indicate a defined range around that value. If “X” is the value, “about X” or “around X” generally indicates a value from 0.90X to 1.10X. Any reference to “about X” indicates at least the 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.O3X, 1.04X, 1.O5X, 1.06X, 1.07X, 1.O8X, 1.09X, and 1.1OX. Thus, “about X” is intended to disclose, e.g., “0.98X.” When “about” is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” is applied to the first value of a set of values, it applies to all values in that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%, about 9%, or about 11%.”

[0051] As used herein, the terms “comprising” and “comprises” are intended to mean that the methods, compounds, compositions, and respective components thereof include the recited elements, but do not exclude others. “Consisting essentially of’ refers to those elements required for a given embodiment. The phrase permits the presence of additional elements that do not materially affect the basic and novel or functional character! stic(s) of the given embodiment e.g., methods, compounds, or compositions). “Consisting of’ refers to methods, compounds, compositions, and respective components thereof, as described herein, which are exclusive of any element not recited in that description of the embodiment. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0052] As used herein, the terms “detect,” “detecting,” or “detection” refer to either the general act of discovering or discerning or the specific observation of carbon monoxide.

[0053] As used herein, the terms “carbon monoxide” or “CO” refer to and •• , as well as other forms of carbon monoxide formed under physiological conditions.

[0054] As used herein, the term “fluorogenic” refers to a property of a substance that is initially not fluorescent and subsequently becomes fluorescent by way of a chemical reaction.

[0055] As used herein, the term “fluorogenic amide-functionalized palladium coordination complex” refers to a non-fluorescent amide-functionalized palladium coordination complex that forms a fluorescent imide upon a sequential CO insertion-carbonylation-amidation- cyclization reaction that occurs between CO and the fluorogenic amide-functionalized palladium coordination complex. The “fluorogenic amide-functionalized palladium coordination complex” refers to a compound having a structure according to Formula I, Formula la, Formula III, or Formula Illa, as described herein, in which a palladium metal center contains a coordinated amide-containing ligand. [0056] As used herein, the term “fluorescent imide” refers to a compound having a structure according to Formula II or Formula IV, as described herein, that forms from the sequential CO insertion-carbonylation-amidation-cyclization reaction between CO and the fluorogenic amide-functionalized palladium coordination complex. The fluorescent imide absorbs light energy of a specific wavelength and re-emits light at a longer wavelength, which is used to detect the presence of carbon monoxide in a test sample.

[0057] As used herein, the term “alkyl,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as Ci-2, C1.3, CM, C1.5, Ci-6, C1.7, Ci-8, Ci-9, Ci-io, Ci-11, Ci-i2, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. For example, Ci-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Unless otherwise specified, alkyl groups can be substituted or unsubstituted. For example, “substituted alkyl” groups can be an alkyl group substituted with one or more groups selected from halo, hydroxy, amino, aminoalkyl, amido, and alkoxy.

[0058] As used herein, the term “alkoxy,” by itself or as part of another substituent, refers to a group having the formula -OR, wherein R is alkyl as described above.

[0059] As used herein, the term “cycloalkyl,” by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated.

Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbomane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene. When cycloalkyl is a saturated monocyclic C3-8 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted cycloalkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0060] As used herein, the term “alkylene” refers to an alkyl group, as defined above, linking at least two other groups (i.e., a divalent alkyl radical). The two moieties linked to the alkylene group can be linked to the same carbon atom or different carbon atoms of the alkylene group.

[0061] As used herein, the terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0062] As used herein, the term “haloalkyl,” by itself or as part of another substituent, refers to an alkyl group where some or all of the hydrogen atoms are replaced with halogen atoms. As for alkyl groups, haloalkyl groups can have any suitable number of carbon atoms, such as Ci-6. For example, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In some instances, the term “perfluoro” can be used to define a compound or radical where all the hydrogens are replaced with fluorine. For example, perfluoromethyl refers to 1,1,1 -trifluoromethyl .

[0063] As used herein, the term “aryl,” by itself 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 can include any suitable number of carbon ring atoms, such as Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15 or Ci6, as well as Ce-io, Ce-12, or Ce-14. Aryl groups can be monocyclic, fused to form bicyclic (e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl and benzyl. Aryl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted aryl” groups, such as substituted phenyl or substituted benzyl groups, can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy. [0064] As used herein, the term “amino” refers to a moiety -NR2, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation. “Dialkylamino” refers to an amino moiety wherein each R group is alkyl.

[0065] As used herein, the term “sulfonyl” refers to a moiety -SO2R, wherein the R group is alkyl, haloalkyl, or aryl. An amino moiety can be ionized to form the corresponding ammonium cation. “Alkyl sulfonyl” refers to an amino moiety wherein the R group is alkyl.

[0066] As used herein, the term “hydroxy” refers to the moiety -OH.

[0067] As used herein, the term “cyano” refers to a carbon atom triple-bonded to a nitrogen atom (z.e., the moiety -ON).

[0068] As used herein, the term “carboxy” refers to the moiety -C(O)OH. A carboxy moiety can be ionized to form the corresponding carboxylate anion.

[0069] As used herein, the term “amido” refers to a moiety -NRC(O)R or -C(O)NR2, wherein each R group is H or alkyl.

[0070] As used herein, the term “nitro” refers to the moiety -NO2.

[0071] As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (z.e., O=).

[0072] 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) capable of attachment to the palladium metal ion. Non-limiting examples of bidentate ligands include alkylenediamine (e.g., ethylenediamine), bipyridine, substituted bipyridine, phenanthroline, and ethylenebis(dimethylphosphine). In some embodiments, a bidentate ligand can be replaced with two monodentate ligands, such as trialkylphosphine or triarylphosphine (e.g., P(CH ₃ ) ₃ , PPh ₃ , etc.). The bidentate ligand moiety that is attached to the palladium metal ion of the fluorogenic amide-functionalized palladium coordination complex of the instant invention is represented by “L ¹ — L ² ” or “L ³ — L ⁴ ” The two monodentate ligand moieties that are attached to the palladium metal ion of the fluorogenic amide-functionalized palladium coordination complex of the instant invention are represented by “L ¹ and L ² ” or “L ³ and L ⁴ ” It will be appreciated by those of ordinary skill in the art that in the fluorogenic amide- functionalized palladium coordination complexes of the instant invention, the bidentate ligands, “L ¹ — L ² ” or “L ³ — L ⁴ ” such as the bidentate ligands shown in Formulae I, la, III, and Illa, can be replaced by two monodentate ligands, “L ¹ and L ² ” or “L ³ and L ⁴ ” z.e., that is to say L ¹ is a monodentate ligand, and L ² is a monodentate ligand, both of which can be the same or different (z.e., independently selected), or L ³ is a monodentate ligand, and L ⁴ is a monodentate ligand, both of which can be the same or different (z.e., independently selected).

[0073] As used herein, the term “test sample” refers to a gas sample (e.g., an environmental air sample, a breathing air sample, a gas sample collected from denatured biological samples such as cell or tissue lysate, or an industrial gas sample) or a variety of biological sample types obtained or isolated from a subject that can be used in any of the methods described herein. Biological sample includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from an animal (e.g., mammal) or extracts thereof; 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, excreted by, or secreted by a subject, wherein “a subject” includes any living organism, including singlecelled microorganisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a human subject). The biological sample can be in any form, including a solid material such as a whole organ, tissue, cells, a cell pellet, a cell extract, cell homogenates, or cell fractions; or a biopsy, or a biological fluid. Examples of sources of such samples include muscle, eye, skin, gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen, thymus, pancreas, solid tumors, macrophages, mammary glands, mesothelium, and the like. The biological fluid may be obtained from any site (e.g. whole blood, saliva or a mouth wash containing buccal cells, tears, plasma, serum, urine, bile, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleural fluid, or cells therefrom, aqueous or vitreous humor, or any bodily secretion), a transudate, an exudate (e.g. fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint. The biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any cell, tissue or organ. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Biological samples also include mixtures of biological molecules including proteins, lipids, carbohydrates and nucleic acids generated by partial or complete fractionation of cell or tissue homogenates. Although the sample is preferably taken from a human subject, biological samples may be from any animal, plant, bacteria, virus, yeast, etc. The term animal, as used herein, refers to humans as well as non-human animals, at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms and single cells. Cell cultures and live tissue samples are considered to be pluralities of animals. In certain exemplary embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). An animal may be a transgenic animal or a human clone. If desired, the biological sample may be subjected to preliminary processing, including preliminary separation techniques.

[0074] As used herein, the term “non-coordinating anion” refers to any negatively charged ion which acts as a counterion to the positively charged fluorogenic amide-functionalized palladium coordination complex of Formula la and Formula Illa. The non-coordinating anion balances the charge of the positively charged fluorogenic amide-functionalized palladium coordination complex, and does not formally bond to or share electrons with the metal center of the fluorogenic amide-functionalized palladium coordination complex in a covalent bond. Examples of suitable non-coordinating anions include, but are not limited to, the following: CH ₃ C ₆ H ₄ SO ₃ -,

III. Fluorogenic amide-functionalized palladium coordination complexes

[0075] In certain embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula I: (I), wherein: each R ¹ is independently selected from the group consisting of-OR ^a , -NR ^a R ^b , and Ci-6 alkyl;

R ² is selected from the group consisting of Ci-6 alkyl, which is optionally substituted with one or more R ^2a , and a functional group containing at least two sulfonic acid or sulfonate moieties; each R ^2a is independently selected from the group consisting of Ci-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 ; each R ^a and R ^b are independently selected from the group consisting of H and Ci-6 alkyl; each R ^c is Ci-6 alkyl; the moiety L ¹ — L ² is a bidentate ligand;

X is an anionic ligand; subscript m is 0 or 1; and subscript n is 0, 1, 2, or 3.

[0076] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula la: (la), wherein A“ is a non-coordinating anion.

[0077] In some embodiments, R ² is a functional group containing at least two sulfonic acid or sulfonate moieties. For example, in some cases, R ² is selected from the group consisting of wherein v is 0, 1, 2, or 3.

[0078] In some embodiments, the fluorescent imide formed from the reaction between carbon monoxide in the test sample and the fluorogenic amide-functionalized palladium coordination complex of Formula I or Formula la in the methods described herein is a compound according to Formula II:

[0079] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula III: wherein: each R ^3a and R ^3b is independently selected from the group consisting of-OR ^a , -NR ^a R ^b , and Ci-6 alkyl, or R ^3a and R ^3b are combined to form a C3-C7 cycloamine ring,

R ⁴ is selected from the group consisting of Ci-6 alkyl, which is optionally substituted with one or more R ^4a , and a functional group containing at least two sulfonic acid or sulfonate moieties, each R ^4a is independently selected from the group consisting of Ci-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 ; each R ^a and R ^b are independently selected from the group consisting of H and Ci-6 alkyl; each R ^c is Ci-6 alkyl; the moiety L ³ — L ⁴ is a bidentate ligand;

X is an anionic ligand; subscript p is 0 or 1; and subscripts q and t are independently 0, 1, 2, or 3. [0080] In some embodiments, the fluorogenic amide-functionalized palladium coordination complex used in the methods for detecting carbon monoxide described herein is a compound according to Formula Illa: (Illa), wherein A is a non-coordinating anion.

[0081] 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 is selected from the group consisting of wherein v is 0, 1, 2, or 3.

In some embodiments, the fluorescent imide formed from the reaction between carbon monoxide in the test sample and the fluorogenic amide-functionalized palladium coordination complex of Formula III or Formula Illa in the methods described herein is a compound according to Formula IV: [0082] In the embodiments disclosed herein wherein X is an anionic ligand, the anionic ligand can independently be selected from the group consisting of a halogen ion and -SO3R, wherein R is selected from the group consisting of H, Ci-Cs alkyl, benzyl, and substituted benzyl. In some embodiments, the anionic ligand, z.e., X, is a halogen ion, such as F", Cl", Br" , and I".

[0083] In the embodiments disclosed herein wherein L-L is a bidentate ligand, such as, for example, alkylenediamine, bipyridine, and phenanthroline, the bidentate ligand can be replaced with 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 pyridine, substituted or unsubstituted triphenylphosphine, tri -tertbutyl - phosphine, bipyridine, substituted bipyridine, 1,10-phenanthroline, 2-aminomethylpyridine, 4-aminomethylimidazole, and its analog such as, for example, histidine.

IV. Examples

[0084] In general, all chemical reagents and solvents used to synthesize compounds described herein were obtained from commercial suppliers (Sigma-Aldrich, Oakwood, and Fisher Scientific) and used without further purification, unless indicated otherwise. Certified carbon monoxide calibration gas was obtained from GASCO. Flash column chromatography was performed, when necessary, using silica obtained from Sigma-Aldrich and carried out on a Biotage SP1 system. HPLC analysis was performed on an Agilent 1100 HPLC system (Column: Kromasil C18 5pm, 4.6 x 150 mm. Mobile phase A: 0.1% trifluoroacetic acid (TFA) in H2O. Mobile phase B: 0.1% TFA in acetonitrile (ACN). Flow rate: 1 mL/min.

Gradient: 5% to 95% B from 0 to 10 min; 95% B from 10 to 12 min; 95% to 5% B from 12 to 12.1 min; 5% B from 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 Agilent 1200 HPLC serving as the LC module (Column: Waters SunFire C18 3.5pm, 3 x 150 mm. Mobile phase A: 0.1% formic acid (FA) in H2O. Mobile phase B: 0.1% FA in ACN. Flow rate: 0.5 ml/min. Gradient: 5% to 95% B from 0 to 10 min; 95% B from 10 to 12 min; 95% to 5% B from 12 to 12.1 min; 5% B from 12.1 to 15 min. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for 'H, and 101 or 151 MHz for ¹³ C on a Bruker AV-400MHz Ultra Shield NMR instrument. Chemical shifts (5 values) and coupling constants (J values) are given in ppm and hertz, respectively, using the respective solvent ( ^X H NMR, ¹³ C NMR) as the internal reference. Fluorescence spectra were recorded on a Shimadzu RF-5301PC Spectrofluorophotometer.

[0085] 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 cell culture experiments was custom-made (250 ppm CO, 5% CO2 with balanced air) by Nexair LLC. Cell incubator: VWR symphony. For the determination of cell viability in cytotoxicity assays, cell counting kit-8 (CCK-8) was purchased from Dojindo and used according to the manufacture’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 450 nm. For fluorescence readings, fluorescence intensity (counts per second, CPS) was measured at 405 nm (excitation) and 535 nm (emission), with a 10 nm bandwidth, normal aperture, and signal collection time of 2 sec per well. Cell imaging was conducted on the Olympus 1X73 inverted fluorescence microscope. Carboxyhemoglobin (COHb) was tested with AVoximeter 4000 according to the manufacture’s manual.

[0086] All data were presented as the mean ± standard deviation (n > 3). Statistical analysis was performed by Student’s t-test for comparison between two groups using GraphPad Prism 9. A p- value of less than 0.05 was considered to be statistically significant.

Example 1. Synthesis of CO probe molecules (Compounds 5, 5a, 6, and 6a).

[0087] Fluorogenic amide-functionalized palladium coordination complexes 5, 5a, 6, and 6a were synthesized as set forth in Scheme 1 below.

Scheme 1

Reagents and conditions: a) HC1, NaNCh, KI, 0 °C then 90 °C, 5 hrs; b) i. oxalyl chloride, DMF (cat.), DCM, 0 °C, 5 minutes then 40 °C, 1 hr; ii. w-butylamine, TEA, 0 °C then rt, 2 hrs; c) BBr ₃ , DCM, -78 °C, 1 hr; d) Pd(0)(dba) ₂ , TMEDA or 2,2’ -bipyridine, DCM, rt, 1.5-2 hrs; e) AgOTf, acetone, rt, 30 minutes.

[0088] 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 HC1 (3 M, 4 mL) and acetone (1 mL), then cooled to 0 °C. To this solution, a mixture 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 before adding KI (30 mg, 2.0 mmol) in 1 mL water with a pipette. The resulting brownish purple mixture was then heated to 90 °C for 5 hrs. The reaction was monitored using thin layer chromatography (TLC, hexane:ethyl acetate = 3: 1, v:v) to completion. After cooling to room temperature, the reaction was quenched with a saturated NH4CI solution (20 mL), extracted with dichloromethane (DCM, 50 mL), and successively washed with brine (3 x 50 mL). The mixture was then dried over Na2SO4 and concentrated under vacuum to give a brownish residue. The residue was purified by flash column chromatography to yield compound 2 as a white solid (376 mg, 75% yield). ^T H NMR (400 MHz, CDCL) 5 9.40 (s-br, 1H), 7.44 (d, J= 8.0 Hz, 1H), 7.08 (t, J= 8.0 Hz, 1H), 6.94 (d, J= 8.0 Hz, 1H), 3.87 (s, 3H). °C NMR (101 MHz, CDCL) 5 172.32, 156.76, 131.94, 131.22, 129.10, 110.89, 92.18, 77.36, 77.04, 76.72, 56.25. The resulting NMR spectra for compound 2 were comparable to those reported in the literature. See, Whyte, A., et al. OrgLett. 2018, 20, 345-348. [0089] Preparation of 7V-butyl-2-iodo-6-methoxybenzamide (3). Under the protection of argon, compound 2 (376 mg, 1.352 mmol) was dissolved in 5 mL of dry DCM and cooled to 0 °C. A catalytic amount (two drops) of anhydrous dimethylformamide (DMF) was then added to the mixture, followed by the addition of oxalyl chloride (0.18 mL, 2.03 mmol). The reaction was heated to 40 °C for 1 hr, and the light-yellow reaction mixture was then concentrated to dryness under vacuum. After dissolving the resulting residue in 2 mL of dry DCM, a mixture of w-butylamine (0.16 mL, 1.62 mmol) and triethylamine (0.223 mL, 1.62 mmol) in 1 mL of DCM was slowly added via syringe at 0 °C and allowed to stir at room temperature for 2 hrs. The reaction progress was monitored by TLC (DCM:methanol = 20: 1, v:v). Upon completion, the reaction was quenched with brine (10 mL), extracted with DCM (50 mL), successively washed with saturated NaHCCh solution (3 x 20 mL) and brine (3 x 20 mL), and dried over Na2SO4. The mixture was then concentrated under vacuum to give compound 3 as a white solid (406 mg, 90% yield) without further purification. ³ H NMR (400 MHz, CDCh) 8 7.38 (dd, J= 8.0, 0.8 Hz, 1H), 7.00 (t, J= 8.1 Hz, 1H), 6.86 (d, J= 8.3 Hz, 1H), 5.66 (s, 1H), 3.79 (s, 3H), 3.45 (td, J= 7.1, 5.8 Hz, 2H), 1.65 - 1.54 (m, 2H), 1.50 - 1.36 (m, 2H), 0.95 (t, J= 13 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 6 167.74, 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 . Calculated for C12H17O2NI [M+H] ⁺ 334.0298, found 334.0292.

[0090] Preparation of \ butyl-2-hydroxy-6-iodobenzaniide (4). A dry 20 mL reaction vial containing a stir bar and a solution of compound 3 (0.377 mg, 1.131 mmol) in 1 mL of dry DCM was cooled to -78 °C using an acetone/dry ice bath. Under the protection of argon, BBn (0.43 mL, 4.526 mmol) was slowly added via syringe to the reaction vial while stirring. The reaction was allowed to stir for 30 minutes at -78 °C, and continued stirring at room temperature overnight. The reaction mixture was then cooled to -78 °C again before quenching with methanol (MeOH, 2 mL). The mixture was then diluted with 50 mL of DCM and washed with brine (3 x 50 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (DCM:MeOH = 50: 1) to yield compound 4 as a white solid (342 mg, 90% yield). 'H NMR (400 MHz, CDCh) 6 10.91 (s, 1H), 7.42 (dd, J= 5.5, 3.5 Hz, 1H), 6.99 - 6.87 (m, 2H), 6.84 - 6.63 (m, 1H), 3.50 (q, J= 6.8 Hz, 2H), 1.65 (p, J= 13 Hz, 2H), 1.46 (h, J= 1A Hz, 2H), 0.97 (t, J= 13 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 6 168.68, 160.53, 133.46, 132.49, 121.65, 118.52, 91.70, 39.98, 31.15, 20.47, 13.85. HRMS (ESI, m/z): Calculated for C11H15O2NI [M+H] ⁺ 320.0142, found 320.0132. [0091] Preparation of (2-(butylcarbamoyl)-3-hydroxyphenyl)( \ L\ U .\ ² - tetramethylethylenediamine) palladium(II) iodide (5). Under protection of argon in a dry 20 mL reaction vial with a sealable septum, 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. Deoxygenated N ^l , N ^l , N ² ,7V ² -tetramethylethylenediamine (TMEDA, 57 pL, 0.375 mmol) was then injected into the sealed reaction vial through the septum with a syringe and the reaction was stirred at room temperature. After 30 minutes of stirring, it was observed that the reaction mixture changed from red to yellow-black in color. The reaction was allowed to stir for an additional 60 minutes until completion, as indicated by TLC. The reaction mixture was filtered through a pad of dry Mg2SO4 and the filtrate was concentrated to dryness, which was triturated with hexane and dry diethyl ether (Et2O) to form an orange solid precipitate that was isolated as compound 5 (151 mg, 89% yield) without further purification. 'H NMR (400 MHz, CDCh) 5 12.29 (s, 1H), 9.39 (s, 1H), 7.12 (d, J= 7.6 Hz, 1H), 6.86 (t, J= 7.8 Hz, 1H), 6.44 (d, J= 8.0 Hz, 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 = 7.4 Hz, 2H), 1.52 (sx, J= 7.3 Hz, 2H), 0.97 (t, J= 7.4 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 5 171.48, 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) Calculated for CnftoNsCbPd [M-I] ⁺ 414.1380, found 414.1396.

[0092] Preparation of (2-(butylcarbamoyl)-3-hydroxyphenyl)(7V ¹ ^V ¹ ^V ² ^V ² - tetramethylethylenediamine) palladacycle trifluoromethanesulfonate salt (5a). In a dry 20 mL reaction vial with a sealable septum and stir bar, compound 5 (120 mg, 0.221 mmol) was dissolved in dry acetone (8 mL). To this solution, silver trifluoromethanesulfonate (AgOTf, 57 mg, 0.222 mmol) was added as a solid, and a precipitate was formed immediately. The resulting mixture was stirred for 30 minutes and filtered through celite. The yellow filtrate was then concentrated, passed through a short silica column (DCM:MeOH = 20: 1), and the main fraction was concentrated to dryness under vacuum to afford compound 5a as a light-yellow solid (108 mg, 87% yield). 'H NMR (400 MHz, CDCh) 8 9.91 (s, 1H), 8.86 (t, J= 5.7 Hz, 1H), 7.08 (t, J= 7.9 Hz, 1H), 6.99 (d, J= 8.2 Hz, 1H), 6.50 (d, J= 7.5 Hz, 1H), 3.37 (q, 6.8 Hz, 2H), 2.99 - 2.83 (m, 8H), 2.83 - 2.64 (m, 8H), 1.57 (quint, 7.2 Hz, 2H), 1.37 (sx, 7.3 Hz, 2H), 0.93 (t, J= 13 Hz, 3H). °C NMR (101 MHz, CDCh) 6 178.89, 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): Calculated for CpftoOzNjPd [M] ⁺ 414.1367, found 414.1367.

[0093] Preparation of (2-(butylcarbamoyl)-3-hydroxyphenyl)(bipyridine) palladium(II) iodide (6) and (2-(butylcarbamoyl)-3-hydroxyphenyl)(bipyridine) palladacycle trifluoromethanesulfonate salt (6a). Under protection of argon in a dry 20 mL reaction vial with a sealable septum, compound 4 (133 mg, 0.417 mmol), Pd(dba)2 (262 mg, 0.458 mmol), and 2,2’-bipyridine (BIPY, 65 mg, 0.417 mmol) were dissolved in 10 mL of DCM. The reaction mixture was stirred at room temperature, changing from a red color to dark green-yellow within 30 minutes of stirring. The reaction was allowed to stir for an additional 30 minutes until completion, as indicated by TLC. The reaction mixture was diluted with DCM (10 mL) and filtered through a pad of dry Mg2SO4 on celite. The filtrate was concentrated to dryness and purified by flash column chromatography to afford compound 6 as a yellow solid intermediate (146 mg, 60% yield), which was then dissolved in dry acetone (12 mL). To this solution, AgOTf (65 mg, 0.251 mmol) was added as a solid, and a precipitate immediately formed. The resulting mixture was stirred for 30 minutes and filtered through celite. The yellow filtrate was concentrated, passed through a short silica column (DCM:MeOH = 20: 1), and the main fraction was collected and concentrated to dryness under vacuum to obtain compound 6a as a bright yellow solid (117 mg, 78% yield). 'H NMR (400 MHz, CD3OD-CDCI3 mixture) 5 8.89 - 8.48 (m, 2H), 8.36 (d, J= 7.9 Hz, 2H), 8.21 (t, J= 7.6 Hz, 2H), 7.71 - 7.64 (m, 2H), 7.09 (t, J= 7.9 Hz, 1H), 6.71 (br-s, 1H), 6.62 (d, J= 8.1 Hz, 1H), 3.47 (t, J= 6.9 Hz, 2H), 1.71 - 1.58 (m, 2H), 1.46 (dd, J= 13 Hz, 2H), 0.97 (br-s, 3H). ¹³ C NMR (151 MHz, CD3OD-CDCI3 mixture) 5 156.33, 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 . Calculated for C2iH ₂ 2O2N ₃ Pd [M+H] ⁺ 454.0741, found 454.0759.

Example 2. Synthesis of CO probe molecules (Compounds 11, Ila and 12-14).

[0094] Fluorogenic amide-functionalized palladium coordination complexes 11, Ila and 12-14 were synthesized as set forth in Scheme 2 below. Scheme 2

Reagents and conditions: a) HC1, NaNCh, KI, 0 °C then 90 °C, 3-5 hr; b) Na2S2C>4, H2O/THF, 50 °C, 3 hrs; c) i. SOCI2, toluene, reflux, 3 hrs; ii. w-butylamine or ^-propylamine, TEA, DCM, 0 °C, 2 hr; d) Pd(0)(dba) ₂ , TMEDA or 2,2’ -bipyridine, DCM, rt, 1.5-2 hrs; e) AgOTf, THF, rt, 30 min.

[0095] 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 HC1 (37%, 5 mL) and acetone (5 mL), then cooled to 0 °C. To this solution, 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 before adding KI (462 mg, 2.8 mmol) in 1 mL water with a pipette. The resulting brownish purple mixture was then heated to 90 °C for 3 hrs. The reaction was monitored with TLC (dichloromethane:methanol = 10: 1, v:v) to completion. After cooling to room temperature, the reaction was quenched with saturated NH4CI solution (30 mL), extracted with DCM (50 mL), then washed with brine (3 x 50 mL). The mixture was then dried over Na2SO4 and concentrated under vacuum to give a brownish residue. The residue was purified by flash column chromatography (DCM:MeOH = 2-10%, with 0.5% acetic acid) to yield compound 8 as an off-white solid (473 mg, 57% yield). ^T H NMR (400 MHz, DMSO-d6) 5 8.26 (d, J= 7.8 Hz, 1H), 8.15 (d, J= 8.2 Hz, 1H), 7.40 (t, J= 8.1 Hz, 1H). It was noted that the resulting ¹ H NMR spectrum for compound 8 was comparable to that reported in the literature. See, Fu, Z., et al. Org. Lett. 2019, 27(9), 3003-3007. [0096] 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 a solution of sodium dithionite (Na2S2C>4, 2.697 g, 15.5 mmol) in 15 mL of deionized (DI) water was added while stirring. The mixture was stirred vigorously at 50 °C for 3 hrs until completion, as indicated by TLC. The reaction mixture was diluted with HC1 (IM, 30 mL), then extracted with ethyl acetate (EtOAc, 80 mL). The organic layer was washed with brine (3 x 80 mL), dried over TsfeSCU, and then concentrated in vacuo to obtain an off-white residue. The residue was purified by flash column chromatography to yield compound 9 as an off-white solid (186 mg, 46% yield). 'H NMR (400 MHz, DMSO-d6): 5 7.03 (d, J= 7.5 Hz, 1H), 6.78 (t, J= 7.9 Hz, 1H), 6.70 (d, J= 8.2 Hz, 1H); ¹³ C NMR (101 MHz, CD ₃ OD) 5 171.86, 147.18, 132.10, 129.52, 128.81, 116.55, 94.34, 40.62, 32.14, 21.41, 14.11. HRMS (ESI, m/zy. Calculated for C7H7N2OI [M+H] ⁺ 263.9516, found 263.9510.

[0097] Preparation of 2-amino-7V-butyl-6-iodobenzamide (10a). To a solution of compound 9 (97 mg, 0.36 mmol) in toluene (10 mL) was added thionyl chloride (SOCI2, 130 pL, 1.8 mmol) at room temperature and the mixture was heated at 110 °C for 3 hrs.

Thereafter, the solvent was removed under vacuum to obtain the crude 2-amino-6- iodobenzoyl chloride as a yellow oil, which was used in the next reaction immediately without purification or characterization. Triethylamine (EtsN, 50 pL, 0.36 mmol) was added to a solution of w-butylamine (35 pL, 0.36 mmol) in DCM (10 mL) at 0 °C and stirred for 10 minutes before adding crude 2-amino-6-iodobenzoyl chloride in DCM (2 mL) drop-wise at 0 °C. The reaction was allowed to stir for 2 hrs until completion, as confirmed by TLC. The solvent was then removed under reduced pressure and the residue was purified by flash column chromatography (hexanes:EtOAc = 1 : 1, v/v) to afford compound 10a as a white solid (85 mg, 75% yield). 'H NMR (400 MHz, CD3OD) 5 7.12 (d, J= 7.7 Hz, 1H), 6.80 (t, J= 7.9 Hz, 1H), 6.73 (d, J= 8.2 Hz, 1H), 3.35 (t, J= 7.1 Hz, 2H), 1.67 - 1.60 (m, 2H), 1.52 - 1.43 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101 MHz, CD3OD) 5 171.86, 147.18, 132.10, 129.52, 128.81, 116.55, 94.34, 40.62, 32.14, 21.41, 14.11. HRMS (ESI, m/z): Calculated for C11H16N2OI ([M+H] ⁺ ) 319.0302, found 319.0295.

[0098] Preparation of 2-amino-7V-propyl-6-iodobenzamide (10b). Compound 10b (95 mg, 87% yield) was prepared according to a similar procedure used to make compound 10a, where ^-propylamine (30 pL, 0.36 mmol) was used instead of w-butylamine. 'H NMR (400 MHz, CDCh) 5 7.15 (d, J= 7.8 Hz, 1H), 6.77 (t, J= 7.9 Hz, 1H), 6.61 (d, J= 8.2 Hz, 1H), 5.96 (s, 1H), 3.38 (q, 7.2 Hz, 2H), 1.72 - 1.58 (m, 2H), 0.99 (t, J= 7.5 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 5 169.02, 145.84, 131.47, 128.99, 127.64, 115.97, 93.37, 41.76, 22.59, 11.77. HRMS (ESI, m/z): Calculated for C10H14N2OI [M+H] ⁺ 305.0151, found 305.0148 .

[0099] Preparation of (2-(butylc:irb:imoyl)-3-aminophenyl)( \ ^l ,\ ^l ,\ ² ,\ ² - tetramethylethylenediamine) palladium(II) iodide (11). Under protection of argon in a dry 20 mL reaction vial with a sealable septum, compound 10a (60 mg, 0.188 mmol) and Pd(dba)2 (107 mg, 0.188 mmol) was dissolved in 3 mL of DCM. Deoxygenated TMEDA (34 pL, 0.225 mmol) was then injected into the sealed reaction vial through the septum with a syringe and the reaction was stirred at room temperature. After 30 minutes of stirring, the reaction mixture changed from red to yellow-black in color. The reaction was allowed to stir for an additional 60 minutes until completion, as indicated by TLC. The reaction mixture was filtered through a pad of dry Mg2SC>4 and the filtrate was concentrated to dryness, which was triturated with hexane and dry Et2O to obtain a yellow solid that was isolated as compound 11 (151 mg, 89% yield) without further purification. 'H NMR (400 MHz, CDCh) 5 8.17 (s, 1H), 6.88 (d, J= 7.3 Hz, 1H), 6.69 (t, J= 7.7 Hz, 1H), 6.18 (d, J= 7.5 Hz, 1H), 3.72 - 3.31 (m, 2H), 2.81 - 2.47 (m, 10H), 2.36 (s, 3H), 2.21 (s, 3H), 1.84 - 1.66 (m, 2H), 1.49 (q, J= 7.5 Hz, 2H), 0.95 (t, J= 7.4 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 8 170.50, 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): Calculated for Ci ₇ H ₃₂ N ₄ OIPd [M+H] ⁺ 541.0656, found 541.0640.

[0100] Preparation of (2-(butylcarbamoyl)-3-aminophenyl)(A4^V ¹ ^V ² ^V ² - tetramethylethylenediamine) palladacycle trifluoromethanesulfonate salt (Ila).

Compound 11 (151 mg, 0.279 mmol) was dissolved in degassed THF (10 mL) and cooled in an ice/water bath before adding AgOTf (75 mg, 0.293 mmol) as a solid under the protection of argon. Precipitate formed immediately and the reaction was stirred for 30 minutes. The reaction mixture was then filtered through celite and the filtrate was concentrated under vacuum. Purification by flash column chromatography (DCM:MeOH = 10: 1) gave compound Ila as a light yellow solid (112 mg, 72% yield). 'H NMR (400 MHz, CDCh) 6 9.32 (s, 1H), 7.06 (t, J= 7.7 Hz, 1H), 6.70 (d, J= 7.8 Hz, 1H), 6.65 (d, J= 7.6 Hz, 1H), 3.99 (s, 2H), 3.43-3.36 (m, 2H), 2.93 (s, 6H), 2.74 (d, J= 5.2 Hz, 2H), 2.70 - 2.25 (m, 8H), 1.63- 1.56 (m, 3H), 1.37 (q, J= 7.3 Hz, 2H), 0.93 (t, J= 7.2 Hz, 3H). 13C NMR (101 MHz, CDC13) 5 179.43, 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): Calculated for Ci ₇ H ₃ iN ₄ OPd [M+H] ⁺ 413.1533, found 413.1551. [0101] Preparation of (2-(propylcarbamoyl)-3-aminophenyl)(A ¹ ^V ¹ ^V ² ^V ² - tetramethylethylenediamine) palladium(II) iodide (12). Under protection of argon in a dry 20 mL reaction vial with a sealable septum, compound 10b (50 mg, 0.164 mmol) and Pd(dba)2 (98 mg, 0.17 mmol) were dissolved in 3 mL of DCM. Deoxygenated TMEDA (30 pL, 0.200 mmol) was then injected into the sealed reaction vial through the septum with a syringe and the reaction was stirred at room temperature. After 30 minutes of stirring, the color of the reaction mixture turned 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 filtered through a pad of dry Mg2SO4 and the filtrate was concentrated to dryness. The dried filtrate was then triturated with hexane and dry Et2O to obtain a yellow solid that was further purified by flash column chromatography (DCM:MeOH = 20: 1) to yield compound

12 (73 mg, 85% yield). ^X H NMR (400 MHz, CD ₃ OD) 5 7.10 (t, J= 7.8 Hz, 1H), 6.76-6.73 (two 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 (CD3OD) 5 181.0, 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.

[0102] Preparation of (2-(butylcarbamoyl)-3-aminophenyl)(bipyridine) palladium(II) iodide (13). Compound 10a (41 mg, 0.13 mmol), Pd(dba)2 (82 mg, 0.142 mmol), and 2,2’- bipyridine (BIPY, 22 mg, 0.130 mmol) were weighed together in a dry 20-mL vial with a sealable septum, which was flushed with argon. Dry and degassed DCM (2 mL) was added via syringe to the reaction vial to fully dissolve the reactants. The reaction mixture was stirred at room temperature, changing from a dark red color to yellow-brown within 30 minutes of stirring. The reaction was allowed to stir for 1 hr until the total consumption of compound 10a, as indicated by TLC. The reaction mixture was then purified by flash column chromatography (DCM/MeOH with a 0%-5% MeOH gradient) to obtain compound

13 as a yellow solid (60 mg, 73% yield). 'H NMR (400 MHz, CDCI3) 8 9.62 (d, J= 3.5 Hz, 1H), 8.07 - 8.01 (m, 3H), 8.00 - 7.91 (m, 2H), 7.60 (d, J= 5.5 Hz, 1H), 7.54 (t, J= 6.4 Hz, 1H), 7.39 (t, J= 5.7 Hz, 1H), 7.01 (d, J= 7.6 Hz, 1H), 6.77 (t, J= 7.7 Hz, 1H), 6.32 (d, J= 7.8 Hz, 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= 7.3 Hz, 3H). °C NMR (101 MHz, CDCh) 5 170.03, 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 . Calculated for C2iH ₂₅ N ₄ OIPd [M+H] ⁺ 581.0030, found 581.0032. [0103] Preparation of (2-(propylcarbamoyl)-3-aminophenyl)(bipyridine) palladium(II) iodide (14). Compound 10b (69 mg, 0.206 mmol), Pd(dba)2 (118 mg, 0.206 mmol), and BIPY (32 mg, 0.206 mmol) were weighed together in a dry 20-mL vial with a sealable septum, which was flushed with argon. Dry and degassed DCM (2 mL) was added via syringe to the reaction vial to fully dissolve the reactants. The reaction mixture was stirred at room temperature, changing from a dark red color to yellow-brown within 30 minutes of stirring. The reaction was allowed to stir for 1 hr until the total consumption of compound 10b, as indicated by TLC. The reaction mixture was then purified by flash column chromatography (DCM/MeOH with a 0%-5% MeOH gradient) to obtain compound 14 as a yellow solid (68 mg, 58% yield). 'H NMR (400 MHz, CDCh) 5 9.62 (d, J= 5.1 Hz, 1H), 8.06 - 8.02 (m, 3H), 7.99 - 7.95 (m, 2H), 7.61 (dd, J= 5.5, 2.1 Hz, 1H), 7.56 - 7.55 (m, 1H), 7.43 - 7.40 (m, 1H), 7.02 (d, J= 7.6 Hz, 1H), 6.79 (t, J= 7.7 Hz, 1H), 6.35 (d, J= 7.9 Hz, 1H), 3.40 (p, J= 6.7 Hz, 1H), 3.25 - 3.10 (m, 1H), 1.53 - 1.37 (m, 2H), 0.74 (t, J= 7.4 Hz, 3H). °C NMR (101 MHZ, CDCh) 5 170.21, 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 . Calculated for C2o H ₂₂ N ₄ OIPd [M+H] ⁺ 566.9868 , found 566.9886

Example 3. Synthesis of protodepalladation products (Compounds 17 and 19).

[0104] The protodepalladation benzamide species 17 (Scheme 3) and 19 (Scheme 4) were synthesized as set forth below.

Scheme 3

O O OH O ci/ 15™ ■ (V 16'®'" » 6^ 17 ”

Reagents and conditions: a) i. oxalyl chloride, DMF (cat.), DCM, 0 °C, 5 minutes then 40 °C, 1 hr; ii. w-butylamine, TEA, 0 °C then rt, overnight; b) BBn, DCM, -78 °C, 1 hr.

[0105] Preparation of V-butyl-2-methoxybenzamide (16). Under the protection of argon in a 20 mL reaction vial, 2-methoxybenzoic acid (15, 200 mg, 1.314 mmol) was dissolved in 10 mL of dry DCM and a catalytic amount (two drops) of anhydrous DMF was added. The mixture was cooled to 0 °C and oxalyl chloride (167 pL, 1.971 mmol) was added via syringe to the reaction vial. The reaction was allowed to stir at 0 °C for 5 minutes, heated to 40 °C and stirred for 1 hr, then the reaction mixture was concentrated to dryness under vacuum. After dissolving the resulting residue in 5 mL of dry DCM, a mixture of w-butylamine (156 pL, 1.577 mmol) and triethylamine (271.5 pL, 1.971 mmol) was added via syringe at 0 °C and allowed to stir at room temperature overnight. The reaction mixture was diluted with DCM (30 mL), washed with saturated NaHCCh solution (3 x 50 mL) and brine (3 x 50 mL), and dried over ISfeSCU. The mixture was then concentrated under vacuum to give compound 16 as a white solid (223 mg, 83% yield). 'H NMR (400 MHz, CDCh) 5 8.20 (dd, J= 7.8, 1.8 Hz, 1H), 7.85 (s, 1H), 7.47 - 7.37 (m, 1H), 7.07 (t, J= 7.5 Hz, 1H), 6.96 (d, J= 8.3 Hz, 1H), 3.46 (q, J= 7.0 Hz, 2H), 1.60 (p, J= 13 Hz, 2H), 1.41 (sx, J= 13 Hz, 2H), 0.96 (t, J= 13 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 5 165.03, 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: 230.1141 (CuHnNChNa, [M+Na] ⁺ ); Found: 230.1148.

[0106] Preparation of V-butyl-2-hydroxybenzaniide (17). A dry 20 mL reaction vial containing a stir bar and a solution of compound 16 (218 mg, 1.063 mmol) in 8 mL of dry DCM was cooled to -78 °C using an acetone/dry ice bath. Under the protection of argon, BBn (420 pL, 4.25 mmol) was slowly added via syringe to the reaction vial while stirring. The reaction was allowed to stir for 30 minutes at -78 °C, and continued stirring at room temperature overnight. The reaction mixture was then cooled to -78 °C again before quenching with MeOH (5 mL). The mixture was then diluted with 50 mL of DCM and washed with brine (3 x 50 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (DCM:MeOH = 50: 1) to yield compound 17 as a light-yellow oil that becomes a waxy solid at -15 °C (186 mg, 92% yield). ^X H NMR (400 MHz, CDCh) 8 12.49 (s, 1H), 7.42 (d, J= 8.0 Hz, 1H), 7.35 (t, J= 8.6 Hz, 1H), 6.94 (d, J= 9.3 Hz, 1H), 6.80 (t, J= 8.2 Hz, 1H), 6.73 (s, 1H), 3.41 (q, J= 7.2 Hz, 2H), 1.57 (p, J= 13 Hz, 2H), 1.36 (sx, J= 13 Hz, 2H), 0.91 (t, J= 13 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 6 170.05, 161.29, 134.09, 125.64, 118.78, 118.39, 114.51, 39.52, 31.50, 20.14, 13.75. HRMS (ESI): Calculated: 216.1000 (CiiHi ₅ NO ₂ Na, [M+Na] ⁺ ); Found: 216.0992. Scheme 4

Reagents and conditions: a) ^-propylamine, H2O, rt, 2 hrs.

[0107] Preparation of A-propyl-2-aminiobenzamide (19). In a 50 mL round bottom flask, isatoic anhydride (18, 200 mg, 1.227 mmol) was suspended in 10 mL of DI water while stirring and ^-propylamine (108.6 mg, 1.84 mmol) was slowly added, causing the suspension to become clear and a white precipitate to form. The reaction was allowed to stir for another 2 hrs before diluting with DI water and filtering. The collected solid was successively washed with water and dried under vacuum to afford compound 19 as white solid (216 mg, 99% yield) without further purification. 'H NMR (400 MHz, CDCL) 8 7.30 (d, J= 7.9 Hz, 1H), 7.20 (t, J = 8.4 Hz, 1H), 6.68 (d, J= 8.2 Hz, 1H), 6.65 (t, J= 7.5 Hz, 1H), 6.10 (s, 1H), 3.37 (q, J= 7.2 Hz, 2H), 1.63 (sx, J= 7.3 Hz, 2H), 0.98 (t, 7.4 Hz, 3H); °C NMR (101

MHz, CDCL) 5 169.45, 148.53, 132.26, 127.13, 117.48, 116.83, 116.68, 41.50, 23.05, 11.60. HRMS (ESI): Calculated for CIOHI ₅ N ₂ 0 [M+H] ⁺ : 179.1184, Found: 179.1187.

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

[0108] The fluorescent imide species 22-23 (Scheme 5) and 24-25 (Scheme 6) were synthesized as set forth below.

Scheme 5

Reagents and conditions: a) AcOH, reflux, 3 hrs.

[0109] Preparation of 3-hydroxy-A-butylphthalimide (22). To a solution of 3- hydroxyphthalic anhydride (20a, 250 mg, 1.52 mmol) in acetic acid (AcOH, 3 mL) was added //-butylamine (21a, 133 mg, 1.82 mmol) dropwise. The resulting mixture was refluxed for 3 hrs, cooled to room temperature, and then poured into 10 mL of purified water. The formed precipitate was filtered and washed with DI water to yield pure compound 22 as a white solid (235 mg, 70% yield). 'H NMR (400 MHz, DMSO) 5 7.60 - 7.49 (m, 1H), 7.22 (d, J= 7.1 Hz, 1H), 7.13 (d, J= 8.4 Hz, 1H), 3.46 (t, J= 7.0 Hz, 2H), 1.52 - 1.39 (m, 2H), 1.24 - 1.10 (m, 2H), 0.81 (t, J= 7.4 Hz, 3H). °C NMR (101 MHz, CDCh) 5 170.74, 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): Calculated for C12H14O3N ([M+H] ⁺ ): 220.0968; Found: 220.0970.

[0110] Preparation of 3-hydroxy-A-propylphthalimide (23). To a solution of 3- hydroxyphthalic anhydride (20a, 150 mg, 0.91 mmol) in AcOH (2 mL) was added n- propylamine (21b, 69 mg, 1.18 mmol) dropwise. The resulting mixture was refluxed for 3 hrs, cooled to room temperature, and then poured into 10 mL of DI water. The precipitate was filtered and washed with purified water to obtain pure compound 23 as a white solid (148 mg, 79% yield). ^X H NMR (400 MHz, DMSO-d6) 5 11.13 (s, 1H), 7.59 (dd, J= 8.3, 7.2 Hz, 1H), 7.25 (d, J= 1A Hz, 1H), 7.17 (d, J= 8.3 Hz, 1H), 3.45 (t, J= 1A Hz, 2H), 1.54 (h, J= 7.4 Hz, 2H), 0.82 (t, J= 7.4 Hz, 3H). ¹³ C NMR (101 MHz, DMSO) 5 167.87, 166.81, 155.13, 135.99, 133.58, 123.24, 114.66, 114.01, 38.73, 21.41, 11.26. HRMS (ESI, m/z): Calculated for C11H12O3N ([M+H] ⁺ ):206.0817; Found: 206.0813.

Scheme 6

Reagents and conditions: a) AcOH, reflux, 3 hrs; b) Na2S2C>4, THF/H2O, rt, 4 hrs.

[0111] Preparation of 3-nitro-A-butylphthalimide (24.1) and 3-amino-A- butylphthalimide (24). To a solution of 3 -nitrophthalic anhydride (20b, 250 mg, 1.29 mmol) in AcOH (3 mL) was added w-butylamine (21a, 122 mg, 1.68 mmol) dropwise. The resulting mixture was refluxed for 3 hrs, cooled to room temperature, and then poured into 10 mL of DI water. The precipitate was filtered and washed with purified water to yield intermediate compound 24.1 as a white solid, which was directly used in the next synthetic step. Sodium dithionite (224 mg, 1.29 mmol) was then added to a solution of intermediate compound 24.1 in a mixture of THF (2 mL) and H2O (2 mL). The reaction was allowed to stir at room temperature for 4 hrs, and then the reaction mixture was diluted with water (40 mL) and extracted with EtOAc (40 mL). The organic layers were combined, dried over anhydrous Na ₂ SO4, and concentrated under reduced pressure to afford crude product, which was then purified by flash column chromatography to obtain pure compound 24 (187 mg, 67% yield). ^X H NMR (400 MHz, CDCh) 5 7.38 (dd, J= 8.1, 7.3 Hz, 1H), 7.12 (d, J= 7.1 Hz, 1H), 6.83 (d, J= 8.3 Hz, 1H), 5.20 (s, 2H), 3.61 (t, J= 7.2 Hz, 2H), 1.67 - 1.55 (m, 2H), 1.40 - 1.28 (m, 2H), 0.93 (t, J= 7.4 Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 5 170.49, 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): Calculated for Ci2Hi ₅ O ₂ N2 ([M+H] ⁺ ): 219.1128; Found: 219.1130.

[0112] Preparation of 3-nitro- \-propylphthaliniide (25.1) and 3-aniino- \- propylphthalimide (25). To a solution of 3 -nitrophthalic anhydride (20b, 250 mg, 1.29 mmol) in AcOH (3 mL) was added ^-propylamine (21b, 98 mg, 1.68 mmol) dropwise. The resulting mixture was refluxed for 3 hrs, cooled to room temperature, and then poured into 10 mL of purified water. The precipitate was filtered and washed with purified water to yield intermediate compound 25.1 as a white solid, which was directly used in the next synthetic step. Sodium dithionite (224 mg, 1.29 mmol) was then added to a solution of intermediate compound 25.1 in a mixture of THF (2 mL) and H ₂ O (2 mL). The reaction mixture was allowed to stir at room temperature for 4 hrs, and then quenched by water (40 mL) and extracted with EtOAc (40 mL). The organic layers were combined, dried over anhydrous Na ₂ SO4, and concentrated under reduced pressure to afford crude product, which was then purified by flash column chromatography to obtain pure compound 25 (181 mg, 69% yield). 'H NMR (400 MHz, DMSO) 5 7.44 - 7.40 (m, 1H), 6.97 - 6.94 (m, 2H), 6.43 (s, 2H), 3.45 (t, J= 7.2 Hz, 2H; collide with H ₂ O peak), 1.56 (p, J= 7.6 Hz, 2H), 0.84 (t, J= 7.4 Hz, 3H). °C NMR (101 MHZ, DMSO) 5 170.01, 168.63, 146.85, 135.56, 132.77, 121.83, 111.13, 109.36, 38.94, 21.89, 11.67. HRMS (ESI, m/z): Calculated for C11H13O2N2 ([M+H] ⁺ ): 205.0972; Found: 205.0973. Example 5. Synthesis of CO probe molecules (Compounds 31 and 31a).

[0113] Fluorogenic amide-functionalized palladium coordination complexes 31 and 31a were synthesized as set forth in Scheme 7 below.

Scheme 7

Reagents and conditions: a) i. SOCh, 60°C, 2 hrs; ii. MeOH, DCM, 0°C, 1 hr; b) i. CuBr, EtAOc, CHsONa/MeOH, reflux, 3 hrs; ii. NaOH, Me0H/H20, reflux, 2 hrs; c) i. SOCh, toluene, reflux, 2 hrs; ii. w-butylamine, TEA, DCM, 0°C, Ihr; d) NBS, H2SO4, AcOH/TFA, 0°C, 30 minutes; e) Pd(dba)2, 2,2’-bipyridine, DCM/toluene, 80°C, 1.5 hrs; f) AgOTf, acetone, rt, 30 minutes.

[0114] Preparation of methyl 5-bromo-l-naphthoate (27). To an oven-dried 100 mL flask charged with 5-bromo-l -naphthoic acid (26, 300 mg, 1.195 mmol) was added SOCh (30 mL, 413 mmol). The cloudy reaction mixture was stirred at 60 °C for 2 hrs. Thereafter, the now clear reaction mixture was dried thoroughly under reduced pressure to obtain the 5- bromo-1 -naphthoyl chloride intermediate as an off-white solid, which was used in the next reaction without purification or characterization. The 5-bromo-l -naphthoyl chloride intermediate was then dissolved in dry DCM (10 mL); the flask was placed in an ice/water bath, cooled to 0 °C, and dry MeOH (5 mL) was added to the reaction mixture via syringe. After stirring at room temperature for 60 minutes, the reaction mixture concentrated in vacuo and the residue was then dissolved in EtOAc (80 mL), successively washed with saturated NaHCCL solution (3 x 80 mL) and brine (3 x 50 mL), and the organic layer was dried over Na2SO4. The mixture was then concentrated under vacuum to afford compound 27 as a white solid (316 mg, 100% yield) without further purification. ^T H NMR (400 MHz, CDCh) 8 8.89 (d, J= 8.7 Hz, 1H), 8.50 (d, J= 8.6 Hz, 1H), 8.21 (d, J= 7.3 Hz, 1H), 7.84 (d, J= 7.4 Hz, 1H), 7.60 (t, J= 8.4 Hz, 1H), 7.44 (t, J= 8.4 Hz, 1H). ¹³ C NMR (101 MHz, CDCh) 5 167.79, 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): calculated for CuHjCLBrNa [M+Na] ⁺ 286.9684, found 286.9673.

[0115] Preparation of 5-methoxy-l-naphthoic acid (28). Compound 28 was synthesized according to methods 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 mixed with CuBr (50.3 mg, 0.351 mmol), EtOAc (115 pL, 1.17 mmol), and a solution of CHsOna in MeOH (25%, 2.2 mL, 10 mmol). The mixture was refluxed for 3 hrs 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 column to isolate intermediate compound methyl-5-methoxy-l -naphthoate for NMR characterization: 'H NMR (400 MHz, CDC13) 5 8.52 (d, J= 8.4 Hz, 1H), 8.45 (d, J= 8.8 Hz, 1H), 8.18 (q, J= 7.2, Hz, 1H), 7.58 - 7.42 (m, 2H), 6.88 (d, J= 7.7 Hz, 1H), 4.01 (s, 3H), 4.00 (s, 3H). ¹³ C NMR (101 MHz, CDCh) 8 168.30, 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 H2O (0.5 mL), the mixture was refluxed for 2 hrs until completion, as confirmed by TLC. When hydrolysis was complete, the reaction mixture was concentrated, acidified with HC1 (IM, 10 mL) to pH 1~2, extracted with EtOAc (100 mL), and washed with brine (3 x 100 mL). The organic layer was dried over Na2SO4, then concentrated under vacuum to obtain compound 28 as an off-white solid (0.22 g, 93% yield after two synthetic steps). TLC confirmed the presence of one product, compound 28 (R _f = 0.2; DCM:MeOH = 20: 1). 'H NMR (400 MHz, CD3OD) 6 8.48 (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). The resulting 'H NMR spectrum for compound 28 was in accordance with the NMR data reported in the literature. See, Meyers, A. I., et al. J. Org. Chem. 1987, 52(20), 4592-4597. HRMS (ESI, m/z): calculated for C12H9O3 [M-H]' 201.0552, found 201.0559.

[0116] Preparation of V-biityl-5-methoxy-l -naphthamide (29, also serves as protodepalladation naphthamide species). In an oven-dried 20 mL scintillation vial, compound 28 (200 mg, 0.99 mmol) was combined with SOCh (10 mL, 138 mmol) and toluene (2 mL). The reaction was refluxed at 80 °C for 2 hrs, changing from cloudy yellow to dark green in color. Thereafter, the solvent was removed under vacuum to obtain intermediate compound 5 -methoxy- 1 -naphthoyl chloride, which was used in the next reaction without characterization or further purification. The 5 -methoxy- 1 -naphthoyl chloride intermediate was dissolved in dry DCM (8 mL) under argon protection. After cooling to 0 °C in an ice/water bath, a mixture of w-butylamine (101 pL, 1 mmol) and EtsN (137 pL, 1 mmol) was slowly added to the reaction mixture via syringe, and the color of the reaction mixture change from dark green to yellow. The reaction was allowed to stir for 1.5 hrs at room temperature until completion, as confirmed by TLC. The reaction mixture was then diluted with EtOAc (50 mL), and washed with saturated NaHCOs (3 x 50 mL) and brine (3 x 50 mL). The organic layer was dried over ISfeSCU then concentrated under vacuum to afford crude compound 29 as a yellowish solid. The crude mixture was recrystallized from Et2O to yield compound 29 as an off-white solid (206 mg, 81% yield). ³ H NMR (400 MHz, CD3OD) 5 8.54 (s, 1H), 8.34 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.45 (t, J = 8.2 Hz, 2H), 6.94 (d, J = 9.3 Hz, 1H), 3.99 (s, 3H), 3.51 - 3.38 (m, 2H), 1.73 - 1.55 (m, 2H), 1.46 7.6 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101 MHz, CD3OD) 5 172.78, 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): calculated for C16H20NO2 [M+H] ⁺ 258.1494, found 258.1500.

[0117] Preparation of 8-bromo-\-biityl-5-methoxy-l -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, A-bromosuccinimide (NBS, 61 mg, 0.340 mmol) was added as solid to the stirring reaction mixture, followed by the addition of concentrated H2SO4 (0.7 mM, 38 pL). 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 NaHCOs solution (3 x 50 mL) and brine (3 x 50 mL), and dried over Na2SO4. The mixture was then concentrated under vacuum and purified by flash column chromatography to yield compound 30 as a dark blue resin-like solid (84.2 mg, 77% yield). 'H NMR (400 MHz, CDCL) 8 8.29 (d, J= 8.3 Hz, 1H), 7.65 (d, J= 8.3 Hz, 1H), 7.46 (d, J= 5.5 Hz, 1H), 7.36 (t, J= 8.3 Hz, 1H), 6.63 (d, J= 8.4 Hz, 1H), 6.03 (t, J= 8.0 Hz, 1H), 3.94 (s, 3H), 3.43-3.33 (m, 2H), 1.55 (p, J= 13 Hz, 2H), 1.36 (sx, J= 13 Hz, 2H), 0.91 (t, J= 13 Hz, 3H). ¹³ C NMR (101 MHz, CDCL) 5 170.88, 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): Calculated for Ci ₆ Hi ₉ N ₂ 0br [M+H] ⁺ 336.0594, found 336.0600.

[0118] Preparation of (8-(butylcarbamoyl)-4-methoxynaphthyl)(bipyridine) palladium(II) bromide (31). Under protection of argon in a dry 20 mL scintillation vial with a sealable septum, compound 30 (230 mg, 0.682 mmol), Pd(dba) ₂ (414 mg, 0.72 mmol), and BIPY (112 mg, 0.72 mmol) were dissolved in DCM (5 mL) and toluene (5 mL). The reaction was slowly heated to 80 °C in about 1 hr while stirring, and the color of the reaction mixture turned 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 (15 mL), filtered through celite, and the filtrate was concentrated to dryness. The residue was triturated with dry Et ₂ O, the supernatant decanted to remove dibenzylideneacetone, and the solids were further purified by flash column chromatography to afford compound 31 as a yellow solid (146 mg, 60% yield). 'H NMR (400 MHz, CDCh) 5 9.44 (d, J= 4.9 Hz, 1H), 8.30 (dd, J= 8.3, 1.5 Hz, 1H), 8.05-7.98 (m, 3H), 7.87 (td, J= 7.9, 1.6 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.62 - 7.48 (m, 2H), 7.42-7.37 (m, 2H), 7.13 - 7.04 (m, 1H), 6.75 (d, J= 7.9 Hz, 1H), 6.46 (t, J= 4.6 Hz, 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 = 7.4 Hz, 3H). HRMS (ESI): Calculated for C ₂₆ H ₂₇ N ₃ O ₂ BrPd [M+H] ⁺ 598.0321, found 598.0347.

[0119] Preparation of (8-(butylcarbamoyl)-4-methoxynaphthyl)(bipyridine) palladacycle trifluoromethanesulfonate salt (31a). In a dry 100 mL round bottom flask, compound 31 (176 mg, 0.294 mmol) was dissolved in dry acetone (50 mL) and AgOTf (76 mg, 0.294 mmol) was added as solid while stirring. White precipitate 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 celite 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 dry and degassed chloroform to obtain pure compound 31a as a yellow solid (118 mg, 60% yield). 'H NMR (400 MHz, DMSO-d6) 5 9.13 - 9.07(m, 2H), 8.72 - 8.68 (m, 2H), 8.40 - 8.30 (m, 3H), 7.94 (s, 1H), 7.77 (d, J = 7.1 Hz, 1H), 7.69 (s, 1H), 7.59 - 7.55 (m, 2H), 7.51 (d, J= 8.0 Hz, 1H), 6.96 (d, J= 7.2 Hz, 1H), 3.98 (s, 3H), 2.97 (q, J = 6.7 Hz, 2H), 1.21 (p, J = 7.1 Hz, 2H), 1.10 (sx, J = 7.4 Hz, 2H), 0.70 (t, 7.3 Hz, 3H).

¹³ C NMR (101 MHz, DMSO) 5 171.85, 155.43, 153.66, 153.45, 151.62, 149.27, 141.02, 134.50, 133.28, 132.99, 127.66, 127.30, 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 (the CH ₂ -NH peak collides in DMSO-d6 peaks). HRMS (ESI): Calculated for C ₂ 6H ₂ 6N ₃ O ₂ Pd ¹⁰⁴ [M+H] ⁺ 516.1065, found 516.1076.

Example 6. Synthesis of fluorescent product (Compound 34).

[0120] The fluorescent imide species 34 was synthesized as set forth in Scheme 8 below.

Scheme 8

Reagents and conditions: a) w-butylamine, EtOH, reflux, 8hrs; b) CH ₃ Ona, Q1SO4 5H ₂ O, MeOH, 80°C, 12hrs.

[0121] Preparation of 4-bromo-7V-butyl-l,8-naphthalimide (33). Compound 33 was synthesized from 4-bromo-l,8-naphthalic anhydride (32) using methods reported in the literature. ⁴ H NMR (400 MHz, CDCh) 5 8.64 (dd, J= 13, 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= 13 Hz, 3H). The resulting ¹ H NMR spectrum for compound 33 was in accordance with the NMR data reported in the literature. See, Feng, L., et al. Anal. Chem. 2018, 90(22), 13341-13347.

[0122] Preparation of 4-methoxy-7V-butyl-l,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 in a 10 mL sealable tube containing a stir bar. Dry MeOH (2 mL) was added and the tube was sealed using an aluminum crimp cap with PTFE/silicon rubber septa. The reaction mixture was stirred at 80 °C for 12 hrs until completion, as confirmed by TLC. The reaction was quenched with HC1 (IM, 30 mL), extracted with EtOAc (50 mL), and washed with brine (3 x 50 mL). The organic layer was then dried over Na ₂ SO4 and concentrated under vacuum. The concentrated residue was then purified by flash column chromatography (25% EtOAc in hexane) to yield compound 34 as a white solid (44 mg, 100% yield). ⁴ H NMR (400 MHz, CDCh) 5 8.51 (dd, J= 13, 1.1 Hz, 1H), 8.48 - 8.43 (m, 2H), 7.62 (dd, J= 8.2, 7.4 Hz, 1H), 6.96 (d, J= 8.3 Hz, 1H), 4.18 - 4.10 (m, 2H), 4.08 (s, 3H), 1.69 (tt, J= 1.1, 6.6 Hz, 2H), 1.43 (sx, J= 7.6 Hz, 2H), 0.96 (t, J= 1A Hz, 3H). ¹³ C NMR (101 MHz, CDCh) 5 164.53, 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 resulting NMR spectra for compound 34 were in accordance with those reported in the literature. See, Feng, L., et al. Anal. Chem. 2018, 90(22), 13341-13347.

Example 7. Sensing chemistry and spectroscopic properties of the CO probe molecules.

[0123] For most reaction-based CO sensing approaches, such as the Tsuji-Trost reactionbased probe and palladacycle-based probe, the sensing mechanism is based on removing a quenching group of a fluorescent molecule with CO. Thus, background fluorescence is inevitable, due to insufficient quenching of the fluorescent molecule and removal of the quenching group via interference by other functional groups, metabolism, and simple chemical degradation. According to some comprehensive reviews in the field, the signal-to- noise ratio (SNR) for most reported probes is less than 200: 1, which is inadequate for quantitative analysis. Sensors with true fluorescence turn-on which create low background noise is needed for quantitative measurements. Selectivity is another issue of reported probes, owing to their sensitivity to various nucleophiles that are ubiquitous in biological samples, including amine and thiol species that can uncage the fluorophore and induce CO- independent fluorescence increase.

[0124] Instead of utilizing CO to remove the quenching group (dequenching), our strategy to sense CO is to achieve sensing through the construction of a fluorophore via two sequential reactions, which essentially eliminate background fluorescence and afford a very high level of selectivity (FIG. 1). The design takes advantage of a palladium mediated CO carbonylation reaction followed by a spontaneous amidation reaction to “construct” the fluorophore de novo (FIG. 2a). Therefore, only upon reacting with CO, the fluorescence can be turned on. To achieve this, a (9-hydroxyl phthalimide (22) with a well-defined excited- state intramolecular proton transfer (ESIPT) fluorescence mechanism was chosen as the fluorescent product with a large Stokes-shift of 114 nm and very minor overlapping between the excitation and emission spectra (FIG. 2d). The quantum yield ( F) of 22 in pH 7.4 phosphate-buffered saline (PBS) was determined to be 0.23 using quinine sulfate as the reference.

[0125] The first probe series was synthesized from 2-amino-6-methoxybenzoic acid (Scheme 1). The palladacycle was not constructed through ortho-direction group as reported for other probes such as COP-1 or CC-CO, because it leads to the formation of a dimer with higher molecular weight, and sometimes may lead to the concern of forming regional isomers. The introduction of an iodo group at the ortho-position of the amide group through diazotization-iodination could direct the oxidative insertion of palladium using tetramethylethylenediamine (TMEDA) as the ligand. Thus, the palladium complex 5 was synthesized under mild conditions and in good yield. TMEDA was chosen as the ligand due to the stability and aqueous solubility of the resulting complex. Indeed, this molecule can react with CO and serve as CO fluorescence probe (COFP) as well. The iodo group was then removed by treating with AgOTf in acetone, yielding the palladocycle 5a. The structure of 5a was characterized with NMR and X-ray crystallography (crystallographic data FIG. 21). The reaction kinetics of 5 towards CO was found to be the same as the 5a (FIG. 2j). Both CO probe molecules 5 and 5a were soluble and stable in PBS solution at pH 7.4 (FIG. 3).

[0126] Incubating 5a with CO gas in a headspace vial led to fluorescence turn-on in a CO- concentration dependent fashion (FIG. 2b and FIG. 2e insert). Compared to other “dequenching” CO probes, major advantages of this strategy include the high SNR and low background of the CO probe molecules 5 and 5a owing to the completely dark nature of the probes, the large Stokes shift of the fluorescent product 22 (114 nm), and non-overlap between the maximum excitation wavelength of the fluorescent product 22 and the protodepalladation species 17 (A,E _X =329 nm, Z,Em=420 nm). Additionally, the CO probe molecules 5 and 5a showed enhanced selectivity and tolerance of the presence of a wide variety of molecules as compared to other “de-quenching” CO probes. Under the excitation wavelength in the range of 385-405 nm for 22, the depalladated product 17 did not show any fluorescence signal (FIG. 2d).

[0127] It is known that similar palladium complexes are reactive toward thiol species. Such reactivity was presumed to cause the response of COP-1 to thiols through protodemetalation, as the depalladation species of the probes may share a similar fluorescence profile with the CO-sensing product. Because thiol species such as H2S and GSH are present in biological samples, the exclusion of such CO-independent responses can significantly enhance the reliability of CO detection. LC-MS studies showed that upon reacting 5a with glutathione (GSH) or H2S (generated from NaHS) in PBS, the depalladation species (17) was formed (FIG. 23 a, b). Directly reacting 5a with two equivalents of NaHS in dimethylacetamide (DMA) gave 17 as the major product (FIG. 23c). However, 17 did not show any fluorescence at the excitation wavelength used for CO detection, preventing a false-positive response due to depalladiation (FIG. 24a-c).

[0128] The SNR of our probe strategy is proportional to the concentration of the probe. For example, the SNR of 5a exceeds 1200: 1 when the concentration of the probe is 100 pM (FIG. 2c). At a concentration of 10 pM, the SNR is 320: 1, which is still higher than most of the existing CO probes. Again, the reason we can use a high concentration of the probe is because of its completely dark nature. Our design allows for a “fail-safe” detection of CO in the presence of other species that could lead to the demetallation (protodepalladation) of the probe and fluorescence turn-on in literature cases. With respect to the selectivity, 5a can only be turned-on by CO gas (FIG. 2e and FIG. 4). No fluorescence change was detected when 5a was exposed to thiol, persulfide, peroxide, NO2’, glutathione, glutathione disulfide, cysteine, CN", hydrogen sulfide, or HC1O. Thus, the CO probe 5a demonstrated a high selectivity compared with other palladacycle based probes, such as COP-1 which responded to some degree to H2S or other thiols that are present in biological samples.

[0129] Regarding the sensing mechanism, the probe was designed in such a way that after CO insertion between the palladium and phenyl carbon followed by hydrolysis, a carboxylic acid group is formed (see, Scheme 9). Owing to the proximity to the amide nitrogen and the kinetically favored formation of a five-membered phthalimide ring, the fluorescent o- hydroxylphthalimide is formed as the final product.

Scheme 9

[0130] To verify the mechanism set forth in Scheme 9, proton NMR, HPLC, and LC-MS were used to study the reaction between 5a and CO gas. In the NMR studies, we monitored the transformation of 5a in the PBS/D2O-DMSO solution, which showed the formation of 22 after adding CO gas in the NMR tube (FIG. 5). [0131] HPLC studies showed that after injecting CO gas into the PBS solution of 5a and incubating at 37 °C for 30 min, the phthalimide fluorescent product 22 was formed as the major product (FIG. 6a). At the same time, black precipitation (presumably palladium) was observed in the reaction mixture. A minor peak at 6.2 minutes showed both in the CO- sensing reaction as well as in the pure 22 sample. LC-MS confirmed it was the ring-open product IM-1 (FIG. 7a), an intermediate of the CO sensing reaction that is present in the hydrolysis equilibrium according to previous studies on the phthalimide hydrolysis. In order to capture the intermediate of the CO sensing reaction, an ethanol solution of 5a was used, since ethanol is not a good leaving group for the lactamization reaction. HPLC and LC-MS showed the ethyl ester intermediate IM-2 along with the cyclized product 22 (FIG. 6b and FIG. 7b-7c). After adding PBS to this reaction mixture, IM-2 was completely converted to 22 almost instantly, indicating the fast intramolecular lactamization reaction in aqueous solution. Such results also indicate the insertion of CO to the palladium complex to be the rate determining step. In addition, after reacting 5a with CO gas, the fluorescence intensity was the same as 22 at the same concentration. To this end, the sensing mechanism is confirmed with a quantitative conversion of the probe to the fluorescent product in PBS solution.

Example 8. Structural optimization of CO probe molecules.

[0132] The CO probes were optimized to achieve better sensitivity, stability, and quantum yield to accommodate biological applications. Sensitivity is important for the probe to detect CO at low concentrations. To achieve this, improvements in the quantum yield and reactivity towards CO were investigated. The pKa of the phenol group is sufficiently close to the physiological pH, which renders the fluorescence pH-sensitive under near physiological conditions (FIG. 8a). To increase the quantum yield and minimize pH-dependency of the fluorescent product, the amino analog 25 was designed as a new reporter product with the quantum yield being substantially increased to 0.33. The maximum excitation and emission wavelengths of 25 are blue-shifted by about 10 nm compared to 22 (FIG. 2d, 2i). Due to the lower pKa of the conjugated acid and better ESIPT effect of aniline, the pH dependency of 25 around physiological pH also largely disappeared (FIG. 8b), indicating improved signal stability. Based on 25, several analogs were designed and synthesized by changing the ligand moiety and the amide chain, and their sensing reaction kinetics were tested under normalized conditions. [0133] First, Ila was synthesized, a complex with TMEDA as the ligand and a butyl chain on the benzamide nitrogen. Compound Ila showed a faster response to CO when compared to the hydroxyl derivative 5a (FIG. 2j). Next, compound 12 was prepared with TMEDA as the ligand and a propyl chain at the benzamide nitrogen. As shown in FIG. 2j, shortening the amide alkyl chain to propyl in compound 12 resulted in a slower response to CO gas compared to Ila and the hydroxyl 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 substantially increased reaction kinetics compared to 12, Ila, and 5a. Still using the BIPY ligand, but shortening the length of the alkyl amide chain from butyl to propyl (compound 14) further increased reaction kinetics with the 2nd order reaction rate constant being 220.6 ± 27.2 M ^-1 s' f The reaction kinetics of 14 allow for almost real-time sensing of the CO gas. CO probe molecule 14 also demonstrated excellent concentration-dependent SNR (FIG. 2g) and selectivity for CO (FIG. 2h and FIG. 9). The quantitative conversion of 14 to the fluorescent product 25 in a mixture of dimethylacetamide (DMA) and PBS solution was also confirmed by the 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 (FIG. 2j). The 2nd order rate constant for compound 6a was determined to be 13.8 ± 1.4 M^s' ¹ . The CO-sensing kinetic parameters generally fall in the region defined by 6a and 14 (FIG. 2j). The results indicate that changing the bidentate ligand and varying the substituents on the benzamide of the Pd complex effect the CO insertion reaction.

[0134] The detection sensitivities of 5a, Ila, and 14 were tested by incubating the CO probe molecules with 1.6-8 ppm CO in air in the headspace vial. Due to their high SNR, it should be feasible to use these probes at high concentrations for increased sensitivity. To test this assumption, detection sensitivity was tested at both low (100 pM) and high (1 mM) concentrations for 5a and Ila and at 1 mM for 14 in DMA. The detection limits for 5a, Ila, and 14 were determined to be about 0.1-0.2 ppm, which is the equivalent of 0.45-0.9 nM in solution according to Henry’s law. As expected, a higher probe concentration gave a higher sensitivity as shown by the lower detection limit in general. The high sensitivity coupled with the high SNR allow for robust determination of CO concentrations in biological samples, as discussed herein.

[0135] Due to the relatively low intracellular accumulation and photostability exhibited by phthalimide fluorophores in live-cell imaging studies, we developed an additional series of probe molecules that are capable of producing 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 naphthamide-based CO probes 31 and 31a were designed to increase photostability and cell retention of the fluorescent product in intracellular CO accumulation imaging applications. The naphthamide-based CO probes were synthesized based on a strategy similar to that used for the benzamide-based CO probes (Scheme 7 and FIG. 11). While the preparation of the naphthamide Pd-complex with TMEDA as the bidentate ligand led to spontaneous decomposition during synthesis, palladium complex 31 was prepared using BIPY without any decomposition issues. As shown in Scheme 7, the bromide in 31 was abstracted using AgOTf to form the sixmembered palladacycle 31a. The structures of both 31 and 31a were characterized with NMR and X-ray crystallography (X-ray crystallography data shown in FIG. 22).

[0136] As shown in FIG. 12, both 31 and 31a were found to be stable for at least 1 hour in PBS solution at 37 °C. The apparent reaction kinetics for compound 31a towards CO was determined to be as fast as 14 (FIG. l id). LC-MS studies confirmed that reacting 31a with CO in PBS led to the formation of the naphthalimide fluorescent product 34 (FIG. 13). The quantum yield of 34 was determined to be 0.82 in water. As shown in FIG. 1 lb, the protodepalladation naphthamide species 29 features an excitation spectrum that is distinct from the excitation spectrum for the naphthalimide CO-sensing product 34, which allows for concentration dependent SNR (FIG. 11c) and high selectivity (FIG. l ie, l id). The CO detection limits of 31 and 31a were determined using 20 pM of 31 in PBS or 20 pM of 31a in PBS. Assuming CO solubility is 1 mM in PBS at 760 mmHg CO partial pressure, the CO detection limits of 31 and 31a were calculated as 2.74 nM and 2.06 nM, respectively, which should allow for endogenous CO detection in cells at micromolar concentrations.

Example 9. Applications of the benzamide-based CO probes and naphthamide-based CO probes.

[0137] CO probe molecules 5a, Ila, 14, and 31a were selected for further studies to determine the feasibility of COFPs in research applications. Generally, there are two types of applications for CO probes: 1) measuring CO content in blood, tissue, and cell culture samples; and 2) imaging intracellular CO accumulation in live cells. Each CO probe molecule has its own physical, chemical, and biological characteristics, which are suited for various applications. [0138] Semi-quantitative determination of CO in blood and cell culture samples.

Because CO sensing uses a turn-on mechanism, the probes are endowed with “fail-proof’ features in three aspects: 1) the probe is completely dark; 2) protodepalladation does not lead to fluorescence; and 3) only CO insertion allows for fluorophore construction. Thus, the probes can give a large linearity range when a biological sample contains high CO concentrations, and the presence of nucleophilic/enzymatic species in the biological samples presents no interference. Compounds 5a and Ila were more water soluble than compounds 14 and 31a due to the coordinated TMEDA ligand and tritiate salt forms of 5a and Ila. Specifically, the solubility in PBS was determined to be about 5 mM for both 5a and Ila, 100 pM for 14, and 50 pM for 31a. Moreover, the fluorescent products of 5a and Ila (i.e., phthalimide compounds 22 and 24, respectively) are stable in serum. Although the fluorescence of 22 was determined to be pH dependent, blood pH is not expected to fluctuate significantly; also, testing fluids were buffered using PBS (lx), which helped prevent pH fluctuations in biological samples. Therefore, both 5a and Ila were selected for measuring carboxyhemoglobin (COHb) levels in mouse blood samples.

[0139] Based on the fact that the blood hemoglobin tetramer concentration is about 2 mM, it was determined that 100% COHb would give a CO concentration of about 8 mM. Thus, testing COHb levels up to 50% would require the CO probe concentration to be at least 4 mM. Owing to the turn-on sensing mechanism via the CO-insertion based construction of a fluorophore, even at this high concentration, the background signal is negligible. By directly incubating 5a at a concentration of 10 mM in blood samples with different COHb levels predetermined by a CO-oximeter, correlation between fluorescence intensity and COHb levels determined with CO-oximeter was established (FIG. 14a). By using this standard curve, the COHb level of an unknown partial CO saturated blood sample with a fluorescence intensity of 117.48 a.u. was calculated to be 7.9%. CO-oximeter reading of the same sample showed a COHb level of 8.2 ± 0.45%, demonstrating good consistency. To make it more feasible in determine COHb levels without the need for CO-oximeter corroboration, a definitive COHb calibration level of the blood was needed. It is reported that pre-saturation blood with pure CO gas led to about a 90% COHb level. Measuring CO-saturated blood with a CO-oximeter by our own hands consistently verified 90% COHb levels in the blood samples collected from five different mice (FIG. 14b). By assigning the COHb level of CO saturated-^ flushed blood to be 90% and diluting serially by normal blood, a calibration curve can be established with COSMs. Thus, the COHb levels of an unknown sample can be determined by the probe with a fluorescence microplate reader without the need of CO-oximeter, GC, or even a fluorometer. This approach was successfully verified in two ex-vivo experiments. After orally administering CO prodrug BW-CO-306 and its activated charcoal formulation BW-AC-306 in mice, blood COHb levels detected by the probe were in good agreement with the one determined with CO-oximeter. Specifically, COHb of the blood samples from the mouse dosed with 100 mg/kg BW-AC-306 was determined to be about 3% when measured with 5a or a CO-oximeter, while COHb of the control mouse was determined to be about 1.5% (FIG. 14c). The blood COHb levels of undosed mice and mice dosed with 200 mg/kg BW-CO-306 was determined to be about 0.9% and 6.1%, respectively, as measured with Ila and confirmed with the CO-oximeter readings (FIG 14d). There is no statistically significant difference between the results of CO probe approach and the CO-oximetry.

[0140] The same methodology can also be used for in vitro cell experiments to determine relative CO saturation changes induced by external CO sources, such as CO gas or endogenous CO induced by an HO-1 activator. To demonstrate, bardoxolone methyl (CDDO-Me) was selected as the HO-1 inducer due to the absence of spectroscopic interference caused by CDDO-Me compared to the chromogenic hemin. HeLa cells were incubated with 250 ppm CO gas for 2 hrs or with 0.3 pM CDDO-Me for 6 hrs followed by collection with a cell scrapper. Incubation of the washed cell pellets with 100 pM Ila followed by fluorescence measurement with a microplate reader showed that fluorescence signal increased by about 28% after CO gas treatment and 7.5% after CDDO-Me activation when compared to the DMSO vehicle control FIG. 14e). Western-blot confirmed that CDDO-Me treatment significantly increased HO-1 expression (FIG. 14f), which presumably accounted for the elevated CO production.

[0141] Determination of absolute amounts of CO in tissue and cell culture samples. The endogenous CO concentration in various organs has been determined to be about 2-10 pmol/mg in mouse tissues using an RGD-GC method by Vreman et al previously.

Theoretically, if about 100 mg tissue releases all bound CO to 1 ml headspace in a headspace vial, it should give at least 4.5 ppm CO, which is well above the detection limit of the probe 14. Because CO-probe molecule 14 had a detection limit of 0.1 ppm and fast reaction kinetics, it was employed in CO concentration quantification experiments. To develop the quantification method, 14 was dissolved in 50 pL degassed dimethylacetamide (DMA) at a concentration of 1 mM and sealed in a 0.5 ml vacuumed headspace vial with PTFE/silicone crimp septum. DMA was chosen for the following reasons: 1) DMA has a relatively high solubility of CO (about 4.5 mM at 760 mmHg), allowing for a higher CO concentration to react with the probe in solution; 2) compound 14 is soluble in DMA, which allows for a high ratio between the probe and the analyte to enable fast reaction and sensitive detection; and 3) DMA is miscible with PBS, which helps to normalize pH and increase accuracy. This probe- charged headspace vial (CO detection vial) was used as a CO “detector” by injecting CO- containing gas, followed by incubation, and then fluorescence measurement using a fluorospectrometer or plate reader. CO concentration was determined by an external standard curve method. Specifically, 250 pL of standard CO-air calibration gas with a CO concentration of 10-100 ppm was injected to the detection vial to establish the standard curve for 14 (FIG 14g). The excellent reproducibility and goodness-of-fit indicated a sound experimental setup. To verify its utility in an ex vivo study of CO donor administration, we used the organ tissues from the same mice dosed with 200 mg/g BW-CO-306 in the aforementioned COHb blood analysis studies. Aliquots of liver and kidney homogenates were concomitantly tested using compound 14 and a GC equipped with a methanizer-FID detector. To release the protein-bound CO in the headspace vial, the tissue was processed and denatured using 3% 5-sulfosalicylic acid (SSA) according to an established procedure. Liver tissue CO concentration was determined to be about 5 pmol/mg for the control and 31 pmol/mg for the BW-CO-306 treated group (FIG. 14h). Kidney tissue CO concentration was determined to be 11 pmol/mg for the control and 26 pmol/mg for the CO-306 treated group (FIG. 14i). There was no statistical difference between the results determined using the GC- methanizer and CO probe molecule 14 (FIG. 14h, 14i, 14j), confirming that our CO detection protocol using 14 can quantify CO in tissue samples with accuracy and reproducibility on par with that of a methanizer-FID-GC system, which is expensive to set up and considered to be the “gold standard” for determining tissue CO concentrations. The tissue CO concentration of the control mouse is also among the similar range tested with RGD-GC method by Vreman et al., demonstrating the reproducibility of our methods.

[0142] Using methods that are similar for CO detection in tissue samples, the CO gas detection tube containing compound 14 can also be used to determine CO concentrations in cell culture samples. The results in FIG. 14k show that after treating HeLa cells with either 250 ppm CO gas or 50 pM of CO prodrug BW-CO-111 for 2 hrs, CO concentration increased substantially from 25 pmol/10 ⁶ cell to 92 pmol/10 ⁶ cell and 203 pmol/10 ⁶ cell, respectively. Since the cells were washed with PBS twice before denaturing and CO determination, the substantially higher CO concentration in the BW-CO-111 treatment group could be attributed to the combined CO amounts from hemoprotein-bound CO and the residual intracellular CO prodrug. To this end, we have demonstrated that 5a, Ila, and 14 can be used to determine CO concentrations in blood, tissue, and cell culture samples, both semi-quantitatively and quantitatively, with excellent reproducibility and accuracy.

[0143] Fluorescence imaging of CO in cell cultures. As a key visualization method in biological research, fluorescence imaging has significantly promoted the understanding of the function of CO and the development of various useful CO donors capable of delivery CO to the intracellular space. The series of naphthamide-based CO probe molecules 31 and 31a are highly selective, highly sensitive, produce low background fluorescence, and are capable of generating a photostable naphthalimide product with pH-independent fluorescence.

Moreover, both 31 and 31a showed no cytotoxicity to HeLa cells at less than 50 pM within 24 hrs (FIG. 15), and, as a trifluoromethanesulfonic salt, compound 31a has improved aqueous solubility. Both 31 and 31a were used in fluorescent imaging of intracellular CO accumulation from various sources, including CO gas and CO prodrug BW-CO-201, which was chosen partly because of the lack of fluorescence interference produced by its CO- released product (FIG. 16). As shown in FIG. 16 and FIG. 17, live HeLa cells showed strong blue fluorescence under DAPI channel after treatment with CO followed by addition of 20 pM 31 or 31a, while the control group without CO treatment did not show any fluorescence under the same imaging conditions. The low background fluorescence in the vehicle control group again demonstrates the advantage of the insertion-tum-on strategy. Further, 31a was also able to sense the increase in endogenous CO production induced by 0.3 pM CDDO-Me using a longer a longer exposure time of 6 seconds (FIG. 17d, 17e, 17f). The line profile region of interest (RO I) of FIG. 17f was used for the calculation of signal intensity and line profile 4 was used for background subtraction (FIG. 17g, 17h).

[0144] Conclusions. 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 generate fluorescent products in the presence of CO with high specificity, high sensitivity, fast response, and high SNR (low background fluorescence). In this strategy, CO is the analyte, as well as the key reactant for de novo synthesis of the fluorophore. By taking advantage of the unprecedented detection features of the CO probes derived from this strategy, detection and quantification of CO in blood, tissue, and cell culture samples has been achieved with high sensitivity, selectivity, accuracy, and reproducibility with benzamide-based CO probe molecules, such as 5a, Ila, and 14. The same design strategy was 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 live cells, as well as endogenous CO production. Overall, this disclosure demonstrates the advantages of the insertion and fluorescence approach in developing fluorescence probes for CO.

Example 10. Kinetics assays of CO probe molecules.

[0145] General procedure for determining the second-order rate constant for 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 (sealed by silicone oil) for over 10 minutes at room temperature (see, Almeida, A.S., el al. J Biol Chem 2012, 287, 10761-10770). The stock solution of probe was prepared as 1 mM solution in DMSO. The second-order kinetic constant of 6a and 14 was assessed by plotting the observed pseudo-first order reaction constant (k _O bs) versus the CO concentration (FIG. 18). The CO concentration was kept greater than 5 -fold of the probe concentration to accommodate the pseudo-first order reaction condition.

[0146] Determination of second-order rate constant for 6a. To a 1 mL cuvette, 890 pL, 790 pL, or 690 pL of PBS were added followed by addition of 10 pL of the 6a probe stock solution. Final concentration of 6a was 10 pM. The cuvette was sealed by a rubber stopper. Under excitation wavelength of 395 nm, 100 pL, 200 pL, or 300 pL of saturated CO PBS solution were injected into the cuvette by 1 mL syringe. Therefore, the final CO concentration was 100 pM, 200 pM, or 300 pM, respectively. The emission fluorescence signal was recorded until reach the plateau. The half-life (Z1/2) was then estimated by plotting the time course of the emission intensity by Graphpad Prism 9, and k _O bs was calculated according to the pseudo-first order reaction kinetic: k _O bs=ln2/ti/2. The experiment was triplicated.

[0147] Determination of second-order rate constant for 14. To a 1 mL cuvette, 940 pL, 890 pL, or 790 pL of PBS was added followed by addition of 10 pL of the 14 probe stock solution. Final concentration of 14 was 10 pM. The cuvette was sealed by a rubber stopper. Under the excitation wavelength of 385 nm, 50 pL, 100 pL, or 200 pL of saturated CO PBS solution were injected into the cuvette by 1 mL syringe. Therefore, the final CO concentration was 62.5 pM, 125 pM, or 250 pM, respectively. The emission fluorescence signal was recorded until reach the plateau. The half-life (Z1/2) was then estimated by plotting the time course of the emission intensity by Graphpad Prism 9, and k _O bs was calculated according to the pseudo-first order reaction kinetic: k _O bs=ln2/ti/2. The experiment was triplicated.

[0148] Kinetics assays for other CO probe molecules (5, 5a, Ila, 12, 13, 31, and 31a).

The other CO probe molecules (5, 5a, Ila, 12, 13, 31, and 31a) were tested under unified conditions: CO probe molecule concentrations = 10 pM; CO concentration = 200 pM. The percentage of the maximum signal intensity (plateaued) was plotted versus time. Therefore, the reaction kinetics can be qualitatively compared under the normalized condition.

Specifically, to a 1 mL cuvette, 790 pL of PBS was added, followed by addition of 10 pL of the CO probe stock solution. The cuvette was sealed by a rubber stopper. Under specific excitation wavelength, 200 pL of saturated CO PBS solution was injected into the cuvette by 1 mL syringe. The emission fluorescence signal was recorded until it reached the plateau.

Example 11. Evaluating quantum yields (OF) of fluorescent products.

[0149] The quantum yields of fluorescent products 22, 23, 24, 25, and 34 were comparatively determined by using well-defined quinine sulfate as the reference standard ( f = 0.55 in 0.5 M H ₂ SO ₄ , r| = 1.346, L= 7040.262: bandwidth=5/5 nm, ex/em, L= 1933.334: bandwidth=3/5 nm, ex/em, A _r =0.0086) and the following equation as reported in the literature (Heller, C.A., et al. Journal of Chemical & Engineering Data 1974, 19, 214-219):: where the subscript r and x denote the reference compound and the testing compound, respectively; is the quantum yield; I is the integrated area of the fluorescence spectrum; A is the UV/Vis absorbance at the excitation wavelength; is the excitation wavelength; and q is the refractive index of the solvent.

[0150] Each fluorescent imide species 22, 23, 24, 25, and 34 was dissolved in the medium indicated in Table 1 below at a concentration of 10-20 pM. UV absorbance at their maximum excitation wavelength was recorded. The fluorescence spectra were recorded and the area under the emission spectra were integrated. Table 1. Quantum yield determination for fluorescent products 22, 23, 24, 25, and 34.

Compound I _x A _x r| (medium) f

22 395 ^a 26702.37 ^a 0.067796 1.3349 (PBS) ^c 0.23

23 395 ^a 25476.39 ^a 0.051658 1.3349 (PBS) ^c 0.29

24 385 ^a 29255.27 ^a 0.047460 1.3349 (PBS) ^c 0.37

24 385 ^a 31176.99 ^a 0.050509 1.333 (H ₂ O) 0.37

25 385 ^a 27745.81 ^a 0.050294 1.3349 (PBS) ^c 0.33

25 385 ^a 29621.90 ^a 0.053998 1.333 (H ₂ O) 0.33

34 377 ^b 37630.31 ^b 0.102224 1.333 (H ₂ O) 0.82

[a] Bandwidth 5/5 nm (Ex/Em); [b] Bandwidth 3/5 nm (Ex/Em); [c] r] value of PBS reported in Hoang, V.T., et al. Applied Sciences 2019, 9, 1145.

Example 12. Detection limit determination of CO probe molecules.

[0151] The detection limits for CO probe molecules 5a, Ila, 14, 31, and 31a were determined by the following equation reported in the literature (Mei, Q. et al. RSC Advances 2015, 5, 74924-74931):

Detection Limit = 3o/K where <5 denotes the standard deviation of the blank measurement, and K denotes the slope of CO concentration against the corresponding fluorescence intensity.

[0152] For benzamide-based CO probe compounds 5a, Ila, and 14, 1 mM or 100 pM of 5a and Ila and 1 mM of 14 were freshly prepared in 50 pL degassed DMA in a 300 pL headspace vial. 0, 20, 40, 60, 80, and 100 pL of CO gas was added by injecting various volumes of 20 ppm CO gas (in air) by a gas-tight syringe. The resulting mixture was incubated on a shaker for 1 hr at room temperature. Then, the fluorescence was measured.

The background signal was measured using a freshly prepared CO probe solution at the same concentrations for detection. The detection limits for 5a, Ila, and 14 were assessed by plotting the CO concentration against the corresponding fluorescence intensity (Table 2 and FIG. 19). Table 2. Detection limits for CO probe molecules 5a, Ila, and 14.

CO probe (concentration) G (SD) ^a K ^b Detection limit (ppm)

5a (100 pM) 0.717 12.856 0.167

5a (l mM) 0.741 22.747 0.099

Ila (100 pM) 1.074 15.817 0.203

Ha (l mM) 2.498 40.098 0.187

14 (l mM) 1.206 33.560 0.108

[a] The standard deviation of the blank measurement; [b] Change in fluorescence intensity per CO concentration.

[0153] For naphthamide-based CO probe compounds 31 and 31a, 20 pM of 31 and 31a were freshly prepared in 4 mL degassed PBS buffer (pH =7.4) in a 6 mL headspace vial. 0, 22, 44, 88, 176 pL of CO gas was added by injecting various volumes of 100 ppm CO gas (in air) by a gas-tight syringe. The resulting mixture was incubated on a shaker for 1 hr at room temperature. Then, the fluorescence was measured. The background signal was measured using a freshly prepared CO probe solution (20 pM). CO concentration was calculated as 1 ppm CO in the air gives 1 nM CO concentration in PBS according to the solubility of CO in PBS (1 mM at 760 mmHg partial pressure). The detection limits for 31 and 31a were assessed by plotting the CO concentration against the corresponding fluorescence intensity (Table 3 and FIG. 20).

Table 3. Detection limits for CO probe molecules 31 and 31a.

CO probe (concentration) G (SD) K Detection limit (nM)

31 (20 pM) 0.431 471.927 2.74

31a (20 pM) 0.307 448.255 2.06

Example 13. Determining the signal-to-noise ratios of CO probe molecules.

[0154] Compounds 5a, 14, and 31a were dissolved in DMSO to make 10 mM stock solution and diluted with PBS in a 6 ml headspace vial (8.8 ml actual volume) and sealed with PTFE-silicone rubber septum. The final concentrations of 5a and 14 were 1, 10, and 100 pM. The final concentrations of 31a were 1, 10, 25, and 50 pM, due to its lower solubility in PBS. At room temperature, for CO-response group, 1 ml pure CO gas (45 pmol) was injected into the headspace vial and incubated for 15 minutes. For blank control group, the probe was incubated in PBS without CO gas injection. The fluorescence spectra were recorded with excitation wavelength of 395 nm, 385 nm, and 377 nm, for 5a, 14, and 31a, respectively. The area under the fluorescence spectra curve (AUC) was calculated by Graphpad Prism 9. The signal -to-noise ratio (SNR) of each concentration level was calculated using the following equation:

Example 14. Administration of CO prodrugs in mice and blood/tissue sample collection protocols.

[0155] CD-I mice (25-30 g) were purchased from Envigo (Indianapolis, USA), and were fed with food and drinking water ad libitum. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. All the animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi (IACUC protocol: 19-012). Mice were randomly separated into two groups (n=3 for each group), untreated control and CO prodrug treatment groups. The CO prodrug treatment group was dosed with BW-CO-306 at 200 mg/kg dosage by oral gavage. See, De La Cruz, L.K., et al. Chemical Science 2021, 12, 10649-10654. BW-CO-306 was prepared by dissolving 13.4 mg BW-CO-306 in 120 pl DMA followed by mixing with 430 pl PEG400 before gavage to minimize the loss of CO yield due to hydrolysis of BW-CO-306 in PEG400. The control group did not undergo any treatment. After CO prodrug administration, the mice were monitored with a CO-oximeter and sacrificed after taking the blood sample (Blood COHb level was measured before and at 10 minutes after oral administration). The blood (0.6-0.8 ml) was collected through retro-orbital bleeding and transferred to a tube containing 30-unit heparin. Then mice were given whole-body perfusion with HBSS buffer after sacrifice. Liver and kidney were collected and snap-freezed by submersing in liquid nitrogen for 30 seconds and stored at -80 °C until CO determination experiments.

Example 15. Relative quantification of COHb levels in blood samples using 5a and Ila.

[0156] Blood collection. CD-I mice (25-30 g) were purchased from Charles River Laboratories (Wilmington, MA, USA), and were fed with food and drinking water ad libitum. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. All the 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 (about 1 ml) was collected via cardiac puncture and transferred to a tube containing 40-unit heparin.

[0157] Standard curve. 50-100 pL blood was saturated with pure CO gas in a headspace vial under normal atmospheric pressure for 20 minutes at room temperature. Then the headspace was flushed with ultra-pure nitrogen for 5 minutes in an ice-bath to remove unbound CO. Since the CO saturated level was tested to be about 90% COHb. Thus, the CO saturated blood was serial diluted with fresh blood from the CO-free control group in an Eppendorf tube and equilibrate for 20 minutes to form 45%, 22.5%, 11.25%, 5.63%, 2.81%, 1.41% COHb calibrators.

[0158] COHb determination. 20 pL of blood was mixed with 2 pL 100 mM DMSO stock solution of 5a or Ila, and incubated at room temperature for 30 minutes. Then the mixture was diluted with 80 pL cold PBS followed by 100 pL cold ACN to precipitate protein. After centrifuging at 20000xg for 5 minutes at 4 °C, 100 pL of the clear supernatant was transferred to a black 96-well plate for fluorescence assay (Coming, New York, USA) and the fluorescence signal was read with the plate reader (filter: Ex:405 nm/Em:535 nm, 10 nm bandwidth). The COHb level was calculated based on the linear regression of the standard curve of the calibrators.

Example 16. Relative quantification of CO in cell culture samples using Ila.

[0159] HeLa cells were seeded in the 6-well plate in DMEM cell culture medium (without phenol red and sodium pyruvate, supplemented with 10% FBS and 100 units penicillin and 100 pg/ml streptomycin) and cultured at 37 °C in 5% CO2 humidified atmosphere. After cells reached about 90% confluency, the culture medium was changed to the same fresh DMEM full culture medium containing 0.5% DMSO as the vehicle control, 0.3% CDDO-Me (DMSO concentration was 0.5%), and 0.5% DMSO for CO gas incubation group. For control and CDDO-Me treatment group, cells were incubated at 37 °C in 5% CO2 humidified atmosphere for 6 hrs. For CO gas treatment group, cell culture plate was placed in a gas-tight chamber filled with 250 ppm CO balanced with 5% CO2 in air, and incubated at 37 °C for 2 hrs. After incubation, all culture medium was removed, and the cells were washed with cold PBS for 3 times and scrapped in PBS. After transferring to 1.5 ml Eppendorf tubes and centrifuged at 250xg for 4 minutes and the cell pellet was lysed with 80 pL ACN containing 100 pM Ila and incubated at room temperature for 1 hr. Then 40 pL PBS was added and incubated for further 15 minutes followed by centrifuging at 20000xg for 5 minutes. The supernatant was transferred to black 96-well plate (Coming, New York, USA) and the fluorescence signal was read with plate reader (filter: Ex:405 nm/Em:535, 10 nm bandwidth).

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

[0160] Preparation of the tissue homogenate and liberation of CO. Tissue in the amount of about 150-300 mg was weighed into an Eppendorf tube depending on the repeat times need for the test. Then ultrapure water was added to the tissue (water volume:tissue weight = 4 pL: 1 mg). While keeping the tube on ice, the tissue was diced with iris scissor into small pieces and then homogenized with Tissue-Tearor homogenizer (Biospec, Bartleville, Oklahoma, USA). 300 pL of the tissue homogenate was transfer into a 2 ml headspace vial (vial volume: 2 mL), and followed by addition of 1200 pL 3.75% 5- sulfosalicylic acid in ultrapure water in one portion and quickly seal the vial by crimp seal cap with PTFE-silicon rubber septa. The headspace vials were incubated at 37 °C for 2 hrs.

[0161] Preparation of the gas detection tube with 14. Compound 14 was dissolved in CO-free DMA at a concentration of 1 mM. CO-free DMA was prepared by freeze-thaw process and purged with ultrapure argon for three times. Fill the 300 pL headspace vial with 50 pL DMA solution of 14 and seal the tube by crimp seal cap (8 mm) PTFE-silicon rubber septa. The pressure inside the vial was reduced by either sealing the tube under vacuum or extracting 200 pL headspace gas out by a syringe with a 28-gauge needle.

[0162] Preparation of the calibration curve and testing CO in the tissue homogenate. CO calibration gas (10, 25, 50, 100 ppm) was filled into the gas sampling bag (Supel™, inert multilayer foil, Sigma- Aldrich, Saint Louis, Missouri, USA). Inject 100 pL headspace gas from the sampling bag or tissue homogenate vial to the gas detection tube by a gas-tight syringe with sample lock valve (Hamilton, Reno, Nevada, USA). Rock the gas detection tube in a tray on an orbital shaker at room temperature for 1 hr. Transfer 25 pL of the solution in the detection vial to the cuvette and dilute with 975 pL ultrapure nitrogen -purged PBS. Measure the fluorescence signal at 499 nm with the excitation wavelength at 385 nm. CO concentration of the calibration gas was against the fluorescence intensity to get the calibration curve. The CO concentration of the tissue sample is determined by using the standard curve and the fluorescence intensity. Example 18. Absolute quantification of CO in cell culture samples using 14.

[0163] Cell treatments. HeLa cells were cultivated in DMEM-based full culture medium (supplemented with 10% FBS and 100 units penicillin and 100 pg/ml streptomycin) in 15 cm Petri dish at 37 °C in 5% CO2 humidified atmosphere to about 90% confluency. Cell culture medium was changed to fresh ones with or without CO prodrug. DMSO concentration for all groups was 0.5%. For CO prodrug, BW-CO-111 (100 pM) with a half-life of 24 minutes were used to match the incubation time of the CO gas treatment group. For CO gas treatment, cells were incubated in a gas-tight chamber containing 250 ppm CO, 5% CO2, and balanced air at 37 °C. All groups were incubated for 2 hrs followed by rinsing with PBS for two times. Cells were lifted from the Petri dish by scrapping in minimum amount of PBS (about 1 ml) After cell counting by haemocytometer, cells were transferred to Eppendorf tube followed by centrifugation at 250xg for 4 minutes to get the cell pellet. After removing the supernatant and rinse once with ultrapure water without disturbing the pellet, cell pellet was transferred to 2 mL headspace vial by resuspending in 300 pL ultrapure water. Then, 1200 pL 3.75% 5- sulfosalicylic acid in ultrapure water was added in one portion and the vial was quickly sealed by crimp seal cap with PTFE-silicon rubber septa. The headspace vials were incubated at 37 °C for 2 hrs.

[0164] Measure CO in the cell lysate. CO concentration in the headspace was measured as described in Example 17, with the calibration curve prepared with CO calibration gas.

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

[0165] Gas chromatography settings. GC was tested on an Agilent 7820a GC system. Purged packed inlet, temperature 150 °C. Column: Restek Molesieve 5A, 80/100 mesh, 0.53 mm x 2 m (Centre County, Pennsylvania, U.S.). Carrier gas: helium. Column flow rate: 4.5 ml/min. Oven temperature: 100 °C isocratic for 5 minutes. Detector: a Restek Methanizer (CH4izer, Centre County, Pennsylvania, U.S) is coupled in between the column and the FID detector; it utilizes a nickel catalyst tube and hydrogen gas to convert CO and CO2 into methane, thus can be detected with FID detector; catalyst tube temperature: 380 °C; catalyst H2 flow rate: 25 ml/min. FID detector: temperature 300 °C; EE flow: 15 ml/min; air flow: 400 ml/min. [0166] Preparation of the calibration curve and testing CO in the tissue homogenate. CO calibration gas (10, 25, 50, 100 ppm) was filled into the gas sampling bag (Supel™, inert multilayer foil, Sigma- Aldrich, Saint Louis, Missouri, USA). Inject 100 pL headspace gas from the sampling bag or tissue homogenate vial (prepared according to Example 17) to the GC. The CO peak area was plotted against the CO concentration of the calibration gas to get the standard curve which can be used to determine the CO concentration in the tissue sample.

Example 20. Cell imaging using 31 and 31a.

[0167] HeLa cells were seeded in the 3.5 cm Cellvis glass bottom culture dish (Mountain View, California, USA) at a density of 5/ I O ⁴ /dish in FluoroBrite DMEM medium (ThermoFisher, Carlsbad, California, USA) supplemented with 10% FBS, 100 units penicillin, and 100 pg/ml streptomycin. After culturing overnight, the cells were treated with designated conditions and incubated for 1 hr: vehicle control: 0.5% DMSO in culture medium; CO donor: 50 pM BW-CO-201 or 50 pM CORM-401; CO gas: 250 ppm CO, 5% CO2, and balanced air in a gas chamber. Then 31 or 31a were added to the culture medium at a concentration of 20 pM and the cells were continuously incubated under the previous conditions for 1 hr. Cells were then washed twice with PBS and incubated in fresh FluoroBrite DMEM medium. The live cells were imaged with Olympus IX-73 inverted fluorescence microscope under DAPI channel and phase-contrast transillumination settings.

Example 21. Western blot for HO-1.

[0168] HeLa cells were cultured in DMEM culture medium (without sodium pyruvate) supplied with 10% fetus bovine serum, 100 units/mL penicillin, and 100 pg/mL streptomycin in a 75 cm ² cell culture flask. Upon 80% confluency, cells were trypsinized and seeded in a 6-well plate at a density of l * I O ⁶ cells/well with 3 mL culture medium containing 0.5% DMSO (vehicle control group) or 0.3 pM CDDO-Me in DMSO (treatment group, DMSO concentration: 0.5%). Cells were incubated in a 37 °C humidified cell incubator under 5% CO2 in air atmosphere for 6 hours. Cells were washed with cold 1 *PBS for 3 times and lysed with 200 pL lx Laemmili loading buffer (contains 2.5% mercaptoethanol). The cell lysate was denatured at 95 °C for 5 minutes. 5 pL denatured cell lysate was separated by 4-15% SDS-PAGE gel and transferred to PTFE membrane. Target proteins were blot with iBind Flex Western-blot system (Invitrogen, USA) according to the manufacture’s manual. Primary antibody (mouse): HO-1 (1 :200, Santa Cruz, USA), P-Actin (1 :400, Bio-rad, USA); HRP conjugated goat anti-mouse 2nd antibody (1 :2000, Bio-rad, USA). Blot bands were detected with Pierce ECL plus Western blotting substrate (Thermo-Fisher, USA) and imaged with GE LAS4000 mini chemiluminescence imager.

Example 22. Cytotoxicity evaluation of 31 and 31a.

[0169] HeLa cells were seeded in a 96-well plate at a density of 1 x 10 ⁵ cells/100 pL/well. The cells were incubated in DMEM full culture medium (supplemented with 10% FBS and 100 unit/ml penicillin and 100 pg/mL streptomycin) in 37 °C cell incubator overnight under humidified condition. After adhesion, the cell culture medium was changed to 100 pL compound-loaded medium containing the 2 x serial dilutions of 31 or 31a with a concentration of 1.56-50 pM. DMSO was used to make the stock solution and the final concentration of DMSO was 0.5% in all wells, including the control group wells. After incubation for 24 hrs, 10 pL of CCK-8 was added to each well followed by further incubation for 2 hrs. Optical density at 450 nm was read with plate reader and cell viability was calculated according to the following equation:

0D _45Q of treatment well

Cell viability (%) x 100%

0D _45Q of control well

[0170] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced 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 was individually incorporated by reference.

Example 23. X-ray crystallography structural determination of the palladium complexes

[0171] 5a, 14, 31, and 31a were recrystallized by vapor-diffusion method (solvent system: dichloromethane-di ethyl ether). X-ray crystallography was conducted at Emory University. A suitable crystal with dimensions was selected and mounted on a loop with paratone on a Rigaku XtaLAB Synergy-S diffractometer (Tokyo, Japan) with Cu K« radiation ( =l.54184 A) or Mo Kct radiation (X=0.71073 A). The crystal was kept at a steady T = 100(1) K during data collection. The structure was solved with the ShelXT solution program using iterative methods and by using Olex2 1.3-alpha as the graphical interface. The model was refined with olex2. refine 1.3-alpha using full matrix least squares minimization on F2. [0172] Crystallographic data of 5a, 14, 31, and 31a have been deposited with the Cambridge Crystallographic Data Center as: CCDC 2181545-2181548.

Table 4. General comparison of the representative Pd-based CO probes and compounds designed herein.

SNR ^# (emission

„ Probe Fluorescence LOD* spectrum area-based

Reference structure profile (nm) (medium) or X _em max intensity¬ based)

Ila

*LOD: limit of detection. ^# SNR: signal to noise ratio (with > 20 equiv. excessive CO)