Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2020 Dec 9;5(50):32507–32514. doi: 10.1021/acsomega.0c04659

A Fluorescent Probe for Selective Facile Detection of H2S in Serum Based on an Albumin-Binding Fluorophore and Effective Masking Reagent

Suji Lee †, Dan-Bi Sung ‡, Jong Seok Lee ‡,§,*, Min Su Han †,*
PMCID: PMC7758950  PMID: 33376888

Abstract

graphic file with name ao0c04659_0008.jpg

A fluorescent probe (4-(2-(4-(diethylamino)phenyl)-4-methyl-5-oxo-4,5-dihydrothieno[3,2-b]pyridin-7-yl)phenyl 2,4-dinitrobenzenesulfonate, KF-DNBS) for facile detection of H2S in serum was developed based on the combination of an environment-sensitive fluorophore (2-(4-(diethylamino)phenyl)-7-(4-hydroxyphenyl)-4-methylthieno[3,2-b]pyridin-5(4H)-one, KF) with albumin and the 2,4-dinitrobenzene sulfonyl (DNBS) group as a recognition unit for H2S. KF-DNBS showed remarkable fluorescence enhancement due to H2S-triggered thiolysis followed by the formation of a fluorescent fluorophore (KF)-albumin complex. The H2S detection limit of KF-DNBS was estimated to be 3.2 μM, and KF-DNBS achieves a high selectivity to H2S over biothiols by employing 2-formyl benzene boronic acid (2-FBBA) as an effective masking reagent. Furthermore, under optimized sensing conditions, KF-DNBS could be applied to accurately determine spiked H2S in human serum without the need for any further procedure for the removal of serum proteins.

Introduction

Recently, hydrogen sulfide (H2S) has emerged as a significant endogenous gaseous mediator, like the already well-known nitric oxide (NO) and carbon monoxide (CO).1−3 H2S is produced through enzymatic reactions of cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST)/cysteine aminotransferase (CAT) in human tissue, and it is widely distributed in the human body in concentrations ranging from 10 to 100 μM.4 H2S has a variety of physiological functions, such as anti-inflammation, neuromodulation, and vasoregulation.5−9 Due to its clinical implications, the perturbed synthesis of endogenous H2S is closely associated with various human diseases. Recent studies have shown that abnormal serum levels of H2S are observed in several physiological disorders, such as Alzheimer’s disease, hypertension, diabetes, and asthma.2,10−14 Hence, the development of a reliable detection method for H2S in serum has great importance in pathology. Moreover, fast and real-time monitoring is desirable considering the rapid metabolism of H2S in physiological processes.

To date, a variety of analytical techniques such as spectrophotometry, electrochemical assay, and chromatography (including gas, ion-exchange, and variants of high-performance liquid chromatography (HPLC)) have been reported for H2S detection.15−19 Among them, two common methods have been widely used for measuring H2S levels in serum: a colorimetric method using methylene blue (MB method) and an ion-selective electrode (ISE)-based sulfide anion (S2–)-specific method.20−22 Both methods are performed under harsh chemical conditions that may lead to overestimation, and they also possess several practical drawbacks, such as tedious sample processing and the requirement of sophisticated instruments.

In contrast, fluorescent small-molecule probes have great potential for real-time monitoring of H2S in terms of their simplicity, rapid response, and high sensitivity.23−26 In past decades, various reaction-based H2S fluorescent probes have been developed based on the intrinsic reactivity of H2S as a good reducing agent or a good nucleophile. The majority of them employed photoinduced electron transfer (PET) modulated by an electron-withdrawing azide or 2,4-dinitrobenzyl group, which are commonly used in H2S probes.27−33 A H2S-triggered reaction releases the fluorophore, and this is followed by a turn-on fluorescence response. Such fluorophores have shown successful performance in the detection of H2S at low levels. However, most of them are focused on the fluorescence imaging of H2S, and it is intricate to be applied for the measurement of H2S levels in serum samples since they suffer signal interference due to nonspecific binding of the fluorophore with serum proteins (Scheme 1a). This problem is found in most prevalent fluorophores, such as coumarin, BODIPY, rosamine, and so on.34−37 To avoid signal interference, which could lead to inaccurate measurement of H2S in serum, an additional process to remove the large amounts of proteins in serum samples before H2S measurement is essential. In addition, many thiolysis-based probes are susceptible to interference from other biothiols present in high concentrations in serum, such as cysteine (Cys) and homocysteine (Hcy), which have similar reactivity to H2S. Thus, it is still necessary to develop a highly selective fluorescent probe that is readily applicable to H2S quantitation in serum samples.

Scheme 1. (a) Summary of Common Problems in Application of Conventional Fluorescent Probes for H2S in Serum. (b) Schematic Illustration of Application of the Fluorescent KF-DNBS Probe for Facile H2S Detection in Serum Using H2S-Triggered Cascade Formation of the Fluorescent KF-Albumin Complex.

Scheme 1

Open in a new tab

Recently, we have reported a new sensing strategy to detect enzyme activity in human serum using a fluorescent probe.38 A caged fluorophore was designed as a probe by conjugation of an enzyme-selective recognition unit with an environment-sensitive fluorophore. The fluorescence of the latter strongly depends on the specific binding of the fluorophore to human serum albumin (HSA), which is present in serum at a very high concentration. The caged fluorophore enabled quantitative detection of the serum enzyme without any pretreatment. In this study, we utilized the strategy to develop a fluorescent chemodosimeter for H2S detection without interference from serum proteins and prepared a caged fluorophore (KF-DNBS) by conjugating an environment-sensitive thienopyridinone derivative (KF) with the 2,4-dinitrobenzene sulfonyl (DNBS) group.39 The fluorescence of KF strongly depends on its specific binding to HSA, and the DNBS group is sensitively cleaved by H2S. KF-DNBS showed weak fluorescence intensity in the absence and presence of albumin. As illustrated in Scheme 1b, in response to H2S, the DNBS group in KF-DNBS was cleaved by thiolysis, thus releasing KF, which immediately combined with albumin, resulting in significant fluorescence enhancement. Based on this H2S-triggered cascade formation of the fluorescent KF-albumin complex, KF-DNBS could be used in the quantitative detection of H2S in physiological conditions and could also be applied to serum samples without additional processing for the removal of serum proteins.

Results and Discussion

Design and Synthesis of KF-DNBS

Serum contains various proteins, such as albumin, globulin, transferrin, and fibrinogen.40 Among them, albumin is present at the highest concentration of 600 μM or more. In the presence of other serum proteins, KF, which is an environment-sensitive fluorophore derived from thienopyridinone, originally exhibited weak fluorescence, but this increased immediately when 2 equiv. of HSA was added, as shown in Figure S1. In addition, a binding constant (Ka) of KF for HSA based on the Benesi–Hildebrand plot was determined to be Ka = 1.9 × 104 M–1 showing strong binding between KF and HSA (Figure S2).41 By utilizing the albumin-specific fluorescence turn-on response of KF, we designed caged KF as a fluorescent probe for facile H2S detection in serum and synthesized the KF-DNBS probe through modification of the phenolic site of KF with a well-known H2S recognition unit, namely, the DNBS group (Scheme 2).

Scheme 2. Synthetic Route for KF-DNBS.

Scheme 2

Open in a new tab

Then, we compared the fluorescence responses of KF and KF-DNBS to HSA. As shown in Figure 1, the fluorescence intensity of KF shifted slightly from 550 to 500 nm by addition of HSA and increased linearly with increasing HSA concentration in the range of 5–50 μM (Figure S3). In contrast to KF, KF-DNBS showed weak fluorescence with or without excess HSA. Also, the fluorescence of KF and KF-DNBS remained constant before and after irradiation or heating showing excellent photo- and thermostability (Figure S4). Based on these results, we envisioned that KF-DNBS with HSA could be utilized as a reaction-based fluorescent probe for H2S using the principle of H2S-triggered cascade formation of the fluorescent KF-HSA complex (Φ = 0.546).

Figure 1.

Figure 1

Open in a new tab

Fluorescence spectra of KF and KF-DNBS (each 25 μM, 10% DMSO) in the absence and presence of HSA (100 μM), λex = 420 nm; fluorescence image of KF in the absence and presence of HSA under handheld UV lamp (365 nm) illumination (inset).

Optimization of Sensing Conditions for Selective H2S Detection

The 2,4-dinitrosulfonyl unit including DNBS has been the most frequently used H2S recognition unit in H2S-reactive fluorescent probes. However, these probes usually possessed moderate selectivity because of interference from other biothiols, such as Cys, Hcy, and GSH. Thus, it is desirable to establish the optimal sensing conditions for highly selective H2S detection by KF-DNBS, especially for reliable application in serum samples containing high concentrations of Cys and Hcy. First, we evaluated the relative reactivity of H2S and other biothiols, including Cys, Hcy, and GSH, to KF-DNBS considering their approximate concentrations in human serum ([H2S] = 100 μM, [Cys] = 250 μM, [Hcy] = 100 μM, [GSH] = 10 μM).42 As shown in Figure 2a, when H2S was added to the sample solution containing KF-DNBS with HSA in SPB (pH 7.4), the fluorescence intensity at 500 nm increased significantly as we expected. However, Cys and Hcy also induced a noticeable fluorescence change.

Figure 2.

Figure 2

Open in a new tab

Change of fluorescence intensity of KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) upon addition of each biothiol, H2S (100 μM), Cys (250 μM), Hcy (100 μM), GSH (10 μM) in SPB (pH 7.4, 20 mM), (a) without 2-FBBA and (b) with 2-FBBA (2 mM), λex = 420 nm, 37 °C.

To solve this problem, we tried to introduce a masking reagent, which can block the nucleophilic reactivity of Cys and Hcy selectively by the formation of a stable covalent bond. Since the cyclization reaction between aldehyde groups and Cys or Hcy has been widely used in selective probe molecule design, we investigated simple aldehydes, and 2-formyl benzene boronic acid (2-FBBA) was chosen as a potential masking reagent. 2-FBBA is a well-known reagent used in facile and selective bioconjugation of N-terminal Cys in proteins at neutral pH.43,44 It enables very rapid formation of a stable thiazolidino boronate complex with the boronic acid moiety by means of a B–N dative bond. We envisioned that the introduction of 2-FBBA would improve the selectivity of KF-DNBS to H2S by blocking the reactivity of Cys and Hcy based on the fast and chemoselective reaction of 2-FBBA with Cys and Hcy.

We confirmed the kinetics and selectivity of the reaction between 2-FBBA and biothiols by monitoring the intrinsic absorption of 2-FBBA, which decreased as the aldehyde in 2-FBBA converted to thiazolidine.43 As shown in Figure S6, the rapid (<100 s) decrease of absorbance at 254 nm was observed only with Cys and Hcy, indicating a rapid and selective reaction with Cys and Hcy. Next, the capability of 2-FBBA as a masking reagent for selective H2S detection using KF-DNBS was investigated. KF-DNBS with HSA in the presence of 2-FBBA showed superior selectivity to H2S over other biothiols (Figure 2b). 2-FBBA was able to completely block the reactivity of Cys and Hcy upon KF-DNBS. These results showed, for the first time, that 2-FBBA could be used as an effective masking reagent for Cys and Hcy in thiolysis-based H2S probes. We investigated further by using KF-DNBS as a H2S probe under the following conditions: 25 μM KF-DNBS, 100 μM HSA, and 2-FBBA (2 mM) in SPB (pH 7.4, 20 mM).

Sensing Behavior of KF-DNBS with HSA as a Selective H2S Probe

The feasibility of KF-DNBS with HSA as a H2S probe was investigated under the optimized measurement conditions. First, we examined the spectral change of KF-DNBS with HSA in response to H2S. When H2S was added to the sensing system containing KF-DNBS with HSA and the masking reagent 2-FBBA in SPB, the intrinsic absorption peak of the probe (λabs = 420 nm) gradually increased and significant enhancement of fluorescence intensity at 500 nm appeared immediately (Figure S7, Figure 3a). The intensity reached the maximum value within 20 min. As shown in Figure 3b, the fluorescence intensity of KF-DNBS with HSA increased linearly with increasing H2S concentration (5–100 μM). The detection limit (3σ/slope) was determined to be 3.2 μM, which is reliable for the measurement of serum H2S levels. In addition, the fluorescence change of the sample solution could be monitored by the naked eye, and it was confirmed that KF-DNBS with HSA worked well in a broad pH range from pH 5 to pH 9 (Figure 3c).

Figure 3.

Figure 3

Open in a new tab

(a) Fluorescence spectral change of KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) in the absence and presence of H2S (100 μM) in SPB (pH 7.4, 20 mM) containing 2-FBBA (2 mM); the corresponding fluorescence image taken under handheld UV lamp (365 nm) illumination after 5 min (inset). (b) Plot of fluorescence intensity at 500 nm of KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) versus different concentrations of H2S (5–250 μM) in SPB (pH 7.4, 20 mM) containing 2-FBBA (2 mM). (c) Fluorescence intensity at 500 nm of KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) in the absence and presence of H2S (100 μM) in various buffer conditions (pH 5–9, 20 mM), λex = 420 nm, 37 °C.

To further confirm that the turn-on fluorescence was due to the H2S-triggered cascade formation of the fluorescent KF-HSA complex, we confirmed the product of the thiolysis reaction of KF-DNBS by HPLC and 1H NMR studies. In the HPLC chromatogram, shown in Figure S8, a peak with a retention time consistent with that of KF appeared when KF-DNBS was incubated with H2S in SPB solution and gradually increased with increasing H2S concentration. Additionally, the 1H NMR spectrum of the sample solution containing KF-DNBS with H2S showed that the peaks of the 2,4-dinitrophenyl by-product (δ 8.95, 8.45) increased (Figure S9). Based on these results, we established that the fluorescence response of KF-DNBS with HSA to H2S was caused by thiolysis-induced KF release followed by the formation of a fluorescent KF-HSA complex.

Next, the selectivity of KF-DNBS with HSA toward H2S was investigated using various biologically relevant species, including biothiols (Cys, Hcy, GSH), reactive sulfur species (RSS) and reactive oxygen species (ROS) (HSO4–, SO42–, SO32–, S2O32–, SCN–, H2O2, ClO–), and anions (CN–, F–, Br–, NO3–, NO2–, HCO3–, CH3CO2–). As shown in Figure 4, the remarkable fluorescence enhancement was observed only for H2S. The specific response to H2S suffered no interference by other analytes (Figure S10), and we could confirm the selectivity for H2S over other biothiols with the naked eye. This result demonstrated that KF-DNBS with HSA and the masking reagent 2-FBBA exhibits a high selectivity toward H2S and would work well in complex serum samples containing many biothiols and other reactive species.

Figure 4.

Figure 4

Open in a new tab

Fluorescence intensity at 500 nm of KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) in the presence of various analytes: 1: blank, 2: H2S (100 μM), 3: Cys (250 μM), 4: Hcy (100 μM), 5: GSH (10 μM), 6: HSO4– (100 μM), 7: SO42– (100 μM), 8: SO32– (100 μM), 9: S2O32– (100 μM), 10: SCN– (100 μM), 11: CN– (100 μM), 12: F– (100 μM), 13: Br– (100 μM), 14: NO3– (100 μM), 15: NO2– (100 μM), 16: HCO3– (100 μM), 17: CH3CO2– (100 μM), 18: H2O2 (100 μM), 19: ClO– (100 μM) in SPB (pH 7.4, 20 mM), λex = 420 nm, 37 °C; the corresponding fluorescence image after 5 min under handheld UV lamp (365 nm) illumination (inset).

Application of KF-DNBS in Quantitative Detection of H2S in Human Serum

To explore the potential applicability of KF-DNBS to facile detection of H2S in serum, the fluorescence response of KF-DNBS to H2S-spiked human serum samples was investigated. Human serum (purchased from Sigma Aldrich) was spiked with different concentrations of H2S (25, 50, 100, 150 μM). The serum samples, without any pretreatment, were directly added to a solution containing 2-FBBA in SPB. Since it has been reported that the concentration of HSA in human serum ranges from 550 to 800 μM, there was no need to add HSA to the sample solutions. After the addition of KF-DNBS, the change in fluorescence intensity of the sample solution was measured. Based on the calibration curve obtained from the plot of the initial rate of fluorescence change for 10 min versus the concentration of H2S using KF-DNBS with HSA (Figure S11), we could determine the spiked H2S level in HSA and the recovery ranged from 95 to 109%, as summarized in Table 1.

Table 1. Determination of Spiked H2S in Human Serum Using KF-DNBS.

  added (μM) found (μM) recovery (%) RSDa (%)
human serum 25 26 106 13
50 51 101 10
100 95 95 11
150 163 109 12
Open in a new tab
a

Relative standard deviation.

This result showed that the fluorescence response of KF-DNBS to spiked H2S was unaffected by the various analytes present in human serum, including high concentrations of biothiols as well as proteins, even though no additional process was performed prior to sample measurement. Thus, the fluorescence response of KF-DNBS could be utilized as an accurate and facile method for measuring H2S levels in serum samples.

Conclusions

We developed a highly selective fluorescent probe KF-DNBS for H2S detection based on a H2S-triggered thiolysis reaction. Upon addition of H2S, KF-DNBS with HSA showed rapid (within 5 min) and remarkable fluorescence enhancement and responded to very low concentrations of H2S (LOD = 3.2 μM). KF-DNBS exhibited high selectivity toward H2S, especially over other biothiols including Cys, Hcy, and GSH, by employing 2-FBBA as an effective masking reagent for Cys and Hcy. 2-FBBA completely blocked the reactivity of Cys and Hcy by a fast and chemoselective reaction. Moreover, KF-DNBS could be applied for the simple, quantitative analysis of spiked H2S in human serum, without the need for any additional protein removal procedure. Consequently, this study presents a facile method to detect H2S in serum samples in various H2S-related diseases. We also demonstrated, for the first time, the application of 2-FBBA as a Cys/Hcy-masking reagent in H2S probes, as a valuable approach to improve the selectivity of many thiolysis-based H2S sensing systems.

Experimental Section

Materials and Instruments

All the biological analytes and other chemical reagents were purchased from commercial suppliers (Sigma Aldrich, Tokyo Chemical Industry, Alfa Aesar, and UChem) and used without further purification. All melting points were recorded on a micromelting point apparatus and are stated uncorrected. Reactions were monitored by thin layer chromatography (TLC) with 0.25 mm precoated silica gel plates (Kieselgel 60F254). Flash column chromatography was performed on silica gel (70–230 mesh) using distilled organic solvents. Fluorescence spectra were recorded using a Cytation 3 Multi-Mode Reader and an Agilent Cary Eclipse fluorescence spectrophotometer. All UV–vis spectra were recorded using an Agilent Cary 8454 UV–vis spectrophotometer. 1H NMR and 13C{1H} NMR spectra were obtained using a Bruker Advance III HD 600 MHz spectrometer. Chemical shifts are reported as δ (ppm) values relative to chloroform (CDCl3, δ 7.260) and dimethyl sulfoxide (DMSO-d6, δ 2.50), and coupling constants are reported in Hz. High-resolution mass spectroscopy (HRMS) data were obtained using a magnetic sector-electric sector double focusing mass analyzer at the Korea Basic Science Institute (KBSI) in electron spray ionization (ESI) mode and measured by time of flight (TOF) in ESI mode on an SCIEX X500R QTOF.

Synthesis of the Probe

The synthetic route of probe KF-DNBS is shown in Scheme 2. The key intermediate compound 5a was synthesized according to the modified method from the previously reported methods.45

Synthesis of Methyl 5-(4-(Diethylamino)phenyl)-3-(methylamino)thiophene-2-carboxylate (3a)

To a solution of 2a (1.04 g, 4.14 mmol, 1.0 equiv.) in 1,2-dimethoxyethane (13.8 mL, 0.3 M), Pd(PPh3)4 (239.4 mg, 0.2072 mmol, 0.05 equiv.), N,N-diethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1.37 g, 4.97 mmol, 1.2 equiv.), K2CO3 (1.72 g, 12.43 mmol, 3.0 equiv.), and H2O (0.3 mL) were added. The reaction mixture was heated at 80 °C and stirred for 12 h. After completion of the reaction, the mixture was filtered through celite and extracted with H2O/EtOAc three times. The organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The crude was purified by flash column chromatography with n-hexane:EtOAc (5:1) on silica to afford 3a (1.26 g, 3.95 mmol, 96%) as a yellow solid. 1H NMR (600 MHz, CDCl3) δ 7.50 (d, J = 9.0 Hz, 2H), 6.70 (s, 1H), 6.65 (d, J = 9.0 Hz, 2H), 3.81 (s, 3H), 3.33 (q, J = 7.0 Hz, 4H), 3.02 (d, J = 4.8 Hz, 3H), 1.19 (t, J = 7.2 Hz, 6H); 13C{1H} NMR (150 MHz, CDCl3) δ 165.3, 158.0, 151.4, 148.3, 127.3, 120.5, 111.4, 108.7, 94.9, 50.9, 44.4, 31.6, 12.6; Data are consistent with those reported in the literature.45

Synthesis of 6-Bromo-2-(4-(diethylamino)phenyl)-7-(4-methoxyphenyl)-4-methylthieno[3,2-b]pyridin-5(4H)-one (7a)

To a solution of 5a (150 mg, 0.5760 mmol, 1.0 equiv.) in DMF (2.3 mL, 0.25 M), 4-methoxycinnamic acid (225.8 mg, 1.267 mmol, 2.2 equiv.), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP, 560 mg, 1.267 mmol, 2.2 equiv.), and DIPEA (0.51 mL, 2.880 mmol, 5.0 equiv.) were added at room temperature. The mixture was stirred at room temperature for 5 h. The reaction was quenched with H2O and extracted with ethyl acetate (EtOAc) three times. The organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The crude was used without further purification. To a solution of mixture 6a (188.0 mg, 0.4813 mmol, 1.0 equiv.) in CH2Cl2 (4.8 mL, 0.1 M), N-bromosuccinimide (128.5 mg, 0.7221 mmol, 1.5 equiv.) was added. The mixture was stirred at room temperature for 1 h. The reaction was quenched with H2O and extracted with EtOAc three times. The organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The crude was purified by flash column chromatography with n-hexane:EtOAc (1:1) on silica to afford 7a as a yellow solid (22.4 mg, 0.0532 mmol, 48%, over two steps); mp: 212–214 °C; 1H NMR (600 MHz, CDCl3) δ 7.45–7.41 (m, 4H), 7.05 (s, 1H), 7.03 (d, J = 8.4 Hz, 2H), 6.62 (d, J = 9.0 Hz, 2H), 3.88 (s, 3H), 3.82 (s, 3H), 3.38 (q, J = 7.0 Hz, 4H), 1.18 (t, J = 6.9 Hz, 6H); 13C{1H} NMR (150 MHz, CDCl3) δ 160.2, 159.3, 150.8, 148.6, 146.4, 143.2, 130.2, 129.9, 127.3, 119.9, 118.4, 114.1, 111.6, 111.3, 108.7, 55.4, 44.5, 33.6, 12.7; HRMS (EI): m/z calcd for C25H25BrN2NaO2S [M + Na]+ 519.0718, found 519.0719.

Synthesis of 2-(4-(Diethylamino)phenyl)-7-(4-methoxyphenyl)-4-methylthieno[3,2-b]pyridin-5(4H)-one (8a)

A solution of 7a (36.1 mg, 0.0726 mmol, 1.0 equiv.) in CH2Cl2 (0.73 mL, 0.1 M) was cooled down to −78 °C. Next, n-butyllithium solution (2.0 M in cyclohexane, 75 μL, 0.109 mmol, 1.5 equiv.) was slowly added and stirred at −78 °C for 1.5 h. The reaction was quenched with H2O. The mixture was extracted with CH2Cl2 three times. The organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The crude was purified by flash column chromatography with n-hexane:EtOAc (1:1 to 100% EtOAc) on silica to afford 8a as a yellow solid (15.8 mg, 0.0377 mmol, 52%); mp: 190–192 °C; 1H NMR (600 MHz, CDCl3) δ 7.64 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.10 (s, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 6.51 (s, 1H), 3.88 (s, 3H), 3.76 (s, 3H), 3.40 (q, J = 7.2 Hz, 4H), 1.19 (t, J = 6.9 Hz, 6H); 13C{1H} NMR (150 MHz, CDCl3) δ 163.0, 160.7, 150.1, 148.5, 146.7, 145.3, 130.1, 129.1, 127.4, 120.3, 116.4, 114.5, 113.3, 111.7, 109.2, 55.5, 44.6, 31.9, 12.7; HRMS (EI): m/z calcd for C25H26N2O2S [M]+ 418.1715, found 418.1711.

Synthesis of 2-(4-(Diethylamino)phenyl)-7-(4-hydroxyphenyl)-4-methylthieno[3,2-b]pyridin-5(4H)-one (KF)

A solution of 8a (109.8 mg, 0.2623 mmol, 1.0 equiv.) in CH2Cl2 (2.6 mL, 0.1 M) was cooled down to −78 °C. After cooling, boron tribromide in CH2Cl2 solution (3.94 mL, 3.935 mmol, 15.0 equiv.) was added slowly, dropwise, to the solution. Next, the mixture was warmed up to room temperature and stirred for 12 h. After the solution was cooled down to −78 °C, the reaction was quenched with iced water and neutralized with NaHCO3. The mixture was extracted with CH2Cl2 three times. The organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The crude was purified by flash column chromatography with n-hexane:EtOAc (1:1 to 100% EtOAc) on silica to afford KF as a yellow solid (83.0 mg, 0.2052 mmol, 78%); mp: 250–251 °C; 1H NMR (600 MHz, DMSO-d6) δ 9.94 (s, 1H), 7.60–7.58 (m, 3H), 7.56 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 9.0 Hz, 2H), 6.27 (s, 1H), 3.65 (s, 3H), 3.38 (q, J = 7.0 Hz, 4H), 1.11 (t, J = 7.2 Hz, 6H); 13C{1H} NMR (150 MHz, DMSO-d6) δ 161.4, 158.8, 148.7, 148.0, 145.9, 145.5, 128.8, 127.6, 127.0, 119.2, 115.8, 114.0, 111.8, 111.4, 110.4, 43.7, 31.4, 12.4; HRMS (EI): m/z calcd for C24H24N2O2S [M]+ 404.1558, found 404.1555.

Synthesis of 4-(2-(4-(Diethylamino)phenyl)-4-methyl-5-oxo-4,5-dihydrothieno[3,2-b]pyridin-7-yl)phenyl 2,4-Dinitrobenzenesulfonate (KF-DNBS)

To a solution of KF (106.5 mg, 0.2633 mmol, 1.0 equiv.) in CH2Cl2 (2.6 mL, 0.1 M), 2,4-dinitrobenzenesulfonyl chloride (119.3 mg, 0.4476 mmol, 1.7 equiv.) and triethylamine (0.13 mL, 1.250 mmol, 4.7 equiv.) were added. The mixture was stirred at room temperature for 16 h. The reaction was quenched with H2O and extracted with CH2Cl2 three times. The organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The crude was purified by flash column chromatography with n-hexane:EtOAc (1:1 to 1:5) on silica to afford KF-DNBS as a dark brown solid (108.6 mg, 0.1711 mmol, 65%, conversion: 82%, borsm yield: 79%); mp: 96–98 °C; 1H NMR (600 MHz, CDCl3) δ 8.67 (s, H), 8.51 (d, J = 8.4 Hz, 2H), 8.25 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 7.2 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 6.65 (br s, 2H), 6.44 (s, 1H), 3.73 (s, 3H), 3.41 (q, J = 7.2 Hz, 4H), 1.12 (t, J = 6.9 Hz, 6H); 13C{1H} NMR (150 MHz, CDCl3) δ 162.6, 151.1, 150.6, 149.4, 149.1, 148.7, 145.6, 137.7, 134.0, 133.6, 129.7, 127.4, 126.7, 122.7, 120.5, 119.7, 115.4, 114.1, 111.6, 109.1, 44.5, 31.9, 12.6; HRMS (ESI): m/z calcd for C30H27N4O8S2 [M + H]+ 635.1270, found 635.1262.

Comparison of the Fluorescence Change of KF and KF-DNBS with HSA

Stock solutions of KF and KF-DNBS were prepared in DMSO, and a stock solution of HSA was prepared in distilled water. Blanks, each containing only KF or KF-DNBS (25 μM, 10% DMSO), and samples, containing each KF or KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) in sodium phosphate buffer (SPB, pH 7.4, 20 mM), were prepared. Fluorescence spectra were then recorded using the fluorescence spectrophotometer under excitation at 420 nm.

Optimization of Sensing Conditions for Selective H2S Detection over Cys and Hcy

The samples containing HSA (100 μM) and each biothiol (H2S 100 μM, Cys 250 μM, Hcy 100 μM, GSH 10 μM) with and without 2-FBBA (2 mM) in SPB (pH 7.4, 20 mM) were incubated for 15 min at 25 °C. KF-DNBS (25 μM, 10% DMSO) was added to the samples followed by the measurement of fluorescence spectra under excitation at 420 nm at 37 °C.

Determination of the Limit of Detection (LOD) for H2S

Fluorescence spectra of KF-DNBS (25 μM, 10% DMSO) with HSA (100 μM) containing various concentrations of H2S (0, 5, 10, 20, 40, 60, 80, 100, 150, 250 μM) in SPB (pH 7.4, 20 mM) were recorded under excitation at 420 nm for 40 min at 5 min intervals at 37 °C. The experiment was repeated three times. The LOD was calculated using 3σ/slope based on the titration experiment, in which σ is the standard deviation of the blank measurements and the slope value is obtained from a plot of the fluorescence intensity versus H2S concentration.

pH Dependency of KF-DNBS with HSA upon H2S

Samples containing HSA (100 μM) and 2-FBBA (2 mM) without and with H2S (100 μM) in different buffer conditions (pH 5 acetate, pH 6–8 SPB, pH 9 Tris; 20 mM) were incubated for 15 min at 25 °C. KF-DNBS (25 μM, 10% DMSO) was added to the samples followed by the measurement of the fluorescence intensity at 500 nm after 10 min under excitation at 420 nm at 37 °C. The experiment was repeated three times.

Selectivity of KF-DNBS toward H2S over Other Biological Analytes

A blank containing no analyte and a sample containing each analyte (H2S 100 μM, Cys 250 μM, Hcy 100 μM, GSH 10 μM, HSO4– 100 μM, SO42– 100 μM, SO32– 100 μM, S2O32– 100 μM, SCN– 100 μM, CN– 100 μM, F– 100 μM, Br– 100 μM, NO3– 100 μM, NO2– 100 μM, HCO3– 100 μM, CH3CO2– 100 μM, H2O2 100 μM, ClO– 100 μM) were prepared followed by the addition of HSA (100 μM) and 2-FBBA (2 mM) in SPB (pH 7.4, 20 mM). After incubation for 15 min at 25 °C, KF-DNBS (25 μM, 10% DMSO) was added to each sample, and then fluorescence intensity at 500 nm was recorded using the fluorescence spectrophotometer under excitation at 420 nm at 37 °C. The experiment was repeated three times.

Quantitative Detection of Spiked H2S in Human Serum

Human serum samples spiked with different concentrations of H2S (25, 50, 100, 150 μM) were prepared. A sample containing HSA (100 μM), 2-FBBA (2 mM), and H2S-spiked serum sample (20%) in SPB (pH 7.4, 20 mM) was incubated for 15 min at 25 °C. KF-DNBS (25 μM, 10% DMSO) was added to the sample, and the fluorescence intensity at 500 nm was recorded using the fluorescence spectrophotometer under excitation at 420 nm at 37 °C. The recovery was calculated based on the standard curve obtained from H2S titration. The experiment was repeated three times.

Acknowledgments

This work was supported by research grants from Korea Institute of Ocean Science and Technology (PE99721).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c04659.

  • Fluorescence-based screening of KF against major serum proteins; comparison of the fluorescence change of KF and KF-DNBS with HSA; photo/thermostability of KF and KF-DNBS; quantum yields of KF and KF-HSA complex; kinetics of the reaction of 2-FBBA with biothiols (H2S, Cys, Hcy, GSH); absorption spectral change of the probe KF-DNBS to H2S; mechanism studies; competition study; calibration curve for quantitative analysis of H2S in real samples; 1H, 13C{1H} NMR, and HRMS spectral data (PDF)

Author Contributions

∥ S.L. and D.-B.S. contributed equally.

The authors declare no competing financial interest.

Supplementary Material

ao0c04659_si_001.pdf (1.9MB, pdf)

References

  1. Olson K. R. Is hydrogen sulfide a circulating “gasotransmitter” in vertebrate blood?. Biochim. Biophys. Acta 2009, 1787, 856–863. 10.1016/j.bbabio.2009.03.019. [DOI] [PubMed] [Google Scholar]
  2. Wang R. Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol. Rev. 2012, 92, 791–896. 10.1152/physrev.00017.2011. [DOI] [PubMed] [Google Scholar]
  3. Olson K. R. A Practical Look at the Chemistry and Biology of Hydrogen Sulfide. Antioxid. Redox Signaling 2012, 17, 32–44. 10.1089/ars.2011.4401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Whiteman M.; Le Trionnaire S.; Chopra M.; Fox B.; Whatmore J. Emerging role of hydrogen sulfide in health and disease: critical appraisal of biomarkers and pharmacological tools. Clin. Sci. 2011, 121, 459–488. 10.1042/CS20110267. [DOI] [PubMed] [Google Scholar]
  5. Lefer D. J. A new gaseus signaling molecule emerges: Cardioprotective role of hydrogen sulfide. Proc. Natl. Acad. Sci. 2007, 104, 17907–17908. 10.1073/pnas.0709010104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Papapetropoulos A.; Pyriochou A.; Altaany Z.; Yang G.; Marazioti A.; Zhou Z.; Jeschke M. G.; Branski L. K.; Herndon D. N.; Wang R.; Szabó C. Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc. Natl. Acad. Sci. 2009, 106, 21972–21977. 10.1073/pnas.0908047106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Li L.; Rose P.; Moore P. K. Hydrogen Sulfide and Cell Signaling. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169–187. 10.1146/annurev-pharmtox-010510-100505. [DOI] [PubMed] [Google Scholar]
  8. Kolluru G. K.; Shen X.; Bir S. C.; Kevil C. G. Hydrogen sulfide chemical biology: Pathophysiological roles and detection. Nitric Oxide 2013, 35, 5–20. 10.1016/j.niox.2013.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Paul B. D.; Snyder S. H. Gasotransmitter hydrogen sulfide signaling in neuronal health and disease. Biochem. Pharmacol. 2018, 149, 101–109. 10.1016/j.bcp.2017.11.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eto K.; Asada T.; Arima K.; Makifuchi T.; Kimura H. Brain hydrogen sulfide is severely decreased in Alzheimer’s disease. Biochem. Biophys. Res. Commun. 2002, 293, 1485–1488. 10.1016/S0006-291X(02)00422-9. [DOI] [PubMed] [Google Scholar]
  11. Yang G.; Wu L.; Jiang B.; Yang W.; Qi J.; Cao K.; Meng Q.; Mustafa A. K.; Mu W.; Zhang S.; Snyder S. H.; Wang R. H2S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine γ-Lyase. Science 2008, 322, 587–590. 10.1126/science.1162667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jain S. K.; Bull R.; Rains J. L.; Bass P. F.; Levine S. N.; Reddy S.; McVie R.; Bocchini J. A. Jr. Low Levels of Hydrogen Sulfide in the Blood of Diabetes Patients and Streptozotocin-Treated Rats Causes Vascular Inflammation?. Antioxid. Redox Signaling 2010, 12, 1333–1337. 10.1089/ars.2009.2956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wang P.; Zhang G.; Wondimu T.; Ross B.; Wang R. Hydrogen sulfide and asthma. Exp. Physiol. 2011, 96, 847–852. 10.1113/expphysiol.2011.057448. [DOI] [PubMed] [Google Scholar]
  14. Zhao K.; Li H.; Li S.; Yang G. Regulation of cystathionine gamma-lyase/H2S system and its pathological implication. Front. Biosci. 2014, 19, 1355–1369. 10.2741/4286. [DOI] [PubMed] [Google Scholar]
  15. Ubuka T.; Abe T.; Kajikawa R.; Morino K. Determination of hydrogen sulfide and acid-labile sulfur in animal tissues by gas chromatography and ion chromatography. J. Chromatogr., B 2001, 757, 31–37. 10.1016/S0378-4347(01)00046-9. [DOI] [PubMed] [Google Scholar]
  16. Shen X.; Pattillo C. B.; Pardue S.; Bir S. C.; Wang R.; Kevil C. G. Measurement of plasma hydrogen sulfide in vivo and in vitro. Free Radical Biol. Med. 2011, 50, 1021–1031. 10.1016/j.freeradbiomed.2011.01.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shen X.; Peter E. A.; Bir S.; Wang R.; Kevil C. G. Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radical Biol. Med. 2012, 52, 2276–2283. 10.1016/j.freeradbiomed.2012.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Deng J.; Lu Q.; Hou Y.; Liu M.; Li H.; Zhang Y.; Yao S. Nanosensor Composed of Nitrogen-Doped Carbon Dots and Gold Nanoparticles for Highly Selective Detection of Cysteine with Multiple Signals. Anal. Chem. 2015, 87, 2195–2203. 10.1021/ac503595y. [DOI] [PubMed] [Google Scholar]
  19. Xu T.; Scafa N.; Xu L.-P.; Zhou S.; Al-Ghanem K. A.; Mahboob S.; Fugetsu B.; Zhang X. Electrochemical hydrogen sulfide biosensors. Analyst 2016, 141, 1185–1195. 10.1039/C5AN02208H. [DOI] [PubMed] [Google Scholar]
  20. Fogo J. K.; Popowsky M. Spectrophotometric Determination of Hydrogen Sulfide. Anal. Chem. 1949, 21, 732–734. 10.1021/ac60030a028. [DOI] [Google Scholar]
  21. Lindell H.; Jäppinen P.; Savolainen H. Determination of Sulphide in Blood with an Ion-selective Electrode by Pre-concentration of Trapped Sulphide in Sodium Hydroxide Solution. Analyst 1988, 113, 839–840. 10.1039/AN9881300839. [DOI] [PubMed] [Google Scholar]
  22. Nagy P.; Pálinkás Z.; Nagy A.; Budai B.; Tóth I.; Vasas A. Chemical aspects of hydrogen sulfide measurements in physiological samples. Biochim. Biophys. Acta 2014, 1840, 876–891. 10.1016/j.bbagen.2013.05.037. [DOI] [PubMed] [Google Scholar]
  23. Jung H. S.; Chen X.; Kim J. S.; Yoon J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem. Soc. Rev. 2013, 42, 6019–6031. 10.1039/c3cs60024f. [DOI] [PubMed] [Google Scholar]
  24. Lin V. S.; Chen W.; Xian M.; Chang C. J. Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems. Chem. Soc. Rev. 2015, 44, 4596–4618. 10.1039/C4CS00298A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wu D.; Sedgwick A. C.; Gunnlaugsson T.; Akkaya E. U.; Yoon J.; James T. D. Fluorescent chemosensors: the past, present and future. Chem. Soc. Rev. 2017, 46, 7105–7123. 10.1039/C7CS00240H. [DOI] [PubMed] [Google Scholar]
  26. Yin G.; Niu T.; Yu T.; Gan Y.; Sun X.; Yin P.; Chen H.; Zhang Y.; Li H.; Yao S. Simultaneous Visualization of Endogenous Homocysteine, Cysteine, Glutathione, and their Transformation through Different Fluorescence Channels. Angew. Chem., Int. Ed. 2019, 58, 4557–4561. 10.1002/anie.201813935. [DOI] [PubMed] [Google Scholar]
  27. Montoya L. A.; Pluth M. D. Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells. Chem. Commun. 2012, 48, 4767–4769. 10.1039/c2cc30730h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Qian M.; Zhang L.; Pu Z.; Xia J.; Chen L.; Xia Y.; Cui H.; Wang J.; Peng X. A NIR fluorescent probe for the detection and visualization of hydrogen sulfide using the aldehyde group assisted thiolysis of dinitrophenyl ether strategy. J. Mater. Chem. B 2018, 6, 7916–7925. 10.1039/C8TB02218F. [DOI] [PubMed] [Google Scholar]
  29. Peng S.; Zhong T.; Guo T.; Shu D.; Meng D.; Liu H.; Guo D. A novel fluorescent probe for selective detection of hydrogen sulfide in living cells. New J. Chem. 2018, 42, 5185–5192. 10.1039/C7NJ04577H. [DOI] [Google Scholar]
  30. Ji Y.; Xia L.-J.; Chen L.; Guo X.-F.; Wang H.; Zhang H.-J. A novel BODIPY-based fluorescent probe for selective detection of hydrogen sulfide in living cells and tissues. Talanta 2018, 181, 104–111. 10.1016/j.talanta.2017.12.067. [DOI] [PubMed] [Google Scholar]
  31. Li Q.; Wang Z.; Zhao M.; Hong Y.; Jin Q.; Yao S.; Zheng C.; Quan Y.-Y.; Ye X.; Huang Z.-S. A NIR fluorescent probe for the detection and visualization of hydrogen sulfide in colorectal cancer cell. Sens. Actuators, B 2019, 298, 126898–126904. 10.1016/j.snb.2019.126898. [DOI] [Google Scholar]
  32. Wang K.-P.; Zhang Q.-L.; Wang X.; Lei Y.; Zheng W.-J.; Chen S.; Zhang Q.; Hu H.-Y.; Hu Z.-Q. A fluorescent probe based on tetrahydro[5]helicene derivative with large Stokes shift for rapid and highly selective recognition of hydrogen sulfide. Spectrochim. Acta, Part A 2019, 214, 487–495. 10.1016/j.saa.2019.02.079. [DOI] [PubMed] [Google Scholar]
  33. Zhu H.; Liang C.; Cai X.; Zhang H.; Liu C.; Jia P.; Li Z.; Yu Y.; Zhang X.; Sheng W.; Zhu B. Rational Design of a Targetable Fluorescent Probe for Visualizing H2S Production under Golgi Stress Response Elicited by Monensin. Anal. Chem. 2020, 92, 1883–1889. 10.1021/acs.analchem.9b04009. [DOI] [PubMed] [Google Scholar]
  34. Sunahara H.; Urano Y.; Kojima H.; Nagano T. Design and Synthesis of a Library of BODIPY-Based Environmental Polarity Sensors Utilizing Photoinduced Electron-Transfer-Controlled Fluorescence ON/OFF Switching. J. Am. Chem. Soc. 2007, 129, 5597–5604. 10.1021/ja068551y. [DOI] [PubMed] [Google Scholar]
  35. Ahn Y.-H.; Lee J.-S.; Chang Y.-T. Selective Human Serum Albumin Sensor from the Screening of a Fluorescent Rosamine Library. J. Comb. Chem. 2008, 10, 376–380. 10.1021/cc800017h. [DOI] [PubMed] [Google Scholar]
  36. Das D. K.; Mondal T.; Mandal A. K.; Bhattacharyya K. Binding of Organic Dyes with Human Serum Albumin: A Single-Molecule Study. Chem. – Asian J. 2011, 6, 3097–3103. 10.1002/asia.201100272. [DOI] [PubMed] [Google Scholar]
  37. Singh I.; Rani R.; Luxami V.; Paul K. Synthesis of 5-(4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzylidene) thiazolidine-2,4-dione as promising DNA and serum albumin-binding agents and evaluation of antitumor activity. Eur. J. Med. Chem. 2019, 166, 267–280. 10.1016/j.ejmech.2019.01.053. [DOI] [PubMed] [Google Scholar]
  38. Yoo S.; Han M. S. A fluorescent probe for butyrylcholinesterase activity in human serum based on a fluorophore with specific binding affinity for human serum albumin. Chem. Commun. 2019, 55, 14574–14577. 10.1039/C9CC07737E. [DOI] [PubMed] [Google Scholar]
  39. Lee S.; Sung D.-B.; Kang S.; Parameswaran S.; Choi J.-H.; Lee J. S.; Han M. S. Development of Human Serum Albumin Selective Fluorescent Probe Using Thieno[3,2-b]pyridine-5(4H)-one Fluorophore Derivatives. Sensors 2019, 19, 5298–5310. 10.3390/s19235298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Adkins J. N.; Varnum S. M.; Auberry K. J.; Moore R. J.; Angell N. H.; Smith R. D.; Springer D. L.; Pounds J. G. Toward a Human Blood Serum Proteome. Mol. Cell. Proteomics 2002, 1, 947–955. 10.1074/mcp.M200066-MCP200. [DOI] [PubMed] [Google Scholar]
  41. Benesi H. A.; Hildebrand J. H. A Spectrophotometric Investigation of the Interaction of Iodine with Aromatic Hydrocarbons. J. Am. Chem. Soc. 1949, 71, 2703–2707. 10.1021/ja01176a030. [DOI] [Google Scholar]
  42. Turell L.; Radi R.; Alvarez B. The thiol pool in human plasma: The central contribution of albumin to redox processes. Free Radical Biol. Med. 2013, 65, 244–253. 10.1016/j.freeradbiomed.2013.05.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Bandyopadhyay A.; Cambray S.; Gao J. Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation. Chem. Sci. 2016, 7, 4589–4593. 10.1039/C6SC00172F. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Faustino H.; Silva M. J. S. A.; Veiros L. F.; Bernardes G. J. L.; Gois P. M. P. Iminoboronates are efficient intermediates for selective, rapid and reversible N-terminal cysteine functionalization. Chem. Sci. 2016, 7, 5052–5058. 10.1039/C6SC01520D. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sung D.-B.; Mun B.; Park S.; Lee H.-S.; Lee J.; Lee Y.-J.; Shin H.-J.; Lee J. S. Synthesis, Molecular Engineering, and Photophysical Properties of Fluorescent Thieno[3,2-b]pyridine-5(4H)-ones. J. Org. Chem. 2019, 84, 379–391. 10.1021/acs.joc.8b01924. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao0c04659_si_001.pdf (1.9MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES