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DOI: 10.1055/s-0043-1775478
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Small Molecules in Medicinal Chemistry

Reactive Probe for Fluorescent Detection of Norepinephrine Based On an Excited-State Intramolecular Proton Transfer (ESIPT) Mechanism

Rajibul Haque
a   Department of Natural Products and Medicinal Chemistry, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, India
c   Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
,
Souma Ghosh 
b   Department of Oils, Lipid Science &Technology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, India
c   Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
,
Rajkumar Banerjee
b   Department of Oils, Lipid Science &Technology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, India
c   Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
,
Debabrata Maity∗
a   Department of Natural Products and Medicinal Chemistry, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, India
c   Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
› Author Affiliations

D.M. gratefully acknowledges the Director of the Council of Scientific and Industrial Research - Indian Institute of Chemical Technology (CSIR-IICT), India (MLP0108) and the Science and Engineering Research Board (SERB), India (SRG/2023/001243) for funding. The IICT Communication No. for this manuscript is IICT/Pubs./2025/028.
› Further Information
 
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Abstract

Norepinephrine is a key neurotransmitter that plays a critical role in the sympathetic nervous system. Its misuse and lack of regulation is closely associated with the progression of different central nervous system syndromes and neurodegenerative disorders. Herein, a reactive probe based on an excited-state intramolecular proton transfer (ESIPT) mechanism is reported for the selective fluorescent detection of norepinephrine under physiological conditions and in live cells.


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Key words

fluorescent probes - norepinephrine - excited-state intramolecular proton transfer - 2-(2′-hydroxyphenyl)benzothiazole - Live-cell imaging

Norepinephrine (NE) or noradrenaline is a catecholamine-based major neurotransmitter in the central and sympathetic nervous systems.[1] NE is produced by a series of enzymatic reactions from tyrosine and released predominantly from the nerve ends of sympathetic nerve fibers.[2] The compound is involved in maintaining various physiological processes related to attention, arousal, learning, memory, stress, heart rate, and blood pressure.[3] Another important responsibility of NE is the ‘fight-or-flight’ response to acute threats.[4] However, the action of NE is closely associated with the development of many neurodegenerative and mental disorders, including depression, anxiety, Alzheimer’s disease, and Parkinson’s disease.[5] Given the importance of NE in different crucial pathophysiological functions, there is an urgent need to develop methods for the reliable and accurate in vivo detection of NE levels. Non-optical techniques such as chromatography, mass spectrometry, and electrochemistry have been used as traditional detection methods for NE.[6] However, they are not suitable for endogenous NE detection. Fluorescence-based techniques have been attracted significant interest in medicinal research because of their higher sensitivity and in vivo applications.[7] Efforts have also focused on developing fluorescent detection systems for NE.[8] Glass and co-workers pioneered the use of small-molecule-based fluorescent probes based on NE amine-aldehyde condensation and NE catechol-phenyl boronic acid group esterification.[9] Another approach is based on a cascade nucleophilic reaction between the unique β-hydroxyethylamine moiety of NE and the protected fluorophores, resulting in deprotected fluorophores.[10] Designed probes based on the ‘protection-deprotection’ approach are found to be very specific for NE. However, practical applications of the probes may be limited due to the need for organic–aqueous mixed media conditions and the prolonged reaction times required for the fluorescence response. Fluorescent systems based on the excited-state intramolecular proton transfer (ESIPT) mechanism have gained considerable attention for detecting biologically important species due to their excellent emissive properties including intense luminescence, photostability, and extensive Stokes shifts.[11] 2-(2′-Hydroxyphenyl)benzothiazole (HBT) is a well-established chromophore that undergoes ESIPT processes.[12]

To our knowledge, ESIPT active fluorophores have not been exploited to develop NE-selective probes. Our design strategy is based on introducing a NE reactive carbonothioate ligand to the ESIPT-based chromophore HBT to obtain probe HBT-NE (Scheme [1]). The carbonothioate ligand in HBT-NE will prevent the ESIPT process and reduce the fluorescence intensity. It is expected that NE will undergo a cascade of nucleophilic attack by the primary amino group and β-hydroxyl group, respectively, at the carbonothioate site of HBT-NE, resulting in the synthesis of a five-membered ring compound with the release of the ESIPT-active HBT fluorophore. The deprotected HBT will undergo enol to keto tautomerization immediately due to ESIPT, and the fluorescence intensity will be restored. The designed probe in this way can be specific for NE over other generic amines and dopamine.

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Scheme 1 Norepinephrine mediated release of ESIPT active fluorophore HBT from HBT-NE
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Figure 1 Time-dependent fluorescence responses of HBT-NE (10 μM) upon addition of 0.5 mM of NE in buffer solution (10 mM HEPES, pH 7.4) (λex = 350 nm)

Herein, we report the synthesis and fluorometric properties of the ESIPT-based probe HBT-NE for NE detection under physiological conditions. HBT-NE was synthesized according to the procedure detailed in the Supporting Information (Scheme S1). In brief, equimolar amounts of 2-(2-hydroxyphenyl)benzothiazole, 4-(dimethylamino)pyridine, and triphosgene were heated at reflux in anhydrous toluene under inert atmosphere for 4 h. The reaction mixture was then cooled to 50 °C, and equimolar amounts of 4-(dimethylamino)pyridine and p-methoxythiophenol, respectively, were added to the reaction mixture. The mixture was stirred at 50 °C for another 4 h, then the reaction was quenched with water and the mixture was extracted with dichloromethane. The organic layer was washed with brine and dried over sodium sulfate. After evaporation in vacuo, the residue was purified by column chromatography using ethyl acetate and hexane solvent mixture as eluent to give HBT-NE as a pale-white solid with 87% yield. Upon 350 nm excitation, HBT-NE showed weak fluorescence around 525 nm in buffer conditions (10 mM HEPES, pH 7.4) (Figure [1]). However, in the presence of NE (0.5 mM), the fluorescence intensity of the system gradually increased by more than 3-fold and saturated in 50 minutes. The relatively rapid fluorescence response time greatly minimizes the interference due to the loss of NE caused by redox reactions. The increase in fluorescence is attributed to the NE-assisted cascade of nucleophilic reaction at the carbonothioate site of HBT-NE to release the ESIPT-active HBT fluorophore. HRMS characterization data validated the probe’s NE-mediated fluorescence enhancement response (see Figure S1 in the Supporting Information). The signals of both products, the deprotected HBT peak at m/z 228.0443 for [C13H10NOS]+ and the five-membered ring compound peak at m/z 194.0459 for [C9H8NO4]–, were found in the HRMS. Subsequently, the UV-Vis absorption spectra of HBT-NE showed an absorption band around 340 nm, which gradually decreased over time in the presence of NE, demonstrating the production of deprotected HBT during the detection process (see Figure S2 in the Supporting Information). Most NE probes face selectivity problems with epinephrine and dopamine. The addition of epinephrine (0.5 mM) caused an increase in fluorescence but less than 2.5-fold and saturated in 50 minutes (Figure [2], Figure S3 in the Supporting Information). However, the addition of a high concentration of dopamine (5 mM) showed minimal effect on fluorescence enhancement (Figure [2], Figure S3 in the Supporting Information). The fluorescence response of HBT-NE for NE is even better compared to epinephrine when they are treated at lower concentrations (100 μM) (Figure S4 in the Supporting Information).

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Figure 2 Top: Time-dependent fluorescence intensity changes of HBT-NE (10 μM) toward 0.5 mM NE, 0.5 mM epinephrine (EP), and 5 mM dopamine (Dop) at 524 nm in buffer solution (10 mM HEPES, pH 7.4) (λex = 350 nm). Bottom: Fluorescence response after 50 min of 10 μM HBT-NE at 524 nm upon addition of 0.5 mM (1) NE, (2) EP, and 5 mM of (3) dopamine, (4) 5-hydroxytryptamine, (5) acetylcholine, (6) urea, (7) alanine, (8) arginine, (9) histidine, (10) glycine, (11) leucine, (12) phenylalanine, (13) tyrosine, (14) proline, (15) isoleucine, (16) methionine, (17) serine, (18) tryptophan, (19) valine, (20) cysteine, (21) aspartic acid, (22) threonine, (23) glutamic acid, (24) lysine, and (25) glutamine.

We checked the fluorescence response of HBT-NE with other neurotransmitters such as 5-hydroxytryptamine, acetylcholine, and all amino acids including lysine, 2-aminoethanols moiety containing threonine, and serine at much higher concentrations (5 mM) (Figure [2], Figure S5–S6, in the Supporting Information). None of them induced major fluorescence intensity enhancement of the probe. These results demonstrate that HBT-NE could be used as a selective probe for NE detection.

Finally, we checked the application of the HBT-NE probe in cultured N2a cells using fluorescence microscopy (Figure [3]). N2a cells were incubated with different concentrations of NE, followed by treatment with 10 μM of HBT-NE. As shown in Figure [3], the fluorescence intensity of cells in the green channel gradually increased with increasing intracellular NE concentration. The results show that probe HBT-NE is cell permeable and reacts with NE in different concentrations to release green-emitting HBT dye in various concentrations. In a control experiment, N2a cells were incubated with 1 mM dopamine, followed by treatment with 10 μM of HBT-NE (Figure S7, the Supporting Information). No fluorescence was observed on the green channel, which indicates that dopamine did not react with the probe HBT-NE to release green-emitting HBT dye. Therefore, there is no potential interference from dopamine for NE detection using the HBT-NE probe. The cytotoxicity of probe HBT-NE toward two cell lines (L-929 and N2a) was measured using the standard MTT assay (Figure S8, the Supporting Information). At concentrations of 3–25 μM, cell viabilities were found to be high (ca. 80%) after incubation for 48 h, suggesting minimal toxicity of HBT-NE.

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Figure 3 CLSM images of N2a cells incubated with NE at different concentrations (100 μM–1 mM), followed by HBT-NE (10 μM). Cells are also treated with DAPI to blue stain the nucleus of cell lines. Panel I shows the bright field images and Panel II captured in the green channel shows NE-responsive green fluorescence. The intensity of the fluorescence increases with increasing NE concentrations. Panel III is captured in a blue channel, showing blue fluorescence emitting from the nucleus of the cells. Panel IV shows the overlay images of Panels II and III. Panel V shows the overlay images of Panels I, II, and III. Scale bar: 25 μm.

In conclusion, a novel ESIPT-based molecular probe, HBT-NE, has been developed for fluorescence turn-on detection of norepinephrine under pure aqueous conditions. A cascade of nucleophilic attack by the primary amino group and β-hydroxyl group of NE at the carbonothioate site of the probe releases the fluorophore HBT. Released HBT undergoes the ESIPT process immediately, which results in enhancement of green fluorescence. The reaction time is relatively fast, which improves the sensitivity of the probe. The probe is highly selective for NE over other relevant neurotransmitters. The probe has been shown to track NE via live cell imaging. Generalizing this design strategy for the development of ESIPT-based fluorescent probes for other neurotransmitters is in progress.


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Conflict of Interest

The authors declare no conflict of interest.

Supporting Information

    Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/s-0043-1775478.
  • Supporting Information

Corresponding Author

Debabrata Maity
Department of Natural Products and Medicinal Chemistry, CSIR-Indian Institute of Chemical Technology (CSIR-IICT)
Hyderabad 500007
India   

Publication History

Received: 25 January 2025

Accepted after revision: 28 March 2025

Article published online:
14 May 2025

© 2025. Thieme. All rights reserved

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Zoom Image
Scheme 1 Norepinephrine mediated release of ESIPT active fluorophore HBT from HBT-NE
Zoom Image
Figure 1 Time-dependent fluorescence responses of HBT-NE (10 μM) upon addition of 0.5 mM of NE in buffer solution (10 mM HEPES, pH 7.4) (λex = 350 nm)
Zoom Image
Figure 2 Top: Time-dependent fluorescence intensity changes of HBT-NE (10 μM) toward 0.5 mM NE, 0.5 mM epinephrine (EP), and 5 mM dopamine (Dop) at 524 nm in buffer solution (10 mM HEPES, pH 7.4) (λex = 350 nm). Bottom: Fluorescence response after 50 min of 10 μM HBT-NE at 524 nm upon addition of 0.5 mM (1) NE, (2) EP, and 5 mM of (3) dopamine, (4) 5-hydroxytryptamine, (5) acetylcholine, (6) urea, (7) alanine, (8) arginine, (9) histidine, (10) glycine, (11) leucine, (12) phenylalanine, (13) tyrosine, (14) proline, (15) isoleucine, (16) methionine, (17) serine, (18) tryptophan, (19) valine, (20) cysteine, (21) aspartic acid, (22) threonine, (23) glutamic acid, (24) lysine, and (25) glutamine.
Zoom Image
Figure 3 CLSM images of N2a cells incubated with NE at different concentrations (100 μM–1 mM), followed by HBT-NE (10 μM). Cells are also treated with DAPI to blue stain the nucleus of cell lines. Panel I shows the bright field images and Panel II captured in the green channel shows NE-responsive green fluorescence. The intensity of the fluorescence increases with increasing NE concentrations. Panel III is captured in a blue channel, showing blue fluorescence emitting from the nucleus of the cells. Panel IV shows the overlay images of Panels II and III. Panel V shows the overlay images of Panels I, II, and III. Scale bar: 25 μm.