View PDF Version
 
DOI: 10.1039/D4OB01086H (Review Article) Org. Biomol. Chem., 2024, Advance Article

Fluorescent N-oxides: applications in bioimaging and sensing

Yogesh Dubey and Sriram Kanvah*
Department of Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar, India. E-mail: sriram@iitgn.ac.in

Received 1st July 2024 , Accepted 16th August 2024

First published on 19th August 2024

Abstract

N-Oxides, due to their zwitterionic nature and ability to form hydrogen bonds through the oxide ion, are highly water-soluble and widely used in biological and pharmacological studies. The N-oxide structural scaffold is introduced into molecules, enabling “turn-on” fluorescence via an intramolecular charge transfer (ICT) process. This process occurs when the N–O bond is cleaved, either through an enzymatic reaction under hypoxic conditions or by using Fe(II), which allows rapid and selective detection of Fe(II) at nanomolar concentrations both in vitro and in vivo. This review focuses on the literature published between 2010 and 2024, particularly emphasising N-oxide fluorophores and their applications in hypoxic cell lines, Fe(II) detection, and bioimaging.

Introduction

Heterocyclic N-oxides are organic compounds featuring an oxygen atom bonded to a nitrogen atom (R–N → O), a structural modification that significantly impacts their chemical and physical properties. This functional group introduces electron delocalization, influencing reactivity, acidity/basicity, and overall stability, often resulting in higher polarity and improved solubility in polar solvents compared to their non-oxidized counterparts. N-oxides are extensively used in organic synthesis as intermediates, participating in nucleophilic substitution, oxidation, and reduction reactions to facilitate the creation of complex molecules.1–3 The natural occurrence of N-oxides in plant and animal tissues has raised questions about their biochemical roles as they exhibit greater activity than their parent amines. N-Oxides are significant in pharmacology and toxicology, with roles in various compounds such as alkaloids, chemotherapeutics, antibiotics, psychotropic drugs, and carcinogens.4 Trimethylamine N-oxide, prevalent in marine species and mammals, has garnered significant attention, and several investigations on their biosynthesis and physiological functions have been performed.5–7 N-Oxides are also crucial in catalysis, functioning as ligands or co-catalysts to enhance the efficiency and selectivity of catalytic reactions, including oxidation and coupling processes.8–10 Examples of widely studied N-oxides include pyridine N-oxide, quinoline N-oxide, acridine N-oxide, and pyrimidine N-oxide, with applications in organic synthesis,11–14 as coformers,15 in supramolecular and coordination chemistry,16–19 as anchoring groups for solar cells,20 and in biological activities.21,22 Current research on N-oxides focuses on developing novel synthetic methodologies, exploring their therapeutic potential, and investigating their roles in advanced materials and catalytic systems. N-Oxides are thus versatile compounds with a broad range of applications, benefiting from their unique electronic properties and reactivity, making them valuable in advancing scientific and technological fields.

In this review, our particular focus is on their utility for sensing or biological imaging applications. The N-oxide functionality undergoes selective bioreduction by hemeproteins under low-oxygen conditions, a process that is inhibited when oxygen binds to the heme iron, allowing for the swift detection of developing hypoxia. Many fluorescent scaffolds incorporating the N-oxide functionality have been established for hypoxia detection (Fig. 1) and sensing applications, particularly Fe(II).


image file: d4ob01086h-f1.tif
Fig. 1 Fluorescent scaffolds for the detection of hypoxia and biological imaging.

Design and mechanism of action

N-Oxide derivatives can be synthesized from aliphatic or benzyl amines using an oxidizing agent, most commonly meta-chloroperoxybenzoic acid (m-CPBA). It yields a non-fluorescent molecular system when attached to a suitable scaffold bearing an electron-withdrawing group. These non-fluorescence properties are utilized for “turn-on” emission studies, particularly in hypoxia or iron sensing in live cells. Under hypoxic conditions, cellular reductase enzymes are more active than they are under normoxic conditions.25 These reductases selectively reduce the N-oxide group to an amine (R3N), initiating the ICT process and leading to “turn-on” fluorescence. The released free amine can then act as an active drug or a toxic species, providing a potential pathway for cancer therapy (Fig. 3).
image file: d4ob01086h-f2.tif
Fig. 2 Molecules 5, 5a, and 5b (left panel) and molecule 6 (right panel) show turn-on fluorescence under hypoxic conditions. Images (5, 5a, and 5b, left panel) are reproduced from ref. 23 with permission from Wiley Publishing 2019; the right panel figures are reproduced from ref. 24 with permission from Elsevier Publishing, 2011.

image file: d4ob01086h-f3.tif
Fig. 3 General synthetic scheme and the mechanism of action.

N-Oxides for hypoxia

This chemical moiety, along with other N-oxide moieties (nitro groups, quinones, aromatic N-oxides, enamines, aliphatic N-oxides, and transition metals), formed the foundation for creating bioreductive prodrugs and fluorophores aimed at targeting or detecting tumour hypoxia.26–28 NIR-emitting fluorophores and caged-staurosporine modified with enamine N-oxides29 were developed for hypoxia-responsive prodrug and imaging applications. The prodrug's time-dependent reduction and dose–response curves were investigated under normoxic and hypoxic conditions in A431 cells. The molecule (1) was developed as a prodrug, and its activity was assessed in multiple cell lines, including those of the skin, pancreas, lung, brain, and cervix. Further prodrug studies were conducted using the A431 epidermoid carcinoma and H460 lung carcinoma cell lines, which exhibited intrinsic sensitivity to the parent drug with IC50 values of 208 and 571 nM, respectively, in cell viability assays. The bioreduction of enamine N-oxides leads to intracellular protein labelling in cells. Also, an N-oxide was conjugated to a near-infrared probe (2), and its hypoxia-responsive bioreduction led to fluorescence imaging of tumours in mice. A BODIPY-based red-emitting N-oxide fluorophore (3)30 was developed to undergo an irreversible two-electron reduction by heme proteins, specifically CYP450 enzymes, under hypoxic conditions. This reduction, which involves competitive binding to the heme iron, produces an enhanced photoacoustic signal upon irradiation at 770 nm. In vitro tests of 3 under normoxic and hypoxic conditions demonstrated its hypoxia selectivity, as evidenced by changes in the photoacoustic signal and a fluorescence turn-on effect. Additionally, ratiometric fluorescence imaging and in vivo imaging of tumor hypoxia and a murine hindlimb ischemia model were successfully demonstrated. Similarly, a conformationally restricted aza-BODIPY (3a) was designed as a NIR absorbing stimulus-responsive dye exhibiting an optimal photoacoustic response and large Stokes shift under hypoxic conditions.28 Photoacoustic dual-mode tumor imaging with N-oxides was also shown using a fluorescent probe (4), a triphenylamine–benzothiadiazole–triphenylamine derivative with four diethylamino N-oxide groups. Within the hypoxic microenvironment, (4) undergoes bioreduction, producing a neutral derivative with near-infrared (NIR) aggregation-induced emission.31 Tetraphenylethylene is an excellent chromophore for aggregation-induced emission (AIE), and this scaffold with diethylamine N-oxide (5) substitution is utilized for the detection of hypoxia and Fe(II), yielding a turn-on emission [Table 1]. The water-soluble molecule is initially weakly luminescent but shows strong emission in aqueous media after interaction with cellular reductases under hypoxic conditions.23 Within HeLa cells, the molecule could respond to oxygen concentrations as low as 8% and visualize lipid droplets (Fig. 2).23 A series of naphthalimide-based aliphatic amine-N-oxides (6) were developed. They were used as biomarkers for hypoxic cells (V79) in a solid tumor, exhibiting turn-on fluorescence (Fig. 2) and distinguishing normoxic from hypoxic cells.24 Furthermore, their binding affinity with ctDNA revealed that the neutral derivatives have a 15-fold lower binding affinity than the N-oxides, attributed to electrostatic and hydrogen bonding interactions. Various hypoxia-selective cytotoxins (HSCs), including tirapazamine (3-amino-1,2,4-benzotriazine 1,4-di-N-oxide), were synthesized as profluorescent substrates (7) to understand the oxygen-poor nature of hypoxic tumor tissue for therapeutic gain. These HSC derivatives, upon interaction with bioreductive enzymes such as NADPH: cytochrome P450 reductase, turn to fluorescent mono-N-oxide metabolites (Fig. 4), serving as indicators for the detection of the enzymes involved in the bioactivation of HSCs.32 The success of tirapazamine led to various other bioreductive drugs with phenazine (8)33 and anthraquinone (9)34 with the demonstration of cellular uptake and DNA binding.
image file: d4ob01086h-f4.tif
Fig. 4 Turn-on fluorescence is shown by 7 upon treatment with one-electron reductases. Reproduced from ref. 32 with permission from the American Chemical Society, 2018.
Table 1 Examples of various N-oxide fluorophores and the representative absorption and emission details
Molecules Mol. wt (g mol−1) Absorption (nm) Emission (nm) Localization Additional studies
a PBS.b CHCl3.c THF.d Solid state.e DCM.f 99% hexane[thin space (1/6-em)]:[thin space (1/6-em)]1% DCM.g EtOH.h CH3CN.i H2O
2 720.04 691a 712a — Imaging of tumors in live mice
3 614.50 670b 697b — Photoacoustic imaging
3a 640.54 691b 713b — Photoacoustic imaging
4 971.27 450c 592c, 600d — Photoacoustic imaging, nanoparticles
5 450.58 312e 444f, 448d Lipid droplets —
5a 506.69 313e 460f, 460d Lipid droplets Solid state emission
5b 542.67 312e 427f, 442d Lipid droplets —
6 329.31 331g 368g — —
6a 299.33 343g 548g — —
6b 390.45 379g 461g — —
6c 390.45 461g 522g — DNA binding
6d 374.39 425g 473g — —
7 (R[double bond, length as m-dash]H) 162.15 407h, 415i 478h, 521i — —
10 274.32 290, 310 377 — Reverse Meisenheimer rearrangement

The N-oxide fragment was attached to fluorescent molecules (10 and 11) and utilized as a turn-on switch through solvent-dependent 2,3-sigmatropic rearrangement and demonstrated a reverse Meisenheimer rearrangement (RMR) through the conversion of alkoxylamines back to N-oxides. In protic solvents, the fluorescence is on, while in organic solvents, the fluorescence is off.35

Imaging, detection and ferroptosis

N-Oxide fluorophores have also been utilized for other biological applications such as imaging and detecting physiological processes such as ferroptosis. Ferroptosis is a form of regulated cell death characterized by iron-dependent accumulation of lipid hydroperoxides to lethal levels in cells and distinct from other forms of cell death, such as apoptosis or necrosis. A naphthalimide fluorophore (12) incorporating cyclohexyl urea was developed for selective targeting of the endoplasmic reticulum (ER), with the pyridine-N-oxide sulfinyl group chosen as the glutathione (GSH)-responsive site. The molecule exhibits a 3900-fold increase in fluorescence intensity, demonstrating high sensitivity and selectivity towards GSH. Fluorescence imaging confirms effective targeting of the ER that can monitor GSH levels during erastin-induced ferroptosis.37 Water-soluble fluorescent derivatives of pyridine N-oxides substituted with various electron-donating amine groups (dimethylamine (13), pyrrolidine (14), piperidine (15), morpholine (16) and triphenylamine (17)) were synthesized by our group.36 The molecules show good ICT and 17 (TNO) shows selective localization with lipid droplets. In contrast, the cationic pyridinium derivative of 17 (TNC) showed selective localization within the mitochondria (Fig. 6).36 The zwitterionic nature of the fluorophores enables greater solubility and hence they have potential for probing the sub-cellular milieu (Fig. 5).
image file: d4ob01086h-f5.tif
Fig. 5 Fluorescent scaffolds of N-oxides utilized for biological imaging.

image file: d4ob01086h-f6.tif
Fig. 6 Confocal images of 17 (TNO) in Cos-7 cells revealing localization in the lipid droplets. The bottom images are for a similar structural scaffold with a pyridinium cation (TNC) instead of an N-oxide exhibiting mitochondrial localization. Reproduced from ref. 36 with permission from the Royal Society of Chemistry, 2023.

Fe(II) responsive N-oxide fluorophores

Iron (Fe) is vital in biological processes, particularly oxygen sensing and metabolism. Maintaining the redox balance of labile iron species is crucial to prevent the formation of reactive oxygen species (ROS). However, detecting specific redox states of iron, especially Fe(II), has been challenging. N-Oxides offer a promising avenue for developing fluorophores that selectively detect Fe(II) ions based on their coordination chemistry and optical properties. In particular, Fe(II) reduces and de-oxygenates N-oxides selectively, leading to a turn-on response.38 Utilising a ratiometric near-infrared (NIR) fluorescent probe (18) comprising a coumarin unit (emitting at short wavelengths) and a dicyanoisophorone (DCI) unit (emitting NIR fluorescence), Fe(II) is detected based on fluorescence resonance energy transfer (FRET) and N-oxide chemistry (Fig. 7).39 Initially, due to the presence of the N-oxide structure, the DCI moiety exhibited no emission, with the emission only from the coumarin unit. In the presence of Fe(II), molecule 18 exhibited notable ratiometric emission shifts from 460 to 675 nm, along with detecting exogenous and endogenous Fe(II) in cells and mice. The molecule was also used to monitor Fe(II) fluctuations during ferroptosis and drug-induced liver injury.39 The fluorescent probe (19) is initially non-emissive, but upon the addition of Fe(II), a turn-on emission is produced owing to the formation of a strong dimethylamino group that induces intramolecular charge transfer.40 The fluorescent probe was also utilized to detect Fe(II) within cells. Such turn-on emission through ICT behavior was also achieved using a DCM derivative (20), which demonstrated strong selectivity, excellent sensitivity (detection limit = 4.5 μM), and a rapid response for real-time monitoring of Fe(II) in living cells.41 Similarly, a dicyanoisophorone scaffold (21) was used to detect Fe(II) in water samples, living cells and zebrafish.42 Labile-heme sensitive H-FluNox (22) through the introduction of an electron-withdrawing 4,4-difluoropiperidine N-oxide into an Fe(II)-inactive rhodol scaffold, was developed as a fluorescent turn-on probe active through biomimetic deoxygenation (Fig. 8).43 The molecule demonstrates high sensitivity and selectivity for labile heme (LH), enabling its detection in living cells. The probe distinguishes LH from labile Fe(II) species and detects LH levels exogenously and endogenously after biological stimulation. Similar observations are also made with the acetylated derivative of H-FluNox (23). The experiments indicate the rise of LH and Fe(II) levels during ferroptosis, suggesting heme involvement. A natural terpene, camphor, with its rigid structure, good biocompatibility and cell permeability, was conjugated to a fluorescent substrate, yielding N,N-dimethyl-4-((4,7,7-trimethyl-3-oxobicyclo[2.2.1]heptan-2-ylidene)methyl)aniline oxide(24).44 The molecule rapidly detects Fe(II) with a low detection limit over a wide pH range. Moreover, the molecule was effectively used for bioimaging in onion cells and living zebrafish, exhibiting turn-on green fluorescence in the presence of Fe(II) by triggering a reduction reaction of the N-oxide group. Dansyl chloride (5-(dimethylamino)naphthalene-1-sulfonyl chloride) is a fluorescent compound widely used as a labelling reagent in biochemical and analytical applications. It reacts with primary and secondary amines to form stable sulfonamide bonds, making it useful for tagging proteins, peptides, and amino acids for detection.45 Utilising this thought process, an intelligent fluorescence “turn-on” probe (25), incorporating a dansyl amide fluorophore with an electron-donating group (the dimethylamino group at the 5-position) and an electron-withdrawing group (the sulfonyl group at the 1-position), was designed. The incorporation of the N-oxide group through mCPBA oxidation yields a non-emissive product and upon treating the molecule with bis(pinacolato)diboron (B2pin2), a turn-on emissive product with 72-fold fluorescence enhancement is noted, indicating successful deoxygenation of the N-oxide group and restoration of the ICT process.46 The probe exhibited good cellular uptake, demonstrating HepG2 live-cell imaging. A related derivative (26), bearing the HaloTag chloroalkane ligand, was designed and conjugated to the expressed 33 kDa HaloTag protein to produce a fluorophore modified protein (Fig. 7).46
image file: d4ob01086h-f7.tif
Fig. 7 Structures of various fluorescent N-oxides and their utility for cellular imaging and Fe(II) sensing.

image file: d4ob01086h-f8.tif
Fig. 8 Confocal fluorescence (top) and DIC images (bottom) of molecule 22 (10 μM) in A549 cells for 30 minutes. ALA: aminolevulinic acid and FAC: ferric ammonium nitrate; Ko143 (ABCG2-selective inhibitor, 1 μM). Reproduced from ref. 43 with permission from the American Chemical Society, 2022.

BODIPY (boron-dipyrromethene) dyes are a class of fluorescent compounds known for their exceptional photophysical properties, which make them highly valuable in various applications.47 Tapping their potential, a dual-responsive NIR fluorescent probe (27) was developed for the simultaneous in vivo detection of Fe(II) and H+. The molecule with an N-oxide strategy through a photoinduced electron transfer (PeT) mechanism shows NIR absorption and emission bands at 650 and 671 nm, respectively. The presence of Fe(II) quenches the NIR fluorescence of 27, leading to a 34-fold decrease in fluorescence intensity, indicating its selectivity.48 The reaction with Fe(II) yields BODIPY-H, which shows excellent sensitivity to H+ and a 22-fold increase in fluorescence intensity. Additionally, 27 is utilised to visualise exogenous Fe(II) and its product to detect H+ in the lysosomes of HeLa cells and mice (Fig. 9). A rhodol N-oxide derivative49 (28) is designed for bilirubin estimation by utilizing the deoxygenation reaction involving the tertiary amine N-oxide group after the reaction in situ generated Fe(II). This strategy was also employed for the synthesis of a water-soluble coumarin N-oxide derivative (29) for bilirubin quantification in human blood serum.50 A related coumarin derivative with a triphenylphosphonium unit for mitochondrial localization and an N-oxide moiety (30) for Fe(II) detection was utilised to determine the iron levels in cosmetics.51 An N-oxide-based rhodamine scaffold (31), RhoNox-4 was developed for detecting intracellular labile Fe(II). The compound demonstrated a 100-fold increase in fluorescence intensity and a rapid response to Fe(II). It also exhibited excellent cell membrane permeability and performed effectively in live-cell imaging. Its fast incubation time made it suitable for high-throughput assays, where a library of compounds was screened. This screening identified lomofungin as a positive hit that increased intracellular Fe(II) levels without affecting total iron levels. Lomofungin was found to elevate intracellular labile Fe(II) and degrade ferritin protein in an unusual manner.52 Inspired by the unique characteristics of the N–O bond and its heterocyclic N-oxidation to enable the formation of new fluorescent scaffolds, coumarin, quinoline and acridine-containing fluorophores (32–36) were developed. The N–O bond functions as a fluorescence switch, activating fluorescence in pyridine (32) and quinoline (33) (Fig. 11) and reducing it in acridines (34). Using quinoline N-oxide as a scaffold, two highly selective and sensitive fluorogenic probes were developed for H2S with an azide group (35) and formaldehyde (36) with a hydrazine group enabling naked-eye detection. Additionally, they are effective in imaging nuclear and cytoplasmic H2S and nuclear and perinuclear formaldehyde (Fig. 9).21


image file: d4ob01086h-f9.tif
Fig. 9 Structures of fluorescent N-oxides utilised for bioimaging and Fe(II) sensing.

image file: d4ob01086h-f10.tif
Fig. 10 Confocal fluorescence imaging of molecule 37 depicting staining of Golgi bodies post Fe(II) turn-on sensing. (a) Without Fe (II), (b) with added Fe(II) and (c) with added Fe (II) and Bipyridine (Bpy). BPy is added to complex with Fe(II). Reproduced from ref. 53 with permission from the Royal Society of Chemistry, 2019.

image file: d4ob01086h-f11.tif
Fig. 11 Optical properties of molecule 33 before and after N-oxidation modification. Reproduced from ref. 21 with permission from the American Chemical Society, 2020.

Targeting sub-cellular organelles through small molecule fluorophores provides a powerful toolset for advancing our understanding of cellular biology and disease mechanisms and developing novel therapeutic strategies. The NOX strategy was extended by adding a myristoyl motif to the Si-rhodamine derivative for Golgi-specific targeting of Fe(II). Divalent metal transporter 1 (DMT1) transports Fe(II) ions, with its delivery regulated partly by a retromer-mediated system involving vacuolar protein-sorting proteins (VPSs) from the trans-Golgi network to the cell membrane. Dysfunction of this system leads to abnormal DMT1 delivery to lysosomes, increasing Fe(II) ions. The molecule (37) (Fig. 10), along with lysoNOX 44, detects Golgi-specific Fe(II) ions, revealing an imbalance in Fe(II) distribution in VPS35-deficient cells, which is corrected by R55 treatment.53 The mem-RhoNox (38) having an N-oxygenated rhodamine scaffold and two arms tethered with palmitoyl groups as membrane-anchoring domains was utilized to monitor local Fe(II) at the surface of the plasma membrane of living cells (Fig. 12).54 Endogenous labile iron detection with a turn-on readout was also achieved using MtFluNox (39) within the iron-rich organelle mitochondria.55 This strategy of selective targeting and monitoring of intracellular iron is possible through the work of Hirayama et al., who are pioneers in developing universal fluorogenic systems responsive to Fe(II) based on N-oxide chemistry. Through the use of different substituents, Fe(II) sensing probes with different emission regions, CoNox-1 (blue) (40), FluNox-1 (green) (41), and SiRhoNox-1 (red) (42), were developed, demonstrating high selectivity and fluorescence enhancement.56 The Fe(II)-selective caging system based on N-oxide chemistry has led to the development of multiple other fluorescent scaffolds. Fluorophores with a hydroxymethylrhodamine scaffold (43) and hydroxymethylrhodol scaffolds (44–47) were developed. Live-cell imaging confirmed their ability to detect intracellular Fe(II) in a dose-dependent manner and enabled imaging of Fe(II) uptake via Tf-induced endocytosis.57 Rhodamine, owing to its strong fluorescence nature, was a go-to scaffold for developing various sensing systems, particularly iron, when substituted with N-oxide (43).58 A probe with rhodamine as a fluorophore component (48) was synthesized with the desired Fe(II) recognition site as a dimethylamine N-oxide functionality and a modified esterase-sensitive unit. Esterase activity, which distinguishes live cells from dead cells by hydrolyzing esters, enables the molecule (48) to provide a highly sensitive and selective response to Fe(II) ions in the presence of an esterase (Fig. 12 and Table 2).59


image file: d4ob01086h-f12.tif
Fig. 12 Structures of various fluorophores that were used for biological imaging and Fe(II) sensing.
Table 2 Listing of various N-oxide fluorophores and their analyte detection
Molecule# Mol. wt (g mol−1) Absorption (nm) Emission (nm) Detection Localization LOD
a DMSO.b Tris buffer.c HEPES.d PBS.e Water.f ACN/PBS (2[thin space (1/6-em)]:[thin space (1/6-em)]1).
12 717.81 281d, 346d — Glutathione ER 0.12 μM
13 240.30 402a 527a — Cytoplasm —
14 266.34 413a 532a — Cytoplasm —
15 280.37 390a 532a — Cytoplasm —
16 282.34 382a 525a — Cytoplasm —
17 364.44 384a 519a — Lipid droplets —
18 548.59 392b 460b Fe(II) — 75 nM
19 347.43 — — Fe(II) — 0.21 μM
20 383.45 415d, 435d 690d Fe(II) — 4.5 μM
21 361.48 380a 675a Fe(II) — 27 nM
22 451.42 455c 535c Hemin, Fe(II) — —
23 493.46 — — Labile heme Mitochondria and ER —
24 299.41 294d - Fe(II) — 15.6 nM
25 412.50 320f 535f B2pin2 — 2 μM
27 823.78 650a 671a Fe(II) Lysosomes 292 nM
28/48 453.49/495.53 580c 623c Bilirubin/Fe(II) — 33 nM/0.15 μM
29 353.37 — — Bilirubin — 76 nM
30 565.62 390c 460c Fe(II) Mitochondria 1.03 μM
31 482.58 493c — Fe(II) Mitochondria and ER 0.1 μM
32 138.17 293d 394d — — —
33 194.61 365d 505d — — —
34 210.23 470d 615d — — —
35 220.61 370d — H2S Nucleus 3.125 μM
36 209.63 355d — HCHO Nucleus 0.78 μM
37 797.16 — — Fe(II) Golgi bodies 50 nM
38 1638.14 — — Fe(II) Plasma membrane —
39 746.79 455c 535c Fe(II) Mitochondria —
40 257.28 295c 495c Fe(II) ER —
41 419.43 450c 530c Fe(II) — —
42 501.72 575c 660c Fe(II) ER —
43 458.55 492c 540c Fe(II) Golgi bodies 0.2 μM
44 388.46 550c 575c Fe(II) Lysosome —
45 444.57 555c 575c Fe(II) Lysosome —
46 361.39 515c 535c Fe(II) ER —
47 389.45 520c 535c Fe(II) ER —
49 240.30 305a 365a Fe(II) Cytoplasm 35 nM
50 264.32 326a 404a Fe(II) Lipid droplets 42 nM
51 385.51 535d 720d Fe(II) Lysosome 3.06 μM
52 523.57 338d 400d Glutathione — —
53 380.29 378b 468b AP — 0.38 U L−1
54 354.40 340e 540e Fe(II) — 1.02 μM

Our group recently demonstrated Fe(II) detection with a styryl N-oxide skeleton. Compounds 49 and 50 have dimethylamine N-oxide at one terminal and pyridine or cyanophenyl at the other terminal, separated by a double-bond spacer. The molecules are non-emissive but show strong ICT behaviour upon Fe(II) deoxygenation (Fig. 13). Interestingly, the molecule (50) shows specific localization within the lipid droplets post-deoxygenation, while the pyridine counterpart (49) has non-specific localization.60 A similar styryl scaffold with julolidine N-oxide (51) (λex/λem = 535/720 nm) was developed and used for highly sensitive detection of Fe(II) (Fig. 14). This molecule was also effectively used for imaging in living cells, monitoring Fe(II) level changes in liver and kidney injuries and in vivo imaging of zebrafish and mice.61 A naphthalimide (52) decorated with thiomorpholine-S-dioxide at one end and ethyl morpholine-N-oxide on the other end is utilized for specific detection of glutathione. Upon encountering glutathione, the sulfonamide bond was cleaved, and a strong green emission was observed due to the formation of a GSH-linked product.62 Another versatile fluorescent probe based on a naphthalimide scaffold bearing dimethylamine oxide and a phosphate group (53) was developed and utilized for ratiometric detection of alkaline phosphatase (AP) activity in vivo. In the presence of an alkaline phosphatase, the compound shows a new emission peak at 554 nm, along with the parent peak at 468 nm. The emission is attributed to the dephosphorylation process by ALP, and the phosphate group is substituted with a hydroxyl group, contributing to emission changes.63


image file: d4ob01086h-f13.tif
Fig. 13 The absorption and emission spectral changes of molecule 50 after the addition of Fe (II) depicting the colorimetric turn-on behavior (top) and visualization of erastin-induced ferroptosis (bottom) using molecule 50. Reproduced from ref. 60 with permission from the Royal Society of Chemistry, 2024.

image file: d4ob01086h-f14.tif
Fig. 14 Fe(II) sensing using molecule 51. Reproduced from ref. 61 with permission from the Royal Society of Chemistry.

Miscellaneous applications

As seen above, N-oxide groups can contribute to designing responsive molecules in the context of fluorescent probes and sensors. They can modulate fluorescence properties or serve as recognition elements in sensing platforms, enabling the detection of specific analytes or biomolecules. Specifically, we summarised those derivatives with potential utility in biological settings. However, other fluorescent N-oxide derivatives are known and have been utilized for various applications.64–66 A unique example is a naphthalimide with an oxidised morpholine component (54) for detecting Fe(II). This molecule showed a significant increase in emission intensity even with just one equivalent of Fe(II) in a frozen state compared to ambient conditions, indicating increased reactivity in ice, likely due to enhanced deoxygenation facilitated by freezing concentration effects. Confocal laser microscopy demonstrated that upon adding Fe(II), the probe localized at the low liquid limit of ice, accompanied by fluorescence enhancement, enabling its potential for tracking environmental Fe(II), especially in polar and cold environments.67 N-Oxide derivatives were also used to monitor the photochemical reaction process. An AIE-active pyridazine N-oxide derivative (55), upon irradiation, undergoes sequential transformations to produce MPZ at room temperature, MF, at −40 °C in the presence of N2, and eventually trans-MDO in the presence of oxygen at −40 °C (Fig. 15). The photoreaction was monitored in real time through fluorescence spectral changes, highlighting the effectiveness of fluorescence techniques in tracking small-molecule reactions.68
image file: d4ob01086h-f15.tif
Fig. 15 Proposed photo-rearrangement for molecule 55.

In conclusion, fluorescent N-oxide derivatives significantly advance chemical and biological sensing. Their unique photophysical properties, including high fluorescence quantum yields and excellent photostability, make them ideal candidates for various applications. These derivatives have shown promising results in multiple areas, such as bioimaging, environmental monitoring, and detecting specific ions and molecules. Their versatility and potential for further functionalisation enable tailored applications, enhancing their efficacy and sensitivity. As research progresses, it is anticipated that fluorescent N-oxide derivatives will continue to play a crucial role in developing innovative diagnostic tools and analytical techniques, contributing to advancements in both scientific research and practical applications.

Data availability

No original data are used in this article. The article is summarized using publicly available or subscription based articles.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

SK acknowledges research funding from SERB (CRG/2022/007048). YD acknowledges an IIT Gandhinagar research fellowship.

References

  1. Y. Wang and L. Zhang, Synthesis, 2015, 289–305 Search PubMed .
  2. J. V. Quagliano, J. Fujita, G. Franz, D. J. Phillips, J. A. Walmsley and S. Y. Tyree, J. Am. Chem. Soc., 1961, 83, 3770–3773 CrossRef CAS .
  3. V. S. Basistyi and J. H. Frederich, Org. Lett., 2022, 24, 1907–1912 CrossRef CAS PubMed .
  4. M. H. Bickel, Pharm. Rev., 1969, 21, 325 CAS .
  5. S. Subramaniam and C. Fletcher, Br. J. Pharmacol., 2018, 175, 1344–1353 CrossRef CAS PubMed .
  6. L. Kong, Q. Zhao, X. Jiang, J. Hu, Q. Jiang, L. Sheng, X. Peng, S. Wang, Y. Chen, Y. W3nan, S. Hou, X. Liu, C. Ma, Y. Li, L. Quan, L. Chen, B. Cui and P. Li, Nat. Commun., 2024, 15, 2526 CrossRef CAS PubMed .
  7. C.-Y. Hou, Y.-W. Chen, S. H. Hazeena, Y.-L. Tain, C.-W. Hsieh, D.-Q. Chen, R.-Y. Liu and M.-K. Shih, FEBS Open Bio, 2024, 14, 358–379 CrossRef CAS PubMed .
  8. H. Xu, C.-G. Wang, Y. Lu and A. Goto, Macromolecules, 2019, 52, 2156–2163 CrossRef CAS .
  9. G. Yan, A. J. Borah and M. Yang, Adv. Synth. Catal., 2014, 356, 2375–2394 CrossRef CAS .
  10. D. Li, P. Wu, N. Sun, Y.-J. Lu, W.-L. Wong, Z. Fang and K. Zhang, Curr. Org. Chem., 2019, 23, 616–627 CrossRef CAS .
  11. I. Habib, K. Singha and M. Hossain, ChemistrySelect, 2023, 8, e202204099 CrossRef CAS .
  12. S. R. Neufeldt, G. Jiménez-Osés, J. R. Huckins, O. R. Thiel and K. N. Houk, J. Am. Chem. Soc., 2015, 137, 9843–9854 CrossRef CAS PubMed .
  13. H. Hwang, J. Kim, J. Jeong and S. Chang, J. Am. Chem. Soc., 2014, 136, 10770–10776 CrossRef CAS PubMed .
  14. V. Satheesh and Y. Deng, J. Org. Chem., 2024 DOI:10.1021/acs.joc.4c01453 .
  15. B. Saikia, R. Khatioda, P. Bora and B. Sarma, CrystEngComm, 2016, 18, 8454–8464 RSC .
  16. W. Byers, B. F.-C. Chou, A. B. P. Lever and R. V. Parish, J. Am. Chem. Soc., 1969, 91, 1329–1333 CrossRef CAS .
  17. R. Puttreddy and P. J. Steel, CrystEngComm, 2014, 16, 556–560 RSC .
  18. Y. P. Nizhnik, A. Sons, M. Zeller and S. V. Rosokha, Cryst. Growth Des., 2018, 18, 1198–1207 CrossRef CAS .
  19. X. Chi, H. Zhang, G. I. Vargas-Zúñiga, G. M. Peters and J. L. Sessler, J. Am. Chem. Soc., 2016, 138, 5829–5832 CrossRef CAS PubMed .
  20. B. Schmitz, B. Li, R. G. W. Jinadasa, S. B. Lalvani, L. L. Kerr and H. Wang, J. Porphyrins Phthalocyanines, 2016, 20, 542–555 CrossRef CAS .
  21. Z. Ma, J. Li, K. Lin, M. Ramachandran, M. Li and Y. Li, Anal. Chem., 2020, 92, 12282–12289 CrossRef CAS PubMed .
  22. K. N. Sedenkova, J. V. Kolodyazhnaya, D. A. Vasilenko, Y. A. Gracheva, E. V. Kharitonoshvili, Y. K. Grishin, A. A. Chistov, V. B. Rybakov, T. Holt and A. G. Kutateladze, Dyes Pigm., 2019, 164, 72–81 CrossRef CAS .
  23. C. Xu, H. Zou, Z. Zhao, P. Zhang, R. T. K. Kwok, J. W. Y. Lam, H. H. Y. Sung, I. D. Williams and B. Z. Tang, Adv. Funct. Mater., 2019, 29, 1903278 CrossRef .
  24. H. Yin, W. Zhu, Y. Xu, M. Dai, X. Qian, Y. Li and J. Liu, Eur. J. Med. Chem., 2011, 46, 3030–3037 CrossRef CAS PubMed .
  25. W. R. Wilson and M. P. Hay, Nat. Rev. Cancer, 2011, 11, 393–410 CrossRef CAS PubMed .
  26. X. Yuan, Z. Xie and T. Zou, Bioorg. Chem., 2024, 144, 107161 CrossRef CAS PubMed .
  27. D. Liu, M. Liang, A. Fan, W. Bing and J. Qi, Luminescence, 2024, 39, e4659 CrossRef CAS PubMed .
  28. E. Y. Zhou, H. J. Knox, C. Liu, W. Zhao and J. Chan, J. Am. Chem. Soc., 2019, 141, 17601–17609 CrossRef CAS PubMed .
  29. D. Kang, S. T. Cheung, A. Wong-Rolle and J. Kim, ACS Cent. Sci., 2021, 7, 631–640 CrossRef CAS PubMed .
  30. H. J. Knox, J. Hedhli, T. W. Kim, K. Khalili, L. W. Dobrucki and J. Chan, Nat. Commun., 2017, 8, 1794 CrossRef PubMed .
  31. M. Li, H. Li, Q. Wu, N. Niu, J. Huang, L. Zhang, Y. Li, D. Wang and B. Z. Tang, iScience, 2021, 24, 102261 CrossRef CAS PubMed .
  32. X. Shen, C. H. Laber, U. Sarkar, F. Galazzi, K. M. Johnson, N. G. Mahieu, R. Hillebrand, T. Fuchs-Knotts, C. L. Barnes, G. A. Baker and K. S. Gates, J. Org. Chem., 2018, 83, 3126–3131 CrossRef CAS PubMed .
  33. M. L. Lavaggi, M. Nieves, M. Cabrera, C. Olea-Azar, A. López de Ceráin, A. Monge, H. Cerecetto and M. González, Eur. J. Med. Chem., 2010, 45, 5362–5369 CrossRef CAS PubMed .
  34. L. H. Patterson and S. R. McKeown, Br. J. Cancer, 2000, 83, 1589–1593 CrossRef CAS PubMed .
  35. C. Spino, M. Latil, R. Lessard, Q. Fevre-Renault and C. Y. Legault, Chem. – Eur. J., 2023, 29, e202301356 CrossRef CAS PubMed .
  36. Y. Dubey, P. Mahalingavelar, D. Rajput, D. J. Shewale, V. Soppina and S. Kanvah, Org. Biomol. Chem., 2023, 21, 8393–8402 RSC .
  37. Q. Chen, Y. Wang, T. Yue, H. Wei, S. Li and B. Dong, Anal. Chem., 2023, 95, 1949–1957 CrossRef CAS PubMed .
  38. T. Hirayama and H. Nagasawa, J. Clin. Biochem. Nutr., 2017, 60, 39–48 CrossRef CAS PubMed .
  39. S. Feng, J. Zheng, J. Zhang, Z. Gui and G. Feng, Sens. Actuators, B, 2022, 371, 132512 CrossRef CAS .
  40. X. Zeng, J. Chen, S. Yu, Z. Liu and M. Ma, J. Lumin., 2022, 250, 119069 CrossRef CAS .
  41. X. Yang, Y. Wang, R. Liu, Y. Zhang, J. Tang, E.-b. Yang, D. Zhang, Y. Zhao and Y. Ye, Sens. Actuators, B, 2019, 288, 217–224 CrossRef CAS .
  42. M. Zhu, Z. Zhao, X. Liu, P. Chen, F. Fan, X. Wu, R. Hua and Y. Wang, J. Hazard. Mater., 2021, 406, 124767 CrossRef CAS PubMed .
  43. K. Kawai, T. Hirayama, H. Imai, T. Murakami, M. Inden, I. Hozumi and H. Nagasawa, J. Am. Chem. Soc., 2022, 144, 3793–3803 CrossRef CAS PubMed .
  44. Y. Liang, Y. Zhang, M. Li, Z. Meng, Y. Gao, J. Yin, Y. Yang, Z. Wang and S. Wang, Anal. Bioanal. Chem., 2021, 413, 6267–6277 CrossRef CAS PubMed .
  45. J. M. Walker, in Basic Protein and Peptide Protocols, ed. J. M. Walker, Humana Press, Totowa, NJ, 1994, pp. 321–328,  DOI:10.1385/0-89603-268-X:321 .
  46. H. Yang, Y. Wang, X. Li, Y. Teng and Y. Tian, ChemistryOpen, 2021, 10, 1013–1019 CrossRef CAS PubMed .
  47. A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891–4932 CrossRef CAS PubMed .
  48. W. Xu, P. Wu, X. Li, S. Liu, L. Feng and H. Xiong, Talanta, 2021, 233, 122601 CrossRef CAS PubMed .
  49. E. Ahmmed, A. Mondal, N. Chandra Saha, K. Dhara and P. Chattopadhyay, Anal. Methods, 2021, 13, 5651–5659 RSC .
  50. E. Ahmmed, A. Mondal, A. Sarkar, S. Chakraborty, S. Lohar, N. C. Saha, K. Dhara and P. Chattopadhyay, ACS Appl. Bio Mater., 2020, 3, 4074–4080 CrossRef CAS PubMed .
  51. S. Khatun, S. Biswas, A. Binoy, A. Podder, N. Mishra and S. Bhuniya, J. Photochem. Photobiol., B, 2020, 209, 111943 CrossRef CAS PubMed .
  52. T. Hirayama, M. Niwa, S. Hirosawa and H. Nagasawa, ACS Sens., 2020, 5, 2950–2958 CrossRef CAS PubMed .
  53. T. Hirayama, M. Inden, H. Tsuboi, M. Niwa, Y. Uchida, Y. Naka, I. Hozumi and H. Nagasawa, Chem. Sci., 2019, 10, 1514–1521 RSC .
  54. M. Niwa, T. Hirayama, I. Oomoto, D. O. Wang and H. Nagasawa, ACS Chem. Biol., 2018, 13, 1853–1861 CrossRef CAS PubMed .
  55. T. Hirayama, S. Kadota, M. Niwa and H. Nagasawa, Metallomics, 2018, 10, 794–801 CrossRef CAS PubMed .
  56. T. Hirayama, H. Tsuboi, M. Niwa, A. Miki, S. Kadota, Y. Ikeshita, K. Okuda and H. Nagasawa, Chem. Sci., 2017, 8, 4858–4866 RSC .
  57. M. Niwa, T. Hirayama, K. Okuda and H. Nagasawa, Org. Biomol. Chem., 2014, 12, 6590–6597 RSC .
  58. T. Hirayama, K. Okuda and H. Nagasawa, Chem. Sci., 2013, 4, 1250–1256 RSC .
  59. B. Dong, W. Song, Y. Lu, M. Tian, X. Kong, A. H. Mehmood and W. Lin, Sens. Actuators, B, 2020, 305, 127470 CrossRef CAS .
  60. Y. Dubey, S. Mansuri and S. Kanvah, J. Mater. Chem. B, 2024, 12, 4962–4974 RSC .
  61. H. Xiang, Y. Song, Y. Wang, W. Fu and N. Xiao, Mater. Adv., 2024, 5, 5624–5631 RSC .
  62. G. Liu, X. Han, J. Zhang, Z. Xu, S. H. Liu, L. Zeng and J. Yin, Dyes Pigm., 2018, 148, 292–297 CrossRef CAS .
  63. X. Hou, Q. Yu, F. Zeng, J. Ye and S. Wu, J. Mater. Chem. B, 2015, 3, 1042–1048 RSC .
  64. Y. Oku, N. Nakajima, M. Hamada and Y. Koyama, Chem. – Eur. J., 2024, 30, e202400092 CrossRef CAS PubMed .
  65. T. Nishida, A. Fukazawa, E. Yamaguchi, H. Oshima, S. Yamaguchi, M. Kanai and Y. Kuninobu, Chem. – Asian J., 2014, 9, 1026–1030 CrossRef CAS PubMed .
  66. A. Szemik-Hojniak, I. Deperasińska, K. Oberda, Y. Erez, D. Huppert and Y. P. Nizhnik, Phys. Chem. Chem. Phys., 2013, 15, 9914–9923 RSC .
  67. Y. H. Lee, P. Verwilst, H. S. Kim, J. Ju, J. S. Kim and K. Kim, Chem. Commun., 2019, 55, 12136–12139 RSC .
  68. J. Gong, J. Gong, Y. He, C. Li, B. Yang, L. Liu, G. Jiang and J. Wang, Org. Chem. Front., 2023, 10, 4581–4587 RSC .
This journal is © The Royal Society of Chemistry 2024