Design and Synthesis of Novel Angular 4,5-Pyranocoumarin Fluorescent Probe for detecting hydrazine and and its applications

Design and Synthesis of Novel Angular 4,5-Pyranocoumarin Fluorescent Probe for detecting hydrazine and and its applications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design and Synthesis of Novel Angular 4,5-Pyranocoumarin Fluorescent Probe for detecting hydrazine and and its applications huafeng zhou, jiayong huang, jian su, xiaowei su, qiujuan chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6756101/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A novel structure of 4,5-pyranocoumarin-based fluorescent probes ( 2b and 3c ) was designed and synthesized for the selective detection of hydrazine hydrate (N₂H₄). The probes feature an extended conjugated system via a 3-aryl-substituted pyranocoumarin skeleton, with ester groups serving as recognition sites for N₂H₄. Probe 3c demonstrated superior performance, exhibiting a 494% fluorescence enhancement at 460 nm with a detection limit of 0.03 µM, surpassing most reported hydrazine probes and the EPA safety threshold. Both probes exhibited high selectivity against 17 interferents (ions/biomolecules) and functioned effectively in complex matrices, including food samples (lettuce, rice) and vapor-phase detection via test strips. Probe 3c achieved rapid (< 5 min), pH-stable (pH 6–10) responses with visible color transitions (blue-green to green), while 2b operated across a broader pH range (1–12). This work highlights pyranocoumarin scaffolds as promising platforms for designing intensity-responsive probes, combining synthetic novelty, sensitivity, and practicality for environmental and food safety. 4 5-Pyranocoumarin Hydrazine Turn-on fluorescence-ICT Real samples Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction As a crucial multifunctional chemical reagent, hydrazine hydrate (N₂H₄) plays an indispensable role in diverse fields, including chemical synthesis, catalytic processes, pesticide manufacturing, and industrial production[1–3]. However, N₂H₄ can enter the organism through skin absorption and inhalation of respiratory mucosa, thereby causing irreversible physiological damage [4–6]. With the acceleration of industrialization, the problem of soil and water pollution caused by improper discharge of hydrazine-containing wastes has become increasingly serious. Consequently, the development of efficient detection methods has as a critical priority for environmental monitoring, industrial safety protocols, and biomedical research[7–8]. Fluorescent probes are widely recognized for their advantages in simple synthesis, strong selectivity, and good biocompatibility[9–13]. The organic fluorophores such as coumarin derivatives have attracted much attention due to their relatively low toxicity, ease of chemical modification and excellent stability[14–16]. These superior attributes have established them as versatile molecular platforms for environmental pollutant monitoring, cellular imaging, and in vivo biosensing applications with promising potential. Recent advancements have focused on coumarin probes for hydrazine detection (Fig. 1 ). For example, Fanyong Yan et al[17]. designed a ratiometric probe ( 1 ) based on a fluorescein-coumarin hybrid structure. Initially, the photoinduced electron transfer (PET) effect quenched fluorescein emission, leaving only coumarin fluorescence. Hydrazine-induced ester bond cleavage eliminated PET, restoring fluorescein emission for dual-channel detection. Xiaoyan Wang et al[18]. developed a near-infrared probe ( 2 ) using a hemicyanine scaffold, where ester bond cleavage by hydrazine restored the excited-state intramolecular proton transfer (ESIPT) process, triggering a 737 nm fluorescence enhancement. This probe enabled real-time monitoring of isoniazid metabolism in vivo due to its pH stability and biocompatibility. Additionally, Xiangdong Geng et al[19]. reported a coumarin-based probe (Probe 3 ) exhibiting a 35-fold fluorescence increase and visible colorimetric changes upon hydrazine exposure, providing a practical solution for detecting hydrazine in environmental water. Despite these advances, challenges remain in improving selectivity, sensitivity, and photostability of hydrazine probes. In the design of hydrazine (N 2 H 4 ) probes, functional groups such as aldehydes, ketones, and esters are strategically incorporated to enable selective detection through nucleophilic addition or substitution reactions with hydrazine, inducing measurable fluorescence changes[17–21]. The ester group, recognized as an efficient recognition moiety, has been widely utilized in the detection of hydrazine hydrate[22–27]. In addition, most of the coumarin-based probes reported to date are derived from simple coumarin skeletons, whereas research on alternative skeleton types remains relatively limited. To enhance the diversity of coumarin-based fluorescent probes and improve their performance, we designed a novel pyranocoumarin skeleton (Fig. 2 ). This skeleton features an aryl group at the 3-position, which effectively extends the conjugated plane of the coumarin. Simultaneously, ester groups are introduced at the 7 or 8 positions as recognition sites for hydrazine, and the effects of monoester and diester groups on hydrazine recognition were systematically investigated. Subsequently, we conducted an in-depth exploration of the spectral properties and reaction mechanisms of the compounds using methods such as fluorescence and UV-Vis spectrophotometry. 2. Experimental 2.1. Materials and apparatus All reagents and metal salts used in the experiments were purchased from Aladdin Industrial Corporation (Shanghai, China) or Sigma-Aldrich Trading Co. LTD (St. Louis, MO, USA).The reagents used in the experiment are all analytically pure. NMR spectra were recorded on a DRX-500 NMR spectrometer (Rheinstetten, Germany), and high-resolution mass spectrometry (HRMS) was performed using an Agilent 6210 ESI/TOF mass spectrometer (Palo Alto, CA, USA). Single-crystal X-ray diffraction (SCXRD) data were collected on an Agilent SuperNova Dual Cu-at-zero AtlasS2 diffractometer (Palo Alto, CA, USA). Fluorescence spectra were measured with a Shimadzu RF-6000 spectrofluorophotometer (Tokyo, Japan). UV-Vis absorption spectra were obtained using a UV-1800P spectrophotometer (Shanghai Meipuda Instrument Co, Ltd., China). pH measurements were conducted with a PHB-1 portable pH meter (Shanghai Shinuo Physical Optical Instrument Co., Ltd., China). 2.2 Preparation of the probe. 2.2.1 Preparation of Intermediate 1 Under the action of electromagnetic stirring, 20 mL of anhydrous dioxane, 2.81 g of 2,4,6-trihydroxybenzoic methylester (10 mmol) and 2.26 g of 3,3-dimethylacrylic acid (22 mmol) were added successively to a 50 mL round-bottom flask and heated in an oil bath at 95 ℃. The reaction was stirred under reflux for 7 h and TLC was used to monitor the reaction (petroleum ether: ethyl acetate = 4:1). After the reaction was completed, the reaction mixture was poured into 100 mL of ice water, saturated potassium carbonate was added to adjust the pH to neutral, and the mixture was filtered. The crude product was obtained and recrystallized in acetone to yield a pale yellow crystal ( Compound 1 ), with a yield of 90% and a melting point of 140.6–144.1 ℃. 1 H NMR (600 MHz, CDCl 3 ), δ (ppm): δ 14.02 (s, 1H), 12.67 (s, 1H), 5.99 (s, 1H), 3.99 (s, 3H, -OCH 3 ), 2.74 (s, 2H), 1.48 (s, 6H,2×CH 3 ). 13 C NMR (101 MHz, CDCl 3 ) δ (ppm): δ 198.07, 168.46, 166.17, 165.85, 164.67, 98.03, 82.75, 82.62, 81.02, 51.11, 48.33, 27.96. HRMS (ESI): calcd For C 13 H 14 O 6 [M + H] + : 267.32 . 2.2.2 Preparation of Probes 2a, 2b , and 2c Under N 2 protection, 4 mL (40 mmol) of acetic anhydride, 0.266 g (1 mmol) of compound 1 and 1 mmol of substituted benzoic acid were successively added to a 50 mL round-bottom flask. The mixture was heated to 100 ℃, then 0.208 mL (1.5 mmol) of triethylamine was added and the temperature was raised to 150 ℃. The reaction was stirred under reflux for 2 hours, and TLC monitoring was carried out (petroleum ether: ethyl acetate = 4:1). After the reaction was completed, the reaction mixture was cooled to room temperature, then 8 mL of water was added. The mixture was extracted with ethyl acetate (3×15 mL), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purification (eluent: petroleum ether: ethyl acetate = 4:1) was performed to obtain compounds 2a, 2b , and 2c , with yields ranging from 39–45%. 2a (2'', 2''-Dimethyl-3-(4'-chlorophenyl)-7-acetoxymethyl-2''H, 3''H-4,5-pyrrolo[3,4-b]oxazole-8-carboxylic acid methyl ester) is a white solid, with a yield of 45% and melting range of 216.3-217.5 ℃. 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): δ 7.44 (d, J = 7.5 Hz, 2H), 7.24 (d, J = 7.5 Hz, 2H), 6.56 (s, 1H), 3.96 (s, 3H), 2.74 (s, 2H), 2.32 (s, 3H), 1.37 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) δ (ppm): δ 168.67, 163.12, 159.37, 155.38, 152.90, 151.66, 142.29, 134.77, 131.47, 130.54, 128.86, 121.83, 107.52, 107.35, 104.56, 77.51, 52.87, 37.77, 26.44, 20.87. HR-MS (ESI): calcd for C 22 H 18 ClO 7 [M + Na] + : 465.0707. 2b (2'', 2''-Dimethyl-3-(3',5'-difluoromethyl phenyl)-7-acetoxy-methyl-2''H, 3''H-4,5-pyrrolo[2,3-b]oxa-zole-8-carboxylic acid methyl ester) is a white solid, with a yield of 40%, melting point ranging from 193.5 to 194.4 ℃. 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): δ 7.94 (s, 1H), 7.76 (s, 2H), 6.60 (s, 1H), 3.97 (s, 3H), 2.73 (s, 2H), 2.33 (s, 3H), 1.40 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) δ (ppm): δ 168.51, 162.91, 158.90, 155.56, 153.56, 151.76, 143.71, 134.32, 130.48, 124.13, 122.68, 121.96, 120.03, 107.79, 107.68, 104.13, 52.89, 37.67, 26.44, 20.85. HR-MS (ESI): calcd for C 25 H 18 F 6 O 7 [M + Na] + : 567.0845. 2c (2'', 2''-Dimethyl-3-(4'-acetoxyphenyl)-7-acetoxy-2''H, 3''-4,5-pyranidinocoumarin-8-methylester) is a white solid, with a yield of 41% and melting range of 176.8–178.9 ℃. 1 H NMR (400 MHz, CDCl 3 ) δ (ppm): δ 7.32 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 6.7 Hz, 2H), 6.56 (s, 1H), 3.96 (s, 3H), 2.78 (s, 2H), 2.34 (s, 3H), 2.32 (s, 3H), 1.37 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) δ (ppm): δ 169.44,168.68, 163.17, 159.50, 155.41, 152.80, 150.75, 142.21, 131.33, 129.62, 122.06, 121.75, 107.43, 107.27, 104.67, 103.31, 52.85, 37.79, 26.42, 21.23. HR-MS (ESI): calcd for C 25 H 22 O 9 [M + Na] + : 489.1143. 2.2.2 Preparation of Probes 3a , 3b , and 3c Under electromagnetic stirring, 100 mL of methanol, compound 2a, 2b, 2c , (3.5 g, 8 mmol) were added successively to a 100 mL round-bottom flask. After dissolving by heating, when the temperature dropped to room temperature, the flask was placed in a low-temperature reactor. Under 0 ℃-5 ℃ conditions, NaBH 4 (1.5 g, 40 mmol) was added in three portions within 1.5 h, and the reaction was stirred for 2 h, followed by TLC monitoring (petroleum ether: ethyl acetate = 3:1). After the reaction was completed, 9–10 drops of 5% HCl were added to the reaction mixture to terminate the reaction. After evaporating the remaining methanol, the organic layer was extracted with ethyl acetate (3×20 mL), and the organic layers were combined. They were successively washed with saturated NaHCO 3 solution, saturated brine, and anhydrous sodium sulfate, dried over a filter, and recrystallized with methanol to obtain compounds 3a, 3b , and 3c . The yield was 65%-71%. 3a (2', 2'-Dimethyl-3-(4'-chlorophenyl)-7-hydroxy-2''H, 3''H-4,5-pyrrolo[4,5-b]chromen-8-carboxylic acid methyl ester) is a white single crystal, with a yield of 68% and melting range of 216.3-217.6 ℃. 1 H NMR (400 MHz, CDCl 3 ) δ (ppm): δ 12.25 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 6.38 (s, 1H), 4.06 (s, 3H), 2.73 (s, 2H), 1.37 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) δ 170.43, 167.25, 159.96, 158.16, 154.47, 143.29, 134.44, 131.61, 130.87, 128.80, 118.76, 100.86, 100.16, 95.71, 52.99, 37.89, 26.52. HR-MS (ESI) : calcd for C 21 H 17 ClO 6 [M + H] + : 401.0784. 3b (2'', 2'-Dimethyl-3-(3', 5'-difluoromethyl phenyl)-7-hydroxy-2''H, 3''H-4,5-pyrrolo[4,5-b]chromen-8-carboxylic acid methyl ester): white needle crystals, with a yield of 67% and melting range of 225.1-226.4 ℃. 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): δ 12.32 (s, 1H), 7.92 (s, 1H), 7.78 (s, 2H), 6.40 (s, 1H), 4.06 (s, 3H), 2.73 (s, 2H), 1.39 (s, 6H). 13 C NMR (126 MHz, CDCl 3 ) δ (ppm): δ 170.27, 167.83, 159.54, 158.25, 154.66, 144.62, 134.66, 130.61, 124.19, 122.36, 122.02, 116.92, 101.21, 99.83, 95.91, 53.03, 37.79, 26.54. HR-MS (ESI) : calcd for C 23 H 16 F 6 O 6 [M + H] + : 503.0923. 3c 2'', 2''-Dimethyl-3-(4'-hydroxyphenyl)-7-hydroxy-2''H, 3''H-4,5-pyrrolo[3,4-b]carbazole-8-carboxylic acid methyl ester is a white solid with a yield of 65% and a melting point of 162.2–163.6 ℃. 1 H NMR (500 MHz, CDCl 3 ) δ (ppm): δ 12.21 (s, 1H), 7.16 (d, J = 7.7 Hz, 2H), 6.89 (d, J = 8.0 Hz, 2H), 6.37 (s, 1H), 5.21 (s, 1H), 4.05 (s, 3H, -OCH 3 ), 2.74 (s, 2H), 1.35 (s, 6H, 2×CH 3 ). 13 C NMR (126 MHz, DMSO) δ (ppm) :δ 165.86, 160.83, 160.32 157.62, 155.63, 151.88, 142.96, 132.04, 123.43, 119.20, 115.33, 101.56, 99.59, 77.76, 52.86, 37.53, 26.41. HR-MS (ESI) : calcd for C 21 H 18 O 7 [M + H] + : 383.1125. 2.2.3 Crystal data and structure determination of the probe compound. To further confirm the structure of the target compound, a saturated solution of 2a in anhydrous ethanol was prepared and allowed to slowly evaporate at room temperature, yielding transparent crystals. A single crystal with dimensions 0.14 × 0.13 × 0.12 mm³ was selected for X-ray diffraction analysis on a Bruker SMART 1000 CCD diffractometer equipped with a graphite-monochromated MoKα radiation source (λ = 0.71073 Å). Data were collected at 100.00(10) K using ω/2θ scans over a range of 4.806° ≤ 2θ ≤ 49.986°, yielding 11,039 reflections (3,150 unique, R int = 0.0310, R sigma = 0.0328). The data were corrected for Lorentz-polarization (LP) and empirical absorption effects. The crystal structure of 2a (C 21 H 17 ClO 6 , Mr = 400.80) crystallizes in the monoclinic C 2/c space group, with unit cell parameters a = 23.9515(14) Å, b = 9.1618(4) Å, c = 17.4802(9) Å, V = 3574.2(3) ų, Dc = 1.490 g/cm³, Z = 8, and F(000) = 1664.0. The linear absorption coefficient (µ) was 0.252 mm − 1 . The structure was refined by full-matrix least-squares methods using SHELXL-97, yielding final residuals of R 1 = 0.0356 and R 2 = 0.0987 [for I > 2σ(I)], with a goodness-of-fit (S) of 1.003. The final difference Fourier map showed maximum/minimum residual electron densities of 0.63 e·Å −3 and − 0.27 e·Å −3 , respectively. 2.3. Preparation and test conditions of experimental materials 2.3.1 Studies on UV-Vis and Fluorescence Spectroscopy Accurately weighed quantities of compounds 2b and 3c were dissolved in an acetonitrile-RNA electrophoresis buffer mixture (v:v = 8:2) with 5 min of sonication to ensure complete dissolution. The resulting solutions were transferred to 10 mL volumetric flasks and brought to volume, yielding stock solutions with concentrations of 0.002 mM and 0.01 mM, respectively. For analysis, aliquots of each compound solution were pipetted into cuvettes, followed by the addition of 10 µL hydrazinium ion solution. After allowing the reaction to proceed at room temperature for a specified duration, the samples were subjected to UV-Vis spectrop-hotometry and fluorescence spectroscopy for characterization. 2.3.2 Anti-interference Detection. Fluorescence intensity responses of probes 2b and 3c toward typical cations (Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Na + ), anions (Br⁻, Cl⁻, ClO 4 ⁻, Ac⁻, HSO 4 ⁻), and biologically relevant species (glutathione, triethylamine) were systematically investigated. 2.4 Practical Application 2.4.1 Detection of Hydrazine Vapor Accurately weighed quantities of compounds 2b and 3c were dissolved in an acetonitrile-RNA electrophoresis buffer mixture (v:v = 8:2) to prepare a 10 mM stock solution, and a series of hydrazine hydrate solutions (0, 10, 20, 40, 60, 100, 300, 500 mM, and 1 M) were prepared. Filter paper strips were immersed in 2b/3c test solution for 10 min, air-dried, then spotted with the hydrazine concentration series. After 20–30 min reaction at ambient conditions, the test strips were photo-documented under UV excitation (λ = 365 nm). 2.4.2 Detection of Hydrazine in Food Samples (Lettuce and Rice) Food samples (rice and lettuce) obtained from local supermarkets were treated by spraying with N₂H₄ solutions (300 mM and 80 mM), followed by surface application of probe solutions 2b and 3c (10 mM each). The samples were then photo-documented under controlled lighting conditions to record the detection response. 2.5 DFT calculations Density functional theory (DFT) was used to optimize the ground state geometry of the probe 2b and 3c using cam-b3lyp/6-311 + g (d,p), m06-2x/def2svp, and pbe0/6311 + g (d,p), respectively. The optimal functional and basis set were selected. Based on the optimized ground state geometric structure, excited state correlation calculations were performed using TD-DFT. All these calculations were performed using Gaussian 16. 3. Results and discussion 3.1 Design and synthesis of probe 2( a-c ) and 3( a-c ) The synthetic route of this study is illustrated in Scheme 2 . Starting from 2,4,6-trihydroxybenzoic acid methyl ester and 3,3-dimethylacrylic acid as the initial raw materials, we successfully constructed a novel class of angular pyranocoumarin skeleton compounds via key reactions such as PPA cyclization, Perkin condensation and reduction reaction, yielding a series of 3-aryl-4,5-pyranocoumarin derivatives with single or dual ester groups ( 2a-2c and 3a-3c ). To date, no literature has reported on the 4,5-pyranocoumarin skeleton or its synthesis methods, representing the first discovery of this study. Additionally, the single crystal of compound 3a was successfully prepared using anhydrous ethanol as the solvent, and its structure was characterized by X-ray single crystal diffraction technology. The molecular structure of 3a is shown in Fig. 3 (A), and the packing diagram of 3a is depicted in Figure (B). Most bond lengths in the system fall in the range of single and double bonds and the C-C bond distances in all rings range between 1.358 (2) and 1.522 (2) Å, almost equal to the values of typical bonds of aromatic structure. 3-aroyl is almost coplanar with the torsion angles of C13-C12-C3-C2 to be -119.45(17). During the structural analysis, it was found that an intramolecular hydrogen bond was formed between O5-H and O6, with a bond length of 2.551 Å. In the crystal packing diagram, the centroid-to-centroid distance of the C5-C10-C9-C8-C7-C6 aromatic plane is 3.632 Å, with a lateral displacement of 1.527 Å. These values suggest a slipped π-π stacking interaction between molecules. The centroid-to-centroid distance falls within the typical range for π-π interactions in aromatic systems (3.3–3.8 Å), while the significant lateral displacement (approximately 42% of the centroid-to-centroid distance) indicates that the molecular planes are not fully overlapped but rather exhibit a partially offset arrangement. This configuration optimizes van der Waals interactions between molecules. Such a packing mode effectively balances π-π attraction and steric repulsion, thereby enhancing the overall stability of the crystal structure. 3.2 Optical responses of 2 ( a-c ) and 3 ( a-c ) to N 2 H 4 Selectivity is a critical parameter for assessing probe performance, as only probes with high selectivity possess practical application value. In this study, we evaluated the potential interference from several common cations (Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Na⁺), anions (Br⁻, Cl⁻, ClO₄⁻, Ac⁻, HSO₄⁻), and small molecules (glutathione, triethylamine) using fluorescence spectroscopy. As shown in Fig. 4 , the fluorescence intensity of probe 2b at 424 nm (~ 8000 a.u.) was dramatically enhanced to 23616 a.u. upon treatment with N 2 H 4 , representing a 195% increase. Probe 3c exhibited even more superior response characteristics, with its fluorescence intensity at 460 nm surging from 4423 a.u. to 26275 a.u. (a 494% increase) in the presence of N 2 H 4 , and a new fluorescence emission peak emerged at 462 nm. No significant responses were observed with other ions or small molecular reagents, and both probes demonstrated excellent selectivity in Ion-selective experiments. Besides, compounds 2a, 2c, 3a , and 3b exhibit extremely low selectivity towards N 2 H 4 . Although they respond to various ions, their selectivity remains relatively low. Specifically, compound 2a displays limited responsiveness, showing only weak quenching activity toward ferric iron (Fe 3+ ), bisulfate (HSO₄⁻) and triethanolamine (TEA), while remaining inert to other tested ions. Notably, 2c, 3a , and 3b demonstrate broad-spectrum sensitivity to multiple anion, with pronounced fluorescence quenching effects observed in the presence of chloride (Cl⁻), bisulfate (HSO₄⁻), perchlorate (ClO4 − ) ion, acetoxy (Ac − ) ion, and bromide (Br⁻) ions. Additionally, 3a shows significant fluorescence quenching in the presence of Zn 2+ . Compound 2c reveals quenching effects for hydrazine (N 2 H 4 ), triethylamine (TEA), Zn 2+ , and Cu 2+ . Compound 3b exhibits a sign-ificant quenching effect on hydrazine (N 2 H 4 ) and triethanolamine (TEA). In conclusion, only compound 2b and compound 3c are suitable for use as hydrazine probes for the next stage of research. UV-Vis spectroscopy of representative compound 2b and 3c (Figs. 4 g, 4 h) also further demonstrated their ability to bind with N 2 H 4 . The results indicate that probe 2b exhibits characteristic absorption peaks at 255 nm and 310 nm. Upon the addition of hydrazine hydrate (N 2 H 4 ), the absorption intensity significantly increases, accompanied by a red shift in the maximum absorption wavelength (310 nm→325 nm), suggesting an interaction between the probe and N 2 H 4 . Additionally, the characteristic absorption peak of probe 3c at 325 nm also demonstrates a pronounced hyperchromic effect. It is noteworthy that the influence of common cations (Mg 2+ , Ca 2+ , Zn 2+ , Cu 2+ , Fe 3+ , Na + ), anions (Br − , Cl − , ClO 4 − , Ac − , HSO 4 − ), and biological reagents (GSH, TEA) on the absorbance of the two probes is significantly lower than that of N 2 H 4 , confirming the probes' high binding selectivity for N 2 H 4 . This paper systematically investigates the relationship between hydrazine concentration and fluorescence intensity (Fig. 5 ). Within the hydrazine concentration range of 0 to 50 µM, the fluorescence intensity of probe 2b demonstrates a strong linear correlation with hydrazine concentration (Fig. 5 a). As hydrazine is gradually added, compound 2b exhibits a concentration-dependent fluorescence enhancement at 424 nm, accompanied by a significant red shift phenomenon (Δλ = 18 nm), with the emission peak shifting to 442 nm. In the hydrazine concentration range of 0 to 10 µM, the fluorescence intensity of probe 3c also displays a significant linear relationship with hydrazine concentration (Fig. 5 b). Compound 3c shows a gradual increase in fluorescence intensity at 457 nm, without any observed change in wavelength. Based on the detection limit calculation formula LOD = 3σ/K (where σ represents the standard deviation and K represents the slope of the calibration curve), it was determined that σ = 7.49. The detection limits for probe 2b and probe 3c were calculated to be 0.12 µmol/L and 0.03 µmol/L, respectively. According to the literature, 24–27 the detection limit of probe 3c is lower than that of most reported hydrazine probes and is significantly below the standard upper limit (10 ppb) established by the United States Environmental Protection Agency (EPA). These results indicate that probes 2b and 3c possess high sensitivity and low detection limits for the recognition of hydrazine ions, making them suitable for detecting trace hydrazine concentrations in actual samples. To further assess whether the selective recognition of hydrazine by probes 2b and 3c would be interfered with by other ions or molecules, we selected a series of typical cations (Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Na⁺), anions (Br⁻, Cl⁻, ClO₄⁻, Ac⁻, HSO₄⁻), and representative biological reagents (glutathione, triethylamine) as potential interferents to evaluate the anti-interference capabilities of probes 2b and 3c . As shown in Fig. 5 (a, c), except for triethylamine, the other analytes had no significant impact on the fluorescence intensity of probe 2b after the addition of hydrazine. It is noteworthy that triethylamine alone can induce a slight fluorescence enhancement in probe 2b ; when triethylamine coexists with hydrazine, the combined effect of the two leads to a further increase in fluorescence signal intensity, suggesting that triethylamine may cause some interference in the quantitative detection of hydrazine through a synergistic effect. In contrast, probe 3c did not exhibit any significant changes in fluorescence response in the presence of all tested interferents, including triethylamine, fully confirming its highly specific recognition capability for hydrazine. This study systematically investigated the stability and detection performance of the hydrazine fluorescent probe compounds 2b and 3c under different pH conditions, and determined the appropriate pH range for their detection (see Fig. 6 ). The results showed that compound 2b exhibited good fluorescence intensity stability within the pH range of 1–12; while in the condition of pH > 12, its fluorescence intensity gradually increased with the rise of pH. This might be attributed to the gradual hydrolysis of the ester group in compound 2b in a strongly alkaline environment, releasing free phenolic hydroxyl groups, which led to the change in fluorescence intensity. After adding hydrazine, the fluorescence intensity of compound 2b significantly increased and remained stable within the pH range of 1–8; in the strongly alkaline condition (pH > 8), its fluorescence intensity increased with the rise of pH and reached a constant value at pH = 12. This phenomenon might be related to the reaction of hydrazine with the ester group in the skeleton of compound 2b to form hydrazone, and then the other ester group hydrolyzed to form phenol under the action of strong base, causing a significant change in fluorescence intensity. For compound 3c , it showed the best fluorescence stability within the pH range of 1–6; however, in the condition of pH > 6, its fluorescence intensity gradually increased with the rise of pH. This might be related to the acidic nature of the phenolic hydroxyl group in the structure, forming salts under alkaline conditions, and possibly involving the hydrolysis of another ester group. After adding hydrazine, the fluorescence intensity of compound 3c did not significantly increase as expected within the pH range of 1–6, but remained relatively stable. This might be because the phenolic hydroxyl group (-OH) was protonated, and the intramolecular conjugated system or charge transfer (ICT) was inhibited. At this time, hydrazine existed in the protonated form (NH 3 ⁺-NH 2 ), the nucleophilicity decreased, and the reaction rate with the ester group decreased, resulting in a smaller amount of hydrazone product formation and the fluorescence was not effectively activated. Therefore, until pH > 6, the phenolic hydroxyl group and hydrazine gradually deprotonated, and the fluorescence intensity gradually increased, reaching a peak and then slightly decreased at pH = 11.5. It is speculated that this phenomenon might be caused by the formation of quinone structure of the phenolic oxygen anion under strongly alkaline conditions, which disrupted the original conjugated system. In conclusion, compound 2b has a wider application range and can achieve the detection of hydrazine within the pH range of 1–12; while compound 3c has a relatively limited application range and can only be used for the detection of hydrazine in weakly acidic or alkaline conditions (pH 6–10). In the acetonitrile-RNA electrophoresis buffer solution (with a volume ratio of 8:2), fluorescence tests were conducted on the probe 2b and 3c at a concentration of 2×10 − 6 mol/L every 10 minutes. The results indicated that the fluorescence intensities of compounds 2b and 3c did not show significant changes within 1 hour when their maximum emission peaks were at 424 nm and 462 nm respectively. This result not only verified the stability of the probe under the experimental conditions, providing a reliable guarantee for the obtained results, but also effectively excluded the errors that might be introduced due to the instability of the probe, thus making it more suitable for complex practical application scenarios. After the addition of N 2 H 4 , the fluorescence intensity of probe 2b continued to increase continuously, but failed to reach a stable state within 140 minutes; while compound 3c exhibited significantly different characteristics. Its fluorescence intensity rapidly reached the maximum value within 5 minutes and remained stable thereafter, without further changes over time. 3.3 The recognition mechanism of N 2 H 4 by probe 2b and 3c The reaction mechanism of the ester-ylhydrazine probes typically involves a specific interaction between the ester bond within the structure and hydrazine (N 2 H 4 ). In this process, the amino group (-NH 2 ) of hydrazine functions as a nucleophile, attacking the two carbonyl carbon atoms of the ester groups in probe 2b . This reaction leads to the cleavage of one ester bond, resulting in the formation of an hydrazide derivative 6 − 1 , while the other ester group undergoes hydrolysis to yield a hydroxyl group to give product 6 − 2 . Ultimately, a fluorescent or chromogenic moiety is released, significantly enhancing fluorescence intensity. High-resolution mass spectrometry (HRMS) analysis (Figs. 7 a and 7 b) reveals that probe 2b exhibits a [M + Na] + peak (e.g., m/z 567.0851) in positive ion mode (ESI + ). Following the reaction with N 2 H 4 , a [M + H] + peak (m/z 503.1966) corresponding to the product 6 − 2 formed. At the same time, probe 3c exhibits a similar reaction mechanism. However, due to the presence of only one ester group, the amino group of N 2 H 4 not only attacks the ester carbonyl carbon atoms of probe 3c , leading to the formation of the coumarin amide hydrazide, but also further interacts with the coumarin lactone ring, resulting in ring rupture. According to the HRMS analysis results (Figs. 7 c and 7 d), probe 3c displays a [M + Na] + peak (m/z 405.0955) in positive ion mode (ESI + ). Upon the addition of hydrazine, the [M + H] + peak (m/z 383.1227) corresponding to the coumarin hydrazide product 6 − 3 and the [M + H] + peak (m/z 415.2096) associated with the product 6 − 4 after ring rupture are generated. Furthermore, under the sustained action of hydrazine, the phenolic hydroxyl group within the structure rearranges into a quinone, which might be the reason why probe 3c shows the fluorescence enhancement among all the compounds in this series. The response mechanism of probe 2b and 3c to hydrazine hydrate are shown in Scheme 2 . To deeply explore the mechanism of fluorescence enhancement, in this study, the interaction between probe 2b and 3c and N 2 H 4 was systematically analyzed through density functional theory (DFT) calculations (Fig. 8 ) . After binding with N 2 H 4 , both the HOMO and LUMO energy levels of probe 2b shifted towards higher energies (HOMO: -2.45→-2.27 eV; LUMO: -6.65→-6.49 eV), indicating that the introduction of N 2 H 4 reconstructed the electronic structure of the probe through electron donor effects, such as lone pair coordination or charge transfer. Although the HOMO-LUMO energy gap only slightly increased from 4.20 eV to 4.21 eV (a change of 0.01 eV), the oscillator strength drastically decreased from 0.194 to 0.0797 (a reduction of 59%), a phenomenon that significantly impacts fluorescence properties. Based on the experimental data, including the red shift of the fluorescence emission wavelength of probe 2b from 424 nm to 442 nm, despite the minimal change in the energy gap, the upward shift of the HOMO/LUMO levels may induce the red shift phenomenon by altering the intramolecular charge transfer (ICT) characteristics. Furthermore, the significant decrease in oscillation strength may be related to the enhanced competition of non-radiative transition pathways in the excited state. This notable reduction suggests that after the probe binds with N 2 H 4 , the probability of electronic transitions has greatly decreased, potentially leading to a weakening of fluorescence emission intensity or a shortening of the excited state lifetime. However, the experimental results show that the fluorescence intensity of probe 2b significantly increases after binding with hydrazine, which may be attributed to changes in solvent polarity or the effects of hydrogen bonding in the experimental system. After the combination of probe 3c with N 2 H 4 , its HOMO level decreased from − 1.25 eV to -1.34 eV, and the LUMO level decreased from − 5.78 eV to -5.82 eV. The HOMO-LUMO gap narrowed by 0.06 eV (from 4.54 eV to 4.48 eV), while the oscillator strength (f-value) increased from 0.0912 to 0.0949. This phenomenon contrasts sharply with the significant decrease in the f-value observed in probe 2b , highlighting the differences in the action mechanisms of various probes. In probe 3c , the simultaneous downward shift of the HOMO and LUMO energy levels enhances intramolecular charge transfer (ICT) from the coumarin donor to the ester acceptor. The contraction of the energy gap reduces the energy difference between the ground state and the excited state, leading to an increased Stokes shift and a red shift in fluorescence emission. However, no significant red shift was observed in either the UV or fluorescence spectra, which may be attributed to the subtle change in the energy gap being masked by solvent effects, instrument resolution, or compensation from other factors. Meanwhile, the increase in the f-value (by 4.1%) enhances the probability of radiative transition, thereby increasing fluorescence intensity, consistent with the experimental results. This study further validated the practical detection capability of probes 2b and 3c through simulated food sample experiments. A 10.0 mM probe solution was uniformly sprayed on pretreated rice and lettuce surfaces, followed by localized application of 80 mM hydrazine hydrate to simulate contamination. Under UV light (365 nm) imaging, hydrazine-exposed areas exhibited a distinct blue-to-cyan fluorescence transition with both probes, generating high-contrast spatially resolved signals. Notably, probe 3c demonstrated significantly higher fluorescence enhancement intensity than 2b, indicating superior detection sensitivity (Fig. 9 ). These experiments confirmed that both probes enable visual monitoring of hydrazine contamination on food surfaces without complex sample pretreatment, with 3c exhibiting greater application value for rapid on-site detection due to its more pronounced optical response characteristics. Based on the aforementioned spectral performance advantages, this study further validated the practical application potential of probes 2b and 3c through a visual test strip experiment. As shown in Fig. 10 , the experiment utilized the impregnation method to prepare probe-loaded test strips, which were then exposed to hydrazine vapor at varying concentrations. The results demonstrated that under 365 nm UV light excitation, the test strip loaded with probe 2b exhibited purple fluorescence, while the strip loaded with probe 3c displayed blue-green fluorescence. As the hydrazine concentration increased, the fluorescence intensity of the test strip loaded with probe 2b gradually intensified, showing a distinct blue fluorescence at a concentration of 10 M. Conversely, the test strip loaded with probe 3c exhibited a color transition from blue-green to blue and then to green. Notably, the test strip loaded with probe 3c displayed a more significant response in terms of fluorescence enhancement, requiring only 250 mM of hydrazine hydrate to induce a noticeable change in the filter paper.(Fig. 10 ) Conclusions In summary, this study successfully designed and synthesized two novel 3-aryl-4,5-pyranocoumarin-based fluorescent probes ( 3c and 2b ) with fully characterized structures by 1 H/ 13 C NMR and HRMS. The structural uniqueness of the 4,5-pyranocoumarin skeleton was confirmed through X-ray crystallography, which revealed intramolecular hydrogen bonding and slipped π-π stacking interactions, both of which stabilize the crystal lattice. Both probes exhibited exceptional selectivity and sensitivity towards hydrazine (N 2 H 4 ), with 3c achieving an ultralow detection limit of 0.03 uM, surpassing most reported probes and meeting the stringent EPA standard of 10 ppb. Mechanistic studies demonstrated that N 2 H 4 induces ester bond cleavage and structural rearrangement, resulting in fluorescence activation and intramolecular charge transfer (ICT) modulation, as corroborated by HRMS analysis and DFT calculations. While 2b displayed broader pH adaptability (pH 1–12) and a red-shifted emission (Δλ = 18 nm), 3c demonstrated superior performance in terms of specificity, rapid response time, and resistance to interferents, including biological thiols and metal ions. Practical validation in food matrices and test strips underscored their potential for on-site hydrazine detection, with 3c demonstrating superior sensitivity and visual signal contrast. This work not only propels the advancement of pyranocoumarin-based optical sensors, but also establishes a versatile platform for environmental and food safety monitoring field-deployable sensors, for accurate hydrazine quantification across environmental and food safety applications. Declarations Funding This work is funded by Natural Science Foundation of Guangxi Province (2023GXNSFAA026476), the Project Program of Guangxi Key Laboratory of Drug Discovery and Optimization (GKLPMDDO2022B02), Qihuang High-level Talent Team Cultivation Project of Guangxi University of Chinese Medicine (202405), Inheritance and Innovation team of Guangxi Traditional Chinese Medicine (2022B005). Competing Interests There are no conflicts to declare. Author contributions Huafeng Zhou and Jiayong Huang: investigation, experimental operation, software, writing – original draft, formal analysis; Jian Su and : investigation, experimental assistance; Xiaowei Su: investigation, data curation, formal analysis; Qiujuan Chen: investigation, data curation, formal analysis; Yuxiao Zheng: experimental assistance, data curation; Rui Chen and Lini Huo: revising the paper and supervising the research. All authors approved the final version of the publication. Data availability Data is provided within the manuscript or supplementary information files. Supplementary Material We have presented all our main data in the form of tables and figures. CCDC 2449340 contains supplementary crystallographic data for compound 3a These datas can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected] . References Ioannidou H A, Koutentis P A, The conversion of isothiazoles into pyrazoles using hydrazine. Tetrahedron . 2009, 65(34): 7023-7037. https:// doi.org/ 10.1016/ j.tet.2009.06.041 Lambert T H. Development of a hydrazine-catalyzed carbonyl-olefin metathesis reaction. Synlett . 2019, 30(17): 1954-1965. https:// doi.org/ 10.1055/ s-0039-1689924 Nakui H, Okitsu K, Maeda Y, et al. Hydrazine degradation by ultrasonic irradiation. J. Hazard. 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USEPA's IRIS . 1999, 3020702: 1-13. Lv Yongzhi, Feng Xueyi, Liu Lusang, et al. Review on the Luminescence Mechanism of Fluorescent Probes. Fujian Analytical Testing . 2017, 26, 25-30. Zhang Tengteng. Construction of a New Hydrazine Hydroxide Fluorescence Probe and Its Application. Guangxi University. 2023. Pršir K, Horak E, Kralj M, et al. Design, synthesis, spectroscopic characterisation and in vitrocytostatic evaluation of novel bis(coumarin-1,2,3-triazolyl)benzenes and hybridcoumarin-1,2,3-triazolyl-aryl derivatives. Molecules . 2022, 27(3): 637-52. https:// doi.org/ 10.3390/ molecules27030637 Sun X Y, Liu T, Sun J, et al. Synthesis and application of coumarin fluorescence probes. RscAdvances . 2020, 10(18): 10826-47. https:// doi.org/ 10.1039/ c9ra10290f Goswami S, Das A K, Maity S. 'PET' vs. 'push-pull' induced ICT: a remarkable coumarinyl-appendedpyrimidine based naked eye colorimetric and fluorimetric sensor for the detection of Hg2+ ions inaqueous media with test trips . Dalton Transactions . 2013, 42(46): 16259-63. https:// doi.org/ 10.1039/ c3dt52252k Hao Tingting. Construction and Application Research of Hydrogen Sulfide Fluorescence Probes Based on Coumarin . Hebei University, 2024. Yang F, Yang L, Xu L, et al. 3D-printed smartphone-based device for fluorimetric diagnosis of ketosis by acetone-responsive dye marker and red emissive carbon dots. Microchimica Acta . 2021, 188(9): 04965. https:// doi.org/ 10.1007/ s00604-021-04965-0 Wang X, Zhang J, Zhu J, et al. Monitoring isoniazid metabolism in vivo using a nearinfrared fluorescent probe. Analytical Methods . 2022, 14(23): 2284-2292. https:// doi.org/ 10.1039/d2ay00185c Xu W, Li X, Yin J, et al. A new fluorescent turn-on dual interaction position probe for determination of hydrazine. Analytical Sciences , 2019, 35(12): 1341-1345. https:// doi.org/ 10.2116/ analsci.19p229 Xia X, Zeng F, Zhang P, et al. An ICT-based ratiometric fluorescent probe for hydrazine detection and its application in living cells and in vivo. Sensors and Actuators B: Chemical . 2016, 227: 411-418. https:// doi.org/ 10.1016/ j.snb.2015.12.046 Tlan G, Zhang Z X, Li H D, et al. Design, synthesis and application in analytical chemistry of photo-sensitive probes based on coumarin. Crit. Rev. Anal. Chem. 2021, 51(6): 565-581. https:// doi.org/ 10.1080/ 10408347.2020.1753163 Liang-Liang G,Shulin P,Ying G, et al. Recent development of organic small-molecule and nanomaterial fluorescent probes for hydrazine. Advanced Agrochem. 2022;1 (1):22-38. https:// doi.org/ 10.1016/ j.aac.2022.08.003 Zuo Ke. Construction and Detection Performance Study of Hydrazine Hydroxide Fluorescence Test Paper. Guangxi University, 2023. Dai Yinghong, Zhao Yanmin, Zhang Meiyuan, et al. Research on the Physiological and Pharmacological Activities of Coumarin Compounds. Shandong Chemical Industry . 2021, 50(4): 30-31.S. https:// doi.org/ 10.3390/ foods11182806 Niyazi S, Pouramiri B, Rabiei K. Functionalized nanoclinoptilote as a novel and green catalyst for thesynthesis of Mannich bases derived from 4-hydroxy coumarin. Journal of Molecular Structure . 2022, 1250: 131908. https:// doi.org/ 10.1016/ j.molstruc.2021.131908 Li M, Chen H, Liu X, et al. Development of three novel benzothiazole-based ratiometric fluorescent chemosensor for detecting of hydrazine in serum and gas phase via ESIPT process and different recognition sites. Tetrahedron Letters . 2019, 60(45): 151219. https:// doi.org/ 10.1016/ j.tetlet.2019.151219 Qu P, Ma X, Chen W, et al. A coumarin-based fluorescent probe for ratiometric detection of hydrazine and its application in living cells. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy . 2019, 210: 381-386. https:// doi.org/ 10.1016/ j.saa.2018.11.007 Li T, Liu J, Song L, et al. A hemicyanine-based fluorescent probe for hydrazine detection in aqueous solution and its application in real time bioimaging of hydrazine as a metabolite in mice. Journal of Materials Chemistry B . 2019, 7(20): 3197-320. https:// doi.org/ 10.1039/ c9tb00132 Xie J H, Wang L, Su X Q, et al. Coumarin-based fluorescent probes for bioimaging: recent applications and developments. Curr. Org. Chem. 2021, 25(18):2142-2154. https:// doi.org/ 10.2174/ 1385272825666210728101823 Scheme Scheme 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supplementarymaterials.docx scheme1.png Scheme 1 Synthesis of the probes (2a-2c) and (3a-3c) scheme2.png Scheme 2 The response mechanism of probe 2b and 3cto hydrazine hydrate Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6756101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467279265,"identity":"737050a9-4775-4723-8f3a-9c8e7474b823","order_by":0,"name":"huafeng zhou","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"huafeng","middleName":"","lastName":"zhou","suffix":""},{"id":467279266,"identity":"deceb2be-85dc-4103-b0f6-664e5dd2f0d7","order_by":1,"name":"jiayong huang","email":"","orcid":"","institution":"Guangxi University of Chinese 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06:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6756101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6756101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84225173,"identity":"b7ff733a-1726-406d-9058-b55d539964a2","added_by":"auto","created_at":"2025-06-09 12:40:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14888,"visible":true,"origin":"","legend":"\u003cp\u003eCoumarin-based metal ion probe\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/15bc9d31dbd76ee2004f381c.png"},{"id":84225174,"identity":"606a9f63-2083-479c-8e48-c99ecadd3697","added_by":"auto","created_at":"2025-06-09 12:40:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64719,"visible":true,"origin":"","legend":"\u003cp\u003eRational Design Strategy for Novel Pyranocoumarin-Based Hydrazine Probes\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/e980037ce4ec89152a7d4b74.png"},{"id":84225523,"identity":"85c81370-a26b-4bd3-9ee8-886343c22f4b","added_by":"auto","created_at":"2025-06-09 12:48:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":291969,"visible":true,"origin":"","legend":"\u003cp\u003e(a) X-ray single crystal diffraction diagram of compound \u003cstrong\u003e3a; \u003c/strong\u003e(b) the packing of \u0026nbsp;\u003cstrong\u003e3a\u003c/strong\u003e, viewed down the b direction. (CCDC 2449340)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/8fd615c5eeaa101956135f27.png"},{"id":84225179,"identity":"d904138d-956f-465e-b5a7-c5fe262817a2","added_by":"auto","created_at":"2025-06-09 12:40:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":260074,"visible":true,"origin":"","legend":"\u003cp\u003e(a-f) IFluorescence changes of different metal ions from \u003cstrong\u003e2a-2c \u003c/strong\u003eto \u003cstrong\u003e3a-3c \u003c/strong\u003e(g) Ion-selective UV-Vis spectrum of \u003cstrong\u003e2b\u003c/strong\u003e. (h) Ion-selective UV-Vis spectrum of \u003cstrong\u003e3c.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/2dfb1592ebcbee1c6d9dc0d0.png"},{"id":84226656,"identity":"4424a884-1005-4ae5-9d5b-c67049df4d32","added_by":"auto","created_at":"2025-06-09 13:04:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":162105,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence spectra of probe\u003cstrong\u003e 2b\u003c/strong\u003e (0.1 mM) in the presence of varying concentrations of [N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e] (0-100 mM). (b) Fluorescence spectra of the probe\u003cstrong\u003e 2b\u003c/strong\u003e (0.1 mM) with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4 \u003c/sub\u003e(0–100 mM). (d) Plot of fluorescence intensity with 0–1.2 mMFe2+. (c) Fluorescence spectra of probe \u003cstrong\u003e3c\u003c/strong\u003e (0.1 mM) with incremental additions of [N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e] (0-50 mM). (d) Fluorescence spectra of the probe \u003cstrong\u003e3c\u003c/strong\u003e (0.1 mM) with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4 \u003c/sub\u003e(0–50 mM).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/d6eb20182193b7fbdd192f76.png"},{"id":84225182,"identity":"9d6239a9-6124-47a2-96f1-bf0aa2ac7b73","added_by":"auto","created_at":"2025-06-09 12:40:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":124200,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Fluorescence intensity profile of probe\u003cstrong\u003e 2b\u003c/strong\u003e toward hydrazine ([N₂H₄]⁺) in the presence of various competing ions (e.g., Mg²⁺, Ca²⁺, Cl⁻, GSH). (b) Time-dependent fluorescence intensity changes of\u003cstrong\u003e 2b\u003c/strong\u003e (control) and \u003cstrong\u003e2b\u003c/strong\u003e+ [N2H4]⁺ over 0–140 min. (c) Corresponding fluorescence response of probe \u003cstrong\u003e3c\u003c/strong\u003efor hydrazine detection under identical interference conditions. (d) Comparative fluorescence intensity profiles of \u003cstrong\u003e3c\u003c/strong\u003e and \u003cstrong\u003e3c\u003c/strong\u003e + [N2H4]⁺ within 0–60 min. (e) pH-dependent fluorescence intensity variations of \u003cstrong\u003e2b\u003c/strong\u003e and\u003cstrong\u003e 2b\u003c/strong\u003e + [N2H4]⁺ across the pH range 0–14. (f) pH-dependent fluore-scence intensity changes of \u003cstrong\u003e3c\u003c/strong\u003e and \u003cstrong\u003e3c\u003c/strong\u003e+ [N2H4]⁺ under the same pH 0–14 conditions.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/b5b1dae4de7836ce44bd1ed9.png"},{"id":84225178,"identity":"dcc940f0-ff0e-4937-bc5a-7bcb870a41f2","added_by":"auto","created_at":"2025-06-09 12:40:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":63414,"visible":true,"origin":"","legend":"\u003cp\u003eHRMS of \u003cstrong\u003e2b\u003c/strong\u003e (a) in the absence and (b) in the presence of hydrazine hydrate; HRMS of \u003cstrong\u003e3c\u003c/strong\u003e (c) in the absence and (d) in the presence of hydrazine hydrate.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/8eceeb4505814fc97b14929f.png"},{"id":84225192,"identity":"28d670d4-ae01-440c-b9fa-fa25c274d285","added_by":"auto","created_at":"2025-06-09 12:40:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":106377,"visible":true,"origin":"","legend":"\u003cp\u003eComputational Analysis of Probes 2b/3c by Density Functional Theory (DFT)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/94ad245113d2978367b127d4.png"},{"id":84225196,"identity":"ca5d51f2-ae52-4d59-82a0-2b869a08ba3f","added_by":"auto","created_at":"2025-06-09 12:40:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":257278,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis and Daylight Response of Probes \u003cstrong\u003e2b\u003c/strong\u003e/\u003cstrong\u003e3c\u003c/strong\u003eToward Hydrazine in Food Matrices Paper\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/7fa31ae308130b05f5c188a4.png"},{"id":84225183,"identity":"2880a9b8-c2ca-4ee0-9fa4-e8a13eb9a20f","added_by":"auto","created_at":"2025-06-09 12:40:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":120937,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Filter Paper Assay of Probe \u003cstrong\u003e2b\u003c/strong\u003e at Graded Concentrations. (b) Filter Assay of Probe \u003cstrong\u003e3c\u003c/strong\u003e at Graded Concentrations.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/fd2ad0f9bc52b0899b8c8e04.png"},{"id":84691941,"identity":"724c67f9-d30a-4c41-85e7-5c361a74e456","added_by":"auto","created_at":"2025-06-16 09:54:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2516452,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/dcf5eff6-1a84-4999-b1d3-13c3aebc1372.pdf"},{"id":84225193,"identity":"a4b66b1e-463a-429a-af2d-f8c88d4e5db4","added_by":"auto","created_at":"2025-06-09 12:40:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6458715,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/e013798da1f702b058e547d0.docx"},{"id":84225175,"identity":"3c63b56f-72bb-4806-8761-9b98f5505b7f","added_by":"auto","created_at":"2025-06-09 12:40:43","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30170,"visible":true,"origin":"","legend":"\u003cp\u003eScheme \u0026nbsp;\u0026nbsp;1 Synthesis of the probes (\u003cstrong\u003e2a-2c\u003c/strong\u003e) and (\u003cstrong\u003e3a-3c\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/ed5be44a7022dc5b229f47cc.png"},{"id":84225522,"identity":"c62926f6-0fb5-4555-a2ea-d35085d38646","added_by":"auto","created_at":"2025-06-09 12:48:43","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":58155,"visible":true,"origin":"","legend":"\u003cp\u003eScheme \u0026nbsp;2 \u0026nbsp;The response mechanism of probe \u003cstrong\u003e2b\u003c/strong\u003e and \u003cstrong\u003e3c\u003c/strong\u003eto hydrazine hydrate\u003c/p\u003e","description":"","filename":"scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-6756101/v1/5b4546a6a66f6beb3d50ee82.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Design and Synthesis of Novel Angular 4,5-Pyranocoumarin Fluorescent Probe for detecting hydrazine and and its applications","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs a crucial multifunctional chemical reagent, hydrazine hydrate (N₂H₄) plays an indispensable role in diverse fields, including chemical synthesis, catalytic processes, pesticide manufacturing, and industrial production[1\u0026ndash;3]. However, N₂H₄ can enter the organism through skin absorption and inhalation of respiratory mucosa, thereby causing irreversible physiological damage [4\u0026ndash;6]. With the acceleration of industrialization, the problem of soil and water pollution caused by improper discharge of hydrazine-containing wastes has become increasingly serious. Consequently, the development of efficient detection methods has as a critical priority for environmental monitoring, industrial safety protocols, and biomedical research[7\u0026ndash;8].\u003c/p\u003e \u003cp\u003eFluorescent probes are widely recognized for their advantages in simple synthesis, strong selectivity, and good biocompatibility[9\u0026ndash;13]. The organic fluorophores such as coumarin derivatives have attracted much attention due to their relatively low toxicity, ease of chemical modification and excellent stability[14\u0026ndash;16]. These superior attributes have established them as versatile molecular platforms for environmental pollutant monitoring, cellular imaging, and in vivo biosensing applications with promising potential. Recent advancements have focused on coumarin probes for hydrazine detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For example, Fanyong Yan et al[17]. designed a ratiometric probe (\u003cb\u003e1\u003c/b\u003e) based on a fluorescein-coumarin hybrid structure. Initially, the photoinduced electron transfer (PET) effect quenched fluorescein emission, leaving only coumarin fluorescence. Hydrazine-induced ester bond cleavage eliminated PET, restoring fluorescein emission for dual-channel detection. Xiaoyan Wang et al[18]. developed a near-infrared probe (\u003cb\u003e2\u003c/b\u003e) using a hemicyanine scaffold, where ester bond cleavage by hydrazine restored the excited-state intramolecular proton transfer (ESIPT) process, triggering a 737 nm fluorescence enhancement. This probe enabled real-time monitoring of isoniazid metabolism in vivo due to its pH stability and biocompatibility. Additionally, Xiangdong Geng et al[19]. reported a coumarin-based probe (Probe \u003cb\u003e3\u003c/b\u003e) exhibiting a 35-fold fluorescence increase and visible colorimetric changes upon hydrazine exposure, providing a practical solution for detecting hydrazine in environmental water. Despite these advances, challenges remain in improving selectivity, sensitivity, and photostability of hydrazine probes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the design of hydrazine (N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e) probes, functional groups such as aldehydes, ketones, and esters are strategically incorporated to enable selective detection through nucleophilic addition or substitution reactions with hydrazine, inducing measurable fluorescence changes[17\u0026ndash;21]. The ester group, recognized as an efficient recognition moiety, has been widely utilized in the detection of hydrazine hydrate[22\u0026ndash;27]. In addition, most of the coumarin-based probes reported to date are derived from simple coumarin skeletons, whereas research on alternative skeleton types remains relatively limited. To enhance the diversity of coumarin-based fluorescent probes and improve their performance, we designed a novel pyranocoumarin skeleton (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This skeleton features an aryl group at the 3-position, which effectively extends the conjugated plane of the coumarin. Simultaneously, ester groups are introduced at the 7 or 8 positions as recognition sites for hydrazine, and the effects of monoester and diester groups on hydrazine recognition were systematically investigated. Subsequently, we conducted an in-depth exploration of the spectral properties and reaction mechanisms of the compounds using methods such as fluorescence and UV-Vis spectrophotometry.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and apparatus\u003c/h2\u003e \u003cp\u003eAll reagents and metal salts used in the experiments were purchased from Aladdin Industrial Corporation (Shanghai, China) or Sigma-Aldrich Trading Co. LTD (St. Louis, MO, USA).The reagents used in the experiment are all analytically pure. NMR spectra were recorded on a DRX-500 NMR spectrometer (Rheinstetten, Germany), and high-resolution mass spectrometry (HRMS) was performed using an Agilent 6210 ESI/TOF mass spectrometer (Palo Alto, CA, USA). Single-crystal X-ray diffraction (SCXRD) data were collected on an Agilent SuperNova Dual Cu-at-zero AtlasS2 diffractometer (Palo Alto, CA, USA). Fluorescence spectra were measured with a Shimadzu RF-6000 spectrofluorophotometer (Tokyo, Japan). UV-Vis absorption spectra were obtained using a UV-1800P spectrophotometer (Shanghai Meipuda Instrument Co, Ltd., China). pH measurements were conducted with a PHB-1 portable pH meter (Shanghai Shinuo Physical Optical Instrument Co., Ltd., China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of the probe.\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Preparation of Intermediate \u003cb\u003e1\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eUnder the action of electromagnetic stirring, 20 mL of anhydrous dioxane, 2.81 g of 2,4,6-trihydroxybenzoic methylester (10 mmol) and 2.26 g of 3,3-dimethylacrylic acid (22 mmol) were added successively to a 50 mL round-bottom flask and heated in an oil bath at 95 ℃. The reaction was stirred under reflux for 7 h and TLC was used to monitor the reaction (petroleum ether: ethyl acetate\u0026thinsp;=\u0026thinsp;4:1). After the reaction was completed, the reaction mixture was poured into 100 mL of ice water, saturated potassium carbonate was added to adjust the pH to neutral, and the mixture was filtered. The crude product was obtained and recrystallized in acetone to yield a pale yellow crystal (\u003cb\u003eCompound 1\u003c/b\u003e), with a yield of 90% and a melting point of 140.6\u0026ndash;144.1 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e), δ (ppm): δ 14.02 (s, 1H), 12.67 (s, 1H), 5.99 (s, 1H), 3.99 (s, 3H, -OCH\u003csub\u003e3\u003c/sub\u003e), 2.74 (s, 2H), 1.48 (s, 6H,2\u0026times;CH\u003csub\u003e3\u003c/sub\u003e). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 198.07, 168.46, 166.17, 165.85, 164.67, 98.03, 82.75, 82.62, 81.02, 51.11, 48.33, 27.96. HRMS (ESI): calcd For C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e : 267.32 .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Preparation of Probes \u003cb\u003e2a, 2b\u003c/b\u003e, and \u003cb\u003e2c\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eUnder N\u003csub\u003e2\u003c/sub\u003e protection, 4 mL (40 mmol) of acetic anhydride, 0.266 g (1 mmol) of \u003cb\u003ecompound 1\u003c/b\u003e and 1 mmol of substituted benzoic acid were successively added to a 50 mL round-bottom flask. The mixture was heated to 100 ℃, then 0.208 mL (1.5 mmol) of triethylamine was added and the temperature was raised to 150 ℃. The reaction was stirred under reflux for 2 hours, and TLC monitoring was carried out (petroleum ether: ethyl acetate\u0026thinsp;=\u0026thinsp;4:1). After the reaction was completed, the reaction mixture was cooled to room temperature, then 8 mL of water was added. The mixture was extracted with ethyl acetate (3\u0026times;15 mL), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography purification (eluent: petroleum ether: ethyl acetate\u0026thinsp;=\u0026thinsp;4:1) was performed to obtain compounds \u003cb\u003e2a, 2b\u003c/b\u003e, and \u003cb\u003e2c\u003c/b\u003e, with yields ranging from 39\u0026ndash;45%.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2a\u003c/b\u003e (2'', 2''-Dimethyl-3-(4'-chlorophenyl)-7-acetoxymethyl-2''H, 3''H-4,5-pyrrolo[3,4-b]oxazole-8-carboxylic acid methyl ester) is a white solid, with a yield of 45% and melting range of 216.3-217.5 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 7.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2H), 7.24 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 2H), 6.56 (s, 1H), 3.96 (s, 3H), 2.74 (s, 2H), 2.32 (s, 3H), 1.37 (s, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 168.67, 163.12, 159.37, 155.38, 152.90, 151.66, 142.29, 134.77, 131.47, 130.54, 128.86, 121.83, 107.52, 107.35, 104.56, 77.51, 52.87, 37.77, 26.44, 20.87. HR-MS (ESI): calcd for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eClO\u003csub\u003e7\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: 465.0707.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2b\u003c/b\u003e (2'', 2''-Dimethyl-3-(3',5'-difluoromethyl phenyl)-7-acetoxy-methyl-2''H, 3''H-4,5-pyrrolo[2,3-b]oxa-zole-8-carboxylic acid methyl ester) is a white solid, with a yield of 40%, melting point ranging from 193.5 to 194.4 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 7.94 (s, 1H), 7.76 (s, 2H), 6.60 (s, 1H), 3.97 (s, 3H), 2.73 (s, 2H), 2.33 (s, 3H), 1.40 (s, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 168.51, 162.91, 158.90, 155.56, 153.56, 151.76, 143.71, 134.32, 130.48, 124.13, 122.68, 121.96, 120.03, 107.79, 107.68, 104.13, 52.89, 37.67, 26.44, 20.85. HR-MS (ESI): calcd for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: 567.0845.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2c\u003c/b\u003e (2'', 2''-Dimethyl-3-(4'-acetoxyphenyl)-7-acetoxy-2''H, 3''-4,5-pyranidinocoumarin-8-methylester) is a white solid, with a yield of 41% and melting range of 176.8\u0026ndash;178.9 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 7.32 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 7.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7 Hz, 2H), 6.56 (s, 1H), 3.96 (s, 3H), 2.78 (s, 2H), 2.34 (s, 3H), 2.32 (s, 3H), 1.37 (s, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 169.44,168.68, 163.17, 159.50, 155.41, 152.80, 150.75, 142.21, 131.33, 129.62, 122.06, 121.75, 107.43, 107.27, 104.67, 103.31, 52.85, 37.79, 26.42, 21.23. HR-MS (ESI): calcd for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e: 489.1143.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Preparation of Probes \u003cb\u003e3a\u003c/b\u003e, \u003cb\u003e3b\u003c/b\u003e, and \u003cb\u003e3c\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eUnder electromagnetic stirring, 100 mL of methanol, compound \u003cb\u003e2a, 2b, 2c\u003c/b\u003e, (3.5 g, 8 mmol) were added successively to a 100 mL round-bottom flask. After dissolving by heating, when the temperature dropped to room temperature, the flask was placed in a low-temperature reactor. Under 0 ℃-5 ℃ conditions, NaBH\u003csub\u003e4\u003c/sub\u003e (1.5 g, 40 mmol) was added in three portions within 1.5 h, and the reaction was stirred for 2 h, followed by TLC monitoring (petroleum ether: ethyl acetate\u0026thinsp;=\u0026thinsp;3:1). After the reaction was completed, 9\u0026ndash;10 drops of 5% HCl were added to the reaction mixture to terminate the reaction. After evaporating the remaining methanol, the organic layer was extracted with ethyl acetate (3\u0026times;20 mL), and the organic layers were combined. They were successively washed with saturated NaHCO\u003csub\u003e3\u003c/sub\u003e solution, saturated brine, and anhydrous sodium sulfate, dried over a filter, and recrystallized with methanol to obtain compounds \u003cb\u003e3a, 3b\u003c/b\u003e, and \u003cb\u003e3c\u003c/b\u003e. The yield was 65%-71%.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3a\u003c/b\u003e (2', 2'-Dimethyl-3-(4'-chlorophenyl)-7-hydroxy-2''H, 3''H-4,5-pyrrolo[4,5-b]chromen-8-carboxylic acid methyl ester) is a white single crystal, with a yield of 68% and melting range of 216.3-217.6 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 12.25 (s, 1H), 7.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 7.26 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 6.38 (s, 1H), 4.06 (s, 3H), 2.73 (s, 2H), 1.37 (s, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 170.43, 167.25, 159.96, 158.16, 154.47, 143.29, 134.44, 131.61, 130.87, 128.80, 118.76, 100.86, 100.16, 95.71, 52.99, 37.89, 26.52. HR-MS (ESI) : calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eClO\u003csub\u003e6\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: 401.0784.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3b\u003c/b\u003e (2'', 2'-Dimethyl-3-(3', 5'-difluoromethyl phenyl)-7-hydroxy-2''H, 3''H-4,5-pyrrolo[4,5-b]chromen-8-carboxylic acid methyl ester): white needle crystals, with a yield of 67% and melting range of 225.1-226.4 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 12.32 (s, 1H), 7.92 (s, 1H), 7.78 (s, 2H), 6.40 (s, 1H), 4.06 (s, 3H), 2.73 (s, 2H), 1.39 (s, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 170.27, 167.83, 159.54, 158.25, 154.66, 144.62, 134.66, 130.61, 124.19, 122.36, 122.02, 116.92, 101.21, 99.83, 95.91, 53.03, 37.79, 26.54. HR-MS (ESI) : calcd for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: 503.0923.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3c\u003c/b\u003e 2'', 2''-Dimethyl-3-(4'-hydroxyphenyl)-7-hydroxy-2''H, 3''H-4,5-pyrrolo[3,4-b]carbazole-8-carboxylic acid methyl ester is a white solid with a yield of 65% and a melting point of 162.2\u0026ndash;163.6 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ (ppm): δ 12.21 (s, 1H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 2H), 6.89 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 6.37 (s, 1H), 5.21 (s, 1H), 4.05 (s, 3H, -OCH\u003csub\u003e3\u003c/sub\u003e), 2.74 (s, 2H), 1.35 (s, 6H, 2\u0026times;CH\u003csub\u003e3\u003c/sub\u003e). \u003csup\u003e13\u003c/sup\u003eC NMR (126 MHz, DMSO) δ (ppm) :δ 165.86, 160.83, 160.32 157.62, 155.63, 151.88, 142.96, 132.04, 123.43, 119.20, 115.33, 101.56, 99.59, 77.76, 52.86, 37.53, 26.41. HR-MS (ESI) : calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: 383.1125.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Crystal data and structure determination of the probe compound.\u003c/h2\u003e \u003cp\u003eTo further confirm the structure of the target compound, a saturated solution of \u003cb\u003e2a\u003c/b\u003e in anhydrous ethanol was prepared and allowed to slowly evaporate at room temperature, yielding transparent crystals. A single crystal with dimensions 0.14 \u0026times; 0.13 \u0026times; 0.12 mm\u0026sup3; was selected for X-ray diffraction analysis on a Bruker SMART 1000 CCD diffractometer equipped with a graphite-monochromated MoKα radiation source (λ\u0026thinsp;=\u0026thinsp;0.71073 \u0026Aring;). Data were collected at 100.00(10) K using ω/2θ scans over a range of 4.806\u0026deg; \u0026le; 2θ\u0026thinsp;\u0026le;\u0026thinsp;49.986\u0026deg;, yielding 11,039 reflections (3,150 unique, R\u003csub\u003eint\u003c/sub\u003e = 0.0310, R\u003csub\u003esigma\u003c/sub\u003e = 0.0328). The data were corrected for Lorentz-polarization (LP) and empirical absorption effects. The crystal structure of \u003cb\u003e2a\u003c/b\u003e (C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eClO\u003csub\u003e6\u003c/sub\u003e, Mr\u0026thinsp;=\u0026thinsp;400.80) crystallizes in the monoclinic C\u003csub\u003e2/c\u003c/sub\u003e space group, with unit cell parameters a\u0026thinsp;=\u0026thinsp;23.9515(14) \u0026Aring;, b\u0026thinsp;=\u0026thinsp;9.1618(4) \u0026Aring;, c\u0026thinsp;=\u0026thinsp;17.4802(9) \u0026Aring;, V\u0026thinsp;=\u0026thinsp;3574.2(3) \u0026Aring;\u0026sup3;, Dc\u0026thinsp;=\u0026thinsp;1.490 g/cm\u0026sup3;, Z\u0026thinsp;=\u0026thinsp;8, and F(000)\u0026thinsp;=\u0026thinsp;1664.0. The linear absorption coefficient (\u0026micro;) was 0.252 mm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The structure was refined by full-matrix least-squares methods using SHELXL-97, yielding final residuals of R\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0356 and R\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0987 [for I\u0026thinsp;\u0026gt;\u0026thinsp;2σ(I)], with a goodness-of-fit (S) of 1.003. The final difference Fourier map showed maximum/minimum residual electron densities of 0.63 e\u0026middot;\u0026Aring;\u003csup\u003e\u0026minus;3\u003c/sup\u003e and \u0026minus;\u0026thinsp;0.27 e\u0026middot;\u0026Aring;\u003csup\u003e\u0026minus;3\u003c/sup\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation and test conditions of experimental materials\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Studies on UV-Vis and Fluorescence Spectroscopy\u003c/h2\u003e \u003cp\u003eAccurately weighed quantities of compounds \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e were dissolved in an acetonitrile-RNA electrophoresis buffer mixture (v:v\u0026thinsp;=\u0026thinsp;8:2) with 5 min of sonication to ensure complete dissolution. The resulting solutions were transferred to 10 mL volumetric flasks and brought to volume, yielding stock solutions with concentrations of 0.002 mM and 0.01 mM, respectively. For analysis, aliquots of each compound solution were pipetted into cuvettes, followed by the addition of 10 \u0026micro;L hydrazinium ion solution. After allowing the reaction to proceed at room temperature for a specified duration, the samples were subjected to UV-Vis spectrop-hotometry and fluorescence spectroscopy for characterization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Anti-interference Detection.\u003c/h2\u003e \u003cp\u003eFluorescence intensity responses of probes \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e toward typical cations (Mg\u0026sup2;⁺, Ca\u0026sup2;⁺, Zn\u0026sup2;⁺, Cu\u0026sup2;⁺, Fe\u0026sup3;⁺, Na\u003csup\u003e+\u003c/sup\u003e), anions (Br⁻, Cl⁻, ClO\u003csub\u003e4\u003c/sub\u003e⁻, Ac⁻, HSO\u003csub\u003e4\u003c/sub\u003e⁻), and biologically relevant species (glutathione, triethylamine) were systematically investigated.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Practical Application\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Detection of Hydrazine Vapor\u003c/h2\u003e \u003cp\u003eAccurately weighed quantities of compounds \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e were dissolved in an acetonitrile-RNA electrophoresis buffer mixture (v:v\u0026thinsp;=\u0026thinsp;8:2) to prepare a 10 mM stock solution, and a series of hydrazine hydrate solutions (0, 10, 20, 40, 60, 100, 300, 500 mM, and 1 M) were prepared. Filter paper strips were immersed in \u003cb\u003e2b/3c\u003c/b\u003e test solution for 10 min, air-dried, then spotted with the hydrazine concentration series. After 20\u0026ndash;30 min reaction at ambient conditions, the test strips were photo-documented under UV excitation (λ\u0026thinsp;=\u0026thinsp;365 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Detection of Hydrazine in Food Samples (Lettuce and Rice)\u003c/h2\u003e \u003cp\u003eFood samples (rice and lettuce) obtained from local supermarkets were treated by spraying with N₂H₄ solutions (300 mM and 80 mM), followed by surface application of probe solutions \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e (10 mM each). The samples were then photo-documented under controlled lighting conditions to record the detection response.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5 DFT calculations\u003c/h2\u003e \u003cp\u003eDensity functional theory (DFT) was used to optimize the ground state geometry of the probe \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e using cam-b3lyp/6-311\u0026thinsp;+\u0026thinsp;g (d,p), m06-2x/def2svp, and pbe0/6311\u0026thinsp;+\u0026thinsp;g (d,p), respectively. The optimal functional and basis set were selected. Based on the optimized ground state geometric structure, excited state correlation calculations were performed using TD-DFT. All these calculations were performed using Gaussian 16.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Design and synthesis of probe 2(\u003cb\u003ea-c\u003c/b\u003e) and 3(\u003cb\u003ea-c\u003c/b\u003e)\u003c/h2\u003e \u003cp\u003eThe synthetic route of this study is illustrated in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Starting from 2,4,6-trihydroxybenzoic acid methyl ester and 3,3-dimethylacrylic acid as the initial raw materials, we successfully constructed a novel class of angular pyranocoumarin skeleton compounds via key reactions such as PPA cyclization, Perkin condensation and reduction reaction, yielding a series of 3-aryl-4,5-pyranocoumarin derivatives with single or dual ester groups (\u003cb\u003e2a-2c\u003c/b\u003e and \u003cb\u003e3a-3c\u003c/b\u003e). To date, no literature has reported on the 4,5-pyranocoumarin skeleton or its synthesis methods, representing the first discovery of this study. Additionally, the single crystal of compound \u003cb\u003e3a\u003c/b\u003e was successfully prepared using anhydrous ethanol as the solvent, and its structure was characterized by X-ray single crystal diffraction technology.\u003c/p\u003e \u003cp\u003eThe molecular structure of \u003cb\u003e3a\u003c/b\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A), and the packing diagram of \u003cb\u003e3a\u003c/b\u003e is depicted in Figure (B). Most bond lengths in the system fall in the range of single and double bonds and the C-C bond distances in all rings range between 1.358 (2) and 1.522 (2) Å, almost equal to the values of typical bonds of aromatic structure. 3-aroyl is almost coplanar with the torsion angles of C13-C12-C3-C2 to be -119.45(17). During the structural analysis, it was found that an intramolecular hydrogen bond was formed between O5-H and O6, with a bond length of 2.551 Å. In the crystal packing diagram, the centroid-to-centroid distance of the C5-C10-C9-C8-C7-C6 aromatic plane is 3.632 Å, with a lateral displacement of 1.527 Å. These values suggest a slipped π-π stacking interaction between molecules. The centroid-to-centroid distance falls within the typical range for π-π interactions in aromatic systems (3.3–3.8 Å), while the significant lateral displacement (approximately 42% of the centroid-to-centroid distance) indicates that the molecular planes are not fully overlapped but rather exhibit a partially offset arrangement. This configuration optimizes van der Waals interactions between molecules. Such a packing mode effectively balances π-π attraction and steric repulsion, thereby enhancing the overall stability of the crystal structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optical responses of 2 (\u003cb\u003ea-c\u003c/b\u003e) and 3 (\u003cb\u003ea-c\u003c/b\u003e) to N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eSelectivity is a critical parameter for assessing probe performance, as only probes with high selectivity possess practical application value. In this study, we evaluated the potential interference from several common cations (Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Na⁺), anions (Br⁻, Cl⁻, ClO₄⁻, Ac⁻, HSO₄⁻), and small molecules (glutathione, triethylamine) using fluorescence spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the fluorescence intensity of probe \u003cb\u003e2b\u003c/b\u003e at 424 nm (~ 8000 a.u.) was dramatically enhanced to 23616 a.u. upon treatment with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, representing a 195% increase. Probe \u003cb\u003e3c\u003c/b\u003e exhibited even more superior response characteristics, with its fluorescence intensity at 460 nm surging from 4423 a.u. to 26275 a.u. (a 494% increase) in the presence of N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, and a new fluorescence emission peak emerged at 462 nm. No significant responses were observed with other ions or small molecular reagents, and both probes demonstrated excellent selectivity in Ion-selective experiments. Besides, compounds \u003cb\u003e2a, 2c, 3a\u003c/b\u003e, and \u003cb\u003e3b\u003c/b\u003e exhibit extremely low selectivity towards N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e. Although they respond to various ions, their selectivity remains relatively low. Specifically, compound \u003cb\u003e2a\u003c/b\u003e displays limited responsiveness, showing only weak quenching activity toward ferric iron (Fe\u003csup\u003e3+\u003c/sup\u003e), bisulfate (HSO₄⁻) and triethanolamine (TEA), while remaining inert to other tested ions. Notably, \u003cb\u003e2c, 3a\u003c/b\u003e, and \u003cb\u003e3b\u003c/b\u003e demonstrate broad-spectrum sensitivity to multiple anion, with pronounced fluorescence quenching effects observed in the presence of chloride (Cl⁻), bisulfate (HSO₄⁻), perchlorate (ClO4\u003csup\u003e−\u003c/sup\u003e) ion, acetoxy (Ac\u003csup\u003e−\u003c/sup\u003e) ion, and bromide (Br⁻) ions. Additionally, \u003cb\u003e3a\u003c/b\u003e shows significant fluorescence quenching in the presence of Zn\u003csup\u003e2+\u003c/sup\u003e. Compound \u003cb\u003e2c\u003c/b\u003e reveals quenching effects for hydrazine (N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e), triethylamine (TEA), Zn\u003csup\u003e2+\u003c/sup\u003e, and Cu\u003csup\u003e2+\u003c/sup\u003e. Compound \u003cb\u003e3b\u003c/b\u003e exhibits a sign-ificant quenching effect on hydrazine (N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e) and triethanolamine (TEA). In conclusion, only compound \u003cb\u003e2b\u003c/b\u003e and compound \u003cb\u003e3c\u003c/b\u003e are suitable for use as hydrazine probes for the next stage of research.\u003c/p\u003e \u003cp\u003eUV-Vis spectroscopy of representative compound \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh) also further demonstrated their ability to bind with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e. The results indicate that probe \u003cb\u003e2b\u003c/b\u003e exhibits characteristic absorption peaks at 255 nm and 310 nm. Upon the addition of hydrazine hydrate (N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e), the absorption intensity significantly increases, accompanied by a red shift in the maximum absorption wavelength (310 nm→325 nm), suggesting an interaction between the probe and N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e. Additionally, the characteristic absorption peak of probe \u003cb\u003e3c\u003c/b\u003e at 325 nm also demonstrates a pronounced hyperchromic effect. It is noteworthy that the influence of common cations (Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e), anions (Br\u003csup\u003e−\u003c/sup\u003e, Cl\u003csup\u003e−\u003c/sup\u003e, ClO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e, Ac\u003csup\u003e−\u003c/sup\u003e, HSO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e), and biological reagents (GSH, TEA) on the absorbance of the two probes is significantly lower than that of N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, confirming the probes' high binding selectivity for N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis paper systematically investigates the relationship between hydrazine concentration and fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Within the hydrazine concentration range of 0 to 50 µM, the fluorescence intensity of probe \u003cb\u003e2b\u003c/b\u003e demonstrates a strong linear correlation with hydrazine concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). As hydrazine is gradually added, compound \u003cb\u003e2b\u003c/b\u003e exhibits a concentration-dependent fluorescence enhancement at 424 nm, accompanied by a significant red shift phenomenon (Δλ = 18 nm), with the emission peak shifting to 442 nm. In the hydrazine concentration range of 0 to 10 µM, the fluorescence intensity of probe 3c also displays a significant linear relationship with hydrazine concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Compound 3c shows a gradual increase in fluorescence intensity at 457 nm, without any observed change in wavelength. Based on the detection limit calculation formula LOD = 3σ/K (where σ represents the standard deviation and K represents the slope of the calibration curve), it was determined that σ = 7.49. The detection limits for probe 2b and probe 3c were calculated to be 0.12 µmol/L and 0.03 µmol/L, respectively. According to the literature,\u003csup\u003e24–27\u003c/sup\u003e the detection limit of probe \u003cb\u003e3c\u003c/b\u003e is lower than that of most reported hydrazine probes and is significantly below the standard upper limit (10 ppb) established by the United States Environmental Protection Agency (EPA). These results indicate that probes \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e possess high sensitivity and low detection limits for the recognition of hydrazine ions, making them suitable for detecting trace hydrazine concentrations in actual samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess whether the selective recognition of hydrazine by probes \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e would be interfered with by other ions or molecules, we selected a series of typical cations (Mg²⁺, Ca²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Na⁺), anions (Br⁻, Cl⁻, ClO₄⁻, Ac⁻, HSO₄⁻), and representative biological reagents (glutathione, triethylamine) as potential interferents to evaluate the anti-interference capabilities of probes \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (a, c), except for triethylamine, the other analytes had no significant impact on the fluorescence intensity of probe 2b after the addition of hydrazine. It is noteworthy that triethylamine alone can induce a slight fluorescence enhancement in probe \u003cb\u003e2b\u003c/b\u003e; when triethylamine coexists with hydrazine, the combined effect of the two leads to a further increase in fluorescence signal intensity, suggesting that triethylamine may cause some interference in the quantitative detection of hydrazine through a synergistic effect. In contrast, probe \u003cb\u003e3c\u003c/b\u003e did not exhibit any significant changes in fluorescence response in the presence of all tested interferents, including triethylamine, fully confirming its highly specific recognition capability for hydrazine.\u003c/p\u003e \u003cp\u003eThis study systematically investigated the stability and detection performance of the hydrazine fluorescent probe compounds \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e under different pH conditions, and determined the appropriate pH range for their detection (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results showed that compound \u003cb\u003e2b\u003c/b\u003e exhibited good fluorescence intensity stability within the pH range of 1–12; while in the condition of pH \u0026gt; 12, its fluorescence intensity gradually increased with the rise of pH. This might be attributed to the gradual hydrolysis of the ester group in compound \u003cb\u003e2b\u003c/b\u003e in a strongly alkaline environment, releasing free phenolic hydroxyl groups, which led to the change in fluorescence intensity. After adding hydrazine, the fluorescence intensity of compound \u003cb\u003e2b\u003c/b\u003e significantly increased and remained stable within the pH range of 1–8; in the strongly alkaline condition (pH \u0026gt; 8), its fluorescence intensity increased with the rise of pH and reached a constant value at pH = 12. This phenomenon might be related to the reaction of hydrazine with the ester group in the skeleton of compound \u003cb\u003e2b\u003c/b\u003e to form hydrazone, and then the other ester group hydrolyzed to form phenol under the action of strong base, causing a significant change in fluorescence intensity. For compound \u003cb\u003e3c\u003c/b\u003e, it showed the best fluorescence stability within the pH range of 1–6; however, in the condition of pH \u0026gt; 6, its fluorescence intensity gradually increased with the rise of pH. This might be related to the acidic nature of the phenolic hydroxyl group in the structure, forming salts under alkaline conditions, and possibly involving the hydrolysis of another ester group. After adding hydrazine, the fluorescence intensity of compound \u003cb\u003e3c\u003c/b\u003e did not significantly increase as expected within the pH range of 1–6, but remained relatively stable. This might be because the phenolic hydroxyl group (-OH) was protonated, and the intramolecular conjugated system or charge transfer (ICT) was inhibited. At this time, hydrazine existed in the protonated form (NH\u003csub\u003e3\u003c/sub\u003e⁺-NH\u003csub\u003e2\u003c/sub\u003e), the nucleophilicity decreased, and the reaction rate with the ester group decreased, resulting in a smaller amount of hydrazone product formation and the fluorescence was not effectively activated. Therefore, until pH \u0026gt; 6, the phenolic hydroxyl group and hydrazine gradually deprotonated, and the fluorescence intensity gradually increased, reaching a peak and then slightly decreased at pH = 11.5. It is speculated that this phenomenon might be caused by the formation of quinone structure of the phenolic oxygen anion under strongly alkaline conditions, which disrupted the original conjugated system. In conclusion, compound \u003cb\u003e2b\u003c/b\u003e has a wider application range and can achieve the detection of hydrazine within the pH range of 1–12; while compound \u003cb\u003e3c\u003c/b\u003e has a relatively limited application range and can only be used for the detection of hydrazine in weakly acidic or alkaline conditions (pH 6–10).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the acetonitrile-RNA electrophoresis buffer solution (with a volume ratio of 8:2), fluorescence tests were conducted on the probe \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e at a concentration of 2×10\u003csup\u003e− 6\u003c/sup\u003e mol/L every 10 minutes. The results indicated that the fluorescence intensities of compounds \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e did not show significant changes within 1 hour when their maximum emission peaks were at 424 nm and 462 nm respectively. This result not only verified the stability of the probe under the experimental conditions, providing a reliable guarantee for the obtained results, but also effectively excluded the errors that might be introduced due to the instability of the probe, thus making it more suitable for complex practical application scenarios. After the addition of N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, the fluorescence intensity of probe \u003cb\u003e2b\u003c/b\u003e continued to increase continuously, but failed to reach a stable state within 140 minutes; while compound \u003cb\u003e3c\u003c/b\u003e exhibited significantly different characteristics. Its fluorescence intensity rapidly reached the maximum value within 5 minutes and remained stable thereafter, without further changes over time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The recognition mechanism of N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e by probe \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe reaction mechanism of the ester-ylhydrazine probes typically involves a specific interaction between the ester bond within the structure and hydrazine (N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e). In this process, the amino group (-NH\u003csub\u003e2\u003c/sub\u003e) of hydrazine functions as a nucleophile, attacking the two carbonyl carbon atoms of the ester groups in probe \u003cb\u003e2b\u003c/b\u003e. This reaction leads to the cleavage of one ester bond, resulting in the formation of an hydrazide derivative \u003cb\u003e6 − 1\u003c/b\u003e, while the other ester group undergoes hydrolysis to yield a hydroxyl group to give product \u003cb\u003e6 − 2\u003c/b\u003e. Ultimately, a fluorescent or chromogenic moiety is released, significantly enhancing fluorescence intensity. High-resolution mass spectrometry (HRMS) analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) reveals that probe \u003cb\u003e2b\u003c/b\u003e exhibits a [M + Na]\u003csup\u003e+\u003c/sup\u003e peak (e.g., m/z 567.0851) in positive ion mode (ESI\u003csup\u003e+\u003c/sup\u003e). Following the reaction with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, a [M + H]\u003csup\u003e+\u003c/sup\u003e peak (m/z 503.1966) corresponding to the product \u003cb\u003e6 − 2\u003c/b\u003e formed.\u003c/p\u003e \u003cp\u003eAt the same time, probe \u003cb\u003e3c\u003c/b\u003e exhibits a similar reaction mechanism. However, due to the presence of only one ester group, the amino group of N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e not only attacks the ester carbonyl carbon atoms of probe \u003cb\u003e3c\u003c/b\u003e, leading to the formation of the coumarin amide hydrazide, but also further interacts with the coumarin lactone ring, resulting in ring rupture. According to the HRMS analysis results (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), probe \u003cb\u003e3c\u003c/b\u003e displays a [M + Na]\u003csup\u003e+\u003c/sup\u003e peak (m/z 405.0955) in positive ion mode (ESI\u003csup\u003e+\u003c/sup\u003e). Upon the addition of hydrazine, the [M + H]\u003csup\u003e+\u003c/sup\u003e peak (m/z 383.1227) corresponding to the coumarin hydrazide product \u003cb\u003e6 − 3\u003c/b\u003e and the [M + H]\u003csup\u003e+\u003c/sup\u003e peak (m/z 415.2096) associated with the product \u003cb\u003e6 − 4\u003c/b\u003e after ring rupture are generated. Furthermore, under the sustained action of hydrazine, the phenolic hydroxyl group within the structure rearranges into a quinone, which might be the reason why probe \u003cb\u003e3c\u003c/b\u003e shows the fluorescence enhancement among all the compounds in this series. The response mechanism of probe \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e to hydrazine hydrate are shown in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo deeply explore the mechanism of fluorescence enhancement, in this study, the interaction between probe \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e and N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e was systematically analyzed through density functional theory (DFT) calculations (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e ) .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter binding with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, both the HOMO and LUMO energy levels of probe \u003cb\u003e2b\u003c/b\u003e shifted towards higher energies (HOMO: -2.45→-2.27 eV; LUMO: -6.65→-6.49 eV), indicating that the introduction of N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e reconstructed the electronic structure of the probe through electron donor effects, such as lone pair coordination or charge transfer. Although the HOMO-LUMO energy gap only slightly increased from 4.20 eV to 4.21 eV (a change of 0.01 eV), the oscillator strength drastically decreased from 0.194 to 0.0797 (a reduction of 59%), a phenomenon that significantly impacts fluorescence properties. Based on the experimental data, including the red shift of the fluorescence emission wavelength of probe \u003cb\u003e2b\u003c/b\u003e from 424 nm to 442 nm, despite the minimal change in the energy gap, the upward shift of the HOMO/LUMO levels may induce the red shift phenomenon by altering the intramolecular charge transfer (ICT) characteristics. Furthermore, the significant decrease in oscillation strength may be related to the enhanced competition of non-radiative transition pathways in the excited state. This notable reduction suggests that after the probe binds with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, the probability of electronic transitions has greatly decreased, potentially leading to a weakening of fluorescence emission intensity or a shortening of the excited state lifetime. However, the experimental results show that the fluorescence intensity of probe \u003cb\u003e2b\u003c/b\u003e significantly increases after binding with hydrazine, which may be attributed to changes in solvent polarity or the effects of hydrogen bonding in the experimental system.\u003c/p\u003e \u003cp\u003eAfter the combination of probe \u003cb\u003e3c\u003c/b\u003e with N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e, its HOMO level decreased from − 1.25 eV to -1.34 eV, and the LUMO level decreased from − 5.78 eV to -5.82 eV. The HOMO-LUMO gap narrowed by 0.06 eV (from 4.54 eV to 4.48 eV), while the oscillator strength (f-value) increased from 0.0912 to 0.0949. This phenomenon contrasts sharply with the significant decrease in the f-value observed in probe \u003cb\u003e2b\u003c/b\u003e, highlighting the differences in the action mechanisms of various probes. In probe \u003cb\u003e3c\u003c/b\u003e, the simultaneous downward shift of the HOMO and LUMO energy levels enhances intramolecular charge transfer (ICT) from the coumarin donor to the ester acceptor. The contraction of the energy gap reduces the energy difference between the ground state and the excited state, leading to an increased Stokes shift and a red shift in fluorescence emission. However, no significant red shift was observed in either the UV or fluorescence spectra, which may be attributed to the subtle change in the energy gap being masked by solvent effects, instrument resolution, or compensation from other factors. Meanwhile, the increase in the f-value (by 4.1%) enhances the probability of radiative transition, thereby increasing fluorescence intensity, consistent with the experimental results.\u003c/p\u003e \u003cp\u003eThis study further validated the practical detection capability of probes \u003cb\u003e2b\u003c/b\u003eand \u003cb\u003e3c\u003c/b\u003e through simulated food sample experiments. A 10.0 mM probe solution was uniformly sprayed on pretreated rice and lettuce surfaces, followed by localized application of 80 mM hydrazine hydrate to simulate contamination. Under UV light (365 nm) imaging, hydrazine-exposed areas exhibited a distinct blue-to-cyan fluorescence transition with both probes, generating high-contrast spatially resolved signals. Notably, probe \u003cb\u003e3c\u003c/b\u003e demonstrated significantly higher fluorescence enhancement intensity than 2b, indicating superior detection sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These experiments confirmed that both probes enable visual monitoring of hydrazine contamination on food surfaces without complex sample pretreatment, with \u003cb\u003e3c\u003c/b\u003e exhibiting greater application value for rapid on-site detection due to its more pronounced optical response characteristics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the aforementioned spectral performance advantages, this study further validated the practical application potential of probes \u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e through a visual test strip experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the experiment utilized the impregnation method to prepare probe-loaded test strips, which were then exposed to hydrazine vapor at varying concentrations. The results demonstrated that under 365 nm UV light excitation, the test strip loaded with probe \u003cb\u003e2b\u003c/b\u003e exhibited purple fluorescence, while the strip loaded with probe \u003cb\u003e3c\u003c/b\u003e displayed blue-green fluorescence. As the hydrazine concentration increased, the fluorescence intensity of the test strip loaded with probe \u003cb\u003e2b\u003c/b\u003e gradually intensified, showing a distinct blue fluorescence at a concentration of 10 M. Conversely, the test strip loaded with probe \u003cb\u003e3c\u003c/b\u003e exhibited a color transition from blue-green to blue and then to green. Notably, the test strip loaded with probe \u003cb\u003e3c\u003c/b\u003e displayed a more significant response in terms of fluorescence enhancement, requiring only 250 mM of hydrazine hydrate to induce a noticeable change in the filter paper.(Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study successfully designed and synthesized two novel 3-aryl-4,5-pyranocoumarin-based fluorescent probes (\u003cb\u003e3c\u003c/b\u003e and \u003cb\u003e2b\u003c/b\u003e) with fully characterized structures by \u003csup\u003e1\u003c/sup\u003eH/\u003csup\u003e13\u003c/sup\u003eC NMR and HRMS. The structural uniqueness of the 4,5-pyranocoumarin skeleton was confirmed through X-ray crystallography, which revealed intramolecular hydrogen bonding and slipped π-π stacking interactions, both of which stabilize the crystal lattice. Both probes exhibited exceptional selectivity and sensitivity towards hydrazine (N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e), with \u003cb\u003e3c\u003c/b\u003e achieving an ultralow detection limit of 0.03 uM, surpassing most reported probes and meeting the stringent EPA standard of 10 ppb. Mechanistic studies demonstrated that N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e induces ester bond cleavage and structural rearrangement, resulting in fluorescence activation and intramolecular charge transfer (ICT) modulation, as corroborated by HRMS analysis and DFT calculations. While \u003cb\u003e2b\u003c/b\u003e displayed broader pH adaptability (pH 1–12) and a red-shifted emission (Δλ = 18 nm), \u003cb\u003e3c\u003c/b\u003e demonstrated superior performance in terms of specificity, rapid response time, and resistance to interferents, including biological thiols and metal ions. Practical validation in food matrices and test strips underscored their potential for on-site hydrazine detection, with \u003cb\u003e3c\u003c/b\u003e demonstrating superior sensitivity and visual signal contrast. This work not only propels the advancement of pyranocoumarin-based optical sensors, but also establishes a versatile platform for environmental and food safety monitoring field-deployable sensors, for accurate hydrazine quantification across environmental and food safety applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is funded by Natural Science Foundation of Guangxi Province (2023GXNSFAA026476), the Project Program of Guangxi Key Laboratory of Drug Discovery and Optimization (GKLPMDDO2022B02), Qihuang High-level Talent Team Cultivation Project of Guangxi University of Chinese Medicine (202405), Inheritance and Innovation team of Guangxi Traditional Chinese Medicine (2022B005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuafeng Zhou and Jiayong Huang: investigation, experimental operation, software, writing \u0026ndash; original draft, formal analysis; Jian Su and : investigation, experimental assistance; Xiaowei Su: investigation, data curation, formal analysis; Qiujuan Chen: investigation, data curation, formal analysis; Yuxiao Zheng: experimental assistance, data curation; Rui Chen and Lini Huo: revising the paper and supervising the research. All authors approved the final version of the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have presented all our main data in the form of tables and figures. CCDC 2449340 contains supplementary crystallographic data for compound 3a These datas can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n \u003cli\u003eIoannidou H A, Koutentis P A, The conversion of isothiazoles into pyrazoles using hydrazine. \u003cem\u003eTetrahedron\u003c/em\u003e. 2009, 65(34): 7023-7037.\u0026nbsp;https://\u0026nbsp;doi.org/\u0026nbsp;10.1016/\u0026nbsp;j.tet.2009.06.041\u003c/li\u003e\n \u003cli\u003eLambert T H. Development of a hydrazine-catalyzed carbonyl-olefin metathesis reaction. \u003cem\u003eSynlett\u003c/em\u003e. 2019, 30(17): 1954-1965.\u0026nbsp;https:// doi.org/\u0026nbsp;10.1055/\u0026nbsp;s-0039-1689924\u003c/li\u003e\n \u003cli\u003eNakui H, Okitsu K, Maeda Y, et al. 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A hemicyanine-based fluorescent probe for hydrazine detection in aqueous solution and its application in real time bioimaging of hydrazine as a metabolite in mice.\u003cem\u003e\u0026nbsp;Journal of Materials Chemistry B\u003c/em\u003e. 2019, 7(20): 3197-320.\u0026nbsp;https:// doi.org/\u0026nbsp;10.1039/\u0026nbsp;c9tb00132\u003c/li\u003e\n \u003cli\u003eXie J H, Wang L, Su X Q, et al. Coumarin-based fluorescent probes for bioimaging: recent applications and developments. \u003cem\u003eCurr. Org. Chem.\u003c/em\u003e 2021, 25(18):2142-2154. https:// doi.org/ 10.2174/ 1385272825666210728101823\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"4,5-Pyranocoumarin, Hydrazine, Turn-on fluorescence-ICT, Real samples","lastPublishedDoi":"10.21203/rs.3.rs-6756101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6756101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel structure of 4,5-pyranocoumarin-based fluorescent probes (\u003cb\u003e2b\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e) was designed and synthesized for the selective detection of hydrazine hydrate (N₂H₄). The probes feature an extended conjugated system via a 3-aryl-substituted pyranocoumarin skeleton, with ester groups serving as recognition sites for N₂H₄. Probe \u003cb\u003e3c\u003c/b\u003e demonstrated superior performance, exhibiting a 494% fluorescence enhancement at 460 nm with a detection limit of 0.03 \u0026micro;M, surpassing most reported hydrazine probes and the EPA safety threshold. Both probes exhibited high selectivity against 17 interferents (ions/biomolecules) and functioned effectively in complex matrices, including food samples (lettuce, rice) and vapor-phase detection via test strips. Probe \u003cb\u003e3c\u003c/b\u003e achieved rapid (\u0026lt;\u0026thinsp;5 min), pH-stable (pH 6\u0026ndash;10) responses with visible color transitions (blue-green to green), while \u003cb\u003e2b\u003c/b\u003e operated across a broader pH range (1\u0026ndash;12). This work highlights pyranocoumarin scaffolds as promising platforms for designing intensity-responsive probes, combining synthetic novelty, sensitivity, and practicality for environmental and food safety.\u003c/p\u003e","manuscriptTitle":"Design and Synthesis of Novel Angular 4,5-Pyranocoumarin Fluorescent Probe for detecting hydrazine and and its applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 12:40:39","doi":"10.21203/rs.3.rs-6756101/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4dbd7d1b-1d46-4f35-af13-27c3a6699b1b","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T09:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 12:40:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6756101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6756101","identity":"rs-6756101","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}