Synthesis method of organic light response probe hbt-cn based on esipt effect, obtained probe and application thereof

Abstract

The application provides a synthesis method of an organic light response probe HBT-CN based on an ESIPT effect, the obtained probe and application thereof, and belongs to the technical field of biological probes.The 1R, 2R-cyclohexanediamine is used as a chiral recognition module, is connected to a 2-(2'-hydroxyphenyl)benzoxazole derivative fluorescent group through a simple amide reaction, and a kind of organic light response probe HBT-CN based on ESIPT effect is successfully designed and synthesized.The probe HBT-CN can realize enantiomer selective fluorescence recognition detection of amino acid ester hydrochloride by direct titration method.In addition, the probe can also be used for enantiomer selective detection through sensitive ratio type ultraviolet absorption change.The fluorescence and ultraviolet response difference of two enantiomers is shown by the research results, and the probe has high sensitivity and enantiomer distinguishing degree.

Description

Technical Field

[0001] This invention belongs to the field of biological probe technology, and particularly relates to a method for synthesizing an organic photoresponsive probe HBT-CN based on the ESIPT effect, the obtained probe and its application. Background Technology

[0002] Modern research on optically active drugs, as well as pharmacodynamic and pharmacokinetic studies, has shown that the two enantiomers of chiral drugs often exhibit different pharmacodynamic and pharmacokinetic behaviors. For example, thalidomide, the sedative used in the infamous thalidomide tragedy, has its active ingredient, the R-form, which has excellent sedative effects, while its enantiomer, the S-form, has embryotoxic and teratogenic effects. Therefore, research on chiral enantiomer detection and separation technologies has attracted considerable attention in recent years, leading to the development of a series of chiral enantiomer recognition systems, mainly including chiral functionalized materials and photoresponsive probes based on small organic molecules. Compared to chiral functionalized materials, photoresponsive probes based on small organic molecules offer better reproducibility and facilitate large-scale applications. However, currently reported chiral recognition probes based on small organic molecules are mostly axially chiral binaphthalene derivatives, with fewer reports on chiral photoresponsive probes based on organic dyes. Summary of the Invention

[0003] This invention provides a method for synthesizing an organic photoresponsive probe HBT-CN based on the ESIPT effect, the obtained probe, and its application. The probe HBT-CN can achieve enantioselective fluorescence recognition and detection of amino acid ester hydrochloride by direct titration, or it can perform enantioselective detection by sensitive ratiometric ultraviolet absorption change, exhibiting high sensitivity and enantioselectivity.

[0004] To achieve the above objectives, this invention provides a method for synthesizing an organic photoresponsive probe HBT-CN based on the ESIPT effect. The probe is synthesized by using 1R,2R-cyclohexanediamine as a chiral recognition module and linking it to a 2-(2'-hydroxyphenyl)benzoazole derivative fluorophore via an amide reaction.

[0005] As a preferred option, the following steps are included:

[0006] 5-Fluorosalicylic acid and hexamethylenetetramine in a molar ratio of 1:2 were added to a reaction flask, followed by the addition of TFA to dissolve them. The reaction mixture was heated to reflux and allowed to react fully before being cooled to room temperature. The reaction solution was then extracted, washed, dried, concentrated, and recrystallized to obtain intermediate 1.

[0007] Intermediate 1 and 2-aminobenzylthiol in a molar ratio of 1:1 were added to a quartz beaker, followed by the addition of 3,6-bis-2-pyridyl-1,2,4,5-tetraazine. The mixture was stirred at a constant temperature under an oxygen atmosphere. After the reaction was completed, the product was dissolved, washed, dried, concentrated, and recrystallized to obtain intermediate 2.

[0008] Intermediate 2 and (1R,2R)-trans-N-Boc-1,2-cyclohexanediamine in a molar ratio of 1:1 were dissolved in DCM solution, followed by the addition of EDC HCl and DMAP. The reaction was carried out at room temperature under argon protection. After the reaction was completed, the reaction solution was extracted, washed, dried, concentrated, and recrystallized to obtain intermediate 3.

[0009] Intermediate 3 was placed in a round-bottom flask, and a CH2Cl2 / TFA solution with a volume ratio of 1:1 was slowly added dropwise to the round-bottom flask. The mixture was stirred at room temperature until the reaction was complete. The reaction was then neutralized, and the reaction solution was extracted, dried, purified, and eluted to obtain HBT-CN.

[0010] The present invention also provides an organic photoresponsive probe HBT-CN prepared by the synthesis method described in any of the above technical solutions.

[0011] The present invention also provides an application of the organic photoresponsive probe HBT-CN according to the above technical solution in achieving enantioselective recognition and detection of amino acid ester hydrochloride.

[0012] Preferably, in application, the HBT-CN probe can be used to determine the concentration of enantiomers by direct titration for identification and detection.

[0013] Preferably, in application, the probe HBT-CN can selectively detect enantiomers through ratiometric ultraviolet absorption changes.

[0014] Preferably, the amino acid ester hydrochloride is selected from at least one of D / L-glutamic acid methyl ester hydrochloride, D / L-tyrosine methyl ester hydrochloride, D / L-phenylalanine methyl ester hydrochloride, D / L-methionine methyl ester hydrochloride, D / L-tryptophan methyl ester hydrochloride, D / L-serine methyl ester hydrochloride, D / L-histidine methyl ester hydrochloride, R / S-phenylglycine methyl ester hydrochloride, and R / S-p-hydroxyphenylglycine methyl ester hydrochloride.

[0015] Preferably, the amino acid ester hydrochloride is R / S-phenylglycine methyl ester hydrochloride.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0017] Organic fluorescent dyes and probes have been widely used in in vivo and in vitro detection of physiologically active small molecules and in cell imaging studies due to their excellent fluorescence properties and sensitive fluorescence responses. Among them, benzoazole fluorescent dyes based on the ESIPT principle are widely used as fluorescent probes, photostabilizers, and organic light-emitting devices because of their advantages such as readily available raw materials, simple synthesis methods, high fluorescence quantum yield, large Stokes shift, and stable fluorescence performance. Based on the different heteroatoms in the five-membered ring of thiazole, benzoazole compounds can be divided into benzothiazoles, benzoxazoles, and benzimidazoles. 2-(2'-hydroxyphenyl)benzoazole compounds are a classic example of this molecular structure. Studies have shown that intramolecular hydrogen bonding gives the molecule a rigid planar structure, which is conducive to electron delocalization, resulting in a stable enol structure. When irradiated with excitation light, the ground-state molecule absorbs photons and is excited to an excited state. The excited-state molecule is unstable and emits enol light in the form of photoradiation. Simultaneously, due to electron energy absorption and electron rearrangement, a proton transfer occurs between the phenolic hydroxyl group and the thiazole N group, forming a transition state keto structure. This structure then returns to the enol ground state via photoradiation, emitting keto fluorescence. This luminescence process involves fluorescence response behavior induced by hydrogen bonding interactions, which can be applied to the study of enantiomeric recognition based on chiral selective hydrogen bonding interactions.

[0018] Following this line of thought, this invention uses a single-configuration 1R,2R-cyclohexanediamine as a chiral recognition module, connecting it to a 2-(2'-hydroxyphenyl)benzoazole derivative fluorophore via a simple amide reaction to design and synthesize an organic small molecule photoresponsive probe, HBT-CN. Probe HBT-CN can achieve enantioselective fluorescence recognition and detection of amino acid ester hydrochloride salts via direct titration. Furthermore, this probe can also achieve enantioselective detection through sensitive ratiometric UV absorption changes. Analysis using fluorescence spectroscopy, UV-vis absorption spectroscopy, time-resolved fluorescence spectroscopy, and DFT theoretical calculations shows that due to differences in enantiomeric spatial structure, different hydrogen bonding interactions exist between the enantiomeric species and HBT-CN, inducing a differential photoresponse in HBT-CN, thereby achieving enantioselective recognition and detection. In addition, the results of the fluorescence and UV response difference ratio studies between the two enantiomeric species indicate that this probe has high sensitivity and enantioselectivity. Attached Figure Description

[0019] Figure 1 The synthesis route of the probe HBT-CN provided in the embodiments of the present invention;

[0020] Figure 2 The design of the HBT-CN probe and the enantioselective recognition principle provided in the embodiments of the present invention;

[0021] Figure 3(a) UV-vis absorption spectrum and fluorescence spectrum of the HBT-CN probe provided for embodiments of the present invention; (b) CD spectrum;

[0022] Figure 4 The amino acid enantiomers (0.1 mmol L) provided in the embodiments of the present invention -1 Effect of aqueous solution on the fluorescence properties of HBT-CN (5.0 × 10⁻⁶) -5 mol L -1 ,λ ex =400nm,λ em,max =472nm);

[0023] Figure 5 For the embodiments of the present invention, under different concentrations of S-PGME (a) or R-PGME (b), HBT-CN (5.0 × 10⁻⁶) -5 (c) The relative fluorescence intensity ((I-I0) / I0) of HBT-CN with S-PGME and R-PGME concentrations in the range of 0.005 to 2.25 mmol / L. -1 The relationship between (d) the relative fluorescence intensity of HBT-CN ((I-I0) / I0) and the concentrations of S-PGME and R-PGME in the range of 0.005 to 2.25 mmol / L. -1 The linear relationship between them;

[0024] Figure 6 For the embodiments of the present invention, under different concentrations of S-PGME (a) or R-PGME (b), HBT-CN (5.0 × 10⁻⁶) -5 (c) The relative absorption intensity A365 / A402 of HBT-CN with S-PGME and R-PGME concentrations in the range of 0.005 to 1.0 mmol / L. -1 The relationship between (d) the relative absorbance of HBT-CN, A365 / A402, and Log[PGME] (0.005 to 2.25 mmol L). -1 A linear relationship between them;

[0025] Figure 7 The linear relationship between the relative fluorescence intensity (I / I0) of HBT-CN and different ee% values ​​of R / S-enantiomers from 0 to 100% provided in the embodiments of the present invention is shown in (a) RH-HPA-OMEHCl, (b) SH-Phe-OMEHCl, (c) S-PGME, with a total concentration of 1.0 mmol / L. -1 ;

[0026] Figure 8 The HBT-CN (5.0×10) provided for embodiments of the present invention-5 mol L -1 ) and fluorescence decay curves under different concentrations of R-PGME(a) or S-PGME(b);

[0027] Figure 9 The HBT-CN (5.0×10) provided for embodiments of the present invention -5 mol L -1 R / S-PGME (1.0 mmol L) -1 A mixture of HBT-CN and S-PGME, and HBT-CN and R-PGME (1.0 mmol L). -1 The UV-vis absorption spectrum of )

[0028] Figure 10 Spatial models of HBT-CN(M) enol, keto, and PGME enantiomers provided in embodiments of the present invention;

[0029] Figure 11 This is a stable conformation of the M-Ketone*+PGME enantiomer provided in the embodiments of the present invention;

[0030] Figure 12 The distribution of the molecular frontier orbital electron cloud of the chiral molecule M in the ground state is provided in an embodiment of the present invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The experimental reagents used in the following examples are shown in Table 1.

[0033] Table 1

[0034]

[0035] The experimental instruments used in the following examples are shown in Table 2.

[0036] Table 2

[0037]

[0038] Example 1: Synthesis of probe HBT-CN

[0039] Figure 1 The synthesis route of the probe HBT-CN provided in this embodiment of the invention is as follows:

[0040] Synthesis of Compound 6-1

[0041] Compound 5-fluorosalicylic acid (1.56 g, 10 mmol) and hexamethylenetetramine (HMT, 2.8 g, 20 mmol) were placed in a 100 mL round-bottom flask, and 20 mL of TFA was added to dissolve them. The reaction mixture was refluxed at 100 °C for 10 h, and the reaction progress was monitored by TLC. After the reaction was completed, it was cooled to room temperature. Then, 10 mL of 1 mol / L hydrochloric acid was carefully added, and stirring was continued for 30 min. The reaction solution was extracted three times with ethyl acetate. The organic layers were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. The crude product was purified by recrystallization from MeOH / CH2Cl2 (1:1) to give 1.12 g of white solid 6-1, with a yield of 61%.

[0042] Characterization data: 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),10.33(s,1H),7.878(d,J=3.2Hz,1H),7.857(d,J=3.2Hz,1H),7.678(d,J=3.2Hz,1H),7.657(d,J=3.2Hz,1H); 13 CNMR(100MHz, CDCl3)δ188.2,170.9,160.4,155.7,153.3,125.1,123.0,119.6; HRMS(TOF-ES+)m / z:[MH]-calcd for C8H4FO4 - 183.0010, found 183.0009.

[0043] Synthesis of compound 6-2

[0044] Compound 2-aminobenzenethiol (0.625 g, 5 mmol) and compound 6-1 (0.92 g, 5 mmol) were placed in a 250 mL double-walled quartz beaker, and 50 mL of ethanol was added. Then, 3,6-bis-2-pyridyl-1,2,4,5-tetraazine (10 mg, 0.0423 mmol) was added, and the mixture was stirred for 8 h in a constant-temperature photoreactor under an oxygen atmosphere. The reaction progress was monitored by TLC. After the reaction was completed, the ethanol was removed by vacuum distillation, and the product was dissolved in ethyl acetate and washed several times with 1 mol / L hydrochloric acid solution. The organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and the crude product was purified by recrystallization from EtOH / CH2Cl2 (1:1) to give 1.16 g of product 6-2 as a yellow solid, with a yield of 80.0%.

[0045] Characterization data: 1H NMR(400MHz, DMSO-d6)δ8.34(dd,J=3.6Hz,J=9.6Hz,1H),8.16(d,J=7.6Hz,1H),8.07(d ,J=4.0Hz,1H),7.75(dd,J=3.2Hz,J=8.0Hz,1H),7.58-7.54(m,1H),7.49-7.45(m,1H); 13 C NMR(100MHz,DMSO-d6)δ171.4,161.0,156.7,155.5,153.1,151.7,136.1,127 .0,125.7,123.0,122.5,120.3,120.0,119.2,118.9; HRMS(TOF-ES+)m / z:[MH] - calcd for C 14 H7FNO3S - 288.0136, found 288.0117.

[0046] Synthesis of compound 6-3

[0047] Compound 6-2 (0.578 g, 2 mmol) and (1R,2R)-trans-N-Boc-1,2-cyclohexanediamine (2.58 g, 2 mmol) were dissolved in 5 mL of DCM solution, followed by the addition of EDC HCl (0.458 g, 2.4 mmol) and DMAP (0.073 g, 0.6 mmol). The reaction was carried out at room temperature under argon protection for 5 h, and the reaction was stopped after TLC monitoring showed complete reaction. The reaction solution was extracted with ethyl acetate (200 mL) and washed with 1N HCl (4 × 60 mL) and saturated brine (60 mL). The organic phase was dried over anhydrous sodium sulfate to remove the solvent and obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 4:1) to give compound 6-3 as a yellowish-white solid, 0.824 g, in 85% yield.

[0048] Characterization data: 1 H NMR (400MHz, CDCl3) δ14.06 (s, 1H), 8.40 (d, J = 3.6Hz, 1H), 8.03 (d, J = 4.0Hz, 1H), 7.94-7.88 (m, 2H), 7.75 (d, J = 3.0Hz, 1H), 7 .55(t,J=7.2Hz,1H),7.45(t,J=7.2Hz,1H),4.89(d,J=4.4Hz,1H),3.88-3,85(m,1H),3.60-3.53(m,1H),2.26-1.29(m,17H); 13C NMR(100MHz,DMSO-d6)δ156.6,156.2,153.9,153.7,151.2,127.0,126.0,122.5,121.6,118.2,117.9,79.6,7 7.3,77.2,55.0,54.3,32.9,32.4,31.4,30.2,29.7,28.2,25.0,24.7,22.7,14.1; HRMS(TOF-ES+)m / z:[M+Na] + calcd for C 25 H 28 FN3NaO4S + 508.1676, found 508.1676.

[0049] Synthesis of probe HBT-CN

[0050] Compound 6-3 (0.485 g, 1.0 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of CH2Cl2 / TFA solution (V / V = 1:1) was slowly added dropwise. The mixture was stirred at room temperature for 2 hours, and the reaction was stopped after TLC monitoring showed complete reaction. The solution was then carefully neutralized to pH 8 using saturated NaHCO3 solution. The reaction mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography, eluting with DCM:MeOH (V / V = 1:1), to give 0.346 g of the final compound, a pale yellow powder, HBT-CN, in 90% yield.

[0051] Characterization data: 1 H NMR (400MHz, DMSO-d6) δ8.15-7.80(m,6H),7.48(t,J=7.2Hz 1H),7.36(t,J=7.6Hz,1H),4.00-3.92(m,1H),3.11-3.05(m,1H),2.05-1.23(m,1H); 13 C NMR (100MHz, DMSO-d6) δ168.6,151.8,136.1,126.2,124.2,122.5,122.4,122. 1,118.3,118.1,54.6,50.8,31.9,30.1,24.7,23.8; HRMS(TOF-ES+)m / z:[M+Na] + calcd for C 20 H 20 FN3NaO2S + 408.1152, found 408.1153.

[0052] Example 2: Design of a chiral HBT-CN probe

[0053] like Figure 2 As shown, under specific wavelength light excitation, intramolecular hydrogen bond interactions in 2-(2'-hydroxyphenyl)benzoazole can induce changes in molecular structure, leading to alterations in UV absorption and fluorescence response. The design is based on the 2-(2'-hydroxyphenyl)benzoazole structure, introducing a sterically less hindered electron-withdrawing fluorine atom at the para-position of the phenolic hydroxyl group to increase its acidity, facilitating proton transfer and enhancing its fluorescence quantum yield. A carboxyl group is introduced at the ortho-position as a linking group, facilitating the introduction of the chiral recognition module cyclohexanediamine via a simple amide reaction. It also provides a hydrogen bond interaction site as close as possible to the phenolic hydroxyl group. With this design, when a chiral enantiomer approaches, the target enantiomer and probe can influence the ESIPT luminescence process between the thiazole imine group and the phenolic hydroxyl group through hydrogen bond interactions, thereby affecting its fluorescence response behavior. Simultaneously, the chiral enantiomer-selective recognition is achieved by relying on the stereoselective hydrogen bond interactions of the chiral recognition module.

[0054] Example 3: Properties of the HBT-CN probe and its enantioselective recognition performance

[0055] Relevant experimental procedures and methods for enantiomeric determination

[0056] Preparation of HBT-CN probe stock solution: Dissolve HBT-CN probe in methanol (MeOH) to prepare a solution of 1.0 × 10⁻⁶. -4 A mol / L solution was prepared and refrigerated at 4°C for later use.

[0057] Preparation of the analyte solution: Weigh appropriate amounts of D / LH-Glu-OMEHCl, D / LH-Phe-OMEHCl, D / LH-Met-OMEHCl, D / LH-Tyr-OMEHCl, D / LH-Trp-OMEHCl, D / LH-Ser-OMEHCl, D / LH-His-OMEHCl, D / L-Met, D / L-Phe, D / L-Trp, and D / L-Tyr enantiomers sequentially into deionized water to prepare a 0.1 mol / L solution for later use.

[0058] Detection Method: Unless otherwise specified, the fluorescence spectroscopy test conditions during the detection process are as follows: excitation wavelength 400 nm, excitation band gap set to 10 nm, emission band gap set to 2.5 nm, and scan range 405-700 nm. The fluorescence peak intensity at 472 nm was selected for fluorescence analysis. All spectral tests were performed in an aqueous system, and the entire experiment was repeated three times to obtain the average value.

[0059] The detection method for amino acid methyl ester hydrochloride and amino acids is as follows: Take a certain amount of HBT-CN probe solution (1.0×10⁻⁶). -4 Add mol / L (5 μL) to 1 mL of aqueous solution, then add S- or R-enantiomers of different concentration gradients, and shake well for subsequent detection.

[0060] Optical properties of HBT-CN probe

[0061] First, the optical properties of the HBT-CN probe were investigated using UV-vis absorption spectroscopy, fluorescence spectroscopy, and CD spectroscopy. For example... Figure 3 As shown in Figure a, the characteristic absorption peaks of the HBT-CN probe in aqueous solution are between 250-450 nm. Significant UV absorption peaks are observed at 294 nm, 365 nm, and 402 nm. The 294 nm absorption peak originates from the UV absorption of benzothiazole in HBT-CN, while the characteristic absorptions at 365 nm and 400 nm are attributed to the coupling between the benzothiazole moiety and the substituted phenyl group. Furthermore, the 365 nm and 400 nm peaks are shoulder peaks, indicating the presence of isomers; therefore, the shoulder peaks originate from the interconversion between the enol and keto forms of HBT-CN in the ground state. Fluorescence spectroscopy shows that its optimal excitation wavelength is 400 nm, and its emission wavelength is 472 nm, primarily exhibiting keto fluorescence. It emits bright blue fluorescence under a 365 nm UV lamp. Figure 3 As shown in b, the circular dichroism spectrum of the HBT-CN probe in the range of 200–600 nm was further recorded using circular dichroism spectroscopy. HBT-CN exhibits a negative absorption band in the range of 200–450 nm. The characteristic peaks of its CD signal mainly include a strong absorption peak near 400 nm and a weak absorption peak near 241 nm. The single spatial conformation of HBT-CN provides a prerequisite for chiral recognition.

[0062] HBT-CN probe's enantioselective recognition performance

[0063] Next, the fluorescence-selective recognition characteristics of the HBT-CN probe for amino acid enantiomeric compounds were preliminarily investigated. For example... Figure 4 As shown, the effect of adding a series of chiral enantiomers of the same concentration to HBT-CN (0.1 mmol L) in aqueous solution was investigated. -1The influence of HBT-CN on the fluorescence properties of 1 mL of HBT-CN was investigated, and the obtained data were quantified by the fluorescence intensity at 472 nm (I-I0) / I0. It can be seen that HBT-CN exhibits different fluorescence quenching responses to some amino acid methyl ester hydrochloride chiral enantiomers. Furthermore, for the same enantiomer, the HBT-CN probe shows significant differences in response to chiral enantiomers with different configurations, especially exhibiting good chiral selectivity and signal differences for R / S-PGME, H-Glu-OMEHCl, and H-Phe-OMEHCl. This demonstrates that the fluorescent probe possesses good chiral selectivity and can be used for enantiomer recognition and detection.

[0064] Fluorescence response analysis

[0065] Furthermore, taking R / S-PGME as an example, the fluorescence response recognition performance of this probe for R / S-PGME was comprehensively studied using titration. Figure 5 As shown in (a, b), the fluorescence signal of HBT-CN decreases with increasing enantiomer concentration when different concentrations of PGME enantiomers are added. The fluorescence signal tends to stabilize at a certain concentration. The quenching ability of S-PGME is much greater than that of R-PGME. According to calculations, in the presence of S-PGME, the fluorescence intensity quenching ratio of HBT-CN ((I-I0) / I0) is approximately -0.9, while the decrease rate of R-PGME is approximately -0.2, and the difference ratio of fluorescence quenching is approximately 4.5. Figure 5 c). Figure 5 Illustration c shows digital electron images of the R- and S-enantiomers under a 365nm UV lamp. It is clearly visible that the color of the S-PGME solution changes from blue to blue-green, while the R-PGME effect is not obvious. The significant color and fluorescence signal differences can be visually distinguished, demonstrating the advantage of this chiral fluorescent probe in chiral recognition. Furthermore, as... Figure 5 As shown in Figure d, within a certain concentration range, linear fitting was performed on the data obtained through the Boltzmann equation, revealing a good linear relationship between fluorescence intensity and enantiomeric concentration (Rd). 2 (All values ​​are greater than 0.99). This indicates that the concentration of PGME enantiomers can be determined by a simple fluorescence titration method using the novel fluorescent probe.

[0066] Ultraviolet absorption response analysis

[0067] In addition, the changes in ultraviolet absorption spectra within the concentration range of 0.005 to 2.25 μM were recorded. For example... Figure 6As shown in Figure a, with increasing S-PGME enantiomer concentration, its absorbance at 402 nm gradually decreases until it disappears completely. Simultaneously, the absorption intensity of the shoulder peak at 365 nm gradually increases, gradually reaching equilibrium at a certain concentration, and a blue shift of the absorption peak occurs to around 348 nm. However, as... Figure 6 As shown in b, R-PGME has a relatively small impact on the UV absorption of the HBT-CN probe at 365 nm and 402 nm. Figure 6 As shown in Figure c, in the presence of S-PGME, the UV absorption intensity ratio (A365 / A402) of HBT-CN at 365 nm and 402 nm is approximately 10.5, while that of R-PGME is approximately 1.0. The difference in their UV absorption signals is approximately 10.5. Furthermore, their UV absorption signals were fitted using the Boltzmann equation in the range of 0.005 to 2.25 mmol / L. -1 The linear correlation between absorbance A365 / A402 and Log[PGME] within the concentration range, as shown in the figure. Figure 6 As shown in d, S-PGME exhibits good linear correlation R. 2 =0.992, while for R-PGME, due to chiral selectivity, R-PGME has a small impact on the HBT-CN probe, therefore its linear correlation is very poor. 2 =0.974. These results indicate that the HBT-CN probe exhibits a good proportional-weighted ultraviolet absorption response to PCME, with a signal-to-difference ratio much greater than that of fluorescence. The self-calibration of the probe molecule itself can overcome the influence of factors such as probe concentration, instrument sensitivity, and environmental factors, improving detection accuracy and offering greater advantages in enantiomeric detection.

[0068] Enantiomer ee% value detection

[0069] Fluorescence and UV-vis absorption spectroscopy indicate that the HBT-CN probe exhibits good enantioselective photoresponse behavior, making it suitable for enantioselective excess detection. Therefore, using RH-HPA-OMEHCl, SH-Phe-OME-HCl, and S-PGME as examples, the relationship between the enantioselective excess value (ee%) and the fluorescence intensity ratio (I / I0) of amino acid methyl ester hydrochloride enantiomeric amino acids was further investigated. Figure 7 As shown, the fluorescence intensity ratio changes systematically with the percentage content of a certain configuration enantiomer, and this correspondence can be used for the determination of the enantiomer composition of amino acid esters. The above results indicate that the excellent chiral selectivity of HBT-CN fluorescence signals can be used as an analytical tool for chiral identification of enantiomers. This is particularly significant for the detection of important chiral precursors for the synthesis of clinical drugs.

[0070] Example 4: Study on the enantioselective fluorescence quenching mechanism of HBT-CN probe

[0071] DFT calculation method

[0072] The calculations were performed using the Gaussian 16 software package. Density functional optimization was performed on all stagnation points in water using a hybrid M062X functional conjugated to the 6-31+G(d) basis set. The excited states were optimized using the TD-DFT method, and the fluorescence properties of the lowest five excited states were calculated. Frequency calculations were performed at the same level to verify whether the stagnation points were minima (0 imaginary frequencies) or saddle points (1 imaginary frequency only). Truhlar's SMD solvent model was used to account for solvation effects in the DFT and TD-DFT optimization calculations. For each stationary point, conformational sampling was performed to locate the global minimum. The calculated structures are illustrated using Gaussian View6.

[0073] Fluorescence lifetime analysis

[0074] Table 3. Average fluorescence lifetime of HBT-CN under different concentrations of PGME enantiomeric enzymes.

[0075]

[0076] Changes in fluorescence lifetime are an important analytical method for exploring fluorescence quenching mechanisms. Firstly, such as... Figure 8 As shown, measurements were recorded and taken at R-PGME ( Figure 3 a) and S-PGME ( Figure 3 b) Time-resolved fluorescence spectroscopy of HBT-CN in the presence of enantiomers to reveal the fluorescence response mechanism of HBT-CN to different PGME enantiomers. The fluorescence decay curves in these cases all conform to a bi-exponential equation, and the intensity decay follows a multi-exponential law. It should be noted that the average decay time, proportional to the steady-state intensity, was used as the evaluation index in the lifetime study; the average value is calculated from ∑b i τ i The sum of the products is given. The average decay time (τ) of HBT-CN. AVThe fluorescence lifetimes of HBT-CN were 3.78 ns (τ1(b1), 0.39±0.063 (5.83%); τ1(b1, 3.99±0.023 (94.17%)), as shown in Table 3. The addition of enantiomers of R-PGME at different concentrations did not affect the fluorescence lifetime of HBT-CN. However, for S-PGME, the average fluorescence lifetime of HBT-CN remained within the acceptable error range, decreasing with increasing concentration. This indicates that the enantiomers interact with HBT-CN through different mechanisms and binding modes. Therefore, it can be inferred that the fluorescence quenching mechanism is due to the hydrogen bonding interaction between the S-PGME enantiomers and HBT-CN affecting the ground and excited states of the HBT-CN probe molecules, resulting in an increase in non-radiative energy loss, thus leading to fluorescence quenching.

[0077] Ultraviolet absorption spectroscopy analysis

[0078] Furthermore, the selective fluorescence quenching mechanism of the HBT-CN probe on the PGME enantiomer was further investigated using UV-vis absorption spectroscopy experiments. Figure 9 As shown, R / S-PGME exhibits no characteristic UV absorption beyond 274 nm, thus ruling out interference with the UV absorption of HBT-CN. The UV absorption peaks at 365 nm and 402 nm originate from the characteristic absorption peaks of the enol and keto isomers of HBT-CN. For S-PGME, compared to the original HBT-CN, it is evident that the UV absorption at 402 nm decreases sharply in aqueous solution, accompanied by a hypochromic effect. This indicates that the interaction between S-PGME and HBT-CN can affect the keto structure of the probe. Simultaneously, the presence of S-PGME increases the UV absorption at 365 nm with a hyperchromic effect, indicating increased absorption of the enol structure in solution. Furthermore, the blue shift of the absorption peak to 348 nm indicates an increase in the electronic transition energy of the HBT-CN molecule, suggesting that the interaction between the enantiomer and the HBT-CN probe can passivate the structure of HBT-CN, making it more stable. Furthermore, the UV absorption response induced by S-PGME is much greater than that of R-PGME, indicating that the interaction mechanisms between the two enantiomers of PGME and HBT-CN are significantly different. Therefore, it can be inferred that the enantiomers can influence the ESIPT process of the HBT-CN probe through chiral selective interactions, thereby affecting its fluorescence response and achieving enantiomer-selective recognition.

[0079] Study on the enantioselective recognition mechanism of HBT-CN probe

[0080] To better understand the enantiomer-selective recognition mechanism of HBT-CN, using PGME enantiomers as a typical case, we optimized the spatial configuration of the enantiomers and HBT-CN probes, as well as the theoretical model of stereoselective hydrogen bonding between them, using DFT theoretical calculations.

[0081] First, such as Figure 10 The spatial models of the enol and keto forms of HBT-CN(M), as well as the S-PGME and R-PGME enantiomers, were optimized, as shown. Next, considering the interaction between the enantiomer and HBT-CN in the form of hydrochloride salts, the protonated amine group is prone to deprotonation. Therefore, two possible phase interaction models, conf1 and conf2, were proposed. As shown in Table 4, the calculation results show that, regardless of the configuration, the conf1 complex has lower energy and a more stable structure than the conf2 complex. Therefore, it can be inferred that during the hydrogen bonding process between the enantiomer and the chiral molecule, protons are transferred to the chiral molecule, forming a more stable complex. Combined with experimental observations in fluorescence spectroscopy, it can be inferred that the protonated amine group plays an important role in the enantiomer recognition process. This may be because ionized H atoms are more easily bound to the enantiomer during intermolecular collisions, and the additional hydrogen bonding interaction makes the binding more stable.

[0082] Table 4. Stability of chiral molecule M and amino ester ion compounds in the ground state (ΔG kcal / mol)

[0083]

[0084] To further understand the chiral selective photoresponse mechanism of the enantiomer and HBT-CN, the dominant spatial conformation in its excited state was calculated, since fluorescence emission is mainly ketone emission. Figure 11 As shown, many weak interactions exist between the R and S enantiomers and the chiral molecule M. Due to differences in spatial configuration, the enantiomers exhibit different spatial hydrogen bonding modes with the M molecule. For the R-type enantiomer, the phenyl group is located directly above the central benzene ring of the chiral molecule. For the S-type enantiomer, the phenyl group is located directly above the nitrogen atom of the thiophene ring. Since fluorescence emission mainly involves the HOMO and LUMO orbitals of the chiral molecule, such as... Figure 12As shown, M-Enol is unrelated to fluorescence emission, while fluorescence emission is mainly related to M-Ketone. From the orbital distribution diagram, the HOMO of M-Ketone is mainly distributed on the intermediate benzene ring, while the LUMO is distributed on both the intermediate benzene ring and the thiazole ring. Therefore, it can be inferred that the benzene ring of the S-enantiomer is located at the top of the intermediate benzene ring of the chiral molecule, serving as the electronic excitation center. The benzene ring of the S-enantiomer significantly influences the HOMO orbitals of the chiral molecule through π-π interactions, thus affecting the electronic transitions during fluorescence excitation and emission. Conversely, the benzene ring of the R-enantiomer is located at the top of the N atom of the thiazole ring, having a smaller impact on its fluorescence excitation and emission. In summary, it can be further inferred that due to differences in spatial bonding structure, the enantiomers have different hydrogen bond interactions with HBT-CN, resulting in different stereochemical bonding modes. Enantiomer side chain groups can influence the ground state electronic transitions of HBT-CN probes and thus affect their excited states through various weak interactions (hydrogen bonds, π-π) with HBT-CN probe molecules, thereby causing changes in the fluorescence properties of HBT-CN probes and achieving selective photoresponse recognition of enantiomers.

Claims

1. A method for synthesizing an organic photoresponsive probe HBT-CN based on the ESIPT effect, characterized in that, Includes the following steps: 5-Fluorosalicylic acid and hexamethylenetetramine in a molar ratio of 1:2 were added to a reaction flask, followed by the addition of TFA to dissolve them. The reaction mixture was heated to reflux and allowed to react fully before being cooled to room temperature. The reaction solution was then extracted, washed, dried, concentrated, and recrystallized to obtain intermediate 1. Intermediate 1 and 2-aminobenzylthiol in a molar ratio of 1:1 were added to a quartz beaker, followed by the addition of 3,6-bis-2-pyridyl-1,2,4,5-tetraazine. The mixture was stirred at a constant temperature under an oxygen atmosphere. After the reaction was completed, the product was dissolved, washed, dried, concentrated, and recrystallized to obtain intermediate 2. Intermediate 2 and (1R,2R)-trans-N-Boc-1,2-cyclohexanediamine in a molar ratio of 1:1 were dissolved in DCM solution, followed by the addition of EDC HCl and DMAP. The reaction was carried out at room temperature under argon protection. After the reaction was completed, the reaction solution was extracted, washed, dried, concentrated, and recrystallized to obtain intermediate 3. Intermediate 3 was placed in a round-bottom flask, and a CH2Cl2 / TFA solution with a volume ratio of 1:1 was slowly added dropwise to the round-bottom flask. The mixture was stirred at room temperature. After the reaction was complete, the reaction was neutralized. The reaction solution was then extracted, dried, purified, and eluted to obtain HBT-CN. The structural formula of intermediate 1 is as follows: The structural formula of intermediate 2 is as follows: The structural formula of intermediate 3 is as follows: The structural formula of the HBT-CN is as follows: 。 2. The organic photoresponsive probe HBT-CN prepared by the synthesis method according to claim 1.

3. The application of the organic photoresponsive probe HBT-CN according to claim 2 in the enantioselective recognition and detection of amino acid ester hydrochloride salts, characterized in that, The amino acid ester hydrochloride is selected from at least one of D / L-glutamic acid methyl ester hydrochloride, D / L-tyrosine methyl ester hydrochloride, D / L-phenylalanine methyl ester hydrochloride, D / L-methionine methyl ester hydrochloride, and R / S-phenylglycine methyl ester hydrochloride.

4. The application according to claim 3, characterized in that, In application, the HBT-CN probe can be used to determine the concentration of enantiomers by direct titration for identification and detection.

5. The application according to claim 3, characterized in that, In application, the probe HBT-CN can selectively detect enantiomers through ratiometric ultraviolet absorption changes.

6. The application according to claim 3, characterized in that, The amino acid ester hydrochloride is R / S-phenylglycine methyl ester hydrochloride.

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