A fluorescent probe specifically recognizing chiral threoninol, preparation and application thereof

Abstract

The application discloses a fluorescent probe for specifically recognizing chiral threoninol, preparation and application of the fluorescent probe, and a chemical structural formula of the probe is as follows: the probe realizes specific selective fluorescence recognition of L-threoninol in DMSO, and shows high fluorescence selectivity for L-threoninol in the presence of 30 equivalent amino alcohols.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis and detection, in particular to a fluorescent probe for specifically recognizing chiral threoninol, preparation and application thereof. BACKGROUND

[0002] With the rapid development of polypeptide drugs, L-threoninol is widely used in the solid-phase synthesis of various alcohol peptides, such as somatostatin and octreotide. It can also be used as a chiral ligand for asymmetric metal catalysis, and is a raw material for nucleotide drugs, used for synthesizing modified oligonucleotides, aliphatic nucleosides or nucleoside analogs, and aminophosphonate containing methyl red fragments. In addition, D-threoninol scaffolds can be used to prepare various pseudo-base pairs, which can simulate the supramolecular properties of natural base pairs. In addition, the modified DNA also has various functions that natural nucleic acids cannot achieve, such as switchable fluorescence, photocrosslinking, insulation, and emission color change. These pseudo-base pairs can be used to prepare various functional nanomaterials. Therefore, we urgently need to develop detection and analysis techniques for threoninol with characteristics such as simplicity, sensitivity and high efficiency.

[0003] At present, there are many methods for detecting L-threoninol. Traditional detection methods include high-performance liquid chromatography, gas chromatography, capillary electrophoresis, etc. The disadvantages of these methods are expensive instruments and equipment, complicated operation, and low sensitivity. Compared with other methods, fluorescence sensing technology has the advantages of high selectivity, high sensitivity, low detection limit, and simple operation. Therefore, using a fluorescent probe to specifically recognize L-threoninol is a method worth promoting. SUMMARY

[0004] The purpose of the present application is to provide a fluorescent probe for specifically recognizing chiral threoninol, preparation and application thereof, so as to realize the specific recognition of L-threoninol from 12 amino alcohols in DMSO by the fluorescent probe. The probe has the advantages of specific selectivity, high sensitivity, and wide detection concentration range.

[0005] The above technical purpose of the present application is achieved by the following technical scheme:

[0006] A fluorescent probe for specifically recognizing chiral threoninol, the chemical formula of which is (S)-3,3'-dicarboxyl-[1,1'-binaphthalene]-2,2'-diacrylic acid diester, and the specific chemical structure formula is as follows:

[0007]

[0008] The present application also provides a preparation method of a fluorescent probe for specifically recognizing chiral threoninol, characterized by comprising the following steps:

[0009] Step 1. Synthesis of (S)-2,2'-bis(methoxymethoxy)-1,1'-binaphthalene

[0010] (S)-BINOL was dispersed in anhydrous THF, added to a solution of NaH dispersed in anhydrous THF at 0°C, reacted at room temperature for 2 hours, added bromomethyl methyl ether at 0°C, reacted at room temperature overnight, added H2O to quench the reaction at 0°C, separated the organic phase from the aqueous phase, and after removing the solvent, the product was recrystallized to obtain a white solid product;

[0011] Step 2. Synthesis of (S)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3,3'-dicarboxaldehyde

[0012] n-BuLi was added to a THF solution of (S)-2,2'-bis(methoxymethoxy)-1,1'-binaphthalene at 0°C, after reacting for 1 hour, DMF was added at 0°C, after reacting at room temperature for 3 hours, saturated ammonium chloride solution was added to quench the reaction, the organic phase was separated from the aqueous phase, and after removing the solvent under reduced pressure, the product was purified by silica gel column to obtain a yellow oil product;

[0013] Step 3. Synthesis of (S)-2,2'-dihydroxy-[1,1'-binaphthalen]-3,3'-dicarboxaldehyde

[0014] (S)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3,3'-dicarboxaldehyde was dissolved in a mixed solution of DCM and anhydrous ethanol, HCl was added, reacted overnight, solid NaHCO3 was added to quench, DCM was added for extraction, the organic phase was separated from the aqueous phase, and after removing the solvent under reduced pressure, a yellow solid product was obtained;

[0015] Step 4. Synthesis of (S)-3,3'-dicarboxy-[1,1'-binaphthalen]-2,2'-diacrylate

[0016] Triethylamine was added to a DCM solution of (S)-2,2'-dihydroxy-[1,1'-binaphthalen]-3,3'-dicarboxaldehyde, reacted for 3h, acryloyl chloride was further added, reacted for 2h, after the reaction was completed, the solvent was removed, and the product was purified by silica gel column to obtain a light yellow solid product.

[0017] Further preferably, in step 1, column chromatography purification is not required.

[0018] Further preferably, in step 2, column chromatography purification is performed, and the eluent used in column chromatography purification is petroleum ether: ethyl acetate = 10:1, v / v.

[0019] Further preferably, in step 3, column chromatography purification is not required.

[0020] Further preferably, in step 4, column chromatography purification is performed, and the eluent used in column chromatography purification is pure dichloromethane.

[0021] The application also provides application of the fluorescent probe specifically recognizing chiral threoninol, for specifically recognizing L-threoninol in DMSO.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] Firstly, the fluorescent probe provided by the application is simple to prepare and low in cost.

[0024] Secondly, the fluorescent probe realizes specific selective fluorescence recognition of low-concentration L-threoninol in DMSO, and shows high fluorescence selectivity for L-threoninol in the presence of 30 equivalents of amino alcohol, and the binaphthol-based dialdehyde and bis-acrylate bifunctional chiral fluorescent probe realizes specific recognition of L-threoninol from 12 kinds of amino alcohol in DMSO.

[0025] Thirdly, the fluorescent probe has the advantages of specific selectivity, high sensitivity, short response time and wide detection concentration range. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The synthesis route of the fluorescent probe of the application is shown in the following figure:

[0027] Figure 2 The principle diagram for specifically detecting L-threoninol is shown in the following figure:

[0028] Figure 3 Selective test of (S)-5; (a) fluorescence spectrum of (S)-5 recognizing 12 kinds of amino alcohol; (b) fluorescence enhancement contrast of (S)-5 recognizing 12 kinds of amino alcohol at 540 nm;

[0029] Figure 4 (a) fluorescence spectrum of (S)-5 reacting with L-threoninol or D-threoninol at different time; (b) fluorescence intensity of (S)-5 reacting with L-threoninol or D-threoninol at 540 nm at different time; (c) fluorescence spectrum of (S)-5 reacting with L-threoninol or D-threoninol for 2 h and then being diluted with THF at different time; (d) fluorescence intensity of (S)-5 reacting with L-threoninol or D-threoninol for 2 h and then being diluted with THF at 540 nm at different time;

[0030] Figure 5 (a) fluorescence spectrum of (S)-5 reacting with L-threoninol or D-threoninol of different concentrations; (b) fluorescence intensity of (S)-5 reacting with L-threoninol or D-threoninol of different concentrations at 540 nm;

[0031] Figure 6 (a) fluorescence spectrum of (S)-5 reacting with 30 equivalents of L-threoninol or D-threoninol of different concentrations; (b) fluorescence intensity of (S)-5 reacting with 30 equivalents of L-threoninol or D-threoninol of different concentrations at 540 nm; 2+Fluorescence spectra of the reaction in the system; (b) NMR kinetic experiments of (S)-5 with 30 equivalents of L- or D-threoninol; 2+ Fluorescence intensity at 540 nm after the reaction in the system;

[0032] Figure 7 (a) NMR kinetic experiments of (S)-5 with 30 equivalents of L- or D-threoninol; (b) NMR titration experiments of (S)-5 with L- or D-threoninol.

[0033] Figure 8 (a) NMR kinetic experiments of (S)-5 with 30 equivalents of L- or D-threoninol; (b) NMR titration experiments of (S)-5 with L- or D-threoninol. DETAILED DESCRIPTION

[0034] In order to make the technical personnel in the art better understand the technical solutions of the present application, the present application will be further described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application.

[0035] Example 1, a preparation method of a fluorescent probe specifically recognizing chiral threoninol:

[0036] Step 1. (S)-BINOL was dispersed in anhydrous THF, and was added to a solution of NaH dispersed in anhydrous THF at 0°C. The reaction was carried out at room temperature for 2 hours, bromomethyl methyl ether was added at 0°C, and the reaction was carried out at room temperature overnight. H2O was added at 0°C to quench the reaction, and the organic phase was separated from the aqueous phase with ethyl acetate. After removing the solvent, the product was recrystallized with n-hexane to obtain a white solid product, and the white solid product was (S)-2,2'-bis(methoxymethoxy)-1,1'-binaphthyl.

[0037] Step 2. n-BuLi was added to a THF solution of (S)-2,2'-bis(methoxymethoxy)-1,1'-binaphthyl at 0°C, and the reaction was carried out for 1 hour. Then, an appropriate amount of DMF was added at 0°C, and the reaction was carried out at room temperature for 3 hours. The reaction was quenched by adding saturated ammonium chloride solution, and the organic phase was separated from the aqueous phase. After removing the solvent under reduced pressure, the yellow oily product was purified by silica gel column chromatography. The eluent used was petroleum ether: ethyl acetate = 10:1, v / v. The yellow oily product was (S)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3,3'-diformyl.

[0038] Step 3. (S)-2,2'-Bis(methoxymethoxy)-[1,1'-binaphthalenyl]-3,3'-diformaldehyde was dissolved in a mixed solution of DCM and absolute ethanol, and an appropriate amount of HCl was added, and the reaction was carried out overnight. The reaction was quenched by adding solid NaHCO3, and DCM was added for extraction. The organic phase was separated from the aqueous phase, and the yellow solid product was obtained after the solvent was removed under reduced pressure. The yellow solid product was (S)-2,2'-dihydroxy-[1,1'-binaphthalenyl]-3,3'-diformaldehyde.

[0039] Step 4. To the DCM solution of (S)-2,2'-dihydroxy-[1,1'-binaphthalenyl]-3,3'-diformaldehyde, triethylamine was added and the reaction was carried out for 3h, and then an appropriate amount of acryloyl chloride was added and the reaction was carried out for 2h. After the reaction was completed, the solvent was removed, and the light yellow solid product was obtained after purification by silica gel column chromatography. The light yellow solid product was (S)-3,3'-diformyl-[1,1'-binaphthalenyl]-2,2'-diacrylate, and the structure is as follows:

[0040]

[0041] The synthesis process of the L-threoninol enantiomer fluorescent probe of the present application is as follows: Figure 1 .

[0042] Example 2, the application of the fluorescent probe of the present application which specifically recognizes chiral threoninol, is to detect L-threoninol in DMSO. The detection method is as follows:

[0043] The L-threoninol fluorescent probe (S)-5 (hereinafter referred to as (S)-5) of the present application is dissolved in DMSO to prepare a 1.6mM stock solution 1, the amino alcohol is dissolved in ultrapure water to prepare a 48mM stock solution 2, and the zinc acetate is dissolved in ultrapure water to prepare a 1.6mM stock solution 3. 50μL of each solution is taken from the stock solution 1, the stock solution 2 and the stock solution 3, and added to a new sample bottle (first add the stock solution 1, then add 250μL of DMSO, and finally add the stock solution 3). Mix immediately and react. After the reaction is completed, dilute to 4mL.

[0044] In the specific fluorescent recognition, 30 equivalents of 12 kinds of amino alcohols are introduced respectively to test the selectivity of (S)-5. In DMSO, (S)-5 shows chemical selective fluorescence enhancement for 30 equivalents of L-threoninol. The time for the probe to recognize L-threoninol is tested at reaction time points of 1min, 3min, 5min, 10min, 15min, 30min, 1h and 2h; and the stability after quenching with THF for 2h is tested.

[0045] Zn 2+ The concentration has a certain influence on the recognition of the probe to L-threoninol. When the concentration of Zn 2+Below 1 equivalent, the fluorescence intensity showed a linear increase with the concentration of Zn 2+ When the equivalent reached 1 equivalent, the fluorescence intensity reached a plateau, and then the fluorescence intensity did not change with the increase of the concentration of Zn 2+ When the equivalent reached 1 equivalent, the fluorescence intensity reached a plateau, and then the fluorescence intensity did not change with the increase of the concentration of Zn 2+ When the equivalent reached 1 equivalent, the fluorescence intensity reached a plateau, and then the fluorescence intensity did not change with the increase of the concentration of Zn

[0046] The principle of the fluorescent probe of the present application for specifically detecting L-threoninol is as follows:

[0047] It can be found from the nuclear magnetic hydrogen spectrum that the aldehyde proton signal disappears after the probe reacts with L-threoninol or D-threoninol, and an imine proton signal is generated at 8.7 ppm. After L-threoninol and D-threoninol react, respectively, the acrylic ester signal is clear and distinguishable between 5.3-6.0 ppm. The structures after L-threoninol and D-threoninol react with the probe, respectively, are similar, as shown in Figure 2 The reason for the fluorescence difference may be that D-threoninol cannot coordinate with Zn 2+ , while L-threoninol can, which can be proved by mass spectrometry.

[0048] The following tests (S)-5, wherein the (S)-5 probe test paper detection result and (R)-5 probe test paper are mirror image relationship, (S)-5 probe test paper detection preparation process and (R)-5 probe test paper are the same.

[0049] Specific selectivity test of (S)-5

[0050] 50 μL of the mother liquor was taken from the standard sample of (S)-5 and transferred to a clean 5 mL centrifuge tube. Then, 250 μL of DMSO was taken and 50 μL of 48 mM (30 eq.) amino alcohol (12 kinds) was added to the centrifuge tube. At the same time, 50 μL of zinc acetate solution was quickly taken and added to the centrifuge tube. After mixing by slight shaking, the reaction solution was left to stand at room temperature, and the volume was made to 4 mL. Immediately after dilution, the solution was transferred to a cuvette for fluorescence spectrum test, and the results are referred to Figure 3 . As shown in Figure 3 (a), (S)-5 only has L-threoninol among the 12 kinds of amino alcohols to produce very strong fluorescence enhancement at 540 nm, and the fluorescence enhancement of other amino alcohols is weak or has no fluorescence enhancement, Figure 3 (b) shows that the fluorescence response of (S)-5 to L-threoninol is much higher than that of other amino alcohols, which means that the (S)-5 probe can specifically recognize L-threoninol.

[0051] Reaction time test of (S)-5

[0052] 50 μL of the stock solution was taken from the (S)-5 standard sample and transferred to a clean 5 mL centrifuge tube. Then, 250 μL of LDMSO and 50 μL of the 48 mM (30 equivalent) L-threonine or D-threonine sample were added to the centrifuge tube. Simultaneously, 50 μL of zinc acetate solution was quickly added to the centrifuge tube. After gentle shaking to mix, the mixture was reacted at room temperature for 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 1 h, and 2 h, respectively. After the reaction was complete, the reaction solution was brought to a final volume of 4 mL. Immediately after dilution, the solution was transferred to a cuvette for fluorescence spectroscopy. The stability after quenching with THF after 2 h of reaction was also tested. Results are referenced... Figure 4 ,like Figure 4 As shown in (a) and (b), the fluorescence intensity increases with time, while Figure 4 (c) and (d) show that after quenching with THF for 2 hours, the fluorescence intensity tended to stabilize, thus determining the reaction time to be 2 hours.

[0053] Equivalent testing of (S)-5 with L-threonine or D-threonine

[0054] Take 50 μL of the stock solution from the (S)-5 standard sample and transfer it to a clean 5 mL centrifuge tube. Then, take 250 μL of LDMSO, followed by 50 μL each of 1.6 mM, 3.2 mM, 4.8 mM, 6.4 mM, 8 mM, 9.6 mM, 11.2 mM, 12.8 mM, 16 mM, 32 mM, 48 mM, 64 mM, 96 mM, 128 mM, 160 mM, and 240 mM L-threonine or D-threonine, and add them to the centrifuge tube. Simultaneously, quickly add 50 μL of zinc acetate solution to the centrifuge tube, gently vortex to mix, allow the reaction to stand, and then dilute the reaction solution to 4 mL. Immediately after dilution, transfer the solution to a cuvette for fluorescence spectroscopy analysis. Figure 5 As shown, the fluorescence intensity increases with the increase of L-threonine or D-threonine concentration until it reaches a plateau at 30 eq, thus determining that the equivalent of threonine should be 30 eq.

[0055] Zn 2+ Test on the effect of concentration on fluorescence intensity

[0056] From the standard sample of (S)-5, 50 μL of mother liquor was pipetted into a clean 5 mL centrifuge tube. Then, 250 μL of DMSO was pipetted, and 50 μL of 48 mM (30 eq) L- or D- threoninol was added into the centrifuge tube. At the same time, 50 μL of 0.8 mM, 1.6 mM, 2.4 mM, 3.2 mM, 4 mM, 4.8 mM, 5.6 mM and 6.4 mM zinc acetate solution was pipetted into the centrifuge tube. After mixing by slight shaking, the solution was left to stand at room temperature, and the reaction solution was diluted to 4 mL. Immediately after dilution, the solution was transferred into a cuvette for fluorescence spectrum test. As shown in Figure 1, the fluorescence intensity increased with the increase of Zn Figure 6 concentration, reached a maximum when the Zn 2+ equivalent reached 1 eq, and then remained stable basically, thus determining that the Zn 2+ equivalent was 1 eq. 2+

[0057] Kinetic study of (S)-5 and L- or D-threoninol

[0058] (S)-5 (2 mg, 0.004 mmol) was dissolved in 0.6 mL of DMSO-d6, and then L- or D- threoninol (12.6 mg, 0.12 mmol) and zinc acetate (0.9 mg, 0.004 mmol) were dissolved in 0.1 mL of heavy water, respectively. The L- or D- threoninol and zinc acetate solutions were added into the DMSO-d6 solution of (S)-5. After mixing well, the solution was analyzed by nuclear magnetic resonance spectrum after reaction for 1 min, 10 min, 30 min, 1 h, 2 h, 4 h and 7 h, respectively. The results are shown in Figure 2. Figure 7 It can be seen from Figure 2 that, no matter the probe reacts with L- or D- threoninol, the aldehyde proton signal disappears after 1 min of reaction, and the reaction is very fast. An imine proton signal is generated at 8.7 ppm, and the imine proton signal becomes more and more obvious with the increase of time. The NMR images after 2 h have little change, indicating that the product structure does not change. The fluorescence spectrum shows that the fluorescence enhancement tends to be stable, which is consistent with the fluorescence change rate, indicating that (S)-5 can be detected quickly.

[0059] Titration experiment of (S)-5 and L- or D-threoninol

[0060] ​(S)-5 (2 mg, 0.004 mmol) was dissolved in 0.6 mL DMSO-d6, then 1 equivalent, 5 equivalent, 15 equivalent, 30 equivalent, 40 equivalent, 60 equivalent, 80 equivalent, 100 equivalent L- or D-threoninol and zinc acetate (0.9 mg, 0.004 mmol) were dissolved in 0.1 mL heavy water, respectively. The L- or D-threoninol and zinc acetate solution was added to the DMSO-d6 solution of (S)-5. After mixing well, the solution was reacted for 2 h before nuclear magnetic resonance spectroscopy analysis. The results are shown in Table 1. Figure 8 The aldehyde proton signal was still strong when the probe reacted with either L- or D-threoninol at 1 equivalent, but the aldehyde proton signal disappeared at 5 equivalent, and an imine proton signal was generated at 8.7 ppm, which became more and more obvious with the increase of the equivalent, indicating that the probe reacted more fully with threoninol with the increase of the equivalent, and fluorescence detection at high concentration can be achieved.

[0061] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A fluorescent probe that specifically recognizes chiral threonine, characterized in that, Its chemical formula is: (S)-3,3'-diformyl-[1,1'-binaphthyl]-2,2'-diacrylate, and its specific chemical structure is shown below:

2. The method for preparing a fluorescent probe that specifically recognizes chiral threonine according to claim 1, characterized in that, Includes the following steps: Step 1. Synthesis of (S)-2,2'-bis(methoxymethoxy)-1,1'-binaphthyl (S)-BINOL was dispersed in anhydrous THF and added to a solution of NaH dispersed in anhydrous THF at 0°C. The reaction was carried out at room temperature for 2 hours. Bromomethyl methyl ether was added at 0°C and the reaction was carried out at room temperature overnight. The reaction was quenched by adding H2O at 0°C. The aqueous and organic phases were separated by ethyl acetate. After removing the solvent, the product was recrystallized to obtain a white solid product. Step 2. Synthesis of (S)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3,3'-dicarboxaldehyde n-BuLi was added to a THF solution of (S)-2,2'-bis(methoxymethoxy)-1,1'-binaphthylene at 0℃. After reacting for 1 hour, DMF was added at 0℃. After reacting for 3 hours at room temperature, a saturated ammonium chloride solution was added to quench the reaction. The aqueous and organic phases were separated, and the solvent was removed under reduced pressure. The product was then purified by silica gel column chromatography to obtain a yellow oily product. Step 3. Synthesis of (S)-2,2'-dihydroxy-[1,1'-binaphthyl]-3,3'-dicarboxaldehyde (S)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3,3'-dicarboxaldehyde was dissolved in a mixed solution of DCM and anhydrous ethanol, HCl was added, the reaction was allowed to proceed overnight, solid NaHCO3 was added to quench the reaction, DCM was added to extract the mixture, the aqueous and organic phases were separated, and the solvent was removed under reduced pressure to obtain a yellow solid product. Step 4. Synthesis of (S)-3,3'-dicarboxylo-[1,1'-binaphthyl]-2,2'-diacrylate Triethylamine was added to a DCM solution of (S)-2,2'-dihydroxy-[1,1'-binaphthyl]-3,3'-dicarboxaldehyde and reacted for 3 h. Acryloyl chloride was then added and reacted for 2 h. After the reaction was completed, the solvent was removed, and the product was purified by silica gel column chromatography to obtain a pale yellow solid product.

3. The method for preparing a fluorescent probe that specifically recognizes chiral threonine according to claim 2, characterized in that: In step 1, column chromatography purification is not required.

4. The method for preparing a fluorescent probe that specifically recognizes chiral threonine according to claim 2, characterized in that: In step 2, column chromatography purification is performed, and the eluent used in column chromatography purification is petroleum ether: ethyl acetate = 10:1, v / v.

5. The method for preparing a fluorescent probe that specifically recognizes chiral threonine according to claim 2, characterized in that: In step 3, column chromatography purification is not required.

6. The method for preparing a fluorescent probe that specifically recognizes chiral threonine according to claim 2, characterized in that: In step 4, column chromatography purification is performed, and the eluent used in column chromatography purification is pure dichloromethane.

7. The application of the fluorescent probe for specifically recognizing chiral threonine according to claim 1, characterized in that: Used for rapid and specific identification of L-threonine in DMSO.

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