Preparation method of fluorescent probe for specifically and selectively recognizing D-cysteine

Abstract

The invention discloses a preparation method of a fluorescent probe for specifically and selectively recognizing D-cysteine, and belongs to the technical field of chiral fluorescence detection. The preparation method comprises the following steps: reacting (S)-binaphthol, triethylamine and trifluoromethanesulfonic anhydride together, removing a solvent, reacting with DIPEA and bromomethyl methyl ether, then acting with NiCl2 (dddp) and MeBrMg respectively, and purifying through a silica gel column to obtain (S)-2-(methoxymethoxy)-2 '-methyl-1, 1'-binaphthalene; the preparation method comprises the following steps: reacting a CCl4 solution of (S)-2-(methoxymethoxy)-2 '-methyl-1, 1'-binaphthalene with azodiisobutyronitrile and N-bromosuccinimide, then adding DMSO (Dimethylsulfoxide) and sodium bicarbonate to react, and purifying through a silica gel column to obtain (S)-2 '-(methoxymethoxy)-[1, 1'-binaphthalene]-2-formaldehyde; (S)-2 '-(methoxymethoxy)-[1, 1'-dinaphthalene]-2-formaldehyde and 3M HCl are mixed and fully reacted, and a target compound (S)-D3 is obtained through silica gel column purification; and replacing (S)-binaphthol with (R)-binaphthol to synchronously synthesize (R)-D3. The fluorescent probe (S)-D3 provided by the invention realizes specific fluorescent recognition of 0-20 equivalents of D-cysteine, has high detection stability, and is not influenced by homocysteine and glutathione.

Description

Technical Field

[0001] The invention belongs to the technical field of chiral fluorescence detection, and particularly relates to a method for preparing a fluorescent probe capable of specifically and selectively recognizing D-cysteine. Background Art

[0002] Cysteine is a sulfur-containing amino acid, and its chiral enantiomers, L-cysteine (L-Cys) and D-cysteine (D-Cys), exhibit significant functional differences in vivo. L-Cys is a building block of natural proteins and is involved in the biosynthesis of glutathione (GSH), the antioxidant defense system, the regulation of metal ion homeostasis, and the sulfur metabolism pathway. It is crucial for maintaining cellular redox balance and detoxification functions. Recent studies have found that although D-Cys is present in very low levels in mammals, its abnormal accumulation is associated with a variety of pathological processes, including neurodegenerative diseases (such as Alzheimer's disease), abnormal metabolism in the tumor microenvironment, and bacterial biofilm formation. For example, D-Cys can cause neuronal damage and enhanced tumor resistance by interfering with redox signaling pathways or directly modifying functional proteins.

[0003] The precise identification of chiral cysteine is of great significance for disease diagnosis and drug development. However, due to interference from a large number of L-amino acids, peptides, and other amines in the body, the analysis of D-amino acids in the human body is extremely difficult. Therefore, there is an urgent need to develop new detection and analysis technologies to achieve high-throughput and accurate detection of D-cysteine. Current methods for the detection and analysis of chiral amino acids include high-performance liquid chromatography, liquid chromatography-mass spectrometry, electrochemical methods, automatic amino acid analyzers, ion chromatography, and gas chromatography-mass spectrometry. However, these methods require pre-column derivatization, which is complex and time-consuming. Moreover, these methods are only suitable for in vitro detection and cannot accurately analyze the physiological activities of chiral amino acids in vivo in real time. In comparison, chiral fluorescence detection technology offers advantages such as good selectivity, high sensitivity, short detection time, low operational difficulty, no need for pre-derivative analysis, simultaneous detection of multiple signal molecules, and applicability to intracellular detection.

[0004] As researchers delve deeper into the study of fluorescent probes for chiral amino acids, a variety of chiral fluorescent scaffolds have been developed. However, the number of chiral fluorescent probes specifically for the enantioselective recognition of D-cysteine remains very limited. Furthermore, the fluorescent recognition of cysteine is susceptible to interference from biological thiols such as homocysteine or glutathione. Therefore, the development of fluorescent probes that specifically and selectively recognize chiral cysteine is of great significance. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a method for preparing a fluorescent probe that specifically and selectively recognizes D-cysteine, so as to achieve specific fluorescent recognition of high-concentration D-cysteine.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing a fluorescent probe that specifically and selectively recognizes D-cysteine comprises the following steps:

[0008] S1. (S)-binaphthol, triethylamine and trifluoromethanesulfonic anhydride are reacted together. After removing the solvent, the reaction is carried out with DIPEA and bromomethyl methyl ether. The reaction is then reacted with NiCl2(dddp) and MeBrMg, respectively, and the reaction is purified on a silica gel column to obtain (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthyl;

[0009] S2. A CCl4 solution of (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthyl was reacted with azobisisobutyronitrile (1 equivalent) and N-bromosuccinimide (1.5 equivalents), and then DMSO and sodium bicarbonate were added to react. The reaction was then purified on a silica gel column to obtain (S)-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-carbaldehyde;

[0010] S3, (S)-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-carbaldehyde and 3M HCl (10 equivalents) were mixed and reacted fully, and purified by silica gel column to obtain the target compound (S)-D3;

[0011] S4. Synchronously replace the (S)-binaphthol in step S1 with (R)-binaphthol, and the remaining steps are the same as those in steps S1-S3 to synthesize (R)-D3.

[0012] Furthermore, in step S1, the equivalent ratio of (S)-binaphthol, triethylamine, and trifluoromethanesulfonic anhydride is 1:3:1.2; the equivalent ratio of DIPEA and bromomethyl methyl ether is 1:2:1.2; and the equivalent ratio of NiCl2 and MeBrMg is 0.1:2.

[0013] Furthermore, in step S1, during the silica gel column purification, the eluent used is composed of petroleum ether and ethyl acetate in a volume ratio of 20:1.

[0014] Furthermore, the eluent used for silica gel column purification consisted of petroleum ether and ethyl acetate in a volume ratio of 50:1.

[0015] Furthermore, in step S3, the eluent used for silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0016] The beneficial effects of the present invention are:

[0017] 1. The present invention successfully prepared a pair of novel chiral fluorescent probes (S)-D3 and (R)-D3 based on a binaphthyl skeleton, and the synthetic route of the fluorescent probes of the present invention is short and the operation difficulty is low.

[0018] 2. The aldehyde group of the fluorescent probe (S)-D3 of the present invention can undergo an imine condensation reaction with the amino group of cysteine. Further, the sulfhydryl group of cysteine can form a thiazolidine with the imine bond. The thiazolidine formed by D-cysteine can form an intramolecular hydrogen bond with the hydroxyl group of the probe, thereby increasing the rigidity of the probe skeleton and producing a specific fluorescence enhancement effect.

[0019] 3. The fluorescent probe of the present invention realizes the specific fluorescent recognition of 0-20 equivalents of D-cysteine, has high detection stability, and is not interfered by biological thiols such as homocysteine and glutathione.

[0020] 4. The fluorescent probe of the present invention can realize the enantiomeric composition analysis of cysteine solution with unknown enantiomeric composition.

[0021] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solutions and beneficial effects of the invention clearer, the present invention is described with the following drawings:

[0023] Figure 1 is the molecular structural formula of the fluorescent probe of the present invention;

[0024] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe (S)-D3 of the present invention;

[0025] Figure 3 is the carbon NMR spectrum of the fluorescent probe (S)-D3 of the present invention;

[0026] Figure 4 The fluorescence enhancement at 361 nm of the fluorescent probe (S)-D3 of the present invention after reacting with different amino acids;

[0027] Figure 5 The effect of the reaction time on the fluorescence recognition of D-Cys by the probe (S)-D3 of the present invention;

[0028] Figure 6 The effect of the reaction time on the fluorescence recognition of L-Cys by the probe (S)-D3 of the present invention;

[0029] Figure 7The fluorescence enhancement at 361 nm changes with the reaction time when the probe (S)-D3 of the present invention recognizes chiral Cys by fluorescence;

[0030] Figure 8 This is a graph showing the fluorescence enhancement results after the fluorescent probe (S)-D3 of the present invention recognizes D-Cys solutions with different concentrations;

[0031] Figure 9 This is a graph showing the fluorescence enhancement results after the fluorescent probe (S)-D3 of the present invention recognizes L-Cys solutions of different concentrations;

[0032] Figure 10 The fluorescence enhancement at 361 nm when the probe (S)-D3 of the present invention recognizes chiral Cys with the change of chiral Cys concentration;

[0033] Figure 11 This is a graph showing the fluorescence enhancement results after the fluorescent probe (R)-D3 of the present invention recognizes D-Cys solutions with different concentrations;

[0034] Figure 12 This is a graph showing the fluorescence enhancement results after the fluorescent probe (R)-D3 of the present invention recognizes L-Cys solutions with different concentrations;

[0035] Figure 13 The fluorescence enhancement at 361 nm when the probe (R)-D3 of the present invention recognizes chiral Cys with the change of chiral Cys concentration;

[0036] Figure 14 This is a graph showing the fluorescence enhancement results of the fluorescent probes (S)-D3 and (R)-D3 of the present invention after acting on Cys solutions with different enantiomeric compositions. DETAILED DESCRIPTION

[0037] The invention discloses a method for preparing a fluorescent probe for specifically and selectively recognizing D-cysteine.

[0038] In the present invention, D-Cys refers to D-cysteine;

[0039] Figure 1 is the molecular structural formula of the fluorescent probe of the present invention.

[0040] Example 1

[0041] S1. Add trifluoromethanesulfonic anhydride to a DCM solution of (S)-binaphthol treated with triethylamine at 0° C. and react for 2 hours. After removing the DCM solution, add anhydrous DCM, and add DIPEA and bromomethyl methyl ether at 0° C., react for 3 hours, then remove the anhydrous DCM, and dissolve in anhydrous THF. Add NiCl2(dddp) and MeBrMg in sequence. After the reaction is completed, water is added to quench the reaction, and the aqueous and organic phases are separated. After removing the solvent from the organic phase under reduced pressure, the organic phase is purified on a silica gel column using an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of 20:1 to obtain a colorless oily compound, which is (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthyl.

[0042] S2. Dissolve (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthyl in CCl4 solution, and add azobisisobutyronitrile and N-bromosuccinimide for reaction. After mixing evenly, heat and reflux until the raw materials are completely reacted. After removing the solvent under reduced pressure, add DMSO to dissolve, and then add sodium bicarbonate and reflux until the reaction is complete. After the reaction, pour the reaction solution into pure water and add DCM. Separate the aqueous and organic phases. After removing the solvent under reduced pressure from the organic phase, purify the organic phase with a silica gel column using an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of 50:1 to obtain a light yellow product (S)-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-carboxaldehyde;

[0043] S3, dissolving (S)-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-carbaldehyde in a mixed solvent of dichloromethane and ethanol, adding 3M HCl and mixing to react thoroughly, adding solid sodium bicarbonate until no bubbles are generated, then adding water and DCM to dissolve the solid, separating the aqueous phase and the organic phase, removing the solvent under reduced pressure from the organic phase, and purifying the mixture on a silica gel column using an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of 10:1 to obtain a light yellow solid product, which is the fluorescent probe (S)-D3;

[0044] S4. Synchronously replace the (S)-binaphthol in step S1 with (R)-binaphthol, and the remaining steps are the same as those in steps S1-S3 to synthesize (R)-D3.

[0045] Figure 2 is the H NMR spectrum of the fluorescent probe (S)-D3, Figure 3 is the carbon NMR spectrum of the fluorescent probe (S)-D3; Figure 2 、 Figure 3 It can be seen that the phenolic hydroxyl proton signal appears at δ = 5.27 ppm, and the aldehyde proton signal appears at δ = 9.56 ppm. The above characteristic peak signals indicate the successful preparation of the fluorescent probe (R)-D3.

[0046] Example 2

[0047] S1, dissolving the fluorescent probe in isopropanol to obtain a 1.6 mM first solution;

[0048] S2, dissolving the amino acid in ultrapure water, and adding 1.5 equivalents of NaOH to obtain a 320 mM second solution;

[0049] S3. Take 50 μL of each of the first solution and the second solution and add them to another sample bottle;

[0050] S4. Add 300 μL of isopropanol to the sample bottle, mix well and react for 1.5 hours;

[0051] S5. After the reaction is completed, dilute to 4 mL with isopropanol to prepare a 0.02 mM third solution;

[0052] S6. Place the third solution as a sample in a fluorescence spectrometer, and detect the sample using a light source with a fixed excitation wavelength, thereby specifically and selectively identifying D-Cys.

[0053] The fluorescent probe (S)-D3 was reacted with L / D-serine (Ser), L / D-cysteine (Cys), L / D-proline (Pro), L / D-leucine (Leu), L / D-valine (Val), L / D-methionine (Met), L / D-alanine (Ala), L / D-arginine (Arg), L / D-phenylalanine (Phe), L / D-threonine (Thr), L / D-glutamate (Glu), L / D-aspartic acid (Asp), L / D-lysine (Lys), L / D-isoleucine (Ile), L / D-asparagine (Asn), L / D-glutamine (Gln), DL-homocysteine (Hcy), reduced glutathione (GSH), oxidized glutathione (GSSG) and glycine (Gly). The results were as follows: Figure 4 As shown: It was found that the fluorescent probe (S)-D3 showed specific and selective fluorescence enhancement for D-Cys under an excitation wavelength of 285 nm (emission wavelength was 361 nm), and other amino acids showed little or no change.

[0054] Example 3

[0055] Different reaction times can affect the recognition effect of fluorescent probes. To demonstrate the effect of reaction time on the recognition of D-Cys by the fluorescent probe (S)-D3, 11 experimental groups were set up with reactions of 10 minutes, 20 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, and 6 hours.

[0056] like Figure 5-7 As shown, from Figure 5It can be seen that during the D-Cys recognition process, the fluorescence intensity gradually increased with the extension of the reaction time, but after 3 hours of reaction, the fluorescence enhancement tended to be stable and was no longer affected by the reaction time;

[0057] from Figure 6 It can be seen that during the L-Cys recognition process, the fluorescence intensity slowly increases with the extension of the reaction time, but after 3 hours of reaction, the fluorescence enhancement tends to be stable and is no longer affected by the reaction time;

[0058] from Figure 7 It can be seen that although both D-Cys and L-Cys reached equilibrium after 3 hours of reaction, the fluorescence enhancement of the probe for L-Cys continued to increase after the reaction time exceeded 1.5 hours, resulting in a decrease in enantioselectivity.

[0059] Therefore, it is ideal to terminate the reaction at 1.5 hours.

[0060] Example 4

[0061] The difference in fluorescence enhancement signal when the probe (S)-D3 recognizes different concentrations of D / L-Cys. To demonstrate the effect of analyte concentration on D-Cys recognition by the fluorescent probe (S)-D3, 11 experimental groups were set up: 1 eq., 2 eq., 4 eq., 6 eq., 8 eq., 10 eq., 12 eq., 14 eq., 16 eq., 18 eq., and 20 eq. The experiments revealed that recognition was effective even at concentrations as low as 0.02 mM (1 eq). The fluorescence enhancement peaked when the D-Cys concentration increased to 10 eq., and the intensity no longer increased with increasing D-Cys concentration.

[0062] from Figure 8 It can be seen that during the recognition process of D-Cys, the fluorescence intensity gradually increases with the increase of D-Cys concentration. When the D-Cys content reaches 10eq., the fluorescence enhancement tends to be stable.

[0063] from Figure 9 It can be seen that: during the recognition process of L-Cys, the fluorescence enhancement is low and is not affected by the L-Cys concentration;

[0064] from Figure 10 It can be seen that when the D-Cys content is lower than 10eq, the fluorescence enhancement at 361nm shows a linear growth trend with the D-Cys concentration. When the D-Cys content is higher than 10eq, the fluorescence enhancement tends to be stable, realizing the high concentration detection of D-Cys; while the fluorescence enhancement of L-Cys is relatively low.

[0065] Example 5

[0066] The above recognition effects on chiral Cys were all demonstrated using S-configuration probes. To further illustrate the chiral recognition ability of the probes, R-configuration probes were selected to replace the S-configuration probes, and the experiment in Example 4 was repeated.

[0067] After the fluorescent probe (R)-D3 reacted with D / L-Cys, it was found that L-Cys produced a fluorescence enhancement effect on (R)-D3, while the fluorescence enhancement when reacting with D-Cys was weaker, and its fluorescence spectrum was a mirror image relationship with the fluorescent probe (S)-D3.

[0068] from Figure 11 It can be seen that: during the recognition process of D-Cys, the fluorescence enhancement is low and is not affected by the D-Cys concentration;

[0069] from Figure 12 It can be seen that during the recognition process of L-Cys, the fluorescence intensity gradually increases with the increase of L-Cys concentration. When the L-Cys content reaches 10eq., the fluorescence enhancement tends to be stable.

[0070] from Figure 13 It can be seen that when the L-Cys content is lower than 10 eq., the fluorescence enhancement at 361 nm increases linearly with the L-Cys concentration. When the L-Cys content is higher than 10 eq., the fluorescence enhancement tends to be stable; while the fluorescence enhancement of D-Cys is relatively low.

[0071] Example 6

[0072] Equal amounts of D / L-Cys mixed solutions but in different proportions were introduced separately:

[0073] (S)-D3 and (R)-D3 were reacted with 9 groups of cysteine solutions respectively. The total amount of D-cysteine and L-cysteine in each group of cysteine solutions was the same, but the composition of D-cysteine and L-cysteine was different.

[0074] The amount of D-cysteine in the first cysteine solution is 0 μmol, and the amount of L-cysteine is 0.8 μmol;

[0075] The amount of D-cysteine in the fifth group of cysteine solutions was 0.4 μmol, and the amount of L-cysteine was 0.4 μmol;

[0076] The amount of D-cysteine in the ninth set of cysteine solutions was 0.8 μmol, and the amount of L-cysteine was 0 μmol.

[0077] Finally, titration experiments of fluorescent probe (S)-D3 and fluorescent probe (R)-D3 were carried out.

[0078] The results are as follows Figure 14As shown: The fluorescence intensity curve after the fluorescent probe (S)-D3 and the mixed solution of D / L-Cys in different ratios is a mirror image relationship with the fluorescent probe (R)-D3.

[0079] The experimental conclusions of Examples 2 to 6 show that the specific fluorescent recognition of chiral Cys by the fluorescent probe is based on the chiral skeleton of the probe, and the enantiomeric composition of any Cys solution can be determined.

[0080] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a fluorescent probe that specifically and selectively recognizes D-cysteine, characterized by: The following steps are included: S1. (S)-binaphthol, triethylamine, and trifluoromethanesulfonic anhydride are added to a DCM solution for reaction, and then the DCM solution is removed. Anhydrous DCM is added to react with DIPEA and bromomethyl methyl ether, and then the anhydrous DCM is removed. NiCl2(dddp) and MeBrMg are added in anhydrous THF for reaction. After the reaction is complete, (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthol is purified by silica gel column to obtain (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthyl; S2. Reacting a CCl4 solution of (S)-2-(methoxymethoxy)-2'-methyl-1,1'-binaphthyl with azobisisobutyronitrile and N-bromosuccinimide, then adding DMSO and sodium bicarbonate to react, and purifying via a silica gel column to obtain (S)-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-carbaldehyde; S3, dissolving (S)-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-carbaldehyde in a mixed solvent prepared by mixing dichloromethane and ethanol, adding 3M HCl and mixing to react fully, and purifying on a silica gel column to obtain the target compound (S)-D3; S4. Synchronously replace the (S)-binaphthol in step S1 with (R)-binaphthol, and the remaining steps are the same as those in steps S1-S3 to synthesize (R)-D3.

2. The method for preparing a fluorescent probe for selectively recognizing D-cysteine according to claim 1, wherein: In step S1, the equivalent ratio of (S)-binaphthol, triethylamine, and trifluoromethanesulfonic anhydride is 1:3:1.2; the equivalent ratio of DIPEA and bromomethyl methyl ether is 1:2:1.2; and the equivalent ratio of NiCl2 and MeBrMg is 0.1:

2.

3. The method for preparing a fluorescent probe for selectively recognizing D-cysteine according to claim 2, characterized in that: In the step S1, during the silica gel column purification, the eluent used is composed of petroleum ether and ethyl acetate in a volume ratio of 20:

1.

4. The method for preparing a fluorescent probe for selectively recognizing D-cysteine according to claim 3, wherein: In step S2, the amount of azobisisobutyronitrile is 1 equivalent, and the amount of N-bromosuccinimide is 1.5 equivalents; and the eluent used for silica gel column purification is composed of petroleum ether and ethyl acetate in a volume ratio of 50:

1.

5. The method for preparing a fluorescent probe for selectively recognizing D-cysteine according to claim 4, characterized in that: In step S3, 3M HCl is 10 equivalents; and the eluent used for silica gel column purification is composed of petroleum ether and ethyl acetate in a volume ratio of 10:1.

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