Chemical-genetic coding probe for NADPH (Nicotinamide Adenine Dinucleotide Phosphate) detection

Abstract

The invention discloses a chemical-genetic coding probe for NADPH (Nicotinamide Adenine Dinucleotide Phosphate) detection, and particularly provides a compound as shown in a formula (I), a compound as shown in a formula (II) or a tautomer of any one of the compounds as shown in the formula (I) and the formula (II). The probe disclosed by the invention is simple to prepare, high in sensitivity, relatively good in selectivity on biomolecules and analogues thereof, and suitable for various pH conditions. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe for detecting reduced nicotinamide adenine dinucleotide phosphate (NADPH). Background Art

[0002] Reduced nicotinamide adenine dinucleotide (NADH) and its phosphate derivative (NADPH) are ubiquitous coenzymes in organisms. The two differ in structure by only one phosphate group and have very similar chemical properties, but their functions are significantly different. NADH is mainly involved in material energy metabolism, while NADPH, as a coenzyme and important electron donor for more than 400 functional proteins, participates in a variety of anabolic reactions, such as the synthesis of lipids, fatty acids and nucleotides. In addition, NADPH can regulate the concentration of reducing molecules such as GSH in cells, and plays an important role in the redox balance inside cells. The concentration and metabolic pathways of NADPH in different organelles are different, and abnormal metabolism of NADPH can lead to changes in cell function, which is closely related to the occurrence of major diseases such as cancer, inflammation, and aging. Cancer cells use high concentrations of NADPH as a coenzyme to regulate key proteins such as glutathione reductase to resist cellular oxidative stress; at the same time, as an electron donor, it regulates functional proteins such as cytochrome p450 reductase and sulfiratin to maintain high metabolic synthesis reactions for the rapid growth of tumor cells. Therefore, developing drugs to regulate abnormal NADPH metabolism is also considered an important research direction for the treatment of diseases such as cancer, and the visual quantitative analysis of NADPH in living cells can also provide an effective tool for evaluating drug efficacy at the molecular level. However, the specific regulatory mechanism of intracellular (especially subcellular level) NADPH in the formation of various diseases and the relationship between NADPH content and disease are still unclear. Therefore, the development of fluorescent probes that can be used to accurately monitor NADPH content in real time is important for the exploration of NADPH function, the study of disease mechanism and drug development.

[0003] For the quantitative detection of NADPH in vitro, there are relatively mature methods such as colorimetry, HPLC-MS, and capillary electrophoresis. However, how to quantitatively analyze NADPH in real time in living cells or in vivo has always been a difficulty in fields such as bioimaging and metabolomics. Michael Duchen's research group can detect NADPH and distinguish NADH signals by analyzing the fluorescence lifetime, realizing the imaging study of NADPH inside living cells. However, this method requires cutting-edge instruments and complex subsequent processing, as well as high-energy short-wavelength excitation light, which makes it difficult to be widely used for the detection of NADPH in living cells. Therefore, NADPH-specific fluorescent probes are urgently needed tools for visualizing the function of NADPH at the cellular and in vivo levels.

[0004] However, due to the complex structure of NADPH and the existence of highly analogous NADPH (such as NADH), fluorescent probes that can be used for quantitative analysis of NADPH in living cells are extremely scarce. 1) Small molecule fluorescent probes have a simple structure and are easy to use. They have significant advantages in the detection of highly reactive substances (such as reactive oxygen species) and metal ions in cells. However, they generally have poor selectivity for the identification of complex biological molecules. The developed small molecule NADPH fluorescent probes mainly rely on the strong reducing properties of NADPH. The redox chemical reaction between NAD(P)H and fluorescent dyes causes changes in the intramolecular charge transfer effect, which in turn causes changes in the fluorescence signal, thereby achieving qualitative detection of NAD(P)H. However, such probes are difficult to reversibly monitor changes in NADPH concentration and cannot distinguish its analog reduced nicotinamide adenine dinucleotide (NADH). 2) Protein probes generally combine fluorescent proteins and recognition proteins, and use the binding of analytes to recognition proteins to cause changes in protein conformation, thereby changing the fluorescent protein signal and achieving specific detection. Although protein probes have high specificity, their design is relatively complex and requires a significant change in protein conformation, making preparation and optimization difficult and time-consuming. It has been reported that the ratio signal response of the NADPH protein probe is pH-sensitive, requiring an additional reference probe to correct the pH effect; at the same time, it requires 420nm short-wavelength excitation, which is highly phototoxic and prone to background fluorescence, so the probe still has a lot of room for improvement. In addition, the research group of Kai Johnsson at the Max Planck Institute reported in the journal Science a semi-synthetic protein probe constructed by combining small molecule dyes, NanoLuc luciferase, label protein (SNAP-Tag) and Escherichia coli dihydrofolate reductase (eDHFR), which successfully achieved instant quantitative detection of NADPH in the blood. Although this design method is simple and does not rely on protein conformational changes, the probe has problems such as poor membrane permeability, resulting in low cell labeling efficiency, and relies on a dual-dye system of resonance energy transfer, and cannot achieve real-time imaging observation of NADPH in living cells. Summary of the invention

[0005] In view of the defects that existing fluorescent probes have poor recognition selectivity for complex biological molecules, are difficult to reversibly monitor changes in NADPH concentration and cannot distinguish its analog NADH, are difficult and require a long period of preparation and optimization, are pH sensitive and easily produce background fluorescence, the present invention provides a chemical-genetic encoding probe for NADPH detection, which is simple to prepare, has high sensitivity, has good selectivity for biological molecules and their analogs, and is suitable for a variety of pH conditions.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The present invention provides a compound as shown in formula (I), a compound as shown in formula (II), or a tautomer of any of the foregoing:

[0008]

[0009] wherein each A, B, C and D is independently -NR1R2, -OR1 or -SR1;

[0010] Each X and Y is independently O, S, SiR1R2, CR1R2, NR1 or Se;

[0011] Each R1 and R2 is independently H, C1-C6 alkyl or C1-C6 alkyl substituted by one or more halogens;

[0012] Each U and V is independently absent, -CH2-, -NH-, a benzene ring, a benzene ring substituted by one or more R3, a 5-7 membered saturated or unsaturated heterocycle, or a 5-7 membered saturated or unsaturated heterocycle substituted by one or more R4;

[0013] Each R3 and R4 is independently hydroxyl, amino, carboxyl, sulfonic acid or halogen;

[0014] L has the following structure:

[0015] * indicates the end connected to V or L2;

[0016] Where Z0 and Z1 are independently m1, m2, m3, m4 and m5 are each independently selected from any integer in the range of 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15);

[0017] L1 is -L A -L A1 ; L2 is -L B -L B1 ;

[0018] Among them, L A1 L is a substrate for the recognition protein that can bind to NADPH. B1 It is a substrate for the tagged protein;

[0019] L A and L B L A1 and L B1 The linkers connected to the mother core structure each independently have the following structure:

[0020] n1, n2 and n3 are independently 0, 1, 2, 3, 4, 5 or 6, and * indicates the end connected to the mother core structure.

[0021] In some embodiments, A, B, C, and D are each independently -NR1R2.

[0022] In some embodiments, A and B are each independently -NR1R2; R1 and R2 are each independently C1-C6 alkyl.

[0023] In some embodiments, A and B are each independently -NR1R2; R1 and R2 are each independently H or C1-C6 alkyl substituted with one or more halogens.

[0024] In some embodiments, C and D are each independently -NR1R2; R1 and R2 are each independently H or C1-C6 alkyl substituted with one or more halogens.

[0025] In some embodiments, A and B are the same.

[0026] In some embodiments, C and D are the same.

[0027] In some embodiments, X and Y are each independently O, CR1R2 or SiR1R2.

[0028] In some embodiments, X and Y are each independently O, CR1R2 or SiR1R2; R1 and R2 are each independently C1-C6 alkyl.

[0029] In some embodiments, U and V are each independently -CH2-, -NH- or a benzene ring.

[0030] In some embodiments, L has any of the structures shown below:

[0031]

[0032] wherein n is independently 1 or 4.

[0033] In some embodiments, L has the structure shown below:

[0034] m6 is 2.

[0035] In some embodiments, in the compound represented by formula (I), -ULV- has the structure shown below:

[0036]

[0037] In some embodiments, in the compound represented by formula (II), -UL- has the structure shown below:

[0038]

[0039] Wherein, n is 1 or 4.

[0040] In some embodiments, L A1 A substrate for the eDHFR protein, such as trimethoprim (TMP).

[0041] In some embodiments, L B1 It is a chloroalkane ligand (Halo-tag substrate), an O6-benzylguanine ligand (SNAP-tag substrate) or an O6-(4-aminomethylbenzyl)cytosine ligand (CLIP-tag substrate), preferably a chloroalkane ligand.

[0042] In some embodiments, in R1 and R2, the "C1-C6 alkyl" and the "C1-C6 alkyl substituted by one or more halogens" are independently C1-C4 alkyl, such as methyl or ethyl.

[0043] In some embodiments, in R1, R2, R3 and R4, the “C1-C6 alkyl substituted by one or more halogens” and the “halogen” in the “halogen” are independently F, Cl, Br or I, such as F.

[0044] In some embodiments, in U and V, the "5-7 membered saturated or unsaturated heterocycle" and the "5-7 membered saturated or unsaturated heterocycle substituted by one or more R4" are "5-7 membered saturated or unsaturated heterocycle" which are independently 1, 2 or 3 heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2 or 3.

[0045] In some embodiments, A, B, C and D are each independently -N(CH3)2 or -NH(CH2CF3).

[0046] In some embodiments, in the compound represented by formula (I), A and B are -N(CH3)2; C and D are -NH(CH2CF3).

[0047] In some embodiments, in the compound represented by formula (II), A and B are independently -N(CH3)2 or -NH(CH2CF3).

[0048] In some embodiments, X is -C(CH3)2-, -O-, or -Si(CH3)2-.

[0049] In some embodiments, Y is -O-.

[0050] In some embodiments, L1 is

[0051] In some embodiments, L2 is

[0052]

[0053] In some embodiments, the structure of the compound represented by formula (I) is as follows:

[0054]

[0055] In some embodiments, the structures of the tautomers of the compound represented by formula (I) are as follows:

[0056] In some embodiments, the compound represented by formula (II) has any of the structures shown below:

[0057]

[0058]

[0059] In some embodiments, the structures of the tautomers of the compound represented by formula (II) are as follows:

[0060] The present invention provides a compound as shown in formula (F), or a tautomer thereof:

[0061]

[0062] Wherein, A, B, C, D, X, Y, U, L, and V are as defined above.

[0063] In some embodiments, the compound represented by formula (F) is the following compound:

[0064]

[0065] In some embodiments, the structures of the tautomers of the compound represented by formula (F) are as follows:

[0066] The present invention also provides a compound as shown below:

[0067]

[0068]

[0069] Each of k1 and k2 is independently 0, 1 or 2.

[0070] The present invention also provides a composition comprising substance A and substance B, wherein:

[0071] Substance A is the compound represented by formula (I) above, the compound represented by formula (II) above, or a tautomer of any of the foregoing;

[0072] Substance B is the tag protein-recognition protein;

[0073] The recognition protein is a protein that can bind to NADPH.

[0074] In some embodiments, the composition is used to detect NADPH.

[0075] In some embodiments, the recognition protein is eDHFR protein.

[0076] In some embodiments, the tag protein is Halo-tag, SNAP-tag or CLIP-tag, preferably Halo-tag.

[0077] In some embodiments, the substance A is the compound represented by the above formula (I) or its tautomer.

[0078] In some embodiments, the composition consists of substance A and substance B.

[0079] The present invention also provides the use of the compound shown in formula (F) or its tautomer; the compound shown in formula (I) or its tautomer; the compound shown in formula (II) or its tautomer; or the use of the above composition in detecting NADPH.

[0080] In some embodiments, the application is for staining, living cell fluorescence imaging or protein function regulation.

[0081] In some embodiments, in the application, NADPH is detected by the following reaction:

[0082]

[0083] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0084] The reagents and raw materials used in the present invention are commercially available.

[0085] The positive improvement effect of the present invention is that the fluorescent probe for NADPH detection of the present invention is simple to prepare, has high sensitivity, good selectivity for biomolecules and their analogs, and is applicable to a variety of pH conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Schematic diagram of the structure of the fluorescent probe, where L1 is the recognition protein substrate, L2 is the label protein substrate, F is the fluorescent dye, Tag is the label protein, and SP is the recognition protein.

[0087] Figure 2 The absorption spectra of T-sCPY-s500R-H in response to NADPH (1 nM and 100 μM) when bound and not bound to the protein eDHFR-Halo.

[0088] Figure 3 The emission spectrum of T-sCPY-s500R-H bound to the target protein after titration with NADPH.

[0089] Figure 4 The fluorescence response kinetics of T-sCPY-s500R-H bound to the target protein to NADPH fluctuations.

[0090] Figure 5 Binding curve of T-sCPY-s500R-H bound to the target protein after titration of NADPH analogs.

[0091] Figure 6 The effect of interferors on the response of T-sCPY-s500R-H to NADPH binding to the target protein.

[0092] Figure 7 To titrate T-sCPY-s500R-H binding to the target protein in different pH ranges, serial dilutions of NADPH were used.

[0093] Figure 8 Live cell imaging of the NADPH biosensor (T-sCPY-s500R-H bound to the target protein).

[0094] Fig. 9 Live cell ratiometric imaging of NADPH fluctuations by T-sCPY-s500R-H bound to target proteins.

[0095] Fig.10 The fluorescence spectrum of T-MAP555-PEG2-H bound to the target protein in response to different concentrations of NADPH and the curve of the change in fluorescence intensity and concentration.

[0096] Fig.11 The fluorescence spectrum of T-MAP555-PEG5-H bound to the target protein in response to different concentrations of NADPH and the curve of the change in fluorescence intensity and concentration. DETAILED DESCRIPTION

[0097] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0098] Example 1: The preparation route of T-Scpy-s500R-H is as follows:

[0099]

[0100] The specific synthesis steps are as follows:

[0101] Allyl 2'-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)sulfonyl)-3,6-bis(dimethylamino)-10,10-dimethyl-3'-oxo-10H-spiro[anthracene-9,1'-isoindoline]-6'-carboxylate (2)

[0102]

[0103] In the dry DCM (1.93mL) solution of compound 1 (5mg, 10μmol, 1.0eq), anhydrous thionyl chloride (10.9μL) was added in a dry capped vial purged three times with argon, followed by dry pyridine (6.5μL). The solution was stirred at 60°C for 1 hour. 20μL of dry MeOH was reacted with 1μL of reaction solution for 10 minutes, and 5μL of the solution was mixed with 300μL of LC-MS solution (10% H2O in MeCN+0.1% TFA solution), and the formation of acyl chloride was monitored by observing the obtained methyl ester (511.6g / mol) by LC-MS. After complete activation, the solvent and thionyl chloride were removed under high vacuum (1 hour). Then, add Fmoc-sulfonamide (10.4mg, 30μmol, 3.0eq) and 4-dimethylaminopyridine (1.23mg, 10μmol, 1.0eq) and DIPEA (49.8μL) in dry acetonitrile (4.23mL) and dry DMF (100μL), and stir for 35 minutes at 60 ℃.After solvent evaporation, add water and DCM, extract the mixture three times with DCM.The combined organic layer is washed with brine, and the solvent is removed under reduced pressure.Then crude compound 2 is used for the next step without further purification.

[0104] 2'-((2-aminoethyl)sulfonyl)-3,6-bis(dimethylamino)-10,10-dimethyl-3'-oxo-10H-spiro[anthracene-9,1'-isoindoline]-6'-carboxylic acid allyl ester (3)

[0105]

[0106] The crude compound 2 (8.3 mg, 10 μmol, 1.0 eq) was dissolved in DMF (800 μL) containing 20% ​​piperidine (200 μL) and stirred at room temperature for 1 hour. Then, the mixture was extracted three times with DCM and purified by preparative HPLC (50 mL / min, 10-70% MeCN / H2O+0.1% TFA) to give a turquoise solid compound 3 (3.2 mg, 5.3 mmol, 52%). 1 H NMR (400MHz, MeOD) δ8.19 (dd, J=8.1, 1.4Hz, 1H), 8.11 (dd, J=8.1, 0.7Hz, 1H), 7.34 (d, J=0 .6Hz,1H),7.10(d,J=2.3Hz,2H),6.83–6.73(m,4H),5.97(ddt,J=17.2,10.5,5.7Hz,1H),5 .32(dq,J=17.2,1.6Hz,1H),5.23(dq,J=10.5,1.3Hz,1H),4.74(dt,J=5.7,1.4Hz,2H),3. 64(t,J=6.8Hz,2H),3.28(t,J=6.8Hz,2H),3.05(s,12H),1.87(d,J=9.1Hz,6H).HR / MS(ESI + )m / z calc.for C 32 H 36 N4O5S[M+H] +

[0107] 603.2636; found 603.2624.

[0108] Allyl 4-aminosulfonylbutyrate (5)

[0109]

[0110] 4-Aminosulfonylbutyric acid (500mg, 2.99mmol, 1.0eq) was dissolved in DMF (38.7mL). Potassium carbonate (827mg, 6.0mmol, 2.0eq) and triethylamine (831μL, 5.98mmol, 2.0eq) were added. Allyl bromide (521μL, 5.98mmol, 2.0eq) was slowly added in an ice bath. Then, the mixture was heated to 60°C and stirred for 4.5 hours. The reaction was diluted with water and extracted 5 times with DCM. The combined organic matter was washed five times with 1M hydrochloric acid, washed once with brine, dried with MgSO4, filtered, and concentrated under reduced pressure to obtain a yellow-brown oily compound 5 (238mg, 1.15mmol, 38%). 1H NMR (400MHz, MeOD) δ5.97 (ddt, J=17.2, 10.5, 5.6Hz, 1H), 5.34 (dq, J=17.2, 1.6Hz, 1H), 5.24 (dq, J=10. 5,1.4Hz,1H),4.62(dt,J=5.7,1.4Hz,2H),3.22–3.12(m,2H),2.59(t,J=7.3Hz,2H),2.20–2.07(m,2H).

[0111] (E)-N-(9-(5-(tert-Butyloxycarbonyl)-2-carboxyphenyl)-6-((2,2,2-trifluoroethyl)amino)-3H-xanthene-3-ylidene)-2,2,2-trifluoroethane-1-ammonium (7)

[0112]

[0113] Compound 6 (115 mg, 165 μmol, 1.0 eq) was dissolved in dry 1,4-dioxane (5.75 mL). Potassium carbonate (110 mg, 792 μmol, 4.8 eq), 2,2,2-trifluoroethylamine (575 μL), tris(dibenzylideneacetone)dipalladium(0) (30.2 mg, 33 μmol, 0.2 eq) and XPhos (23.6 mg, 49.5 μmol, 0.3 eq) were added. The reaction mixture was heated to 100 ° C and stirred at this temperature for 4 hours. In turn, the mixture was cooled to room temperature and the solvent was evaporated under high vacuum overnight while temporarily heated to 40 ° C. The crude product was purified by flash column chromatography (DCM / MeOH, 0-9%) to give a dark red solid 7 (89 mg, 150 μmol, 90%). 1 H NMR (400MHz, MeOD) δ8.26(dd,J=8.1,1.6Hz,1H),8.13(dd,J=8.1Hz,1.6Hz,1H),7.77(d,J=1.5Hz,1H),6.98(d, J=9.0Hz,2H),6.93(d,J=2.3Hz,2H),6.80(dd,J=9.0,2.3Hz,2H),4.10(q,J=9.2Hz,4H),1.58(s,9H).HR / MS(ESI + )m / z calc.forC 29 H 25 F6N2O5 + [M] +

[0114] 595.1662; found 595.1661.

[0115] 2-((4-(allyloxy)-4-oxobutyl)sulfonyl)-3-oxo-3',6'-bis((2,2,2-trifluoroethyl)amino)spiro[isoindoline-1,9'-xanthene]-6-carboxylic acid (9)

[0116]

[0117] A solution of compound 7 (60 mg, 101 μmol, 1.0 eq), compound 5 (104 mg, 504 μmol, 5.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (77.3 mg, 403 μmol, 4.0 eq) and 4-dimethylaminopyridine (49.2 mg, 403 μmol, 4.0 eq) in DCM (15 mL) was heated to 60°C and stirred at this temperature for 22 hours to generate the tert-butyl ester of the product as an intermediate (784.75 g / mol), which was saponified after water treatment: water (15 mL) was added, the aqueous layer was extracted three times with DCM, the combined organic layers were dried with MgSO4, filtered, and concentrated under reduced pressure. The residue was dissolved in TFA / DCM (1:4, 9 mL) and stirred at room temperature for 3 hours. After evaporation of the solvent, the crude product was purified by preparative HPLC (8 mL / min, 30-90% MeCN / H2O+0.1% TFA) within 60 min to afford 9 (43 mg, 59 μmol, 59%) as a light red solid. 1 H NMR (400MHz, MeOD) δ8.25 (dd, J=8.0, 1.4Hz, 1H), 8.07 (dd, J=8.1, 0.7Hz, 1H), 7.64 (dd, J=1.3, 0. 7Hz,1H),6.57–6.51(m,4H),6.42(dd,J=8.7,2.4Hz,2H),5.92(ddt,J=17.2,10.5,5.6Hz,1H),5. 29(dq,J=17.2,1.6Hz,1H),5.21(dq,J=10.5,1.4Hz,1H),4.55(dt,J=5.6,1.5Hz,2H),3.86(qd,J =9.1,1.6Hz,4H),3.32(m,J=3.2,1.6Hz,2H),2.30(t,J=7.2Hz,2H),1.84–1.72(m,2H).HR / MS(ESI + )m / z calc.for C 32 H 27 F6N3O8S[M+H] + 728.1496; found 728.1496.

[0118] Allyl 4-((6-((2-((6-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3',6'-bis((2,2,2-trifluoroethyl)amino)spiro[isoindoline-1,9'-xanthene]-2-yl)sulfonyl)butyrate (10)

[0119]

[0120] Tert-butyl 2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (Boc-Halo, 29 mg, 90 μM) was dissolved in a mixture of DCM / TFA (1:1, 5 mL) and stirred for 15 minutes, and (2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)ethan-1-amine (Halo-NH2) was prepared and stirred for 15 minutes. Compound 9 (31 mg, 42.5 μmol, 1.0 eq) and prepared Halo-NH2 (19 mg, 85.1 μmol, 2.0 eq) were dissolved in DMF (987 μL), and DIPEA (14.8 μL, 89.3 μmol, 2.1. eq) was added. Then, benzotriazol-1-yloxytris(dimethylamino)-phosphine hexafluorophosphate (24.5 mg, 55.3 μmol, 1.3 eq) was added. The reaction mixture was stirred at room temperature for 1 h and purified continuously by preparative HPLC to give 10 (36 mg, 39 mmol, 91%) as a light red solid. 1 H NMR (400MHz, MeOD) δ8.09 (qd, J=8.0, 1.1Hz, 2H), 7.56 (dd, J=8.1, 0.7Hz, 1H), 6.58–6.51 (m, 4H), 6.42 (dd,J=8.7,2.5Hz,2H),5.92(ddt,J=17.2,10.5,5.6Hz,1H),5.29(dq,J=17.2,1.6Hz,1H),5.21(dq,J =10.5,1.4Hz,1H),4.55(dt,J=5.7,1.4Hz,2H),3.86(qd,J=9.3,3.3Hz,4H),3.60–3.47(m,9H),3.40( t,J=6.5Hz,2H),3.33(m,2H),2.30(t,J=7.2Hz,2H),1.84–1.67(m,4H),1.56–1.25(m,7H).HR / MS(ESI + )m / zcalc.for C 42 H 47 ClF6N4O9S[M+H] + 933.2729; found 933.2732.

[0121] 4-((6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3',6'-bis((2,2,2-trifluoroethyl)amino)spiro[isoindoline-1,9'-xanthene]-2-yl)sulfonyl)butanoic acid (11)

[0122]

[0123] Compound 10 (35 mg, 37.5 μmol, 1.0 eq) was mixed with 1,3-dimethylbarbituric acid (43.9 mg) and tetrakis(triphenylphosphine)palladium(0) (32.5 mg, 28.1 μmol, 0.75 eq) in MeOH / DCM (5:1, 2 mL / 10 mmol) and stirred at room temperature for 1.5 h. The mixture was purified by preparative HPLC to give 11 (30.9 mg, 34.5 mmol, 92%) as a light red solid. 1 H NMR(400MHz,MeOD)δ8.08(qd,J=8.0,1.1Hz,2H),7.56(dd,J=8.1,0.7Hz,1H),6.59–6.51(m,4H),6.42(dd,J=8.7,2.4Hz,2H),3.86(qd,J=9.2,2.7Hz ,4H),3.62–3.45(m,9H),3.40(t,J=6.5Hz,2H),3.33(dq,J=3.3,1.4Hz,2H ),2.23(t,J=7.2Hz,2H),1.81–1.67(m,4H),1.54–1.26(m,7H).HR / MS(ESI + )m / z calc.for C 39 H 43 ClF6N4O9S,[M+H] + 893.2416; found 893.2417.

[0124] Allyl 2'-((2-(4-((6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3',6'-bis((2,2,2-trifluoroethyl)amino)spiro[isoindoline-1,9'-xanthene]-2-yl)sulfonyl)butyramido)ethyl)sulfonyl)-3,6-bis(dimethylamino)-10,10-dimethyl-3'-oxo-10H-spiro[anthracene-9,1'-isoindoline]-6'-carboxylate (12)

[0125]

[0126] Compound 11 (6.5 mg, 10.8 μmol, 1.0 eq) and compound 15 (14.4 mg, 16.1 μmol, 1.5 eq) were dissolved in DMF (650 μL), and DIPEA (5.34 μL, 32.3 μmol, 3.0 eq) was added. Then, benzotriazol-1-yloxy tris (dimethylamino)-phosphine hexafluorophosphate (7.14 mg, 16.1 μmol, 1.5 eq) was added and stirred at room temperature for 2.5 h. Purification by preparative HPLC (20 mL / min, 30–70% MeCN / H2O+0.1% TFA) gave a light blue solid 12 (3.7 mg, 2.5 mmol, 23%). 1 H NMR(400MHz,MeOD)δ8.67(t,J=5.4Hz,1H),8.17(dd,J=8.0,1.4Hz,1H),8.12–8.01(m,3H),7.66(t,J=5.5Hz,1H),7.56(s,1H),7.33(s,1H), 7.07–7.03(m,2H),6.80–6.68(m,4H),6.59–6.49(m,4H),6.38(dd,J=8.7,2.4Hz,2H),5.97(ddt,J=17.4,10.5,5.7Hz,1H),5.32(dq,J=17.2, 1.6Hz,1H),5.23(dq,J=10.5,1.3Hz,1H),4.73(d,J=5.7Hz,1H),3.85(qd,J=9.2,2.6Hz,4H),3.61–3.46(m,11H),3.39(t,J=6.5Hz,2H),3.3 2(m,2H),3.27–3.18(m,2H),3.01(s,12H),2.04(t,J=7.2Hz,2H),1.86(s,3H),1.84(s,3H),1.77–1.65(m,4H),1.54–1.31(m,7H).HR / MS(ESI + )m / z calc.for C 72 H 79 ClF6N8O 13 S2[M+2H] 2+ 739.2473; found 739.2468.

[0127] 2'-((2-(4-((6-((2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-3-oxo-3',6'-bis((2,2,2-trifluoroethyl)amino)spiro[isoindolin-1,9'-xanthene]-2-yl)sulfonyl)butyramido)ethyl)sulfonyl)-3,6-bis(dimethylamino)-10,10-dimethyl-3'-oxo-10H-spiro[anthracene-9,1'-isoindolin]-6'-carboxylic acid (13)

[0128]

[0129] Compound 15 (3 mg, 2.03 μmol, 1.0 eq) was mixed with 1,3-dimethyl-1,3-diazine ring-2,4,6-trione (2.38 mg) and tetrakis(triphenylphosphine)palladium(0) (1.76 mg, 1.52 μmol, 0.75 eq) in MeOH / DCM (390 uL; 5:1, 2 mL / 10 mmol) and stirred at room temperature for 1.5 hours. After removing the solvent in vacuo, it was purified by preparative HPLC (8 mL / min, 20-70% MeCN / H2O+0.1% TFA) to give 13 (2.8 mg, 1.9 mmol, 96%) as a light blue solid. 1 H NMR(400MHz,MeOD)δ8.21(m,1H),8.11–8.01(m,3H),7.56(s,1H),7.32(m,3H),6. 97–6.84(m,4H),6.62–6.47(m,4H),6.41(m,2H),3.94–3.80(m,4H),3.65–3.45(m ,11H),3.41(m,3H),3.32(m,2H),3.23(q,J=8.7,8.2Hz,1H),3.11(s,12H),2.14– 2.00(m,2H),1.91(s,3H),1.87(s,3H),1.73(m,4H),1.56–1.34(m,7H).HR / MS(ESI + )m / z calc.for C 69 H 75 ClF6N8O 13 S2[M+2H] 2+ 719.2317; found 719.2314.

[0130] T-Scpy-s500R-H

[0131]

[0132] Compound 13 (3 mg, 2.09 μmol, 1.0 eq) and TMP-C4-NH2 (2.9 mg, 8.35 μmol, 4 equiv) were dissolved in DMF (126 μL), and DIPEA (1.03 μL, 6.26 μmol, 3.0 eq) was added. Then, benzotriazol-1-yloxy tris(dimethylamino)-phosphine hexafluorophosphate (1.38 mg, 3.13 μmol, 1.5 eq) was added to 20 μL DMF of the stock solution (13.8 mg per 200 μL DMF), and the mixture was stirred at room temperature for 1 hour. Preparative HPLC (8 mL / min, 30–70% MeCN / H2O+0.1% TFA) was used to obtain T-CPY-500R-H (3.4 mg, 1.9 mmol, 92%) as a light blue solid. 1 H NMR(400MHz,MeOD)δ8.67(t,J=5.5Hz,1H),8.63(t,J=5.6Hz,1H),8.12–7.99(m,3H),7.93(dd,J=8.1,1.4Hz,1H),7 .73(t,J=5.5Hz,1H),7.56(s,1H),7.24–7.18(m,2H),7.14(s,2H),6.79(s,4H),6.59–6.49(m,6H),6.39(dd,J=8.7, 2.5Hz,2H),3.94–3.78(m,6H),3.72(s,6H),3.65(s,2H),3.62–3.44(m,12H),3.44–3.33(m,6H),3.23(t,J=8.6,6. 6Hz,2H),3.04(s,12H),2.04(t,J=7.1Hz,2H),1.89(s,3H),1.84(s,3H),1.71(m,8H),1.55–1.36(m,7H).HR / MS(ESI + )m / z calc.for C 86 H 98 ClF6N 13 O 15 S2[M+3H] 3+ 589.5519; found 589.5512.

[0133] Example 2: The preparation route of the compound shown in 20 is as follows:

[0134]

[0135] Step 1: Preparation of compound 15

[0136]

[0137] Compound 14 (1.0 eq) was dissolved in dry acetonitrile. Heat to 80 ° C, and N, N-dimethylformamide di-tert-butyl acetal (6.0 eq) was added dropwise within 5 minutes. The reactant was stirred at 80 ° C for 15 minutes. After the mixture was cooled to room temperature, it was diluted with saturated NaHCO3 and extracted with DCM. The combined organic extracts were dried (MgSO4), filtered and evaporated. The product was purified by flash column chromatography (DCM / MeOH system) to obtain compound 15.

[0138] Steps 2 and 3: Preparation of compounds 16 and 17

[0139]

[0140] Compound 15 (1.0 eq), aminosulfonylglycine allyl ester (5.0 eq), benzotriazole-1-yl-oxy-tris-pyrrolidine-phosphine hexafluorophosphate (2.0 eq) and N,N-diisopropylethylamine (3.0 eq) were stirred in acetonitrile at room temperature for 5 hours and concentrated under reduced pressure. The residue was dissolved in TFA / DCM (1:1) and stirred at room temperature for 3 hours. After evaporating the solvent, the product was purified by flash column chromatography (DCM / MeOH system) to obtain compound 17.

[0141] Step 4: Preparation of compound 18

[0142]

[0143] Compound 17 (1.0 eq) and TMP-C4-NH2 (4.0 eq) were dissolved in acetonitrile, and N,N-diisopropylethylamine (3.0 eq) was added. Then, benzotriazole-1-yloxy tris (dimethylamino)-phosphine hexafluorophosphate (2 eq) was added and the mixture was stirred at room temperature for 1 hour. Purification by flash column chromatography (DCM / MeOH system) or preparative HPLC gave compound 18.

[0144] Step 5: Preparation of compound 19

[0145]

[0146] Compound 18 (35 mg, 37.5 μmol, 1.0 eq) was mixed with 1,3-dimethylbarbituric acid (7.5 eq) and tetrakis(triphenylphosphine)palladium(0) (0.75 eq) in MeOH / DCM (5:1, 2 mL / 10 mmol), stirred at room temperature for 1.5 h, and purified by preparative HPLC to give the product compound 19.

[0147] Step 6: Compound Halo-PEG n Preparation of -NH2

[0148] 2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)ethan-1-amine (Halo-NH2) (1.0 eq) and the previously prepared Fmoc-PEG n -COOH (1.1 eq, commercially available reagent Fmoc-PEG n -carboxyl) was dissolved in acetonitrile and DIPEA (3.0 eq) was added. Then, benzotriazol-1-yloxy tris(dimethylamino)-phosphine hexafluorophosphate (2.0 eq) was added. The reaction mixture was stirred at room temperature for 1 hour, and the product was purified by flash column chromatography (DCM / MeOH system). The purified intermediate was dissolved in DMF containing 20% ​​piperidine and stirred at room temperature for 1 hour. Then, the mixture was extracted three times with DCM. After evaporating the solvent, the compound Halo-PEG was obtained. n The crude -NH2 was used directly in the subsequent reaction.

[0149] Step 7: Preparation of compound 20

[0150]

[0151] Compound 20 (1.0 eq) and Halo-PEG n -NH2 (2.0 eq) was dissolved in acetonitrile and DIPEA (3.0 eq) was added. Then, benzotriazole-1-yloxy tris(dimethylamino)-phosphine hexafluorophosphate (2.0 eq) was added. The reaction mixture was stirred at room temperature for 1 hour and purified continuously by preparative HPLC to obtain the final product, compound 20.

[0152] Referring to the method of Example 2, the following compound was prepared:

[0153]

[0154] T-MAP555-PEG2-H 1 H NMR (600MHz, MeOD) δ8.07 (s, 1H), 7.91 (s, 1H), 7.68 (dd, J = 6, 3Hz,

[0155] 2H),7.55(dd,J=6,3Hz,2H),7.41(m,3H),6.42(d,J=2.4Hz,1H),6.40(s,2H),4.52(s,2H),4.25(t,J =6Hz,2H),3.77(s,6H),3.67-3.55(m,8H),3.50(s,8H),2.88(s,12H),1.77-1.42(m,12H).HR / MS(ESI + )m / z calc.for C60 H 80 C1N 11 O 14 S[M+2H] 2+ 623.7721; found 623.7683.

[0156] T-MAP555-PEG5-H 1 H NMR (600MHz, MeOD) δ8.10 (s, 1H), 7.93 (s, 1H), 7.70 (dd, J = 5.8, 2.8

[0157] Hz,2H),7.58(dd,J=5.8,2.8Hz,2H),7.40(m,3H),6.42(d,J=2.4Hz,1H),6.40(s,2H),4.50(s,2H),4.22( t,J=6Hz,2H),3.78(s,6H),3.63-3.51(m,8H),3.52(s,20H),2.85(s,12H),1.73-1.39(m,12H).HR / MS(ESI + )m / z calc.for C 66 H 92 C1N 11 O 17 S[M+3H] 3+ 460.2100; found 460.2549.

[0158] Effect Example 1

[0159] T-sCPY-s500R-H was mixed with the target protein in labeling buffer (50mM HEPES, 50mM NaCl, 0.1% Triton X-100, pH7.3) at the following concentrations: eDHFR-Halo (6μM) and probe T-sCPY-s500R-H (5μM). The mixture was shaken at 500rpm, 25°C for 3 hours in a Thermo Mixer (Thermo Fisher Scientific) and then diluted 5 times in HEPES buffer (50mM HEPES, 50mM NaCl, pH 7.3) containing 20% ​​mg / μL Bio-Beads SM-2 (Bio-Rad, biotechnology grade). The mixture was centrifuged at 99rpm for 1h with a rotary mixer (Kisker Biotech GmbH) to remove Triton and excess dye. The mixture was then washed twice in HEPES buffer, after which the Triton was removed using a 10kDa Amicon centrifugal filter. The concentration was then measured using a NanoDrop 2000c spectrophotometer. According to the Lambert-Beer Law, A=εcl, the molar extinction coefficient of the probe is 33733L×mol -1 ×cm -1 ) and the absorbance at 520 nm were used to calculate the concentration. Finally, about 4 μM of T-sCPY-s500R-H+target protein was obtained, which was then further diluted to a suitable concentration for in vitro titration detection.

[0160] After labeling, 500nM T-sCPY-s500R-H bound to the target protein or 500nM T-sCPY-s500R-H not bound to the target protein was added to a black, transparent and flat-bottom 384-well plate (Greiner) with different concentrations of NADPH. After incubation at room temperature for 30 minutes, the plate was placed in the BioTek Cytation 5 multi-function microplate reader, and then the absorption spectrum of the fluorescent probe T-sCPY-s500R-H in response to NADPH (1nM and 100μM) in the case of binding and not binding to the protein eDHFR-Halo was detected. Figure 2 As shown in the figure, after T-sCPY-s500R-H binds to eDHFR-Halo, the absorbance at 510nm is increased by 25 times, and a 22-fold fluorescence enhancement effect is presented at 535nm. These enhancement effects indicate the conversion process of the fluorescent dye (donor) from the spirocyclic isomer to the diionic isomer.

[0161] Example 2

[0162] After 100nM T-sCPY-s500R-H bound to the target protein was mixed and incubated with different concentrations of NADPH (10nM-128μM) for 30 minutes, the well plate was placed in the BioTek Cytation 5 multi-function microplate reader, and then the fluorescence spectrum of T-sCPY-s500R-H in response to a series of gradient dilutions of NADPH in the case of binding to the protein eDHFR-Halo was detected. Excitation wavelength: 490nm, emission wavelength: 515-750nm.

[0163] like Figure 3 As shown, the fluorescence intensity (I 535 ) is reduced, while at 635nm (I 635 ), which is caused by the FRET effect. These significant changes in the labeling and sensing process verify the working principle of the biosensor.

[0164] Example 3

[0165] The target protein-bound T-sCPY-s500R-H was added to a black, transparent and flat-bottomed 384-well plate (Greiner), and then placed in a BioTek Cytation 5 multifunctional microplate reader. The fluorescence signal was excited at a wavelength of 490 nm, and the emission fluorescence signal was detected at wavelengths of 535 nm and 635 nm, respectively, every 20 s to obtain the fluorescence ratio value (I 635 / I 535 During the assay, 10 μM NADPH was first added, and then NADPH levels were reduced (oxidized) by adding glutathione disulfide (GSSG 100 μM) and glutathione reductase (GR, 1 U).

[0166] like Figure 4 As shown, T-sCPY-s500R-H bound to the target protein was able to monitor the fluctuation of NADPH with a time resolution of seconds, indicating that the binding ligand TMP has rapid binding and dissociation capabilities.

[0167] Example 4

[0168] A series of different gradient dilution concentrations (1nM-1mM) of NADPH and NADP + , NADH and NAD + 100 nM T-sCPY-s500R-H binding to target protein was titrated respectively. Excitation wavelength: 490 nm, emission wavelength: 515-750 nm. After the detection was completed, the fluorescence ratio value (I 635 / I 535 ) as the analyte concentration changes.

[0169] like Figure 5 As shown, T-sCPY-s500R-H bound to the target protein can inhibit NADH and NAD even at a concentration of up to 1 mM. + and NADP + No response was detected. For NADPH, the maximum FRET ratio change was 4.86 ± 0.24-fold, C 50 It is 2.15±0.27μM.

[0170] Example 5

[0171] A series of different gradient dilution concentrations (1nM-100μM) of NADPH were used to titrate 100nM T-sCPY-s500R-H bound to the target protein, and different interferents were added to the system. ATP: 1mM, ADP: 1mM, nicotinamide, NADH, NADP + , GTP, NAD + , G6PDi-1, NMN and glucose: 100 μM. Excitation wavelength: 490 nm, emission wavelength: 515-750 nm. After the detection, the fluorescence ratio value (I 635 / I 535 ) is a fitted curve of the change of analyte concentration, where the ordinate R is I 635 / I 535 , R max For I 635 / I 535 The maximum value, R min For I 635 / I 535 The minimum value of . Figure 6 As shown, the results showed that these analogs or metabolites had little interference with the measurement of NADPH.

[0172] Example 6

[0173] In different pH buffer environments (pH 7.0-8.0), a series of different gradient dilution concentrations (1nM-1mM) of NADPH were used to titrate 100nM T-sCPY-s500R-H bound to the target protein. Excitation wavelength: 490nm, emission wavelength: 515-750nm. After the detection was completed, the fluorescence ratio value (I 635 / I 535 ) is a fitted curve of the change of analyte concentration, where the ordinate R is I 635 / I 535 , R max For I 635 / I 535 The maximum value, Rmin For I 635 / I 535 The minimum value of .

[0174] like Figure 7 As shown, T-sCPY-s500R-H bound to the target protein exhibited a consistent response to NADPH in the cytoplasmic pH range of 7.0 to 8.0, suggesting the excellent stability of the biosensor for dynamic detection of cytoplasmic NADPH.

[0175] Example 7

[0176] HEK293T cells were cultured in DMEM medium (4.5 g / L glucose) supplemented with 10% (v / v) fetal bovine serum, 1 mM GlutaMAX and 1 mM sodium pyruvate. The cells were placed in a humidified 5% CO2 (v / v) incubator at 37°C for culture. 2-3 days before imaging, the cells were inoculated in 8-well culture dishes with a transparent bottom (μ-Slide 8Well Glass Bottom dishes, ibidi, Martinsried, Germany). The plasmid encoding DHFRmt-Halo was transfected into the cells by Lipofectamine 3000 (Invitrogen, Thermofisher, USA). The cells were then incubated with a mixture containing 500 nM T-sCPY-s500R-H and 0.1% Pluronic F-127 for 12 hours. To evaluate the specific imaging of NADPH, live cells were imaged under no-wash and HBSS buffer 2 wash conditions. Confocal imaging of live cells was performed on a Laser confocal scanning microscopy FV3000 (Olympus) microscope. Imaging conditions: HC PL APO CS2 20.0x.λ ex :488nm, green fluorescence detection range: 500–580nm, red fluorescence detection range 600–700nm. The images were pseudo-colored in the HSB color space using the pixel ratio of the 600–700nm emission image and the 500–590nm emission image of the same cell.

[0177] like Figure 8As shown, in HEK293T live cell imaging, cells expressing eDHFR-Halo labeled with T-sCPY-s500R-H were observed to have strong fluorescence in both the green channel (500-580 nm) and the red channel (600-700 nm). In contrast, the signal obtained in HEK293T cells that did not express the eDHFR-Halo fusion protein was negligible, indicating that the fluorescence measurement of the unbound probe had little interference.

[0178] Example 8

[0179] To evaluate the dynamic changes of NADPH, the transfected HEK293T cells were washed twice with HBSS buffer (136.7mM NaCl, 5.4mM KCl, 0.35mM Na2HPO4, 0.44mM KH2PO4, 4.2mM NaHCO3, 1.26mM CaCl2, 0.81mM MgSO4, pH 7.4) and incubated in the buffer for 1h. In the drug functional analysis experiment, the cells were incubated in HBSS buffer without glucose, containing glucose (5mM) or containing the drug G6PD inhibitor G6PDi-1 (inhibiting the generation of NADPH) for 1h. After that, during the long-term imaging of live cells, 250μM H2O2 was first added to break the intracellular NADPH homeostasis, and then 5mM glucose was added to increase the NADPH level in the cells.

[0180] like Fig. 9 As shown, HEK293T cells were cultured in glucose-fueled and glucose-free medium for 1 hour, and the fluorescence intensity ratio (red / green) showed similar values, probably due to NADPH homeostasis in living cells. After the addition of oxidants (250μM H2O2) to glucose-removed cells, a rapid decrease in the fluorescence ratio was observed, indicating a rapid decrease in NADPH levels in the cytoplasm. In contrast, subsequent glucose treatment (5mM) led to a rapid recovery of NADPH levels (<1 minute). However, during oxidative stress, NADPH levels in cells that were always supplied with glucose showed minimal changes. The fluctuations in NADPH levels after oxidative stress treatment reveal the key role of glucose in maintaining NADPH homeostasis in the cytoplasm.

[0181] Example 9

[0182] After 1 μM T-MAP555-PEG2-H or T-MAP555-PEG5-H bound to the target protein was mixed and incubated with different concentrations of NADPH (0-100 μM) for 30 minutes, the well plate was placed in the BioTek Cytation 5 multi-function microplate reader, and then the fluorescence spectrum of T-MAP555-PEG2-H or T-MAP555-PEG5-H in response to a series of gradient dilutions of NADPH in the case of binding to the protein eDHFR-Halo was detected. Excitation wavelength: 545 nm, emission wavelength: 560-700 nm.

[0183] like Fig.10 (T-MAP555-PEG2-H), Fig.11 As shown in (T-MAP555-PEG5-H), the fluorescence intensity of the two probes at 578nm changes with the concentration of NADPH, and the signal-to-noise ratio of T-MAP555-PEG5-H is higher and the detection effect is better. These significant changes in the labeling and sensing process verify the working principle of the biosensor.

Claims

1. A compound represented by formula (I), a compound represented by formula (II), or a tautomer of any of the foregoing: in, Each A, B, C and D is independently -NR1R2, -OR1 or -SR1; Each X and Y is independently O, S, SiR1R2, CR1R2, NR1 or Se; Each R1 and R2 is independently H, C1-C6 alkyl or C1-C6 alkyl substituted by one or more halogens; Each U and V is independently absent, -CH2-, -NH-, a benzene ring, a benzene ring substituted by one or more R3, a 5-7 membered saturated or unsaturated heterocycle, or a 5-7 membered saturated or unsaturated heterocycle substituted by one or more R4; Each R3 and R4 is independently hydroxyl, amino, carboxyl, sulfonic acid or halogen; L has the following structure: * indicates the end connected to V or L2; Where Z0 and Z1 are independently m1, m2, m3, m4 and m5 are each independently selected from any integer between 0 and 15; L1 is -L A -L A1 ; L2 is -L B -L B1 ; Among them, L A1 L is a substrate for the recognition protein that can bind to NADPH. B1 It is a substrate for the tagged protein; L A and L B L A1 and L B1 The linkers connected to the mother core structure each independently have the following structure: n1, n2 and n3 are independently 0, 1, 2, 3, 4, 5 or 6, and * indicates the end connected to the mother core structure.

2. The compound of formula (I), the compound of formula (II), or a tautomer of any of the foregoing according to claim 1, characterized in that: It meets one or more of the following conditions: (1) A, B, C and D are each independently -NR1R2; (2) A and B are the same; (3) C and D are the same; (4) X and Y are independently O, CR1R2 or SiR1R2; (4) U and V are independently -CH2-, -NH- or a benzene ring; (5) L has any of the following structures: Wherein, n is 1 or 4; m6 is 2; (6)L A1 It is a substrate of eDHFR protein; and (7)L B1 It is a chloroalkane ligand, an O6-benzylguanine ligand or an O6-(4-aminomethylbenzyl)cytosine ligand.

3. The compound of formula (I), the compound of formula (II), or a tautomer of any of the foregoing according to claim 1, characterized in that: It meets one or more of the following conditions: (1) A and B are each independently -NR1R2; R1 and R2 are each independently C1-C6 alkyl; or, A and B are each independently -NR1R2; R1 and R2 are each independently H or C1-C6 alkyl substituted with one or more halogens; (2) C and D are each independently -NR1R2; R1 and R2 are each independently H or a C1-C6 alkyl group substituted with one or more halogens; (3) X and Y are each independently O, CR1R2 or SiR1R2; R1 and R2 are each independently C1-C6 alkyl; (4) In the compound represented by formula (I), -ULV- has the structure shown below: (5) In the compound represented by formula (II), -UL- has the structure shown below: wherein n is independently 1 or 4; (6)L A1 for trimethoprim; and (7)L B1 It is a chloroalkane ligand.

4. The compound of formula (I), the compound of formula (II), or a tautomer of any of the foregoing according to claim 1, characterized in that: It meets one or more of the following conditions: (1) In R1 and R2, the "C1-C6 alkyl" and the "C1-C6 alkyl substituted by one or more halogens" are independently C1-C4 alkyl, such as methyl or ethyl; (2) In R1, R2, R3 and R4, the "C1-C6 alkyl group substituted by one or more halogens" and the "halogen" in the "halogen" are independently F, Cl, Br or I, for example, F; and (3) in U and V, the "5-7 membered saturated or unsaturated heterocyclic ring" and the "5-7 membered saturated or unsaturated heterocyclic ring substituted by one or more R4" are "5-7 membered saturated or unsaturated heterocyclic ring" which are independently selected from 1, 2 or 3 heteroatoms selected from N, O and S, and the number of heteroatoms is 1, 2 or 3.

5. The compound of formula (I), the compound of formula (II), or a tautomer of any of the foregoing according to claim 1, characterized in that: It meets one or more of the following conditions: (1) A, B, C and D are independently -N(CH3)2 or -NH(CH2CF3); preferably, in the compound represented by formula (I), A and B are -N(CH3)2; C and D are -NH(CH2CF3); preferably, in the compound represented by formula (II), A and B are independently -N(CH3)2 or -NH(CH2CF3); (2) X is -C(CH3)2-, -O- or -Si(CH3)2-; (3) Y is -O-; (4) L1 is and (5)L2 is 6. The compound of formula (I), the compound of formula (II), or a tautomer of any of the foregoing according to claim 1, characterized in that: It meets one or more of the following conditions: (1) The structure of the compound represented by formula (I) is as follows: (2) The structures of the tautomers of the compound represented by formula (I) are shown below: (3) The compound represented by formula (II) has any of the following structures: and (4) the structures of the tautomers of the compound represented by formula (II) are shown below:

7. A compound represented by formula (F) or its tautomer: in, A, B, C, D, X, Y, U, L, V are as defined in claim 1.

8. The compound as shown in formula (F) or its tautomer according to claim 7, characterized in that: The compound shown in formula (F) is the following compound: And / or, the structures of the tautomers of the compound represented by formula (F) are as follows:

9. The compound shown below: Each k1 and k2 is independently 0, 1 or 2; A, B, C, D, X, Y, U, V are as defined in claim 1.

10. A composition comprising substance A and substance B, wherein: Substance A is a compound of formula (I) as described in any one of claims 1 to 6, a compound of formula (II) or a tautomer of any of the foregoing; Substance B is the tag protein-recognition protein; The recognition protein is a protein that can bind to NADPH.

11. The composition according to claim 10, characterized in that It meets one or more of the following conditions: (1) The composition is used to detect NADPH; (2) The recognition protein is eDHFR protein; (3) The tag protein is Halo-tag, SNAP-tag or CLIP-tag, preferably Halo-tag; (4) The substance A is a compound represented by formula (I) or its tautomer; and (5) the composition consists of substance A and substance B.

12. Use of the compound of formula (I) according to any one of claims 1 to 6, the compound of formula (II) or a tautomer of any of the foregoing; the compound of formula (F) according to claim 7 or 8 or a tautomer thereof; or the composition according to claim 10 or 11 in detecting NADPH; Preferably, the application is used for staining, live cell fluorescence imaging or protein function regulation; Preferably, in the application, NADPH is detected by the following reaction:

Images