Preparation and application of sulfonamide fluorescent probes with permeability

Abstract

The application discloses preparation and application of a sulfonamide fluorescent probe with permeability, and specifically provides a compound as shown in formula (I) or a tautomer thereof. The fluorescent probe is simple in synthesis, and is used for wash-free imaging of microtubules and F-actin in living cells by regulating balance between a non-fluorescent, cell-permeable spirocyclic lactam form and a fluorescent zwitterionic state to enhance fluorescence and cell permeability. The method is applied to commercial tetramethyl rhodamine (TMR) and rhodamine 110 (R110), and is 100 times cheaper than previously used dyes.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of a permeable sulfonamide fluorescent probe. Background Technology

[0002] Fluorescent probes play a crucial role in cytoskeleton research. The cytoskeleton is a complex network of cytoskeletal proteins, including microfilaments, microtubules, and intermediate filaments, which play a vital role in various cellular processes, such as maintaining cell shape, cell division, endocytosis, cell movement, and signal transduction. Fluorescent probe technology allows scientists to observe and analyze the dynamics and structure of the cytoskeleton with high spatiotemporal resolution. First, fluorescently labeled proteins or compounds can bind to cytoskeletal proteins, making cytoskeleton components visible and enabling real-time tracking of dynamic processes such as cytoskeleton assembly, disassembly, movement, and morphological changes. Second, it allows for in-depth study of subcellular structures of the cytoskeleton, such as the distribution and organization of microfilament bundles, microtubule networks, and intermediate filaments, contributing to a better understanding of the function and cooperation of the cytoskeleton in different cellular regions. Fluorescent probes can also be used to track cell movement and migration, revealing the key roles of the cytoskeleton in cell movement, directional migration, and tissue development. They also provide a powerful tool for drug development, helping to develop drugs that affect the cytoskeleton, such as new therapies for cancer treatment and neurological diseases. Fluorescent probe technology provides an indispensable tool for cytoskeleton research.

[0003] While several cell-permeable fluorescent probes for cytoskeleton imaging have been reported, their complex synthesis and expensive parent dyes result in considerable costs (US$400-900 / 100 nmole). In previously reported rhodamine-based dyes for cytoskeleton imaging, an additional carboxylic acid is required at position 4, 5, or 6 to link the ligand; since isomers possess different imaging properties, isomer separation is typically necessary before imaging. When using expensive commercial dyes (e.g., ... 488 and At a cost of approximately $1000 per milligram (594), this problem becomes even more pronounced. On the other hand, many commercially available rhodamine dyes lack additional carboxylic acid functional groups (e.g., ATTO 550, ATTO 647N). Furthermore, many novel dyes with excellent photophysical properties, photostability, or large Stokes shifts often have complex chemical structures, making the introduction of additional carboxylic acid groups and the isolation of the resulting isomers extremely difficult and expensive. Therefore, there is an urgent need for simple and direct synthetic routes to convert affordable monocarboxylated rhodamine into novel probes with good cell permeability, fluorescence, and solubility. Summary of the Invention

[0004] To address the shortcomings of existing fluorescent probes, such as complex synthesis, expensive dyes, and difficult separation, this invention provides a permeable sulfonamide-based fluorescent probe. The fluorescent probe of this invention is simple to synthesize and enhances fluorescence and cell permeability by regulating the balance between a non-fluorescent, cell-permeable spironolactone form and a fluorescent zwitterionic state, enabling wash-free imaging of microtubules and F-actin in live cells. Furthermore, this method has been applied to commercially available tetramethylrhodamine (TMR) and rhodamine 110 (R110), and is 100 times cheaper than previously used dyes.

[0005] This invention provides a compound or a tautomer of formula (I):

[0006]

[0007] Where A and B are independently -NR A R B -OR A or -SR A ;

[0008] X represents O, S, or SiR. A R B CR A R B NR A Or Se;

[0009] Each R A and R B Each is independently H, C1-C6 alkyl, or C1-C6 alkyl substituted with one or more halogens;

[0010] R1, R2, R3, R4, R5 and R6 are each independently selected from H, halogen, C1-C6 alkyl, nitro, cyano or C1-C6 alkoxy;

[0011] U and V, along with the carbon atom in between, are linked to form a benzene ring or a 5-7 membered heterocycle.

[0012] W represents a single bond, -CH2-, -NH-, a benzene ring, or is bonded by one or more R groups. C Substituted benzene ring, 5-7 membered heterocycle, or ring with one or more R D Substituted 5-7 membered heterocycles;

[0013] Each R C and R D Each can be independently a hydroxyl, amino, carboxyl, sulfonic acid, or halogen group;

[0014] Linker has the following structure:

[0015] (where the amide bond is connected to L);

[0016] Z0 and Z1 are independently m0, m1, m2, m3, m4 and m5 are each independently selected from any integer from 0 to 15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15);

[0017] L is a ligand fragment that can bind to microtubules, F-actin, folate receptors, DNA, or dihydrofolate reductase.

[0018] In some implementations, A and B are the same.

[0019] In some implementations, A and B are independently -NR. A R B .

[0020] In some implementations, A and B are independently -NR. A R B Each R A and R B Each is independently H or C1-C6 alkyl.

[0021] In some implementations, X is 0.

[0022] In some embodiments, R1, R2, R3, R4, R5 and R6 are each independently selected from H or halogens, preferably H.

[0023] In some implementations, U and V, along with the carbon atom in between, are linked to form a benzene ring.

[0024] In some implementations, W represents a connection key.

[0025] In some implementations, the Linker has one of the structures shown in the following formula:

[0026]

[0027] Z is -O-; m and n are independent integers between 0 and 7;

[0028] Preferably, the Linker has the following structure:

[0029]

[0030] m is 2, 5, or 7 independently; n is 1 or 2;

[0031] More preferably, the Linker has the following structure:

[0032]

[0033] In some implementations, L is a ligand fragment that can bind to microtubules or F-actin, such as Jasplakinolide. or Cabazitaxel

[0034] In some implementations, L is a ligand fragment that can bind to the folic acid receptor, such as a folic acid molecule.

[0035] In some implementations, L is a ligand fragment that can bind to DNA, such as hochest.

[0036] In some implementations, L is a ligand fragment that can bind to dihydrofolate reductase, such as trimethoprim.

[0037] In some implementations, R A R B In R1, R2, R3, R4, R5 and R6, the “C1-C6 alkyl” and the “C1-C6 alkyl substituted with one or more halogens” are each independently C1-C4 alkyl, such as methyl or ethyl.

[0038] In some implementations, R A R B R C R D In R1, R2, R3, R4, R5 and R6, the “C1-C6 alkyl group substituted with one or more halogens” and the “halogen” in the “halogen” are independently F, Cl, Br or I, for example Br.

[0039] In some embodiments, in R1, R2, R3, R4, R5 and R6, the “C1-C6 alkoxy” is independently C1-C4 alkoxy, such as methoxy or ethoxy.

[0040] In some implementations, in U, V, and W, the "5-7 member heterocycle" and the "performed by one or more R" D The “5-7 membered heterocycle” in the phrase “substituted 5-7 membered heterocycle” refers to a 5-7 membered saturated or unsaturated heterocycle with 1, 2 or 3 heteroatoms selected from N, O and S, and having 1, 2 or 3 heteroatoms.

[0041] In some implementations, A and B are independently -NH2 or -N(CH3)2.

[0042] In some implementations, R2, R3, R5, and R6 are H.

[0043] In some embodiments, R1 and R4 are independently selected from H or Br, preferably H.

[0044] In some implementations, the Linker is n is 5 or 7;

[0045] n is 5 or 7;

[0046] n is 2;

[0047] or n is 5.

[0048] In some embodiments, the compound represented by formula (I) has any of the following structures:

[0049]

[0050]

[0051] In some embodiments, the tautomers of the compound shown in formula (I) have the following structures:

[0052]

[0053] The present invention also provides the following compounds:

[0054]

[0055]

[0056] The present invention also provides the use of the compounds shown in formula (I) or their tautomers in the detection of tubulin or F-actin; the use can be used in staining, live-cell fluorescence imaging and protein function regulation.

[0057] In some embodiments, in the applications described, the compound of formula (I) or its tautomers detects tubulin or F-actin via the following reaction:

[0058]

[0059] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0060] The reagents and raw materials used in this invention are all commercially available.

[0061] The positive and progressive effects of this invention are as follows: the fluorescent probe of this invention is simple to synthesize, and its fluorescence and cell permeability are enhanced by regulating the balance between the non-fluorescent, cell-permeable spironolactone form and the fluorescent zwitterionic state, which can be used for wash-free imaging of microtubules and F-actin in live cells; and this method has been applied to commercial tetramethylrhodamine (TMR) and rhodamine 110 (R110), which is 100 times cheaper than the dyes used previously. Attached Figure Description

[0062] Figure 1 This is a TMR derivative-type actin probe used for live-cell actin protein imaging.

[0063] Figure 2 A TMR derivative-type tubulin probe for live-cell tubulin protein imaging.

[0064] Figure 3 Tubulin and actin probes, which are TMR derivatives, are used for live cell scaffold imaging. Detailed Implementation

[0065] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0066] Example 1: The preparation routes of TMR-SC9-actin and TMR-SC11-tubulin are shown below:

[0067]

[0068] The specific synthesis steps are as follows:

[0069] 2-(4-(dimethylamino)-2-hydroxybenzoyl)benzoic acid (1)

[0070]

[0071] 3-Dimethylaminophenol (1.93 g, 14.1 mmol, 1.0 eq) and phthalic anhydride (2.19 g, 14.8 mmol, 1.05 eq) were dissolved in 15 mL of toluene, refluxed under argon for 3 hours, and cooled to 50–60 °C. 15 mL of 35% NaOH aqueous solution was added, and the mixture was heated to 90 °C and stirred at this temperature for 6 hours. The resulting reaction mixture was poured into water, the pH was adjusted to 1–2 with dilute hydrochloric acid, and the mixture was allowed to stand at room temperature for 2 hours. The suspension was filtered to give brown solid 1 (1.9 g, 6.66 mmol, 47%), which was used directly in the next step.

[0072] Tetramethylrhodamine (2)

[0073]

[0074] Compound 1 (1.9 g, 6.66 mmol, 1 eq) was dissolved in 30 mL of methanesulfonic acid, and 3-dimethylaminophenol (914 mg, 6.66 mmol, 1 eq) was added. The reaction mixture was stirred for 24 hours under an argon atmosphere at 90 °C. After cooling to room temperature, 5 drops of perchloric acid were added to precipitate the product. The precipitate was dissolved in MeOH, dried over MgSO4, filtered, concentrated, and purified by silica gel chromatography (DCM: MeOH 0-20%) to give a deep red solid 2 (1.49 g, 3.86 mmol, 58%). 1 H NMR (400MHz, CDCl3) δ = 8.23 ​​(d, J = 7.7Hz, 1H), 7.87 (td, J = 7.5, 1.2Hz, 1H), 7.81 (td, J = 7.6, 1 .1Hz,1H),7.47(dd,J=7.5,1.4Hz,1H),7.09-7.01(m,4H),7.02-6.94(m,2H),2.55(s,12H). 13 CNMR(101MHz, methanol-d4)δ=159.3,159.1,158.7,134.9,132.7,131.7,131.5,131.1,130.1,115.0,106.3,105.6,101.5,97.5,41.0.HRMS(ESI + )m / z calcd387.1709[C 24 H 23 N2O3] + (M+H + ),387.1702found.

[0075] 3-((N-((benzyloxy)carbonyl)amino)tert-butyl propionate (3)

[0076]

[0077] Chlorosulfonyl isocyanate (615 μL, 7.7 mmol, 1.05 eq) was dissolved in 20 mL of DCM solution. Benzyl alcohol (697 μL, 6.73 mmol, 1 eq) was added under argon atmosphere at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. Pyridine (1.09 mL, 13.5 mmol, 2.0 eq) was added, and the mixture was stirred at 0 °C for another hour. After adding 4-amino-1,1-dimethyl ethyl butyrate hydrochloride (2.0 g, 10.1 mmol, 1.5 eq) and DIPEA (2.22 mL, 13.5 mmol, 2.0 eq), the reaction mixture was heated to room temperature and stirred for 3 h. The pH of the reaction mixture was adjusted to 1 using dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were washed with 1 N HCl and saturated brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (1-80% ethyl acetate-hexane solution) to give a pale yellow oil 3 (1.5 g, 4.17 mmol, 62%). 1 H NMR (400MHz, CDCl3) δ = 7.39-7.34 (m, 5H), 5.77 (t, J = 6.4Hz, 1H), 5.20 (s, 2H), 3.30 (q, J = 6.2Hz, 2H), 2.51 (t, J = 6.1Hz, 2H), 1.61 (s, 1H), 1.45 (s, 9H). 13 C NMR (101MHz, CDCl3) δ = 171.1, 151.3, 134.8, 128.7, 82.0, 68.8, 39.8, 34.9, 28.3. HRMS (ESI + )m / z calcd 359.1277[C 15 H 23 N2O6S] + (M+H + ),359.1271found.

[0078] 3-(aminosulfonamide)propionate tert-butyl ester (4)

[0079]

[0080] Intermediate 3 (1.5 g, 4.15 mmol, 1.0 eq) was dissolved in 20 mL of a 1:1 mixture of THF and EtOH, and activated palladium on carbon (1.5 mg, 1.40 mmol, 0.3 eq) was added. The reaction mixture was stirred under a hydrogen atmosphere for 12 hours and monitored by LC-MS. After the reaction was complete, the catalyst was removed by diatomaceous earth filtration, and the filtrate was concentrated under reduced pressure to give a pale yellow oily intermediate 4 (0.85 g, 3.82 mmol, 92%). 1H NMR (400MHz, CDCl3) δ = 4.60 (s, 2H), 3.35 (t, J = 5.9Hz, 2H), 2.56 (t, J = 5.9Hz, 2H), 1.46 (s, 9H). 13 C NMR (101MHz, CDCl3) δ = 171.9, 81.9, 39.5, 35.0, 28.3.

[0081] 4-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanthon])-2-sulfonamido)propionic acid (5)

[0082]

[0083] Dissolve 2 (50 mg, 129 μmol, 1.0 eq.) in 30 mL of anhydrous DCM. Add 3 (87 mg, 386 μmol, 3.0 eq.), DMAP (126 mg, 1.03 mmol, 8.0 eq.), and EDC (197 mg, 1.03 mmol, 8.0 eq.) to the reaction mixture and reflux at 60 °C for 3 h (monitored by LC-MS). After the reaction is complete, remove the solvent and dissolve the residue in a 3:1 DCM:TFA mixture and stir for 20 min (monitored by LC-MS; add more TFA if necessary). After evaporating the solvent, purify the crude product by preparative chromatography. Obtain a deep red solid 5 (22 mg, 46.0 μmol, 36%). 1 H NMR (400MHz, Methanol-d4) δ = 7.99 (dd, J = 7.6, 1.5Hz, 1H), 7.91–7.69 (m, 2H), 7.51–7.23 (m, 1H), 7. 19–6.44(m,6H),3.37(s,1H),3.25(s,11H),2.78(t,J=7.2Hz,2H),2.28(t,J=7.3Hz,2H).HRMS(ESI + )m / zcalcd 537.1802[C 27 H 29 N4O6S] + (M+H + ),537.1797found.

[0084] 8-(3-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanton])-2-sulfonamido)propamido)octanoic acid (6)

[0085]

[0086] 5 (5.0 mg, 9.30 μmol, 1.0 eq.), 8-aminooctanoic acid (2.23 mg, 14 μmol, 1.5 eq.), PyBOP (5.3 mg, 10.3 μmol, 1.1 eq.), and DIPEA (12.3 μl, 74.5 μmol, 8 eq.) were dissolved in 1 mL of dry DMF and stirred at room temperature for 30 min. After evaporating the solvent under reduced pressure, the crude product was purified by preparative chromatography to obtain a deep red solid 6 (1.4 mg, 2.00 μmol, 22%). 1 H NMR(400MHz, methanol-d4)δ=7.93 -7.88(m,1H),7.77-7.65(m,2H),7.42-7.25(m,1H),7.15-6.65(m,6H),3.24(s,12H),3.04(t,J=7.0Hz,2H),2.73(t,J=7. 0Hz,2H),2.20(t,J=7.4Hz,2H),2.11(t,J=7.0Hz,2H),1.56-1.48(m,2H),1.43-1.45(m,2H),1.30-1.20(m,6H).HRMS(ESI + )m / z calcd 678.2961[C35H44N5O7S] + (M+H) + ,678.2956found.

[0087] 5-(3-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanton])-2-sulfonamido)propamido)hexanoic acid (7)

[0088]

[0089] The synthesis method was the same as that for compound 6. Compound 5 (5.0 mg, 9.32 μmol, 1.0 eq), 6-aminohexanoic acid (1.8 mg, 13.98 μmol, 1.5 eq), PyBOP (5.3 mg, 10.25 μmol, 1.1 eq.), and DIPEA (12.3 μl, 74.5 μmol, 8 eq.) were reacted to give a deep red solid 7 (4.5 mg, 6.90 μmol, 74%). 1H NMR (400MHz, CDCl3) δ = 8.03-7.98 (m, 1H), 7.74 (p, J = 7.3Hz, 2H), 7.52-7.43 ( m,1H),7.21-7.12(m,2H),7.09-7.01(m,2H),6.93(s,2H),3.30-3.28(m,12H) ,3.14-3.08(m,2H),2.81(t,J=7.0Hz,2H),2.19(t,J=7.4Hz,2H),2.11(t,J=7 .0Hz,2H),1.64-1.46(m,2H),1.52-1.44(m,2H),1.38-1.29(m,2H).HRMS(ESI + )m / z calcd 650.2648[C33H40N5O7S] + (M+H) + ,783.2652found.

[0090] TMR-SC11-tubulin

[0091]

[0092] BOC-cabazitaxel (3.5 mg, 4.13 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was evaporated to dryness, and the solution was dissolved together with 6 (1.4 mg, 2.07 μmol, 1.0 eq.), PyBOP (3.2 mg, 6.20 μmol, 3.0 eq.), and DIPEA (6.83 μL / μmol, 20.0 eq.) in 2 mL of anhydrous DMF. The solution was stirred at room temperature for 1 h and the solvent was evaporated to dryness. The red solid TMR-SC11-tubulin (1.2 mg, 1.65 μmol, 40%) was obtained by preparative chromatography. HRMS (ESI) + )m / z calcd698.3095[C 75 H 91 N6O 18 S] 2+ (M+H) 2+ ,698.3089found.

[0093] TMR-SC9-actin

[0094]

[0095] BOC-jasplakinolide (2.9 mg, 9.23 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was evaporated to dryness, and the solution was dissolved together with 7 (1.4 mg, 4.62 μmol, 1.0 eq.), PyBOP (1.7 mg, 6.93 μmol, 1.5 eq.), and DIPEA (7.15 μL 18.5 μmol 20.0 eq.) in 2 mL of anhydrous DMF. The solution was stirred at room temperature for 1 h and the solvent was evaporated to dryness. A red solid (2.5 mg, 1.94 μmol, 42%) was obtained by preparative chromatography. 1 H NMR (400MHz, Methanol-d4) δ = 8.30 (d, J = 8.5Hz, 1H), 7.99 (dd, J = 7.8, 1.4Hz, 1H), 7.82 (t, J = 9.0Hz, 2H),7.59(d,J=7.9Hz,2H),7.44(s,1H),7.32(d,J=8.1Hz,1H),7.19–7.13(m,1H),7.09–7.04(m,3H ),7.02–6.96(m,3H),6.85(d,J=20.5Hz,2H),6.74–6.69(m,2H),5.64(dd,J=10.4,6.1Hz,1H),5.25 (td,J=9.1,3.8Hz,1H),5.04(t,J=7.0Hz,1H),4.83(d,J=7.6Hz,2H),4.76(q,J=7.0Hz,1H),3.31–3. 19(m,12H),3.18–3.10(m,5H),3.09(s,3H),2.96(dp,J=19.8,6.7Hz,3H),2.84(dd,J=6.9,3.5Hz,2 H),2.80–2.56(m,4H),2.32–2.26(m,1H),2.21(t,J=6.9Hz,2H),2.16(t,J=7.3Hz,2H),1.90(q,J=7. 3Hz,3H),1.64–1.57(m,3H),1.54(s,3H),1.49(t,J=7.4Hz,2H),1.44–1.37(m,1H),1.36–1.23(m,3 H),1.18(d,J=6.3Hz,5H),1.09(d,J=6.8Hz,3H),1.05–0.84(m,4H),0.80(d,J=8.6Hz,1H).HRMS(ESI + )m / z calcd 653.3230[C 71 H 90 N 10 O 12 S] 2+(M+H) 2+ ,653.3225found.

[0096] Example 2: The preparation routes of TMR-SC6-actin and TMR-SC8-tubulin are shown below:

[0097]

[0098] tert-butyl 6-aminohexanoate (8)

[0099]

[0100] Compound 6-aminohexanoic acid (200 mg, 1.52 mmol, 1.0 eq.) was dissolved in thionyl chloride (2.0 mL, 27.6 mmol, 20.0 eq.) and stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure, and tert-butanol (5.0 mL, 52.6 mmol, 35 eq.) and NaHCO3 (281.8 mg, 3.35 mmol, 2.0 eq.) were added. The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was dissolved in 1 M NaOH aqueous solution and ethyl acetate. The organic layer was washed with water (1 × 20 mL) and brine (3 × 10 mL), dried over MgSO4, filtered, concentrated, and purified by silica gel chromatography (pentane: ethyl acetate 0-80%) to give compound 8 (190 mg, 1.02 mmol, 67%) as a colorless oil. 1 H NMR (400MHz, methanol-d4) δ = 2.65-2.61 (m, 2H), 2.17 (t, J = 7.4Hz, 2H), 1.51 (p, J = 7.4Hz, 2H), 1.47-1.40 (m, 2H), 1.36 (s, 9H), 1.34-1.26 (m, 2H). 13 C NMR (101MHz, methanol-d4)δ=175.1,81.5,42.0,36.4,32.2,28.5,27.4,26.0.HRMS (ESI + )m / z calcd 188.1651[C 10 H 22 NO2] + (M+H) + ,188.1645found.

[0101] 8-Aminooctanoic acid tert-butyl ester (9)

[0102]

[0103] Intermediate 9 was synthesized in the same manner as intermediate 8, yielding a colorless oily compound 9 (201 mg, 933 μmol, 99%). 1 HNMR(400MHz, methanol-d4)δ=3.31-3.30(m,2H),2.66-2.60(m,2H),2.61(t,J=7 .4Hz,2H),1.61-1.54(m,2H),1.51-1.45(m,2H),1.44(s,9H),1.36-1.31(m,6H). 13 C NMR (101MHz, methanol-d4) δ = 175.3, 81.5, 42.6, 36.6, 33.7, 30.4, 30.2, 28.6, 28.0, 26.3. HRMS (ESI + )m / z calcd 216.1964[C 12 H 26 NO2] + (M+H) + ,216.1958found.

[0104] 6-((N-((benzyloxy)carbonyl)amino)tert-butyl hexanoate (10)

[0105]

[0106] Intermediate 10 was synthesized in the same way as intermediate 3, with intermediate 8 (175 mg, 933 μmol, 1.5 eq.) participating in the reaction, finally yielding yellow oily intermediate 10 (215 mg, 802 μmol, 86%). 1 H NMR (400MHz, CDCl3) δ = 7.41 (s, 1H) 7.38 -7.33(m,5H),5.19(s,2H),3.04(q,J=6.9Hz,2H),2.20(t,J=7.4Hz,2H),1.59-1.50(m,4H),1.44(s,9H),1.37-1.29(m,2H). 13 C NMR (101MHz, CDCl3) δ = 173.1, 151.4, 134.8, 129.1, 129.0, 128.7, 80.5, 68.8, 43.9, 35.4, 29.0, 28.3, 26.1, 24.6.

[0107] 8-((N-((benzyloxy)carbonyl)amino)octanoate tert-butyl ester (11)

[0108]

[0109] Intermediate 11 was synthesized using the same method as intermediate 3, with intermediate 9 (190 mg, 883 μmol, 1.5 eq.) participating in the reaction, ultimately yielding a yellow oily intermediate 11 (205 mg, 724 μmol, 82%). 1 H NMR (400MHz, CDCl3) δ = 7.56 (s, 1H) 7.40 -7.32(m,5H),5.19(s,2H),3.02(q,J=6.9Hz,2H),2.19(t,J=7.5Hz,2H),1.60-1.48(m,4H),1.34-1.24(m,6H). 13 C NMR (101MHz, CDCl3) δ = 173.5, 151.5, 134.8, 129.1, 129.0, 128.7, 80.3, 68.8, 44.1, 35.7, 29.1, 29.0, 28.9, 28.3, 26.5, 25.1.

[0110] 6-(aminosulfonamide)hexanoate tert-butyl ester (12)

[0111]

[0112] Intermediate 12 was synthesized in the same way as intermediate 4, with intermediate 10 (215 mg, 537 μmol, 1.0 eq.) participating in the reaction, ultimately yielding colorless oily intermediate 12 (140 mg, 526 μmol, 98%). 1 H NMR (400MHz, CDCl3) δ = 3.11 (t, J = 7.0Hz, 2H), 2.20 (t, J = 7.3Hz, 2H), 1.61-1.53 ​​(m, 4H), 1.41 (s, 9H), 1.39-1.32 (m, 2H). 13 CNMR(101MHz, CDCl3)δ=173.4,80.5,43.4,35.4,29.2,28.3,26.1,24.5.

[0113] 8-(aminosulfonamide)octanoate tert-butyl ester (13)

[0114]

[0115] Intermediate 13 was synthesized in the same way as intermediate 4, with intermediate 11 (185 mg, 432 μmol, 1.0 eq.) participating in the reaction, ultimately yielding colorless oily intermediate 13 (121 mg, 410 μmol, 95%). 1H NMR (400MHz, CDCl3) δ = 3.10 (t, J = 7.1Hz, 2H), 2.18 (t, J = 7.4Hz, 2H), 1.59-1.52 (m, 4H), 1.42 (s, 9H), 1.35-1.26 (m, 6H). 13 CNMR(101MHz, CDCl3)δ=173.5,80.3,43.8,35.7,29.5,29.0,28.9,28.3,26.6,25.1.

[0116] 6-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanthon])-2-sulfonamido)hexanoic acid (14)

[0117]

[0118] Intermediate 14 was synthesized in the same manner as intermediate 5, with intermediates 2 (15 mg, 38.6 μmol, 1.0 eq.) and 12 (31 mg, 116 μmol, 3.0 eq.) participating in the reaction, ultimately yielding red solid 14 (6.0 mg, 10.4 μmol, 27%). 1 H NMR(400MHz, methanol-d4)δ=7.7-7.94(m,1H),7.83-7.73(m,2H),7.48-7.31(m,1H),7.23-6.63(m,6H),3.30(s,12 H),2.47(t,J=7.0Hz,2H),2.23(t,J=7.4Hz,2H),1.52-1.43(m,2H),1.30-1.23(m,2H),1.12-1.04(m,2H).HRMS(ESI + )m / z calcd 579.2277[C 30 H 35 N4O6S] + (M+H) + ,579.2272found.

[0119] 8-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanton])-2-sulfonamido)octanoic acid (15)

[0120]

[0121] Intermediate 15 was synthesized in the same manner as intermediate 5, with intermediates 2 (15 mg, 38.6 μmol, 1.0 eq.) and 13 (34.1 mg, 116 μmol, 3.0 eq.) participating in the reaction, ultimately yielding red solid 15 (8.0 mg, 13.1 μmol, 34%). 1 HNMR(400MHz, methanol-d4)δ=8.98-8.93(m,1H),8.85-8.74(m,2H),8.40(s,1H),8.25-7.81(m,6H),4.19(s,12H) ,3.49(t,J=7.0Hz,2H),3.27(t,J=3.20Hz,2H),2.59-2.52(m,2H),2.32-2.17(m,6H),2.15-2.07(m,2H).HRMS(ESI + )m / z calcd 607.2590[C 32 H 39 N4O6S] + (M+H) + ,607.2585found.

[0122] TMR-SC6-actin

[0123]

[0124] BOC-jasplakinolide (7.0 mg, 10.4 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was evaporated to dryness, and the solution was dissolved together with 14 (3.0 mg, 5.18 μmol, 1.0 eq.), PyBOP (8.0 mg, 15.54 μmol, 3.0 eq.), and DIPEA (18.11 μL, 103.6 μmol, 20.0 eq.) in 2 mL of anhydrous DMF. The solution was stirred at room temperature for 1 h and the solvent was evaporated to dryness. The red solid TMR-SC6-actin (2.88 mg, 4.68 μmol, 45%) was obtained by preparative chromatography. 1H NMR(400MHz,Methanol-d4)δ8.21(d,J=8.5Hz,1H),7.95–7.92(m,1H),7.80(p,J=7.3Hz,2H),7.57(dd,J=7.9,1.1Hz,1H),7.42(s,1H),7.30(dt,J=8.1,0.9Hz,1H),7.13(s,1H),7.09–7.01(m,4H),7.00–6.95(m,2H),6.93(s,1H),6.89(s,1H),6.78(d,J=2.4Hz,1H),6.71–6.67(m,2H),5.62(dd,J=10.6,5.9Hz,1H),5.21(td,J=9.2,3.7Hz,1H),5.02(t,J=6.9Hz,1H),4.80(ddd,J=7.8,4.5,3.1Hz,1H),4.73(dd,J=7.0,4.4Hz,1H),3.22(d,J=21.1Hz,13H),3.16–3.07(m,2H),3.05(s,4H),2.97(t,J=7.0Hz,2H),2.76–2.66(m,2H),2.66–2.47(m,4H),2.27(dd,J=14.2,11.7Hz,1H),2.11(t,J=7.2Hz,2H),1.92–1.86(m,3H),1.65–1.45(m,7H),1.43–1.36(m,1H),1.33–1.23(m,3H),1.15(d,J=6.3Hz,8H),1.07(d,J=6.8Hz,4H),1.04–0.81(m,3H),0.70(d,J=10.4Hz,1H).HRMS(ESI + )m / z calcd617.8044[C 68 H 85 N9O 11 S] 2+ (M+H) 2+ ,617.8039found.

[0125] TMR-SC8-tubulin

[0126]

[0127] BOC-cabazitaxel (3.5 mg, 4.13 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was evaporated to dryness, and the solution was then dissolved together with 15 (1.4 mg, 2.07 μmol, 1.0 eq.), PyBOP (3.2 mg, 6.20 μmol, 3.0 eq.), and DIPEA (6.83 μL, 41.3 μmol, 20.0 eq.) in 2 mL of anhydrous DMF. The solution was stirred at room temperature for 1 h and the solvent was evaporated to dryness. The red solid TMR-SC8-tubulin (1.2 mg, 1.65 μmol, 40%) was obtained by preparative chromatography. HRMS (ESI) + )m / z calcd698.3095[C 75 H 91 N6O 18 S] 2+ (M+H) 2+ ,698.3089found.

[0128] Example 3: The preparation routes of TMR-SPEG2-actin, TMR-SPEG2-tubulin, TMR-SPEG5-actin and TMR-SPEG5-tubulin are shown below:

[0129]

[0130] 1-(9H-fluorene-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecane-12-oic acid tert-butyl ester (16)

[0131]

[0132] Intermediate 16 was synthesized in the same manner as intermediate 8, with the compound Fmoc-8-amino-3,6-dioxooctanoic acid (300 mg, 778 μmol, 1.0 eq.) participating in the reaction, ultimately yielding a yellow oily intermediate 16 (270 mg, 615 μmol, 79%). 1 H NMR (400MHz, CDCl3) δ = 7.74 (d, J = 7.5Hz, 2H), 7.59 (d, J = 7.4Hz, 2H), 7.38 (t, J = 7.4Hz, 2H), 7.32-7.27 (m, 2H), 5.42 (s, 1H), 4.36 ( d,J=7.0Hz,2H),4.24-4.17(m,1H),4.00(s,2H),3.72-3.62(m,4H),3.60-3.55(m,2H),3.43-3.36(m,2H),1.46(s,9H).HRMS(ESI +)m / zcalcd 464.2044[C 25 H 31 [NNaO6] + (M+Na) + ,464.2039found.

[0133] 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19-hexaoxa-4-azaeicosane-22-tert-butyl agar (17)

[0134]

[0135] Intermediate 17 was synthesized in the same manner as intermediate 8, with the compound Fmoc-1-amino-3,6,9,12,15-pentaoctadecane-18-oleic acid (300 mg, 564 μmol, 1.0 eq.) participating in the reaction, ultimately yielding a yellow oily intermediate 17 (270 mg, 462 μmol, 82%). 1 H NMR (400MHz, Chloroform) δ = 7.74 (d, J = 7.5Hz, 2H), 7.58 (d, J = 7.4Hz, 2H), 7.3 8(t,J=7.4Hz,2H),7.32-7.26(m,2H),5.42(s,1H),5.51-5.35(m,1H),4.38(d, J=7.0Hz,2H),4.20(t,J=6.9Hz,1H),3.67(t,J=6.6Hz,2H),3.63-3.54(m,17H) ,3.39-3.34(m,2H),2.47(t,J=6.6Hz,2H),1.80(s,1H),1.42(s,9H).HRMS(ESI + )m / z calcd532.2547[C 28 H 38 NO6] + (M+H) + ,532.2538found.

[0136] 2-(2-(2-((N-((benzyloxy)carbonyl)aminosulfonyl)amino)ethoxy)ethoxy)tert-butyl acetate (18)

[0137]

[0138] The synthesis method of intermediate 18 is the same as that of intermediate 3, wherein intermediate 16 (77 mg, 174 μmol, 1.5 eq.) is dissolved in DCM / DBU (95:5, 100 μL), stirred at room temperature for 5 minutes, the solvent is evaporated under reduced pressure and used directly in subsequent reactions to obtain a yellow viscous oil 18 (33 mg, 76.7 μmol, 66%). 1 H NMR (400MHz, CDCl3) δ=7.36-7.29(m,5H),5.75(s,1H),5.16(s,2H),3.94(s,2H),3.63-3.56(m,6H),3.34-3.29(m,2H),1.45(s,9H). 13 C NMR (101MHz, CDCl3) δ = 169.9, 151.8, 135.1, 128.9, 128.9, 128.6, 82.3, 70.9, 70.2, 69.7, 69.0, 68.5, 44.0, 28.3.

[0139] 1-((N-((benzyloxy)carbonyl)amino)-3,6,9,12,15-pentoxane-octadecane-18-oic acid tert-butyl ester (19)

[0140]

[0141] Intermediate 19 was synthesized in the same way as intermediate 3, wherein intermediate 17 (45 mg, 76.6 μmol, 1.1 eq.) was dissolved in DCM / DBU (95:5, 100 μL), stirred at room temperature for 5 minutes, the solvent was evaporated under reduced pressure and used directly in subsequent reactions to obtain a yellow viscous oil 19 (40 mg, 69.0 μmol, 99%). 1 H NMR (400MHz, CDCl3) δ = 7.7-7.30 (m, 5H), 5.25 (s, 2H), 3.66 (t, J = 6.6Hz, 2H) ,3.63-3.52(m,16H),3.34-3.29(m,2H),2.46(t,J=6.6Hz,2H),1.41(s,9H). 13 C NMR(101MHz, CDCl3)δ=177.3,171.2,135.3,128.8,128.8,128.7,128.6,80.8 ,70.7,70.7,70.7,70.6,70.6,70.5,70.2,69.7,68.2,67.0,44.0,36.4,28.3.

[0142] 2-(2-(2-(aminosulfonamide)ethoxy)ethoxy)tert-butyl acetate (20)

[0143]

[0144] Intermediate 20 was synthesized in the same manner as intermediate 4, with compound 18 (33 mg, 76.3 μmol, 1.0 eq.) participating in the reaction, ultimately yielding a yellow oily product, 20 (23 mg, 76.1 μmol, 100%). 1 H NMR (400MHz, CDCl3) δ=5.14-5.08(m,1H),4.97(s,2H),3.98(s,2H),3.69-3.64(m,6H),3.37-3.31(m,2H),1.45(s,9H). 13 C NMR (101MHz, CDCl3) δ = 169.9, 82.4, 70.8, 70.1, 68.9, 43.8.30.5, 28.3.

[0145] 1-(aminosulfonamide)-3,6,9,12,15-pentaoctadecane-18-oic acid tert-butyl ester (21)

[0146]

[0147] Intermediate 21 was synthesized in the same manner as intermediate 4, with compound 19 (40 mg, 69.1 μmol, 1.0 eq.) participating in the reaction, ultimately yielding a yellow oily product 21 (23 mg, 76.1 μmol, 100%). 1 H NMR (400MHz, CDCl3) δ = 5.45 (s, 2H), 5.25-5.19 (m, 1H), 3.70 (t, J = 6.5Hz, 2H), 3.67-3. 58(m,14H),3.36-3.31(m,2H),2.51(t,J=6.5Hz,2H),1.72-1.60(m,4H),1,43(s,9H). 13 C NMR (101MHz, CDCl3) δ = 171.3, 125.7, 80.9, 70.6, 70.52, 70.48, 70.47, 70.4, 70.0, 69.6, 67.0, 43.7, 36.34, 30.5, 28.3.

[0148] 2-(2-(2-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanton])-2-sulfonamide)ethoxy)ethoxy)tert-butyl acetate (22)

[0149]

[0150] Intermediate 22 was synthesized using the same method as intermediate 5, with intermediate 2 (6.5 mg, 16.8 μmol, 1.0 eq.) and intermediate 20 (10.0 mg, 33.5 μmol, 2 eq.) participating in the reaction, ultimately yielding red solid 22 (3.1 mg, 4.69 μmol, 28%). HRMS (ESI) + )m / z calcd 667.2802[C 34 H 43 N4O8S] + (M+H) + ,667.2796found.

[0151] 1-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-xanton])-2-sulfonamido)-3,6,9,12,15-pentaoctadecane-18-olate tert-butyl ester (23)

[0152]

[0153] Intermediate 23 was synthesized using the same method as intermediate 5, with intermediates 2 (4.4 mg, 11.3 μmol, 1.0 eq.) and 21 (10.0 mg, 22.5 μmol, 2.0 eq.) participating in the reaction, ultimately yielding red solid 23 (5.0 mg, 6.15 μmol, 55%). HRMS (ESI) + )m / z calcd 813.3745[C 41 H 57 N4O 11 S] + (M+H) + ,813.3739found.

[0154] TMR-SPEG2-actin

[0155]

[0156] Intermediate 22 (2.0 mg, 3.00 μmol, 1.0 eq.) was dissolved in DCM / TFA (3:1, 100 μL) to remove the tert-butyl group. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated under reduced pressure. The solution was then ready for use. BOC-jasplakinolide (4.0 mg, 6.00 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated. The products obtained from the removal of the tert-butyl group from intermediate 22, PyBOP (3.1 mg, 6.00 μmol, 2.0 eq.) and DIPEA (4.96 μL 30.0 μmol, 10.0 eq.), were dissolved in 2 mL of anhydrous DMF. The mixture was stirred at room temperature for 1 h, and the solvent was evaporated. Preparative chromatography yielded a red solid, TMR-SPEG2-actin (2.5 mg, 1.97 μmol, 67%). 1 H NMR(400MHz, Methanol-d4)δ8.25(d,J=8.5Hz,1H),8.01–7.96(m,1H),7.79(s,2H),7 .57(d,J=7.9Hz,1H),7.31–7.28(m,1H),7.08–7.04(m,3H),6.99–6.94(m,2H),6.73– 6.70(m,2H),5.62(t,J=8.2Hz,1H),5.24(ddt,J=9.3,7.0,3.6Hz,1H),5.04(t,J=7.0 Hz,1H),4.85–4.79(m,2H),4.75(dd,J=7.0,4.5Hz,1H),3.89(s,2H),3.59–3.53(m,2H ),3.46(dd,J=5.9,2.9Hz,2H),3.41–3.34(m,3H),3.31–3.08(m,13H),3.07(s,3H),3 .04–2.68(m,9H),2.67–2.54(m,2H),2.29(dd,J=14.3,11.6Hz,2H),2.05(s,1H),1.9 0(q,J=5.7Hz,4H),1.60(dd,J=13.9,7.2Hz,1H),1.56–1.51(m,3H),1.48–1.33(m,2H ),1.12(dd,J=39.9,6.6Hz,10H),1.03–0.84(m,3H),0.73(d,J=28.9Hz,2H).HRMS(ESI + )m / z calcd 633.7994[C 68 H 85 N9O 13 S] 2+ (M+H) 2+,633.7988found.

[0157] TMR-SPEG2-tubulin

[0158]

[0159] Intermediate 22 (7.0 mg, 10.5 μmol, 1.0 eq.) was dissolved in DCM / TFA (3:1, 100 μL) to remove the tert-butyl group. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated under reduced pressure. The solution was then ready for use. BOC-cabazitaxel (8.8 mg, 10.5 μmol, 1.0 eq.) was dissolved in 1 mL of formic acid. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated. The products obtained from the removal of the tert-butyl group from intermediate 22, PyBOP (16 mg, 31.5 μmol, 3.0 eq.) and DIPEA (34.7 μL, 210 μmol, 20.0 eq.), were dissolved in 2 mL of anhydrous DMF. The mixture was stirred at room temperature for 1 h, and the solvent was evaporated. Preparative chromatography yielded a red solid, TMR-SPEG2-tubulin (7.4 mg, 5.57 μmol, 53%). 1H NMR(400MHz,Methanol-d4)δ8.12(d,J=1.2Hz,1H),8.10(d,J=1.5Hz,1H),7.95–7.92(m,1H),7.86–7.77(m,2H),7.69–7.65(m,1H),7.57(dd,J=8.3,7.0Hz,2H),7.49–7.35(m,6H),7.32–7.28(m,1H),7.15(s,2H),7.00(s,2H),6.93(s,2H),6.18(td,J=9.2,1.8Hz,1H),5.61(d,J=7.1Hz,1H),5.50(d,J=4.2Hz,1H),5.01(dd,J=9.7,2.0Hz,1H),4.64(d,J=4.2Hz,1H),4.18(s,2H),3.99(d,J=5.5Hz,2H),3.90(dd,J=10.7,6.5Hz,1H),3.81(d,J=7.1Hz,1H),3.68–3.58(m,2H),3.54–3.51(m,2H),3.41(s,3H),3.36(d,J=5.6Hz,2H),3.28(d,J=11.6Hz,17H),3.18(td,J=6.6,3.7Hz,1H),2.82–2.72(m,3H),2.33(s,3H),2.29–2.23(m,1H),2.12–2.06(m,1H),1.93(d,J=1.5Hz,3H),1.89–1.85(m,1H),1.68(s,4H),1.15(s,3H),1.12(s,3H).HRMS(ESI + )m / z calcd1328.5325[C 70 H 82 N5O 19 S] + (M+H) + ,1328.5319found.

[0160] TMR-SPEG5-actin

[0161]

[0162] Intermediate 23 (2.3 mg, 2.83 μmol, 1.0 eq.) was dissolved in DCM / TFA (3:1, 100 μL) to remove the tert-butyl group. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated under reduced pressure. The solution was then ready for use. BOC-jasplakinolide (3.8 mg, 5.66 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated. The products obtained from the removal of the tert-butyl group from intermediate 23, PyBOP (3.0 mg, 5.66 μmol, 2.0 eq.) and DIPEA (4.96 μL, 28.3 μmol, 10.0 eq.), were dissolved in 2 mL of anhydrous DMF. The mixture was stirred at room temperature for 1 h, and the solvent was evaporated. Preparative chromatography yielded a red solid, TMR-SPEG5-actin (2.5 mg, 1.77 μmol, 63%). 1H NMR(400MHz,Methanol-d4)δ8.31(d,J=8.5Hz,1H),8.02–7.97(m,1H),7.83(d,J=8.0Hz,2H),7.58(d,J=7.9Hz,1H),7.46(s,1H),7.33(d,J=8.1Hz,1H),7.18(s,2H),7.10–7.06(m,3H),7.00(d,J=3.5Hz,2H),6.99–6.96(m,1H),6.92(s,1H),6.86(s,1H),6.72(d,J=8.6Hz,2H),5.64(dd,J=10.6,5.9Hz,1H),5.28–5.22(m,1H),5.04(t,J=6.9Hz,1H),4.83(d,J=7.7Hz,2H),4.75(dd,J=6.9,4.5Hz,1H),3.73(t,J=6.1Hz,2H),3.66–3.53(m,12H),3.53–3.49(m,2H),3.45(dd,J=5.8,3.1Hz,2H),3.37(s,1H),3.32–3.22(m,12H),3.20–3.10(m,3H),3.09(s,3H),3.05–2.87(m,3H),2.74(ddd,J=19.9,10.3,5.0Hz,4H),2.68–2.57(m,2H),2.43(t,J=6.1Hz,2H),2.29(dd,J=14.2,11.8Hz,2H),1.91(q,J=7.2Hz,3H),1.61(dd,J=13.8,7.2Hz,1H),1.54(d,J=1.2Hz,3H),1.47–1.35(m,2H),1.18(d,J=6.3Hz,5H),1.09(d,J=6.8Hz,3H),1.03–0.90(m,3H),0.78(t,J=8.7Hz,2H).HRMS(ESI + )m / z calcd 706.8465[C 75 H 99 N9O 16 S] 2+ (M+H) 2+ ,706.8460found.

[0163] TMR-SPEG5-tubulin

[0164]

[0165] Intermediate 23 (2.0 mg, 2.46 μmol, 1.0 eq.) was dissolved in DCM / TFA (3:1, 100 μL) to remove the tert-butyl group. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated under reduced pressure. The solution was then ready for use. BOC-cabazitaxel (4.1 mg, 4.92 μmol, 2.0 eq.) was dissolved in 1 mL of formic acid. The mixture was stirred at room temperature for 30 minutes, and the solvent was evaporated. The products obtained from the removal of the tert-butyl group from intermediate 23, PyBOP (3.8 mg, 7.38 μmol, 3.0 eq.) and DIPEA (8.13 μL 49.2 μmol, 20.0 eq.), were dissolved in 2 mL of anhydrous DMF. The mixture was stirred at room temperature for 1 h, and the solvent was evaporated. Preparative chromatography yielded a red solid, TMR-SPEG5-tubulin (2.9 mg, 1.70 μmol, 69%). 1 H NMR(400MHz, Methanol-d4)δ8.13(d,J=1.2Hz,1H),8.10(d,J=1.5Hz,1H),8.01–7.97(m,1H ),7.90–7.80(m,3H),7.68–7.64(m,1H),7.58–7.54(m,2H),7.50(s,1H),7.47–7.38(m,5H), 7.30(td,J=5.4,2.5Hz,1H),7.21(s,2H),7.07(s,2H),6.98(s,2H),6.24–6.19(m,1H),5.63 (d,J=7.1Hz,1H),5.50(d,J=4.4Hz,1H),5.04–5.01(m,1H),4.90(s,2H),4.63(d,J=4.3Hz,1 H),4.19(s,2H),3.91(dd,J=10.7,6.5Hz,2H),3.83(d,J=7.1Hz,1H),3.72(t,J=6.2Hz,2H) ,3.64–3.54(m,13H),3.46–3.44(m,2H),3.42(s,3H),3.31(s,10H),2.93(s,2H),2.90(s,2H ),2.74(t,J=5.6Hz,3H),2.67(s,1H),2.65(s,1H),2.55(td,J=6.2,4.6Hz,2H),2.36(s,2H) ,1.97(d,J=1.5Hz,3H),1.89–1.85(m,6H),1.69(s,3H),1.19(s,3H),1.13(s,3H).HRMS(ESI + )m / z calcd 737.8173[C 77 H 97 N5O22 S] 2+ (M+H) 2+ ,737.8167found.

[0166] Example 4: The preparation routes of R110-SPEG2-tubulin and R110-SPEG2-actin are shown below:

[0167]

[0168] 2-(2-(2-((3',6'-bis(diallylamino)-3-oxospiro[isoindoline-1,9'-xanthon])-2-sulfonamido)ethoxy)ethoxy)acetic acid(25)

[0169]

[0170] 24 (5.5 mg, 11.2 μmol, 1.0 eq.) was dissolved in 3 mL of dry DCM and POCl3 (10.4 μL, 112 μmol, 10 eq.) was added. The reaction mixture was refluxed for 1–1.5 h, and the formation of the acyl chloride was analyzed by the reaction of a small sample with MeOH and subsequent LC-MS. After successful formation of the acyl chloride, the solvent was evaporated under reduced pressure. Intermediate 20 (10 mg, 33.5 μmol, 3.0 wq.) and DIPEA (36.9 μL, 223 μmol, 20 eq.) were dissolved in 3 mL of acetonitrile, added to the acyl chloride, and stirred at 60 °C for 2 h. After the reaction was complete, all solvent was removed under vacuum. The residue was dissolved in 200 μL of DCM:TFA (3:1) and stirred at room temperature for 30 min or until complete. After removing the solvent under vacuum, the crude product was purified by preparative HPLC to obtain 25 (2.9 mg, 4.25 μmol, 38%) as a red solid. HRMS (ESI) + )m / z calcd 715.2802[C 38 H 43 N4O8S] + (M+H) + ,715.2795found.

[0171] (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-acetoxy-9-(((2R,3S)-3-(2-(2-(2-((3',6'-bis(diallylamino)-3-oxospiro[isoindoline-1,9'-xanthan])-2-sulfonamido)ethoxy)ethoxy)acetamido)-2-hydroxy (26)-(3-phenylpropionyl)oxy)-11-hydroxy-4,6-dimethoxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecylhydro-1H-[7,11]methylenecyclodeca[3,4]benzo[1,2-b]oxacyclohexane-12-ylbenzoate (26)

[0172]

[0173] BOC-cabazitaxel (2.6 mg, 3.15 μmol, 1.5 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was then evaporated to dryness. This, along with intermediate 25 (1.5 mg, 2.10 μmol, 1.0 eq.), PyBOP (2.2 mg, 4.20 μmol, 2.0 eq.), and DIPEA (3.47 μL 21.0 μmol, 10 eq.), was dissolved in 2 mL of anhydrous DMF and stirred at room temperature for 1 h. The solvent was then evaporated to dryness. Preparative chromatography yielded a red solid 26 (1.5 mg, 1.01 μmol, 49%). HRMS (ESI) + )m / z calcd685.8307[C 76 H 93 N9O 13 S] 2+ (M+H) 2+ ,685.8301found.

[0174] N-(4-((4R,7R,13S,19S,E)-7-((1H-indol-3-yl)methyl)-4-(4-hydroxyphenyl)-8,13,15,19-tetramethyl-2,6,9,12-tetraoxo-1-oxa-5,8,11-triazacyclononadecano-15-en-10-yl)butyl)-2-(2-(2-((3',6'-bis(diallylamino)-3-oxospiro[isoindolin-1,9'-xanthon])-2-sulfonamido)ethoxy)ethoxy)acetamide (27)

[0175]

[0176] BOC-jasplakinolide (2.1 mg, 3.15 μmol, 1.5 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was then evaporated to dryness. This solution, along with intermediate 25 (1.5 mg, 2.10 μmol, 1.0 eq.), PyBOP (2.2 mg, 4.20 μmol, 2.0 eq.), and DIPEA (6.94 μL 42.0 μmol, 20 eq.), was dissolved in 2 mL of anhydrous DMF and stirred at room temperature for 1 h. The solvent was then evaporated to dryness. Preparative chromatography yielded a red solid 27 (1.0 mg, 730 nmol, 35%). HRMS (ESI) was used. + )m / z calcd1432.5951[C 78 H 90 N5O 19 S] + (M+H) + ,1432.5951found.

[0177] R110-PEG2-tubulin

[0178]

[0179] 26 (2.0 mg, 1.40 μmol, 1.0 eq.) was dissolved in 1 mL of dry DCM:MeOH (5:1), and fresh tetrakis(triphenylphosphine)palladium(0) (1.61 mg, 1.40 μmol, 1.0 eq.) was added, followed by 1,3-dimethyl-1,3-diazine-2,4,6-trione (1.31 mg, 8.38 μmol, 6.0 eq.). The reaction mixture was stirred at room temperature under an argon atmosphere for 15 minutes or until complete. After removing all volatiles under vacuum, the crude product was purified by preparative HPLC to obtain a light orange solid of R110-PEG2-tubulin (1.0 mg, 790 nmol, 56%). HRMS (ESI) + )m / z calcd 1272.4699[C 66 H 74 N5O 19 S] + (M+H) + ,1272.4695found.

[0180] R110-PEG2-actin

[0181]

[0182] 27 (1.0 mg, 730 nmol, 1.0 eq.) was dissolved in 1 mL of dry DCM:MeOH (5:1), and fresh tetrakis(triphenylphosphine)palladium(0) (0.84 mg, 0.73 μmol, 1.0 eq.) was added, followed by 1,3-dimethyl-1,3-diazine-2,4,6-trione (0.68 mg, 4.38 μmol, 6.0 eq.). The reaction mixture was stirred at room temperature under an argon atmosphere for 15 minutes or until complete. After removing all volatiles under vacuum, the crude product was purified by preparative HPLC to obtain a light orange solid of R110-PEG2-actin (200 μg, 153 nmol, 21%). HRMS (ESI) + )m / z calcd 605.7681[C 64 H 77 N9O 13 S] 2+ (M+H) 2+ ,605.7672found.

[0183] Example 5: The preparation route of STMR-SPEG2-tubulin is shown below:

[0184]

[0185] 2-(2-((3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-1,9'-oxanthracene])-2-sulfonamide)ethoxy)ethoxy)acetic acid(29)

[0186] 28 (7.0 mg, 17.4 μmol, 1.0 eq.) was dissolved in 5 mL of dry DCM and POCl3 (15.9 μL, 174 μmol, 10 eq.) was added. The reaction mixture was refluxed for 1–1.5 h, and the formation of the acyl chloride was analyzed by the reaction of a small sample with MeOH and subsequent LC-MS. After successful formation of the acyl chloride, the solvent was evaporated under reduced pressure. Intermediate 20 (15.5 mg, 52.2 μmol, 3.0 eq.) and DIPEA (60.6 μL, 348 μmol, 20 eq.) were dissolved in 3 mL of acetonitrile, added to the acyl chloride, and stirred at 60 °C for 2 h. After the reaction was complete, all solvent was removed under vacuum. The residue was dissolved in 500 μL of DCM:TFA (3:1) and stirred at room temperature for 30 min or until complete. After removing the solvent under vacuum, the crude product was purified by preparative HPLC to obtain 29 (3.2 mg, 5.24 μmol, 30.2%) as a red solid. HRMS (ESI) + )m / zcalcd611.2170[C 30 H 35 N4O8S] +(M+H) + ,611.2158found.

[0187] STMR-SPEG2-tubulin

[0188]

[0189] BOC-cabazitaxel (4.0 mg, 4.92 μmol, 1.5 eq.) was dissolved in 1 mL of formic acid and stirred at room temperature for 30 min. The solvent was then evaporated to dryness. Together with intermediate 29 (2 mg, 3.28 μmol, 1.0 eq.), PyBOP (3.4 mg, 6.56 μmol, 2.0 eq.), and DIPEA (5.7 μL, 32.8 μmol, 10 eq.), the solution was dissolved in 2 mL of anhydrous DMF and stirred at room temperature for 1 h. The solvent was then evaporated to dryness. Preparative chromatography yielded solid 26 (1.2 mg, 0.89 μmol, 27%). HRMS (ESI) + )m / z calcd 1344.5091[C 70 H 82 N5O 18 S2] + (M+H) + ,1344.5066found.

[0190] Effect Examples: Application of Probes in Cell Imaging

[0191] Normal U2OS cells were cultured in DMEM (4.5 g / L glucose) supplemented with 10% (v / v) fetal bovine serum, 1 mM GlutaMAX, and 1 mM sodium pyruvate, and maintained at 37°C in a humidified 5% CO2 (v / v) environment. One to two days prior to imaging, cells were incubated in 10-well dishes with clear bottoms. For imaging, clear DMEM medium containing 10% FBS, 1 mM pyruvate, and 2 mM L-glutamine was used. Live-cell confocal imaging was performed on a Leica TCS SP8 microscope. Cells were incubated with a 1 μM probe for 2 hours. For colocalization experiments, cells were co-incubated with the corresponding SPY650-tubulin or SiR-actin probe at a concentration of 1 μM for 2 hours. For TMR, the excitation wavelength for routine live-cell confocal imaging was 555 nm (em. 565–750 nm). For the colocalization experiment of the TMR probe, the excitation wavelength of the TMR channel is 540 nm (em. 550-640 nm), and the excitation wavelength of the SiR channel is 651 nm (em. 661-750 nm).

[0192] (1) Performance of Actin protein probes in live cell imaging

[0193] To evaluate the performance of actin protein probes in live-cell imaging, U2OS cells were incubated with TMR-SC6-actin and TMR-SC9-actin for 2 hours, respectively. TMR-SC9-actin showed mild F-actin staining and produced bright spots of probe aggregates. Figure 1 a) The presence of numerous aggregates indicates poor solubility of TMR-SC9-actin in aqueous media. Subsequently, we attempted to shorten the alkyl linker length to obtain TMR-SC6-actin, which did improve labeling quality, but nonspecific bright spots remained and could not be eliminated. Figure 1 b).

[0194] To improve the solubility of actin protein probes, we synthesized TMR-SPEG2-actin and TMR-SPEG5-actin probes. Confocal imaging of live U2OS cells showed that both TMR-SPEG2-actin and TMR-SPEG5-actin exhibited bright and high-contrast F-actin protein staining. Figure 1 c and 1d). To further confirm the specificity of TMR-SPEG2-actin and TMR-SPEG5-actin for F-actin staining, co-localization experiments were performed using SiR-actin ( Figure 1 (e and 1f). The excellent labeling performance of TMR-SPEG2-actin and TMR-SPEG5-actin demonstrates that sulfonamide-PEG linkers can simultaneously improve the fluorescence, cell permeability, and solubility of rhodamine.

[0195] (2) Performance of Tubulin protein probe in live cell imaging

[0196] To evaluate the performance of tubulin protein probes in live-cell imaging, U2OS cells were incubated with TMR-SC8-tubulin, TMR-SC11-tubulin, TMR-SPEG2-tubulin, and TMR-SPEG5-tubulin. TMR-SC8-tubulin provided clear staining of the target microtubules, while TMR-SC11-tubulin, with its longer alkyl linker, did not produce more aggregates but instead showed high-contrast microtubule staining. Figure 2 (a and 2b). TMR-SPEG2-tubulin and TMR-SPEG5-tubulin exhibited clearer microtubule structures and higher imaging contrast. Figure 2 c and 2d). Co-localization experiments were performed using the SPY650-tubulin ( Figure 2 e and 2f).

[0197] Live U2OS cells were incubated with 1 μM R110-SPEG2-tubulin and R110-SPEG5-tubulin in the presence of 10 μM verapamil for 1 and 2.5 hours, respectively, and then imaged under 510 nm irradiation. Figure 3 As shown, after 1 and 2.5 hours of incubation, R110-SPEG2-tubulin and R110-SPEG5-tubulin provided high-contrast labeling of microtubules and F-actin in live U2OS cells.

Claims

1. A compound or a tautomer of formula (I): Formula I in, A and B are independently -NR A R B ; X represents O, S, or SiR. A R B CR A R B NR A Or Se; Each R A and R B Each is independently H or C1-C6 alkyl; R1, R2, R3, R4, R5, and R6 are H; U and V, along with the carbon atom in between, are linked to form a benzene ring; W connects to a single bond or -CH2-; Linker has any of the following structures: (1) , where m is any integer between 5 and 7; (2) , where m is any integer between 2 and 5, n is 1 or 2, and Z is -0-; Or (3) , where m is any integer between 5 and 7; L is or .

2. The compound or its tautomer as claimed in claim 1, characterized in that, It meets one or more of the following conditions: (1) A and B are the same; (2) X is O; (3)W is a connecting single key.

3. The compound or its tautomer as described in claim 2, characterized in that, It meets one or more of the following conditions: (1) A and B are independently -NR A R B Each R A and R B Each is independently H or C1-C6 alkyl; (2) The Linker has any of the following structures: 1) m is 5 or 7; 2) m is 2 or 5, and n is 1 or 2; Or 3) m is 5 or 7.

4. The compound or its tautomer as claimed in claim 1, characterized in that, R A and R B In this context, "C1-C6 alkyl" refers to C1-C4 alkyl.

5. The compound or its tautomer as claimed in claim 1, characterized in that, R A and R B In this context, "C1-C6 alkyl" refers to methyl or ethyl.

6. The compound or its tautomer as claimed in claim 1, characterized in that, It meets one or more of the following conditions: (1) A and B are independently -NH2 or -N(CH3)2; and (2) Linker is n is 2; or n is 5.

7. The compound or its tautomer as claimed in claim 1, characterized in that, The compound shown in formula (I) has any of the following structures: , , , , , , , , , or .

8. The compound or its tautomer as claimed in claim 1, characterized in that, The tautomers of the compound shown in formula (I) have the following structures: 。 9. The following compounds: , , , , , or .

10. The use of the compound or its tautomer as claimed in any one of claims 1-7 in the detection of tubulin or F-actin, wherein the use is for non-diagnostic and non-therapeutic purposes.

11. The application as described in claim 10, characterized in that, The applications are used in staining, live-cell fluorescence imaging, and protein function regulation.

12. The application as described in claim 10, characterized in that, In the aforementioned applications, the compound of formula (I) or its tautomers detects tubulin or F-actin via the following reaction: 。

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