A SNAP-tag probe and its preparation and application

Abstract

This invention relates to a method for preparing and applying a SNAP-tag probe that can be excited at 405 nm. The probe has the following structure: azacyclobutanone is introduced at the 4-position of a naphthalimide, and benzylguanine, which is specifically recognized by the SNAP-tag, is introduced at the N-terminus. This probe can specifically label target proteins fused with SNAP-tags in living cells under 405 nm excitation light, enabling fluorescence imaging of the target protein within living cells.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a SNAP-tag probe that can be excited by a 405nm laser. Background Technology

[0002] Compared to fluorescent proteins, small organic fluorescent dyes offer advantages such as small size, rich color variety, and ease of modification. Therefore, they are widely used in protein fluorescence imaging, including dynamic protein tracking and protein-protein interactions. However, poor specificity has long been a limiting factor in the further application of small organic fluorescent dyes in protein research. To address this issue, researchers have proposed protein tagging technology, where protein tags can specifically recognize substrate derivatives. SNAP-tags, currently the most widely used protein tagging technology, can specifically recognize benzylguanine derivatives and form stable covalent bonds with small organic dyes. Consequently, in recent years, numerous small organic fluorescent probes based on SNAP-tags have emerged and have been widely applied in the field of protein labeling.

[0003] Currently, commercial SNAP-tag probes typically use excitation wavelengths above 500 nm, including SNAP-Cell 505-Star (excitation wavelength 504 nm), SNAP-Cell TMR-Star (excitation wavelength 554 nm), SNAP-Cell 647-SiR (excitation wavelength 645 nm), SNAP-Surface 488 (excitation wavelength 506 nm), SNAP-Surface Alexa Fluor 546 (excitation wavelength 558 nm), SNAP-Surface 549 (excitation wavelength 560 nm), and SNAP-Surface Alexa Fluor 647 (excitation wavelength 652 nm). These dyes are difficult to match with commonly used 405 nm lasers, which limits multicolor labeling of proteins during fluorescence imaging. Therefore, there is an urgent need to develop SNAP-tag fluorescent probes with excitation wavelengths around 400 nm to help study the dynamic behavior and interactions of proteins. Summary of the Invention

[0004] One of the objectives of this invention is to provide a SNAP-tag probe that can be excited by a 405nm laser, enabling fluorescence imaging within live cells and thus applying it to multicolor imaging.

[0005] Another objective of this invention is to provide a method for preparing a SNAP-tag probe that can be excited by a 405nm laser. This synthesis method has the advantages of simple synthesis steps, convenient operation, and easy purification.

[0006] This invention provides a SNAP-tag probe that can be excited by a 405nm laser. Using 1,8-naphthalimide as the parent compound and introducing azacyclobutanone at the 4-position, it achieves super-resolution fluorescence imaging of SNAP-tag proteins at 405nm, which can be applied to multicolor imaging.

[0007] A SNAP-tag probe that can be excited at 405 nm has the following structure:

[0008]

[0009] The synthesis route for this fluorescent probe is as follows:

[0010]

[0011] The specific synthesis steps are as follows:

[0012] (1) Synthesis of intermediate AO-4:

[0013] Under N2 protection, 0.5-2.0 g of 4-bromo-1,8-naphthalene anhydride, 0.19-2.57 g of 2-azacyclobutanone, 0.88-11.76 g of cesium carbonate, and 0.085-0.86 g of methanesulfonic acid [9,9-dimethyl-4,5-bis(diphenylphosphine)xanthon][2'-amino-1,1'-biphenyl]palladium(II)dichloromethane adduct (XantPhos Pd G3) were dissolved in 15-100 mL of dry 1,4-dioxane. The reaction solution was slowly heated to 70-130 °C and stirred for 1-5 h. After heating was stopped and the reaction solution was cooled to room temperature, the solvent was removed under reduced pressure. The residue was separated by silica gel column chromatography, initially using dichloromethane:petroleum ether at a volume ratio of 1:1 as the eluent, and then replaced with dichloromethane:methanol at a volume ratio of 400:1. The solvent was removed by distillation under reduced pressure to obtain a pale yellow solid AO-4.

[0014] (2) Synthesis of SNAP-tag probes

[0015] Dissolve 0.01-0.5 g of intermediate AO-4 and 0.01-2.53 g of 6-((4-(aminomethyl)benzyl)oxy)-7H-purine-2-amine in a mixed solvent of 2-20 mL anhydrous ethanol and 0.2-2 mL dichloromethane. Slowly heat the reaction solution to 30-90 °C and stir for 9-15 h. Stop heating and allow the reaction solution to cool to room temperature. Remove the solvent under reduced pressure. Separate the residue by silica gel column chromatography. Using dichloromethane:methanol at a volume ratio of 30:1 as the eluent, remove the solvent by reduced pressure distillation to obtain a fluorescent probe targeting the SNAP-tag protein. In step (1), 0.5-2.0 g of 4-bromo-1,8-naphthalene anhydride, 0.19-2.57 g of 2-azacyclobutanone, 0.88-11.76 g of cesium carbonate, 0.085-0.86 g of methanesulfonic acid [9,9-dimethyl-4,5-bis(diphenylphosphine)xanthan][2'-amino-1,1'-biphenyl]palladium(II) dichloromethane adduct (XantPhos Pd G3), and 15-100 mL of dry 1,4-dioxane.

[0016] In step (2), a mixed solvent of 0.01-0.5 g intermediate AO-4, 0.01-2.53 g 6-((4-(aminomethyl)benzyl)oxy)-7H-purine-2-amine, 2-20 mL anhydrous ethanol, and 0.2-2 mL dichloromethane is used. The above-mentioned SNAP-tag probe, which can be excited at 405 nm, exhibits high selectivity for SNAP-tag proteins and can specifically recognize SNAP-tags in complex environments such as living cells.

[0017] This invention incorporates azacyclobutanone at the 4-position of a naphthalimide and a benzylguanine SNAP-tag at the N-terminus for specific recognition. This probe can specifically label target proteins fused with SNAP-tags within living cells under 405 nm excitation light, enabling fluorescence imaging of the target proteins within living cells.

[0018] This invention has the following features:

[0019] This SNAP-tag probe has advantages such as inexpensive and readily available raw materials, simple and universal synthesis methods, and simple synthesis steps.

[0020] This SNAP-tag probe can achieve fluorescence emission under 405nm excitation.

[0021] This SNAP-tag probe can specifically recognize SNAP-tag proteins in living cells, enabling fluorescence imaging. Attached Figure Description

[0022] Figure 1 The hydrogen NMR spectrum of AO-4 prepared in Example 1.

[0023] Figure 2 The carbon NMR spectrum of AO-4 prepared in Example 1.

[0024] Figure 3 High-resolution mass spectrometry of AO-4 prepared in Example 1.

[0025] Figure 4 The proton NMR spectrum of the SNAP-tag probe prepared in Example 1.

[0026] Figure 5 The carbon NMR spectrum of the SNAP-tag probe prepared in Example 1.

[0027] Figure 6 High-resolution mass spectrometry of the SNAP-tag probe prepared in Example 1.

[0028] Figure 7 The SNAP-tag probe prepared in Example 1 was used in transfected pSNAP f -H2B fluorescence confocal imaging of HeLa cells, with a fluorescent probe concentration of 1 μM.

[0029] Figure 8 The SNAP-tag probe prepared in Example 1 was used in transfected pSNAP f In situ fluorescence spectrum of -H2B in HeLa cells, with a fluorescent probe concentration of 1 μM. Detailed Implementation

[0030] Example 1. Synthesis method of SNAP-tag probe.

[0031] Synthesis of intermediate AO-4:

[0032]

[0033] Under N2 protection, 4-bromo-1,8-naphthalene anhydride (0.50 g, 1.81 mmol), 2-azacyclobutanone (190 mg, 2.72 mmol), cesium carbonate (880 mg, 2.72 mmol), and methanesulfonic acid [9,9-dimethyl-4,5-bis(diphenylphosphine)xanthones][2'-amino-1,1'-biphenyl]palladium(II)dichloromethane adduct (XantPhos Pd G3) (85 mg, 0.09 mmol) were dissolved in 15 mL of dry 1,4-dioxane. The reaction mixture was slowly heated to 70 °C and stirred for 1 h. Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography. Initially, dichloromethane:petroleum ether (v / v) = 1:1 was used as the eluent, and then the eluent was replaced with dichloromethane:methanol (v / v) = 400:1. The solvent was removed by distillation under reduced pressure to obtain 300 mg of pale yellow solid AO-4, with a yield of 62%.

[0034] 1 H NMR(700MHz,DMSO-d6)δ8.93(dd,J=8.6,0.8Hz,1H),8.54(dd,J=7.2,0.8Hz,1H),8.49(d ,J=8.1Hz,1H),7.88(dt,J=7.2,3.8Hz,1H),4.14(t,J=4.9Hz,2H),3.30(t,J=4.9Hz,2H). 13 C10 NMR (176MHz, DMSO-d6) δ 166.80, 161.43, 160.64, 142.33, 134.03, 133.47, 132.96, 131.67, 126.97, 123.27, 119.49, 116.78, 114.62, 41.95, 36.75. Theoretical C10 values ​​from high-resolution mass spectrometry. 15 H9NO4[M+H] + 268.0610, actual value 268.0588.

[0035] Upon testing, its structure is shown in equation AO-4 above.

[0036] Synthesis of SNAP-tag probes:

[0037]

[0038] Intermediate AO-4 (10 mg, 0.04 mmol) and 6-((4-(aminomethyl)benzyl)oxy)-7H-purine-2-amine (10.8 mg, 0.04 mmol) were dissolved in a mixed solvent of 2 mL anhydrous ethanol and 0.2 mL dichloromethane. The reaction solution was slowly heated to 30 °C and stirred for 9 h. Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solvent was removed under reduced pressure. The residue was separated by silica gel column chromatography. Using dichloromethane:methanol (v / v) = 30:1, the solvent was removed by reduced pressure distillation to obtain 10.8 mg of a fluorescent probe targeting the SNAP-tag protein, with a yield of 52%.

[0039] 1H NMR (700MHz, DMSO-d6) δ8.87(dd,J=8.6,0.9Hz,1H),8.55(dd,J=7.3,0.9Hz,1H),8.50(d,J=8.1Hz,1H),7.86(dt,J=8.5,3.8Hz,2H) ,7.44(d,J=8.2Hz,2H),7.39(d,J=8.2Hz,2H),6.25(s,2H),5.43(s,2H),5.25(s,2H),4.12(t,J=4.8Hz,2H),3.28(t,J=4.8Hz,2H). 13 C NMR (176MHz, DMSO-d6) δ 166.69, 164.00, 163.36, 160.04, 141.60, 137.70, 136.05, 134.04, 133.48, 132.98, 132.46, 132.08, 132.04, 130.11, 129.29, 129.08, 128.88, 128.11, 126.98, 126.78, 123.47, 122.46, 118.19, 116.89, 66.95, 43.17, 41.82, 36.62. Theoretical C values ​​from high-resolution mass spectrometry. 28 H 21 N7O4[M+H] + 520.1733, actual value 520.1705.

[0040] Upon testing, its structure is shown in the above formula SNAP-tag.

[0041] Example 2. Synthesis method of SNAP-tag probe.

[0042] Synthesis of intermediate AO-4:

[0043]

[0044] Under N2 protection, 4-bromo-1,8-naphthalene anhydride (1.25 g, 4.51 mmol), 2-azacyclobutanone (1.04 g, 14.66 mmol), cesium carbonate (4.78 g, 14.66 mmol), and methanesulfonic acid [9,9-dimethyl-4,5-bis(diphenylphosphine)xanthones][2'-amino-1,1'-biphenyl]palladium(II)dichloromethane adduct (XantPhos Pd G3) (1.18 g, 1.24 mmol) were dissolved in 60 mL of dry 1,4-dioxane. The reaction mixture was slowly heated to 100 °C and stirred for 3 h. Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography. Initially, dichloromethane:petroleum ether (v / v) = 1:1 was used as the eluent, and then the eluent was replaced with dichloromethane:methanol (v / v) = 400:1. The solvent was removed by distillation under reduced pressure, yielding 843 mg of pale yellow solid AO-4, with a yield of 70%.

[0045] Upon testing, its structure is shown in equation AO-4 above.

[0046] Synthesis of SNAP-tag probes:

[0047]

[0048] Intermediate AO-4 (255 mg, 0.95 mmol) and 6-((4-(aminomethyl)benzyl)oxy)-7H-purine-2-amine (774 mg, 2.86 mmol) were dissolved in a mixed solvent of 11 mL anhydrous ethanol and 1.1 mL dichloromethane. The reaction solution was slowly heated to 60 °C and stirred for 12 h. Heating was stopped, and after the reaction solution cooled to room temperature, the solvent was removed under reduced pressure. The residue was separated by silica gel column chromatography using dichloromethane:methanol (v / v) = 30:1, and the solvent was removed by reduced pressure distillation to obtain 222 mg of a fluorescent probe targeting the SNAP-tag protein, with a yield of 45%.

[0049] Upon testing, its structure is shown in the above formula SNAP-tag.

[0050] Example 3. Synthesis method of SNAP-tag probe.

[0051] Synthesis of intermediate AO-4:

[0052]

[0053] Under N2 protection, 4-bromo-1,8-naphthalene anhydride (2.0 g, 7.22 mmol), 2-azacyclobutanone (2.57 g, 36.1 mmol), cesium carbonate (11.76 g, 36.1 mmol), and methanesulfonic acid [9,9-dimethyl-4,5-bis(diphenylphosphine)xanthones][2'-amino-1,1'-biphenyl]palladium(II)dichloromethane adduct (XantPhos Pd G3) (3.42 g, 3.61 mmol) were dissolved in 100 mL of dry 1,4-dioxane. The reaction mixture was slowly heated to 130 °C and stirred for 5 h. Heating was stopped, and the reaction solution was allowed to cool to room temperature. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography. Initially, dichloromethane:petroleum ether (v / v) = 1:1 was used as the eluent, and then the eluent was replaced with dichloromethane:methanol (v / v) = 400:1. The solvent was removed by distillation under reduced pressure, yielding 1.06 g of pale yellow solid AO-4, with a yield of 55%.

[0054] Upon testing, its structure is shown in equation AO-4 above.

[0055] Synthesis of SNAP-tag probes:

[0056]

[0057] Intermediate AO-4 (500 mg, 1.87 mmol) and 6-((4-(aminomethyl)benzyl)oxy)-7H-purine-2-amine (2.53 g, 9.35 mmol) were dissolved in a mixed solvent of 20 mL anhydrous ethanol and 2 mL dichloromethane. The reaction solution was slowly heated to 90 °C and stirred for 15 h. Heating was stopped, and after the reaction solution cooled to room temperature, the solvent was removed under reduced pressure. The residue was separated by silica gel column chromatography, using dichloromethane:methanol (v / v) = 30:1 as the eluent, and the solvent was removed by reduced pressure distillation to obtain 466 mg of a fluorescent probe targeting the SNAP-tag protein, with a yield of 48%.

[0058] Upon testing, its structure is shown in the above formula SNAP-tag.

[0059] Example 4: Fluorescence confocal imaging and in situ fluorescence spectroscopy of the SNAP-tag probe in transfected cells. The probe was dissolved in DMSO solution to prepare a 2 mM stock solution. 0.5 μL of the stock solution was dissolved in 1 mL of HeLa cell DMEM culture medium, and then incubated at 37°C for 30 minutes before fluorescence confocal imaging. The imaging image is shown below. Figure 7 As shown, Figure 7 via pSNAP f -H2B induces HeLa cells to express H2B fused with a SNAP-tag (plasmid purchased from NEB). The intracellular in situ fluorescence spectrum under 405 nm excitation light is as follows: Figure 8 As shown.

Claims

1. A SNAP-tag probe, the structure of which is as follows: 。 2. A method for synthesizing the SNAP-tag probe according to claim 1, characterized by the following steps: (1) Synthesis of intermediate AO-4: Dissolve 0.5-2.0 g of 4-bromo-1,8-naphthalene anhydride, 0.19-2.57 g of 2-azacyclobutanone, 0.88-11.76 g of cesium carbonate, and 0.085-0.86 g of methanesulfonic acid [9,9-dimethyl-4,5-bis(diphenylphosphine)xanthon][2'-amino-1,1'-biphenyl]palladium(II)dichloromethane adduct (XantPhos Pd G3) in 15-100 mL of 1,4-dioxane; heat the reaction solution to 70-130 °C and stir for 1-5 h; Purification yielded the solid intermediate AO-4; (2) Synthesis of SNAP-tag probes Dissolve 0.01-0.5 g of intermediate AO-4 and 0.01-2.53 g of 6-((4-(aminomethyl)benzyl)oxy)-7H-purine-2-amine in a mixed solvent of 2-20 mL ethanol and 0.2-2 mL dichloromethane. Heat the reaction solution to 30-90 °C and stir for 9-15 h. After purification, a fluorescent probe targeting SNAP-tag protein is obtained.

3. The use of the SNAP-tag probe of claim 1 in a reagent for the preparation of SNAP-tag protein recognition and / or detection processes.

4. The application according to claim 3, characterized in that: The SNAP-tag probe is excited by 405 nm light to achieve fluorescence imaging of the SNAP-tag protein at 405 nm.

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