CN110143975B - Fluorescent-labeled O of naphthalimide or perylene bisimide6-benzylguanine derivatives and process for the preparation thereof - Google Patents

Abstract

The invention provides O of fluorescent labeling of naphthalimide or perylene bisimide⁶-benzylguanine derivatives and processes for their preparation. The derivative of the invention is chemically modified by taurine, and the naphthalimide and the perylene bisimide with excellent water solubility are used for preparing O with high fluorescence yield and good water solubility⁶Benzyl guanine derivatives for improving the Tag-specific substrate O in the SNAP-Tag protein labeling technique⁶The problem of poor water solubility of Benzylguanine derivatives (BG) and insufficient interference of nonspecific staining background by washing with water. The invention uses taurine to react with the naphthalic anhydride and the perylene anhydride to obtain the sulfonic acid salt of the naphthalimide and the perylene imide, and the product has good water solubility.

Description

Naphthalimide or peryleneimide fluorescently labeled O6-benzylguanine derivatives and preparation method thereof

technical field

The invention belongs to the synthesis of protein markers, and relates to O ⁶ -benzylguanine derivatives labeled with naphthalimide or peryleneimide fluorescence and a preparation method thereof.

Background technique

Protein-specific small molecule fluorescent dye labeling technology overcomes many limitations of fluorescent protein (fluorescentprotein FP) in the field of protein research. This type of technology uses standard genetic engineering methods to construct proteins and polypeptides, and recombinant proteins with protein tags, and uses fluorescent probe ligands to specifically and efficiently identify corresponding peptides and protein tags to achieve site-specific fluorescent labeling of target proteins. SNAP-Tag stands out from many targeted labeling technologies due to its unique advantages such as high specificity and stability to target proteins, as well as functional diversity of ligands. It has become one of the best fusion labels and has been widely used in many fields. For example, in vivo imaging, cell imaging, protein in vitro research, protein in vivo research, microbial pathogenic mechanism research, disease diagnosis, etc.

SNAP-Tag was developed by johnsson et al. in 2003. It is a specific protein tag obtained by modifying human O ^{6 -alkylguanine} -DNA alkyitransferase (hAGC). hAGT is a monomeric protein composed of 207 amino acids, which can transfer the methyl group at the O ⁶ position of methylated DNA guanine to its own hAGT-active cysteine thiol (Cys145), thereby achieving Repair of DNA. The modification of hAGT structure reduces the interaction between the original protein structure and methylated DNA on the one hand, and improves its specific reactivity with O ⁶ modified benzyiguanine (BG) on the other hand. The cysteine sulfhydryl group (Cys145) located in the active site of SNAP-Tag can react with the benzyl group of BG to form a very stable covalent bond between the protein and the substrate to achieve labeling. The process is as follows:

The unique advantages of SNAP-Tag are: the protein tag reacts with the substrate quickly and with high specificity; the protein bound by covalent bond has good stability, and can be stably labeled even under the denaturing conditions of SDS-PAGE in vitro analysis; The chemical design combines the label-specific substrate BG with functional dyes, with high flexibility; it is easy to derive a variety of fluorescent dyes, and has strong versatility, which can meet the needs of various fluorescence imaging. At present, SNAP-Tag has been widely used in the research of intracellular protein labeling, in vitro analysis and targeted fluorescence detection. However, this method also has certain shortcomings; the synthesis of the label-specific substrate BG is complicated, and there may be a problem of poor solubility and dispersion after being combined with the dye; when most dyes are used, there is no significant difference in the fluorescence signal before and after labeling. , requires extensive washing to remove non-specific staining, and there may be background interference. However, the present invention provides a novel protein-specific molecular fluorescent marker for improving the deficiencies in the SNAP-Tag technology.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a fluorescently labeled O ⁶ -benzylguanine derivative of naphthalimide and peryleneimide.

Another object of the present invention is to provide a preparation method of the derivative.

In order to achieve above-mentioned purpose ID, the present invention adopts following reaction mechanism:

According to above-mentioned reaction mechanism, the present invention adopts following technical scheme:

A naphthalimide or peryleneimide fluorescently labeled O ⁶ -benzylguanine derivative, characterized in that the general structural formula of the compound is one of the following:

a.

b.

c.

d.

where R = alkyl, cycloalkyl, aryl, heteroaryl or polyoxyethylene ether chain.

A method for preparing the O6- ^{benzylguanine} derivative a of the fluorescently labeled naphthalimide according to claim 1, wherein the specific steps of the method are:

a. Dissolve 6-chloroguanine and N-methylpyrrolidine in N,N-dimethylformamide at a molar ratio of 1:1.5-1:2.1, 60℃-100℃, react for 24h-48h, The reaction finishes suction filtration, and the filter residue is washed with acetone, and suction filtration obtains compound M, and its structural formula is:

b. Dissolve 4-bromo-1,8-naphthalene anhydride and organic sulfonate in absolute ethanol at a molar ratio of 1:1.2-1:1.5, react under reflux conditions for 6h-12h, cool, and filter out the solid crude The product is separated and purified to obtain compound Ib, and its structural formula is:

所述的有机磺酸盐的结构式为：NH₂RSO₃H；

The structural formula of the organic sulfonate is: NH ₂ RSO ₃ H;

c. Dissolve the intermediate product Ib, aminomethylbenzyl alcohol and potassium carbonate obtained in step b in N,N-dimethylformamide in a molar ratio of 1:1:1.2-1; 1.5:2.0, and perform a reflux reaction for 6h～8h, A dark brown reaction solution was obtained, the pH was adjusted to 4-5, and most of the solvent was removed to obtain a paste-like substance, which was filtered with suction to obtain a crude product; and then separated and purified to obtain compound Ic, whose structural formula is:

d. The intermediate product Ic obtained in step c and the intermediate M and t-BuOK obtained in step a are dissolved in N,N-dimethylformamide in a molar ratio of 1:2:5-1:3:5, inert Under the protection of atmosphere, the reaction is carried out for 3h-8h, and the naphthalimide fluorescently labeled O ⁶ -benzylguanine derivative a is obtained by separation and purification, and its structural formula is:

A method for preparing the fluorescent protein marker c of naphthalimide fluorescently labeled O ⁶ -benzylguanine homomolecular twins according to claim 1, characterized in that the specific steps of the method are:

e. Dissolve 4-bromo-1,8-naphthalene anhydride and aminomethylbenzyl alcohol in absolute ethanol at a molar ratio of 1:1.2-1:1.5, react under reflux conditions for 6h-12h, cool, and filter out the solid The crude product is separated and purified to obtain compound IIIa, whose structural formula is:

f. The intermediate product IIIa, aminomethylbenzyl alcohol and potassium carbonate obtained in step e were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1.2-1; 1.5:2.0, and the reflux reaction was carried out for 6h～8h, A dark brown reaction solution was obtained, the pH was adjusted to 4-5, and most of the solvent was removed to obtain a paste-like substance, which was filtered with suction to obtain a crude product; and then separated and purified to obtain compound IIIb, whose structural formula is:

g. Dissolve the product IIIb obtained in step f, product M obtained in step a, and t-BuOK in N,N-dimethylformamide in a molar ratio of 1:2:5-1:3:5, under the protection of an inert atmosphere , react for 3h-8h, and separate and obtain the fluorescent protein marker c of O ⁶ -benzylguanine homomolecular twinned with naphthalimide fluorescently labeled, and its structural formula is:

A method for preparing the peryleneimide fluorescently labeled O ⁶ -benzylguanine derivative according to claim 1, wherein the specific steps of the method are:

a. Dissolve tetrachloroperylene anhydride and organic sulfonate in ethylene glycol methyl ether in a molar ratio of 1:1.2-1:1.5, and react at 100℃-120℃

6h-12h, through separation and purification, compound IIa is obtained, and its structural formula is:

The structural formula of described tetrachloroperylene anhydride is:

The structural formula of the organic sulfonate is: NH ₂ RSO ₃ H;

b. Dissolve the intermediate product IIa and aminomethylbenzyl alcohol obtained in step a in absolute ethanol according to the molar ratio of 1:1.2-1:1.5, stir and react under reflux conditions for 6h-12h, remove the solvent, and separate the obtained crude product Purification to obtain solid compound IIb, its structural formula is:

c. The intermediate product IIb obtained in step b, p-tert-butylphenol and potassium carbonate are dissolved in N,N-dimethylformamide in a molar ratio of 1:4:4-1:5:6, and the reaction is stirred under reflux conditions After 6 to 8 hours, the obtained crude product is separated and purified to obtain compound IIc, and its structural formula is:

d. The product IIc obtained in step c, product M and t-BuOK in claim 2, step a, are reacted for 3h-5h in a molar ratio of 1:1.2:3-1:2.1:5 under the protection of an inert atmosphere, and are separated and purified The O ⁶ -benzylguanine derivative b, which is fluorescently labeled with peryleneimide, is obtained, and its structural formula is:

A preparation method for preparing the fluorescently labeled O ⁶ -benzylguanine homomolecular fluorescent twinned label of the peryleneimide according to claim 1, characterized in that the specific steps of the method are:

e. Dissolve tetrachloroperylene anhydride and aminomethylbenzyl alcohol in ethylene glycol methyl ether at a molar ratio of 1:2-1:3, react at 100°C-140°C for 6h-12h, and obtain compound IVa through separation and purification, Its structural formula is:

f. product IVa obtained in step e, p-tert-butylphenol and potassium carbonate are dissolved in N,N-dimethylformamide in a molar ratio of 1:4:5-1:5:5, and the reaction is stirred under reflux conditions for 6 ~8 hours, then the solvent is evaporated, and the obtained crude product is separated and purified to obtain compound IVb, and its structural formula is:

g. product M and t-BuOK obtained in step f and product M and t-BuOK in step a of claim 2 are in a molar ratio of 1:2:5-1:2.5:5, under the protection of an inert atmosphere, react for 3h-5h, and are separated and purified The peryleneimide fluorescently labeled O ⁶ -benzylguanine homomolecular fluorescent twin label d is obtained, and its structural formula is:

The taurine chemically modified naphthalene imide and perylene imide of the present invention have excellent water solubility, and prepare O ⁶ -benzylguanine derivatives with high fluorescence yield and good water solubility, so as to improve SNAP-Tag In protein labeling technology, the label-specific substrate O ⁶ -benzylguanine derivative (Benzylguanine, BG) has poor water solubility and insufficient washing to remove non-specific staining background interference. In the present invention, taurine is reacted with naphthalene anhydride and perylene anhydride to obtain sulfonates of naphthalene imide and perylene imide, and the product has good water solubility, further reacts with aminomethylbenzyl alcohol, and then reacts with guanine The reactive intermediates are coupled to obtain the target product. Simultaneously synthesizing dimers of target molecules (using the synergistic effect of homomolecular twinning) is expected to improve the targeting performance of such specific labeled proteins.

Description of drawings

Fig. 1 is the normalized fluorescence excitation emission spectrum of O ⁶ -benzylguanine derivative A ₁ fluorescently labeled with naphthalimide in Example 1;

FIG. 2 is the normalized fluorescence excitation emission spectrum of the peryleneimide fluorescently labeled O ⁶ -benzylguanine derivative c in Example 2. FIG.

Detailed ways

The present invention is further described with reference to the examples, but the present invention is not limited by the following examples.

Example 1: Preparation of Naphthalimide Fluorescently Labeled O ⁶ -benzylguanine Derivative A ₁

a. In a 250mL round bottom flask, add 1.69g (10mmol) of 6-chloroguanine, 80mL N,N-dimethylformamide as solvent, add 1.75g (21mmol) of N-methylpyrrole, stir at 60°C for 24 hours . After the reaction was completed, it was cooled, and the precipitate was washed with 20 mL of acetone, and then purified by filtration to obtain 1.94 g of a white solid with a yield of 76.5%. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 13.41 (s, 1H), 8.35 (s, 1H), 7.12 (s, 2H), 4.67–4.48 (m, 2H), 4.03–3.89 (m , 2H), 3.65(s, 3H), 2.30–2.19(m, 2H), 2.06(dd, J=7.4, 4.7Hz, 2H). Its structural formula is;

b. In a 50mL round-bottomed flask, 277.0mg (1.0mmol) of 4-bromo-1,8-naphthalene anhydride, 125.1mg (1.0mmol) of taurine, and 20mL of EtOH were added as solvent, and the mixture was stirred at 78°C under reflux for 6 hours. After the reaction was completed, the crude product was obtained by suction filtration, and then separated by column chromatography. Using dichloromethane: methanol = 60: 1 as the eluent, column chromatography was used to separate and purify the brown solid Iba; 279.6 mg, the yield was 79% . ¹ H NMR (500 MHz, D ₂ O, ppm): δ 7.83 (d, J=7.2 Hz, 2H), 7.59 (d, J=7.9 Hz, 1H), 7.35 (t, J=7.9 Hz, 1H) ,7.26(d,J=7.9Hz,1H),4.06(t,J=5.6Hz,2H),3.15(t,J=5.7Hz,2H). Its structural formula is;

c. In a 50mL round-bottomed flask, add 355.0mg (1.0mmol) of Intermediate Iba, 137.0mg (1.0mmol) of aminomethyl benzyl alcohol, 20mL of N,N-dimethylformamide as a solvent, add 138.0mg of potassium carbonate ( 1 mmol), and stirred at reflux for 8 hours. After the completion of the reaction, the pH was adjusted to 4-5, the solvent was distilled under reduced pressure, the residue was separated by column chromatography, and dichloromethane:methanol=40:1 was used as the eluent, and the column chromatography was separated and purified to obtain a brown solid Ica 264.0 mg, the yield was 60%. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 8.57 (d, J=7.2Hz, 2H), 8.33 (d, J=7.9Hz, 1H), 8.20 (d, J=7.9Hz, 1H) ,7.98(t,J＝7.9Hz,1H),7.35-7.36(m,1H),7.33(d,J＝7.9Hz,2H),7.25(d,J＝8.0Hz,2H),5.22(s, 2H), 5.14(t, J＝5.6Hz, 1H), 4.44(d, J＝5.6Hz, 2H). 3.15(t, J＝5.6Hz, 2H), 4.06(t, J＝5.6Hz, 2H) . Its structural formula is;

d. Add 440.0 mg (1.0 mmol) of Intermediate Ica, 635.1 mg (2.5 mmol) of Intermediate M, 10 mL of dry N,N-dimethylformamide to 50 mL, add 684.80 mg (5 mmol) of t-BuOK, and Stir at room temperature for 3 hours under _N2 protection. TLC spot plate tracking, after the completion of the reaction, the solvent was distilled under reduced pressure to adjust the pH to 4-5, the residue was separated by column chromatography, using dichloromethane: methanol = 20:1 as the eluent, column chromatography separation and purification A dark brown solid, 303.7 mg, was obtained with a yield of 53%. ¹ HNMR (500MHz, DMSO-d6, ppm): δ 12.41 (s, 1H), 8.57 (d, J=7.2Hz, 2H), 8.44 (d, J=7.2Hz, 1H) 8.33 (d, J= 7.9Hz, 1H), 8.20 (d, J=7.9Hz, 1H), 7.98 (t, J=7.9Hz, 1H), 7.35 (m, 1H), 7.33 (d, J=7.9 Hz, 2H), 7.25 (d, J＝8.0Hz, 2H), 5.36(s, 2H), 5.22(s, 2H), 5.14(t, J＝5.6Hz, 1H), 4.44(d, J＝5.6 Hz, 2H), 4.06 (t, J=5.6Hz, 2H), 3.15 (t, J=5.7Hz, 2H). Its structural formula is:

Example 2: Preparation of Peryleneimide Fluorescently Labeled O ⁶ -benzylguanine Derivative B ₁ ;

a. Add 532.0 mg (1.0 mmol) of tetrachloroperylene anhydride, 20 mL of ethylene glycol methyl ether as solvent, 125.1 mg (1.0 mmol) of taurine into a 50 ml round-bottomed flask, and stir under reflux at 100°C for 6 hours. After the reaction was completed, the crude product was obtained by suction filtration, and then separated by column chromatography, with dichloromethane: methanol=60:1 as the eluent, and the column chromatography was separated and purified to obtain a dark red solid compound IIaa; 477.9 mg, the yield was 477.9 mg. 75%. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 7.92 (s, 2H), 6.28 (s, 2H), 3.64 (t, J = 5.6 Hz, 2H), 3.33 (t, J = 5.7 Hz, 2H). Its structural formula is:

b. Add 637.0 mg (1.0 mmol) of Intermediate IIaa, 137.0 mg (1.0 mmol) of aminomethylbenzyl alcohol, and 20 mL of DMF as solvent to a 50 mL round-bottomed flask, and stir at reflux for 8 hours. After the reaction was completed, the solvent was distilled under reduced pressure, and the residue was separated by column chromatography. Using dichloromethane: methanol = 40:1 as the eluent, column chromatography was used to separate and purify a dark purple-red solid IIba 612.4 mg, with a yield of 81 %. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 7.92 (s, 2H), 7.33 (d, J=7.9 Hz, 2H), 7.25 (d, J=8.0 Hz, 2H), 6.28 (s, 2H), 5.22(s, 2H), 5.14(t, J＝5.7Hz, 1H), 4.44(d, J＝5.6Hz, 2H), 3.64(t, J＝5.6 Hz, 2H), 3.33(t, J=5.6Hz, 2H). Its structural formula is:

c. Add 756.0 mg (1.0 mmol) of Intermediate IIba, 750 mg (5 mmol) of p-tert-butylphenol, 20 mL of N,N-dimethylformamide to 50 mL, add 414.0 mg (3 mmol) of potassium carbonate, and react at 130°C for 6 h. TLC spot plate tracking, after the completion of the reaction, the solvent was distilled under reduced pressure to adjust the pH to 4-5, the residue was separated by column chromatography, using dichloromethane: methanol = 30:1 as the eluent, column chromatography separation and purification The deep purple-red solid compound IIca 1.0302g was obtained, and the yield was 85%. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 7.92 (s, 2H), 7.33 (d, J=7.9Hz, 2H), 7.29 (d, J=8.5Hz, 8H), 7.25 (d, J＝8.0 Hz, 2H), 6.74(d, J＝8.0Hz, 8H), 6.28(s, J＝8.5Hz, 2H), 5.22(s, 2H), 5.14(t, J＝5.7Hz, 1H) ,4.44(d,J＝5.6Hz,2H),3.33(t,J＝5.7Hz,2H,),3.64(t,J＝5.6Hz,2H),1.42(s,36H). Its structural formula is:

d. Add 1213.5 mg (1.0 mmol) of Intermediate IIca, 635.1 mg (2.5 mmol) of Intermediate M, 10 mL of dry N,N-dimethylformamide to 50 mL, add 684.80 mg (5 mmol) of t-BuOK, Stir at room temperature for 3 hours under _N2 protection. TLC spot plate tracking, after the completion of the reaction, the solvent was distilled under reduced pressure to adjust the pH to 4-5, the residue was separated by column chromatography, using dichloromethane: methanol = 20:1 as the eluent, column chromatography separation and purification 874.2 mg of pure dark brown product compound was obtained in a yield of 63%. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 12.41 (s, 1H), 8.44 (d, J=7.2Hz, 1H), 7.92 (s, 2H), 7.33 (d, J=7.9Hz, 2H), 7.25(d, J＝8.0Hz, 2H), 7.29(d, J＝8.5Hz, 8H), 6.74(d, J＝8.0Hz, 8H), 6.28(s, 2H), 5.36(s, 2H), 5.22(s, 2H), 4.44(d, J=5.6Hz, 2H), 3.33(t, J=5.7Hz, 2H), 3.64(t, J= 5.6Hz, 2H), 1.42(s, 36H)

Example 3: The method for fluorescently labeling O ⁶ -benzylguanine homomolecular twinned fluorescent protein label c with naphthalimide:

a. In a 50mL round-bottomed flask, add 554.0mg (2.0mmol) of 4-bromo-1,8-naphthalene anhydride, 274.0mg (2.0 mmol) of aminomethyl benzyl alcohol, 20mL of absolute ethanol as solvent, and reflux at 78°C with stirring for 6 Hour. After the reaction was completed, the crude product was obtained by suction filtration, and then separated by column chromatography. With dichloromethane: methanol=100:1 as the eluent, the white solid was separated and purified by column chromatography to obtain a pure product compound IIIa 636.6 mg. The yield was 636.6 mg. 82%. ¹ H NMR (400MHz, DMSO-d6, ppm): δ 8.57 (d, J=7.2Hz, 2H), 8.33 (d, J=7.9Hz, 1H), 8.20 (d, J=7.9Hz, 1H) ,7.98(t,J＝7.9Hz,1H),7.33(d,J＝7.9Hz,2H),7.25(d,J＝8.0Hz,2H),5.22(s,2H),5.14(t,J＝ 5.7Hz, 1H), 4.44 (d, J=5.6Hz, 2H). Its structural formula is:

In a 50mL round-bottomed flask, 395.0mg (1.0mmol) of Intermediate IIIa, 137.0mg (1.0mmol) of aminomethyl benzyl alcohol, 20mL of N,N-dimethylformamide were added as a solvent, and 138.0mg (1mmol) of potassium carbonate was added. , stirring at reflux for 8 hours. After the completion of the reaction, the pH was adjusted to 4-5, the solvent was distilled under reduced pressure, and the residue was separated by column chromatography. Using dichloromethane:methanol=40:1 as the eluent, column chromatography was used to separate and purify the light brown solid IIIb. 327.6 mg, 70% yield. ¹ H NMR (400MHz, DMSO-d6, ppm): δ 8.57 (d, J=7.2Hz, 1H), 8.53 (d, J=8.5Hz, 1H), 8.33 (d, J=7.9Hz, 1H) ,8.20(d,J＝7.9Hz,1H),7.98(t,J＝7.9Hz,1H),7.35-7.36(m,1H),7.33(d,J＝7.9Hz,4H),7.25(d, J＝8.0Hz, 4H), 5.22(s, 4H), 5.14(t, J＝5.7Hz, 2H), 4.44(d, J＝5.6Hz, 4H). Its structural formula is:

Add 440.0 mg (1.0 mmol) of intermediate Ica, 1016 mg (4 mmol) of intermediate M, 10 mL of dry DMF to 50 mL, add 1088 mg (8 mmol) of t-BuOK, and stir at room temperature for 3 hours under the protection of N ₂ . TLC spot plate tracking, after the completion of the reaction, the solvent was distilled under reduced pressure to adjust the pH to 4-5, the residue was separated by column chromatography, using dichloromethane: methanol = 20: 1 as eluent, column chromatography separation and purification 574.4 mg of pure dark brown product compound was obtained in 80% yield. ¹ H NMR (500 MHz, DMSO-d6, ppm): δ 12.41 (s, 2H), 8.57 (d, J=7.2 Hz, 2H), 8.44 (d, J=7.2 Hz, 2H) 8.33 (d, J=7.9Hz, 1H), 8.20(d, J=7.9Hz, 1H), 7.98(t, J=7.9Hz, 1H), 7.35-7.36(m, 1H), 7.33(d, J=7.9 Hz, 4H), 7.25(d, J=8.0Hz, 4H), 5.36(s, 4H), 5.22(s, 4H), 4.44(d, J=5.6Hz, 4H). Its structural formula is:

Example 4: Preparation of peryleneimide fluorescently labeled O ⁶ -benzylguanine homomolecular fluorescent twin label d:

a. In a 50 mL round-bottomed flask, add 532.0 mg (1.0 mmol) of tetrachloroperylene anhydride, 20 mL of absolute ethanol as solvent, and 274.0 mg (2.0 mmol) of aminomethylbenzyl alcohol, and stir at 100° C. for 6 hours under reflux. After the reaction was completed, the crude product was obtained by suction filtration, and then separated by column chromatography, with petroleum ether: ethyl acetate=1:1 as the eluent, and the column chromatography was separated and purified to obtain 477.9 mg of dark red solid compound IVa, with a yield of 75 %. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 7.92 (s, J=8.5Hz, 4H), 7.33 (d, J=7.9Hz, 4H), 7.25 (d, J=8.0Hz, 4H) ,5.22(s,4H),5.14(t,J＝5.7Hz,2H),4.44(d,J＝5.6Hz,4H). Its structural formula is:

In a 25mL round-bottomed flask, add 230.4mg (0.3mmol) of Intermediate IVa, 225mg (1.5mmol) of p-tert-butylphenol, 10mL of N,N-dimethylformamide, add 124.2mg (0.9mmol) of potassium carbonate, 130 ℃ reaction 6h. TLC spot plate tracking, after the completion of the reaction, the solvent was distilled under reduced pressure, the pH was adjusted to 4-5, the residue was separated by column chromatography, and dichloromethane: methanol = 30: 1 was used as the eluent, and the column chromatography was separated and purified. 293.8 mg of dark purple-red solid IIca was obtained with a yield of 80%. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 7.92(s, 4H), 7.29(d, J=8.5Hz, 8H), 7.33(d, J=7.9Hz, 4H), 6.74(d, J＝8.0 Hz, 8H), 7.25(d, J＝8.0Hz, 4H), 5.22(s, 4H), 5.14(t, J＝5.7Hz, 2H), 4.44(d, J＝5.6Hz, 4H) , 1.42(s,36H). Its structural formula is:

In 50mL, add Intermediate IVb 244.8mg (0.2mmol), Intermediate M 203.2mg (0.8mmol), 10mL dry N,N-dimethylformamide, add t-BuOK 217.6mg (1.6mmol), in N ₂ Stir at room temperature for 3 hours under the protection. TLC spot plate tracking, after the completion of the reaction, the solvent was distilled under reduced pressure, the residue was separated by column chromatography, using dichloromethane: methanol = 20:1 as the eluent, column chromatography separation and purification to obtain a dark purple pure product compound 232.2 mg, 78% yield. ¹ H NMR (500MHz, DMSO-d6, ppm): δ 12.41 (s, 2H), 8.44 (d, J=7.2Hz, 2H), 7.92 (s, 4H), 7.29 (d, J=8.5Hz, 8H), 7.33(d, J＝7.9Hz, 4H), 6.74(d, J＝8.0Hz, 8H), 7.25(d, J＝8.0Hz, 4H), 5.36(s, 4H), 5.22(s, 4H), 4.44(d, J=5.6Hz, 4H), 1.42(s, 36H). Its structural formula is:

Claims (3)

1. a O ⁶ -benzylguanine derivative of naphthalimide fluorescent labeling is characterized in that, the general structural formula of this derivative is one of the following: a.

c.

wherein R is an alkyl group. 2. a kind of preparation method of the O ⁶ -benzylguanine derivative of naphthalimide fluorescent labeling according to claim 1, is characterized in that, the concrete steps of this method are: a. Dissolve 6-chloroguanine and N-methylpyrrolidine in N,N-dimethylformamide in a molar ratio of 1:1.5-1:2.1, at 60℃-100℃, react for 24h-48h, The reaction finishes suction filtration, and the filter residue is washed with acetone, and suction filtration obtains compound M, and its structural formula is:

b. Dissolve 4-bromo-1,8-naphthalene anhydride and organic sulfonate in absolute ethanol at a molar ratio of 1:1.2-1:1.5, react under reflux conditions for 6h-12h, cool, and filter out the solid crude The product is separated and purified to obtain compound Ib, and its structural formula is:

The structural formula of the organic sulfonate is: NH ₂ RSO ₃ H; c. Dissolve the intermediate product Compound 1b, aminomethylbenzyl alcohol and potassium carbonate obtained in step b in N,N-dimethylformamide in a molar ratio of 1:1:1.2-1; 1.5:2.0, and react under reflux for 6-8h , obtain a dark brown reaction solution, adjust the pH to 4-5, remove most of the solvent to obtain a paste-like substance, suction filtration to obtain a crude product; and then separate and purify to obtain compound Ic, whose structural formula is:

d. The intermediate product Compound Ic obtained in step c and compound M and t-BuOK obtained in step a are dissolved in N,N-dimethylformamide in a molar ratio of 1:2:5-1:3:5, inert Under the protection of atmosphere, the reaction is carried out for 3h-8h, and the naphthalimide fluorescently labeled O ⁶ -benzylguanine derivative a is obtained by separation and purification, and its structural formula is:

3. a preparation method of the O ⁶ -benzylguanine derivative of naphthalimide fluorescent labeling according to claim 1, is characterized in that, the concrete steps of this method are: e. Dissolve 4-bromo-1,8-naphthalene anhydride and aminomethylbenzyl alcohol in absolute ethanol at a molar ratio of 1:1.2-1:1.5, react under reflux conditions for 6h-12h, cool, and filter out the solid The crude product is separated and purified to obtain compound IIIa, whose structural formula is:

f. The intermediate product compound IIIa, aminomethylbenzyl alcohol and potassium carbonate obtained in step e were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1.2-1; 1.5:2.0, and the reaction was refluxed for 6-8h , obtain a dark brown reaction solution, adjust the pH to 4-5, remove most of the solvent to obtain a paste-like substance, suction filtration to obtain a crude product; and then separate and purify to obtain compound IIIb, whose structural formula is:

g. Dissolve the product compound IIIb, compound M, t-BuOK obtained in step f in N,N-dimethylformamide at a molar ratio of 1:2:5-1:3:5. The chemical formula of the compound M is for:

Under the protection of an inert atmosphere, the reaction is carried out for 3h-8h, and the fluorescent protein marker c of the naphthalimide fluorescently labeled O ⁶ -benzylguanine homomolecular twin is obtained, and its structural formula is:

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