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

Fluorescent-labeled O of naphthalimide or perylene bisimide6-benzylguanine derivatives and process for the preparation thereof Download PDF

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CN110143975B
CN110143975B CN201910416208.4A CN201910416208A CN110143975B CN 110143975 B CN110143975 B CN 110143975B CN 201910416208 A CN201910416208 A CN 201910416208A CN 110143975 B CN110143975 B CN 110143975B
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周祎迪
沈卫平
于静
宋力平
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University of Shanghai for Science and Technology
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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

Fluorescent-labeled O of naphthalimide or perylene bisimide6-benzylguanine derivatives and process for the preparation thereof
Technical Field
The invention belongs to the synthesis of protein markers, and relates to O fluorescently labeled with naphthalimide or perylene bisimide6-benzylguanine derivatives and processes for their preparation.
Background
The protein-specific small-molecule fluorescent dye labeling technology overcomes many limitations of fluorescent protein (fluorescent protein FP) in the field of protein research. The technology constructs protein, polypeptide and protein-labeled recombinant protein through a standard genetic engineering means, and realizes the fixed-point fluorescent labeling of target protein by utilizing the specificity and high-efficiency recognition of a fluorescent probe ligand and corresponding polypeptide and protein labels. The SNAP-Tag is distinguished from a plurality of targeting labeling technologies by virtue of the unique advantages of high specificity and stability of target protein, functional diversity of ligand and the like. Becomes one of the best fusion labels and is widely applied in a plurality of fields. For example, in vivo imaging, cell imaging, in vitro protein studies, in vivo protein studies, microbial pathogenesis studies, disease diagnosis, and the like.
SNAP-Tag was developed by johnsson, 2003, by modifying the artificial O6-guanine-DNA alkyltransferase (human O)6-alkylguanine-DNA alkylitransferase, hAGC). hAGT is a monomeric protein consisting of 207 amino acids which converts methylated DNA, guanine O6The positional methyl group is transferred to the native hAGT active cysteine thiol group (Cys145) to effect DNA repair. Aiming at the modification of hAGT structure, on one hand, the interaction of the original protein structure in methylated DNA is reduced, and on the other hand, the interaction with O is improved6Specific reactivity of modified Benzylguanine (BG). The cysteine sulfydryl (Cys145) positioned at the active site of the SNAP-Tag can react with the benzyl group of BG to form a very stable covalent bond between the protein and a substrate to realize labeling, and the process is shown as follows:
Figure BDA0002064516160000011
the SNAP-Tag has the following unique advantages: the protein label and the substrate have high reaction speed and high specificity; the protein combined by the covalent bond has good stability, and can be stably marked even under the denaturation condition of SDS-PAGE analysis in vitro; the label specific substrate BG is combined with the functional dye through a modular design, so that the flexibility is high; the method is convenient for deriving diversified fluorescent dyes, has strong universality and can meet the requirements of various fluorescent imaging. At present, SNAP-Tag is widely applied to the research of intracellular protein labeling, in-vitro analysis and targeted fluorescence detection. However, this method also has certain disadvantages; the synthesis of a label specific substrate BG is complex, and the problem of poor solubility and dispersibility after the label specific substrate BG is combined with a dye possibly exists; most dyes are used, the fluorescence signals before and after labeling have no obvious difference, and the dyes need to be washed sufficiently to remove non-specific staining, and background interference can exist. The invention provides a novel protein-specific molecular fluorescent marker for overcoming the defects in the SNAP-Tag technology.
Disclosure of Invention
One object of the present invention is to provide a fluorescent-labeled O of naphthalimide and perylene bisimide6-benzylguanine derivatives.
The second purpose of the invention is to provide a preparation method of the derivative.
To achieve the above object ID, the present invention employs the following reaction mechanism:
Figure BDA0002064516160000021
Figure BDA0002064516160000031
Figure BDA0002064516160000041
Figure BDA0002064516160000051
according to the reaction mechanism, the invention adopts the following technical scheme:
o fluorescently labeled with naphthalimide or perylene bisimide6-benzyl guanine derivatives, characterized in that the compound has the general structural formula one of:
a.
Figure BDA0002064516160000061
b.
Figure BDA0002064516160000062
c.
Figure BDA0002064516160000063
d.
Figure BDA0002064516160000064
wherein R ═ alkyl, cycloalkyl, aryl, heteroaryl, or polyoxyethylene ether chains.
The naphthalimide fluorescently labeled O prepared according to claim 16-benzylguanine derivative a, characterized in that the process comprises the specific steps of:
a. dissolving 6-chloroguanine and N-methylpyrrolidine in a molar ratio of 1:1.5-1:2.1 in N, N-dimethylformamide, reacting for 24-48 h at 60-100 ℃, filtering after the reaction is finished, washing filter residue with acetone, and filtering to obtain a compound M, wherein the structural formula of the compound M is as follows:
Figure BDA0002064516160000071
b. dissolving 4-bromo-1, 8-naphthalic anhydride and organic sulfonate in absolute ethyl alcohol according to a molar ratio of 1:1.2-1:1.5, reacting for 6-12 h under a reflux condition, cooling, filtering out a solid crude product, and separating and purifying to obtain a compound Ib, wherein the structural formula of the compound Ib is as follows:
Figure BDA0002064516160000072
the structural formula of the organic sulfonate is as follows: NH (NH)2RSO3H；
c. B, mixing the intermediate product Ib obtained in the step b, aminomethyl benzyl alcohol and potassium carbonate according to a ratio of 1:1: 1.2-1; dissolving the raw materials in N, N-dimethylformamide according to a molar ratio of 1.5:2.0, performing reflux reaction for 6-8 hours to obtain dark brown reaction liquid, adjusting the pH to 4-5, removing most of solvent to obtain pasty substances, and performing suction filtration to obtain a crude product; then separating and purifying to obtain a compound ic, wherein the structural formula is as follows:
Figure BDA0002064516160000073
d. dissolving the intermediate product IC obtained in the step c and the intermediate M and t-BuOK obtained in the step a in N, N-dimethylformamide according to the molar ratio of 1:2:5-1:3:5, reacting for 3h-8h under the protection of inert atmosphere, and separating and purifying to obtain O fluorescently labeled with naphthalimide6-benzylguanine derivative a of formula:
Figure BDA0002064516160000081
preparation of the fluorescent-labeled O of naphthalimide according to claim 16-benzylguanine with a molecular geminal fluorescent protein marker c, characterized in that it comprises the following steps:
e. dissolving 4-bromo-1, 8-naphthalic anhydride and aminomethyl benzyl alcohol in absolute ethyl alcohol according to a molar ratio of 1:1.2-1:1.5, reacting for 6-12 h under a reflux condition, cooling, filtering out a solid crude product, and separating and purifying to obtain a compound IIIa, wherein the structural formula of the compound IIIa is as follows:
Figure BDA0002064516160000082
f. e, mixing the intermediate IIIa obtained in the step e, aminomethyl benzyl alcohol and potassium carbonate according to a ratio of 1:1: 1.2-1; dissolving the raw materials in N, N-dimethylformamide in a molar ratio of 1.5:2.0, performing reflux reaction for 6-8 hours to obtain dark brown reaction liquid, adjusting the pH to 4-5, removing most of solvent to obtain pasty substances, and performing suction filtration to obtain a crude product; then separating and purifying to obtain a compound IIIb, wherein the structural formula is as follows:
Figure BDA0002064516160000083
g. dissolving the product IIIb obtained in the step f and the product M, t-BuOK obtained in the step a in N, N-dimethylformamide according to the molar ratio of 1:2:5-1:3:5, reacting for 3h-8h under the protection of inert atmosphere, and separating to obtain O fluorescently labeled with naphthalimide6-benzylguanine syntolecular geminal fluorescent protein marker c having the structural formula:
Figure BDA0002064516160000084
preparation of perylene bisimide fluorescent labeled O according to claim 16-benzylguanine derivatives, characterized in that the process comprises the specific steps of:
a. dissolving tetrachloroperylene anhydride and organic sulfonate in the molar ratio of 1:1.2-1:1.5 in ethylene glycol monomethyl ether, and reacting at 100-120 DEG C
And (4) separating and purifying for 6h-12h to obtain a compound IIa, wherein the structural formula of the compound IIa is as follows:
Figure BDA0002064516160000091
the structural formula of the tetrachloroperylene anhydride is as follows:
Figure BDA0002064516160000092
the structural formula of the organic sulfonate is as follows: NH (NH)2RSO3H；
b. Dissolving the intermediate IIa obtained in the step a and aminomethyl benzyl alcohol in absolute ethyl alcohol according to the molar ratio of 1:1.2-1:1.5, stirring and reacting for 6-12 h under the reflux condition, removing the solvent, and separating and purifying the obtained crude product to obtain a solid compound IIb, wherein the structural formula of the solid compound IIb is as follows:
Figure BDA0002064516160000101
c. dissolving the intermediate product IIb obtained in the step b, p-tert-butylphenol and potassium carbonate in N, N-dimethylformamide according to the molar ratio of 1:4:4-1:5:6, stirring and reacting for 6-8 hours under the reflux condition, and separating and purifying the obtained crude product to obtain a compound IIc, wherein the structural formula of the compound IIc is as follows:
Figure BDA0002064516160000102
d. reacting the product IIc obtained in the step c, the product M in the step a of claim 2 and t-BuOK for 3 to 5 hours according to the molar ratio of 1:1.2:3 to 1:2.1:5 under the protection of inert atmosphere, and separating and purifying to obtain the perylene imide fluorescent labeled O6-benzylguanine derivative b of formula:
Figure BDA0002064516160000111
preparation of the perylene bisimide fluorescent labeled O prepared according to claim 16The preparation method of the benzyl guanine homomolecular fluorescence twin marker is characterized by comprising the following specific steps:
e. dissolving tetrachloroperylene anhydride and aminomethyl benzyl alcohol in a molar ratio of 1:2-1:3 in ethylene glycol monomethyl ether, reacting at 100-140 ℃ for 6-12 h, and separating and purifying to obtain a compound IVa, wherein the structural formula is as follows:
Figure BDA0002064516160000112
f. dissolving the product IVa obtained in the step e, p-tert-butylphenol and potassium carbonate in N, N-dimethylformamide according to the molar ratio of 1:4:5-1:5:5, stirring and reacting for 6-8 hours under the reflux condition, then evaporating the solvent, and separating and purifying the obtained crude product to obtain a compound IVb, wherein the structural formula of the compound IVb is as follows:
Figure BDA0002064516160000121
g. reacting the product IVb obtained in the step f with the product M and t-BuOK obtained in the step a of claim 2 for 3 to 5 hours in the presence of inert atmosphere according to the molar ratio of 1:2:5 to 1:2.5:5, and separating and purifying to obtain the perylene imide fluorescence labeled O6-benzylguanine homomolecular fluorescent twin marker d, having the structural formula:
Figure BDA0002064516160000122
the taurine chemically modified naphthalimide and perylene bisimide with excellent water solubility of the invention can prepare O with high fluorescence yield and excellent water solubility6Benzyl guanine derivatives for improving the Tag-specific substrate O in the SNAP-Tag protein labeling technique6-Benzylguanine derivatives (BG) have poor water solubility and insufficient interference with non-specific 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, the product presents good water solubility, and the product further uses aminomethyl benzyl alcohol to react and then is coupled with the guanine active intermediate to obtain the target product. Meanwhile, the dimer of the target molecule is synthesized (by utilizing the same molecule twin synergistic effect), and the targeting performance of the specific marker protein is expected to be improved.
Drawings
FIG. 1 is the fluorescent O-labeled naphthalimide of example one6-benzylguanine derivative A1Normalized fluorescence excitation emission spectrum of (a);
FIG. 2 is a perylene imide fluorescent labelled O of example 26Normalized fluorescence excitation emission spectrum of benzylguanine derivative c.
Detailed Description
The present invention is further illustrated by the following examples, but the present invention is not limited to the following examples.
The first embodiment is as follows: naphthalimide fluorescence labeled O6-benzylguanine derivative A1Preparation of
a. Adding 6-chlorobird into a 250mL round-bottom flaskPurine 1.69g (10mmol), 80mL of N, N-dimethylformamide as a solvent, N-methylpyrrole 1.75g (21mmol) were added, and the mixture was stirred at 60 ℃ for 24 hours. After completion of the reaction, it was cooled, and the precipitate was washed with 20mL of acetone and then purified by filtration to obtain 1.94g of a white solid with a yield of 76.5%.1H 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;
Figure BDA0002064516160000131
b. 277.0mg (1.0mmol) of 4-bromo-1, 8-naphthalic anhydride, 125.1mg (1.0mmol) of taurine and 20mL of EtOH as a solvent were added to a 50mL round-bottomed flask, and the mixture was stirred at 78 ℃ under reflux for 6 hours. After the reaction is finished, a crude product is obtained by suction filtration and then is separated by column chromatography, and the reaction is carried out by using dichloromethane: methanol 60: 1 as eluent, and performing column chromatography separation and purification to obtain a brown solid I ba; 279.6mg, yield 79%.1H NMR(500MHz,D2O, ppm): δ 7.83(d, J ═ 7.2Hz,2H),7.59(d, J ═ 7.9Hz,1H),7.35(t, J ═ 7.9Hz,1H),7.26(d, J ═ 7.9Hz,1H),4.06(t, J ═ 5.6Hz,2H),3.15(t, J ═ 5.7Hz,2H), formula (la);
Figure BDA0002064516160000141
c. a50 mL round-bottomed flask was charged with 355.0mg (1.0mmol) of intermediate I ba, 137.0mg (1.0mmol) of aminomethyl benzyl alcohol, and 20mL of N, N-dimethylformamide as a solvent, and 138.0mg (1mmol) of potassium carbonate was added, followed by stirring under reflux for 8 hours. After the reaction is finished, adjusting the pH value to be 4-5, distilling the solvent under reduced pressure, and separating residues through column chromatography by using dichloromethane: methanol 40: 1 as eluent, column chromatography separation and purification to obtain a brown solid I ca 264.0mg with a yield of 60%.1H NMR(500MHz, DMSO-d6,ppm):δ8.57(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) and the structural formula is shown in the specification;
Figure BDA0002064516160000142
d. to 50mL was added 440.0mg (1.0mmol) of intermediate I ca, 635.1mg (2.5mmol) of intermediate M, 10mL of dry N, N-dimethylformamide, and t-BuOK 684.80mg (5mmol) in N2Stirring for 3 hours at normal temperature under protection. TLC point plate tracking, after the reaction is finished, the solvent is decompressed and distilled, the pH is adjusted to 4-5, and the residue is separated by column chromatography and is mixed with dichloromethane: methanol 20: 1 as eluent, column chromatography separation and purification gave 303.7mg of dark brown solid with 53% yield.1H NMR (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 ═ 7.9Hz,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 ═ 5.6Hz,2H), 4.6 (t, 6, 5.6H), 5.15H, 15 (J ═ 7.7H), and their structural formulae:
Figure BDA0002064516160000151
example two: perylene bisimide fluorescent labelled O6-benzylguanine derivative B1Preparing;
a. a50 mL round-bottomed flask was charged with 532.0mg (1.0mmol) of tetrachloroperylene anhydride, 20mL of ethylene glycol methyl ether as a solvent, and 125.1mg (1.0mmol) of taurine, and the mixture was stirred under reflux at 100 ℃ for 6 hours. After the reaction is finished, a crude product is obtained by suction filtration and then is separated by column chromatography, and the reaction is carried out by using dichloromethane: methanol 60: 1 as eluent, and performing column chromatography separation and purification to obtain a deep red solid compound IIaa; 477.9mg, yield 75%.1H NMR (500MHz, DMSO-d6, ppm): δ 7.92(s,2H),6.28(s,2H),3.64(t, J ═ 5.6Hz,2H),3.33(t, J ═ 5.7Hz,2H), which has the structural formula:
Figure BDA0002064516160000152
b. a50 mL round bottom flask was charged with intermediate IIaa 637.0mg (1.0mmol), aminomethyl benzyl alcohol 137.0mg (1.0mmol), and 20mL of DMF as solvent, and stirred under reflux for 8 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was separated by column chromatography using dichloromethane: 40 parts of methanol: 1 as eluent, column chromatography separation and purification to obtain dark purple solid II ba 612.4mg with 81% yield.1H NMR (500MHz, DMSO-d6, ppm): δ 7.92(s,2H),7.33(d, J ═ 7.9Hz,2H),7.25(d, J ═ 8.0Hz,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 ═ 5.6Hz,2H),3.33(t, J ═ 5.6Hz,2H), the structural formula of which is:
Figure BDA0002064516160000161
c. 50mL of intermediate II ba 756.0mg (1.0mmol), p-tert-butylphenol 750mg (5mmol), and 20mL of N, N-dimethylformamide were added thereto, potassium carbonate 414.0mg (3mmol) were added, and the reaction was carried out at 130 ℃ for 6 hours. TLC point plate tracking, after the reaction is finished, the solvent is decompressed and distilled, the pH is adjusted to 4-5, and the residue is separated by column chromatography and is mixed with dichloromethane: methanol 30: 1 as eluent, column chromatography separation and purification are carried out to obtain 1.0302g of a dark purple solid compound II ca with the yield of 85 percent.1H 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 ═ 8.0Hz,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) and the structural formula:
Figure BDA0002064516160000162
d. 50mL of intermediate IIca 1213.5mg (1.0mmol), intermediate M635.1 mg (2.5mmol),10mL of dry N, N-dimethylformamide were added, t-BuOK 684.80mg (5mmol) was added, and the reaction mixture was stirred at room temperature under N2Stirring for 3 hours at normal temperature under protection. TLC point plate tracking, after the reaction is finished, the solvent is decompressed and distilled, the pH is adjusted to 4-5, and the residue is separated by column chromatography and is mixed with dichloromethane: methanol 20: 1 as eluent, and separating and purifying by column chromatography to obtain dark brown pure product compound874.2mg, 63% yield.1H NMR(500MHz,DMSO-d6,ppm):δ12.41(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)
Figure BDA0002064516160000171
Example three: naphthalimide fluorescence labeled O6-benzylguanine with molecular geminal fluorescent protein marker c method:
a. a50 mL round-bottomed flask was charged with 554.0mg (2.0mmol) of 4-bromo-1, 8-naphthalic anhydride, 274.0mg (2.0mmol) of aminomethylbenzyl alcohol, and 20mL of absolute ethanol as a solvent, and the mixture was stirred under reflux at 78 ℃ for 6 hours. After the reaction is finished, a crude product is obtained by suction filtration, and then is separated by column chromatography, and the reaction is carried out by using dichloromethane: methanol 100: 1 as eluent, and the white solid obtained by column chromatography separation and purification is pure compound IIIa 636.6mg with 82% yield.1H 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), formula:
Figure BDA0002064516160000172
a50 mL round-bottomed flask was charged with 395.0mg (1.0mmol) of intermediate IIIa, 137.0mg (1.0mmol) of aminomethyl benzyl alcohol, and 20mL of N, N-dimethylformamide as a solvent, and 138.0mg (1mmol) of potassium carbonate was added, followed by stirring under reflux for 8 hours. After the reaction is finished, adjusting the pH value to be 4-5, distilling the solvent under reduced pressure, and separating residues through column chromatography by using dichloromethane: 40 parts of methanol: 1 as eluent, column chromatography separation and purification to obtain light brown solid IIIb 327.6mg, the yield is 70%.1H NMR(400MHz, DMSO-d6,ppm):δ8.57(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(m1H),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) the formula is:
Figure BDA0002064516160000181
to 50mL was added 440.0mg (1.0mmol) of intermediate I ca, 1016mg (4mmol) of intermediate M, 10mL of dry DMF, t-BuOK1088mg (8mmol) in N2Stirring for 3 hours at normal temperature under protection. TLC point plate tracking, after the reaction is finished, the solvent is decompressed and distilled, the pH is adjusted to 4-5, and the residue is separated by column chromatography and is mixed with dichloromethane: methanol 20: 1 as eluent, column chromatography separation and purification to obtain the dark brown pure product compound 574.4mg with 80% yield.1H NMR (500MHz, DMSO-d6, ppm): δ 12.41(s,2H),8.57(d, J ═ 7.2Hz,2H),8.44(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 to 7.36(m, 1H),7.33(d, J ═ 7.9Hz,4H),7.25(d, J ═ 8.0Hz,4H),5.36(s,4H),5.22(s,4H),4.44(d, J ═ 5.6Hz,4H), and the structural formulae thereof:
Figure BDA0002064516160000182
example four: perylene bisimide fluorescent labelled O6-preparation of benzyl guanine homomolecular fluorescent twin marker d:
a. a50 mL round-bottomed flask was charged with 532.0mg (1.0mmol) of tetrachloroperylene anhydride, 20mL of anhydrous ethanol as a solvent, and 274.0mg (2.0mmol) of aminomethylbenzyl alcohol, and the mixture was stirred under reflux at 100 ℃ for 6 hours. After the reaction is finished, a crude product is obtained by suction filtration, and then is separated by column chromatography, and the weight ratio of petroleum ether: ethyl acetate ═ 1:1 as eluent, column chromatography separation and purification are carried out to obtain a dark red solid compound IVa 477.9mg with 75 percent of yield.1H 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), structural formulae of which are:
Figure BDA0002064516160000191
a25 mL round-bottom flask was charged with 230.4mg (0.3mmol) of intermediate IVa, 225mg (1.5mmol) of p-tert-butylphenol, 10mL of N, N-dimethylformamide, 124.2mg (0.9mmol) of potassium carbonate and reacted at 130 ℃ for 6 hours. TLC point plate tracking, after the reaction is finished, the solvent is decompressed and distilled, the pH is adjusted to 4-5, and the residue is separated by column chromatography and is mixed with dichloromethane: methanol 30: 1 as eluent, column chromatography separation and purification to obtain dark purple solid IIca 293.8mg with 80% yield.1H 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 ═ 8.0Hz,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) having the structural formula:
Figure BDA0002064516160000192
50mL of the intermediate IV b244.8mg (0.2mmol), the intermediate M203.2 mg (0.8mmol), and 10mL of dry N, N-dimethylformamide were added thereto, and t-BuOK 217.6mg (1.6mmol) was added thereto under stirring2Stirring for 3 hours at normal temperature under protection. TLC plates were followed and after completion of the reaction, the solvent was distilled under reduced pressure and the residue was isolated by column chromatography with dichloromethane: methanol 20: 1 as eluent, column chromatography separation and purification to obtain the deep purple pure product compound 232.2mg with 78% yield.1H 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), which has the structural formula:
Figure BDA0002064516160000201

Claims (3)

1. naphthalimide fluorescence labeled O6-benzylguanine derivatives, characterized in that they have the general structural formula one of the following:
a.
Figure FDA0003532136910000011
c.
Figure FDA0003532136910000012
wherein R is an alkyl group.
2. The naphthalimide fluorescently labeled O of claim 16A process for the preparation of (E) -benzylguanine derivatives, characterized in that it comprises the following steps:
a. dissolving 6-chloroguanine and N-methylpyrrolidine in a molar ratio of 1:1.5-1:2.1 in N, N-dimethylformamide, reacting for 24-48 h at 60-100 ℃, filtering after the reaction is finished, washing filter residue with acetone, and filtering to obtain a compound M, wherein the structural formula of the compound M is as follows:
Figure FDA0003532136910000013
b. dissolving 4-bromo-1, 8-naphthalic anhydride and organic sulfonate in absolute ethyl alcohol according to a molar ratio of 1:1.2-1:1.5, reacting for 6-12 h under a reflux condition, cooling, filtering out a solid crude product, and separating and purifying to obtain a compound Ib, wherein the structural formula of the compound Ib is as follows:
Figure FDA0003532136910000014
the structural formula of the organic sulfonate is as follows: NH (NH)2RSO3H；
c. B, mixing the intermediate compound Ib obtained in the step b, aminomethyl benzyl alcohol and potassium carbonate according to a ratio of 1:1: 1.2-1; dissolving the raw materials in N, N-dimethylformamide in a molar ratio of 1.5:2.0, performing reflux reaction for 6-8 hours to obtain a dark brown reaction solution, adjusting the pH to 4-5, removing most of the solvent to obtain a pasty substance, and performing suction filtration to obtain a crude product; then separating and purifying to obtain a compound ic, wherein the structural formula is as follows:
Figure FDA0003532136910000021
d. dissolving the intermediate compound IC obtained in the step c and the compound M and t-BuOK obtained in the step a in N, N-dimethylformamide according to the molar ratio of 1:2:5-1:3:5, reacting for 3h-8h under the protection of inert atmosphere, and separating and purifying to obtain O fluorescently labeled with naphthalimide6-benzylguanine derivative a of formula:
Figure FDA0003532136910000022
3. the naphthalimide fluorescently labeled O of claim 16A process for the preparation of (E) -benzylguanine derivatives, characterized in that it comprises the following steps:
e. dissolving 4-bromo-1, 8-naphthalic anhydride and aminomethyl benzyl alcohol in absolute ethyl alcohol according to a molar ratio of 1:1.2-1:1.5, reacting for 6-12 h under a reflux condition, cooling, filtering out a solid crude product, and separating and purifying to obtain a compound IIIa, wherein the structural formula of the compound IIIa is as follows:
Figure FDA0003532136910000023
f. e, mixing the intermediate compound IIIa obtained in the step e, aminomethyl benzyl alcohol and potassium carbonate according to the ratio of 1:1: 1.2-1; dissolving the raw materials in N, N-dimethylformamide in a molar ratio of 1.5:2.0, performing reflux reaction for 6-8 hours to obtain a dark brown reaction solution, adjusting the pH to 4-5, removing most of the solvent to obtain a pasty substance, and performing suction filtration to obtain a crude product; then separating and purifying to obtain a compound IIIb, wherein the structural formula is as follows:
Figure FDA0003532136910000031
g. dissolving the product obtained in the step f, namely a compound IIIb and a compound M, t-BuOK in N, N-dimethylformamide according to a molar ratio of 1:2:5-1:3:5, wherein the compound M has a chemical formula:
Figure FDA0003532136910000032
reacting for 3-8 h under the protection of inert atmosphere, and separating to obtain naphthalimide fluorescence labeled O6-benzylguanine syntolecular geminal fluorescent protein marker c having the structural formula:
Figure FDA0003532136910000033
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