Environment-sensitive switch fluorescent nucleoside as well as preparation method and application thereof

Abstract

The invention discloses environment-sensitive switch fluorescent nucleosides as well as a preparation method and application thereof, and belongs to the technical field of molecular biology. The environment-sensitive switch fluorescent nucleoside comprises 12 2-amino-2 '-deoxyadenosine substituted by aromatic vinyl at C-8 sites and 7 2'-deoxyisoguanosine derivatives substituted by aryl vinyl at C-8 sites. Through the designed synthesis method, the aromatic vinyl modified environment-sensitive switch fluorescent nucleosides can be obtained with high yield. All the new compounds have fluorescence characteristics and show different degrees of sensitivity to solvent polarity. Wherein the compound 4f and the compound 4e both show high fluorescence quantum efficiency in a non-polar solvent and a polar solvent; the compound 5a and the compound 5b have remarkable fluorescence quantum efficiency in a non-polar solvent, and are expected to be used as switching type fluorescence probes. The compound 5a can be further processed and linked into a deoxyoligonucleotide chain to realize on and off of fluorescence, and the fluorescent nucleosides are suitable for the fields of nucleic acid labeling, fluorescent probes, biomedicine and the like.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a class of environmentally sensitive switchable fluorescent nucleosides, their preparation methods, and applications. Background Technology

[0002] The dynamic structure and functional diversity of DNA and RNA are the core foundation of life activities. Their conformational changes, base pairing, and interactions with proteins directly regulate key processes such as gene expression and signal transduction. However, current technologies struggle to analyze these dynamic events in real time and in situ, especially lacking molecular probes capable of precisely responding to changes in the microenvironment (such as polarity, pH, viscosity, and ionic strength). Fluorescent nucleoside analogs, due to their ability to directly intercalate into nucleic acid chains while retaining fluorescence properties, have become ideal tools for monitoring nucleic acid dynamics, but their development still faces significant challenges.

[0003] Most fluorescent nucleoside analogs exhibit strong fluorescence in their free state, but after incorporation into nucleic acids, they are quenched by neighboring bases due to electron transfer effects (such as photoinduced electron transfer, PET) or energy transfer (such as Förster resonance energy transfer, FRET), leading to a significant decrease in fluorescence intensity or even complete quenching. For example, in 1963, Stryer first reported the emission-bearing adenine analog 2'-aminopurine (2-AP). When 2-AP entered the DNA chain, the fluorescence quantum efficiency showed a significant decrease or even quenching effect, with a maximum decrease of 68-fold. Traditional fluorescent probes (such as organic dyes and quantum dots) have the following drawbacks: short-wavelength excitation easily induces autofluorescence in cells, resulting in a low signal-to-noise ratio (SNR); the fluorescence signal is sluggish in response to changes in the microenvironment and cannot distinguish multi-parameter changes in complex biological systems; some probes, due to hydrophobicity or uneven charge distribution, destroy the natural structure and function of nucleic acids after insertion.

[0004] Although "environmentally sensitive" fluorescent nucleosides can respond to changes in the microenvironment through fluorescence "on-off" (such as fluorescence enhancement when polarity decreases), existing designs still have contradictions: most probes have high fluorescence in the free state and low fluorescence in the nucleic acid state, which is the opposite of the ideal state (high fluorescence in the nucleic acid state); they are only sensitive to a single parameter (such as pH), making it difficult to fully reflect the dynamics of the microenvironment.

[0005] In summary, developing novel fluorescent nucleosides that are sensitive to the microenvironment, exhibit weak fluorescence at the nucleoside level, and show significant fluorescence enhancement or even switching effects after incorporation into nucleic acids has become one of the urgent problems to be solved in the field of nucleic acid fluorescent probes. Summary of the Invention

[0006] To address the problems of low quantum efficiency and weak fluorescence in purine ring ethynyl-modified fluorescent nucleosides, and the continued short emission wavelength and interference with spectral analysis due to background light interference even after iterative extension of the benzene ring conjugated structure, this invention aims to provide a class of environmentally sensitive switchable fluorescent nucleosides, their preparation method, and applications. By replacing the ethynyl group with a vinyl group, a new fluorescent nucleoside is prepared, resulting in a redshift of the emission wavelength and yielding purine nucleoside derivatives emitting visible fluorescence, thus enhancing their advantages in nucleic acid research.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a class of environmentally sensitive, switchable fluorescent nucleosides having the structure shown in Formula 1 or Formula 2: or ; Where R is selected from , , , , , , , , , , and Any one of them.

[0008] Furthermore, the aforementioned environmentally sensitive switchable fluorescent nucleotide has the following structural formula: 4e, 4f, 5a, 5b

[0009] This invention provides a method for preparing the above-mentioned type of environmentally sensitive switchable fluorescent nucleosides, comprising: Step 1: Mix 8-bromo-2-amino-2'-deoxyadenosine, base, and catalyst evenly under nitrogen protection. Add potassium trifluorovinylborate to the mixed solution for coupling reaction. After the reaction is complete, concentrate under reduced pressure and purify by column chromatography to obtain compound 1. Step 2: Compound 1 is mixed with a base and a catalyst under nitrogen protection. After adding compound 2 and reacting, the mixture is concentrated and purified by column chromatography to obtain the environmentally sensitive switch fluorescent nucleoside shown in Formula 1. The structural formula of compound 1 is as follows: ; Compound 2 is Br-R, wherein R is selected from... , , , , , , , , , , and Any one of the following; The environmentally sensitive switching fluorescent nucleoside shown in Formula 2 was obtained by reacting the compound shown in Formula 1 with sodium nitrite and glacial acetic acid, adjusting the pH value, filtering, washing and recrystallizing. The structural formula of compound 1 is as follows: ; The alkali is potassium carbonate or cesium carbonate; The catalyst is any one of bis(triphenylphosphine)palladium dichloride, palladium chloride, tetra(triphenylphosphine)palladium, and palladium acetate.

[0010] In step 1, the molar ratio of 8-bromo-2-amino-2'-deoxyadenosine to the base and catalyst is 1:1:0.1~0.2, the reaction temperature is 70~90℃, and the reaction time is 1~3h.

[0011] Furthermore, the solvent used is N,N-dimethylformamide.

[0012] Furthermore, the alkali is cesium carbonate, and the catalyst is palladium dichloride bis(triphenylphosphine).

[0013] Furthermore, the eluent used in the purification is dichloromethane and methanol in a volume ratio of 10:1.

[0014] In step 2, the molar ratio of compound 1, base, catalyst and compound 2 is 1:1~2:0.1~0.2:1~2, the reaction temperature is 100~120℃, and the reaction time is 4~6h.

[0015] Furthermore, the solvent used is N,N-dimethylformamide.

[0016] Furthermore, the alkali is potassium carbonate, and the catalyst is palladium dichloride bis(triphenylphosphine).

[0017] Furthermore, the eluent used in the purification is dichloromethane and methanol in a volume ratio of 25~30:1.

[0018] In step 2, the molar ratio of the compound of formula 1, sodium nitrite, and glacial acetic acid is 1:3~5:7~9; the reaction temperature is 50~70℃, and the reaction time is 1~3h.

[0019] Furthermore, the solvent used is a mixture of DMF and H2O with a volume ratio of 1:2.

[0020] Furthermore, the pH value is adjusted to 7-8, and the recrystallization is carried out using a mixed solvent of acetonitrile and water with a volume ratio of 1:1.

[0021] This invention provides a fluorescent nucleoside phosphoramide monomer, which is synthesized from the aforementioned type of environmentally sensitive switchable fluorescent nucleoside.

[0022] Furthermore, the structural formula of the fluorescent nucleoside phosphoramide monomer is as follows: .

[0023] Furthermore, the method for preparing the fluorescent nucleoside phosphoramide monomer includes: (1) Using compounds After reacting with N,N-dimethylacetamide dimethyl acetal at room temperature with stirring, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain the compound. The compound The molar ratio of N,N-dimethylacetamide to dimethyl acetal is 1~2:1; the reaction time is 1~3h; (2) Ice bath will contain the compound The mixture was stirred at room temperature with N,N-diphenylcarbamoyl chloride and N,N-diisopropylethylamine, washed, extracted, dried, concentrated under reduced pressure, and purified by column chromatography to obtain the compound. The compound The molar ratio of N,N-diphenylcarbamoyl chloride to N,N-diisopropylethylamine is 1:2~3; the reaction time is 1~2 h; (3) Compounds After reacting with 4,4-dimethoxytriphenylchloro at room temperature under nitrogen protection, the mixture was washed, extracted, dried, concentrated under reduced pressure, and purified by column chromatography to obtain the compound. The compound The molar ratio of 4,4-bismethoxytriphenylchloro to 4,4-bismethoxytriphenylchloro is 1:1~2; the reaction time is 1~3 h; (4) The compound N,N-diisopropylethylamine and 2-cyanoethyldiisopropylphosphonic chloride were added, and the mixture was stirred at room temperature. The mixture was then washed, extracted, dried, concentrated under reduced pressure, and purified by column chromatography to obtain the fluorescent nucleoside phosphorimide monomer. The compound The molar ratio of N,N-diisopropylethylamine and 2-cyanoethyldiisopropylphosphoryl chloride is 1:1~2:1~2; the reaction time is 0.5~1h.

[0024] This invention provides a fluorescent nucleoside triphosphate, which is synthesized from the aforementioned type of environmentally sensitive switch fluorescent nucleoside.

[0025] This invention provides the above-mentioned type of environmentally sensitive switchable fluorescent nucleosides, or the application of the above-mentioned fluorescent nucleoside phosphoramide monomer in the preparation of nucleic acids; Furthermore, the nucleic acid includes a fluorescent probe.

[0026] This invention provides the application of the above-mentioned environmentally sensitive switchable fluorescent nucleoside, or one of the above-mentioned fluorescent nucleoside phosphoramide monomers, in the preparation of bioimaging reagents.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an environmentally sensitive switchable fluorescent nucleoside that, through structural innovation and mechanism optimization, successfully solves the core problems of traditional fluorescent nucleosides, such as fluorescence quenching and high background interference in nucleic acids. It develops a class of environmentally sensitive switchable fluorescent nucleosides with "nucleic acid-state enhanced" fluorescence response and multi-parameter sensitivity. This technology provides a highly sensitive and selective molecular tool for nucleic acid dynamics research and has broad application prospects in basic life sciences and clinical medicine.

[0028] Furthermore, 4e, 4f, 5a, and 5b exhibit excellent sensitivity to environmental factors, particularly solvent polarity. Moreover, fluorescent nucleoside 5a is a novel environmentally sensitive on-off fluorescent nucleoside; at the nucleoside level, it exhibits weak fluorescence in aqueous solution, but shows significant fluorescence enhancement after incorporation into nucleic acids, making it potentially applicable in the biomedical field.

[0029] The present invention provides a method for preparing environment-sensitive switchable fluorescent nucleosides. Through optimized reaction conditions, modular design, and efficient purification strategies, it achieves highly selective and high-yield synthesis of environment-sensitive switchable fluorescent nucleosides. The yields of compounds shown in Formula 1 all reach over 70%, and the compounds shown in Formula 2 are obtained in recrystallization form without the need for silica gel column purification, demonstrating potential for industrial scale-up. This technology provides a key tool for nucleic acid dynamic research, tumor diagnosis, and drug development, possessing significant scientific value and promising clinical translation prospects.

[0030] Furthermore, using 8-bromo-2-amino-2'-deoxyadenosine as the starting material, cesium carbonate was used as HBr generated by the base neutralization reaction, and palladium dichloride of bis(triphenylphosphine) chloride was used as a catalyst to efficiently catalyze the activation of the C-Br bond, providing an active site for subsequent boric acid coupling. Potassium trifluorovinylborate was introduced to introduce trifluorovinyl groups at the 8-position of the nucleoside via the Suzuki-Miyaura coupling reaction. Its strong electron-withdrawing properties (high electronegativity of fluorine atoms) can significantly enhance the intramolecular charge transfer (ICT) effect, thereby increasing the sensitivity of the fluorescent nucleoside to changes in environmental polarity. Through the coupling reaction of compound 1 with a 4-bromoaromatic compound (al), a series of fluorescent nucleosides with different electronic effects can be rapidly constructed. The amino group of the compound is oxidized by sodium nitrite under acidic conditions to generate a diazonium salt intermediate, which is then subjected to intramolecular rearrangement after treatment with ammonia water to form a nucleoside with the ability to switch between a "dark-fluorescence state and a bright state".

[0031] The nucleoside phosphoramidide monomer designed in this invention is obtained by further conversion of an environmentally sensitive switch fluorescent nucleoside, and can be effectively applied to biomedical applications such as nucleic acid fluorescent labeling and detection technology, intracellular DNA fluorescent labeling and tracking. Attached Figure Description

[0032] Figure 1 The UV absorption and fluorescence spectra of an environmentally sensitive switchable fluorescent nucleoside compound 4f in four solvents are shown below. Figure 2 This is a comparison of the fluorescence intensity of the single-stranded deoxy oligonucleotide and 5a doped with 5a in an aqueous buffer according to the present invention. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0034] The present invention will now be described in further detail with reference to the accompanying drawings: I. Specific Implementation Methods Example 1 This embodiment provides a class of environmentally sensitive, switchable fluorescent nucleosides, and the specific preparation process is as follows: (1) Preparation of compound 1 2-Amino-2'-deoxyadenosine (1.00 g, 3.80 mmol) was dissolved in a mixture of glacial acetic acid (33.00 mL) and anhydrous sodium acetate (0.81 g, 12.30 mmol). The mixture was stirred at room temperature until completely dissolved. Liquid bromine (0.60 mL, 11.70 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was transferred to an oil bath at 40 °C and reacted for 0.5 h. The reaction was monitored by TLC until complete. The solvent was evaporated under reduced pressure. The crude product was purified by column chromatography with DCM:MeOH = 12:1 as the eluent, yielding 1.07 g of a white solid, with a yield of 82.5%. The white solid was identified as 8-bromo-2-amino-2'-deoxyadenosine.

[0035] 8-Bromo-2-amino-2'-deoxyadenosine (1.0 g, 2.89 mmol) was dissolved in N,N-dimethylformamide (15.0 mL), followed by the addition of cesium carbonate (0.94 g, 2.89 mmol) and palladium dichloride bis(triphenylphosphine) (0.2 g, 0.28 mmol). After stirring until homogeneous, potassium trifluorovinylborate (0.47 g, 3.47 mmol) was added. Nitrogen gas was introduced to purge oxygen from the reaction system, and the reaction mixture was transferred to an oil bath preheated to 80 °C and refluxed for 2.0 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the reaction solution was allowed to return to room temperature, concentrated under reduced pressure, and purified by column chromatography using dichloromethane and methanol in a volume ratio of 10:1. The solution was then concentrated to obtain a pale yellow solid. 8-Vinyl-2-diamino-2'-deoxyadenosine (1), pale yellow solid, yield 82%, TLC silica gel, (CH2Cl2 / MeOH 15:1) Rf 0.45. The structural identification data of this compound are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 7.02 (q, J = 16.0 Hz, 1H), 6.85 (s, 2H), 6.30 (dd, J = 8.0, 4.0 Hz), 6.13 (dd, J = 16.0, 4.0 Hz, 1H), 5.76 (s, 2H), 5.50 (t, J = 4.0 Hz, 1H), 5.45 (dd, J = 12.0, 4.0 Hz, 1H), 5.26 (s, 1H), 4.39(d, J = 4.0 Hz, 1H), 3.83 (q, J = 4.0 Hz, 1H), 3.67-3.53 (m, 2H), 2.77 (m,1H), 2.06 (m, 1H). 13 HRMS calcd for: C12H16N6O3[M+H + ] 293.1357, found 293.1362. The structural formula of compound 1 is confirmed as follows: .

[0036] (2) Synthesis of environmentally sensitive fluorescent switch nucleosides ① Synthesis of the environmentally sensitive switchable fluorescent nucleoside (compound 4a-l) shown in Formula 1 Compound 1 (1.0 g, 1.0 eq) was dissolved in N,N-dimethylformamide (5.0 mL), potassium carbonate (0.47 g, 1.0 eq) and palladium dichloride bis(triphenylphosphine) (0.2 g, 0.1 eq) were added, and the mixture was stirred until homogeneous. Nitrogen gas was then introduced, and a DMF solution of the corresponding 4-bromoaromatic compound (1.5 eq) was slowly added. The reaction was carried out at 110 °C. The reaction was monitored by TLC until it ended. The mixture was concentrated under reduced pressure and purified by column chromatography with dichloromethane:methanol = 30:1 - 25:1 as the eluent. The concentrated solution yielded a series of target compounds 4a-l.

[0037] 8-Styryl-2-amino-2'-deoxyadenosine (4a), yellow solid, yield 76%, TLC silica gel, (CH2Cl2 / MeOH 10:1) Rf 0.5. 1 H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 8.0 Hz,2H), 7.53 (s, 2H), 7.41 - 7.37 (m, 2H), 7.34-7.29 (m, 1H), 6.86 (s, 2H), 6.45(dd, J = 8.0, 6.1 Hz, 1H), 5.80 (s, 2H), 5.60 (q, J = 6.6 Hz, 1H), 5.28 (d, J= 4.0 Hz, 1H), 4.49 - 4.45 (m, 1H), 3.87 (q, J = 4.0 Hz, 1H), 3.75 - 3.70 (m,1H), 3.67 - 3.61 (m, 1H), 2.75 - 2.67 (m, 1H), 2.13 - 2.07 (m, 1H). 13 C NMR (100 MHz, DMSO) δ 159.76, 155.86, 152.13, 144.32, 136.11,134.56, 134.45, 133.10, 130.47, 130.34, 128.78, 127.13, 115.89, 113.30,87.51, 82.83, 70.67, 61.59. HRMS calcd for: C 18 H 20 N6O3[M+H +369.1670, found369.1677. The structural formula of compound 4a is confirmed as follows: ; 8-p-Fluorophenylvinyl-2-amino-2'-deoxyadenosine (4b), yellow solid, yield 74%, TLC silicagel, (CH2Cl2 / MeOH 10:1) Rf 0.48. 1 H NMR (400 MHz, DMSO-d6) δ 7.77 - 7.68 (m,2H), 7.52 (m, 2H), 7.21 (t, J = 16.0 Hz, 2H), 6.87 (s, 2H), 6.46 (dd, J =8.0, 6.0 Hz, 1H), 5.82 (s, 2H), 5.65 - 5.60 (m, 1H), 5.29 (d, J = 4.0 Hz,1H), 4.50 - 4.46 (m, 1H), 3.88 (q, J = 4.0 Hz, 1H), 3.79 - 3.58 (m, 2H),2.81-2.67 (m, 1H), 2.17 - 2.02 (m, 1H). 13C NMR (100 MHz, DMSO) δ 160.83,159.74, 155.86, 152.12, 144.27, 132.80, 131.85, 129.20, 115.87, 115.55,113.30, 87.55, 82.88, 70.68, 61.61. HRMS calcd for: C 18 H 19 N6O3F [M+H + ] 387.1575, found 387.1573. The structural formula of compound 4b is confirmed as follows: ; 8-Formylstyryl-2-amino-2'-deoxyadenosine (4c), pale red solid, yield 79%, TLC silicagel, (CH2Cl2 / MeOH 10:1) Rf 0.45. 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H),7.95 - 7.88 (m, 4H), 7.75 (d, J = 16.0 Hz, 1H, =CH), 7.61 (d, J = 16.0 Hz,1H, =CH), 6.92 (s, 2H, NH2), 6.48 (dd, J = 8.0, 8.0 Hz, 1H), 5.88 (s, 2H), 5.62 (br, 1H), 5.30 (d, J = 4.0 Hz, 1H), 4.51 - 4.47 (m, 1H), 3.89 (q, J =4.0 Hz, 1H), 3.76 - 3.64 (m, 2H), 2.76 - 2.69 (m, 1H), 2.18 - 2.09 (m, 1H). 13 CNMR (100 MHz, DMSO) δ 192.41, 159.97, 156.01, 152.22, 143.72, 142.07, 135.40,131.38, 129.96, 129.96, 127.67, 127.63, 119.28, 113.66, 87.53, 70.60, 61.51.HRMS calcd for: C 19 H 20 N6O4[M+H + ] 397.1619, found 397.1617. The structural formula of compound 4c is confirmed as follows: ; 8-Ethoxyformylstyryl-2-amino-2'-deoxyadenosine (4d), yellow solid, yield 82%, TLC silica gel, (CH2Cl2 / MeOH 10:1) Rf 0.55. 1H NMR (400 MHz, DMSO-d6) δ 7.97 -7.93 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 16.0 Hz, 1H), 7.58 (d, J = 16.0 Hz, 1H), 6.92 (s, 2H), 6.47 (dd, J = 8.0, 8.0 Hz, 1H),5.88 (s, 2H), 5.61 (br, 1H), 5.30 (d, J = 4.0 Hz, 1H), 4.47 - 4.49 (m, 1H),4.32 (q, J = 8.0 Hz, 2H), 3.89 (t, J = 4.0 Hz, 1H), 3.76-3.65 (m, 2H), 2.74-2.67 (m, 1H), 2.14-2.09 (m, 1H), 1.33 (t, J = 6.0 Hz, 3H). 13 C NMR (100 MHz, DMSO) δ 165.37, 159.93, 155.97, 152.21, 143.79, 140.78, 131.42, 129.54,128.99, 127.21, 118.72, 113.56, 87.55, 82.86, 70.63, 61.51, 60.70, 14.15.HRMS calcd for: C 21 H 24 N6O5[M+H + ] 441.1881, found 441.1883. The structural formula of compound 4d has been confirmed as follows: ; 8-P-acetylstyryl-2-amino-2'-deoxyadenosine (4e), yellow solid, 80% yield, TLC silicagel, (CH2Cl2 / MeOH 10:1) Rf 0.52. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.0Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 16.0 Hz, 1H), 7.59 (d, J =16.0 Hz, 1H), 6.92 (s, 2H), 6.48 (dd, J = 8.0, 6.1 Hz, 1H), 5.88 (s, 2H), 5.62 (br, 1H), 5.30 (d, J = 4.0 Hz, 1H), 4.50 - 4.47 (m, 1H), 3.89 (q, J =4.0 Hz, 1H), 3.77 - 3.63 (m, 2H), 2.76 - 2.68 (m, 1H), 2.58 (s, 3H), 2.15-2.09 (m, 1H). 13 C NMR (100 MHz, DMSO) δ 197.19, 159.94, 155.99, 152.21, 143.84,140.69, 135.97, 131.55, 128.69, 127.20, 118.62, 113.58, 87.59, 82.82, 70.66,61.56, 26.63. HRMS calcd for: C 20 H 22 N6O4[M+H + ] 411.1775, found 411.1777. The structural formula of compound 4e has been confirmed as follows: ; 8-Cyanostylenyl-2-amino-2'-deoxyadenosine (4f), yellow solid, 70% yield, TLC silica gel, (CH2Cl2 / MeOH 10:1) R f 0.48. 1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 8.0Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 16.0 Hz, 1H), 7.57 (d, J = 16Hz, 1H), 6.93 (s, 2H), 6.48 (dd, J = 8.0, 6.0 Hz, 1H), 5.89 (s, 2H), 5.62(dd, J = 8.0, 4.0 Hz, 1H), 5.29 (d, J = 4.0 Hz, 1H), 4.50-4.46 (m, 1H), 3.89(q, J = 4.0 Hz, 1H), 3.76 - 3.63 (m, 2H), 2.71 (m, 1H), 2.12 (m,1H). 13 C NMR(100 MHz, DMSO) δ 160.00, 156.05, 152.21, 143.56, 140.83, 132.57, 130.85,127.74, 119.59, 118.94, 113.68, 110.01, 87.57, 82.90, 70.65, 70.61, 61.52.HRMS calcd for: C 19 H 19 N7O3[M+Na + ] 416.1442, found 416.1438. The structural formula of compound 4f has been confirmed as follows: ; 8-2-Methoxypyridylvinyl-2-amino-2'-deoxyadenosine (4 g), yellow solid, 70% yield, TLCsilica gel, (CH2Cl2 / MeOH 9:1) Rf 0.49. 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J= 5.4 Hz, 1H), 7.76 (d, J = 15.8 Hz, 1H), 7.45 (d, J = 15.8 Hz, 1H), 7.33 (d,J = 5.4 Hz, 1H), 7.04 (s, 1H), 6.92 (s, 2H), 6.50 - 6.44 (m, 1H), 5.89 (s,2H), 5.58 (s, 1H), 5.29 (s, 1H), 4.49 (s, 1H), 3.86 (s, 4H), 3.69 (d, J =20.5 Hz, 2H), 2.70 (dt, J = 14.6, 7.1 Hz, 1H), 2.12 (dd, J = 13.4, 6.1 Hz, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 164.94, 160.57, 156.60, 152.75, 147.63,146.85, 143.83, 130.55, 121.01, 115.13, 114.18, 108.69, 88.06, 83.35, 71.10,62.03, 53.64. HRMS calcd for: C 18 H 21 N7O4[M+H + ] 338.1575, found 338.1568. The structural formula of compound 4g has been confirmed as follows: ; 8-Pyridinylstyryl-2-amino-2'-deoxyadenosine (4h), yellow solid, 70% yield, TLC silica gel, (CH2Cl2 / MeOH 10:1) R f 0.38. 1H NMR (400 MHz, DMSO-d6) δ 8.66-8.64 (m, 2H),7.85 (s, 4H), 7.77-7.74 (m, 2H), 7.65 (d, J=16.0 Hz, 1H), 7.59 (d, J=16.0 Hz,1H), 6.98 (s, 2H), 6.48 (dd, J = 8.0, 6.0 Hz, 1H), 5.91 (s, 2H), 5.64(br,1H), 5.30 (d, J = 4.0 Hz, 1H), 4.50-4.47 (m, 1H), 3.88 (t, J = 4.0 Hz, 1H),3.76-3.64 (m, 2H), 2.77 - 2.66 (m, 1H), 2.15-2.09 (m, 1H). 13 C NMR (100 MHz, DMSO) δ 159.84, 155.94, 152.18, 150.25, 146.24, 144.19, 137.14, 136.62,133.13, 132.02, 131.49, 128.66, 127.91, 127.12, 120.88, 116.90, 113.50,87.58, 82.93, 70.71, 61.63. HRMS calcd for: C 23 H 23 N7O3[M+H + ] 446.1935, found446.1947. The structural formula of compound 4h has been confirmed as follows: ; 8-(6-quinoxalolinevinyl)-2-amino-2'-deoxyadenosine (4i), yellowish-brown solid, yield 76%, TLCsilica gel, (CH2Cl2 / MeOH 10:1) Rf 0.45. 1H NMR (400 MHz, DMSO-d6) δ 8.92 (dd,J = 20.0, 4.0 Hz, 2H), 8.36 (dd, J = 9.0, 2.0 Hz, 1H), 8.26 (d, J = 4.0 Hz,1H), 8.06 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 16.0 Hz, 1H), 7.78 (d, J = 16.0Hz, 1H), 6.92 (s, 2H), 6.52 (dd, J = 8.0 Hz, 1H), 5.87 (s, 2H), 5.67 (dd, J =6.0, 4.0 Hz, 1H), 5.31 (d, J = 4.1 Hz, 1H), 4.53-4.49 (m, 1H), 3.90 (q, J =4.0 Hz, 1H), 3.78-3.65 (m, 2H), 2.80-2.73 (m, 1H), 2.16-2.11 (m, 1H). 13 C NMR(100 MHz, DMSO) δ 159.93, 156.02, 152.21, 146.11, 145.31, 143.90, 142.69,142.31, 138.05, 131.58, 129.40, 128.31, 127.69, 118.81, 113.64, 87.60, 82.98,70.72, 70.70, 61.62. HRMS calcd for: C 20 H 20 N8O3[M+H + ] 421.1731, found 421.1737. The structural formula of compound 4i has been confirmed as follows: ; 8,6-Quinolinylvinyl-2-amino-2'-deoxyadenosine (4j), yellow solid, yield 64%, TLC silicagel, (CH2Cl2 / MeOH 9:1) Rf 0.49. 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 8.88 (dd, J = 4.2, 1.8 Hz, 1H), 8.40 (dd, J=8.4, 1.8 Hz, 1H), 8.13 - 7.97 (m, 4H),7.67 - 7.51 (m, 3H), 7.20 (d, J=16.6 Hz, 1H), 6.74 (s, 2H), 5.83 (s, 2H), 3.36 (s, 3H), 2.51 (q, J = 1.9 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 161.04,156.23, 154.10, 151.00, 148.12, 145.01, 136.58, 134.85, 130.76, 130.03,128.71, 127.47, 127.01, 122.48, 120.06, 115.18. HRMS calcd for: C 21 H 21 N7O3[M+H + ] 418.1626, found 418.1638. The structural formula of compound 4j has been confirmed as follows: ; 8,3-pyridylvinyl-2-amino-2'-deoxyadenosine (4k), pale yellow solid, 67% yield. TLC silica gel, (CH2Cl2 / MeOH 10:1) Rf 0.53. 1H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.49 (d, J = 4.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 16.0 Hz, 1H), 7.55 (d, J = 15.9 Hz, 1H), 7.40 (dd, J = 8.0, 4.7 Hz, 1H), 6.91 (s, 2H), 6.47 (dd, J = 8.9, 6.0 Hz, 1H), 5.86 (s, 2H), 5.63 (dd, J = 6.6, 4.4 Hz, 1H), 5.29 (d, J = 4.0 Hz, 1H), 4.49 (s, 1H), 3.89 (s, 1H), 3.76-3.63 (m, 2H), 2.77-2.68 (m, 1H), 2.17-2.09 (m, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 158.28,154.36, 150.55, 147.32, 142.25, 131.63, 130.32, 127.69, 122.17, 116.38,111.82, 85.93, 81.25, 69.00, 59.91. HRMS calcd for: C 17 H 19 N7O3[M+H + 368.1469, found 368.1480. The structural formula of compound 4k has been confirmed as follows: ; 8,3-Thienylvinyl-2-amino-2'-deoxyadenosine (4l), pale yellow solid, yield 68%, TLC silicagel, (CH2Cl2 / MeOH 10:1) Rf 0.46. 1H NMR (400 MHz, DMSO-d6) δ 7.74 (s, 1H),7.63-7.61 (m, 1H), 7.59-7.57 (m, 1H), 7.53 (s, 1H), 7.34 (d, J = 15.8 Hz,1H), 6.82 (d, J = 14.5 Hz, 2H), 6.42 (dd, J = 8.8, 6.1 Hz, 1H), 5.77 (d, J =9.1 Hz, 2H), 5.62 - 5.59 (m, 1H), 5.27 (d, J = 4.0 Hz, 1H), 4.47 (s, 1H), 3.86 (s, 1H), 3.74 - 3.63 (m, 2H), 2.82 - 2.62 (m, 1H), 2.12 - 2.05 (m, 1H). 13 C NMR (100 MHz, DMSO-d6) δ 160.19, 156.28, 152.65, 144.96, 139.79, 129.29,127.78, 125.94, 116.12, 113.76, 88.00, 83.30, 71.10, 62.07. HRMS calcd for:C 16 H 18 N6O3S [M+H + ] 373.1781, found 373.1710. The structural formula of compound 4l is confirmed as follows: ; ② Synthesis of the environmentally sensitive switchable fluorescent nucleoside (compound 5a-g) shown in Formula 2 Weigh 1.0 eq of compound 4a-g into a dry flask, add 15 mL of mixed solvent (DMF:H2O (v / v) = 1:2) and stir until completely dissolved. Add sodium nitrite (NaNO2, 4.0 eq), then slowly add glacial acetic acid (8.0 eq). React in an oil bath at 60 °C for 1-3 hours, monitoring the reaction progress by thin-layer chromatography (TLC). After the reaction is complete, remove the oil bath and allow the reaction system to return to room temperature. Slowly add 25% ammonia to adjust the pH of the reaction system to 7.0-8.0. Pour the reaction solution into a Buchner funnel and filter to obtain a crude solid product. Wash with ethyl acetate and methanol, respectively. Transfer the crude product to a round-bottom flask, add a mixed solvent (acetonitrile and water in a 1:1 volume ratio), and recrystallize to obtain compound 5a-g.

[0038] 8-Styryl-2'-deoxyguanosine (5a), yellow solid, 54% yield, TLC silica gel, (CH2Cl2 / MeOH 10:1) R f 0.12. 1 H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 7.73-7.68 (d, J=12.0 Hz, 2H), 7.57-7.45 (q, J=16.0 Hz, 2H), 7.42-7.32 (m, 3H), 6.40 (t, , 2.12-2.08 (m, 1H). 13 C NMR (100 MHz, DMSO) δ 135.83, 134.21, 128.80, 128.70, 127.28, 115.06, 87.77, 70.68, 61.49.HRMS calcd for: C 18 H 19 N5O4[M+H + ] 370.1510, found 370.1512. The structural formula of compound 5a has been confirmed as follows: ; 8-Fluorostyryl-2'-deoxyguanosine (5b), pale yellow solid, yield 61%, TLC silica gel, (CH2Cl2 / MeOH 10:1) R f 0.12. 1 H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.78 -7.75 (m, 2H), 7.53 (d, J = 16.0 Hz, 1H), 7.44 (d, J = 16.0 Hz, 1H) 7.24 -7.19 (m, 2H), 6.42 (t, J = 8.0 Hz, 1H), 5.80(s, 1H), 5.34 (br, 1H), 4.48 -4.44 (m, 1H), 3.89 (s, 1H), 3.74 - 3.62 (m, 2H), 2.74-2.67 (m, 1H), 2.15-2.10(m, 1H).13 HRMS calcd for:C 18 H 18 FN5O4[M+H + ] 388.1416, found 388.1418. The structural formula of compound 5b has been confirmed as follows: ; 8-Formylstyryl-2'-deoxyguanosine (5c), yellowish-brown solid, yield 60%, TLC silica gel, (CH2Cl2 / MeOH 10:1) Rf 0.18. 1 H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J =8.0 Hz, 2H), 7.83 (d, J=8.0 Hz, 2H), 7.61 (q, J=16.0 Hz, 2H), 6.44 (t, J=6.0 Hz, 1H), 5.33 (s, 1H), 4.47 (br, 1H), 4.31 (d, J = 8.0 Hz, 1H), 3.91 (br, 1H), 3.75-3.63 (m, 2H), 2.72-2.67 (m, 1H), 2.16-2.11 (m, 1H). 13 HRMS calcd for: C 19 H 19 N5O5[M+H + ] 398.1459, found 398.1466. The structural formula of compound 5c has been confirmed as follows: ; 8-Ethoxyformylstyryl-2'-deoxyguanosine (5d), yellow solid, yield 58%, TLC silicagel, (CH2Cl2 / MeOH 10:1) Rf 0.15. 1H NMR (400 MHz, DMSO-d6) δ 11.51 (s, 1H), 7.94 (d, J=8.0 Hz, 2H), 7.83 (d, J =8.0 Hz, 2H), 7.66 (d, J=16.0 Hz, 1H), 7.57 (d, J=16.0 Hz, 1H), 6.44 (t, J=8.0 Hz, 1H), 5.35 (br, 1H), 4.48-4.46 (m,1H), 4.31 (q, J=8.0 Hz, 2H), 3.91-3.89 (m, 1H), 3.76-3.63 (m, 2H), 2.72-2.64(m, 1H), 2.17-2.12 (m, 1H), 1.32 (t, J=6.0 Hz, 3H). 13 C NMR (100 MHz, DMSO) δ165.32, 155.56, 140.41, 132.57, 129.52, 129.25, 127.38, 117.81, 87.79, 83.35,70.64, 61.45, 60.73, 21.03, 14.11. HRMS calcd for: C 21 H 23 N5O6[M+H + 442.1721, found 442.1723. The structural formula of compound 5d has been confirmed as follows: ; 8-P-acetylstyryl-2'-deoxyguanosine (5e), ​​yellowish-brown solid, yield 65%, TLC silica gel, (CH2Cl2 / MeOH 10:1) Rf 0.25. 1 H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.0 Hz,2H), 7.83 (d, J = 8.0 Hz, 2H), 7.62 (q, J = 16.0 Hz, 2H), 6.45 - 6.41 (m,1H), 5.32 (br, 1H), 4.48 - 4.46 (m, 1H), 3.90-3.88 (m, 1H), 3.75 - 3.62 (m,2H), 2.74-2.67 (m, 1H), 2.58 (s, 3H), 2.18 - 2.08 (m, 1H). 13C NMR (100 MHz, DMSO) δ 197.22, 155.45, 154.68, 140.33, 136.21, 132.68, 128.68, 127.37,126.93, 117.79, 112.42, 111.82, 87.68, 83.24, 70.66, 61.46, 26.74. HRMS calcdfor: C 20 H 21 N5O5[M+H + ] 412.1615, found 412.1623. The structural formula of compound 5e has been confirmed as follows: ; 8-Cyanostylenyl-2'-deoxyguanosine (5f), yellow solid, yield 59%, TLC silica gel, (CH2Cl2 / MeOH 10:1) Rf 0.22. 1 H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.89 (d,J = 8.0 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.69 (d, J=16.0 Hz, 1H), 7.56 (d,J = 16.0 Hz, 1H), 6.45 (t, J = 8.0 Hz, 1H), 5.33 (br, 1H), 4.47-4.46 (m, 1H), 3.90 (m, 1H), 3.75 - 3.63 (m, 2H), 2.74-2.67 (m, 1H), 2.14 - 2.12 (m, 1H). 13 HRMS calcd for: C 19 H 18 N6O4[M+Na + ]417.1282, found 417.1289. The structural formula of compound 5f has been confirmed as follows: ; 8-Methoxypyridylvinyl-2'-deoxyguanosine (5 g), pale yellow solid, yield 58%, TLC silica gel, (CH2Cl2 / MeOH 7:1) R f 0.48. 1 H NMR (400 MHz, DMSO- d 6) δ 8.15 (s, 1H), 7.72(d, J = 15.8 Hz, 1H), 7.44 (d, J=15.7 Hz, 1H), 7.34 (d, J=5.3 Hz, 2H), 7.06(s, 1H), 6.42 (s, 1H), 5.30 (s, 1H), 4.46 (s, 4H), 3.87 (s, 3H), 2.15-2.09(m, 2H), 1.23 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 164.93, 147.69, 146.55,131.64, 115.21, 108.85, 88.31, 71.10, 62.02, 53.67. HRMS calcd for: C 16 H 18 N6O3S[M+H + ] 399.1415, found 339.1415. The structural formula of compound 5g is confirmed as follows: ; (3) Synthesis of nucleoside phosphoramide monomer (compound 5A)

[0039] In a dry 10 mL round-bottom flask, compound 5a (500 mg, 1.35 mmol) was suspended in methanol (5.0 mL) to obtain a suspension. N,N-dimethylacetamide dimethyl acetal (DMAc-DMA, 1.2 eq) was slowly added to the suspension, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by column chromatography with dichloromethane:methanol = 10:1 as the eluent to obtain a bright yellow solid compound 3.

[0040]

[0041] Compound 3 (500 mg, 1.14 mmol) was dissolved in pyridine (5.0 mL). N,N-diphenylcarbamoyl chloride (624 mg, 2.69 mmol) and N,N-diisopropylethylamine (279 mg, 2.15 mmol) were added under ice bath conditions. The ice bath was removed, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated under reduced pressure, and after shaking with an appropriate amount of dichloromethane, it was poured into a separatory funnel. The mixture was washed with 5% sodium bicarbonate aqueous solution (20 mL) and extracted with dichloromethane (3 × 10 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by silica gel column chromatography with petroleum ether and ethyl acetate in a volume ratio of 5:1 to give yellow foamy compound 4.

[0042]

[0043] Compound 4 (662 mg, 1.04 mmol) was dissolved in anhydrous pyridine (5.0 mL), and 4,4-bismethoxytriphenylchloro (424 mg, 1.25 mmol) was added to replace N2. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was washed with 5% sodium bicarbonate aqueous solution (20 mL), extracted with dichloromethane (3 × 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product, and the crude product was subjected to rapid column chromatography with dichloromethane and methanol in a volume ratio of 40:1 to obtain a pale yellow foamy solid compound 5.

[0044]

[0045] Compound 5 (235 mg, 0.25 mmol) was dissolved in anhydrous dichloromethane (5.0 mL), and N,N-diisopropylethylamine (64.6 mg, 0.50 mmol) and 2-cyanoethyl N,N-diisopropylphosphonamide (105 mg, 0.44 mmol) were added. The mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was diluted with dichloromethane (30.0 mL), washed with 5% sodium bicarbonate aqueous solution (20 mL), extracted with dichloromethane (3 × 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure to obtain the crude product, and purified by silica gel column chromatography with petroleum ether and ethyl acetate in a volume ratio of 4:1 to obtain a white foamy compound 5A.

[0046] 8-(styryl)-6-N,N-dimethylacetamidine-2'-deoxyguanosine (compound 3), pale yellow solid, 74% yield. TLC silica gel, (CH2Cl2 / MeOH=10:1) Rf 0.46. 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.75 (d, J = 7.1 Hz, 2H), 7.61 (s, 1H), 7.50–7.32 (m, 4H), 6.41 (dd, J = 8.7, 6.2 Hz, 1H), 5.31 (d, J = 4.4 Hz, 1H), 4.53–4.41 (m, 1H), 3.88 (d, J = 3.0 Hz, 1H), 3.72 (dd, J = 12.1, 2.9 Hz, 1H), 3.62 (dd, J =12.1, 3.5 Hz, 1H), 3.20 (s, 2H), 3.09 (d, J = 21.1 Hz, 3H), 2.83-2.72 (m,1H), 2.20 (s, 3H), 2.15-2.08 (m, 1H), 1.27-1.15 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 163.14, 155.39, 152.15, 147.34, 135.31, 134.30, 128.16, 126.89,114.47, 112.62, 87.17, 82.40, 70.10, 61.07, 44.89, 37.50, 18.22, 7.91. HRMScalcd for: C 22 H 26 N6O4[M+H + ] 437.1935, found 437.0956. 8-(Styryl)-6-N,N-dimethylacetamidine-2-O-diphenylcarbamoyl-2'-deoxyguanosine (compound 4), yellow foamy solid, 70% yield, TLC silica gel, (CH2Cl2 / MeOH = 30:1:) R f 0.49. 1 HNMR (400 MHz, DMSO- d6) δ 7.80 (d, J = 7.1 Hz, 3H), 7.63 (d, J = 15.9 Hz, 1H), 7.48-7.36 (m, 10H), 7.32-7.28 (m, 1H), 6.59 (dd, J = 8.1, 6.5 Hz, 2H), 5.43 -5.23 (m, 1H), 4.55 (d, J = 3.5 Hz, 2H), 3.91 (d, J = 3.6 Hz, 1H), 3.83-3.63(m, 2H), 3.35 (s, 1H), 2.87-2.74 (m, 1H), 2.30-2.19 (m, 1H), 2.16 (s, 3H),2.08 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 160.72, 158.26, 153.14, 150.91,150.00, 148.02, 140.31, 134.97, 134.08, 127.62, 127.16, 126.08, 125.44,125.24, 122.30, 116.42, 113.34, 86.06, 81.45, 68.80, 59.83, 36.71, 36.16,15.61. HRMS calcd for: C 35 H 35 N7O5[M+H - ] 632.2619, found 632.2622. 8-(styryl)-6-N,N-dimethylacetamidine-2-diphenylcarbamoyl-5'-O-(4,4'-dimethoxytriphenylmethyl)-3'-O-((bis(1-methylethyl)amino)(2-cyanoethoxy)phosphine))-2'-deoxyguanosine (compound 5A), white foamy solid, 80% yield, TLC silica gel, (PE / EA = 4:1) Rf 0.4. 1H NMR (400MHz, DMSO-d6) δ 7.90-7.83 (m, 1H), 7.76 (t, J = 6.4 Hz, 2H), 7.52 (dd, J =15.9, 3.4 Hz, 1H), 7.40 (dd, J = 14.6, 7.2 Hz, 12H), 7.28 (t, J = 7.1 Hz, 1H), 7.23-7.12 (m, 1H), 7.10-7.00 (m, 6H), 6.65 (dt, J = 8.4, 4.3 Hz, 5H), 5.76 (s, 1H), 5.08 (ddd, J = 29.4, 14.0, 7.4 Hz, 1H), 4.04 (dd, J = 6.3, 3.2Hz, 1H), 3.68 (dd, J = 9.5, 5.5 Hz, 1H), 3.65-3.55 (m, 6H), 3.50 (dq, J =9.5, 5.9, 3.3 Hz, 4H), 3.26 (ddd, J = 15.1, 10.7, 2.8 Hz, 1H), 3.14 (s, 7H), 2.72-2.61 (m, 1H), 2.55 (t, J = 6.0 Hz, 1H), 2.12-2.02 (m, 3H), 1.20-1.00 (m,9H), 0.93 (d, J = 6.7 Hz, 3H). 31 P NMR (162 MHz, DMSO- d 6) δ 147.71, 147.04.HRMS calcd for: C 65 H 70 N9O7P [M+H + ] 1118.5055, found 1118.5051. II. Performance Testing 1. Performance testing of environmentally sensitive switch-type fluorescent nucleosides Fluorescent nucleoside molecules with excellent properties possess considerable fluorescence quantum efficiency, emission wavelengths entering the visible light region, and photophysical properties sensitive to environmental conditions. For living organisms, biochemical reactions such as DNA transcription and translation occur in complex and variable liquid environments. In this example, solutions of different polarities were used to simulate the environment. The photophysical properties of the novel fluorescent nucleoside compounds 4e, 4f, 5a, and 5b synthesized in Example 1 in solutions of different polarities were characterized using ultraviolet spectrophotometry and fluorescence spectrophotometry. The effect of solvent polarity changes on the ultraviolet absorption intensity and fluorescence properties of the fluorescent nucleosides was tested. All characterizations were performed at 20°C.

[0047] Accurately weigh fluorescent nucleoside compounds 4e, 4f, 5a, and 5b into 10 mL volumetric flasks, dissolve them in dry dimethyl sulfoxide and dilute to volume to prepare a stock solution with a molar concentration of 1.0 mM.

[0048] Four solvents were selected based on their polarity parameter ET (30) values: water (ET (30) = 63.1 kcal / mol), methanol (ET (30) = 55.4 kcal / mol), acetonitrile (ET (30) = 45.6 kcal / mol), and 1,4-dioxane (ET (30) = 36.0 kcal / mol). The fluorescence properties of the compounds were measured, and their sensitivity to polarity changes was investigated.

[0049] Measure 100 μL of the prepared fluorescent nucleoside stock solution into 10 mL volumetric flasks, and dilute to volume with the above four solvents to prepare 10 μM dilutions. Then, use a fluorescence spectrophotometer and a UV spectrophotometer to scan the fluorescence spectrum and UV absorption spectrum in different solvents to obtain the fluorescence emission wavelength and UV absorption wavelength. Calculate the absorption coefficient ε of the corresponding fluorescent nucleoside according to the formula ε = A / 1 * c.

[0050] Fluorescence quantum efficiency was calculated using quinine sulfate as a reference. Samples and quinine sulfate solutions of specific concentrations were prepared, and their UV absorption spectra were scanned. A wavelength (320 nm, where the absorption wavelength of the sample and the reference solution showed the same trend, indicating a fluorescence quantum efficiency of quinine sulfate excited at 320 nm is 0.55) was selected. The fluorescence intensity corresponding to different absorbances was then measured, and a standard curve was plotted (vertical axis: light intensity, horizontal axis: absorbance). The slope was obtained, and then the formula Φd = Φstandard × (Gd / Gstandard) × (ηd) was used. 2 Waiting / η 2The fluorescence quantum efficiency Φ is calculated using the following formula: Φto is the fluorescence quantum efficiency of the sample to be tested, which is the ratio of the number of photons emitted after the sample absorbs photons to the number of photons absorbed; Φto is the fluorescence quantum efficiency of the reference substance (quinine sulfate), used as the calculation benchmark; Grad is the linear slope of the fluorescence intensity-absorbance of the sample to be tested, i.e., the rate at which the fluorescence intensity (I) changes with the absorbance (A); Grad is the linear slope of the fluorescence intensity-absorbance of the reference substance (quinine sulfate); ηto is the refractive index of the solvent in which the sample to be tested is located, and ηto is the refractive index of the solvent in which the reference substance (quinine sulfate) is located; ηto 2 Waiting / η 2 Mark: Corrects the effect of solvent refractive index on fluorescence collection efficiency; Grad to / Grad mark: Corrects the difference in fluorescence emission efficiency between the sample and the reference material, brightness (b) = Φ × ε, where ε represents the molar absorptivity. The final photophysical data of fluorescent nucleosides 4e, 4f, 5a, and 5b in the above four solvents are shown in Tables 1-4 and appendices. Figure 1 As shown.

[0051] Table 1: Photophysical data of fluorescent nucleoside 4e measured in different polar solvents

[0052] εa[* 10 5 M -1 cm -1 Brightness b [* 10] 5 L*M -1 cm -1 ] Table 2: Photophysical data of fluorescent nucleoside 4f measured in different polar solvents

[0053] Table 3: Photophysical data of fluorescent nucleoside 5a measured in different polar solvents

[0054] Table 4: Photophysical data of fluorescent nucleoside 5b measured in different polar solvents

[0055] 2. Performance testing of phosphorus amide monomer To verify the ability of phosphoramide monomer 5A as a labeling agent for environmentally sensitive switch fluorescent nucleosides, we incorporated it into deoxy oligonucleotide single chains and tested the changes in its fluorescence properties, exploring its application potential in nucleic acid fluorescent labeling and biomedicine.

[0056] (1) Preparation of fluorescent deoxy oligonucleotide single strands Oligonucleotides were synthesized by Hunan Aikerui Biotechnology Co., Ltd. using a solid-phase synthesis standard method with phosphoramide compound 5A on a YB-192S DNA synthesizer. The phosphoramide monomer compound 5A was precisely incorporated into single-stranded DNA with four different ortho- and ortho-base pairs (T / A / C / G) at a 1 μM scale on an automated DNA synthesizer. After lysis from the solid carrier, the oligonucleotides were deprotected in concentrated ammonia solution at 55 °C for 16 hours, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by MALDI-TOF. Specific sequence information and characterization data are shown in Table 5.

[0057] Table 5: Information and characterization of single-stranded deoxyoligonucleotides containing 5a

[0058] (2) Fluorescence property test of fluorescent deoxy oligonucleotide single strands Compound 5a and the four fluorescent oligonucleotide single strands (deoxy oligonucleotide single strands containing 5a) from Table 5 were dissolved in pure water to prepare 3.0 μM solutions. The ultraviolet absorption spectrum (λabs) and fluorescence emission spectrum (λfl) were scanned using an ultraviolet spectrometer and a fluorescence spectrometer, respectively, and the fluorescence quantum efficiency (Φ) was measured. According to the brightness formula: Brightness = Molar absorptivity (λabs / λfl), the fluorescence quantum efficiency was determined. The value of Φ represents the overall efficiency of fluorescent labeling. See Table 6 and Appendix for specific results. Figure 2 As shown.

[0059] From Table 6 and Appendix Figure 2 Data shows that compound 5a exhibits almost no fluorescence in buffer solution, while the four oligonucleotide single strands containing 5a prepared all possess fluorescence emission properties and good fluorescence intensity, with a maximum emission wavelength at 450 nm. The fluorescence intensity and fluorescence quantum efficiency of compound 5a show significant changes after it is used as a monomer and after incorporation into the oligonucleotide chain, with a maximum increase of 395-fold in fluorescence intensity and a maximum increase of 38-fold in fluorescence quantum efficiency (A5aA). Even thymine, which has the least effect on fluorescence enhancement, shows a 17-fold change in fluorescence quantum efficiency; guanine typically quenches fluorescence, but G5aG did not exhibit quenching and its fluorescence was also significantly enhanced.

[0060] Table 6: Optical parameters of 5a and deoxyoligonucleotide single strands containing 5a in buffer solution

[0061] In summary, this invention presents a method for synthesizing environmentally sensitive switchable fluorescent nucleosides. Using 8-bromo-2-amino-2'-deoxyadenosine as a raw material, numerous fluorescent nucleosides can be synthesized. Furthermore, 5a can be efficiently and conveniently obtained through recrystallization. Additionally, 5a can be effectively converted into the phosphoramidite monomer 5A. Combined with a DNA synthesizer, fluorescent nucleosides can be incorporated into any designated position in nucleic acids for modification. This method is suitable for fluorescent labeling of nucleic acids and biomedical applications, providing an efficient and flexible tool for nucleic acid fluorescent labeling with broad prospects for biomedical applications.

[0062] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A class of environmentally sensitive switchable fluorescent nucleosides, characterized in that, having a structure as shown in Formula 1 or Formula 2: or ; wherein R is selected from , , , , , , , , , , and any of the above.

2. The class of environmentally sensitive switchable fluorescent nucleosides according to claim 1, wherein The environment-sensitive switch fluorescent nucleoside has the following structural formula: 4e, 4f, 5a, 5b 。 3. A method of preparing a class of environmentally sensitive switchable fluorescent nucleosides according to claim 1 or 2, characterized in that, comprising: Step 1, uniformly mix 8-bromo-2-amino-2'-deoxyadenosine, base, catalyst, protect with nitrogen, add potassium trifluorovinylborate to the mixed solution for coupling reaction, after the reaction is completed, concentrate under reduced pressure, and purify by column chromatography to obtain compound 1; Step 2, mix compound 1 with base and catalyst, protect with nitrogen, add compound 2 after the reaction, concentrate, and purify by column chromatography to obtain the environment-sensitive switch fluorescent nucleoside shown in Formula 1; The structural formula of the compound 1 is ; The compound 2 is Br-R, wherein R is selected from any one of , , , , , , , , , , and . Use the compound shown in Formula 1 to react with sodium nitrite and glacial acetic acid, adjust the pH value after filtration, washing and recrystallization to obtain the environment-sensitive switch fluorescent nucleoside shown in Formula 2; The base is potassium carbonate or cesium carbonate; The catalyst is any one of dichlorobis(triphenylphosphine)palladium, palladium chloride, tetrakis(triphenylphosphine)palladium and palladium acetate.

4. The method for preparing a type of environmentally sensitive switchable fluorescent nucleoside according to claim 3, characterized in that, In step 1, the molar ratio of 8-bromo-2-amino-2'-deoxyadenosine to base and catalyst is 1:1:0.1-0.2, the reaction temperature is 70-90°C, and the reaction time is 1-3h.

5. The method for preparing a type of environmentally sensitive switchable fluorescent nucleoside according to claim 3, characterized in that, In step 2, the molar ratio of compound 1, base, catalyst and compound 2 is 1:1-2:0.1-0.2:1-2, the reaction temperature is 100-120°C, and the reaction time is 4-6h.

6. The method for preparing a class of environmentally sensitive switchable fluorescent nucleosides according to claim 3, characterized in that, In step 2, the molar ratio of the compound of Formula 1, sodium nitrite and glacial acetic acid is 1:3-5:7-9; the reaction temperature is 50-70°C, and the reaction time is 1-3h.

7. A fluorescent nucleoside phosphoramidite monomer, characterized in that, The fluorescent nucleoside phosphoramidite monomer is synthesized from the environment-sensitive switch fluorescent nucleoside of claim 1.

8. A fluorescent nucleoside triphosphate, characterized in that, The fluorescent nucleoside triphosphate is synthesized from the environment-sensitive switch fluorescent nucleoside of claim 1.

9. Use of the environment-sensitive switch fluorescent nucleoside of claim 1 or 2, or the fluorescent nucleoside phosphoramidite monomer of claim 7, or the fluorescent nucleoside triphosphate of claim 8 in the preparation of nucleic acids. Preferably, the nucleic acids include fluorescent probes.

10. Use of the environment-sensitive switch fluorescent nucleoside of claim 1 or 2, or the fluorescent nucleoside phosphoramidite monomer of claim 7, or the fluorescent nucleoside triphosphate of claim 8 in the preparation of biological imaging reagents.

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