Use of fluorescently labeled nucleotides in DNA synthesis sequencing and single molecule sequencing

Abstract

The application discloses application of fluorescently labeled nucleotides in DNA synthesis sequencing and single molecule sequencing, and the typical structure of the fluorescently labeled nucleotides is shown in the figure, wherein Base is four different bases of U, C, A and G; R is a benzene ring, an alkyne group or a vinyl group; Cleavable Linker is a triazene connecting unit; R1 is a chemical molecular group for connecting the triazene connecting unit and the fluorescein together; and Dye is a fluorescent dye. After the fluorescently labeled nucleotides participate in DNA extension and break, the groups remaining on the bases are only π-electron-containing groups such as benzene (amine) groups, alkyne groups and vinyl groups. In the DNA synthesis sequencing and single molecule sequencing, after the fluorescently labeled nucleotides participate in extension and break, the residues on the DNA chain are only benzene (amine) groups, alkyne groups and vinyl groups, which is helpful to longer DNA sequencing read length and lower error rate.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to the application of fluorescently labeled nucleotides in DNA synthesis sequencing and single-molecule sequencing. Background Technology

[0002] Following the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing refers to the analysis of the base sequence of a specific DNA fragment, specifically the arrangement of adenine (A), thymine (T), cytosine (C), and guanine (G). Developing accurate, high-throughput, and low-cost DNA sequencing methods is of great significance for biology, medicine, and other fields.

[0003] DNA synthesis and sequencing based on fluorescently labeled nucleotides, as well as single-molecule synthesis and sequencing technologies, have been widely used. However, existing core fluorescently labeled nucleotides inevitably retain a residue after participating in DNA strand elongation and breakage. This residue accumulates with repeated sequencing cycles, leading to changes in the conformation and configuration of the DNA double helix. This is the fundamental reason for the limited read length and high error rate in DNA sequencing. For example, CN 104003902A discloses the synthesis of a triazine linker and its application in DNA sequencing; the reversible terminal structure formed by linking the triazine linker with a nucleotide and a fluorescein is shown below:

[0004] Its structural group between the triazine and the base is relatively large, and the steric hindrance caused by the residue after breakage is greater, which affects the DNA synthesis and sequencing effect; moreover, the synthesis steps are complicated and the synthesis cost is high.

[0005] Generally, natural nucleotides are better elongation reactants than base-modified nucleotides when participating in DNA elongation. Therefore, most research directions in DNA synthesis sequencing technology are to minimize the influence of residues or even eliminate residues to achieve traceless sequencing, thereby increasing sequencing read length and reducing error rate.

[0006] The applicant's prior research, CN 112390839 A, proposed triazine-free sequencing. Theoretically, this type of reversible terminating nucleotide is almost perfect. However, in reality, it suffers from a series of drawbacks. First, side reactions occur during DNA extension product breakage, resulting in incomplete and ineffective reactions. This leads to severe DNA strand damage during actual sequencing, severely limiting read length and error rate. Furthermore, our experiments revealed that the impact of these side reactions is actually greater than the impact of residues generated after linker unit breakage. Second, the synthetic conditions for directly modifying triazine linker units onto bases are extremely demanding, making synthesis very difficult. The products are also difficult to purify, requiring repeated HPLC purification to barely obtain usable compounds. Finally, when this type of reversible terminating nucleotide breaks the linker unit under the combined action of acid and reducing agents, the presence of active groups such as hydroxyl and amino groups on the bases, due to the direct attachment of triazine to the bases, easily leads to side reactions during triazine structure synthesis. The formation of complex isomers between triazine and the bases results in significant loss of reversible terminating nucleotides and severe DNA double-strand damage, making it difficult to apply to commercial sequencing. Additionally, the sequencing costs, including reagent synthesis, are very high. Summary of the Invention

[0007] To address the shortcomings of existing sequencing technologies, the purpose of this invention is to provide a novel application of fluorescently labeled nucleotides in DNA synthesis sequencing and single-molecule sequencing.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention provides a novel four-color fluorescent reversible nucleotide sequencing termination reagent, the overall structural formula of which is shown below: The overall structural formula is as follows: Base consists of four different bases: U, C, A, and G; R represents a benzene ring, alkynyl group, or vinyl group; Cleavable Linker is a triazine linking unit; R1 is a chemical molecular group that links the triazine linking unit to the fluorescein; and Dye is the fluorescent dye. Specific Dyes include Cy3, Cy5, Cy2, Cy3.5, TAMRA, FITC, sulfo-Cy3, sulfo-Cy2, sulfo-Cy5, and sulfo-Cy3.5.

[0010] This invention provides a novel fluorescently labeled reversible termination nucleotide, which can be classified into the following structures according to the type of linker unit:

[0011]

[0012] The novel reversible nucleotide DNA sequencing reagent (structural formula 1) provided by this invention has four different bases: U, C, A, and G; R is a benzene ring, alkynyl, or vinyl; R2 is methyl, ethyl, propyl, butyl, pentyl, or hexyl; and R3 is an aliphatic carbon chain of 2-6 carbon atoms or an aliphatic carbon chain of 10 carbon atoms containing an N heterocyclic structure.

[0013] Preferably, the specific structure of structural formula 1 is as follows:

[0014]

[0015]

[0016] This invention also provides a method for synthesizing a triazine-reversibly terminated nucleotide; the method includes the following steps:

[0017] S1,4-Aminophenylboronic acid pinacol ester and compounds (n=1-3) The reaction yields

[0018] S2, and The reaction yielded

[0019] S3 The fluorescently labeled reversible termination nucleotide is obtained by reacting with Sulf-Cy3-NHS, Cy3-NHS, Cy5-NHS, FITC-NHS, or Cy3.5-NHS.

[0020] As one embodiment of the present invention, the compound (n = 1-3) were prepared by a method including the following steps: compound The reaction with ethyl trifluoroacetate yields a compound The compound was further deprotected by removing the Boc protecting group in the presence of trifluoroacetic acid to obtain the compound.

[0021] As one embodiment of the present invention, the compound The compound was prepared by a method including the following steps: It reacts with 2-chloro-1,3,2-benzodioxophosphazenecyclohexane-4-one and tri-n-butylamine pyrophosphate. The reactant is precipitated as an alcohol solid, which is then reacted with concentrated ammonia. After the solvent is evaporated, water is added to dissolve the solid, and the mixture is separated and purified to obtain the final product.

[0022] In one embodiment of the present invention, step S1 is carried out in the presence of hydrochloric acid and sodium nitrite.

[0023] In one embodiment of the present invention, step S2 is carried out in the presence of cesium carbonate, palladium acetate and TPPTS.

[0024] The triazine fluorescently labeled nucleotides provided by this invention, after participating in DNA chain elongation and breakage, leave only phenyl, alkynyl, and vinyl groups on the bases.

[0025] This invention also provides the application of this type of fluorescently labeled nucleotide in DNA synthesis sequencing and single-molecule sequencing.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The triazine four-color fluorescently labeled reversible termination nucleotide proposed in this invention leaves only phenyl, alkyne or vinyl groups on the bases after participating in DNA chain elongation and breakage, which is more conducive to achieving longer read lengths and lower error rates in DNA synthesis sequencing and single-molecule sequencing.

[0028] 2. In the practice of DNA synthesis sequencing and single-molecule sequencing, the triazine reversible termination nucleotide provided by this invention has been found to be clean and thorough in both extension and fragmentation experiments, with no byproducts observed.

[0029] 3. Compared to reversible terminating nucleotides generated by directly linking the bases of nucleoside triphosphates to triazine fluorescently labeled nucleotides (Patent 202011286386.9), the reversible terminating nucleotides of this invention, in which the bases of nucleoside triphosphates are linked to triazine fluorescently labeled nucleotides via phenyl, alkynyl, or vinyl groups, require commercially available and readily available raw materials. Furthermore, the reaction process and methods are simple, efficient, and yield high-purity products. The final product only requires one HPLC purification to obtain high-purity (>99.9%) reversible terminating nucleotides, and can be synthesized in large quantities. Therefore, this type of reversible terminating nucleotide can be used for more experimental verifications, thereby obtaining more experimental data and possessing greater practical value and application development value. In contrast, the reversible terminating nucleotides based on triazine-based traceless linker units developed by the inventors (Invention Patent 202011286386.9) for DNA sequencing are theoretically almost perfect. However, in reality, they suffer from a series of drawbacks. First, side reactions occur during DNA extension product breakage, resulting in incomplete and unclean reactions. This leads to severe DNA strand damage during actual sequencing, severely limiting read length and error rate. Furthermore, our experiments revealed that the impact of these side reactions is actually greater than the impact of residues generated after linker unit breakage. Second, the compounds described in Patent 202011286386.9 are very difficult to synthesize, and the products are hard to purify, requiring repeated HPLC purification to barely obtain usable compounds. Finally, when the linker units of the compounds described in the above patents are broken under the combined action of acid and reducing agents, because triazine is directly linked to the bases, and the bases contain active groups such as hydroxyl and amino groups, side reactions easily occur during the synthesis of the triazine structure. The formation of complex isomers between triazine and the bases leads to significant loss of the reversible terminating nucleotides, with a considerable portion of the nucleotides being destroyed in the process. In this context, we developed a novel fluorescently labeled nucleotide that first synthesizes triazaene on a benzene ring, alkynyl group, or vinyl group, and then connects the triazaene to the base via the benzene ring, alkynyl group, or vinyl group. This avoids the problem of active groups on the base participating in the reaction during the triazaene synthesis process, and avoids a large number of side reactions and the formation of isomers. The reaction is clean and thorough, with almost no side reactions observed. Moreover, the synthesis of this type of compound is simple and convenient, and it can be synthesized in large quantities. Attached Figure Description

[0030] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0031] Figure 1 The overall structural formula of the fluorescently labeled nucleotides described in this invention is as follows;

[0032] Figure 2The structure of the triazine reversible termination nucleotide described in this invention is as follows;

[0033] Figure 3 The dUTP fluorescently labeled nucleotide and its synthesis method described in Example 1 of this invention;

[0034] Figure 4 The triazine dUTP fluorescently labeled nucleotide and its synthesis method described in Example 2 of this invention;

[0035] Figure 5 This refers to the triazine dUTP fluorescently labeled nucleotide and its synthesis method as described in Example 3 of the present invention;

[0036] Figure 6 This refers to the triazine dUTP fluorescently labeled nucleotide and its synthesis method as described in Example 4 of the present invention;

[0037] Figure 7 This refers to the triazine dATP fluorescently labeled nucleotide and its synthesis method as described in Example 5 of the present invention;

[0038] Figure 8 This refers to the triazine dCTP fluorescently labeled nucleotide and its synthesis method as described in Example 6 of the present invention;

[0039] Figure 9 This refers to the triazine dGTP fluorescently labeled nucleotide and its synthesis method as described in Example 7 of the present invention;

[0040] Figure 10 This is a route diagram of the rapid and complete cleavage of the four-color fluorescently labeled nucleotides described in Example 4 of the present invention under the action of hypophosphite;

[0041] Figure 11 The results of the rapid and complete cleavage of the four-color fluorescently labeled nucleotides described in Example 4 of this invention under hypophosphite are shown; where Line 1: 24bp, Line 2: 25bp, Line 3: C4+Bst+(-A-) first extension, Line 4: C4+Bst+(-A-) cleavage after the first extension, and Line 5: Ph+Bst+(-A-).

[0042] Figure 12The results of the fluorescently labeled nucleotides described in Example 4 of this invention were characterized by denaturing gel for their participation in extension and breakage; wherein, Line 1: Primer (24bp), Line 2: Primer (25bp), Line 3: C4+Bst+Template1, Line 4: C4+Bst+Template2 for the first extension, Line 5: C4+Bst+Template2 for breakage after the first extension, Line 6: C4+Bst+Template2 for the second extension, and Line 7: C4+Bst+Template2 for breakage after the second extension;

[0043] Figure 13 The fluorescent image shown is of the fluorescently labeled nucleotides participating in DNA elongation as described in Example 4 of this invention.

[0044] Figure 14 The 1H NMR spectrum of the fluorescently labeled nucleotide described in Example 4;

[0045] Figure 15 This is the phosphorus NMR spectrum of the fluorescently labeled nucleotide described in Example 4. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0047] The overall structural formula of the fluorescently labeled nucleotides involved in this invention is as follows: Figure 1 As shown, the following examples are all triazine reversible termination nucleotides, and their structural formulas are as follows: Figure 2 As shown.

[0048] Example 1: Synthetic route of fluorescently labeled nucleotide (Structure II)

[0049] Synthetic routes such as Figure 3 As shown, it specifically includes:

[0050] Synthesis of Compound 43

[0051]

[0052] Add 180 ml of tetrahydrofuran to a 500 ml single-necked flask. While stirring in an ice-water bath, add 4.6 g (120 mmol) of lithium aluminum hydride in fractions. Weigh out 11.56 g (50 mmol) of compound 42N-Boc-6-aminohexanoic acid, dissolve it in 50 ml of tetrahydrofuran, and slowly add it dropwise to the reaction mixture. After the addition is complete, stir at room temperature for 0.5 h, then reflux at 65 °C for 2 h. After cooling to room temperature, quench the reaction by slowly adding 4.6 ml of distilled water and 4.6 ml of 15% sodium hydroxide solution in a water bath. Once the solid in the system has completely turned white, filter under reduced pressure. Concentrate the filtrate, and elute the residue with 5:1 DCM / MeOH column chromatography to give 6.1 g of compound 43, with a yield of 93%. 1 H NMR (400MHz, CDCl3) δ3.60 (t, J = 6Hz, 2H), 2.56 (t, J = 7Hz, 2H), 2.40 (s, 3H), 2.05 (s, 2H), 1.52-1.36 (m, 8H).

[0053] Synthesis of Compound 44

[0054]

[0055] Compound 43 (6.1 g, 46.5 mmol) was weighed into a 250 mL single-necked flask and dissolved in 60 mL of ethanol. Boc₂O (15.2 g, 69.7 mmol) was weighed, dissolved in 50 mL of ethanol, and added dropwise to the reaction mixture. The mixture was stirred at 25 °C for 2 h. After removing the solvent by evaporation, the residue was subjected to column chromatography using 10:1 DCM / MeOH as eluent to give compound 44 (7.8 g, yield 73%). 1 H NMR (400MHz, CDCl3) δ3.64(t,J=6.4Hz,2H),3.21(m,2H),2.83(s,3H),1.63–1.50(m,6H),1.46(s,9H),1.42–1.36(m,2H).

[0056] Synthesis of Compound 45

[0057]

[0058] Compound 44 (6.5 g, 28 mmol) was placed in a 250 mL single-necked flask, and carbon tetrabromide (11.1 g, 33.5 mmol) was added. After evacuation under nitrogen protection, 60 mL of anhydrous DCM was injected to dissolve the compound. The mixture was stirred in an ice bath, and then triphenylphosphine solution (8.8 g, 33.5 mmol, dissolved in 50 mL of anhydrous DCM, with the temperature controlled at 0 °C) was added dropwise. After the addition was complete, the mixture was stirred overnight at 25 °C. The reaction solution was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by column chromatography using 10:1 PE / EA as the eluent to obtain compound 45 (8.2 g), with a yield of 99%. 1 H NMR (400MHz, CDCl3) δ3.40(t,J=6.4Hz,2H),3.20(m,2H),2.83(s,3H),1.86(m,2H),1.52(m,2H),1.45(s,9H),1.29(m,2H).

[0059] Synthesis of Compound 47

[0060]

[0061] Compound 45 (5 g, 17 mmol) and potassium phthalimide (4.7 g, 25 mmol) were weighed into a 250 mL single-necked flask. After evacuation under nitrogen protection, 100 mL of anhydrous DMF was added to dissolve the compound. The mixture was heated and stirred at 100 °C for 24 h. After removing the solvent by evaporation, 100 mL of dichloromethane was added and stirred. The mixture was filtered, and after removing the solvent by evaporation from the filtrate, 8 mL of hydrazine hydrate and 50 mL of ethanol were added. The mixture was heated and refluxed at 90 °C for 2 h, and the solvent was removed by evaporation. Column chromatography was performed using 5:1 DCM / MeOH as the eluent to obtain 2.5 g of compound 47, with a yield of 64%.

[0062] Compound 46 1 H NMR (400MHz, CDCl3) δ7.84(m,2H),7.71(m,2H),3.67(t,J=7.6Hz,2H),3.17(t,J=7.6Hz, 2H),2.81(s,3H),1.74–1.62(m,2H),1.54–1.45(m,2H),1.44(s,9H),1.39–1.26(m,4H).

[0063] Compound 47 1 H NMR (400MHz, CDCl3) δ3.18(t,J=4.8Hz,2H),2.82(s,3H),2.69(t,J=6.8Hz,2H),1.55–1.41(m,4H),1.45(s,9H),1.39–1.23(m,4H).

[0064] Synthesis of Compound 48

[0065]

[0066] Compound 47 (2.5 g, 11 mmol) and triethylamine (2.2 g, 22 mmol) were weighed into a 150 mL single-necked flask and dissolved in 50 mL of methanol by stirring. Ethyl trifluoroacetate (3.3 g, 16 mmol) was added dropwise to the reaction mixture, and the mixture was stirred at 25 °C for 4 h. After removing the solvent by evaporation, 50 mL of dichloromethane was added, and the mixture was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The residue was subjected to column chromatography using 5:1 PE / EA as eluent to give compound 48 2.3 g, with a yield of 65%. 1 H NMR (400MHz, CDCl3) δ3.17(t,J=4.8Hz,2H),2.92(t,J=7.6Hz,2H),2.82(s, 3H),1.86–1.75(m,2H),1.56–1.34(m,4H),1.45(s,9H),1.34–1.21(m,2H).

[0067] Synthesis of Compound 49

[0068]

[0069] Compound 48 (2.3 g, 7 mmol) was weighed into a 100 mL single-necked flask, and 15 mL of dichloromethane and 15 mL of trifluoroacetic acid were added. The mixture was stirred at 25 °C for 2 h. After removing the solvent by vortexing, the product was used directly in the next reaction without purification. 1 H NMR (400MHz, DMSO) δ3.18(q,J=6.4Hz,6.8Hz,2H),2.86(m,2H),2.55(t,J=5.2Hz,3H),1.60–1.42(m,4H),1.35–1.21(m,4H).

[0070] Synthesis of Compound 50

[0071]

[0072] Compound 6,4-aminophenylboronic acid pinacol ester (1.4 g, 6.4 mmol) was placed in a 150 mL double-necked flask, and 15 mL of acetone and 15 mL of HCl aqueous solution (diluted 1 / 2 with 7.5 mL of concentrated HCl) were added. The mixture was stirred, and sodium nitrite (442 mg, 6.4 mmol) was added under ice bath stirring at 0–5 °C. The mixture was stirred at 0 °C for 1 h, and then compound 49 (1.6 g, 7 mmol) was added under 0–5 °C. Et3N was added to adjust the pH to 8–9, and the mixture was stirred at 0 °C for 0.5 h, then at room temperature for 1 h. After the reaction was complete, acetone was removed by rotary evaporation, and the mixture was extracted with EA (30 mL * 5). The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography was performed using a 5:1 PE / EA eluent to obtain compound 50 (2.1 g, 71% yield). 1 H NMR (400MHz, CDCl3) δ7.77(d,J=8.4Hz,2H),7.39(d,J=8.4Hz,2H),3.76(t,J=6.8Hz ,2H),3.34(m,2H),3.22(s,3H),1.62-1.53(m,4H),1.41–1.35(m,4H),1.34(s,12H).

[0073] Synthesis of Compound 39

[0074]

[0075] Weigh out 5-iodo-2-deoxyuridine (306 mg, 67 mmol), 2-chloro-1,3,2-benzodioxophosphazenecyclohexane-4-one (162 mg, 80 mmol), and tri-n-butylamine pyrophosphate (440 mg, 80 mmol) into three 25 ml single-necked flasks, numbered 1, 2, and 3 respectively. Under nitrogen protection, add 1 ml of anhydrous DMF to each flask. Stir at room temperature, then add 1.5 ml of anhydrous tri-n-butylamine to flask 1. After stirring at room temperature for 0.5 h, transfer the solution from flask 1 to flask 2. Continue stirring at room temperature for 0.5 h, then transfer the solution from flask 2 to flask 3. Continue stirring at room temperature for 2 h, then add 5 ml of 3% iodine solution (Py / H₂O = 9:1), and maintain the color for 15 minutes. Add 8 ml of distilled water, stir at room temperature for 2 h, and then add 6 ml of saturated sodium chloride solution. Add the reaction solution to 120 ml of ethanol, shake well, mix and allow to settle. Let stand at -20℃ for 2 h, then centrifuge at 3200 r / min for 20 min. Discard the supernatant, transfer the solid to a 25 ml single-necked flask, add 6 ml of concentrated ammonia, and stir overnight at room temperature. After evaporating the solvent, dissolve in a small amount of distilled water, filter using a syringe filter, and add to a sample vial for preparative HPLC purification. Separation conditions: Agilent Prep-C 18 column (10 μm, 21.2 × 250 mm), mobile phase: 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient: 0%–8% methanol (2 min), 8%–11% methanol (23 min), overall mobile phase flow rate: 8 mL / min, UV detection wavelength: 254 nm, retention time: approximately 20 min. After removing methanol and most of the water by rotary evaporation, the product obtained by preparative HPLC was freeze-dried to obtain 120 mg of white solid. The purity was determined to be 93% by analytical HPLC. The analytical HPLC purity determination conditions were as follows: Elite Supersil ODS2 (5 μm, 4.6 × 250 mm), mobile phase consisting of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade ethanol, elution gradient of 0%–20% ethanol (35 min), overall mobile phase flow rate of 1 mL / min, UV detection wavelength of 254 nm, and retention time of approximately 17 min. 1H NMR (400MHz, D2O) δ8.16(s,1H),6.17(t,J=6.9Hz,1H),4.53(s,1H),4.09(d,J=5.1Hz,3H),2.28(dd,J=6.9,4.7Hz,2H).31P NMR(162MHz,D2O)δ-10.96(d,J=19.6Hz),-11.76(d,J=20.3Hz),-23.37(t,J=20.0Hz).

[0076] Synthesis of Compound 51

[0077]

[0078] Weigh compound 39 (50 mg, 84 μmol), compound 50 (38 mg, 84 μmol), and cesium carbonate (137 mg, 0.4 mmol) into a 25 ml single-necked flask. After evacuating the flask under nitrogen protection, add 4 ml of a water / acetonitrile (2:1) mixed solvent (purged with nitrogen to remove oxygen) and stir at room temperature. Palladium acetate (1.9 mg, 8 μmol) and TPPTS (24 mg, 42 mmol) were weighed into a 10 mL single-necked flask. After evacuation and nitrogen protection, 2 mL of a water / acetonitrile (2:1) mixed solvent was added. After the solid dissolved, it was transferred to a 25 mL single-necked flask and refluxed at 90 °C for 0.5 h. After cooling, acetonitrile and most of the water were removed by rotary evaporation. The residue was filtered using a syringe filter and added to a sample vial for preparative HPLC separation and purification. The separation conditions were: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer system and chromatographic grade methanol, elution gradient of 0%-50% methanol (50 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 293 nm, and retention time of approximately 32 min. After rotary evaporation to remove methanol and most of the water from the product separated by preparative HPLC, it was freeze-dried to obtain 7 mg of orange solid. Analytical HPLC purity detection conditions: Elite Supersil ODS2 (5μm, 4.6×250mm), mobile phase was 20mM triethylamine-acetic acid (TEAA) buffer system and chromatographic grade ethanol, elution gradient was 0%-40% ethanol (70min), overall flow rate of mobile phase was 1mL / min, UV detection wavelength was 293nm, and retention time was 32min.

[0079] Synthesis of Compound 52

[0080]

[0081] Compound 51 (7 mg, 10 μmol) was weighed and added to a 10 mL single-necked flask with 2 mL of 0.5 M Na₂CO₃-NaHCO₃ buffer solution. Compound 41 (Sulf-Cy₃-NHS, 5 mg, 6.8 μmol) was weighed and dissolved in 0.1 mL of anhydrous triethylamine and 1 mL of acetonitrile, and then added to the reaction flask. The mixture was stirred overnight at room temperature in the dark. After removing the acetonitrile by rotary evaporation, the solution was filtered through a syringe filter and added to a sample vial. The solution was then purified by preparative HPLC under the following conditions: Elite Supersil ODS₂ (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0%–50% methanol (50 min), overall flow rate of mobile phase of 4 mL / min, UV detection wavelength of 546 nm, and retention time of approximately 42 min. The product obtained by preparative HPLC was rotary evaporated to remove methanol and most of the water, and then freeze-dried to obtain 1.8 mg of red solid. 1 H NMR(700MHz,D2O)δ8.19(t,J=13.3Hz,1H),7.74-7.57(m,5H),7.27-7.15(m,3H),7. 12-7.01(m,3H),6.11(t,J=13.3Hz,1H),6.09-5.99(m,2H),4.02(m,3H),3.89(m,3H) ,3.79(m,2H),3.48(m,2H),2.96-2.85(m,5H),2.19(s,2H),2.09(m,2H),1.54(s,2H) ,1.47(s,6H),1.45(s,6H),1.21-1.17(m,2H),1.09-1.05(m,2H),1.04-0.98(m,2H). 31 P NMR(283MHz,D2O)δ-5.47,-10.67,-19.21.

[0082] The fluorescently labeled nucleotides of the other three bases (C, A, G) (structures shown in formulas II, III, and IV) were synthesized using a similar method. All final products underwent HPLC purification and lyophilization.

[0083] Example 2: Synthetic route of fluorescently labeled nucleotide (Structural Formula I)

[0084] Synthetic routes such as Figure 4 As shown, it specifically includes:

[0085] Weigh the compound (10.6 mg, 13.5 μmol), compound Sulf-Cy3-NHS (5 mg, 6.8 μmol) was placed in a 10 ml single-necked flask, and after evacuation and nitrogen protection, 1 ml of anhydrous DMF and 0.1 ml of anhydrous triethylamine were added. The mixture was stirred overnight at room temperature in the dark. After diluting with 4 ml of water, the product was freeze-dried to remove DMF. Then, 2 ml of water was added to dissolve the solid. The solution was filtered through a syringe filter and added to a sample vial. Preparative HPLC was used for separation and purification. The separation conditions were: Agilent Eclipse Plus C18 column (10 μm, 21.2 × 250 mm), mobile phase was 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient was 0%-30% methanol (0-5 min), 30%-50% methanol (5-35 min), 50%-80% methanol (35-55 min), overall mobile phase flow rate was 8 mL / min, UV detection wavelength was 546 nm, and retention time was approximately 36 min. After rotary evaporation to remove methanol and most of the water from the product separated by preparative HPLC, it was freeze-dried to obtain 2.3 mg of red solid. 1 H NMR(700MHz,D2O)δ8.34(t,J=13.3Hz,1H),8.28-8.22(m,2H),7.80-7.64(m,7H),7.23(t,J=7.7Hz,1H),7.20-7.10(m,2H),6. 29-6.19(m,3H),4.46(s,1H),4.39(s,1H),4.13(t,J=6.2Hz,1H),4.08(t,J=6.2Hz,1H),4.01-3.95(m,2H),3.94-3.81(m,5H) ,3.00-2.94(m,1H),2.80-2.73(m,2H),2.36(t,J=7.7Hz,1H),2.23(t,J=6.3Hz,1H),2.19(t,J=7.0Hz,1H),2.06-1.94(m,2H) ,1.74-1.67(m,1H),1.57(s,6H),1.48(s,6H),1.45-1.34(m,4H),1.14-1.07(m,5H),0.91-0.87(m,2H),0.80(t,J=7.0Hz,2H).

[0086] 31 P NMR(283MHz,D2O)δ-5.44,-10.68,-19.24.

[0087] The fluorescently labeled nucleotides of the other three bases (C, A, G) (structures shown in formulas II, III, and IV) were synthesized using a similar method. All final products underwent HPLC purification and lyophilization.

[0088] Example 3: Synthetic route of fluorescently labeled nucleotide (Structure III)

[0089] Synthetic routes such as Figure 5 As shown, it specifically includes:

[0090] Synthesis of Compound 54

[0091]

[0092] Compound 53N-Boc-N-methylethylenediamine (3.5 g, 20 mmol) and triethylamine (4 mL) were weighed into a 150 mL single-necked flask and dissolved in 50 mL of methanol. Ethyl trifluoroacetate (4.3 g, 30 mmol) was added dropwise to the reaction mixture with stirring in an ice bath, and the mixture was stirred at 25 °C for 4 h. After removing the solvent by evaporation, 50 mL of dichloromethane was added, and the mixture was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The residue was eluented by column chromatography using 5:1 PE / EA to give compound 54 4.6 g, with a yield of 85%. 1 H NMR (400MHz, CDCl3) δ3.48(s,,4H),2.90(s,3H),1.46(s,9H).

[0093] Synthesis of Compound 55

[0094]

[0095] Compound 54 (4.6 g, 17 mmol) was weighed into a 100 mL single-necked flask, and 25 mL of dichloromethane and 25 mL of trifluoroacetic acid were added. The mixture was stirred at 25 °C for 2 h. After removing the solvent by vortexing, the product was used directly in the next reaction without purification.

[0096] Synthesis of Compound 56

[0097]

[0098] 3.4 g (15.5 mmol) of compound 4-aminophenylboronic acid pinacol ester was placed in a 250 mL double-necked flask, and 20 mL of acetone and 20 mL of HCl aqueous solution (diluted with 10 mL of concentrated HCl) were added. The mixture was stirred, and sodium nitrite (1.1 g, 15.5 mmol) was added under ice bath stirring and temperature control at 0-5 °C. The mixture was stirred at 0 °C for 1 h, and then compound 55 (2.9 g, 17 mmol) was added under temperature control at 0-5 °C. Et3N was added to adjust the pH to 8-9, and the mixture was stirred at 0 °C for 0.5 h, then at room temperature for 1 h. After the reaction was complete, acetone was removed by rotary evaporation, and the mixture was extracted with EA (50 mL x 5). The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography was performed using a 4:1 PE / EA eluent to obtain compound 56 (4.2 g), yield 67%.1 H NMR (400MHz, CDCl3) δ7.80 (d, J = 4.8 Hz, 2H), 7.39 (d, J = 4.8 Hz, 2H), 3.96 (t, J = 2.8 Hz, 2H), 3.67 (s, 2H), 3.39 (s, 3H), 1.34 (s, 12H).

[0099] Synthesis of Compound 57

[0100]

[0101] Compound 39 (50 mg, 84 μmol), compound 56 (33.7 mg, 84 μmol), and cesium carbonate (137 mg, 0.4 mmol) were weighed into a 25 mL single-necked flask. After evacuation under nitrogen protection, 4 mL of a 2:1 water / acetonitrile mixture was added (nitrogen was used to purge oxygen), and the mixture was stirred at room temperature. Palladium acetate (1.9 mg, 8 μmol) and TPPTS (24 mg, 42 mmol) were weighed into a 10 mL single-necked flask. After evacuation under nitrogen protection, 2 mL of a 2:1 water / acetonitrile mixture was added. After the solid dissolved, the mixture was transferred to a 25 mL single-necked flask and refluxed at 90 °C for 0.5 h. After cooling, acetonitrile and most of the water were removed by vortexing. The residue was filtered using a syringe filter and added to a sample vial for preparative HPLC purification. ODS2 (5 μm, 10 × 250 mm) was used with a mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol. The elution gradient was 0% methanol (0-2 min), 10%-20% methanol (2-7 min), and 20%-40% methanol (7-37 min). The overall flow rate of the mobile phase was 4 mL / min. The UV detection wavelength was 293 nm, and the retention time was approximately 15 min. After removing methanol and most of the water by rotary evaporation, the product obtained by preparative HPLC was freeze-dried to obtain 5 mg of orange solid.

[0102] Synthesis of Compound 59

[0103]

[0104] Weigh out compound 57 (5 mg, 7.8 μmol) and add 2 ml of 0.5 M Na2CO3-NaHCO3 buffer solution to a 10 ml single-necked flask. Weigh out Cy3-NHS (2 mg, 3.5 μmol), dissolve it in 0.1 ml of anhydrous triethylamine and 1 ml of acetonitrile, and then add it to the reaction flask. Stir overnight at room temperature in the dark. After removing acetonitrile by rotary evaporation, the solution was filtered through a syringe filter and added to a sample vial. Preparative HPLC was used for separation and purification under the following conditions: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0% methanol (0–2 min), 40%–60% methanol (2–12 min), and 60%–78% methanol (12–30 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 546 nm, and retention time of approximately 17 min. The product obtained by preparative HPLC was rotary evaporated to remove methanol and most of the water, and then freeze-dried to obtain 1.2 mg of a red solid. 1 ¹H NMR (700 MHz, D₂O) δ 8.46 (t, J = 13.3 Hz, 1H), 7.86 (s, 1H), 7.63–7.45 (m, 8H), 7.43–7.36 (m, 2H), 7.21 (d, J = 8.4 Hz, 1H), 6.32 (d, J = 14.0 Hz, 1H), 6.22 (t, J = 7.0 Hz, 1H), 6.12 (d, J = 13.3 Hz, 1H). Alkyl regions were not analyzed.

[0105] The fluorescently labeled nucleotides of the other three bases (C, A, G) (structures shown in formulas II, III, and IV) were synthesized using a similar method. All final products underwent HPLC purification and lyophilization.

[0106] Example 4: Synthetic route of fluorescently labeled nucleotide (structure IV)

[0107] Synthetic routes such as Figure 6 As shown, it specifically includes:

[0108] Synthesis of Compound 61

[0109]

[0110] 2 g (10 mmol) of tert-butyl(4-aminobutyl)(methyl)carbamate and 2 mL of triethylamine were weighed into a 150 mL single-necked flask and dissolved in 30 mL of methanol. Ethyl trifluoroacetate (2.1 g, 15 mmol) was added dropwise to the reaction mixture with stirring in an ice bath, and the mixture was stirred at 25 °C for 4 h. After removing the solvent by evaporation, 30 mL of dichloromethane was added, and the mixture was washed successively with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. The residue was eluent by column chromatography using 5:1 PE / EA to give 2.9 g of compound 61, with a yield of 97%. 1 H NMR (400MHz, CDCl3) δ3.27 (t, J = 4.8Hz, 2H), 3.02-2.94 (m, 4H), 2.87 (s, 3H), 1.58-1.46 (m, 4H), 1.42 (s, 9H).

[0111] Synthesis of Compound 62

[0112]

[0113] Compound 61 (2.9 g, 9.7 mmol) was weighed into a 100 mL single-necked flask, and 15 mL of dichloromethane and 15 mL of trifluoroacetic acid were added. The mixture was stirred at 25 °C for 2 h. After removing the solvent by vortexing, the product was used directly in the next reaction without purification.

[0114] Synthesis of Compound 63

[0115]

[0116] 2 g (9 mmol) of 4-aminophenylboronic acid pinacol ester was placed in a 250 mL double-necked flask. 10 mL of acetone and 10 mL of HCl aqueous solution (diluted 1 / 2 with 5 mL of concentrated HCl) were added and stirred. Sodium nitrite (630 mg, 9 mmol) was added under ice bath stirring and temperature control at 0-5 °C. The mixture was stirred at 0 °C for 1 h, and compound 62 (2 g, 10 mmol) was added under temperature control at 0-5 °C. Et3N was added to adjust the pH to 8-9, and the mixture was stirred at 0 °C for 0.5 h, then at room temperature for 1 h. After the reaction was complete, acetone was removed by rotary evaporation, and the mixture was extracted with EA (30 mL * 5). The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography was performed using a 3:1 PE / EA eluent to obtain 3 g of compound 63, with a yield of 77%. 1 H NMR (400MHz, CDCl3) δ7.78(d,J=8.4Hz,2H),7.37(d,J=8.4Hz,2H),3.76(t,J= 4.8Hz,2H),3.51-3.43(m,4H),3.36(s,3H),2.07-1.95(m,4H),1.34(s,12H).

[0117] Synthesis of Compound 64

[0118]

[0119] Compound 39 (93 mg, 156 μmol), compound 63 (67 mg, 156 μmol), and cesium carbonate (255 mg, 0.8 mmol) were weighed into a 25 mL single-necked flask. After evacuating the flask under nitrogen protection, 4 mL of a water / acetonitrile (2:1) mixed solvent was added and stirred at room temperature. Palladium acetate (3.5 mg, 16 μmol) and TPPTS (45 mg, 78 μmol) were weighed into a 10 mL single-necked flask. After evacuation and nitrogen protection, 3 mL of a water / acetonitrile (2:1) mixed solvent was added. After the solid dissolved, it was transferred to a 25 mL single-necked flask and refluxed at 90 °C for 0.5 h. After cooling, acetonitrile and most of the water were removed by rotary evaporation. The residue was filtered using a syringe filter and added to a sample vial for preparative HPLC separation and purification. The separation conditions were: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer system and chromatographic grade methanol, elution gradient of 0%-50% methanol (50 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 293 nm, and retention time of approximately 30 min. After rotary evaporation to remove methanol and most of the water from the product separated by preparative HPLC, it was freeze-dried to obtain 5 mg of orange solid.

[0120] Synthesis of Compound 65

[0121]

[0122] Weigh out compound 64 (11 mg, 16 μmol) and add 2 ml of 0.5 M Na2CO3-NaHCO3 buffer solution to a 10 ml single-necked flask. Weigh out Cy3-NHS (2 mg, 3.5 μmol), dissolve it in 0.2 ml of anhydrous triethylamine and 2 ml of acetonitrile, and then add it to the reaction flask. Stir overnight at room temperature in the dark. After removing acetonitrile by rotary evaporation, the solution was filtered through a syringe filter and added to a sample vial. Preparative HPLC was used for separation and purification under the following conditions: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0% methanol (0–2 min), 40%–60% methanol (2–12 min), and 60%–78% methanol (12–30 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 546 nm, and retention time of approximately 19 min. The product obtained by preparative HPLC was rotary evaporated to remove methanol and most of the water, then freeze-dried to obtain 1.2 mg of a red solid. The 1H NMR spectrum is shown below. Figure 14 The phosphorus NMR spectrum is shown below. Figure 15 .

[0123] Example 5: Synthetic route of fluorescently labeled nucleotide (structure V)

[0124] Synthetic routes such as Figure 7 As shown, it specifically includes:

[0125] Weigh 67 mmol of 7-deazon-7-iodo-2'-deoxyadenosine, 80 mmol of 2-chloro-1,3,2-benzodioxane-4-one, and 80 mmol of tri-n-butylamine pyrophosphate into three 25 ml single-necked flasks, numbered 1, 2, and 3 respectively. Under nitrogen protection, add 1 ml of anhydrous DMF to each flask. Stir at room temperature, then add 1.5 ml of anhydrous tri-n-butylamine to flask 1. After stirring at room temperature for 0.5 h, transfer the solution from flask 1 to flask 2. Continue stirring at room temperature for 0.5 h, then transfer the solution from flask 2 to flask 3. Continue stirring at room temperature for 2 h, then add 5 ml of 3% iodine solution (Py / H2O = 9:1), and maintain the color for 15 minutes. Add 8 ml of distilled water, stir at room temperature for 2 h, then add 6 ml of saturated sodium chloride solution. Add the reaction solution to 120 ml of ethanol, shake well to mix, and allow to settle. The mixture was allowed to stand at -20℃ for 2 hours, then centrifuged at 3200 rpm for 20 minutes. The supernatant was discarded, and the solid was transferred to a 25 mL single-necked flask. 6 mL of concentrated ammonia was added, and the mixture was stirred overnight at room temperature. After the solvent was evaporated, a small amount of distilled water was added to dissolve the solid. The solution was filtered through a syringe filter and then transferred to a sample vial for preparative HPLC purification. The purification conditions were: Agilent Prep-C 18 column (10 μm, 21.2 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0%–8% methanol (2 min), 8%–11% methanol (23 min), overall mobile phase flow rate of 8 mL / min, UV detection wavelength of 254 nm, and retention time of approximately 20 min. After removing methanol and most of the water by rotary evaporation, the product obtained by preparative HPLC was freeze-dried to obtain 120 mg of a white solid. The purity was determined to be 93% by analytical HPLC. Analytical HPLC purity determination conditions: Elite Supersil ODS2 (5 μm, 4.6 × 250 mm), mobile phase: 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade ethanol, elution gradient: 0%–20% ethanol (35 min), overall mobile phase flow rate: 1 mL / min, UV detection wavelength: 254 nm, retention time: approximately 20 min. Lyophilization yielded compound dA-I.

[0126] Weigh 159 μmol of compound dA-I, 63 (67 mg, 156 μmol), and cesium carbonate (255 mg, 0.8 mmol) into a 25 mL single-necked flask. After evacuating the flask under nitrogen protection, add 4 mL of a water / acetonitrile (2:1) mixed solvent and stir at room temperature. Palladium acetate (3.5 mg, 16 μmol) and TPPTS (45 mg, 78 μmol) were weighed into a 10 mL single-necked flask. After evacuation and nitrogen protection, 3 mL of a water / acetonitrile (2:1) mixed solvent was added (nitrogen was used to purge oxygen). After the solid dissolved, it was transferred to a 25 mL single-necked flask and refluxed at 90 °C for 0.5 h. After cooling, acetonitrile and most of the water were removed by rotary evaporation. The residue was filtered using a syringe filter and added to a sample vial for preparative HPLC separation and purification. The separation conditions were: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase was 20 mM triethylamine-acetic acid (TEAA) buffer system and chromatographic grade methanol, elution gradient was 0%-50% methanol (50 min), overall mobile phase flow rate was 4 mL / min, UV detection wavelength was 293 nm, and retention time was approximately 38 min. After rotary evaporation to remove methanol and most of the water from the product separated by preparative HPLC, it was freeze-dried to obtain 7 mg of solid.

[0127]

[0128] Weigh 66 16 μmol of compound and add 2 ml of 0.5 M Na2CO3-NaHCO3 buffer solution to a 10 ml single-necked flask. Weigh Cy5-NHS (2 mg, 3.5 μmol), dissolve it in 0.2 ml of anhydrous triethylamine and 2 ml of acetonitrile, and add it to the reaction flask. Stir overnight at room temperature in the dark. After removing acetonitrile by rotary evaporation, the solution was filtered through a syringe filter and added to a sample vial. Preparative HPLC was used for separation and purification under the following conditions: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0% methanol (0–2 min), 40%–60% methanol (2–12 min), and 60%–78% methanol (12–30 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 546 nm, and retention time of approximately 28 min. The product separated by preparative HPLC was rotary evaporated to remove methanol and most of the water, then freeze-dried to obtain 1.8 mg of a red solid. The final product was characterized by EESI-MS, showing a correct structure, a measured molecular weight of 1160.24, and a theoretical molecular weight of 1160.13.

[0129] Example 6: Synthetic route of fluorescently labeled nucleotide (structure VI)

[0130] Synthetic routes such as Figure 8 As shown, it specifically includes:

[0131] Using 5-iodo-2'-deoxycytosine nucleoside as a starting material, dC-I was synthesized following the synthetic routes and methods for compounds 39 and dA-I. 160 μmol of compound dC-I, 67 mg (156 μmol) of compound 63, and 255 mg (0.8 mmol) of cesium carbonate were weighed into a 25 mL single-necked flask. After evacuation under nitrogen protection, 4 mL of a water / acetonitrile (2:1) mixed solvent was added, and the mixture was stirred at room temperature. Palladium acetate (3.5 mg, 16 μmol) and TPPTS (45 mg, 78 μmol) were weighed into a 10 mL single-necked flask. After evacuation and nitrogen protection, 3 mL of a water / acetonitrile (2:1) mixed solvent was added. After the solid dissolved, it was transferred to a 25 mL single-necked flask and refluxed at 90 °C for 0.5 h. After cooling, acetonitrile and most of the water were removed by rotary evaporation. The residue was filtered using a syringe filter and added to a sample vial for preparative HPLC separation and purification. The separation conditions were: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase was 20 mM triethylamine-acetic acid (TEAA) buffer system and chromatographic grade methanol, elution gradient was 0%-50% methanol (50 min), overall mobile phase flow rate was 4 mL / min, UV detection wavelength was 293 nm, and retention time was approximately 43 min. After rotary evaporation to remove methanol and most of the water from the product separated by preparative HPLC, it was freeze-dried to obtain 9 mg of solid.

[0132]

[0133] Weigh 68 16 μmol of compound and add 2 ml of 0.5 M Na2CO3-NaHCO3 buffer solution to a 10 ml single-necked flask. Weigh FITC-NHS (2 mg, 3.5 μmol), dissolve it in 0.2 ml of anhydrous triethylamine and 2 ml of acetonitrile, and add it to the reaction flask. Stir overnight at room temperature in the dark. After removing acetonitrile by rotary evaporation, the solution was filtered through a syringe filter and added to a sample vial. Preparative HPLC was used for separation and purification under the following conditions: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0% methanol (0–2 min), 40%–60% methanol (2–12 min), and 60%–78% methanol (12–30 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 546 nm, and retention time of approximately 19 min. The product obtained by preparative HPLC was rotary evaporated to remove methanol and most of the water, then freeze-dried to obtain 1.6 mg of a red solid. The final product was characterized by ESI-MS, showing a correct structure, a measured molecular weight of 1159.37, and a theoretical molecular weight of 1159.23.

[0134] Example 7: Synthetic route of fluorescently labeled nucleotide (structure VII)

[0135] Synthetic routes such as Figure 9 As shown, it specifically includes:

[0136]

[0137] Using 7-denitr-7-iodo-2'-deoxyguanosine as a starting material, it was synthesized according to the synthetic routes and methods of compound 39 and dA-I.

[0138] 0.8 mmol) was placed in a 25 ml single-necked flask, and after evacuation and nitrogen protection, 4 ml of water / acetonitrile (2:1) mixed solvent was added and stirred at room temperature. Palladium acetate (3.5 mg, 16 μmol) and TPPTS (45 mg, 78 μmol) were weighed into a 10 mL single-necked flask. After evacuation and nitrogen protection, 3 mL of a water / acetonitrile (2:1) mixed solvent was added. After the solid dissolved, it was transferred to a 25 mL single-necked flask and refluxed at 90 °C for 0.5 h. After cooling, acetonitrile and most of the water were removed by rotary evaporation. The residue was filtered using a syringe filter and added to a sample vial for preparative HPLC separation and purification. The separation conditions were: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase was 20 mM triethylamine-acetic acid (TEAA) buffer system and chromatographic grade methanol, elution gradient was 0%-50% methanol (50 min), overall mobile phase flow rate was 4 mL / min, UV detection wavelength was 293 nm, and retention time was approximately 49 min. After rotary evaporation to remove methanol and most of the water from the product separated by preparative HPLC, it was freeze-dried to obtain 8 mg of solid.

[0139]

[0140] Weigh 70 16 μmol of the compound and add 2 ml of 0.5 M Na2CO3-NaHCO3 buffer solution to a 10 ml single-necked flask. Weigh Cy3.5-NHS (2 mg, 3.5 μmol), dissolve it in 0.2 ml of anhydrous triethylamine and 2 ml of acetonitrile, and then add it to the reaction flask. Stir overnight at room temperature in the dark. After removing acetonitrile by rotary evaporation, the solution was filtered through a syringe filter and added to a sample vial. Preparative HPLC was used for separation and purification under the following conditions: Elite Supersil ODS2 (5 μm, 10 × 250 mm), mobile phase of 20 mM triethylamine-acetic acid (TEAA) buffer and chromatographic grade methanol, elution gradient of 0% methanol (0–2 min), 40%–60% methanol (2–12 min), and 60%–78% methanol (12–30 min), overall mobile phase flow rate of 4 mL / min, UV detection wavelength of 546 nm, and retention time of approximately 31 min. The product separated by preparative HPLC was rotary evaporated to remove methanol and most of the water, then freeze-dried to obtain 2.0 mg of a red solid. The final product was characterized by ESI-MS, showing a correct structure, a measured molecular weight of 1150.26, and a theoretical molecular weight of 1150.45.

[0141] Example 8: Rapid and complete cleavage of four-color fluorescently labeled nucleotides under hypophosphite.

[0142] Fracture test route as follows Figure 10 As shown. The four-color fluorescently labeled nucleotides (Formulas I-IV) prepared in Examples 4-7 were dissolved in sodium hypophosphite solution at pH 5 and reacted at room temperature for 5 min. The products after the reaction (characterized by 1H-NMR and HRMS) showed a cleavage efficiency of 100%, indicating that this type of compound cleaves DNA strands cleanly and completely after participating in DNA strand elongation, without the formation of byproducts. Moreover, denaturing gel PAGE confirmed ( Figure 11 In this embodiment, the product line 4 after cleavage was separated and purified, and was correctly characterized by denaturing PAGE with the in-situ cleavage product.

[0143] Example 9: DNA strand elongation reaction with reversible terminator

[0144] The reversible termination nucleotides IV, V, VI, and VII synthesized in Examples 4-7 were confirmed by sequencing gels to successfully participate in DNA strand elongation and breakage, demonstrating their potential for DNA sequencing and meeting the requirements of DNA synthesis sequencing.

[0145] (1) DNA strand annealing and extension

[0146] Take a template and primer (24) with a molar ratio of 1:1 (as shown in Table 1), mix them well, keep them at 95℃ for min, and then slowly lower them to room temperature at a rate of 0.1℃ / min to obtain double-stranded DNA (dsDNA); prepare a 20μL extension reaction system according to the components listed in Table 2, mix the prepared reaction solution well, and perform the chain extension reaction on a PCR instrument (Bio-rad T100, US). The reaction conditions are: 65℃ for 5 min → keep at 16℃.

[0147] (2) Fracture

[0148] Dilute the extended DNA solution with 180 μL of dd H2O, then add 28 μL of 0.24 M HCl to adjust the pH to 2.95. Shake at room temperature for 1 min, then add 16 μL of 0.1 M NaH2PO2 and shake at room temperature for another 1 min. Finally, adjust the pH to 8.0 with 28 μL of 1 M Tris.

[0149] (3) Gel electrophoresis detection

[0150] The extended and fragmented samples were analyzed by gel electrophoresis using 12% denaturing polyacrylamide containing 7M urea. Before electrophoresis, the prepared gel was run at a constant voltage of 2000V and a constant power of 40W for 30 min. Simultaneously, the sequencing reaction sample was mixed with a small amount of 0.1M sodium hydroxide, heated to 95℃ for 3 min, and then rapidly cooled to room temperature to denature it into single strands. 1 μL of sample (approximately 15 ng / μL DNA) was mixed with 2 μL of loading buffer containing labeled dye, and then electrophoresis was performed at a constant voltage of 2000V and a constant power of 40W for 3 h. The gel was then observed under 785nm laser excitation using an Odyssey infrared imaging system (LI-CORBiosciences, US). Figure 12 , Figure 13 Experimental results show that the reversible termination nucleotide can achieve 100% extension in the sequencing cycle, and the extended product can be 100% cleaved. After the first extension product is cleaved, the second extension can still achieve 100% extension and 100% cleavage. Furthermore, when there are multiple consecutive identical bases in the template, the reversible termination nucleotide we synthesized only extends one base per sequencing cycle.

[0151] Table 1 DNA Synthesis Sequencing Templates and Primers

[0152]

[0153] Table 2 Buffer systems for DNA synthesis and sequencing

[0154]

[0155] Example 10: Four-color fluorescently labeled reversible termination nucleotide DNA single-molecule sequencing system

[0156] This embodiment provides a DNA single-molecule sequencing system and sequencing method. In this embodiment, the reversible termination nucleotides IV, V, VI, and VII prepared in Examples 4-7 are selected as four-color fluorescent reversible termination nucleotide sequencing reagents.

[0157]

[0158] The four different template sequences to be tested are as follows: 5'-CTACGTTCGAACTACTAACTTGATGTAGCTTCGTAGTAATTTTTTTTTTTTTTTTTTTTTT-3' (Sequence 1),

[0159] 5'-CTACGTTCGAACTACTAATGGCCAACTTTAGGTACAGGCTTTTTTTTTTTTTTTTT TTT-3' (sequence 2),

[0160] 5'-CTACGTTCGAACTACTAAGCAATCCGGCAGATCGTCACTTTTTTTTTTTTTTTTTTT TTT-3' (sequence 3),

[0161] 5'-CTACGTTCGAACTACTAAAACTGGTACAGCCAACGTCTGTTTTTTTTTTTTTTTT TTT-3' (sequence 4)

[0162] First, the templates with the four different sequences were hybridized with primers fixed on the surface of the flow cell reactor at 65°C for 5 min. Then, under the action of DNA polymerase, four different fluorescently labeled reversible termination nucleotide primers were used for extension reaction for 15 min at 60°C. After the first extension reaction, the information of the target bases could be obtained by detecting the fluorescence signal of the extension product. Then, the fluorescein labeled on the bases was removed under hypophosphorous acid (pH=5). The fluorescence image after the first extension was used as the localization fluorescence, and the same steps were used for the second extension cycle, and so on, for multiple sequencing cycles. In this embodiment, the fluorescence information of the previous extension product is used as the localization information of the next extension product, and it is not necessary to label the 3′-end of the target template with specific localization fluorescence information. Furthermore, in our preliminary experiments, we found that the four-color fluorescence single-molecule sequencing system does not have fluorescence quenching due to localization information without the need to specifically label the target template with localization information.

[0163] Therefore, the single-molecule sequencing system described in this invention is a high-throughput single-molecule sequencing system that achieves long read lengths and low error rates. These experimental results were obtained using our own designed sequencing chip and device.

[0164] In summary, the triazine four-color fluorescently labeled nucleotides described in this invention offer advantages such as longer read lengths and lower error rates for both DNA synthesis sequencing and single-molecule sequencing. Furthermore, they exhibit high sequencing efficiency, requiring only one base read in a single sequencing cycle. In contrast, existing monochrome single-molecule sequencing systems require four sequencing cycles to determine a single base read, thus improving sequencing efficiency by four times. In addition, Michal Hocek et al. investigated the extension effects of natural nucleotides with nucleotides modified with π-electron groups such as benzene rings, alkynes, or vinyl groups. They concluded that compared to natural nucleotides, nucleotides modified with π-electron groups such as benzene rings, alkynes, or vinyl groups exhibit better extension effects due to their superior site binding to DNA polymerases such as Bst (Angew. Chem. Int. Ed. 2014, 53, 7552-7555; ACS Chem. Biol. 2016, 11, 3165-3171). However, it only compared the results of natural nucleotides with nucleotides modified with π-electron groups such as benzene rings, alkynyl groups, or vinyl groups in the first extension of the DNA chain. DNA sequencing requires hundreds of extensions to be meaningful. Whether the changes in DNA chain configuration and conformation caused by multiple extensions still have better binding sites with polymerase, or whether they are still better DNA chain extension reactants, are unknown and unpredictable. Furthermore, it does not involve the structure of triazine, and naturally does not involve the study of the breakage of triazine-related structures.

[0165] It should be noted that the four-color fluorescent single-molecule sequencing system provided by this invention is not limited to the currently proposed types of reversible terminators, but is also applicable to other types of reversible terminators.

[0166] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A fluorescently labeled reversible termination nucleotide, the structural formula of which is: 、 、 、 、 or .

2. A fluorescently labeled reversible termination nucleotide, the structural formula of which is: 、 or .

3. The use of a fluorescently labeled reversible termination nucleotide according to any one of claims 1-2 in DNA synthesis sequencing or single-molecule sequencing.

Images