Compounds, preparation methods and uses thereof

Abstract

The present invention provides a compound, a preparation method and use thereof. The compound has a structure shown in formula (I). The compound is used as a linker and can complete splicing through two-step reactions. The resulting spliced ​​compound has good chemical stability and optical properties and is used for gene sequencing. The synthetic route is fast, direct, and efficient, and has high atomic economy, simple operation, low cost, and is easy to mass produce, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the biological field. Specifically, the present invention relates to a compound, a preparation method thereof and use thereof. Background Art

[0002] The first generation of gene sequencing is the Sanger method, which uses direct sequencing. Its basic principle is that dideoxynucleoside triphosphate (ddNTP) lacks the 3'-OH required for PCR extension. Therefore, every time the DNA chain adds a molecule of ddNTP, the extension is terminated. Each DNA sequencing is composed of 4 independent reactions. The template, primers and 4 kinds of ddNTP containing different radioactive isotope-labeled nucleotides are mixed with DNA polymerase to form fragments of different lengths. A large number of DNA fragments with the same starting point and different end points exist in the reaction system. DNA sequences with a single base difference can be separated by polyacrylamide denaturing gel electrophoresis to obtain radioactive isotope autoradiographic bands, and the base sequence of the double-stranded DNA is read based on the electrophoresis bands.

[0003] ABI further developed a fluorescently labeled dideoxy sequencing kit based on Sanger sequencing, namely BigDye TM Reagents, and then combined with capillary electrophoresis, produced very successful test instruments such as ABI3730 and ABI3500, the main features of which are sequencing read lengths of up to 1000bp and accuracy of up to 99.999%. ABI3730, ABI3500 and its essential BigDye sequencing reagents based on Sanger principle of capillary electrophoresis are still recognized as the gold standard for ultra-high precision sequencing in the industry.

[0004] BigDye TM The reagent consists of three parts: Acceptor Dye, Linker, and ddNTP. Taking ddTTP-Linker-dTMRA as an example, its current synthesis route is as follows:

[0005]

[0006] Synthesis steps: First, ddTTP is synthesized, and then ddTTP-Linker is obtained through four steps of amidation, hydrolysis, amidation, and hydrolysis. Then, it is reacted with dTMRA to complete the final splicing to obtain BigDye. TM Reagents.

[0007] BigDye TMIn the current synthesis method of the reagent, ddNTP is used as the starting material, and it takes five steps of reaction to complete the final splicing with Dye. Considering the instability of ddNTP, the reaction process is complicated, the yield is extremely low, and the separation and purification are difficult. It cannot be used for large-scale synthesis, so this method has a large room for improvement. Summary of the invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention proposes a compound, a preparation method and use thereof, wherein the compound is used as a linker and can complete splicing through two-step reactions. The resulting spliced ​​compound has good chemical stability and optical properties and is used for gene sequencing. The synthetic route is fast, direct, and efficient, and has high atomic economy, simple operation, low cost, and is easy to mass produce, and has broad application prospects.

[0009] In one aspect of the present invention, the present invention provides a compound. According to an embodiment of the present invention, the compound has a structure shown in formula (I),

[0010]

[0011] Among them, each R1~R 10 are independently H, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, C 1-6 Cycloalkyl, heterocyclic group composed of 3-8 atoms or R1~R 10 The heterocyclic group is formed by connecting 5 to 8 ring atoms, wherein the heterocyclic group formed by the 5 to 8 ring atoms is unsubstituted or substituted by at least one group selected from the following: 1 to 4 halogen atoms, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino substituted; X is H, halogen or amino.

[0012] The compounds according to the embodiments of the present invention can be used as intermediates to prepare fluorescently labeled nucleotides for gene sequencing, making the synthesis route fast, direct, and efficient, with high atom economy, simple operation, low cost, and easy large-scale production, and having broad application prospects.

[0013] According to an embodiment of the present invention, the composition may also have the following additional technical features:

[0014] According to an embodiment of the present invention, each R1~R 10 are independently H, halogen, C 1-6 alkyl.

[0015] According to an embodiment of the present invention, each R1~R 10 are independently H; X is Cl.

[0016] In another aspect of the present invention, the present invention provides a compound. According to an embodiment of the present invention, the compound has a structure shown in formula (II),

[0017]

[0018] Among them, R1~R 10 and X are defined as above; R 11 is -ddNTP.

[0019] The compounds according to the embodiments of the present invention contain amino groups and can be used as intermediates to prepare fluorescently labeled nucleotides for gene sequencing, making the synthesis route fast, direct, and efficient, with high atom economy, simple operation, low cost, and easy large-scale production, and having broad application prospects.

[0020] According to an embodiment of the present invention, the ddNTP is selected from any one of ddATP, ddGTP, ddCTP and ddTTP.

[0021] In another aspect of the present invention, the present invention provides a compound. According to an embodiment of the present invention, the compound has a structure shown in formula (III),

[0022]

[0023] Among them, R1~R 10 , X and R 11 The definition of R is the same as above; 12 Connected with -OH to form fluorescein.

[0024] The compounds according to the embodiments of the present invention have good chemical stability and optical properties, can be used for DNA sequencing and termination of DNA chain extension, and have broad application prospects.

[0025] According to an embodiment of the present invention, the fluorescein is selected from any one of dTMRA, dRox, dR6G, and dR110.

[0026] According to an embodiment of the present invention, the compound is at least one of the following:

[0027]

[0028]

[0029]

[0030] The inventors found that the above six compounds, when used for DNA sequencing, can better meet various conditions such as machines and enzymes and can effectively terminate DNA chain extension.

[0031] In another aspect of the present invention, the present invention provides a method for preparing the compound represented by the above formula (I). According to an embodiment of the present invention, the method comprises:

[0032]

[0033] The inventors have conducted in-depth research on the synthesis route of the compound represented by formula (I), and the compound represented by formula (I-a) is subjected to a first acid-amine condensation reaction with the compound represented by formula (I-b) in a first solvent under the action of a condensation agent and an acid binding agent, thereby efficiently obtaining the compound represented by formula (I) and reducing the occurrence of side reactions.

[0034] According to an embodiment of the present invention, the condensing agent is TNTU, the acid binding agent is DIPEA, and the first solvent is dimethylformamide. The inventors have optimized the conditions of the first acid-amine condensation reaction in large quantities and found that under the above conditions, the reaction can be well promoted, the reaction efficiency can be improved, and the side reactions can be reduced.

[0035] According to an embodiment of the present invention, the method further comprises: subjecting the compound represented by formula (I) to liquid chromatography purification. The inventors have found that liquid chromatography purification can effectively separate and purify the product and improve the product yield and purity.

[0036] According to an embodiment of the present invention, the method for preparing the compound represented by formula (I-a) comprises:

[0037]

[0038] The inventors have conducted in-depth research on the synthesis route of the compound represented by formula (Ⅰ-a). The compound represented by formula (Ⅰ-aa) undergoes a second acid-amine condensation reaction with the compound represented by formula (Ⅰ-ab) in a second solvent under the action of a condensation agent and an acid binding agent, thereby effectively obtaining the compound represented by formula (Ⅰ-a) and reducing the occurrence of side reactions.

[0039] According to an embodiment of the present invention, the condensing agent is TNTU, the acid binding agent is DIPEA, and the second solvent is dimethylformamide. The inventors have optimized the conditions of the second acid-amine condensation reaction in large quantities and found that under the above conditions, the reaction can be well promoted, the reaction efficiency can be improved, and the side reactions can be reduced.

[0040] According to an embodiment of the present invention, the method further comprises: subjecting the compound represented by formula (I-a) to liquid chromatography purification. The inventors have found that liquid chromatography purification can effectively separate and purify the product and improve the product yield and purity.

[0041] According to an embodiment of the present invention, the method for preparing the compound represented by formula (I-b) comprises:

[0042]

[0043] Under alkaline conditions, one of the amino groups of the compound represented by formula (I-ba) is subjected to Boc protection, and then the other amino group is subjected to trifluoroacetyl protection, and the Boc protection is removed, and HCl gas is passed to obtain the compound represented by formula (I-b).

[0044] According to the method of an embodiment of the present invention, the two amino groups of the compound represented by formula (I-ba) are successively subjected to Boc protection and trifluoroacetyl protection, and the Boc protection is removed under acidic conditions, and the trifluoroacetyl-protected amino group is not affected, so that when the first acid-amine condensation reaction is carried out with the compound represented by formula (I-a), the target product can be obtained, thereby improving the reaction yield.

[0045] In another aspect of the present invention, the present invention provides a method for preparing a compound represented by formula (II). According to an embodiment of the present invention, the method comprises: reacting the compound represented by formula (I) with a condensing agent and an acid-binding agent in a third solvent; A third acid amine condensation reaction is carried out, wherein R 11 The definition of is the same as described above. The acid-amine condensation reaction can effectively obtain the compound represented by formula (II) and reduce the occurrence of side reactions.

[0046] According to an embodiment of the present invention, the condensing agent includes TNTU and / or HBTU, the acid binding agent is DIPEA, and the third solvent is dimethylformamide. The inventors have optimized the conditions of the third acid-amine condensation reaction in large quantities and found that under the above conditions, the reaction can be better promoted, the reaction efficiency can be improved, and the side reactions can be reduced.

[0047] According to an embodiment of the present invention, the method further comprises: subjecting the compound represented by formula (II) to liquid chromatography purification. The inventors have found that liquid chromatography purification can effectively separate and purify the product and improve the product yield and purity.

[0048] In another aspect of the present invention, the present invention proposes a method for preparing a compound shown in formula (III). According to an embodiment of the present invention, the method comprises: the compound shown in formula (II) undergoes a fourth acid amine condensation reaction with fluorescein in a fourth solvent under the action of a condensing agent and an acid binding agent. The acid amine condensation reaction can effectively obtain the compound of formula (III) and reduce the occurrence of side reactions. As mentioned above, the compound shown in formula (I) contains a carboxyl group and a trifluoroacetamide group. As a linker compound, the compound undergoes an acid amine condensation reaction with ddNTP under the action of a condensing agent and an acid binding agent, and then removes the corresponding amino protecting group (such as trifluoroacetyl) under alkaline conditions to obtain an amino-containing compound (formula (II)). The compound further undergoes an acid amine condensation reaction with fluorescein under the action of a condensing agent and an acid binding agent to obtain a compound shown in formula (III). The overall reaction route is reasonably designed, reducing the number of times a compound having a ddNTP structure participates in the reaction. The synthetic route is fast, direct, efficient, and has high atom economy, simple operation, low cost, and is easy to mass produce, and has broad application prospects.

[0049] According to an embodiment of the present invention, the condensing agent includes TNTU, TSTU and / or DSC, the acid binding agent includes DIPEA and / or DMAP, and the fourth solvent includes dimethylformamide and / or dimethyl sulfoxide. The inventor has optimized the conditions of the acid-amine condensation reaction in large quantities and found that under the above conditions, the reaction can be promoted well, the reaction efficiency can be improved, and the side reactions can be reduced.

[0050] According to an embodiment of the present invention, the method further comprises: subjecting the compound represented by formula (III) to liquid chromatography purification. The inventors have found that liquid chromatography purification can effectively separate and purify the product and improve the product yield and purity.

[0051] In another aspect of the present invention, the present invention proposes the use of the compound represented by the above formula (III) in gene sequencing. As mentioned above, the compound represented by formula (III) has good chemical stability and optical properties, can be used for DNA sequencing, terminates DNA chain extension, and the entire synthetic route has high atomic economy, simple operation, low cost, easy large-scale production, and has broad application prospects.

[0052] In another aspect of the present invention, the present invention proposes a gene sequencing kit. According to an embodiment of the present invention, the gene sequencing kit contains the compounds shown in the above formula (I), formula (II), and formula (III). As mentioned above, the compound shown in formula (III) has good chemical stability and optical properties, can be applied to DNA sequencing, terminate DNA chain extension, and the entire synthesis route has high atomic economy, simple operation, low cost, easy large-scale production, and has broad application prospects. In the kit, the compound shown in formula (III) can be directly provided, and the compound shown in formula (I) or formula (II) can also be provided. Before it is needed for sequencing, the compound shown in formula (III) is synthesized according to the method described above.

[0053] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. DETAILED DESCRIPTION

[0054] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0055] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0056] Example 1: Synthesis of V3-linker-TFA (corresponding to each R1 to R 10 are independently H; X is Cl)

[0057] The synthetic route is as follows:

[0058]

[0059] a) a-1. (Boc)2 di-tert-butyl dicarbonate, TEA (triethylamine) / DCM (dichloromethane); a-2. CF3COOEt; a-3. HCl / EA (ethyl acetate); b) TNTU, DIPEA / DMF; c) TNTU, DIPEA / DMF

[0060] Compound 1 (5 g, 36.8 mmol, 3.0 eq) and triethylamine (7.43 g, 73.6 mmol, 6.0 eq.) were added to a 500 mL flask, and completely dissolved with dichloromethane (150 mL). The reaction solution was cooled to 0°C. Di-tert-butyl dicarbonate (2.5 g, 11.5 mmol, 1.0 eq.) was slowly dripped into the reaction solution, and the reaction temperature was kept at 0°C and stirred for 16 hours. CF3COOEt (2.85 g, 23 mmol, 2.0 eq.) was added dropwise at room temperature, and then the reaction was stirred at room temperature for 2 hours. The crude product was concentrated under reduced pressure and purified by column chromatography (elution with a volume ratio of petroleum ether and ethyl acetate of 2:1) to obtain 2.1 g of white solid. It was completely dissolved with ethyl acetate (50 mL); HCl gas was passed through at room temperature for 2 hours, and then the reaction was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was filtered to obtain white solid compound 2 (1.6 g).

[0061] 1 H-NMR (400M, DMSO-D6): δ (ppm) 8.93 (s, 1H), 8.71 (s, 2H), 7.21-7.31 (m, 4H), 4.3 (s, 2H), 4.2 (s, 2H).

[0062] Compound 4 (816 mg, 3.2 mmol, 1.2 eq.) was added to a 100 mL single-necked flask and completely dissolved with anhydrous DMF (10 mL); TNTU (1.272 g, 3.2 mmol, 1.2 eq.) and DIPEA (1.344 g, 9.6 mmol, 3.6 eq.) were added at room temperature and reacted at 30°C for 60-90 min (TLC: DCM: MeOH: formic acid = 30:1:1 (v / v / v)); then compound 3 (1.18 g, 2.67 mmol, 1 eq.) was added to the reaction solution and reacted at 30°C overnight. After the reaction was completed, the mixture was filtered with an organic filter membrane and sent to preparative HPLC for separation. The chromatographic conditions were as follows:

[0063] Chromatographic column: YMC–Actus ODS-A-HG 250*30mm C18 column; mobile phase: A acetonitrile, B 0.1mol / L TEAB (pH=7.5-7.8); HPLC gradient setting see Table 1.

[0064] Table 1

[0065] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 3 30 5 95 5 30 8 92 16 30 10 90 19.5 30 10 90 20 30 40 60 23 30 60 40 23.5 30 5 95 27 30 5 95

[0066] The target product had an elution time of about 18 min and was freeze-dried to obtain compound 5 (867 mg).

[0067] 1H-NMR (400M, DMSO-D6): δ (ppm) 13.11 (s, 1H), 12.68 (s, 1H), 10.45 (s, 1H), 9.78-9.81 (t, 1H), 9.09-9.12 (t, 1H), 8.55 -8.58(t,1H),8.22(s,1H),8.11-8.13(t,1H),7.54(s,1H),7.51(s,1H),6.74(s,1H),6.3-6.55(m,4H),3.93(s,2H).

[0068] Compound 5 (867 mg, 1.4 mmol, 1.0 eq.) was added to a 50 mL single-necked flask and completely dissolved in anhydrous DMF (10 mL); TNTU (509 mg, 1.4 mmol, 1.0 eq.) and DIPEA (540 mg, 4.2 mmol, 3.0 eq.) were added at room temperature, reacted at 30 ° C for 90 min, and TLC spot plate detection (DCM: MeOH: formic acid = 10: 1: 0.5); then compound 2 (449 mg, 1.68 mmol, 1.2 eq.) was added to the reaction solution and reacted at 30 ° C for 4 h. After the reaction was completed, it was filtered with an organic filter membrane and sent to preparative HPLC for separation. Chromatographic conditions: chromatographic column: YMC-Actus ODS-A-HG 250*30 mm C18 column; mobile phase: A acetonitrile, B 0.1 mol / L TEAB (pH = 7.5-7.8); HPLC gradient settings are shown in Table 2.

[0069] Table 2

[0070]

[0071]

[0072] The target product had a peak time of about 22 min and was freeze-dried to obtain V3-Linker-TFA (386 mg).

[0073] 1 H-NMR (400M, DMSO-D6): δ (ppm) 9.94-9.97 (t, 1H), 9.35-9.38 (t, 1H), 8.77-8.80 (t, 1H), 8.48 (s, 1H), 8.25-8.27 (t, 1H), 7.82 ( s,1H),7.51(s,1H),7.21-7.32(m,4H),6.74(s,1H),6.5-6.63(m,4H),4.64-4.66(m,2H),4.47-4.49(d,2H),4.33-4.34(d,2H).

[0074] Example 2: Synthesis of ddTTP-V3-linker-dTMRA (corresponding to formula (III-1))

[0075] The synthetic route is as follows:

[0076]

[0077] Dissolve the compound V3-Linker-TFA (100 mg, 1.0 eq) in 3 mL of anhydrous DMF, add HBTU (54 mg, 1.2 eq) and DIPEA (18.6 mg, 1.2 eq) in turn, and activate at 30°C for 3 h. Add ddTTP (72 mg, 1.2 eq) to the activated V3-Linker-TFA and react at 30°C overnight. Send to LC-MS for monitoring, it shows that some of the conversion is not complete, add 2.4 eq DIPEA, and react again overnight. Then add 20 mL of 25-28% ammonia water, stir and react at room temperature for 1 h, concentrate under reduced pressure until no solvent is evaporated, and purify by preparative HPLC. Chromatographic conditions: Chromatographic column: Actus ODS-AHG 30*25010um; mobile phase: A acetonitrile, B 0.1mol / LTEAB (pH=7.5-7.8); HPLC gradient setting see Table 3.

[0078] Table 3

[0079] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 3 30 5 95 5 30 20 80 17 30 30 70 20 30 35 65 23 30 40 60 23.5 30 5 95 26 30 5 95

[0080] The target product elutes at about 19 min, and lyophilization yields ddTTP-V3-linker (250 mg, purity 93%, yield 68%). MS: [M+1] + =1227.4.

[0081] Compounds dTMRA (20 mg, 0.04 mmol, 1.0 eq), DSC (12.3 mg, 0.047 mmol, 1.2 eq) and DMAP (5.8 mg, 0.047 mmol, 1.2 eq) were added to a 5 mL brown bottle, dissolved completely in 1 mL DMF, and reacted at 25 ° C for 4 h; TLC monitoring showed that the activated product was generated, and the compound ddTTP-V3-linker (49 mg, 0.04 mmol, 1.0 eq) was dissolved in 1 mL of water and added to the reaction solution, and reacted at 25 ° C for 12 h; LCMS monitoring: Take a small amount of the reaction solution, dilute with water, and LCMS shows that the product is generated. After the reaction is completed, dilute with water to obtain a crude product, which is separated and purified by preparative HPLC. Chromatographic conditions: Chromatographic column: Actus ODS-AHG 30*25010um; mobile phase: A acetonitrile, B 0.1mol / L TEAB (pH=7.5-7.8); HPLC gradient setting see Table 4.

[0082] Table 4

[0083]

[0084]

[0085] The target product elutes at about 16 min and is freeze-dried to obtain a dark red solid, namely ddTTP-V3-linker-dTMRA (2 mg, yield 3%). + =1710.1.

[0086] Example 3: Synthesis of ddCTP-V3-linker-dRox (corresponding to formula (III-2))

[0087] The synthetic route is as follows:

[0088]

[0089] a) 1. HBTU, DIPEA, DMF, 30℃; 2. Ammonia, 30℃. b) DSC, DMAP, DMF, 25℃

[0090] Compound V3-Linker-TFA (100 mg, 1.0 eq) was dissolved in 3 mL of anhydrous DMF, and HBTU (54 mg, 1.2 eq) and DIPEA (18.6 mg, 1.2 eq) were added in sequence, and activated at 30°C for 3 h. ddCTP (72 mg, 1.2 eq) was added to the reaction solution 1, stirred at 30°C for 2 h, and then 200 mL of 25-28% ammonia water was added, and stirring was continued for 1 h. After reduced pressure concentration, preparative HPLC purification was performed, and the chromatographic conditions were: chromatographic column: Actus ODS-AHG 30*250 10um mobile phase: A acetonitrile, B 0.1mol / L TEAB (pH=7.5-7.8); HPLC gradient setting see Table 5.

[0091] Table 5

[0092]

[0093]

[0094] The target product elutes at about 15.5 min, and ddCTP-V3-Linker (about 180 mg, yield 60%, purity 99%) is obtained by freeze-drying. MS: [M+1] + =1226.3.

[0095] Compound dRox (20 mg, 0.033 mmol, 1.0 eq), DSC (10 mg, 0.039 mmol, 1.2 eq) and DMAP (4.76 mg, 0.039 mmol, 1.2 eq) were added to a 5 mL brown bottle, dissolved completely in 1 mL DMF, and reacted at 25 ° C for 4 h; TLC monitoring showed that the activated product was generated, and the compound ddCTP-V3-Linker (40.5 mg, 0.033 mmol, 1.0 eq) was dissolved in 1 mL of water and added to the reaction solution, and reacted at 25 ° C for 12 h; LCMS monitoring: Take a small amount of the reaction solution, dilute with water, and LCMS shows that the product is generated. After the reaction is completed, dilute with water to obtain a crude product; the crude product is separated and purified by preparative HPLC, chromatographic conditions: chromatographic column: Actus ODS-AHG 30*250 10um; mobile phase: A acetonitrile, B 0.1mol / L TEAB (pH=7.5-7.8); HPLC gradient setting see Table 6.

[0096] Table 6

[0097] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 3 30 5 95 5 30 32 68 24 30 32 68 26 30 40 60 27 30 40 60 28 30 5 95 32 30 5 95

[0098] The target product elutes at about 25 min and is freeze-dried to obtain a dark purple solid, namely ddCTP-V3-linker-dRox (7 mg, yield 11.6%). + =1811.8.

[0099] Example 4: Synthesis of ddATP-V3-linker-dR6G (corresponding to formula (III-3))

[0100] The synthetic route is as follows:

[0101]

[0102] V3-Linker-TFA (200 mg, 0.24 mmol, 1.0 eq) was dissolved in 5 mL of anhydrous DMF, and HBTU (110 mg, 0.29 mmol, 1.2 eq) and DIPEA (38 mg, 0.29 mmol, 1.2 eq) were added in sequence, and activated at 30°C for 3 h; then ddATP (157 mg, 0.29 mmol, 1.2 eq) was added, and the reaction was carried out at 30°C for 12 h. Finally, 10 mL of 25-28% ammonia water was added, and the reaction was stirred at room temperature for 1 h. After concentrating under reduced pressure, the product was purified by preparative HPLC. Chromatographic conditions: chromatographic column: YMC-Actus prep C18 30×250 mm, mobile phase: A acetonitrile, B 0.1 mol / L TEAB (pH=7.5-7.8), and the HPLC gradient setting is shown in Table 7.

[0103] Table 7

[0104]

[0105]

[0106] The target product elutes at about 15 min, and ddATP-V3-Linker (180 mg, yield 60%, purity 99%) is obtained by freeze-drying. MS: [M+1] + =1250.7.

[0107] Compound dR6G (30 mg, 0.057 mmol, 1.0 eq), TSTU (20.7 mg, 0.069 mmol, 1.2 eq) and DIPEA (9.0 mg, 0.069 mmol, 1.2 eq) were added to a 10 mL brown bottle, 5 mL DMSO was added to dissolve completely, and activated at 25 ° C for 4 h; TLC monitoring showed that the activated product was generated, and compound ddATP-V3-Linker (86 mg, 0.069 mmol, 1.2 eq) was added, and the reaction was carried out at 25 ° C for 12 h; LCMS monitoring: a small amount of the reaction solution was taken and diluted with water, and LCMS showed that the product was generated. After the reaction was completed, it was diluted with water to obtain a crude product; the crude product was separated and purified by preparative HPLC, and the chromatographic conditions were as follows: Chromatographic column: Actus ODS-AHG 30*250 10 μm; mobile phase: A acetonitrile, B 0.1 mol / L TEAB (pH=7.5-7.8); HPLC gradient setting see Table 8.

[0108] Table 8

[0109] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 2 30 5 95 3 30 29 71 19 30 32 68 21.5 30 32 68 22 30 5 95 26 30 5 95

[0110] The target product elutes at about 18.5 min and is freeze-dried to obtain a dark purple solid, namely ddATP-V3-linker-dR6G (18 mg, yield 18%). + =1760.3.

[0111] Example 5: Synthesis of ddGTP-V3-linker-dR6G (corresponding to formula (III-4)

[0112] The synthetic route is as follows:

[0113]

[0114] Compound V3-Linker-TFA (300 mg, 0.36 mmol, 1.0 eq) was dissolved in 5 mL of anhydrous DMF, and HBTU (163 mg, 0.43 mmol, 1.2 eq) and DIPEA (56 mg, 0.43 mmol, 1.2 eq) were added in sequence, and activated at 30°C for 3 h; ddGTP (234 mg, 0.43 mmol, 1.2 eq) was then added, and stirred at 30°C for 12 h. Finally, 10 mL of 25-28% ammonia water was added, and the reaction was stirred at room temperature for 1 h. After reduced pressure concentration, the product was purified by preparative HPLC, chromatographic column: YMC-Actus prep C18 30×250 mm, mobile phase: A acetonitrile, B 0.1 mol / L TEAB (pH=7.5-7.8), HPLC gradient settings are shown in Table 9.

[0115] Table 9

[0116] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 2 30 5 95 3 30 17 83 12 30 17 83 12.5 30 40 60 15 30 40 60 16 30 5 95 18 30 5 95

[0117] The target product elutes at about 15 min and is freeze-dried to obtain ddGTP-V3-Linker (220 mg, purity 99.15%, MS: [M+1] + =1249.2), yield 51%, maximum absorption wavelength 495.5nm.

[0118] Weigh compound R6G (20 mg, 0.038 mmol, 1.0 eq) into a 10 mL brown bottle, add 5 mL DMSO to dissolve, add TSTU (13.8 mg, 0.046 mmol, 1.2 eq) and DIPEA (6.0 mg, 0.046 mmol, 1.2 eq) in sequence, activate at 25 ° C for 4 h; TLC monitoring, activated product is generated, add compound ddGTP-V3-Linker (58 mg, 0.046 mmol, 1.2 eq), react at 25 ° C for 12 h; LCMS monitoring: take a small amount of reaction solution, dilute with water, LCMS shows that the product is generated. After the reaction is completed, dilute with water to obtain a crude product; prepare HPLC purification, the chromatographic conditions are as follows: Chromatographic column: Actus ODS-AHG 30*250 10 μm; mobile phase A: acetonitrile, mobile phase B: 0.1 mol / L TEAB (pH=7.5-7.8); HPLC gradient setting see Table 10.

[0119] Table 10

[0120] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 3 30 5 95 5 30 13 87 8 30 15 85 17 30 17 83 19 30 17 83 21 30 20 80 22 30 5 95 25 30 5 95

[0121] The target product elutes at about 21 min and is freeze-dried to obtain a dark purple compound, namely ddGTP-V3-linker-dR6G (12 mg, yield 15%). + =1775.3.

[0122] Example 6: Synthesis of ddGTP-V3-linker-dR110 (corresponding to formula (III-5))

[0123] The synthetic route is as follows:

[0124]

[0125] Compound 2 (20 mg, 0.045 mmol, 1.0 eq), TSTU (16.4 mg, 0.054 mmol, 1.2 eq) and DIPEA (6.8 mg, 0.054 mmol, 1.2 eq) were added to a 10 mL brown bottle, dissolved with 2.5 mL DMSO, activated at 25 ° C for 4 h; TLC monitoring showed that the activated product was generated, and compound 1 (62 mg, 0.054 mmol, 1.2 eq) was added, and the reaction was carried out at 25 ° C for 12 h; LCMS monitoring: a small amount of the reaction solution was taken and diluted with water, and LCMS showed that the product was generated. After the reaction was completed, the crude product was diluted with water to obtain the crude product; the crude product was separated and purified by preparative HPLC, and the chromatographic conditions were as follows: Chromatographic column: Actus Triant-C18 30*250 5 μm; mobile phase: A acetonitrile, B 0.1 mol / L TEAB (pH=7.5-7.8); HPLC gradient settings are shown in Table 11 below.

[0126] Table 11

[0127] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 2 30 5 95 3 30 25 85 22 30 27 83 24 30 45 55 25 30 5 95 26 30 5 95

[0128] The target product elutes at about 12.5 min and is freeze-dried to obtain a dark purple compound, namely ddGTP-V3-linker-dR110 (15 mg, yield 20%). + =1705.2.

[0129] Example 7: Synthesis of ddATP-V3-linker-dR110 (corresponding to formula (III-6))

[0130] The synthetic route is as follows:

[0131]

[0132] Compound 2 (20 mg, 0.045 mmol, 1.0 eq), TSTU (16.25 mg, 0.054 mmol, 1.2 eq) and DIPEA (6.9 mg, 0.054 mmol, 1.2 eq) were added to a 10 mL brown bottle, 5 mL DMSO was added to dissolve completely, and activated at 25 ° C for 4 h; TLC monitoring showed that the activated product was generated, and compound 1 (56.2 mg, 0.045 mmol, 1.0 eq) was added, and the reaction was carried out at 25 ° C for 12 h; LCMS monitoring: a small amount of the reaction solution was taken and diluted with water, and LCMS showed that the product was generated. After the reaction was completed, the crude product was diluted with water to obtain the crude product; the crude product was separated and purified by preparative HPLC, and the chromatographic conditions were as follows: Chromatographic column: Actus Triant-C18 30*250 5 μm; mobile phase: A acetonitrile, B 0.1 mol / L TEAB (pH=7.5-7.8); HPLC gradient settings are shown in Table 12 below.

[0133] Table 12

[0134] Time / min Flow rate (mL / min) A％ B％ 0 30 5 95 3 30 5 95 5 30 15 85 8 30 17 83 12 30 19 81 15 30 19 81 15.5 30 30 70 18 30 5 95 20 30 5 95

[0135] The target product elutes at about 17 min and is freeze-dried to obtain a dark purple solid, namely ddATP-V3-linker-dR110 (5 mg, yield 6.7%). + =1675.3.

[0136] Example 8: Fluorescence and absorption spectra

[0137] The maximum emission wavelength and maximum absorption wavelength of the compound were detected using a Hitachi F-4600 fluorescence spectrophotometer. The results are shown in Table 13 below.

[0138] Table 13

[0139] Compound Maximum emission wavelength (nm) Maximum absorption wavelength (nm) ddATP-V3-linker-dR6G 584 552.5nm ddCTP-V3-linker-dRox 529.6 605.5nm ddGTP-V3-linker-dR6G 579 553.5nm ddTTP-V3-linker-dTMRA 603.6 577.5nm ddATP-V3-linker-dR110 545.8 500.5nm ddGTP-V3-linker-dR110 564.6 621.5nm

[0140] From the contents of Table 13, it can be seen that the compound of the present invention has a good energy resonance transfer efficiency. TM The reagent-related parameters were compared and it was found that the two had similar characteristics. At the same time, according to the principle of DNA sequencing, the reagent excites a detectable fluorescent signal after the reaction. If the wavelength is appropriate, it can be detected, and if the wavelength is not appropriate, it cannot be detected. The experiment verified that the compounds of the present invention can all be detected. Therefore, it is shown that the compounds of the present invention can be preferably used for DNA sequencing.

[0141] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0142] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A compound, characterized in that The compound has a structure shown in formula (I), (Ⅰ) in, Each R1~R 10 are independently H; X is Cl.

2. A compound, characterized in that The compound has a structure shown in formula (II), (Ⅱ) Among them, R1~R 10 and X has the same definition as in claim 1; R 11 is -ddNTP.

3. The compound according to claim 2, characterized in that The ddNTP is selected from any one of ddATP, ddGTP, ddCTP and ddTTP.

4. A compound, characterized in that The compound has a structure shown in formula (III), (Ⅲ) Among them, R1~R 10 , X and R 11 The definition is the same as that of claim 2; R 12 It is the fluorescein group.

5. The compound according to claim 4, characterized in that The fluorescein is selected from any one of dTMRA, dRox, dR6G, and dR110.

6. The compound according to claim 4, characterized in that The compound is at least one of the following: (Ⅲ-1) (Ⅲ-2) (Ⅲ-3) (Ⅲ-4) (Ⅲ-5) (Ⅲ-6)。 7. A method for preparing the compound according to claim 1, characterized in that: include: (Ⅰ-a) (Ⅰ-b) The compound represented by formula (I-a) undergoes a first acid-amine condensation reaction with the compound represented by formula (I-b) in a first solvent under the action of a condensing agent and an acid-binding agent.

8. The method according to claim 7, characterized in that The condensing agent is TNTU, the acid binding agent is DIPEA, and the first solvent is dimethylformamide.

9. The method according to claim 7, characterized in that: The method further comprises: subjecting the compound represented by formula (I) to liquid chromatography purification.

10. The method according to claim 7, characterized in that The method further comprises the following steps of preparing the compound represented by formula (I-a): (Ⅰ-a-a) (Ⅰ-a-b) The compound represented by formula (I-aa) undergoes a second acid-amine condensation reaction with the compound represented by formula (I-ab) in the second solvent under the action of a condensing agent and an acid-binding agent.

11. The method according to claim 10, characterized in that The condensing agent is TNTU, the acid binding agent is DIPEA, and the second solvent is dimethylformamide.

12. The method according to claim 10, characterized in that The method further comprises: subjecting the compound represented by formula (I-a) to liquid chromatography purification.

13. The method according to claim 7, characterized in that The method further comprises the following steps of preparing the compound represented by formula (I-b): (Ⅰ-b-a) In the compound represented by formula (I-ba), one of the amino groups is subjected to Boc protection under alkaline conditions, and then the other amino group is subjected to trifluoroacetyl protection, and HCl gas is passed through to remove the Boc protection to obtain the compound represented by formula (I-b).

14. A method for preparing the compound according to claim 2, characterized in that: include: The compound represented by formula (I) as claimed in claim 1 is reacted with A third acid amine condensation reaction is carried out, wherein R 11 The definition is the same as that of claim 2.

15. The method according to claim 14, characterized in that The condensing agent is selected from TNTU and / or HBTU, the acid binding agent is DIPEA, and the third solvent is dimethylformamide.

16. The method according to claim 14, characterized in that The method further comprises: subjecting the compound represented by formula (II) to liquid chromatography purification.

17. A method for preparing the compound according to claim 4, characterized in that: include: The compound represented by formula (II) as claimed in claim 2 undergoes a quaternary acid amine condensation reaction with fluorescein in a fourth solvent under the action of a condensing agent and an acid binding agent.

18. The method according to claim 17, characterized in that The condensing agent is selected from TNTU, TSTU and / or DSC, the acid binding agent is selected from DIPEA and / or DMAP, and the fourth solvent is selected from dimethylformamide and / or dimethyl sulfoxide.

19. The method according to claim 17, characterized in that The method further comprises: purifying the compound represented by formula (III) by liquid chromatography.

20. Use of the compound according to any one of claims 4 to 6 in gene sequencing.

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