Alkynyl-substituted azaindole polymethine cyanine dye as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of biological detection and fluorescence labeling, and particularly discloses an alkynyl-substituted azaindole polymethine cyanine dye and a synthesis method and application thereof, the alkynyl-substituted azaindole polymethine cyanine dye has a structure as shown in the following general formula I: in the general formula I, X is selected from H, F, Cl, Br or I; y is a C or N atom; n is an integer of 0, 1 or 2; r1, R3, R5 and R6 are respectively and independently selected from hydrogen, C-Calkyl, C-Ccarboxy alkyl, aryl, aryl carboxylic ester, C-Calkyl sulfonate, aryl sulfonate, C-Calkyl sulfonamide or aryl sulfonamide; r2 and R4 are respectively and independently selected from hydrogen, halogen,-COOH,-NHH,-SOH,-SONHH, C-Calkyl carboxylic ester, C-Camino alkyl, aryl, aryl carboxylic ester, C-Calkyl sulfonate, aryl sulfonate, C-Calkyl sulfonamide or aryl sulfonamide.

Description

Technical Field

[0001] This invention relates to the field of biological detection and fluorescent labeling technology, specifically to an alkynyl-substituted aza-indole polymethyl cyanine dye, its synthesis method, and its application. Background Technology

[0002] The evolution of gene sequencing technology has always revolved around the demand for higher throughput and lower costs. Since the Sanger chain termination method was pioneered in the 1970s, first-generation sequencing technology has long been regarded as the gold standard. However, its inherent throughput limitations have driven the rise of second-generation high-throughput sequencing technology, which is based on "sequencing while synthesis".

[0003] In next-generation sequencing, fluorescent labeling technology is crucial. By labeling four different dNTPs with various fluorescent groups and monitoring the fluorescence signal in real time during synthesis, the results are ultimately converted into sequence information. The efficiency of this process is highly dependent on the performance of the fluorescent dyes, including their optical properties, photostability, water solubility, and impact on enzyme activity. While carbocyanine dyes are widely used in sequencing, they often face problems such as fluorescence quenching and molecular aggregation, limiting sequencing read length and accuracy. Existing improvements mainly focus on structural modification of the indole ring, but these still fall short of fully meeting the high requirements of signal intensity and stability for high-throughput sequencing.

[0004] Therefore, there is an urgent need in this field to develop novel fluorescent dyes that combine high fluorescence quantum yield, excellent photostability, and good water solubility to meet the stringent requirements of modern high-throughput sequencing systems. To this end, an alkynyl-substituted aza-indole polymethyl cyanine dye, its synthesis method, and its applications are provided. Summary of the Invention

[0005] The purpose of this invention is to address the deficiencies of the prior art by providing an alkynyl-substituted aza-indole polymethyl cyanine dye, its synthesis method, and its applications, thereby resolving the problems raised in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an alkynyl-substituted aza-indole polymethyl cyanine dye having the structure shown in general formula I: General Formula I in: X is selected from H, F, Cl, Br, or I; Y is a C or N atom; n is an integer of 0, 1, or 2; R1, R3, R5, and R6 are each independently selected from: hydrogen, C1-C 12 Alkyl, C1-C 12 Carboxylalkyl, aryl, aryl carboxylic acid esters, C1-C 12 Alkyl sulfonates, aryl sulfonates, C1-C 12Alkyl sulfonamides or aryl sulfonamides; R2 and R4 are each independently selected from: hydrogen, halogen, -COOH, -NH2, -SO3H, -SO2NH2, Cl-C 12 Alkyl carboxylic esters, C1-C 12 Aminoalkyl, aryl, aryl carboxylic acid esters, C1-C 12 Alkyl sulfonates, aryl sulfonates, C1-C 12 Alkyl sulfonamides or aryl sulfonamides.

[0007] A method for synthesizing alkynyl-substituted aza-indole polymethyl cyanine dye as described above. The synthesis steps include the following: , , , ;

[0008] Synthesis steps: Step 1: In an organic solvent, at 90–160 °C, 2-hydrazinopyridine S1 with halogen substitution at the 5-position is heated under reflux in an acidic environment to generate an hydrazone intermediate; this intermediate undergoes further rearrangement and elimination of ammonia under acid catalysis to obtain aza-indole compound C1, wherein the molar ratio of S1 to R1-substituted 2-butanone is 1:2–10; Step 2: Dissolve the azidoindole compound C1 and the R2-substituted alkyne in a suitable solvent, add an equimolar amount of amine and a catalytic amount of monovalent copper salt and palladium catalyst, and react at 25 to 80 °C for 1 to 4 hours under nitrogen protection; after the reaction is completed, remove the solvent by rotary evaporation, and purify by silica gel column chromatography to obtain the alkyne-substituted azidoindole intermediate C2. Step 3: Subsequently, in an organic solvent, C2 is reacted with an N-alkylating agent at 60–120 °C for 3–24 hours to introduce N–R3 substitution side chain, yielding an azidoindole derivative C3 containing an R2 alkynyl group at the 5-position. Step 4: In an organic solvent, at 90–160°C, the hydrazine raw material S2 containing R4 substitution and the 2-butanone containing R5 substitution are heated under acidic conditions and refluxed to form an hydrazone intermediate. This intermediate undergoes acid-catalyzed rearrangement and deamination to generate indole intermediate C5. C5 is dissolved in an organic solvent and reacted with an N-alkylating agent at 60–120°C for 3–24 hours to obtain an N–R6 substituted indole intermediate C6 with R4 at the 5-position. Step 5: In an organic solvent, at 90–120°C, intermediate C6 is reacted with condensing agent S3 for 1–2 hours. After the reaction, the mixture is purified by column chromatography or directly fed as crude product to obtain hemicyanine compound C7. The molar ratio of C6 to S3 is 1:1–1.2. Step 6: In an organic solvent, at 25 to 120°C, under the catalysis of an organic base, react the hemicyanine compound C7 with azirindole C3 for 2–8 hours to obtain the final product, alkynyl-substituted azirindole polymethyl cyanine dye I; the molar ratio of C7 to C3 is 1:1–2.

[0009] Furthermore, in steps 1, 3, and 4, the organic solvents are selected from any one or a mixture of two or more of the following: benzene, toluene, xylene, o-dichlorobenzene, sulfolane, and N,N-dimethylformamide (DMF).

[0010] Furthermore, in steps 1 and 4, the acid catalyst is selected from any one of zinc chloride, boron trifluoride, polyphosphoric acid, acetic acid, hydrochloric acid, trifluoroacetic acid, and sulfuric acid.

[0011] Furthermore, in steps 2 and 4, the amine is selected from any one or a mixture of two or more of diethylamine, triethylamine, piperidine, diisopropylamine, morpholine, and pyridine; the monovalent copper salt is selected from any one of cuprous iodide, cuprous bromide, cuprous chloride, cuprous trifluoromethanesulfonate, and cuprous sulfate; and the palladium catalyst is selected from any one of tetrakis(triphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, tris(dibenzylideneacetone)palladium, palladium acetate, palladium chloride, and palladium trifluoroacetate.

[0012] Furthermore, in step 5, the organic solvent is selected from any one or a mixture of two or more of ethanol, acetic acid, acetic anhydride, and DMF; the condensing agent is selected from any one of malondialdehyde diphenylamine hydrochloride, 1-anilino-2-bromo-3-phenyliminopropene, 1-anilino-2-chloro-3-phenyliminopropene, and N,N'-diphenylmethanediamine.

[0013] Furthermore, in step 6: the organic solvent is selected from any one of acetonitrile, dimethyl sulfoxide, and DMF; the organic base is selected from any one of triethylamine, diethylamine, pyridine, 2,6-dimethylpyridine, and N,N-diisopropylethylamine.

[0014] A fluorescently labeled nucleotide prepared using the alkynyl-substituted aza-indole polymethyl cyanine dye described above has the following general structural formula II: Formula II.

[0015] A sequencing reagent comprising at least one fluorescently labeled nucleotide as described above, the reagent being used in a four-color sequencing system or in a single-color sequencing system based on chemical cleavage and signal activation.

[0016] A method for monochrome sequencing using the sequencing reagents described above specifically includes the following steps: Step 1: Provide four nucleotides with their 3' ends blocked, labeled with A, T, C, and G bases respectively, wherein: dTTP derivatives are linked to fluorescent groups; dATP derivatives are linked to fluorescent groups connected via disulfide bonds (-SS-); dCTP derivatives are linked to fluorescent group-quencher group pairs (via -SS-linking); dGTP derivatives are not linked to fluorescent groups; Step 2: Each sequencing cycle includes: nucleotide incorporation, first fluorescence imaging, disulfide bond cleavage, second fluorescence imaging, and complete cleavage to remove all labeled groups; Step 3: Determine the base type by combining the two imaging signals.

[0017] The fluorescently labeled dCTP and dTTP of this invention, together with existing AF532 or ATTO532-labeled dATP and Cy5-labeled dGTP, form DNA sequencing reagents. This combination not only maintains the spectral resolution of traditional four-color sequencing systems but also significantly improves the quality of sequencing signals and the accuracy of sequencing results by enhancing the water solubility and fluorescence quantum yield of the labels. Particularly in high-throughput sequencing, this improvement can extend the effective read length, reduce the error rate, and provide more reliable technical support for genomics research.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention requires only a single excitation wavelength and receiving channel, significantly reducing the complexity of the optical system and the cost of the instrument; the dye of this invention uses azaindole as the parent core and introduces an alkyne functional group onto its pyridine ring, achieving a performance breakthrough through dual structural modification: 1) The introduction of nitrogen heteroatoms effectively modulates the frontier molecular orbital energy levels (HOMO / LUMO) of dye molecules, reduces excited-state reactivity, and significantly enhances their photobleaching resistance. The nitrogen atom in the pyridine ring acts as a hydrogen bond acceptor, increasing the hydrophilicity of the entire molecule and effectively inhibiting molecular aggregation (such as H-aggregation and J-aggregation) and non-specific surface adsorption caused by hydrophobic interactions, thereby reducing background noise.

[0019] 2) The introduction of the alkyne unit enhances the rigidity of the conjugated system, effectively suppresses nonradiative transitions in the excited state, and improves the fluorescence quantum yield. Simultaneously, as a π-electron bridge, it extends the molecular conjugated structure, enabling a redshift in the absorption / emission spectrum, providing a regulatory mechanism for multicolor sequencing and FRET applications. Furthermore, this alkyne group can carry additional groups, serving as a universal linker site for efficient and directional coupling with nucleotides or other biomolecules, significantly enhancing the flexibility and application potential of labeled probes. Attached Figure Description

[0020] Figure 1 The fluorescence absorption spectra of MED690Hy of the present invention were measured in buffer solutions of different pH values. Figure 2 These are photographs taken twice in Embodiment 18 of the present invention. Detailed Implementation

[0021] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0022] Example 1: Synthesis of dye MED690Hy

[0023] Step 1: Synthesis of 7-methyl-8-oxononanoic acid

[0024] Add 200 mL of anhydrous ethanol to a 500 mL single-necked flask. Under ice bath conditions, add 3.5 g of metallic sodium in portions and stir for 1 hour. Add 20 g of methyl 2-methyl-3-oxobutyrate dropwise to the above solution under ice bath conditions and stir for 0.5 hours. After a large amount of white solid precipitates, add 30 g of ethyl 6-bromohexanoate. React at 80°C for 16 hours. Rotate the solvent to dryness, adjust the pH to 1 with 1N hydrochloric acid, and extract with DCM. Dissolve the oily residue after rotary evaporation of the organic phase in methanol, add a pre-prepared aqueous sodium hydroxide solution, and react at 50°C for 16 hours. Remove methanol under reduced pressure, back-extract with ethyl acetate, adjust the pH of the aqueous solution to 1 with 1N hydrochloric acid, extract three times with EA, dry to sodium sulfate, filter, and rotary evaporate to dryness. Silica gel column chromatography yields 35 g of product.

[0025] Step 2: Synthesis of 6-(5-bromo-2,3-dimethyl-3H-pyrrolo[2,3-b]pyridin-3-yl)hexanoic acid

[0026] 15 g of 7-methyl-8-oxononanoic acid, 10 g of 2-hydrazino-5-bromopyridine, and 150 mL of acetic acid were added to a 500 mL single-necked flask and reacted at 120°C for 5 hours under nitrogen protection. After cooling to room temperature, a large amount of EA was added, and the mixture was stirred until the solid precipitated. The supernatant was poured off, washed twice with EA, and dried under vacuum to obtain 12 g of crude product, which was used directly in the next reaction.

[0027] Step 3: Synthesis of 6-(5-ethynyl-2,3-dimethyl-3H-pyrrolo[2,3-b]pyridin-3-yl)hexanoic acid

[0028] 4.1 g (12 mmol) of 6-(5-bromo-2,3-dimethyl-3H-pyrrolo[2,3-b]pyridin-3-yl)hexanoic acid, 3.55 g (36 mmol) of trimethylsilylacetylene, 0.6 mmol (420 mg) of bis(triphenylphosphine)palladium dichloride, 110 mg of copper iodide, 10 mL of triethylamine, and 50 mL of dioxane were added to a 100 mL single-necked flask. The mixture was reacted at 60°C for 1 hour under nitrogen protection. The solvent was removed by rotary evaporation, and the crude product was dissolved in water and methanol. An aqueous solution of potassium hydroxide was added to adjust the pH to 11, and the mixture was reacted at room temperature for 2 hours. The pH was then adjusted to 3 with dilute hydrochloric acid, and the mixture was extracted three times with EA. The organic phase was collected, dried over anhydrous sodium sulfate, and then evaporated to dryness. Silica gel column chromatography yielded 2.1 g of product (61% yield). ESI - :283.2.

[0029] Step 4: Synthesis of 6-[5-ethynyl-2,3-dimethyl-1-(3-sulfopropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]hexanoic acid

[0030] 6-(5-ethynyl-2,3-dimethyl-3H-pyrrolo[2,3-b]pyridin-3-yl)hexanoic acid (2.85 g, 10 mmol), 1,3-propanesulfonyl lactone (4.9 g, 40 mmol), and 1,2-dichlorobenzene (60 mL) were added to a single-necked flask and reacted at 65°C for 5 hours, resulting in the precipitation of a large amount of precipitate. After the reaction was completed, the solution was cooled to room temperature, and a large amount of EA was added. The mixture was stirred to precipitate the precipitate, filtered, dried, and subjected to silica gel column chromatography to obtain 3.1 g of product, with a yield of 76%. ESI - :407.1.

[0031] Step 5: Synthesis of 2,3,3-trimethyl-5-sulfonyl-3H-indole

[0032] 4-Hydroxybenzenesulfonic acid hydrate (22 g, 106 mmol) and 3-methyl-2-butanone (35 mL) were dissolved in acetic acid (60 mL) and heated under reflux with stirring for 4 hours. After the reaction was complete, the reaction solution was cooled to room temperature, filtered, and the filtrate was slurried with ethyl acetate to precipitate a red solid. The filtrate was then filtered again, and the filter cake was washed 2-3 times with ethyl acetate. All solids were then combined and dried to give 20.8 g of a pale red solid product, 2,3,3-trimethyl-5-sulfonic-3H-indole, with a yield of 83.2%. ESI - :238.4.

[0033] Step 6: Synthesis of 1-anilino-2-bromo-3-phenyliminopropene

[0034] Mucilaginous bromic acid (94 mmol) was dissolved in anhydrous ethanol (150 mL), and a solution of aniline (20 g) in anhydrous ethanol (50 mL) was added dropwise using a constant pressure dropping funnel. The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the solvent was evaporated to dryness, dissolved in a small amount of methanol, and then 300 mL of methyl tert-butyl ether was added and stirred. A yellow precipitate was formed, filtered, and dried to give 22.8 g of the solid product 1-anilino-2-bromo-3-phenyliminopropene (yield 81.7%).

[0035] Step 7: Synthesize MED690Hy 2,3,3-Trimethyl-5-sulfono-3H-indole (239 mg, 1 mmol) and 1-anilino-2-bromo-3-phenyliminopropene (281 mg, 1.1 mmol) were dissolved in 5 mL of acetic anhydride. Under nitrogen protection, the mixture was heated and stirred at 110 °C for 2 hours. Then, 6-[5-ethynyl-2,3-dimethyl-1-(3-sulfopropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]hexanoic acid (410 mg, 1 mmol) dissolved in 10 mL of pyridine was added, and the mixture was heated and stirred at 110 °C for another 2 hours. After the reaction was complete, a large amount of frozen diethyl ether was added to the reaction solution, precipitating a blue solid. The solid was filtered, and the crude product was separated by C18 column chromatography to obtain 284 mg of a brown solid, yield 36%, λex: 691 (acetic acid buffer, pH 6.5), ESI. - : 759.5.

[0036] MED690Hy exhibits typical pH sensitivity; its absorption and emission spectra shift significantly at different pH levels (e.g., ...). Figure 1 As shown in the figure, its fluorescence is extremely weak at alkaline pH, but significantly enhanced when the pH is decreased (acidification). This indicates that its fluorescence properties are closely related to the environmental pH value, making it suitable for fluorescent probes or labeling applications in acidic environments. It can also be used as a monochromatic fluorescent MRT gene sequencing reagent based on environmentally sensitive dyes.

[0037] Figure 1 (A) Shows the absorption curve of dye MED690Hy in Tris-HCl acetate buffer (pH 7.4). Figure 1 (B) shows the absorption curve of dye MED690Hy in acetate buffer (pH 6.5).

[0038] Example 2: Synthesis of MED690SO4

[0039] Step 1: Synthesis of 2-(2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-5-yl)acetylen-1-sulfonic acid

[0040] 5-Bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine (3 g, 12.5 mmol), sodium alkynylpropanesulfonate (3.55 g, 25 mmol), bis(triphenylphosphine)palladium dichloride (430 mg), copper iodide (51 mg), triethylamine (8 mL), and 25 mL of LDMF were added to a 100 mL single-necked flask. Under nitrogen protection, the mixture was reacted at 60°C for 1 hour. After cooling to room temperature, EA was added while stirring. The mixture was stirred thoroughly until a solid precipitated. The supernatant was poured off, leaving the precipitate to obtain the crude product, which was dried and used for the next reaction.

[0041] Step 2: Synthesis of 2,3,3-trimethyl-5-(sulfoethynyl)-1-(3-sulfopropyl)-3H-pyrrolo[2,3-b]pyridine-1-onium

[0042] The synthesis method refers to step 3 of Example 1, except that the starting material is replaced with 2-(2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-5-yl)acetylenic acid, with a yield of 78% and ESI. - : 400.1, HPLC purity: 93%.

[0043] Step 3: Synthesis of 5-methyl-6-oxoheptanoic acid

[0044] The synthesis method is the same as step 1 in Example 1.

[0045] Step 4: Synthesis of 4-(2,2,3-trimethyl-5-sulfono-3H-2-indol-3-yl)butyric acid

[0046] The synthesis method is the same as step 2 in Example 1, except that the raw materials are replaced with 5-methyl-6-oxoheptanoic acid and 4-hydrazinobenzenesulfonic acid hydrate, with a yield of 81%.

[0047] Step 5: Synthesis of 4-[2,3-dimethyl-5-sulfon-1-(3-sulfopropyl)-3H-indol-3-yl]butyric acid

[0048] The synthesis method is the same as step 3 in Example 1, except that the raw material was replaced with 4-(2,2,3-trimethyl-5-sulfono-3H-2-indol-3-yl)butyric acid, with a yield of 69%, ESI: 433.1, and HPLC purity: 89%.

[0049] Step 6: Synthesize dye MED690SO4 The synthesis method is the same as step 7 in Example 1. C18 column chromatography yielded 74 mg of product (29% yield). MS: ESI - : 869.4, λex: 688, λem: 710, HPLC purity: 94%. Example 3: MED580SO

[0050] Step 1: Synthesis of 4-[2,3-dimethyl-5-sulfon-1-(3-sulfopropyl)-3H-indol-3-yl]butyric acid

[0051] The synthesis method refers to the synthesis of 4-[2,3-dimethyl-5-sulfon-1-(3-sulfopropyl)-3H-indole-3-yl]butyric acid in Implementation 2, ESI - 353.6.

[0052] Step 2: Synthesis of 5-ethynyl-2,3,3-trimethyl-1-(3-sulfopropyl)-3H-pyrrolo[2,3-B]pyridine

[0053] 2.1 g of 5-ethynyl-2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine, 3.3 g of 1,3-propanesulfonyl lactone, and 10 mL of 1,2-dichlorobenzene were added to a single-necked flask and reacted at 65°C for 5 hours. After cooling to room temperature, PE was added, the supernatant was poured off, and the precipitate was retained to give 3.2 g of crude product, which was dried and used for the next reaction.

[0054] Step 3: Synthesize dye MED580SO 300 mg of 5-ethynyl-2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine, 200 mg of N,N'-dibenzomidine, 8 mL of acetic anhydride, and 2 mL of acetic acid were added to a single-necked flask and reacted at 120°C for 1 hour. Then, 420 mg of 4-[2,3-dimethyl-5-sulfono-1-(3-sulfopropyl)-3H-indol-3-yl]butyric acid was dissolved in pyridine and added to the above solution, and reacted at 120°C for 2 hours. Ethyl acetate was added to precipitate the product, and the precipitate was separated. C18 column chromatography yielded 69 mg of the product (42% yield). ESI - : 669, λex: 581, λem: 604, HPLC purity: 95%.

[0055] Example 4: MED630NH2

[0056] Step 1: Synthesis of 3-[2,3,3-trimethyl-5-(3-((2,2,2-trifluoroacetyl)amino)prop-1-yn-1-yl)-3H-1λ 4 -pyrrolo[2,3-b]pyridin-1-yl]propane-1-sulfonic acid

[0057] 5-Bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-B]pyridine (1.53 g, 6.2 mmol), 2,2,2-trifluoro-N-(prop-2-yn-1-yl)acetamide (2.7 g, 18 mmol), bis(triphenylphosphine)palladium dichloride (210 mg), copper iodide (20 mg), triethylamine (4 mL), and 15 mL of dioxane were added to a 100 mL single-necked flask. The mixture was reacted at 60°C for 1 hour under nitrogen protection. After cooling to room temperature, the solvent was evaporated, and silica gel column chromatography yielded 1.7 g of the intermediate. This intermediate, along with 2.3 g of 1,3-propanesulfonyl lactone and 10 mL of 1,2-dichlorobenzene, was added to a single-necked flask and reacted at 110°C for 2 hours. After cooling to room temperature, EA was added and stirred. The supernatant was discarded, leaving a precipitate, which was then subjected to silica gel column chromatography to give 1.3 g of the product (49% yield). ESI - :431.5.

[0058] Step 2: Synthesis of MED630NH2 430 mg of 3-[2,3,3-trimethyl-5-(3-((2,2,2-trifluoroacetyl)amino)prop-1-yn-1-yl)-3H-1λ 4 200 mg N,N'-dibenzomidine, 8 mL acetic anhydride, and 2 mL acetic acid were added to a single-necked flask and reacted at 120°C for 1 hour. Then, 420 mg of 4-[2,3-dimethyl-5-sulfonyl-1-(3-sulfopropyl)-3H-indol-3-yl]butyric acid was dissolved in pyridine and added to the above solution. The reaction was carried out at 120°C for 2 hours. Ethyl acetate was added to precipitate the product. The precipitate was separated, collected, and mixed with 25 mL of methanol. 5 mL of 0.1 M potassium hydroxide solution was added, and the mixture was stirred at room temperature. HPLC monitoring was performed. After the reaction was complete, a large amount of water was added for dilution. C18 column chromatography yielded 142 mg of the product (19% yield). ESI - :751.2,λex:633.

[0059] The present invention also provides the following embodiments, with the synthesis method referring to Embodiments 1-4, and their structures and maximum absorption wavelengths are shown in Table 1 below: Table 1: Structural Formulas and Maximum Absorption Wavelengths of Examples 5-14

[0060] Quantum yield determination: The fluorescence quantum yields (QY) of the representative compounds described in this paper, MED690SOII, MED690SO4, control dyes (MED680H, MED680Br, synthesized as described in Examples 1-4), monosulfonic acid Cy5 (mono SO3 Cy5, a traditional indocyanine dye), and AF680 (an unmodified alkyne-based aza-indole dye), were determined using a HORIBA Fluoromax instrument. Each compound was dissolved in aqueous solution, and its absorbance at the maximum absorption wavelength was adjusted to below 0.1 before quantum yield determination.

[0061] Table 2: Results of dye fluorescence quantum yield determination

[0062] Note: The relative fluorescence quantum yield was determined using the Fluoromax system in this experiment, and the data are the average of three repeated experiments. The measurement results differ from the absolute fluorescence quantum yield (AF680: 0.36) stated in the manufacturer's instructions. This difference is due to variations in the measurement solvent, instrument parameters, and observation conditions, which is consistent with expectations in fluorescence metrology.

[0063] Experimental results show that: 1) Replacing the indole skeleton with an azaindole structure can redshift the absorption and emission spectra by about 30 nm and increase the quantum yield by about 35%; 2) Introducing an alkyne functional group into the azaindole system can further achieve a redshift of the absorption spectrum (about 20 nm) and further increase the quantum yield by about 7-10%. The introduction of halogen groups can achieve a redshift of the absorption spectrum, but will reduce the quantum yield; 3) Even if additional sulfonic acid groups and alkyl sulfonic acid groups are introduced to enhance water solubility, the compound can still maintain excellent fluorescence brightness characteristics.

[0064] Example 15: This example provides a fluorescently modified nucleotide prepared from the compound shown in general formula I. The preferred embodiment has the following structural formula (3'-N3-5-NL-dTTP-MED630SO).

[0065] The synthesis method is referenced in the patent publication (announcement) number CN114250282A, which describes a gene sequencing reagent and method based on a pH-sensitive dye. Example 16: This example provides a fluorescently modified nucleotide prepared from a compound of general formula I, characterized in that its fluorescent group is linked to the base through two different cleavable chains, such as (azoline chain NL and disulfide bond SS). The preferred embodiment has the following structural formula (3'-N3-7-NL-dATP-SS-MED630SO).

[0066] The synthesis method is referenced in the patent publication (announcement) number CN114250282A, which describes a gene sequencing reagent and method based on a pH-sensitive dye. Example 17: This example provides a fluorescently modified nucleotide (structure: 3'-N3-5-NL-dCTP-MED630NH2-SS-BHQ3) prepared based on a compound of general formula I. Its core lies in the use of an activateable fluorescent reporter system. The core of this system is the bifunctional fluorescent group MED630NH2, whose molecular structure simultaneously contains two reactive functional groups: a carboxyl group (-COOH) and an amino group (-NH2). This unique design enables highly modular construction. 1) The carboxyl terminus forms a stable amide bond, which covalently binds to the amino group on the nucleotide linker arm, anchoring it to the molecular backbone; 2) The amino terminus serves as a functional interface, specifically linked to the quenching group BHQ3 via a cleavable disulfide bond (-SS-).

[0067]

[0068] This structure places the fluorophore and quencher groups in close spatial proximity, efficiently quenching fluorescence through the fluorescence resonance energy transfer (FRET) effect, resulting in no detectable signal initially. Upon the addition of a specific cleavage reagent to break the disulfide bond, the BHQ3 quencher group detaches, the FRET effect is released, and the fluorescence of MED630NH2 is restored, producing a strong detectable signal.

[0069] This design achieves the "off-on" switching of fluorescence signals through precise chemical control, which not only provides key technical support for monochrome sequencing, but also provides a universal platform for the customized development of fluorescently labeled molecules through its modular design concept.

[0070] Example 18: This example describes a sequencing method based on monochromatic fluorescence detection. This method uses four specially modified chemical nucleotides and achieves DNA sequence determination through a cycle of two imaging and chemical cutting steps. Its core principle is to distinguish the four base types by utilizing the combination of changes in fluorescence signals (present / absent fluorescence) of different base derivatives before and after treatment with the cutting reagent.

[0071] (1) Sequencing reagents: This method uses four specially modified nucleotides whose fluorescent groups emit light at the same wavelength, but whose chemical structures are different: dTTP derivative (3'-N3-5-NL-dTTP-MED630SO): with a fluorescent group (MED630SO) attached.

[0072] dATP derivative (3'-N3-7-NL-dATP-SS-MED630SO): with a fluorescent group (MED630SO) linked by a disulfide bond (-SS-).

[0073] The dCTP derivative (3'-N3-5-NL-dCTP-MED630NH2-SS-BHQ3) consists of a fluorescent group (MED630NH2) and a quencher group (BHQ3) linked by a disulfide bond. Fluorescence is quenched in the presence of the quencher group; fluorescence is restored upon removal of the quencher group.

[0074] dGTP derivatives (3'-N3-dGTP in patent publication number CN 114250282 B): without any fluorescent groups attached, they never emit light.

[0075] The 3' end of all nucleotides is sealed with a protecting group to ensure that only one base is incorporated in each cycle.

[0076] (2) Workflow (per cycle): Incorporation: Polymerases incorporate complementary modified nucleotides into the DNA chain.

[0077] First time taking photos Figure 2 Photo 1: Detection of initial fluorescence signal. At this point, sites doped with T or A emit light, while sites doped with C or G do not emit light.

[0078] Selective cleavage: A cleaving agent is added to break the disulfide bond. This step enables: The fluorophore of the A base is detached and it no longer emits light.

[0079] The quenching group of the C base is removed, and it begins to emit light.

[0080] The T and G bases are unaffected.

[0081] Second photo Figure 2 Photo 2: After cleaning, the signal changes after cutting are detected.

[0082] Complete cut-off and zeroing: Using another reagent, all fluorescent groups and the 3' end protecting group are simultaneously removed to prepare for the next round of reaction and ensure that no signal remains.

[0083] (3) Base determination logic By comparing the signals from the two photographs, the base type can be uniquely determined: Table 3: Detection Results and Corresponding Bases in Example 18

[0084] (4) Validation experiment: Three sequencing tests were performed on the Uniseq 100 sequencing platform using the sequencing reagents from Example 18. The validation results are as follows: Table 4: Sequencing Validation Results of Example 18

[0085] The results show that Example 18 demonstrates an innovative monochromatic fluorescence sequencing technology. Its core lies in utilizing the dyes and ingenious chemical design of this invention to differentiate four bases by detecting signal changes over time twice, before and after disulfide bond cleavage, replacing the traditional four-color fluorescence system. Validation experiments demonstrate that this is not merely a theoretical design, but a mature, high-performance sequencing solution. The entire sequencing process requires only one wavelength of excitation light and one wavelength of detection channel, greatly simplifying the complexity of the optical system and reducing instrument costs.

[0086] Example 19: This example provides a fluorescently modified nucleotide prepared from the compound shown in general formula I. The preferred embodiment has the following structural formula (3'-N3-5-NL-dCTP-MED690SO4):

[0087] The synthesis method is referenced in the patent publication (announcement) number CN114250282A, which describes a gene sequencing reagent and method based on a pH-sensitive dye. Example 20: This example provides a fluorescently modified nucleotide prepared from the compound shown in general formula I. A preferred embodiment of this nucleotide has the following structural formula (3'-N3-5-NL-dTTP-MED580SO):

[0088] The synthesis method is referenced in the patent publication (announcement) number CN114250282A, which describes a gene sequencing reagent and method based on a pH-sensitive dye. Example 21: This example provides a four-color sequencing reagent, which replaces the 3'-N3-5-NL-dCTP-MED690SO4 and 3'-N3-5-NL-dTTP-MED580SO in the existing four-color sequencing reagent of a certain commercial sequencing platform with fluorescent modified nucleotides 3'-N3-5-NL-dCTP-IF700 and 3'-N3-5-NL-dTTP-ROX.

[0089] In four-color DNA sequencing systems, different fluorescently labeled nucleotides are crucial for base recognition. Commonly used labeling dyes include rhodamine-based dyes (e.g., AF532, ROX) and cyanine-based dyes (e.g., Cy5, IF700). ROX, as a red fluorescent label, is frequently used for labeling adenine (A) nucleotides; however, it suffers from poor water solubility, easily agglomerating in pure water or low-salt buffers, affecting labeling efficiency and elution efficiency during sequencing. Furthermore, ROX readily binds non-specifically, leading to enhanced background signals and consequently impacting sequencing signal quality. In contrast, while Cy3-based dyes have better water solubility, their excitation and emission spectral characteristics differ from ROX, making them unsuitable for sequencing systems requiring specific spectral properties.

[0090] The alkynyl-substituted nitrogen-based polymethoxyl indocyanine dye structure used in this embodiment has the following advantages: 1) Alkyne-substituted azidoindole trimethylcyanine dyes (such as MED580SO) have excitation and emission wavelengths similar to ROX; alkyne-substituted azidoindole pentamethylcyanine dyes (such as MED690SO4) have excitation and emission wavelengths similar to IF700, and can be compatible with existing optical detection systems.

[0091] 2) It has excellent fluorescence quantum yield and water solubility, which significantly improves the elution performance of labeled nucleotides during sequencing. It is also negatively charged, which reduces the binding with non-specific proteins, resulting in lower background and improved signal-to-noise ratio. 3) As a fluorescently labeled nucleotide, it exhibits good stability. In solution, it can be stored at low temperature (-20 degrees Celsius) for 12 months, and in lyophilized powder, it can be stored at room temperature for one month with a degradation rate of less than 0.05%. It can be stored at low temperature (-20 degrees Celsius) for more than 24 months.

[0092] In practical applications, the fluorescently modified nucleotides of this invention can be combined with existing AF532 or ATTO532-labeled dATP, Cy5-labeled dGTP, etc., to form DNA sequencing incorporation reagents. This combination not only maintains the spectral resolution of traditional four-color sequencing systems but also significantly improves the quality of sequencing signals and the accuracy of sequencing results by improving the water solubility and stability of the labels. Especially in high-throughput sequencing, this improvement can extend the effective read length, reduce the error rate, and provide more reliable technical support for genomics research.

[0093] During sequencing, the incorporation reagent in the commercially available kit was replaced with a mixture containing 3'-N3-5-NL-dCTP-MED690SO4, 3'-N3-5-NL-dTTP-MED580SO4, and the dye-labeled dGTP and dATP from the original incorporation reagent. The experiment used a standard 2*150 sequencing procedure and the results were compared with those obtained using the commercially available kit. The summary of the indicators is shown in Table 5 below.

[0094] Table 5: Sequencing Validation Results of Example 21

[0095] Note: The signal-to-noise ratio (SNR) is measured for the last loop (C150). The methods for obtaining S (bright spot intensity) and N (background intensity) are as follows: Using kernel=(25, 25), move the kernel window to obtain the foreground and background values ​​within the window. Average the foreground and background values ​​to obtain S (bright spot intensity) and N (background intensity). The signal-to-noise ratio (SNR) is calculated using the formula: SNR = 10 * log 10 * (S² / N²) where S is the bright spot intensity and N is the background intensity.

[0096] Experimental results show that replacing the corresponding components in commercial sequencing reagents with the MED690SO4 and MED580SO-labeled dCTP and dTTP of this invention demonstrates superior performance in a 2×150 standard sequencing pipeline. Specifically, this is reflected in improved signal-to-noise ratio (SNR) and effectively reduced sequencing error rate.

[0097] This result is attributed to the unique structural advantages of the alkynyl-substituted aza-indocyanine dyes used: 1) excellent photostability ensures the maintenance of fluorescence signal intensity during long-term detection; 2) enhanced water solubility significantly improves the elution efficiency of labeled nucleotides in sequencing cycles; and 3) the negative charge of the dye molecules effectively reduces adsorption to non-specific substances, thereby significantly reducing background signal. These characteristics work together to enable the labeled nucleotides of this invention to exhibit superior performance in high-throughput sequencing.

[0098] The alkynyl-substituted aza-indole polymethyl cyanine dye and its derivatives provided by this invention exhibit excellent fluorescence properties, water solubility, and stability, making them suitable for high-throughput DNA sequencing and possessing significant practical application value and market prospects.

[0099] The above embodiments merely illustrate implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An alkynyl-substituted azaindolespiromacrocyanine dye, characterized by: having the following general formula I: Formula I wherein: X is selected from H, F, Cl, Br or I; Y is a C or N atom; n is an integer of 0, 1 or 2; R1, R3, R5, and R6 are each independently selected from: hydrogen, C1-C 12 Alkyl, C1-C 12 Carboxylalkyl, aryl, aryl carboxylic acid esters, C1-C 12 Alkyl sulfonates, aryl sulfonates, C1-C 12 Alkyl sulfonamides or aryl sulfonamides; R2, R4are each independently selected from the group consisting of: hydrogen, halogen, -COOH, -NH2, -SO3H, -SO2NH2, Ci-C 12 alkylcarboxylate, Ci-C 12 aminoalkyl, aryl, arylcarboxylate, Ci-C 12 alkylsulfonate, arylsulfonate, Ci-C 12 alkylsulfonamide or arylsulfonamide.

2. A method for synthesizing the alkynyl-substituted azaindole polymethine cyanine dye according to claim 1, characterized in that: comprising the following synthesis steps: 、 、 、 ； synthesis steps: Step 1: heating and refluxing 5-halogen-substituted 2-hydrazinylpyridine S1 with R1-substituted 2-butanone in an acid environment at 90-160°C in an organic solvent to form a hydrazone intermediate; the intermediate further undergoes rearrangement and ammonia elimination under acid catalysis to obtain azaindole compound C1, wherein the molar ratio of S1 and R1-substituted 2-butanone is 1:2-10; Step 2: dissolving azaindole compound C1 and R2-substituted alkyne in a suitable solvent, adding equimolar amount of amine and catalytic amount of monovalent copper salt and palladium catalyst, and reacting under nitrogen protection at 25-80°C for 1-4 hours; after the reaction is completed, the solvent is removed by rotary evaporation, and the product is purified by silica gel column chromatography to obtain alkynyl-substituted azaindole compound intermediate C2; Step 3: then reacting C2 with N-alkylating agent in an organic solvent at 60-120°C for 3-24 hours to introduce N-R3-substituted side chain and obtain 5-R2-alkynyl-containing azaindole derivative C3; Step 4: heating and refluxing R4-substituted hydrazine S2 and R5-substituted 2-butanone in an acid environment at 90-160°C in an organic solvent to form a hydrazone intermediate; the intermediate further undergoes rearrangement and ammonia elimination under acid catalysis to obtain indole intermediate C5; dissolving C5 in an organic solvent, and reacting with N-alkylating agent at 60-120°C for 3-24 hours to obtain N-R6-substituted, 5-R4-indole intermediate C6; Step 5: reacting intermediate C6 with condensing agent S3 in an organic solvent at 90-120°C for 1-2 hours; after the reaction is completed, the product is purified by column chromatography or directly used as crude product; thus obtaining semi-cyanine compound C7; the molar ratio of C6 to S3 is 1:1-1.2; Step 6: reacting semi-cyanine compound C7 with azaindole C3 in an organic solvent at 25-120°C under catalysis of an organic base for 2-8 hours to obtain the final product, alkynyl-substituted azaindole polymethine cyanine dye I; the molar ratio of C7 to C3 is 1:1-2.

3. The method of synthesis of alkynyl substituted azaindoles polymethine cyanine dyes according to claim 2, characterized by that: In Step 1, Step 3 and Step 4, the organic solvent is selected from any one or a mixture of two or more of benzene, toluene, xylene, o-dichlorobenzene, sulfolane, N,N-dimethylformamide (DMF).

4. The method of synthesis of alkynyl substituted azaindoles polymethine cyanine dyes according to claim 2, characterized by: In Step 1 and Step 4, the acid catalyst is selected from any one of zinc chloride, boron trifluoride, polyphosphoric acid, acetic acid, hydrochloric acid, trifluoroacetic acid, sulfuric acid.

5. The method of synthesis of alkynyl substituted azaindoles polymethine cyanine dyes according to claim 2, characterized by that: In step 2 and step 4, the amine is selected from any one or mixture of two or more of diethylamine, triethylamine, piperidine, diisopropylamine, morpholine, pyridine; the monovalent copper salt is selected from any one of cuprous iodide, cuprous bromide, cuprous chloride, cuprous triflate, cuprous sulfate; the palladium catalyst is selected from any one of tetrakis(triphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, tris(dibenzylideneacetone)dipalladium, palladium acetate, palladium chloride, palladium trifluoroacetate.

6. The method of synthesis of alkynyl substituted azaindoles polymethine cyanine dyes according to claim 2, characterized by that: In step 5, the organic solvent is selected from any one or mixture of two or more of ethanol, acetic acid, acetic anhydride, DMF; the condensing agent is selected from any one of malondialdehyde diphenylamine salt hydrochloride, 1-anilino-2-bromo-3-phenylimino propene, 1-anilino-2-chloro-3-phenylimino propene, N,N'-dibenzformamidine.

7. The method of synthesis of alkynyl substituted azaindoles polymethine cyanine dyes according to claim 2, characterized by that: In step 6, the organic solvent is selected from any one of acetonitrile, dimethyl sulfoxide, DMF; the organic base is selected from any one of triethylamine, diethylamine, pyridine, 2,6-dimethylpyridine, N,N-diisopropylethylamine.

8. A fluorescently labeled nucleotide prepared using the alkynyl-substituted aza-indole polymethyl cyanine dye according to claim 1, characterized in that: The general structure is shown in formula II below: Formula II.

9. A sequencing reagent comprising at least one fluorescently labeled nucleotide according to claim 8, wherein: The reagent is used in a four-color sequencing system or in a single-color sequencing system based on chemical cleavage and signal activation.

10. A method of performing single-color sequencing with the sequencing reagent of claim 9, characterized by: Specifically comprising the following steps: Step 1: providing four kinds of 3' end blocked nucleotides, respectively labeled A, T, C, G base, wherein: the dTTP derivative is connected with a fluorescent group; the dATP derivative is connected with a fluorescent group connected by a disulfide bond (-SS-); the dCTP derivative is connected with a fluorescent group-quenching group pair (connected by -SS-); the dGTP derivative is not connected with a fluorescent group; Step 2: each round of sequencing cycle includes: nucleotide incorporation, first fluorescence imaging, disulfide bond cleavage, second fluorescence imaging, and complete cleavage to remove all labeled groups; Step 3: determining the base type by combining the signals of the two images.

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