Preparation method of nucleotide colloidal dye
The synthesis process of SYBR GREEN I was simplified by using the coupling reaction of compound A and compound B and the halopropane substitution reaction, which solved the problem that the synthesis method was not reported in the prior art and achieved high yield and low cost.
Patent Information
- Application Number
- CN202511013627.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-11-04
AI Technical Summary
There are no reported methods for synthesizing SYBR GREEN I in the existing technology, which limits its industrial production. Furthermore, the synthesis process is complex and has high professional barriers.
A method for preparing the nucleotide colloidal dye SYBR GREEN I is provided, which involves a coupling reaction of compound A and compound B, followed by a substitution reaction with a halopropane. Commercially available reagents are used, simplifying the process steps and improving the yield.
A high-yield synthesis of SYBR GREEN I was achieved. The process is simple, the raw materials are readily available, and the production cost is reduced, which supports large-scale promotion.
Smart Images
Figure BDA0005512543480000021 
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Figure BDA0005512543480000041
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic synthesis, specifically relating to a method for preparing a nucleotide colloidal dye. Background Technology
[0002] In molecular biology research, highly sensitive and specific nucleic acid detection technologies are core tools driving the development of quantitative gene analysis and diagnosis. SYBR GREEN I, a nucleotide colloidal dye, has rapidly become an important alternative to traditional nucleic acid dyes (such as ethidium bromide, EtBr) since its development in the 1990s due to its superior double-stranded DNA (dsDNA) binding characteristics and low toxicity. Furthermore, multiple toxicological studies have confirmed that SYBR GREEN I has significantly lower mutagenicity than EtBr, reducing health risks to laboratory personnel and environmental pollution.
[0003] Synbran Green I, with its high sensitivity, low toxicity, and ease of use, is widely used in real-time quantitative PCR, nucleic acid electrophoresis visualization, and other fields. Furthermore, it has shown potential in emerging technologies such as microfluidic chips and high-throughput nucleic acid quantification, making SYBR Green I a cornerstone tool in the field of molecular detection. However, due to professional barriers and process complexity, industrial production is limited to specialized manufacturers, and no synthetic methods for SYBR Green I have been reported.
[0004] Therefore, developing a method for preparing the nucleotide colloidal dye SYBR GREEN I has become an urgent problem to be solved. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing nucleotide colloidal dyes. The preparation method provided by the present invention uses readily available and simple raw materials, is low in cost, and yields high results.
[0006] To achieve this objective, the present invention employs the following technical solution:
[0007] A method for preparing a nucleotide colloidal dye, the method comprising the following steps: a coupling reaction of compound A and compound B to obtain compound C; and a substitution reaction of compound C with a halopropane to obtain the nucleotide colloidal dye SYBR GREEN I.
[0008] The reaction route is as follows:
[0009]
[0010] Specifically, in the preparation of compound C, the coupling reaction requires the addition of a base. The molar ratio of compound A, compound B, and the base is 1:(1-1.2):(1.1-1.5). The temperature of the coupling reaction is 25-50℃, and the time is 2-10h. Preferably, the temperature of the coupling reaction can be 25℃, 30℃, 35℃, 40℃, 45℃, or 50℃, and the time can be 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, or 10h, but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0011] Preferably, the alkali includes one of potassium carbonate, cesium carbonate, sodium hydroxide, and DBU.
[0012] Specifically, the molar ratio of compound C to halopropane is 1:(1.2-1.5); the temperature of the substitution reaction is 25-85℃, and the time is 4-16h; preferably, the temperature of the substitution reaction can be 25℃, 35℃, 45℃, 55℃, 65℃, 75℃ or 85℃, etc., and the time can be 4h, 6h, 8h, 10h, 12h, 14h or 16h, etc., but is not limited to the values listed above, and other unlisted values within the above range are also applicable.
[0013] Preferably, the halopropane includes one of fluoropropane, chloropropane, bromopropane, and iodopropane;
[0014] Preferably, the substitution reaction also requires the addition of a solvent;
[0015] More preferably, the solvent includes one of DMF, DMSO, THF, ethanol, and methanol.
[0016] Specifically, the preparation of compound B includes the following steps: methylating 2-hydroxybenzothiazole with a methylating agent to obtain compound F; reacting compound F with methyltriphenylphosphine bromide via a Wittig reaction to obtain compound B;
[0017] The reaction route is as follows:
[0018]
[0019] Preferably, the preparation of compound F is further carried out under the action of alkali and solvent, and the molar ratio of 2-hydroxybenzothiazole, methylating agent and alkali is 1:(1.1-1.5):(1.2-1.6). The methylation reaction is carried out at reflux temperature for 8-14 hours. Preferably, the methylation reaction time can be 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours or 14 hours, etc., but is not limited to the values listed above. Other values not listed in the above range are also applicable.
[0020] Preferably, the methylating agent includes one of iodomethane and dimethyl sulfate, the base includes one of potassium carbonate, cesium carbonate, and sodium hydride, and the solvent includes one of acetonitrile, acetone, and DMF.
[0021] Preferably, the preparation of compound B is further carried out under the action of alkali and solvent, and the molar ratio of compound F, methyltriphenylphosphine bromide, and alkali is 1:(1.1-1.3):(1.2-1.4). The Wittig reaction temperature is -78-0℃ and the time is 4-16h. Preferably, the Wittig reaction temperature can be -78℃, -70℃, -60℃, -50℃, -40℃, -30℃, -20℃, -10℃, or -0℃, and the time can be 4h, 6h, 8h, 10h, 12h, 14h, or 16h, but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0022] Preferably, the alkali includes one of potassium tert-butoxide, sodium hydride, potassium hydride, n-butyllithium, and sodium tert-butoxide, and the solvent includes tetrahydrofuran.
[0023] Specifically, the preparation steps of compound A are as follows: 2-hydroxyquinoline is coupled with a halobenzene to obtain compound D; compound D undergoes a nucleophilic reaction with N,N-dimethyl-1,3-propanediamine to obtain compound E; compound E undergoes an oxidation reaction with an oxidizing agent to obtain compound A.
[0024] The reaction route is as follows:
[0025]
[0026] Preferably, in the preparation of compound D, the coupling reaction also requires the addition of a catalyst, a base, and a solvent. The molar ratio of 2-hydroxyquinoline, halobenzene, catalyst, and base is 1:(1.1-1.2):(0.05-0.2):(1.1-1.3). The coupling reaction is carried out at reflux temperature for 8-16 hours. More preferably, the coupling reaction time can be 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours, but is not limited to the values listed above. Other values not listed within the above range are also applicable.
[0027] Preferably, the catalyst includes one of cuprous iodide and cuprous bromide, the base includes one of potassium carbonate and cesium carbonate, the solvent includes one of DMF and DMSO, and the halobenzene includes one of bromobenzene, chlorobenzene, and iodobenzene.
[0028] Preferably, in the preparation of compound E, the nucleophilic reaction further requires the addition of a base and a solvent. The molar ratio of compound D, N,N-dimethyl-1,3-propanediamine, and the base is 1:(1.1-1.3):(1.2-1.4). The temperature of the nucleophilic reaction is 10-30°C, and the time is 2-6 hours. More preferably, the temperature of the nucleophilic reaction can be 10°C, 15°C, 20°C, 25°C, or 30°C, and the time can be 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, etc., but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0029] Preferably, the alkali includes one of potassium carbonate, cesium carbonate, and sodium hydroxide, and the solvent includes one of ethanol, methanol, and THF.
[0030] Preferably, in the preparation of compound A, the molar ratio of compound E to oxidant is 1:(1.5-10), the oxidation reaction temperature is 0-40℃, and the time is 8-16h; more preferably, the oxidation reaction temperature can be 0℃, 10℃, 20℃, 30℃ or 40℃, etc., and the time can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h or 16h, etc., but is not limited to the values listed above, and other unlisted values within the above range are also applicable.
[0031] Preferably, the oxidation reaction also requires a solvent, which includes one of acetone, ethyl acetate, and THF;
[0032] Preferably, the oxidant comprises a combination of 70% aqueous tert-butanol peroxide and a catalyst, manganese dioxide, and chromic anhydride, and the catalyst comprises cobalt acetylacetonate and iron acetylacetonate.
[0033] More preferably, in the combination, the molar ratio of the 70% tert-butanol peroxide aqueous solution to the catalyst is (20-100):1.
[0034] Compared with the prior art, the present invention has the following beneficial effects:
[0035] This invention provides a method for preparing nucleotide colloidal dyes and reports for the first time a synthetic method for SYBR GREEN I. This process route has a high yield and is simple. All raw materials used are commercially available reagents that are inexpensive and readily available, which can provide theoretical support for the large-scale promotion of SYBR GREEN I. Detailed Implementation
[0036] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.
[0037] Example 1
[0038] This embodiment provides a method for preparing nucleotide colloidal dyes, including the following steps:
[0039] Preparation of compound F:
[0040] In a reaction flask, 2-hydroxybenzothiazole (50.0 g, 151.18 g, 0.33 mol, 1.0 eq), methyl iodoform (51.6 g, 141.94 g, 0.36 mol, 1.1 eq), and potassium carbonate (54.9 g, 138.21 g, 0.40 mol, 1.2 eq) were added to 250 mL of acetonitrile. The reaction solution was heated to reflux and reacted for 12 h. After the reaction was complete, waste salts were removed by filtration, and the filtrate was collected and concentrated to obtain a crude product. The crude product was slurried using a mixed solvent of ethyl acetate / petroleum ether to obtain 47.6 g of a white solid product, compound F, namely 3-methyl-2(3H)-benzothiazole, with a yield of 87%.
[0041] Preparation of compound B:
[0042] Under nitrogen protection, methyltriphenylphosphine bromide (77.8 g, 357.2 g, 0.22 mol, 1.2 eq) was added to a reaction flask containing 350 mL of anhydrous tetrahydrofuran. Stirring was started, and the temperature was lowered to -20 °C. Potassium tert-butoxide solid (26.5 g, 112.2 g, 0.24 mol, 1.3 eq) was slowly added. After the addition was complete, the reaction system changed from white to orange. The mixture was kept at this temperature for 30 min to obtain a phosphorus ylide solution. Compound F (30 g, 165.2 g, 0.18 mol, 1.0 eq) was dissolved in 100 mL of anhydrous tetrahydrofuran and slowly added dropwise to the above phosphorus ylide solution. The temperature was maintained at -20 °C. After the addition was complete, the temperature was slowly raised to room temperature, and the reaction was continued for 12 h. After the reaction was complete, the above reaction solution was poured into 300 mL of water, 200 mL of ethyl acetate was added, and the mixture was extracted and separated. The organic phase was collected, and the aqueous phase was extracted three more times with 100 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:10) to give 27.2 g of a colorless oily product, compound B, with a yield of 92%.
[0043] Preparation of compound D:
[0044] In a reaction flask, 2-hydroxyquinoline (50.0 g, 145.16 g, 0.34 mol, 1.0 eq), cuprous iodide (6.6 g, 190.45 g, 0.034 mol, 0.1 eq), potassium carbonate (57.1 g, 138.21 g, 0.41 mol, 1.2 eq), and bromobenzene (59.5 g, 157.01 g, 0.38 mol, 1.1 eq) were dissolved in 250 mL of DMF. The mixture was stirred and heated to 120 °C for 12 h until the reaction was complete. The mixture was filtered, and the filtrate was collected. The filtrate was poured into 1000 mL of water, and 200 mL of ethyl acetate was added. The mixture was extracted, separated, and the organic phase was collected. The aqueous phase was extracted three times with 200 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated and subjected to column chromatography (ethyl acetate: petroleum ether = 1:8) to give 63.9 g of a white solid product, compound D, with a yield of 84%.
[0045] Preparation of compound E:
[0046] In a reaction flask, compound D (30.0 g, 221.26 g, 0.14 mol, 1 e.q.), N,N-dimethyl-1,3-propanediamine (15.2 g, 102.18 g, 0.15 mol, 1.1 eq.), and potassium carbonate (22.5 g, 138.21 g, 0.16 mol, 1.2 eq.) were dissolved in 250 mL of ethanol. The mixture was stirred and reacted at 25 °C for 4 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate / petroleum ether to give 36.3 g of a yellow solid product, compound E, with a yield of 88%.
[0047] Preparation of compound A:
[0048] In a reaction flask, compound E (20.0 g, 305.43 g, 65.5 mmol, 1.0 eq) and cobalt acetylacetonate catalyst (2.3 g, 356.26 g, 6.6 mmol, 0.1 eq) were dissolved in 200 mL of acetone. The mixture was stirred and cooled to 0 °C. A 70% aqueous solution of tert-butanol peroxide (42.2 g, 90.12 g, 0.33 mol, 5.0 eq) was slowly added dropwise while maintaining the temperature at 0 °C. After the addition was complete, the mixture was allowed to react at room temperature for 12 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:4) to give a yellow solid product, compound A17.5 g, with a yield of 83%.
[0049] Preparation of compound C:
[0050] In a reaction flask, compound A (10.0 g, 321.42 g, 31.1 mmol, 1.0 eq), compound B (5.1 g, 163.24 g, 31.1 mmol, 1.0 eq), and potassium carbonate (4.7 g, 138.21 g, 34.2 mmol, 1.1 eq) were dissolved in 100 mL of tetrahydrofuran. The mixture was stirred and reacted at room temperature for 4 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate to give a yellow solid product, compound C11.5 g, with a yield of 79%.
[0051] Preparation of SYBR GREEN I:
[0052] In a reaction flask, compound C (10.0 g, 466.65 g, 21.4 mmol, 1.0 eq) and bromopropane (3.2 g, 122.99 g, 25.7 mmol, 1.2 eq) were dissolved in 100 mL of DMF. The mixture was stirred and reacted at 80 °C for 12 h until the reaction was complete. 200 mL of anhydrous ethanol was added to the reaction solution, and the mixture was cooled to 0 °C. Crystallization was allowed to occur for 2 h. The mixture was filtered, washed with a small amount of anhydrous ethanol, and the filter cake was collected to obtain the crude product. The crude product was recrystallized from the ethanol / water mixture to obtain a dark green solid product, namely SYBR GREEN I, 9.9 g, with a yield of 91%.
[0053] Example 2
[0054] This embodiment provides a method for preparing nucleotide colloidal dyes, including the following steps:
[0055] Preparation of compound F:
[0056] In a reaction flask, 50.0 g (0.33 mol, 1.0 eq) of 2-hydroxybenzothiazole, 61.0 g (0.43 mol, 1.3 eq) of iodomethane, and 64.0 g (0.46 mol, 1.4 eq) of potassium carbonate were added to 250 mL of acetonitrile. The reaction solution was heated to reflux and reacted for 12 h. After the reaction was complete, waste salts were removed by filtration, and the filtrate was collected and concentrated to obtain a crude product. The crude product was slurried using a mixed solvent of ethyl acetate / petroleum ether to obtain 48.7 g of a white solid product, compound F, namely 3-methyl-2(3H)-benzothiazole, with a yield of 89%.
[0057] Preparation of compound B:
[0058] Under nitrogen protection, methyltriphenylphosphine bromide (71.4 g, 0.20 mol, 1.1 eq) was added to a reaction flask containing 350 mL of anhydrous tetrahydrofuran. Stirring was started, and the temperature was lowered to -20 °C. Potassium tert-butoxide solid (24.5 g, 0.22 mol, 1.2 eq) was slowly added. After the addition was complete, the reaction system changed from white to orange. The mixture was kept at this temperature for 30 min to obtain a phosphorus ylide solution. Compound F (30 g, 0.18 mol, 1.0 eq) was dissolved in 100 mL of anhydrous tetrahydrofuran and slowly added dropwise to the above phosphorus ylide solution. The temperature was maintained at -20 °C. After the addition was complete, the temperature was slowly raised to room temperature, and the reaction was continued for 12 h. After the reaction was complete, the above reaction solution was poured into 300 mL of water, 200 mL of ethyl acetate was added, and the mixture was extracted and separated. The organic phase was collected, and the aqueous phase was extracted three more times with 100 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:10) to give 26.3 g of a colorless oily product, compound B, with a yield of 89%.
[0059] Preparation of compound D:
[0060] In a reaction flask, 2-hydroxyquinoline (50.0 g, 0.34 mol, 1.0 eq), cuprous iodide (3.3 g, 0.017 mol, 0.05 eq), potassium carbonate (51.5 g, 0.38 mol, 1.1 eq), and bromobenzene (64.9 g, 0.41 mol, 1.2 eq) were dissolved in 250 mL of DMF. The mixture was stirred and heated to 120 °C for 12 h until the reaction was complete. The mixture was filtered, and the filtrate was collected. The filtrate was poured into 1000 mL of water, and 200 mL of ethyl acetate was added. The mixture was extracted, separated, and the organic phase was collected. The aqueous phase was extracted three times with 200 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated and subjected to column chromatography (ethyl acetate: petroleum ether = 1:8) to give 61.6 g of a white solid product, compound D, with a yield of 81%.
[0061] Preparation of compound E:
[0062] In a reaction flask, compound D (30.0 g, 0.14 mol, 1 e. q.), N,N-dimethyl-1,3-propanediamine (18.0 g, 0.18 mol, 1.3 eq.), and potassium carbonate (26.2 g, 0.19 mol, 1.4 eq.) were dissolved in 250 mL of ethanol. The mixture was stirred and reacted at 25 °C for 4 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate / petroleum ether to give 37.5 g of a yellow solid product, compound E, with a yield of 91%.
[0063] Preparation of compound A:
[0064] In a reaction flask, compound E (20.0 g, 65.5 mmol, 1.0 eq) and cobalt acetylacetonate catalyst (2.3 g, 6.6 mmol, 0.1 eq) were dissolved in 200 mL of acetone. The mixture was stirred and cooled to 0 °C. 70% tert-butanol peroxide aqueous solution (84.3 g, 0.66 mol, 10 e.q.) was slowly added dropwise while maintaining the temperature at 0 °C. After the addition was complete, the mixture was allowed to react at room temperature for 12 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:4) to give a yellow solid product, compound A17.9 g, yield: 85%.
[0065] Preparation of compound C:
[0066] In a reaction flask, compound A (10.0 g, 31.1 mmol, 1.0 eq), compound B (6.1 g, 37.3 mmol, 1.2 eq), and potassium carbonate (6.5 g, 46.7 mmol, 1.5 eq) were dissolved in 100 mL of tetrahydrofuran. The mixture was stirred and reacted at room temperature for 4 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate to give a yellow solid product, compound C12.1 g, with a yield of 83%.
[0067] Preparation of SYBR GREEN I:
[0068] In a reaction flask, compound C (10.0 g, 21.4 mmol, 1.0 eq) and bromopropane (4.0 g, 32.1 mmol, 1.5 eq) were dissolved in 100 mL of DMF. The mixture was stirred and reacted at 80 °C for 12 h until the reaction was complete. 200 mL of anhydrous ethanol was added to the reaction solution, and the mixture was cooled to 0 °C and allowed to crystallize for 2 h. The mixture was then filtered, washed with a small amount of anhydrous ethanol, and the filter cake was collected to obtain the crude product. The crude product was recrystallized from the ethanol / water mixture to obtain 10.2 g of the dark green solid product, namely SYBR GREEN I, with a yield of 93%.
[0069] Example 3
[0070] This embodiment provides a method for preparing nucleotide colloidal dyes, including the following steps:
[0071] Preparation of compound F:
[0072] In a reaction flask, 50.0 g (0.33 mol, 1.0 eq) of 2-hydroxybenzothiazole, 70.4 g (0.50 mol, 1.5 eq) of iodomethane, and 73.1 g (0.53 mol, 1.6 eq) of potassium carbonate were added to 250 mL of acetonitrile. The reaction solution was heated to reflux and reacted for 12 h. After the reaction was complete, waste salts were removed by filtration, and the filtrate was collected and concentrated to obtain a crude product. The crude product was slurried using a mixed solvent of ethyl acetate / petroleum ether to obtain 50.3 g of a white solid product, compound F, namely 3-methyl-2(3H)-benzothiazole, with a yield of 92%.
[0073] Preparation of compound B:
[0074] Under nitrogen protection, methyltriphenylphosphine bromide (84.3 g, 0.24 mol, 1.3 eq) was added to a reaction flask containing 350 mL of anhydrous tetrahydrofuran. Stirring was started, and the temperature was lowered to -20 °C. Potassium tert-butoxide solid (28.5 g, 0.25 mol, 1.4 eq) was slowly added. After the addition was complete, the reaction system changed from white to orange. The mixture was kept at this temperature for 30 min to obtain a phosphorus ylide solution. Compound F (30 g, 0.18 mol, 1.0 eq) was dissolved in 100 mL of anhydrous tetrahydrofuran and slowly added dropwise to the above phosphorus ylide solution. The temperature was maintained at -20 °C. After the addition was complete, the temperature was slowly raised to room temperature, and the reaction was continued for 12 h. After the reaction was complete, the above reaction solution was poured into 300 mL of water, 200 mL of ethyl acetate was added, and the mixture was extracted and separated. The organic phase was collected, and the aqueous phase was extracted three more times with 100 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:10) to give 27.2 g of a colorless oily product, compound B, with a yield of 92%.
[0075] Preparation of compound D:
[0076] In a reaction flask, 2-hydroxyquinoline (50.0 g, 0.34 mol, 1.0 eq), cuprous iodide (13.1 g, 0.068 mol, 0.2 eq), potassium carbonate (61.5 g, 0.45 mol, 1.3 eq), and bromobenzene (62.2 g, 0.40 mol, 1.15 eq) were dissolved in 250 mL of DMF. The mixture was stirred and heated to 120 °C for 12 h until the reaction was complete. The mixture was filtered, and the filtrate was collected. The filtrate was poured into 1000 mL of water, and 200 mL of ethyl acetate was added. The mixture was extracted, separated, and the organic phase was collected. The aqueous phase was extracted three times with 200 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated and subjected to column chromatography (ethyl acetate: petroleum ether = 1:8) to give 67.7 g of a white solid product, compound D, with a yield of 89%.
[0077] Preparation of compound E:
[0078] In a reaction flask, compound D (30.0 g, 0.14 mol, 1 e. q.), N,N-dimethyl-1,3-propanediamine (16.6 g, 0.16 mol, 1.2 eq.), and potassium carbonate (24.4 g, 0.18 mol, 1.3 eq.) were dissolved in 250 mL of ethanol. The mixture was stirred and reacted at 25 °C for 4 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate / petroleum ether to give a yellow solid product, compound E, in 37.1 g (yield: 90%).
[0079] Preparation of compound A:
[0080] In a reaction flask, compound E (20.0 g, 65.5 mmol, 1.0 eq) and cobalt acetylacetonate catalyst (2.3 g, 6.6 mmol, 0.1 eq) were dissolved in 200 mL of acetone. The mixture was stirred and cooled to 0 °C. 70% tert-butanol peroxide aqueous solution (16.9 g, 0.13 mol, 2 e. q.) was slowly added dropwise while maintaining the temperature at 0 °C. After the addition was complete, the mixture was allowed to react at room temperature for 12 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:4) to give a yellow solid product, compound A, 16.0 g, yield: 76%.
[0081] Preparation of compound C:
[0082] In a reaction flask, compound A (10.0 g, 31.1 mmol, 1.0 eq), compound B (5.6 g, 34.2 mmol, 1.1 eq), and potassium carbonate (5.6 g, 40.5 mmol, 1.3 eq) were dissolved in 100 mL of tetrahydrofuran. The mixture was stirred and reacted at room temperature for 4 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate to give a yellow solid product, compound C11.8 g, with a yield of 81%.
[0083] Preparation of SYBR GREEN I:
[0084] In a reaction flask, compound C (10.0 g, 21.4 mmol, 1.0 eq) and bromopropane (3.6 g, 28.9 mmol, 1.35 eq) were dissolved in 100 mL of DMF. The mixture was stirred and reacted at 80 °C for 12 h until the reaction was complete. 200 mL of anhydrous ethanol was added to the reaction solution, and the mixture was cooled to 0 °C and allowed to crystallize for 2 h. The mixture was then filtered, washed with a small amount of anhydrous ethanol, and the filter cake was collected to obtain the crude product. The crude product was recrystallized from the ethanol / water mixture to obtain a dark green solid product, namely SYBR GREEN I, 9.9 g, with a yield of 91%.
[0085] Example 4
[0086] This embodiment provides a method for preparing nucleotide colloidal dyes, including the following steps:
[0087] Preparation of compound F:
[0088] In a reaction flask, 50.0 g (0.33 mol, 1.0 eq) of 2-hydroxybenzothiazole, 45.8 g (0.36 mol, 1.1 eq) of dimethyl sulfate, and 129.3 g (0.40 mol, 1.2 eq) of cesium carbonate were added to 250 mL of DMF. The reaction solution was heated to reflux and reacted for 8 h. After the reaction was complete, waste salts were removed by filtration, and the filtrate was collected and concentrated to obtain a crude product. The crude product was slurried using a mixed solvent of ethyl acetate / petroleum ether to obtain 44.9 g of a white solid product, compound F, namely 3-methyl-2(3H)-benzothiazole, with a yield of 82%.
[0089] Preparation of compound B:
[0090] Under nitrogen protection, methyltriphenylphosphine bromide (77.8 g, 0.22 mol, 1.2 eq) was added to a reaction flask containing 350 mL of anhydrous tetrahydrofuran. Stirring was started, and the temperature was lowered to -20 °C. A 2.5 M n-butyllithium solution (94.4 mL, 0.24 mol, 1.3 eq) was slowly added. After the addition was complete, the reaction system changed from white to orange. The mixture was kept at this temperature for 30 min to obtain a phosphorus ylide solution. Compound F (30 g, 0.18 mol, 1.0 eq) was dissolved in 100 mL of anhydrous tetrahydrofuran and slowly added dropwise to the above phosphorus ylide solution. The temperature was controlled at -50 °C. After the addition was complete, the temperature was slowly raised to room temperature, and the reaction was continued for 8 h. After the reaction was complete, the above reaction solution was poured into 300 mL of water, 200 mL of ethyl acetate was added, and the mixture was extracted and separated. The organic phase was collected, and the aqueous phase was extracted three more times with 100 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:10) to give 26.3 g of a colorless oily product, compound B, with a yield of 89%.
[0091] Preparation of compound D:
[0092] In a reaction flask, 2-hydroxyquinoline (50.0 g, 0.34 mol, 1.0 eq), cuprous bromide (4.9 g, 0.03 mol, 0.1 eq), cesium carbonate (134.7 g, 0.41 mol, 1.2 eq), and chlorobenzene (42.7 g, 0.38 mol, 1.1 eq) were dissolved in 250 mL of DMSO. The mixture was stirred and heated to 120 °C for 16 h until the reaction was complete. The mixture was filtered, and the filtrate was collected. The filtrate was poured into 1000 mL of water, and 200 mL of ethyl acetate was added. The mixture was extracted, separated, and the organic phase was collected. The aqueous phase was extracted three times with 200 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated and subjected to column chromatography (ethyl acetate: petroleum ether = 1:8) to give 59.3 g of a white solid product, compound D, with a yield of 78%.
[0093] Preparation of compound E:
[0094] In a reaction flask, compound D (30.0 g, 0.14 mol, 1 e.q.), N,N-dimethyl-1,3-propanediamine (15.2 g, 0.15 mol, 1.1 eq.), and sodium hydroxide (6.5 g, 0.16 mol, 1.2 eq.) were dissolved in 250 mL of methanol. The mixture was stirred and reacted at 20 °C for 6 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate / petroleum ether to give a yellow solid product, compound E, in 36.7 g (yield: 89%).
[0095] Preparation of compound A:
[0096] In a reaction flask, compound E (20.0 g, 65.5 mmol, 1.0 eq) was dissolved in 200 mL of acetone. The mixture was stirred and cooled to 0 °C. Manganese dioxide (28.5 g, 0.33 mol, 5.0 eq) was slowly added while maintaining the temperature at 0 °C. After the addition was complete, the mixture was allowed to react at room temperature for 10 h until the reaction was complete. The mixture was then filtered, and the filtrate was collected, concentrated, and subjected to column chromatography (ethyl acetate: petroleum ether = 1:4) to give a yellow solid product, compound A, 16.0 g, with a yield of 76%.
[0097] Preparation of compound C:
[0098] In a reaction flask, compound A (10.0 g, 31.1 mmol, 1.0 eq), compound B (5.1 g, 31.1 mmol, 1.0 eq), and DBU (5.2 g, 34.2 mmol, 1.1 eq) were dissolved in 100 mL of tetrahydrofuran. The mixture was stirred and reacted at room temperature for 6 h until the reaction was complete. The mixture was filtered, the filtrate was collected, concentrated, and recrystallized from ethyl acetate to give a yellow solid product, compound C1, 0.5 g, with a yield of 72%.
[0099] Preparation of SYBR GREEN I:
[0100] In a reaction flask, compound C (10.0 g, 21.4 mmol, 1.0 eq) and chloropropane (2.0 g, 25.7 mmol, 1.2 eq) were dissolved in 100 mL of DMF. The mixture was stirred and reacted at 60 °C for 8 h until the reaction was complete. 200 mL of anhydrous ethanol was added to the reaction solution, and the mixture was cooled to 0 °C and allowed to crystallize for 2 h. The mixture was filtered, washed with a small amount of anhydrous ethanol, and the filter cake was collected to obtain the crude product. The crude product was recrystallized from the ethanol / water mixture to obtain 9.2 g of SYBR GREEN I, a dark green solid product, with a yield of 84%.
[0101] The applicant declares that the present invention illustrates the preparation method of the nucleotide colloidal dye through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
[0102] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0103] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
Claims
1. A method for preparing a nucleotide colloidal dye, characterized in that, The preparation method includes the following steps: compound A undergoes a coupling reaction with compound B to obtain compound C; compound C undergoes a substitution reaction with a halopropane to obtain the nucleotide colloidal dye SYBR GREEN I; The reaction route is as follows:
2. The preparation method according to claim 1, characterized in that, In the preparation of compound C, the coupling reaction requires the addition of a base. The molar ratio of compound A, compound B, and the base is 1:(1-1.2):(1.1-1.5). The coupling reaction is carried out at a temperature of 25-50℃ for 2-10 hours. Preferably, the alkali includes one of potassium carbonate, cesium carbonate, sodium hydroxide, and DBU.
3. The preparation method according to claim 1, characterized in that, The molar ratio of compound C to halopropane is 1:(1.2-1.5); the substitution reaction is carried out at a temperature of 25-85℃ for 4-16 hours. Preferably, the halopropane includes one of fluoropropane, chloropropane, bromopropane, and iodopropane; Preferably, the substitution reaction also requires the addition of a solvent; More preferably, the solvent includes one of DMF, DMSO, THF, ethanol, and methanol.
4. The preparation method according to claim 1, characterized in that, The preparation of compound B includes the following steps: methylating 2-hydroxybenzothiazole with a methylating agent to obtain compound F; reacting compound F with methyltriphenylphosphine bromide via a Wittig reaction to obtain compound B; The reaction route is as follows:
5. The preparation method according to claim 4, characterized in that, The preparation of compound F also needs to be carried out under the action of alkali and solvent. The molar ratio of 2-hydroxybenzothiazole, methylating agent and alkali is 1:(1.1-1.5):(1.2-1.6). The methylation reaction is carried out at reflux temperature for 8-14 hours. Preferably, the methylating agent includes one of iodomethane and dimethyl sulfate, the base includes one of potassium carbonate, cesium carbonate, and sodium hydride, and the solvent includes one of acetonitrile, acetone, and DMF.
6. The preparation method according to claim 4, characterized in that, The preparation of compound B also requires the action of a base and a solvent. The molar ratio of compound F, methyltriphenylphosphine bromide, and base is 1:(1.1-1.3):(1.2-1.4). The Wittig reaction is carried out at a temperature of -78 to 0°C for 4 to 16 hours. Preferably, the alkali includes one of potassium tert-butoxide, sodium hydride, potassium hydride, n-butyllithium, and sodium tert-butoxide, and the solvent includes tetrahydrofuran.
7. The preparation method according to claim 1, characterized in that, The preparation steps of compound A are as follows: 2-hydroxyquinoline is coupled with a halobenzene to obtain compound D; compound D undergoes a nucleophilic reaction with N,N-dimethyl-1,3-propanediamine to obtain compound E; compound E undergoes an oxidation reaction with an oxidizing agent to obtain compound A. The reaction route is as follows:
8. The preparation method according to claim 7, characterized in that, In the preparation of compound D, the coupling reaction also requires the addition of a catalyst, a base and a solvent. The molar ratio of 2-hydroxyquinoline, halobenzene, catalyst and base is 1:(1.1-1.2):(0.05-0.2):(1.1-1.3). The coupling reaction is carried out at reflux temperature for 8-16 hours. Preferably, the catalyst includes one of cuprous iodide and cuprous bromide, the base includes one of potassium carbonate and cesium carbonate, the solvent includes one of DMF and DMSO, and the halobenzene includes one of bromobenzene, chlorobenzene, and iodobenzene.
9. The preparation method according to claim 7, characterized in that, In the preparation of compound E, the nucleophilic reaction also requires the addition of a base and a solvent. The molar ratio of compound D, N,N-dimethyl-1,3-propanediamine, and base is 1:(1.1-1.3):(1.2-1.4). The temperature of the nucleophilic reaction is 10-30℃, and the time is 2-6h. Preferably, the alkali includes one of potassium carbonate, cesium carbonate, and sodium hydroxide, and the solvent includes one of ethanol, methanol, and THF.
10. The preparation method according to claim 7, characterized in that, In the preparation of compound A, the molar ratio of compound E to oxidant is 1:(1.5-10), the oxidation reaction temperature is 0-40℃, and the time is 8-16h; Preferably, the oxidation reaction also requires a solvent, which includes one of acetone, ethyl acetate, and THF; Preferably, the oxidant comprises a combination of 70% aqueous tert-butanol peroxide and a catalyst, manganese dioxide, and chromic anhydride, and the catalyst comprises cobalt acetylacetonate and iron acetylacetonate. More preferably, in the combination, the molar ratio of the 70% tert-butanol peroxide aqueous solution to the catalyst is (20-100):1.