CN103819523B - The synthetic method of 7-denitrification-7-halogen guanosine- - Google Patents

Abstract

The invention discloses a method for synthesizing 7-deaza-7-haloguanosine; the method comprises the following steps: the compound of formula (III) is deprotected under alkaline conditions to obtain the compound of formula (IV1) or (IV2) Compound; further demethylation to obtain the compound of formula (I), that is, the 7-deaza-7-halogenated guanosine; Wherein, R ₁ is H or OH, R ₂ is I, Br or Cl, R ₃ is H or The 7-deaza-7-halogen-guanosine synthesized by the present invention is a basic raw material widely used in biological fields such as DNA sequencing, labeling, and extension. At present, its sales price is very high, and the synthesis method is complicated and difficult to control; However, the raw materials required by the synthesis method of the present invention are simple and easy to obtain, and the synthesis process is all conventional chemical reactions, which can be used for large-scale popularization.

Description

Synthetic method of 7-deaza-7-halogen guanosine

technical field

The invention relates to the fields of chemical synthesis and biochemistry, in particular to a synthesis method of 7-deaza-7-halogen guanosine (guanosine for short, including dG-X and G-X).

Background technique

DNA sequencing technology is one of the important means of modern life science and medical research. DNA sequencing started with Sanger sequencing technology (generation sequencing) in 1977, and has developed rapidly in the past thirty years. The throughput of sequencing has been greatly increased and the cost has dropped sharply. Some people even think that the speed of its development has broken the existing Moore's Law budget in the semiconductor industry. The emergence of the second-generation high-throughput parallel sequencing technology is a concentrated expression of the rapid development of sequencing technology. Using first-generation sequencing technology, the Human Genome Project (HGP) spent $3 billion to sequence the entire human genome (3 billion bases). The current latest technology of next-generation sequencing can complete the sequencing of the entire human genome for only about US$5,000.

Even so, the cost and technical aspects of next-generation sequencing are still insufficient, which cannot meet the requirements of basic science and clinical medicine for sequencing. Single-molecule sequencing technology (third-generation sequencing technology) came into being. The core of the third-generation sequencing technology is to directly sequence a single DNA molecule without any DNA amplification reaction, thereby reducing costs and increasing throughput. Although single-molecule sequencing technologies have been commercialized, they still have technical difficulties and cannot be applied on a large scale.

At present, the high-throughput sequencing platform on the market is monopolized by a few foreign products. What is particularly worrying is that foreign companies have almost completely controlled the domestic sequencing market by virtue of their control over sequencing reagents. If we make breakthroughs, we will still be constrained by others in terms of sequencing reagents and other supporting products. Therefore, independent research and development of sequencing reagents that can be applied to second-generation sequencing or even third-generation sequencing platforms will have strategic significance for changing the current market structure and establishing my country's own independent sequencing platform. For this reason, the national 863, 973 and "Twelfth Five-Year Plan" biotechnology development plans all list the research and development of next-generation sequencing technology and supporting products as key development objects.

The reversible terminal for sequencing generally selects nucleotides of four bases U, C, A, and G. In actual work, we found that the starting materials for the synthesis of four different base nucleotides, namely four Substituent nucleosides with different bases (U, C, A, G) are expensive, especially 7-deaza-7-halogen guanosine (including guanosine dG-X and G-X) is not only very expensive but also synthetic The method is very complicated, so many researchers try to avoid using guanosine (J.Org.Chem.2011, 76, 3457–3462), which makes the original perfect research work a bit regrettable.

Contents of the invention

The object of the present invention is to provide a kind of synthetic method of 7-deaza-7-halogenated guanosine (guanosine dG-X and G-X for short); Suitable for mass production.

The purpose of the present invention is achieved through the following technical solutions:

In a first aspect, the present invention relates to a synthetic method of 7-deaza-7-halogenated guanosine, the method comprising the following steps:

A. The compound of formula (Ⅲ) is deprotected under basic conditions to obtain the compound of formula (Ⅳ1) or (Ⅳ2);

B. The compound of formula (IV1) or (IV2) is demethylated under alkaline conditions to obtain the compound of formula (I), that is, the 7-deaza-7-halogenoguanosine;

Wherein, R ₁ is H or OH, R ₂ is I, Br or Cl, R ₃ is H or

Preferably, in step A, when R ₃ is H, a compound of formula (IV1) is generated; R ₃ is When, the compound of formula (Ⅳ2) is generated.

Preferably, the compound of formula (III) is obtained through the compound of formula (II) Prepared by connecting a halogen atom to the 7-position of the purine base.

Preferably, the compound of formula (II) is combined with compound of formula G007 or Prepared by glycosylation reaction,

Preferably, the compound G007 is prepared through the following steps:

A. Compound G005 Synthesis of: Compound Sm-1 with Sm-2 Reaction, obtain compound G005;

B. Compound G006 Synthesis of: Compound G005 Under the action of phosphorus oxychloride, react to obtain compound G006;

C. Compound G007 Synthesis of: Compound G006 React with isobutyryl chloride under basic conditions to obtain the compound G007.

In the second aspect, the present invention also relates to a method for synthesizing 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide, the method comprising formula (I) synthesized by the aforementioned method The compound is further synthesized from the 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide; in the formula (I), R1 is H.

Preferably, the following steps are included:

A. Synthesis of compound dG(AP ₃ ): in the presence of CuI, Pd(PPh ₃ ) ₄ (tetrakis(triphenylphosphine) palladium) and TEA (triethylamine), trifluoroacetylpropynylamine and formula (I) compound Reaction, get compound dG(AP ₃ ) The molar ratio of the compound of formula (I), trifluoroacetylpropynylamine, CuI, Pd(PPh ₃ ) ₄ and TEA is 1:(1.5～3):(0.05～0.10):(0.02～0.05):( 1.5～3);

B. Synthesis of compound dGTP (AP ₃ ): compound dG (AP ₃ ) and tri-n-butylamine pyrophosphate, 2-chloro-4H-1,3,2-benzodioxophosphor-4-one in triethyl In the presence of amine and iodine, the reaction product is deprotected to obtain the compound dGTP (AP ₃ ), that is, the 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide; the tri-n-butylamine pyrophosphate, 2-chloro-4H-1,3 , the molar ratio of 2-benzodioxophosphor-4-one to dG(AP ₃ ) was 2:2:1.

In the third aspect, the present invention also relates to a method for synthesizing 7-deaza-7-propynylamine-guanosine, the method comprising further synthesizing the 7 - deaza-7-propynylamine-guanosine; R1 is OH in the formula (I).

Preferably, the following steps are included:

In the presence of CuI, Pd(PPh ₃ ) ₄ and TEA, trifluoroacetylpropargylamine and the compound of formula (I) reaction to obtain compound G(AP ₃ ) That is, the 7-deaza-7-propynylamine-guanosine; the molar ratio of the formula (I) compound, trifluoroacetylpropynylamine, CuI, Pd(PPh ₃ ) ₄ and TEA is 1: (1.5～3):(0.05～0.10):(0.02～0.05):(1.5～3).

In the fourth aspect, the present invention also relates to the use of 7-deaza-7-halogenoguanosine prepared by the aforementioned synthesis method in the synthesis of 7-deaza-7-propynylamine-guanosine.

The present invention has following beneficial effects:

(1) The present invention synthesized 7-deaza-7-halogen-2'-deoxyguanosine (abbreviated as dG-I) and 7-deaza-7-halogenoguanosine (abbreviated as G-I); the Compounds are basic raw materials widely used in biological fields such as DNA sequencing, labeling, and extension;

(2) Compared with the patent CN201310489397.0, the synthetic method provided by the present invention, the core raw materials required in the patent 201310489397.0 can only be imported, but the raw materials required by the present invention are very cheap. Compared with the patent 201310489350.4, the first step of the patent 201310489350.4 The reaction yield is only 10%, and the method used in the present invention can reach 86%; the raw material used when generating G009 in patent 201310489350.4 contains benzyl-protected ribose, which is expensive, and the yield is only 45%. The price of ribose used in the present invention It is extremely cheap, and the yield of the experimental method used can reach 76%. Therefore, while the synthetic method of the present invention greatly improves the reaction yield, it also greatly reduces the cost of the experimental raw materials. The reaction raw materials are conventional chemical reagents, and finally In one-step reaction treatment, the method for removing inorganic salts of the present invention is simpler and more efficient.

(3) The purity of the final product obtained by the method of the present invention is high. Compared with the previous patent, the last step of purification adopts the method of washing with organic solvent, which is difficult to remove the inorganic salts. , thereby avoiding the existence of inorganic salts in the product, greatly improving the purity of the product, the raw materials required for the synthesis method of the present invention are simple and easy to obtain, and the synthesis process is a conventional chemical reaction, which can be used for large-scale promotion and use.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings.

Fig. 1 is the synthetic process schematic diagram of 7-deaza-7-iodo-2'-deoxyguanosine (abbreviated as dG-I);

Fig. 2 is a schematic diagram of the synthesis process of 7-deaza-7-iodine guanosine (abbreviated as G-I);

Figure 3 is the use of 7-deaza-7-iodo-2'-deoxyguanosine dG-I in the synthesis of dGTP (AP ₃ );

Figure 4 is the use of 7-deaza-7-iodoguanosine GI in the synthesis of GTP (AP ₃ );

Figure 5 is the ¹ H-NMR of 7-deaza-7-iodo-2'-deoxyguanosine dG-I;

Figure 6 is the ¹ H-NMR of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ );

Figure 7 is the ³¹ P-NMR of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ );

Figure 8 is the HRMS spectrum of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ );

Fig. 9 is a diagram of fluorescence scanning results of DNA chain extension reaction; Lane1: Primer (Oligo1); Lane2: chain extension product containing dGTP (AP3).

detailed description

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make some adjustments and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available AR and CP grades. The obtained intermediate product and final product of the present invention are characterized by NMR etc.;

The synthetic method of embodiment 1,7-deaza-7-iodo-2'-deoxyguanosine dG-I

The synthesis schematic diagram of dG-I in this embodiment is shown in Figure 1, and the specific synthesis method includes the following steps respectively:

step one,

Dissolve Sm-2 (12.6g, 100mmol) in a mixture of 120mL DMF and 20mL water, stir at room temperature for 15min, add Sm-1 (12.7mL, 100mmol), and stir at room temperature for 46h. The solvent was spun out, and the solid was dissolved in 2.5 mL of water, filtered, and dried in vacuo to obtain 12.9 g with a yield of 86%. ¹ HNMR(400MHz,DMSO-d ₆ ):δ=10.94(s,1H),10.35(s,1H),6.58(dd,J=3.4,2.2Hz,1H),6.15(dd,J=3.4,2.1 Hz,1H),6.09(s,2H).

Step two,

Add G005 (10.0g, 66.6mmol) into 100mL POCl ₃ , reflux for 2h, cool to room temperature and spin off the solvent, add 120mL ice water to the reaction, filter the solid, and adjust the filtrate to pH=2 with ammonia water , and the precipitate was placed in an ice bath for 2 hours and then filtered. The filtered solid was washed with 10 mL of ice water for the first time, and washed with 30 mL of ice ether for the second time. After drying, 8.7 g was obtained, with a yield of 78%. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ=11.43 (s, 1H, NH), 7.07 (d, 1H, NHCHCH), 6.46 (s, 2H, NH2), 6.22 (d, 1H, CHNH).

Step three,

Add G006 (6.7g, 39.88mmol) into 280mL of anhydrous pyridine, slowly add isobutyryl chloride (4.6mL, 43.87mmol) dropwise under ice-water bath, stir for 20min under ice-water bath, add 600μL methanol to quench, and continue stirring for 10min Then spin out the solvent, add 20 mL of diethyl ether, filter, collect the solid, wash with 50 mL of 80% cold methanol/20% diethyl ether, 10 mL of cold methanol, 10 mL of diethyl ether, and dry in vacuo to obtain 6.68 g with a yield of 70%. ¹ HNMR(400MHz,DMSO-d ₆ ):δ(ppm)12.34(bs,1H),10.54(bs,1H),7.49(dd,J=2.4Hz,3.2Hz,1H),6.49(dd,J= 1.6Hz, 3.2Hz, 1H), 2.78(sept, J=6.8Hz, 1H), 1.08(d, J=6.8Hz, 6H).

Step four,

Potassium hydroxide (3.47g, 61.8mmol) powder was dissolved in 500mL of acetonitrile, G007 (6.66g, 28.0mmol) was slowly added, stirred at room temperature for 5min, Sm-1 (14.98g, 35mmol) was slowly added, and stirred at room temperature for 20min. Filter, wash the solid with 10 mL of dichloromethane, and dry. Column chromatography DCM:MeOH=10:1 gave 13.4 g of white solid with a yield of 76%. ¹ HNMR(400MHz,DMSO-d ₆ ):δ=10.29(s,1H),8.07-7.93(m,5H),7.66-7.56(m,4H),6.62(t,J=7.2Hz,1H,) ,5.84-5.81(m,1H),4.71-4.67(m,1H),4.59-4.44(m,3H),3.30-3.22(m,1H),2.79-2.72(m,1H),1.06(d, J=6.8Hz,6H).

Step five,

Dissolve G008 (8.23g, 13.0mmol) in 60mLTHF, protect it under nitrogen, wrap it in tinfoil, add NIS (3.04g, 13.51mmol) and stir at room temperature for 1h, add 500mL DCM, wash with 200mL water, spin off the solvent, Column chromatography DCM:MeOH=10:1, 7.98g was obtained, the yield was 81%. ¹ HNMR(400MHz,DMSO-d ₆ ):δ=10.29(s,1H),8.07-7.93(m,5H),7.66-7.56(m,4H),5.84-5.81(m,1H),4.71-4.67 (m,1H),4.59-4.44(m,3H),3.30-3.22(m,1H),2.79-2.72(m,1H),1.05(d,J=6.8Hz,6H).

Step six,

G009 (1.14g, 1.5mmol) was added to 0.5MMeONa/MeOH (20.0mL), refluxed for 3h, neutralized to neutral with glacial acetic acid, followed by column chromatography DCM:MeOH=10:1, the compound dG1-D490mg was obtained, The yield is 80%. ¹ H-NMR (400MHz, CD ₃ OD)δ7.17(s,1H),6.36(dd,J=6.0,8.4Hz,1H),4.47(m,1H),3.99(s,3H),3.96( m,1H),3.77(dd,J=3.4,12.0Hz,1H),3.70(dd,J=3.7,12.0Hz,1H),2.55-2.64(ddd,J=6.0,8.4,13.4Hz,1H) ,2.20-2.26(ddd,J=2.4,5.9,13.4Hz,1H).

Step seven,

Compound dG ₁ -D (490mg, 1.20mmol) was placed in 25mL sodium hydroxide solution (2N) and refluxed for 5h. After cooling, it was neutralized by adding acetic acid solution, filtered, washed with a small amount of water, and dried to obtain 428mg of white solid, namely dG-I. Yield 91%. See Figure 5: ¹ H-NMR (400MHz, MeOD) δ7.09(s, 1H), 6.35(dd, J=6.0Hz, J=8.0Hz, 1H), 4.42-4.44(m, 1H), 3.89-3.92(m,1H), 3.65-3.74(m,2H), 2.43–2.50(m,1H), 2.19–2.24(m,1H). Note: This method is also suitable for 7-denitrogen-7-bromo Compared with the synthesis of 7-deaza-7-chloro-2'-deoxyguanosine dG-Br/Cl, the difference is that in the fifth step of the reaction, NBS or BCS can be used instead of NIS, and all other reaction steps and methods are the same.

The synthetic method of embodiment 2,7-deaza-7-iodine-guanosine G-I

The synthesizing schematic diagram of G-I in the present embodiment is as shown in Figure 2, and specific synthesizing method comprises the following steps respectively:

step one,

Potassium hydroxide (3.47g, 61.8mmol) powder was dissolved in 500mL of acetonitrile, G007 (6.66g, 28.0mmol) was slowly added, stirred at room temperature for 5min, Sm-1 (20.44g, 35mmol) was slowly added, and stirred at room temperature for 20min. Filter, wash the solid with 10 mL of dichloromethane, and dry. Column chromatography DCM:MeOH=10:1, 16.77g white solid was obtained, the yield was 76%. ¹ H-NMR(400MHz,DMSO-d ₆ ):δ=10.25(s,1H),8.11-7.95(m,9H),7.76-7.58(m,8H),6.64(t,J=7.2Hz,1H ,),5.88-5.82(m,1H),4.74-4.66(m,1H),4.61-4.40(m,4H),1.09(d,J=6.8Hz,6H).

Step two,

Dissolve G008 (10.24g, 13.0mmol) in 60mLTHF, protect it under nitrogen, wrap it in tinfoil, add NIS (3.04g, 13.51mmol) and stir at room temperature for 1h, add 500mL DCM, wash with 200mL water, and spin off the solvent. Chromatography DCM:MeOH=10:1, to obtain 9.62g, yield 81%. ¹ HNMR(400MHz,DMSO-d ₆ ):δ=10.29(s,1H),8.19-7.96(m,9H),7.68-7.52(m,8H),5.87-5.82(m,1H),4.75-4.65 (m,1H),4.58-4.35(m,4H),1.10(d,J=6.8Hz,6H).

Step three,

G009 (1.37g, 1.5mmol) was added to 0.5MMeONa/MeOH (20.0mL), refluxed for 3h, neutralized to neutral with glacial acetic acid, followed by column chromatography DCM:MeOH=10:1 to obtain 506mg of compound G-D, The yield is 80%. ¹ H-NMR(250MHz,DMSO-d ₆ ):δ3.49-3.57(m,2H,HC(5')),3.80-3.82(m,1H,HC(4')),3.93(s,3H ,OMe),4.01-4.03(m,1H,HC(3')),4.23-4.28(m,1H,HC(2')),5.01(t,J=5.5Hz,1H,OH-C(5 ')),5.05(d,J=4.4Hz,1H,OH-C(3')),5.25(d,J=6.2Hz,1H,OH-C(2')),5.94(d,J= 6.5Hz,1H,HC(1')),6.38(s,2H,NH2),7.31(s,1H,HC(6)).

Step four,

Compound GD (506 mg, 1.20 mmol) was placed in 25 mL of sodium hydroxide solution (2N) and refluxed for 5 h. After cooling, it was neutralized by adding acetic acid solution, filtered, washed with a small amount of water, and dried to obtain 445 mg of a white solid, namely GI, with a yield of 91%. ¹ HNMR(600MHz,DMSO-d ₆ ):4.99(brs,1H,5'-OH),5.04(d,1H,J=3.2,3'-OH),5.26(d,1H,J=5.9,2 '-OH), 6.33 (s, 2H, NH2), 7.14 (s, 1H, 6-H), 10.48 (s, 1H, NH). Note: This method is also suitable for 7-deaza-7-bromo and 7 - the synthesis of deaza-7-chloro-guanosine dG-Br/Cl, the difference is that in the second step of reaction, NBS or BCS can be used instead of NIS, and all other reaction steps and methods are the same.

Example 3, 7-deaza-7-iodo-2'-deoxyguanosine dG-I in the synthesis of dGTP (AP ₃ ) used in way

The synthesizing schematic diagram of dGTP (AP ₃ ) in the present embodiment is shown in Figure 3, and specific synthesizing method comprises the following steps respectively:

step one,

Add compound dG-I (0.25g, 0.4mmol) to a single-necked flask, then weigh CuI (22mg; 1mmol) and Pd(PPh ₃ ) ₄ (48mg; 0.04mmol) into the reaction flask, vacuumize and protect with nitrogen , wrapped in aluminum foil, add 10ml of DMF, stir to dissolve, inject TEA (0.088g; 0.8mmol) and trifluoroacetylpropargylamine (0.2g; 1.2mmol), stir at 50°C for 13 hours, the reaction is over, spin out the solvent, and remove the remaining The substance was dissolved in 100ml ethyl acetate, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, column chromatography [V (ethyl acetate): V (n-hexane) = 1: 3] to obtain 0.1 g of white solid, namely dG(AP ₃ ), with a yield of 39%. See Figure 6: ¹ HNMR (400MHz, MeOD) δ7.25(s,1H),6.38-6.42(m,1H),4.47–4.50(m,1H),4.33(s,2H),3.96(dd ,J=3.6Hz,J=6.8Hz,1H),3.70–3.80(m,2H),2.48–2.55(m,1H),2.26–2.32(m,1H).

Step two,

Compound dG(AP ₃ ) was vacuum-dried for 12 hours, and compound dG(AP ₃ ) (30mg, 0.072mmol), tri-n-butylamine pyrophosphate (80mg, 0.145mmol), 2-chloro-4H - 1,3,2-Benzophosphor-4-one (30 mg, 0.15 mmol) was placed in three reaction tubes. Dissolve tri-n-butylamine pyrophosphate in 0.25 mL of anhydrous DMF, then add 0.3 mL of freshly distilled tri-n-butylamine, stir at room temperature for half an hour, then inject the reaction solution into 2-chloro-4H-1,3,2 - In anhydrous DMF (0.25 mL) solution of benzodioxophosphor-4-one, stir at room temperature for half an hour. Then the mixture was injected into 2 and stirred for 1.5h. Add 1mL of 3% iodine (9:1Py/H2O) solution, and keep the color of the iodine solution for 15min without fading. After 15 minutes, add 2 mL of water, and after 2 hours, add 0.75 mL of 3M NaCl solution and 20 mL of absolute ethanol, freeze at -20°C for 12 hours, and centrifuge (20 minutes, 3200 rpm). Pour off the supernatant, drain the solvent after the precipitation, add concentrated ammonia water, and stir at room temperature for 5 hours. The solvent was spinned out under reduced pressure, and a brown solid appeared, analyzed by RP-HPLC [conditions: column: C18, 5 μm, 4.6 × 250 mm; flow rate: 1 mL/min; mobile phase: 20 mM TEAA and EtOH, 0-20% EtOH (35 min), visible Detector wavelength: 650nm], retention time t=18min. RP-HPLC separation [conditions: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 20mM TEAA and MeOH, 0-15%MeOH (25min), UV detector wavelength: 254nm], retention time t =15min. NaCl/EtOH was used to remove the triethylamine acetate salt to obtain 12 mg of white solid, dGTP(AP ₃ ). Yield 26%. The ¹ H-NMR, ³¹ P-NMR, and HRMS spectra of dGTP(AP ₃ ) are shown in Figures 8 and 9 respectively, ¹ HNMR (400MHz, D ₂ O) δ7.45(s,1H), 6.34(t, J=6.8Hz,1H),4.73(s,1H),4.11–4.20(m,3H),4.06(s,2H),2.53–2.58(m,1H),2.41–2.46(m,1H); see As shown in Figure 7: ³¹ PNMR(D ₂ O,162MHz):-10.59(t,J=9.9Hz,1P),-11.24(d,J=17.3Hz,1P),-22.98(d,J=20.7Hz ,1P).ESI-HRMS: calcforC ₁₄ H ₁₉ N ₅ O ₁₃ P ₃ [MH] ^- 558.0192, found558.0179. Note: This method is also suitable for 7-deaza-7-bromo/chloro-2'-deoxybird The use of purine nucleoside dG-Br/Cl in the synthesis of dGTP (AP ₃ ), the difference is that in the first step of the reaction, dG-Br/Cl can be used instead of dG-I, and all other reaction steps and methods are the same .

Embodiment 4, 7-deaza-7-iodine guanosine G-I is synthesizing G(AP ₃ ) uses in

In the present embodiment, the synthetic schematic diagram of G(AP ₃ ) is shown in Figure 4, and the specific synthetic method comprises the following steps respectively:

Add compound GI (0.25g, 0.4mmol) to a single-necked flask, then weigh CuI (22mg; 1mmol) and Pd(PPh ₃ ) ₄ (48mg; 0.04mmol) into the reaction flask, vacuumize, nitrogen protection, aluminum foil Wrap, add 10ml of DMF, stir to dissolve, inject TEA (0.088g; 0.8mmol) and trifluoroacetylpropargylamine (0.2g; 1.2mmol), stir at 50°C for 13 hours, the reaction is over, spin out the solvent, and dissolve the residue In 100ml of ethyl acetate, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, column chromatography [V (ethyl acetate): V (n-hexane) = 1:3] , to obtain 0.1 g of white solid, namely G(AP ₃ ), with a yield of 39%. ¹ HNMR (400MHz, CDCl ₃ )δ7.24(s,1H),6.38(t,J=0.8Hz,1H),4.49–4.46(m,1H),4.31(s,2H),3.94(d,J =1.6Hz,1H), 3.78–3.68(m,1H), 3.54–2.47(m,1H), 2.3–2.24(m,1H). Note: This method is also suitable for 7-deaza-7-iodoguanine The use of nucleoside GI in the synthesis of G(AP ₃ ) is different in that in the first step of reaction, G-Br/Cl can be used instead of GI, and all other reaction steps and methods are the same.

Embodiment 5, the synthetic dGTP (AP ₃ ) to participate in the DNA chain extension reaction test

The sequencing template sequences used are as follows:

5'GAGGAAAGGGAAGGGAAAGGAAGGOligo1Primer

3' CTCCTTTTCCCTTCCCTTTCCTTCCCATGATCGCCATGTGCOligo2

The 5' end of Oligo1 was labeled with fluorescein Dylight800.

1) Set up a DNA chain extension reaction with reversible terminal in an eppendorf tube according to the following system: 10×Klenowbuffer10uL, BSA(10mg/mL)1uL, DMSO20uL, NaCl(1M)25uL, Klenow(exo-)pol(5U/uL)1.32 uL, dUTP (10uM) 6uL, template DNA (853ng/uL) 1.25uL, ddH ₂ O 35.43uL, total volume 100uL.

The reaction system was treated in a 30°C water bath for 15 minutes, and then placed in a 75°C water bath for 10 minutes to inactivate the DNA polymerase.

2) Separation and purification: phenol chloroform extraction, ethanol precipitation and concentration to solid, add 20uLddH ₂ O and 1uL 0.1M NaOH, treat at 95°C for 5min, immediately cool in ice-water bath for 2min, and then perform electrophoresis analysis.

3) Electrophoresis analysis: The results of fluorescence scanning of the DNA chain extension reaction are shown in Figure 9. From the results in Figure 9, it can be seen that dGTP (AP3) can be recognized by DNA polymerase and participate in the extension of the DNA chain as its substrate. This further proves that the structure of the synthesized dGTP (AP3) is correct.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (2)

1. a synthetic method of 7-deaza-7-halogen guanosine, characterized in that, the method comprises the steps: A. The compound of formula (Ⅲ) is deprotected under basic conditions to obtain the compound of formula (Ⅳ1) or (Ⅳ2); B. The compound of formula (IV1) or (IV2) is demethylated under alkaline conditions to obtain the compound of formula (I), that is, the 7-deaza-7-halogenoguanosine; Wherein, R ₁ is H or OH, R ₂ is I, R ₃ is H or In step A, when R ₃ is H, the compound of formula (Ⅳ1) is generated; R ₃ is When, generate formula (Ⅳ2) compound; The compound of the formula (III) is prepared by connecting a halogen atom to the 7-position of the purine base of the compound of the formula (II), Described formula (II) compound passes formula G007 compound with compound or Prepared by glycosylation reaction. 2. the synthetic method of 7-deaza-7-halogen guanosine as claimed in claim 1, is characterized in that, described compound G007 is prepared by following steps: A. Compound G005 Synthesis of: Sm-1 and React under acidic conditions to obtain compound G005; B. Compound G006 Synthesis of: Compound G005 Under the action of phosphorus oxychloride, react to obtain compound G006; C. Compound G007 Synthesis of: Compound G006 React with isobutyryl chloride under basic conditions to obtain the compound G007.