CN103601779B - The synthetic method of 7-denitrification-2 '-deoxidation-7-halogen 9 substituted guanine nucleosides - Google Patents

Abstract

The invention discloses a synthesis method of 7-deaza-2'-deoxy-7-halogen substituted guanosine; the method comprises demethylating the compound of the following formula (V) under alkaline conditions to obtain the following formula ( I) a compound, that is, the 7-deaza-2'-deoxy-7-halogen substituted guanosine; (I), (V), wherein R ₂ is I, Br or Cl. The 7-deaza-7-iodo-2'-deoxyguanosine synthesized by the present invention is a basic raw material widely used in biological fields such as DNA sequencing, labeling, and extension. Currently, its selling price is very high, and its synthesis method is complicated ; And the raw materials needed for the synthetic method provided by the invention are simple and easy to get, and the synthetic process is a conventional chemical reaction, which can be used for large-scale popularization.

Description

Synthetic method of 7-deaza-2'-deoxy-7-halogen substituted guanosine

technical field

The invention relates to the fields of chemical synthesis and biochemistry, in particular to a synthesis method of 7-deaza-2'-deoxy-7-halogen substituted guanosine (guanosine dG-X for short).

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 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). However, the current state-of-the-art 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 used for sequencing generally selects nucleotides with four bases U, C, A, and G. We have found in practical work that the starting materials for the synthesis of four different base nucleotides, that is, four Substituent nucleosides with different bases (U, C, A, G) are expensive, especially 7-deaza-2'-deoxy-7-halogen substituted guanosine (guanosine dG-X for short) Not only is it very expensive, but also the synthesis 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. If it is in the case of four bases, you have to spend a lot of money to buy them. The present invention synthesizes the compound by a simple and convenient method, and the synthesis method is simple, the reaction condition is mild, the reaction process is controllable, and it is suitable for large-scale production. And we successfully synthesized 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ ) with our own guanosine.

Contents of the invention

The object of the present invention is to provide a kind of synthetic method of 7-deaza-2'-deoxy-7-halogen substituted guanosine (guanosine dG-X for short); The synthetic raw material of this method is simple, cheap, and reaction condition is gentle, The operation is simple and suitable for mass production.

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

The present invention relates to a kind of synthetic method of 7-deaza-2'-deoxy-7-halogen substituted guanosine, said method comprises demethylating the compound of following formula (V) to obtain following formula (I ) compound, that is, the 7-deaza-2'-deoxy-7-halogen substituted guanosine;

(I),

(V), wherein R ₂ is I, Br or Cl.

Preferably, the compound of formula (V) is prepared by deprotecting the compound of formula (IV) under basic conditions:

(IV), wherein, R is a protecting group for protecting a hydroxyl group or an amino group.

Preferably, the protecting group is pivaloyl or isobutyryl.

Preferably, the compound of formula (IV) is prepared by attaching a halogen to the 7-position of the purine base of the compound of formula (III):

(III). Iodine can be connected to the 7-position of the purine base with NIS or other iodination reagents; bromine or chlorine can also be connected to the 7-position of the purine base with NBS or NCS and other reagents.

Preferably, the compound of formula (III) is prepared by protecting two hydroxyl groups and one amino group of the compound of formula (II) with a protecting group under basic conditions:

(II). The name of the compound of formula (II) is 7-Deaza-2'-deoxy-6-methoxyguanosine, CAS number: 86392-74-7, molecular formula: C ₁₂ H ₁₆ N ₄ 0 ₄ , molecular weight: 280.29.

The present invention also relates to a method for synthesizing 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide, said method comprising 7-deaza-2'- Deoxy-7-halogen substituted guanosine to further synthesize the 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide.

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), trifluoroacetylpropargylamine and formula (I) compound Reaction, get compound dG(AP3)

B. Synthesis of compound dGTP (AP ₃ ): compound dG (AP ₃ ) and tri-n-butylamine pyrophosphate (E-4), 2-chloro-4H-1,3,2-benzodioxophosphorus-4 - Ketone (E-3) reacts in the presence of triethylamine and iodine, and the reaction product is deprotected to obtain the compound dGTP (AP ₃ ), that is, the 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide.

Preferably, in step A, the molar ratio of the compound of formula (I), trifluoroacetylpropargylamine, CuI, Pd(PPh ₃ ) ₄ and TEA is 1: (2-3): 0.072: 0.025: (1.5 ~2).

Preferably, in step B, the molar ratio of tri-n-butylamine pyrophosphate, 2-chloro-4H-1,3,2-benzodioxophosphor-4-one and dG(AP ₃ ) is 2: 2:1.

The present invention also relates to the 7-deaza-7-halogen substituted guanosine prepared by the aforementioned synthetic method in the synthesis of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide the use of.

The present invention has the following beneficial effects: the present invention synthesizes 7-deaza-7-halogen-2'-deoxyguanosine (abbreviated as dG-X); The widely used basic raw materials are currently sold at a high price, and the synthesis method is complicated and difficult to control. 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 of drawings

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

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

Fig. 2 is the synthesizing process schematic diagram of the synthetic method 2 of 7-deaza-7-iodo-2'-deoxyguanosine (dG-I for short).

3 is a schematic diagram of the synthesis process of 7-deaza-7-iodoguanosine (G-I for short).

Figure 4 is a schematic diagram of the synthesis process of 7-deaza-7-bromo/chloro-2'-deoxyguanosine (dG-G for short).

Figure 5 shows the use of 7-deaza-7-iodo-2'-deoxyguanosine dG-I in the synthesis of dGTP (AP ₃ ).

Fig. 6 is the use of 7-deaza-7-iodoguanosine GI in the synthesis of GTP (AP ₃ ).

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

Fig. 8 is ¹ H-NMR of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ ).

Fig. 9 is the ³¹ P-NMR of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ ).

Fig. 10 is the HRMS spectrum of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide dGTP (AP ₃ ).

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.;

One of the synthetic methods 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 compound dG ₁ -A (0.20g; 0.714mmol) in anhydrous pyridine, slowly add pivaloyl chloride (0.75mL; 7.14mmol) dropwise at 0°C, stir at 0°C for 1h, add 2ml of methanol, and stir for 10min , spin out the solvent, add ethyl acetate (200ml) and saturated sodium bicarbonate solution (50ml) for extraction, separate the organic phase, add saturated sodium bicarbonate solution and saturated brine successively for washing, dry over anhydrous sodium sulfate, spin out the solvent, Silica gel column chromatography [V (ethyl acetate): V (petroleum ether) = 1: 1] gave 0.39 g of white solid, compound dG ₁ -B, with a yield of 92%. ¹ HNMR (400MHz, CD ₃ OD) δ7.28(d, J=3.7Hz, 1H), 6.66(dd, J=5.9, 8.6Hz, 1H), 6.51(d, J=3.7Hz, 1H), 5.41 (m, 1H), 4.33-4.36(m, 2H), 4.22(m, 1H), 4.08(s, 3H), 2.83-2.96(m, 2H), 2.54-2.70(m, 2H), 2.48-2.54 (ddd, J=2.0, 5.9, 14.2Hz, 1H), 1.15-1.23(m, 27H).

Step two,

The compound dG1-B (0.42g; 0.84mmol) was dissolved in anhydrous DMF, under vigorous stirring, 4-iodosuccinimide (220mg; 0.9mmol) was added, stirred at room temperature for 22h, the solvent was spun out, and 100ml was added Diethyl ether and 50ml sodium bicarbonate solution extraction, separate the organic phase, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, spin off the solvent, silica gel column chromatography [V (ethyl acetate): V (petroleum ether) = 1: 1] to obtain 0.5 g of white solid, namely compound dG ₁ -C, with a yield of 91%. ¹ H-NMR (400MHz, CD ₃ OD) δ7.43(s, 1H), 6.63(dd, J=6.0, 8.2Hz, 1H), 5.41(m, 1H), 4.33-4.36(m, 2H), 4.23(m, 1H), 4.09(s, 3H), 2.78-2.94(m, 2H), 2.57-2.70(m, 2H), 2.50-2.57(ddd, J=2.3, 6.0, 14.2Hz, 1H), 1.17-1.24(m, 27H).

Step three,

Dissolve compound dG ₁ -C in 0.5M methanol/sodium methoxide (10ml), stir at 65°C for 12h, then add 10ml of saturated sodium bicarbonate solution, continue stirring for 10min, spin out methanol, add 50ml of ethyl acetate for extraction, The organic layer was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively, dried over anhydrous sodium sulfate, concentrated, and the residue was subjected to silica gel column chromatography [V (methanol): V (dichloromethane) = 1: 10] to obtain 0.24 g of white solid is compound dG ₁ -D, yield 74%. 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 four,

The compound dG ₁ -D was placed in a sodium hydroxide solution (2N) and refluxed for 4 hours. After cooling, 2N hydrochloric acid solution was added to adjust the pH of the solution to 6. Concentrate, add 100ml of dichloromethane and methanol mixture (V:V=1:1) for washing, combine the organic phases, and concentrate to obtain 255mg of white solid, namely dG-I, with a yield of 98%. The ¹ H-NMR of dG-I is shown in Figure 7, ¹ HNMR (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- The synthesis of deaza-7-bromo and chloro-2'-deoxyguanosine dG-Br/Cl, the difference is that in the second step reaction, NBS or BCS can be used instead of NIS, and all other reaction steps and The methods are the same.

Embodiment 2, the second synthetic method of 7-deaza-7-iodo-2'-deoxyguanosine dG-I

The synthesis schematic diagram of dG-I in this embodiment is shown in Figure 2, and the specific synthesis method includes the following steps respectively:

step one,

After Sm-1 (27.3g, 138mmol) was added to 70mL of water, 3.0mL of concentrated hydrochloric acid was added and stirred at 90°C for 0.5h. After cooling to room temperature, sodium acetate (13.6g, 165mmol) was added and stirred, and Sm-2 ( 20.0g, 159mmol) and sodium acetate (7.0g, 85.4mmol) were dissolved in 150mL water and added to the reaction, stirred at 80°C for 2h, then moved to zero°C and stirred for 1.5h, filtered, and washed with ice water and acetone, It was sucked dry to obtain 15.4 g, and the yield was 74%. ¹ HNMR (400MHz, DMSO): δ=10.94(s, 1H), 10.35(s, 1H), 6.58(dd, J=3.4, 2.2Hz, 1H), 6.15(dd, J=3.4, 2.1Hz, 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): δ=11.43 (s, 1H, NH), 7.07 (d, 1H, NHCHCH), 6.46 (s, 2H, NH2), 6.22 (d, 1H, CHNH).

Step three,

G006 (8.5g, 50.42mmol) was added to 120mL of anhydrous pyridine, then pivaloyl chloride (21.68mL, 176.20mmol) was added and stirred at room temperature for 2h, then the solvent was removed, dissolved in 1.7L of dichloromethane, organic phase After washing with 0.1M hydrochloric acid solution (2*350mL), spin off the solvent and perform column chromatography DCM:MeOH10:1 to obtain 8.15g, yield 64%. ¹ HNMR (400MHz, [D6]-DMSO): δ=9.98(s, 1H, NHC(0)), 7.50(d, J=3.6Hz, 1H, NHCHCH), 6.49(d, J=3.6Hz, 1H , CHNH), 1.20 (s, 9H, C(CH3)3).

Step four,

G007 (3.10g, 12.27mmol) was dissolved in 60mLTHF, protected by nitrogen, wrapped in foil paper, added NIS (3.04g, 13.51mmol) and stirred at room temperature for 1h, added 500mL of DCM, washed with 200mL of water, and after the solvent was removed, Column chromatography DCM:MeOH99:1 yielded 3.76g, yield 81%. ¹ HNMR (400MHz, DMSO): δ=12.65(s, 1H, CHNH), 10.06(s, 1H, NHC(0)), 7.73(d, J=2.4Hz, 1H, CH), 1.19(s, 9H , C(CH ₃ ) ₃ ).

Step five,

G008 (1.5g, 4.0mmol) and ammonium sulfate (15mg, 0.11mmol) were refluxed in hexamethyldisilazane (15mL, 72.76mmol) for 20h under the protection of argon, and 40mL of dichloro Ethane, add Sm-1 (2.304, 6.0mmol) and TMSOTf (1.25mL, 6.47mmol) and stir at room temperature until clear, then stir at 50°C for 24h, add 60mL DCM, and wash with 30mL saturated sodium bicarbonate and saturated saline After washing and spinning off the organic phase, 1.48 g was obtained by column chromatography with a yield of 45%. ¹ HNMR (400MHz, D6-DMSO): δ=10.29(s, 1H), 8.02(s, 1H,), 7.90-7.41(m, 10H), 6.35(s, 1H), 6.26(d, J=0.8 Hz, 1H), 4.27(s, 1H), 3.75(s, 1H), 3.49(t, J=0.8Hz, 1H), 2.35-2.28(m, 1H), 2.09-2.01(m, 1H), 1.15 (s, 9H).

Step six,

G009 (1.056g, 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:MeOH5:1 to obtain 490mg of compound dG1-D, yield 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,

The compound dG ₁ -D was placed in a sodium hydroxide solution (2N) and refluxed for 4 hours. After cooling, 2N hydrochloric acid solution was added to adjust the pH of the solution to 6. Concentrate, add 100ml of dichloromethane and methanol mixture (V:V=1:1) for washing, combine the organic phases, and concentrate to obtain 255mg of white solid, namely dG-I, with a yield of 98%. ¹ HNMR (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-deaza-7-bromo and chloro-2'-deoxy The difference in the synthesis of guanosine dG-Br/Cl is that in the fourth step, NBS or BCS can be used instead of NIS, and all other reaction steps and methods are the same.

The synthetic method of embodiment 3,7-deaza-7-iodine-guanosine G-I

The synthesizing schematic diagram of G-I in the present embodiment is as shown in Figure 3, and specific synthesizing method comprises the following steps respectively:

step one,

Reflux G008 (1.5g, 4.0mmol) and ammonium sulfate (15mg, 0.11mmol) in hexamethyldisilazane (15mL, 72.76mmol) for 20h under the protection of argon, spin off the solvent and add 40mL of dichloro Ethane, add G-I-0 (6.0mmol) and TMSOTf (1.25mL, 6.47mmol) and stir at room temperature until clear, then stir at 50°C for 24h, add 60mL DCM, and wash with 30mL saturated sodium bicarbonate and saturated brine, After spinning off the organic phase, 1.6 g of G-I-B compound was obtained by column chromatography. 1HNMR (600MHz, DMSO): δ=10.29(s, 1H), 8.02(s, 1H), 7.91-7.85(m, 6H), 7.64-7.58(m, 3H), 7.46-7.39(m, 6H), 6.48(d, J=3.9Hz, 1H), 6.41(t, J=6.1, 6.1Hz, 1H), 6.32(dd, J=6.0, 4.0Hz, 1H), 4.82(dd, J=10.7, 5.0Hz , 1H), 4.75(dd, J=11.8, 4.4Hz, 1H), 4.63(dd, J=11.8, 5.7Hz, 1H), 1.15(s, 9H).

Step two,

G-I-B (1.056g, 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:MeOH5:1 to obtain 490mg of G-I-A compound, yield 80% . 1H-NMR (DMSO-d6, 250MHz): δ3.49-3.57(m, 2H, H-C(5')), 3.80-3.82(m, 1H, H-C(4')), 3.93(s, 3H, OMe ), 4.01-4.03(m, 1H, H-C(3')), 4.23-4.28(m, 1H, H-C(2')), 5.01(t, J=5.5Hz, 1H, 0H-C(5') ), 5.05(d, J=4.4Hz, 1H, 0H-C(3')), 5.25(d, J=6.2Hz, 1H, 0H-C(2')), 5.94(d, J=6.5Hz , 1H, H-C(1')), 6.38(s, 2H, NH2), 7.31(s, 1H, H-C(6)).

Step three,

Compound G-I-A was placed in sodium hydroxide solution (2N) and refluxed for 4 h, and after cooling, 2N hydrochloric acid solution was added to adjust the pH of the solution to 6. Concentrate, add 100ml of dichloromethane and methanol mixture (V:V=1:1) for washing, combine the organic phases, and concentrate to obtain 255mg of white solid, namely G-I, with a yield of 98%. 1HNMR (600MHz, DMSO): 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 chloroguanosine The difference in the synthesis of G-Br/Cl is that the 7-position of the guanine base of the raw material G008 used in the first step reaction can be bromine or chlorine, and all other reaction steps and methods are the same.

One of the synthetic methods of embodiment 4, 7-deaza-7-bromo/chloro-2-deoxyguanosine dG-I

The synthesis schematic diagram of dG-G in this embodiment is shown in Figure 4, and the specific synthesis method includes the following steps respectively:

step one,

Compound dG1-A (0.20g; 0.714mmol) was dissolved in anhydrous pyridine, pivaloyl chloride (0.75mL; 7.14mmol) was slowly added dropwise at 0°C, stirred at 0°C for 1h, 2ml of methanol was added, and stirred for 10min, Spin out the solvent, add ethyl acetate (200ml) and saturated sodium bicarbonate solution (50ml) for extraction, separate the organic phase, add saturated sodium bicarbonate solution and saturated brine successively to wash, dry over anhydrous sodium sulfate, spin out the solvent, silica gel Column chromatography [V (ethyl acetate): V (petroleum ether) = 1: 1] gave 0.39 g of white solid, compound dG ₁ -B, with a yield of 92%. ¹ HNMR (400MHz, CD ₃ OD) δ7.28(d, J=3.7Hz, 1H), 6.66(dd, J=5.9, 8.6Hz, 1H), 6.51(d, J=3.7Hz, 1H), 5.41 (m, 1H), 4.33-4.36(m, 2H), 4.22(m, 1H), 4.08(s, 3H), 2.83-2.96(m, 2H), 2.54-2.70(m, 2H), 2.48-2.54 (ddd, J=2.0, 5.9, 14.2Hz, 1H), 1.15-1.23(m, 27H).

Step two,

Compound dG ₁ -B (0.42g; 0.84mmol) was dissolved in anhydrous DMF, under vigorous stirring, 4-bromosuccinimide or 4-chlorosuccinimide (220mg; 0.9mmol) was added , stir at room temperature for 22h, spin out the solvent, add 100ml ether and 50ml sodium bicarbonate solution for extraction, separate the organic phase, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, spin out the solvent, silica gel column chromatography [V (ethyl acetate ester): V (petroleum ether) = 1:1] to obtain 0.5 g of white solid, compound dG ₁ -E, with a yield of 91%. ¹ H-NMR (400MHz, CD ₃ OD) δ7.43(s, 1H), 6.63(dd, J=6.0, 8.2Hz, 1H), 5.41(m, 1H), 4.33-4.36(m, 2H), 4.23(m, 1H), 4.09(s, 3H), 2.78-2.94(m, 2H), 2.57-2.70(m, 2H), 2.50-2.57(ddd, J=2.3, 6.0, 14.2Hz, 1H), 1.17-1.24(m, 27H).

Step three,

Dissolve compound dG1-E in 0.5M methanol/sodium methoxide (10ml), stir at 65°C for 12h, then add 10ml of saturated sodium bicarbonate solution, continue stirring for 10min, spin out methanol, add 50ml of ethyl acetate to extract, organic The layers were washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and the residue was subjected to silica gel column chromatography [V (methanol): V (dichloromethane) = 1: 10] to obtain 0.24 g white solid is the compound dG ₁ -F, the yield is 74%. 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 four,

The compound dG ₁ -F was placed in a sodium hydroxide solution (2N) and refluxed for 4 hours. After cooling, 2N hydrochloric acid solution was added to adjust the pH of the solution to 6. Concentrate, add 100ml of dichloromethane and methanol mixture (V:V=1:1) for washing, combine the organic phases, and concentrate to obtain 255mg of white solid, namely dG ₁ -G, with a yield of 98%. ¹ HNMR (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).

Example 5, 7-deaza-7-iodo-2'-deoxyguanosine dG-I in the synthesis of dGTP (AP ₃ ) uses in

The synthesizing schematic diagram of dGTP (AP3) in the present embodiment is shown in Figure 5, 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(PPh3)4 (48mg; 0.04mmol) into the reaction flask, vacuumize, nitrogen protection, Wrap 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 the residue Dissolve in 100ml ethyl acetate, wash with saturated sodium bicarbonate solution and saturated sodium chloride solution successively, dry over anhydrous sodium sulfate, concentrate, column chromatography [V (ethyl acetate): V (n-hexane) = 1:3 ] to obtain 0.1 g of white solid, ie, dG(AP ₃ ), with a yield of 39%. 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, inject the reaction solution into 2-chloro-4H-1,3,2 - in anhydrous DMF (0.25 mL) solution of benzodioxophosphor-4-one, stirred at room temperature for half an hour. Then the mixture was injected into 2 and stirred for 1.5h. Add 1 mL of 3% iodine (9:1 Py/H20) solution, and keep the color of the iodine solution for 15 minutes without fading. After 15 min, 2 mL of water was added, and after 2 h, 0.75 mL of 3M NaCl solution and 20 mL of absolute ethanol were added, frozen at -20°C for 12 h, and centrifuged (20 min, 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(AP3) are shown in Figures 8, 9, and 10, 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); 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'-deoxyguanosine dG- The use of Br/Cl in the synthesis of dGTP (AP3) is different in 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 6, 7-deaza-7-iodine guanosine G-I is synthesizing G(AP ₃ ) uses in

The schematic diagram of the synthesis of G(AP ₃ ) in this example is shown in Figure 6, and the specific synthesis method includes the following steps:

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.

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 (4)

1. A synthetic method for 7-deaza-2'-deoxy-7-halogen substituted guanine nucleotides, characterized in that the method comprises the steps: step one, Add 0.4mmol of compound dG-I to a single-necked bottle, then weigh 1mmol of CuI and 0.04mmol of Pd(PPh ₃ ) ₄ into the reaction bottle, vacuumize, protect with nitrogen, wrap with aluminum foil, add 10ml of DMF, stir to dissolve, and inject 0.8mmol of TEA and trifluoroacetylpropargylamine 1.2mmol, stirred at 50°C for 13 hours, the reaction was completed, the solvent was spun out, the residue was dissolved in 100ml of ethyl acetate, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution successively, no Dry over sodium sulfate, concentrate, and perform column chromatography with ethyl acetate-n-hexane mixed solvent with a volume ratio of 1:3 to obtain 0.1 g of white solid, namely dG(AP ₃ ), with a yield of 39%; Step two, The compound dG(AP ₃ ) was vacuum-dried for 12 hours, and the compound dG(AP ₃ ) 0.072mmol, tri-n-butylamine pyrophosphate 0.145mmol, 2-chloro-4H-1,3,2-benzene Put 0.15mmol of phospho-4-one in three reaction tubes; dissolve tri-n-butylamine pyrophosphate in 0.25mL of anhydrous DMF, then add 0.3mL of freshly steamed tri-n-butylamine, stir at room temperature for half After 1 hour, inject the reaction solution into 0.25mL solution of 2-chloro-4H-1,3,2-benzodioxophosphor-4-one in anhydrous DMF, stir at room temperature for half an hour; then inject the mixture into dG (AP ₃ ), stirred for 1.5 h; added 1 mL of 3% iodine in Py-H ₂ O solution, kept the color of iodine solution for 15 min without fading. In the Py-H ₂ O solution, the volume ratio of Py-H ₂ O The ratio is 9:1; after 15 minutes, add 2 mL of water, after 2 hours, add 0.75 mL of 3M NaCl solution, 20 mL of absolute ethanol, freeze at -20°C for 12 hours, centrifuge at 3200 rpm for 20 minutes; pour off the supernatant, drain the solvent from the precipitate, and then add concentrated ammonia water , stirred at room temperature for 5 hours; spin out the solvent under reduced pressure, and a brown solid appeared, and carried out RP-HPLC analysis. The conditions of this step RP-HPLC are: column: C18, 5μm, 4.6×250mm; flow rate: 1mL/min; mobile phase: 20mM TEAA and EtOH, 0-20%EtOH35min, visible detector wavelength: 650nm, retention time t=18min; perform RP-HPLC separation, the conditions of this step RP-HPLC are: column: C18, 5μm, 9.4×250mm; flow rate: 4mL/min; mobile phase: 20mM TEAA and MeOH, 0-15% MeOH for 25min, UV detector wavelength: 254nm, retention time t=15min, NaCl/EtOH to remove triethylamine acetate salt, to obtain 12mg white solid namely dGTP(AP ₃ ) , yield 26%. 2. the synthetic method of 7-deaza-2'-deoxy-7-halogen substituted guanine nucleotides as claimed in claim 1, is characterized in that, the preparation specific steps of described compound dG-1 are: the following The compound of formula (V) is demethylated under basic conditions, that is, the 7-deaza-2'-deoxy-7-halogen substituted guanosine; Wherein R ₂ is 1; The compound of formula (V) is prepared by deprotecting the compound of formula (IV) under basic conditions: Wherein, R is a protecting group for protecting a hydroxyl group or an amino group; The protecting group is pivaloyl or isobutyryl; The compound of the formula (IV) is prepared by connecting a halogen to the 7-position of the purine base of the compound of the formula (III): The compound of formula (III) is prepared by protecting two hydroxyl groups and one amino group of the compound of formula (II) with a protecting group under basic conditions: 3. the synthetic method of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide as claimed in claim 1, is characterized in that, in step 1, described dG-I compound, three The molar ratio of fluoroacetylpropargylamine, CuI, Pd(PPh ₃ ) ₄ and TEA is 1:(2~3):0.072:0.025:(1.5~2). 4. the synthetic method of 7-deaza-7-propynylamine-2'-deoxyguanine nucleotide as claimed in claim 1, is characterized in that, in step 2, described tri-n-butylamine pyrophosphate The molar ratio of salt, 2-chloro-4H-1,3,2-benzodioxophosphor-4-one and dG(AP ₃ ) was 2:2:1.