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CN111909197B - Preparation method of triphosphate compound and deoxynucleotide - Google Patents

Preparation method of triphosphate compound and deoxynucleotide Download PDF

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CN111909197B
CN111909197B CN202010639823.4A CN202010639823A CN111909197B CN 111909197 B CN111909197 B CN 111909197B CN 202010639823 A CN202010639823 A CN 202010639823A CN 111909197 B CN111909197 B CN 111909197B
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triphosphate compound
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CN111909197A (en
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秦龙
杨照亮
何筠
田晖
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Anxuyuan Biotechnology Shenzhen Co ltd
Shenzhen Research Institute Tsinghua University
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Abstract

The invention discloses a preparation method of a triphosphate compound and deoxynucleotide. According to the preparation method of the triphosphate compound, tetrahydrofuran is used for replacing trimethyl phosphate/triethyl phosphate, tri-N-propylamine is used for replacing tri-N-butylamine, and acetonitrile is used for replacing N, N-dimethylformamide, so that the triphosphate compound has the advantages of high yield, few byproducts, easiness in removing a solvent, no toxicity, safety and the like. According to the deoxynucleotide method, morpholine-dimethylformamide solution is used for replacing triethylamine solution to remove the-Fmoc group, so that the reaction time is greatly shortened, the generation of byproducts is reduced, and the yield is improved.

Description

Preparation method of triphosphate compound and deoxynucleotide
Technical Field
The invention relates to a preparation method of a triphosphate compound and deoxynucleotide, belonging to the technical field of organic synthesis.
Background
Terminal deoxynucleotidyl transferase is a template-free DNA polymerase that catalyzes the binding of deoxynucleotides to the 3' hydroxyl end of DNA molecules and plays an important role in the development of immune reactions in humans and animals. 2 '-deoxynucleoside-5' -triphosphates are natural substrates for terminal deoxynucleotidyl transferases, although studies have found that the presence of nucleoside fragments does not affect the properties of triphosphates as substrates, i.e., triphosphates can be used as substrates for certain terminal deoxynucleotidyl transferases (e.g., calf thymic terminal deoxynucleotidyl transferase) in place of 2 '-deoxynucleoside-5' -triphosphates. However, the general reaction yield of the synthesis method of the triphosphoric acid compounds reported in the literature is only 7% -12%, and tri-n-butylamine, which is a highly toxic substance, is also needed, so that the synthesis method cannot be popularized and applied on a large scale.
The nucleotide is a compound consisting of purine base or pyrimidine base, ribose or deoxyribose and phosphate. The most common modification means of nucleotides is to label fluorescent dye molecules at the 5-position or modify some groups for detection at the 7-position base for various genomics including DNA labeling and sequencing, while the molecules modified at the 7-position base are inserted into the extended DNA strand after reacting with DNA polymerase, so as to change the new DNA structure. In recent years, researchers have discovered that 5-position modifications, which typically extend the 5-position phosphate linkage and throw away a functional group that readily attaches to some labeling molecule, such as-NH, can also be used for DNA sequencing when studying single molecule sequencing 2 And the like, so that the synthesis research of the compounds is crucial to single-molecule sequencing, the diversity of the compounds can increase the sample size of single-molecule sequencing substrate screening, and more possibilities are provided for optimizing the screening of sequencing substrates. However, the method for synthesizing nucleotides by triphosphate compounds reported in the literature generally has the problems of low yield of target products, more byproducts, long preparation period, difficult removal of solvents and the like, and cannot be popularized and applied on a large scale.
Therefore, it is necessary to develop a method for synthesizing a triphosphate compound in a higher yield and with higher safety, and further synthesize deoxynucleotides using the triphosphate compound.
Disclosure of Invention
The invention aims to provide a preparation method of a triphosphate compound and deoxynucleotides.
The technical scheme adopted by the invention is as follows:
a method for preparing a triphosphate compound, comprising the steps of:
1) Fluorenylmethoxycarbonylcarbonyl chloride and
Figure BDA0002570540740000011
dispersed in a solvent, X is C 2 ～C 9 Alkylene of (3), further adding Na 2 CO 3 Reacting the solution at normal temperature, extracting and crystallizing to obtain the product
Figure BDA0002570540740000021
2) Phosphorus oxychloride and
Figure BDA0002570540740000022
dispersing in tetrahydrofuran, reacting at 30-50 deg.C to obtain
Figure BDA0002570540740000023
3) Adding an acetonitrile solution of tri-n-propyl ammonium pyrophosphate and tri-n-propylamine into the reaction solution obtained in the step 2), and reacting at room temperature to obtain the intermediate
Figure BDA0002570540740000024
4) Adding triethylamine-carbonic acid buffer solution into the reaction solution obtained in the step 3), reacting at room temperature, and concentrating to obtain a crude product of the triphosphate compound;
5) Purifying the crude product of the triphosphate compound by reversed-phase high performance liquid chromatography to obtain the triphosphate compound
Figure BDA0002570540740000025
Preferably, the fluorenylmethoxycarbonyl chloride in the step 1),
Figure BDA0002570540740000026
The molar ratio of (1.1-1.5): 1.
preferably, the solvent in step 1) is at least one of 1, 4-dioxane, acetonitrile, dimethylformamide and dichloromethane.
Preferably, said Na of step 1) 2 CO 3 The mass fraction of the solution is 5-15%.
Preferably, the reaction time in step 1) is 10 to 15 hours.
Preferably, the phosphorus oxychloride in the step 2),
Figure BDA0002570540740000027
The molar ratio of (1.2-2.1): 1.
preferably, the reaction time in the step 2) is 3-5 h.
Preferably, said step 2) is
Figure BDA0002570540740000028
The molar ratio of the tri-n-propyl ammonium pyrophosphate in the step 3) is 1: (5-8).
Preferably, step 2) is as described
Figure BDA0002570540740000029
Step 3) the molar ratio of the tri-n-propylamine is 1: (5-8).
Preferably, the reaction time in step 3) is 1-2 h.
Preferably, the reaction time of the step 4) is 20-50 h.
A method for preparing deoxynucleotides, comprising the following steps:
1) Will be provided with
Figure BDA00025705407400000210
And N, N' -carbonyldiimidazole in a solvent, X is C 2 ～C 9 Reacting at room temperature, adding methanol, and reacting at room temperature to obtain
Figure BDA0002570540740000031
2) Dissolving tri-n-butyl ammonium salt of 2 '-deoxynucleoside-5' -polyphosphate in a solvent, adding the solution into the reaction solution obtained in the step 1), and adding MgCl 2 Reacting at room temperature, and rotary steaming to obtain
Figure BDA0002570540740000032
Y is one of four basic groups of A, G, C and T, and n is a natural number of 1-4;
3) Will be provided with
Figure BDA0002570540740000033
Adding the mixture into a morpholine-dimethylformamide solution, reacting at room temperature, and concentrating to obtain a deoxynucleotide crude product;
4) Purifying the crude deoxynucleotide product by reversed phase high performance liquid chromatography to obtain the deoxynucleotide
Figure BDA0002570540740000034
Preferably, step 1) is as described
Figure BDA0002570540740000035
The mol ratio of N, N' -carbonyl diimidazole to methanol is 1: (2-5): (4-7).
Preferably, the solvent in step 1) is at least one of dimethylformamide, acetonitrile and dimethyl sulfoxide.
Preferably, the time of the first reaction in the step 1) is 3 to 5 hours, and the time of the second reaction is 20 to 40min.
Preferably, step 1) is as described
Figure BDA0002570540740000036
Step 2) tri-n-butylammonium salt of the 2 '-deoxynucleoside-5' -polyphosphate, step 2) MgCl 2 In a molar ratio of 1: (1.1-1.5): (8 to 20).
Preferably, the reaction time in the step 2) is 10-20 h.
Preferably, the reaction time of the step 3) is 2-3 h.
The beneficial effects of the invention are: the preparation method of the triphosphate compound has the advantages of high yield, few byproducts, easy removal of solvent, no toxicity, safety and the like, and the method for preparing the deoxynucleotide by the triphosphate compound has the advantages of short reaction time, few byproducts, high yield and the like, and is suitable for large-scale popularization and application.
Specifically, the method comprises the following steps:
1) The yield of the preparation method of the triphosphate compound is more than 40 percent, which is about 4 times of that of the traditional method, and the yield of the preparation method of the deoxynucleotide is more than 80 percent, while the yield of the traditional method is lower than 60 percent;
2) The invention uses tetrahydrofuran to replace trimethyl phosphate/triethyl phosphate as solvent to carry out phosphorus oxychloride and
Figure BDA0002570540740000041
the reaction effectively reduces the generation of the diphosphoric acid methyl esterification by-product and finally improves the yield of the triphosphoric acid compound;
3) The invention uses tri-n-propylamine to replace tri-n-butylamine (a class of highly toxic products) and
Figure BDA0002570540740000042
the reaction is safer;
4) According to the invention, acetonitrile is used for replacing N, N-dimethylformamide to disperse tri-N-propyl ammonium pyrophosphate, and the solvent is easier to remove after the reaction is finished;
5) According to the method, the morpholine-dimethylformamide solution is used for replacing triethylamine solution to remove the-Fmoc group, so that the reaction time is greatly shortened, the generation of byproducts is reduced, and the yield of deoxynucleotide is improved.
Drawings
FIG. 1 is a liquid chromatogram of the crude triphosphate compound of step 4) of example 1.
FIG. 2 is a NMR chart of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate in example 1.
FIG. 3 is a NMR phosphorus spectrum of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate in example 1.
FIG. 4 shows dA6P-NH in example 1 2 Nuclear magnetic resonance hydrogen spectra of Nucleotides.
FIG. 5 shows dA6P-NH in example 1 2 Nuclear magnetic resonance phosphograms of Nucleotides.
FIG. 6 shows dA4P-NH in example 2 2 Nuclear magnetic resonance hydrogen spectra of nucleosides.
FIG. 7 shows dA4P-NH in example 2 2 Nuclear magnetic resonance phosphograms of Nucleotides.
FIG. 8 shows dT4P-NH in example 3 2 Nuclear magnetic resonance hydrogen spectra of Nucleotides.
FIG. 9 shows dT4P-NH in example 3 2 Nuclear magnetic resonance phosphograms of Nucleotides.
FIG. 10 is a NMR hydrogen spectrum of N- (9-fluorenylmethoxycarbonyl) -2-aminoethyl triphosphate in example 4.
FIG. 11 is a NMR phosphogram of N- (9-fluorenylmethoxycarbonyl) -2-aminoethyl triphosphate in example 4.
FIG. 12 is a liquid chromatogram of the crude triphosphate compound in comparative example step 4).
Detailed Description
The invention will be further explained and illustrated with reference to specific examples.
Example 1:
synthesis of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate:
1) 5.3g of fluorenylmethoxycarbonylcarbonyl chloride was dissolved in 40mL of 1, 4-dioxane, 2g of 6-amino-1-hexanol was added at 0 ℃ to dissolve sufficiently, and 20mL of 10% Na was slowly added 2 CO 3 The solution is reacted at 25 ℃ for 12h, the product is extracted with methanol-dichloromethane (methanol, dichloromethane volume ratio 1: 19), the organic phases are combined, the solvent is spun dry on a rotary evaporator, and slurried with a mixed solvent (consisting of dichloromethane, ethyl acetate and petroleum ether in a volume ratio of 1
Figure BDA0002570540740000051
(yield: 98%);
2) Mixing 1g of
Figure BDA0002570540740000052
Adding 10mL of anhydrous acetonitrile, evaporating to dryness under reduced pressure by rotary evaporator, repeating the above steps to remove water, dissolving 0.9034g of phosphorus oxychloride in 10mL of tetrahydrofuran, and dissolving at 35 deg.C
Figure BDA0002570540740000053
Adding in batches, stirring for 4h to obtain
Figure BDA0002570540740000054
3) Adding tri-n-propyl ammonium pyrophosphate and tri-n-propylamine into the reaction solution obtained in the step 2), and reacting for 1h at 25 ℃ to obtainTo
Figure BDA0002570540740000055
4) Adding 50mL of 0.1mol/L triethylamine-carbonic acid buffer solution into the reaction solution obtained in the step 3), adjusting the pH value to about 7.5, reacting for 48 hours at 25 ℃, and concentrating to obtain a crude triphosphate compound;
5) The crude triphosphate compound was purified by reverse phase high performance liquid chromatography to give N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate (yield: 42%);
the specific operation of purifying the crude triphosphate compound by reverse phase high performance liquid chromatography is as follows:
a) Dissolving the crude product of the triphosphate compound in methanol, and filtering by using a filter membrane;
b) Purifying the filtrate by reversed phase high performance liquid chromatography with column specification C 18 (Innoval ODS-2X 250mm,5 μm), mobile phase A was 0.1mol/L triethylamine-carbonate buffer, mobile phase B was methanol, column chromatography was performed with mobile phase B: after 62% equilibration, the mixture was separated with mobile phase B: gradient elution of 62-75%, collecting purified compound and freeze drying.
And (3) performance testing:
the liquid chromatogram of the crude triphosphate compound in step 4) is shown in FIG. 1.
The NMR hydrogen spectrum of the N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate is shown in figure 2, the NMR phosphorus spectrum is shown in figure 3, and the spectrum analysis is as follows:
1 H NMR(D 2 O，400MHz)：δ＝7.47(s,2H),7.30(s,2H),7.14-7.08(m,4H),4.08(s,2H),3.81-3.73(m,3H),2.71(s,2H),1.37-0.71(m,8H)。
31 P NMR(D 2 O)：δ＝-11.0(m,2P),-23.6(t,1P)。
the structural formula of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate is as follows:
Figure BDA0002570540740000061
dA6P-NH 2 -synthesis of Nucleotides:
1) Adding 0.1g of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate into 10mL of anhydrous acetonitrile, evaporating to dryness under reduced pressure by using a rotary evaporator, repeating the operation again to remove water in the compound, dissolving N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate into 3mL of dimethylformamide, adding 0.7mmol of N, N' -carbonyldiimidazole, stirring for 4h at the temperature of 25 ℃, adding 1.05mmol of methanol, and continuing to stir for 30min to obtain the N-aminohexyl triphosphate
Figure BDA0002570540740000062
2) 0.193mmol of tri-n-butylammonium salt of 2 '-deoxyadenosine-5' -triphosphate was dissolved in 5mL of dimethylformamide, and added to the reaction solution of step 1), and 1.75mmol of MgCl was further added 2 Stirring at 25 deg.C for 18h, and rotary steaming to obtain
Figure BDA0002570540740000063
3) Will be provided with
Figure BDA0002570540740000064
Adding the mixture into a morpholine-dimethylformamide solution (the volume ratio of morpholine to dimethylformamide is 3);
4) Preliminary separation and purification are carried out on the crude deoxynucleotide product by medium-pressure preparative chromatography, the peak position of the product is about 2min, and the gradient condition is as follows: 0-100 min, further purifying the product after the preliminary separation and purification by reversed-phase high performance liquid chromatography, wherein the mobile phase uses 0.1mol/L triethylamine-carbonic acid buffer solution/acetonitrile, the prepared column filler is reversed-phase C 18 The preparation gradient is 3-15 percent, the peak position of the product is about 20min, and the deoxynucleotide dA6P-NH is obtained 2 Nucleotides (yield: 83%).
And (4) performance testing:
dA6P-NH 2 nuclear magnetic resonance hydrogen spectra of Nucleotides are shown in fig. 4, nuclear magnetic resonance phosphorus spectra are shown in fig. 5, and the solution spectrum analysis is as follows:
1 H NMR(D 2 O,400MHz)：δ＝8.37(s,1H),8.12(s,H),6.39(t,H),4.17-4.44–4.00(m,3H),3.85(t,2H),2.69(m,H),2.46(m,H),1.53(m,4H),1.53(m,H),1.29(m,4H)。
31 P NMR(D 2 O)：δ＝-10.91(bs,P),-10.45(bs,P),-23.35(bm,4P)。
dA6P-NH 2 -structural formula of Nucleotides:
Figure BDA0002570540740000071
example 2:
dA4P-NH 2 -synthesis of Nucleotides:
1) Adding 0.1g of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate into 10mL of anhydrous acetonitrile, evaporating to dryness under reduced pressure by using a rotary evaporator, repeating the operation again to remove water in the compound, dissolving N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate into 3mL of dimethylformamide, adding 0.7mmol of N, N' -carbonyldiimidazole, stirring for 4h at the temperature of 25 ℃, adding 1.05mmol of methanol, and continuing to stir for 30min to obtain the N-aminohexyl triphosphate
Figure BDA0002570540740000072
2) Dissolving 0.193mmol of tri-n-butylammonium salt of 2 '-deoxyadenosine-5' -monophosphate in 5mL of dimethylformamide, adding the solution to the reaction solution in step 1), and adding 1.75mmol of MgCl 2 Stirring at 25 deg.C for 18h, and rotary steaming to obtain
Figure BDA0002570540740000081
3) Will be provided with
Figure BDA0002570540740000082
Adding morpholine-dimethylformamide solution (the volume ratio of morpholine to dimethylformamide is 3;
4) Deoxyribonucleotides by medium pressure preparative chromatographyThe crude product is primarily separated and purified (operation is the same as example 1), and the primarily separated and purified product is further purified by reversed-phase high performance liquid chromatography (operation is the same as example 1), so as to obtain deoxynucleotide dA4P-NH 2 Nucleotides (yield: 85%).
And (3) performance testing:
dA4P-NH 2 nuclear magnetic resonance hydrogen spectra of Nucleotides are shown in fig. 6, nuclear magnetic resonance phosphorus spectra are shown in fig. 7, and the solution spectrum analysis is as follows:
1 H NMR(D 2 O,400MHz)：δ＝8.36(s,1H),8.11(s,H),6.39(t,H),4.21-4.08(m,3H),3.85(m,2H),2.88(m,2H),2.73(m,H),2.51(m,H),1.53(m,4H),1.53(m,H),1.29(m,4H)。
31 P NMR(D 2 O)：δ＝-10.84(bs,P),-11.49(bs,P),-21.98(bm,2P)。
dA4P-NH 2 -structural formula of Nucleotides:
Figure BDA0002570540740000083
example 3:
dT4P-NH 2 -synthesis of Nucleotides:
1) Adding 0.1g of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate into 10mL of anhydrous acetonitrile, evaporating to dryness under reduced pressure by using a rotary evaporator, repeating the operation again to remove water in the compound, dissolving the N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate into 3mL of dimethylformamide, adding 0.7mmol of N, N' -carbonyldiimidazole, stirring for 4 hours at the temperature of 25 ℃, adding 1.05mmol of methanol, and continuously stirring for 30 minutes to obtain the compound
Figure BDA0002570540740000091
2) Dissolving 0.193mmol of tri-n-butylammonium salt of 2 '-deoxythymidine-5' -monophosphate in 5mL of dimethylformamide, adding the solution to the reaction solution in step 1), and adding 1.75mmol of MgCl 2 Stirring at 25 deg.C for 18h, and rotary steaming to obtain
Figure BDA0002570540740000092
3) Will be provided with
Figure BDA0002570540740000093
Adding morpholine-dimethylformamide solution (the volume ratio of morpholine to dimethylformamide is 3;
4) The crude deoxynucleotide is primarily separated and purified by medium-pressure preparative chromatography (the operation is the same as the example 1), and the primarily separated and purified product is further purified by reversed-phase high-performance liquid chromatography (the operation is the same as the example 1) to obtain the deoxynucleotide dT4P-NH 2 Nucleosides (yield: 80%).
And (4) performance testing:
dT4P-NH 2 nuclear magnetic resonance hydrogen spectra of Nucleotides are shown in fig. 8, nuclear magnetic resonance phosphorus spectra are shown in fig. 9, and the solution spectrum analysis is as follows:
1 H NMR(D 2 O,400MHz)：δ＝7.65(s,1H),6.29(t,H),4.56(t,H),4.15(m,3H),3.93(m,2H),2.94(m,2H),2.30(m,2H),1.86(s，3H),1.61(m,4H),1.36(m,4H)。
31 P NMR(D 2 O)：δ＝-10.79(bs,P),-11.51(bs,P),-21.92(bm,2P)。
dT4P-NH 2 -structural formula of Nucleotides:
Figure BDA0002570540740000101
example 4:
synthesis of N- (9-fluorenylmethoxycarbonyl) -2-aminoethyl triphosphate:
1) Dissolving 5.1g of fluorenylmethoxycarbonylcarbonyl chloride in 40mL of 1, 4-dioxane, adding 1g of 2-hydroxyethylamine at 0 ℃, fully dissolving, and slowly adding 20mL of 10% Na with concentration 2 CO 3 The solution was reacted at 25 ℃ for 12h, the product was extracted with methanol-dichloromethane (methanol, dichloromethane volume ratio 1Methyl chloride, ethyl acetate and petroleum ether are pulped according to the volume ratio of 1
Figure BDA0002570540740000102
(yield: 98%);
2) Mixing 1g of
Figure BDA0002570540740000103
Adding 10mL of anhydrous acetonitrile, evaporating to dryness under reduced pressure by using a rotary evaporator, repeating the operation again to remove water in the compound, dissolving 0.9724g of phosphorus oxychloride in 10mL of tetrahydrofuran, and dissolving the solution at 35 DEG C
Figure BDA0002570540740000104
Adding in batches, stirring for 4h to obtain
Figure BDA0002570540740000105
3) Adding tri-n-propyl ammonium pyrophosphate and tri-n-propylamine into the reaction solution obtained in the step 2), and reacting for 1h at 25 ℃ to obtain
Figure BDA0002570540740000106
4) Adding 50mL of 0.1mol/L triethylamine-carbonic acid buffer solution into the reaction solution obtained in the step 3), adjusting the pH value to about 7.5, reacting for 48 hours at 25 ℃, and concentrating to obtain a crude product of the triphosphate compound;
5) The crude triphosphate compound was purified by reverse phase high performance liquid chromatography to give N- (9-fluorenylmethoxycarbonyl) -2-aminoethyl triphosphate (yield: 41%);
the specific operation of purifying the crude triphosphate compound by reverse phase high performance liquid chromatography is as follows:
a) Dissolving the crude product of the triphosphate compound in methanol, and filtering by using a filter membrane;
b) Purifying the filtrate by reversed phase high performance liquid chromatography with column specification of C 18 (Innoval ODS-2X 250mm,5 μm), mobile phase A was 0.1mol/L triethylamine-carbonate buffer, mobile phase B was methanol, column chromatography was performed with mobile phase B:62After% equilibration, with mobile phase B: gradient elution is carried out by 62 percent to 75 percent, and the purified compound is collected and freeze-dried.
And (3) performance testing:
the NMR hydrogen spectrum of N- (9-fluorenylmethoxycarbonyl) -2-aminoethyl triphosphate is shown in FIG. 10, the NMR phosphorus spectrum is shown in FIG. 11, and the spectrum analysis is as follows:
1 H NMR(D 2 O，400MHz)：δ＝7.82(d,J＝7.5Hz,2H),7.62(d,J＝7.4Hz,2H),7.41(m,2H),7.38-7.31(m,2H),4.39(d,J＝6.0Hz,2H),4.22(d,J＝5.6Hz,1H),3.89(d,J＝5.7Hz,2H),3.26(d,J＝5.8Hz,2H)。
31 P NMR(D 2 O)：δ＝-9.88(m,1P),-10.97(m,1P),-23.06(t,1P)。
the structural formula of the N- (9-fluorenylmethoxycarbonyl) -2-aminoethyl triphosphate is as follows:
Figure BDA0002570540740000111
comparative example:
synthesis of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate:
1) 5.3g of fluorenylmethoxycarbonylcarbonyl chloride was dissolved in 40mL of 1, 4-dioxane, 2g of 6-amino-1-hexanol was added at 0 ℃ to dissolve sufficiently, and 20mL of 10% Na was slowly added 2 CO 3 The solution was reacted at 25 ℃ for 12h, the product was extracted with methanol-dichloromethane (methanol, dichloromethane volume ratio 1
Figure BDA0002570540740000112
2) Mixing 1g of
Figure BDA0002570540740000113
Adding 10mL anhydrous acetonitrile, evaporating to dryness under reduced pressure with rotary evaporator, repeating the operation again to remove water from the compound, and adding 0.9034gDissolving phosphorus oxychloride in 10mL trimethyl phosphate, and reacting at 35 deg.C
Figure BDA0002570540740000114
Adding in portions, stirring for 4h to obtain
Figure BDA0002570540740000115
3) Adding tri-n-propyl ammonium pyrophosphate and tri-n-propylamine into the reaction solution obtained in the step 2), and reacting for 1h at 25 ℃ to obtain
Figure BDA0002570540740000121
4) Adding 50mL of 0.1mol/L triethylamine-carbonic acid buffer solution into the reaction solution obtained in the step 3), adjusting the pH value to about 7.5, reacting for 48 hours at 25 ℃, and concentrating to obtain a crude triphosphate compound;
5) The crude triphosphate compound was purified by reverse phase high performance liquid chromatography (the same procedure as in example 1) to give N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate (yield: 29%).
And (4) performance testing:
the liquid chromatogram of the crude triphosphate compound in step 4) is shown in FIG. 12.
As can be seen from fig. 1: in the step 2), tetrahydrofuran is used as a solvent, N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphoric acid is used for 9.15min in a liquid chromatogram, and a phosphoric acid by-product is used for 8.90 min.
As can be seen from fig. 12: in the step 2), trimethyl phosphate is taken as a solvent, N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphoric acid is obtained at 9.13min, a pentaphosphoric acid by-product is obtained at 8.87min, and dimethyl diphosphate is obtained at 11.1min in a liquid chromatogram.
Therefore, the tetrahydrofuran used as the solvent in the step 2) can obviously reduce the impurity content of the prepared triphosphoric acid product, and further can improve the yield of the triphosphoric acid product.
dA6P-NH 2 -synthesis of Nucleotides:
1) 0.1g of N- (9-fluorenylmethoxycarbonyl) -6-aminohexyl triphosphate was added to 10mL of anhydrous acetonitrileEvaporating under reduced pressure, repeating the operation again to remove water, dissolving N- (9-fluorenylmethoxycarbonyl) -6-aminohexyltriphosphoric acid in 3mL of dimethylformamide, adding 0.7mmol of N, N' -carbonyldiimidazole, stirring at 25 deg.C for 4h, adding 1.05mmol of methanol, and stirring for 30min to obtain the final product
Figure BDA0002570540740000122
2) Dissolving 0.193mmol of tri-n-butylammonium salt of 2 '-deoxyadenosine-5' -triphosphate in 5mL of dimethylformamide, adding the resulting solution to the reaction solution in step 1), and adding 1.75mmol of MgCl 2 Stirring at 25 deg.C for 18h, and rotary steaming to obtain
Figure BDA0002570540740000131
3) Will be provided with
Figure BDA0002570540740000132
Adding the mixture into triethylamine solution with the mass fraction of 10%, stirring for 18 hours at 25 ℃, and concentrating to obtain a deoxynucleotide crude product;
4) The crude deoxynucleotide is primarily separated and purified by medium-pressure preparative chromatography (the operation is the same as that in example 1), and the primarily separated and purified product is further purified by reversed-phase high performance liquid chromatography (the operation is the same as that in example 1), so that the deoxynucleotide dA6P-NH is obtained 2 Nucleotides (yield: 55%).
Therefore, the morpholine-dimethylformamide solution is adopted to replace the triethylamine solution in the step 3), so that the reaction time can be greatly shortened, and the product yield can be greatly improved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims (10)

1. A method for producing a triphosphate compound, characterized by: the method comprises the following steps:
1) Fluorenylmethoxycarbonylcarbonyl chloride and
Figure FDA0002570540730000011
dispersed in a solvent, X is C 2 ～C 9 Alkylene of (3), further adding Na 2 CO 3 Reacting the solution at normal temperature, extracting and crystallizing to obtain the product
Figure FDA0002570540730000012
2) Phosphorus oxychloride and
Figure FDA0002570540730000013
dispersing in tetrahydrofuran, reacting at 30-50 deg.C to obtain
Figure FDA0002570540730000014
3) Adding an acetonitrile solution of tri-n-propyl ammonium pyrophosphate and tri-n-propylamine into the reaction solution obtained in the step 2), and reacting at room temperature to obtain the intermediate
Figure FDA0002570540730000015
4) Adding triethylamine-carbonic acid buffer solution into the reaction solution obtained in the step 3), reacting at room temperature, and concentrating to obtain a crude product of the triphosphate compound;
5) Purifying the crude product of the triphosphate compound by reversed-phase high performance liquid chromatography to obtain the triphosphate compound
Figure FDA0002570540730000016
2. The method for producing a triphosphate compound according to claim 1, characterized in that: step 1) the fluorenylmethoxycarbonyl chloride,
Figure FDA0002570540730000017
The molar ratio of (1.1-1.5):1。
3. the method for producing a triphosphate compound according to claim 1 or 2, characterized in that: the solvent in the step 1) is at least one of 1, 4-dioxane, acetonitrile, dimethylformamide and dichloromethane.
4. The method for producing a triphosphate compound according to claim 1, characterized in that: step 2) the phosphorus oxychloride,
Figure FDA0002570540730000018
The molar ratio of (1.2-2.1): 1.
5. the method for producing a triphosphate compound according to claim 1 or 4, characterized by: step 2) the
Figure FDA0002570540730000019
The molar ratio of the tri-n-propyl ammonium pyrophosphate in the step 3) is 1: (5-8).
6. The method for producing a triphosphate compound according to claim 1 or 4, characterized by: step 2) the
Figure FDA00025705407300000110
Step 3) the molar ratio of the tri-n-propylamine is 1: (5-8).
7. A method for preparing deoxynucleotides, which is characterized by comprising the following steps: the method comprises the following steps:
1) Will be provided with
Figure FDA0002570540730000021
And N, N' -carbonyldiimidazole in a solvent, X is C 2 ～C 9 The alkylene of (A) is reacted at room temperature, then methanol is added for reaction at room temperature, and the obtained product is obtained
Figure FDA0002570540730000022
2) Dissolving tri-n-butyl ammonium salt of 2 '-deoxynucleoside-5' -polyphosphate in a solvent, adding the solution into the reaction solution obtained in the step 1), and adding MgCl 2 Reacting at room temperature, and rotary steaming to obtain
Figure FDA0002570540730000023
Y is one of four basic groups of A, G, C and T, and n is a natural number of 1-4;
3) Will be provided with
Figure FDA0002570540730000024
Adding the mixture into a morpholine-dimethylformamide solution, reacting at room temperature, and concentrating to obtain a deoxynucleotide crude product;
4) Purifying the crude deoxynucleotide product by reversed phase high performance liquid chromatography to obtain the deoxynucleotide
Figure FDA0002570540730000025
8. The method for producing deoxynucleotides according to claim 7, wherein: step 1) of
Figure FDA0002570540730000026
The molar ratio of N, N' -carbonyl diimidazole to methanol is 1: (2-5): (4-7).
9. The method for producing deoxynucleotides according to claim 7 or 8, characterized in that: the solvent in the step 1) is at least one of dimethylformamide, acetonitrile and dimethyl sulfoxide.
10. The method for producing deoxynucleotides according to claim 7 or 8, characterized in that: step 1) the
Figure FDA0002570540730000027
Step 2) the 2 '-deoxynucleoside-5' -polyphosphateTri-n-butylammonium salt of ester, the MgCl of step 2) 2 In a molar ratio of 1: (1.1-1.5): (8 to 20).
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