CN113461743A - 6-methylthioethyl purine-2' -deoxynucleoside and preparation method and application thereof - Google Patents

6-methylthioethyl purine-2' -deoxynucleoside and preparation method and application thereof Download PDF

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CN113461743A
CN113461743A CN202110777242.1A CN202110777242A CN113461743A CN 113461743 A CN113461743 A CN 113461743A CN 202110777242 A CN202110777242 A CN 202110777242A CN 113461743 A CN113461743 A CN 113461743A
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deoxynucleoside
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CN113461743B (en
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孙亚伟
马星光
许永娣
王栋
李慧
王继乾
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China University of Petroleum East China
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Abstract

The invention discloses 6-methylthioethyl purine-2' -deoxynucleoside and a preparation method and application thereof, belonging to the technical field of organic synthesis. In the process of DNA synthesis, methylthioethyl in 6-methylthioethyl purine-2' -deoxynucleoside can be converted into alkenyl, and has crosslinkable property, thereby playing an important role in preparation of functional nucleic acid-based materials, preparation of nucleic acid drugs, DNA encryption and other applications. The preparation method has the advantages of simple route, convenient operation, simple purification mode of the intermediate and the product, no need of column chromatography purification, realization of high-efficiency mass preparation of the target product by using a simple crystallization mode, safer and more reliable synthesis process, and environmental protection.

Description

6-methylthioethyl purine-2' -deoxynucleoside and preparation method and application thereof
Technical Field
The invention belongs to the technical field of organic synthesis, and particularly relates to 6-methylthioethyl purine-2' -deoxynucleoside and a preparation method and application thereof.
Background
The concept of crosslinkable nucleosides is proposed in the last 90 th century, vinyl groups are introduced on bases of nucleosides or deoxynucleosides, michael addition is carried out on the vinyl groups and amino groups on the bases in a complementary sequence of nucleic acid to carry out interchain crosslinking reaction, and site-directed mutagenesis of genes is realized.
In recent years, the market demand of crosslinkable nucleosides and related derivatives is gradually increased, so that the process improvement and optimization of the original synthetic route and the development of a new synthetic process have very important practical significance. The prior reported synthesis method of the cross-linkable nucleoside is realized by taking deoxyguanosine as a raw material and carrying out multi-step reaction (J.Am.chem.Soc.1999,121,6753-6754), in the document, technical personnel adopt deoxyguanosine of which the 3 'end and the 5' end are protected by tert-butyldimethylsilane as a starting material for reaction, the CAS number of the deoxyguanosine of which the 3 'end and the 5' end are protected by the tert-butyldimethylsilane is 51549-35-0, the compound is not sold in the international and domestic markets at present, and the compound is prepared by the deoxyguanosine and tert-butyldimethylsilane under the catalysis of organic base. Deoxyguanosine is expensive and poorly soluble in various solvents, so that the operability when using this nucleoside is poor and the reaction steps are complicated.
Disclosure of Invention
The invention provides a preparation method of 6-methylthioethyl purine-2' -deoxynucleoside, and the synthetic route is as follows:
Figure BDA0003156058400000011
wherein, the reaction conditions in the synthesis process are as follows: a. sodium bis (trimethylsilyl) amide, 2-amino-6-chloro-purine, tetrahydrofuran, room temperature; b. tetrakis (triphenylphosphine) palladium, lithium chloride, ethyl acetate and tributyltin ethylene are heated and refluxed; c. acetonitrile, 20% sodium methyl mercaptide aqueous solution, room temperature; d. dichloromethane, ammonia in methanol, 40-100 ℃.
The above-mentioned production method is carried out by reacting 1-chloro-2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose (Compound 1, CAS No.: 21740-23-8) as a starting material with 2-amino-6-chloro-purine to give 1- (2-amino-6-chloropurine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose (Compound 2), then converting the chlorine atom at the 6-position of purine into vinyl group by Stille reaction to give 1- (2-amino-6-vinylpurine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose (Compound 3), then reducing the vinyl group into methyl ethyl thioether to generate 1- (2-amino-6-thiomethyl ethyl purine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose (compound 4), and finally removing the 4-methylbenzoyl groups at the 3-position and the 5-position to obtain the 6-methylthioethyl purine-2' -deoxynucleoside (compound 5, namely the compound shown in the formula I).
On the basis of the scheme, the preparation method of the 6-methylthioethyl purine-2' -deoxynucleoside comprises the following specific steps:
(1) reacting sodium bis (trimethylsilyl) amide with 2-amino-6-chloro-purine in an ether solvent for 1-12h, adding a tetrahydrofuran solution of 1-chloro-2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose (compound 1) into the reaction system, stirring for reacting for 1-12h, quenching the reaction, and separating to obtain an off-white crystal (compound 2);
(2) dispersing the grey white crystal, the palladium tetrakis (triphenylphosphine) and the lithium chloride in the step (1) in ethyl acetate, stirring for 1-120min, adding tributyl tin ethylene, reacting, heating and refluxing for 1-24h, and separating to obtain a crude product of a compound 3 after the reaction is finished;
(3) dissolving the crude product prepared in the step (2) in acetonitrile, adding 20% sodium methyl mercaptide aqueous solution, reacting for 1-24h, and separating after the reaction is finished to obtain a crude product of a compound 4;
(4) and (3) dissolving the crude product prepared in the step (3) in a mixed solution of dichloromethane and 5mol/L ammonia methanol solution, stirring and reacting for 1-24h at 40-100 ℃, and separating to obtain the 6-methylthioethyl purine-2' -deoxynucleoside (compound 5) after the reaction is finished.
On the basis of the scheme, the ether solvent in the step (1) is selected from one of tetrahydrofuran, isopropyl ether, anisole, 1, 4-dioxane and methyl tert-butyl ether.
On the basis of the scheme, the separation process of the compound 2 in the step (1) comprises the steps of adding water and a saturated ammonium chloride solution into a reaction system to quench the reaction, removing a solvent in the reaction system, then adding ethyl acetate and water into the reaction system, separating an organic phase, concentrating, and crystallizing a concentrated crude product in a mixed solvent of ethyl acetate and n-hexane with a volume ratio of 1:3 to obtain an off-white crystal.
On the basis of the scheme, the separation process of the crude product of the compound 3 in the step (2) is that a saturated sodium fluoride aqueous solution is added into the system and is continuously stirred for 1-24 hours, insoluble substances are removed by suction filtration, a water phase is separated, the organic phase is washed by saturated saline solution to obtain the crude product, and the crude product is directly subjected to the next reaction without purification.
On the basis of the above scheme, the crude product of the compound 4 in the step (3) is separated by removing acetonitrile, adding ethyl acetate and water to the reaction system, separating the organic phase, washing the organic phase with saturated saline solution, and drying with anhydrous sodium sulfate to obtain the crude product.
On the basis of the scheme, the separation process of the compound 5 in the step (4) comprises the steps of dispersing the system after the reaction is finished in ethyl acetate, carrying out ultrasonic treatment for 1-3h, carrying out suction filtration to obtain a crude product, and crystallizing the crude product in isopropyl ether to obtain the 6-methylthioethyl purine-2' -deoxynucleoside.
The 6-methylthioethyl purine-2' -deoxynucleoside (compound 5) prepared by the method has a structure shown in formula I:
Figure BDA0003156058400000031
the 6-methylthioethyl purine-2' -deoxynucleoside can be prepared into a phosphoramidite unit shown in a structure of a formula II by protecting hydroxyl (3 site and 5 site) and amino, so that the phosphoramidite unit is applied to a DNA synthesis process to prepare cross-linked DNA; the methylthioethyl group can be converted into an alkenyl group and has a crosslinkable property, so that the methylthioethyl group plays an important role in the preparation of functional nucleic acid-based materials, the preparation of nucleic acid medicaments, DNA encryption and other applications.
Figure BDA0003156058400000032
The invention has the beneficial effects that:
1-chloro-2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose is used as a raw material to prepare the crosslinkable nucleoside, the cost is low, and the price of the raw material is far lower than that of deoxyguanosine; in the reaction process, when a double bond is introduced on a purine ring, the using amount of the organic tin reagent is lower and even far lower than that in the prior art (Tetrahedron, Vol.53, No.9, pp.3035-3044,1997: 3040, lines 33-36), the synthesis cost is greatly reduced, and the organic tin reagent has higher toxicity, so that the synthesis process is safer and more reliable, green and environment-friendly;
compared with the preparation method of the crosslinkable purine deoxynucleoside analogue in the prior art, the preparation method has the advantages that the reaction steps are only four steps, the route of the method is simple, the operation is convenient and fast, the purification mode of the intermediate and the product is simple, column chromatography purification is not needed, and the target product can be efficiently prepared in a large amount by using a simple crystallization mode; therefore, the invention provides a method with controllable cost, simplicity and high efficiency for synthesizing the crosslinkable purine deoxynucleoside analogue (6-methylthioethyl purine-2' -deoxynucleoside).
Drawings
FIG. 1 is a NMR spectrum of Compound 2;
FIG. 2 is a mass spectrum of Compound 2;
FIG. 3 is a NMR spectrum of Compound 3;
FIG. 4 is a mass spectrum of Compound 3;
FIG. 5 is a NMR spectrum of Compound 4;
FIG. 6 is a mass spectrum of Compound 4;
FIG. 7 is a NMR spectrum of Compound 5;
FIG. 8 is a mass spectrum of Compound 5.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
The 6-methylthioethyl purine-2' -deoxynucleoside is synthesized by taking 1-chloro-2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose as a raw material, and the synthetic route is shown as follows:
Figure BDA0003156058400000041
wherein, the reaction conditions in the synthesis process are shown as follows: a. sodium bis (trimethylsilyl) amide, 2-amino-6-chloro-purine, tetrahydrofuran; b. tetrakis (triphenylphosphine) palladium, lithium chloride, ethyl acetate, tributyltin ethylene; c. acetonitrile, 20% sodium methyl mercaptide aqueous solution; d. dichloromethane, ammonia in methanol, 40-100 ℃.
The specific synthetic process is as follows:
(1) 60mL (2M) of sodium bis (trimethylsilyl) amide was added to 100mL of tetrahydrofuran, and 16.9g (100mmol) of 2-amino-6-chloro-purine was added under stirring to react for 8 hours; dissolving 44.6g (120mmol) of 1-chloro-2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose (compound 1) in 200mL of tetrahydrofuran, adding the solution into the reaction system, and stirring for reacting for 6 hours; adding 10mL of water and 5mL of saturated ammonium chloride solution into the reaction solution to quench the reaction; after the solvent was removed by rotary evaporation, 400mL of ethyl acetate and 200mL of water were added to the reaction system, the organic phase was separated and concentrated, and the concentrated crude product was crystallized from a mixed solvent of 100mL of ethyl acetate and 300mL of n-hexane to obtain 39.4g of off-white crystals (Compound 2) in a yield of 78%.
(2) 10.1g (200mmol) of off-white crystals, 78mg (0.07mmol) of tetrakis (triphenylphosphine) palladium and 2.7g (64mmol) of lithium chloride are dispersed in 300mL of ethyl acetate, stirred for 45min, 100mL of tributylstannylene are added, and the reaction is refluxed at elevated temperature for 18 h. After the reaction was completed, 100mL of a saturated aqueous solution of sodium fluoride was added to the system and the mixture was further stirred for 2 hours, then insoluble matter was removed by suction filtration, the aqueous phase was separated, and the organic phase was washed with saturated brine to obtain a crude product (containing Compound 3), which was used in the next reaction.
(3) The crude product prepared in step (2) was dissolved in 200mL of anhydrous acetonitrile, then 30mL of 20% aqueous solution of sodium methanethiol was added, reacted for 2 hours, the acetonitrile solvent was removed by rotary evaporation, then 500mL of ethyl acetate and 500mL of water were added to the system, the organic phase was separated, and the organic phase was washed with 500mL of saturated brine and then dried over anhydrous sodium sulfate to obtain a crude product (containing compound 4).
(4) Dissolving the crude product prepared in the step (3) in 10mL of dichloromethane and 4mL of methanolic ammonia, then placing the system in a pressure-sealed tube, stirring and reacting at 60 ℃ for 4h, then dispersing the reaction system in 20mL of ethyl acetate, carrying out 50mW ultrasonic treatment for 1h, carrying out suction filtration to obtain a crude product, and placing the crude product in 60mL of isopropyl ether for crystallization to obtain 6-methylthioethyl purine-2' -deoxynucleoside (compound 5) with the yield of 72%.
Identification data for compounds 1-5, as follows:
compound 1
Figure BDA0003156058400000051
Purchased from biotech ltd, ribo, beijing.
Compound 2
Figure BDA0003156058400000052
Fomular C26H24ClN5O5,calc 521.1460,found 522.1539[M+H]+,544.1358[M+Na]+；1H NMR(DMSO-d6，400MHz)，δ：2.33(s，CH3，3H)；2.36(s，CH3，3H)；2.69～2.73(m，ArH，1H)；3.17～3.22(m，ArH，1H)；4.49～4.53(m，CH2，2H)；4.58～4.62(m，ArH，1H)；5.71～5.72(t，ArH，1H)；6.36～6.38(q，ArH，1H)；6.99(s，CH2，2H)；7.25～7.27(d，ArH，2H)；7.32～7.33(d，ArH，2H)；7.79～7.81(d，ArH，2H)；7.89～7.90(d，ArH，2H)；8.32(s，ArH，1H)。
The NMR spectrum of Compound 2 is shown in FIG. 1, and the mass spectrum is shown in FIG. 2.
Compound 3
Figure BDA0003156058400000061
Fomular C28H27N5O5,calc 513.2007,found 514.2085[M+H]+,536.1904[M+Na]+；1H NMR(DMSO-d6，400MHz)，δ：2.33(s，CH3，3H)；2.37(s，CH3，3H)；2.46～2.47(t，ArH，1H)；2.68～2.72(m，ArH，1H)；3.19～3.24(m，ArH，1H)；5.71～5.72(q，ArH，1H)；5.80～5.83(q，ArH，1H)；6.52(s，CH2，2H)；6.77～6.81(q，ArH，1H)；6.90～6.95(q，ArH，1H)；7.26～7.28(d，CH2，2H)；7.33～7.34(d，CH2，2H)；7.51～7.53(q，ArH，1H)；7.57～7.64(td，ArH，H)；7.81～7.82(d，ArH，2H)；7.90～7.91(d，ArH，2H)；8.27(s，ArH，1H)。
The NMR spectrum of Compound 3 is shown in FIG. 3, and the mass spectrum is shown in FIG. 4.
Compound 4
Figure BDA0003156058400000062
Fomular C29H31N5O5S,calc 561.2040,found 562.2119[M+H]+,584.1938[M+Na]+；1H NMR(DMSO-d6，400MHz)，δ：2.05(s，CH3，3H)；2.33(s，CH3，3H)；2.36(s，CH3，3H)；2.66～2.70(m，ArH，1H)；2.88～2.90(t，CH2，2H)；3.07～3.10(t，CH2，2H)；3.18～3.23(m，ArH，1H)；4.49～4.52(t，CH2，2H)；4.59～4.62(m，ArH，1H)；5.70～5.72(m，ArH，1H)；7.27～7.28(t，ArH，2H)；7.33～7.34(d，ArH，2H)；7.82～7.83(d，ArH，2H)；7.90～7.91(d，ArH，2H)；8.19(s，ArH，1H)。
The NMR spectrum of Compound 4 is shown in FIG. 5, and the mass spectrum is shown in FIG. 6.
Compound 5
Figure BDA0003156058400000071
Fomular C13H19N5O3S,calc 325.1203,found 326.1281[M+H]+,348.1101[M+Na]+；1H NMR(DMSO-d6，400MHz)，δ：2.05(s，CH3，3H)；2.17～2.21(m，ArH，1H)；2.46～2.47(t，ArH，1H)；2.57～2.62(m，ArH，1H)；2.88～2.91(t，CH2，2H)；3.07～3.09(t，CH2，2H)；3.45～3.49(m，ArH，1H)；3.52～3.55(m，ArH，1H)；3.78～3.80(m，ArH，1H)；4.32～4.34(m，ArH，1H)；4.94～4.96(m，ArH，1H)；5.26(d，ArH，1H)；6.19～6.22(q，，ArH，1H)；6.44(s，CH2，2H)；8.18(s，ArH，1H)。
The NMR spectrum of Compound 5 is shown in FIG. 7, and the mass spectrum is shown in FIG. 8.
Example 26 application of methylthioethyl purine-2' -deoxynucleoside to DNA Cross-linking
The 6-methylthioethyl purine-2' -deoxynucleoside does not contain a functional group capable of being crosslinked with amino groups on DNA bases, but in DNA synthesis, methylthioethyl groups on the chemical structure can be oxidized into methylsulfonyl ethyl units by an oxidation step inherent in DNA synthesis; then under the reaction condition of high-temperature ammonolysis, the methylsulfonyl ethyl unit can be hydrolyzed into terminal olefin; the terminal olefin can generate Michael addition with amine groups in other bases in a DNA chain under specific conditions to realize interchain crosslinking.
In the application process, hydroxyl (3 position and 5 position) and amino in the 6-methylthioethyl purine-2 '-deoxynucleoside need to be protected to form a phosphoramidite unit shown as a structure in a formula II, wherein the chemical name of the phosphoramidite unit is 5' - (4, 4-dimethoxytrityl) -6-methylthioethyl purine-3 '- [ N, N-diisopropylamino-O-cyanoethyl phosphoramidite ] -2' -deoxynucleoside.
Figure BDA0003156058400000072
The synthetic route for formula II (Compound 8) is as follows:
Figure BDA0003156058400000073
wherein, the reaction conditions in the synthesis process are shown as follows: a. chlorotrimethylsilane, pyridine, phenoxyacetyl chloride, 25 ℃; 4,4' -dimethoxytriphenylchloromethane, pyridine, 25 ℃; 2-O-cyanoethyl N, N-diisopropyl chlorophosphite amide, anhydrous tetrahydrofuran, triethylamine at 0 ℃.
The reaction process for realizing the cross-linking between DNA chains by taking the formula II as a raw material is as follows:
(1) synthesis process of methylsulfonylethyl unit
The synthetic route is as follows:
Figure BDA0003156058400000081
reaction conditions of the steps: step a: 2mol/L trichloroacetic acid in dichloromethane at room temperature; step b: 0.2mol/L of 5 ' - (4, 4-dimethoxytrityl) -6-methylthioethylpurine-3 ' - [ N, N-diisopropylamino-O-cyanoethylphosphoramidate ] -2' -deoxynucleoside, 0.45mol/L of 5-mercaptoethyltetrazole, anhydrous acetonitrile, room temperature; step c: 1mol/L iodine in tetrahydrofuran at room temperature.
1) Removing the 4,4' -dimethoxytrityl protecting group from the 5 ' hydroxyl group of the deoxynucleoside on the solid support under acidic conditions to expose the 5 ' hydroxyl group of the deoxynucleoside (corresponding to step a in the synthetic route);
2) the exposed 5' hydroxyl and the compound shown in the formula II are subjected to nucleophilic substitution under the catalysis of 5-mercaptoethyltetrazole to generate a trivalent phosphite ester structure (corresponding to the step b in the synthetic route);
3) the trivalent phosphite unit is oxidized by iodine to the pentavalent phosphate unit, i.e., to form a DNA containing 2-amino-6-methylsulfonyl (ethyl) purine (corresponding to step c in the synthetic scheme).
The above reaction is a basic operation for synthesizing one deoxynucleoside unit in DNA solid phase synthesis.
(2) Synthesis of terminal olefin units
After the methyl sulfonyl ethyl unit is formed, ammonia water or ammonia water-methylamine mixed solution is adopted to remove protecting groups of DNA under the heating condition of 50-80 ℃, and the removing specifically comprises the removing of amino protecting groups on basic groups, the removing of cyanoethyl on phosphotriester bonds and the cutting of connecting ester bonds of the DNA and a solid phase carrier. Then under the reaction condition of high-temperature ammonolysis, the methylsulfonyl ethyl unit can be hydrolyzed into terminal olefin, namely 2-amino-6-methylsulfonyl (ethyl) purine can be hydrolyzed into 2-amino-6-vinyl purine, and the synthetic route is as follows:
Figure BDA0003156058400000091
the mechanism that the methylsulfonyl ethyl unit can be hydrolyzed into terminal olefin is that the methylsulfonyl ethyl firstly generates carbanions in an alkaline environment, then intramolecular rearrangement is carried out to generate a ternary cyclic sulfone structure, the cyclic sulfone unit is decomposed at high temperature to release sulfur dioxide molecules while terminal olefin is obtained, and the mechanism is as follows:
Figure BDA0003156058400000092
(3) crosslinking of terminal olefins
As shown in the following synthetic route, the vinyl group can generate Michael addition with amine groups in other bases in a DNA chain in a neutral to weakly alkaline aqueous solution, so that the DNA inter-chain crosslinking is realized. Neutral to slightly alkaline aqueous solutions include, but are not limited to, phosphate buffer solutions, Tris (hydroxymethyl) aminomethane (Tris) buffer solutions, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solutions, and Tris (hydroxymethyl) aminomethane-boronic acid-sodium ethylenediaminetetraacetate (TBE) buffer solutions.
Figure BDA0003156058400000093
In the step i, non-covalent hydrogen bond interaction occurs in the reaction process; in step ii, Michael addition is carried out on the 4-amine group on the deoxycytidine and the alkene to form a covalent bond.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (10)

  1. A process for producing 6-methylthioethylpurine-2' -deoxynucleoside, characterized by comprising the steps of:
    (1) reacting sodium bis (trimethylsilyl) amide with 2-amino-6-chloro-purine in an ether solvent for 1-12h, adding an ether solvent solution of 1-chloro-2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose into the reaction system, stirring for reacting for 1-12h, quenching the reaction, and separating to obtain an off-white crystal of 1- (2-amino-6-chloropurine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose;
    (2) dispersing the grey white crystal, the palladium tetrakis (triphenylphosphine) and the lithium chloride in the step (1) in ethyl acetate, stirring for 1-120min, adding tributyl tin ethylene, reacting, heating and refluxing for 1-24h, and separating after the reaction is finished to obtain a crude product of 1- (2-amino-6-vinylpurine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose;
    (3) dissolving the crude product prepared in the step (2) in acetonitrile, adding 20% sodium methyl mercaptide aqueous solution, reacting for 1-24h, and separating after the reaction is finished to obtain a crude product of 1- (2-amino-6-thiomethyl purine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose;
    (4) and (3) dissolving the crude product prepared in the step (3) in a mixed solution of dichloromethane and 5mol/L methanol ammonia solution, stirring and reacting for 1-24h at 40-100 ℃, and separating to obtain the 6-methylthioethyl purine-2' -deoxynucleoside after the reaction is finished.
  2. 2. The method according to claim 1, wherein the ethereal solvent in step (1) is one selected from tetrahydrofuran, isopropyl ether, anisole, 1, 4-dioxane and methyl tert-butyl ether.
  3. 3. The process according to claim 1, wherein the 1- (2-amino-6-chloropurine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose in the step (1) is isolated by adding water and a saturated ammonium chloride solution to the reaction system to quench the reaction, removing the solvent from the reaction system, adding ethyl acetate and water to the reaction system, separating the organic phase and concentrating, and crystallizing the concentrated crude product in a mixed solvent of ethyl acetate and n-hexane at a volume ratio of 1:3 to obtain off-white crystals.
  4. 4. The process according to claim 1, wherein the crude product of 1- (2-amino-6-vinylpurine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose in the step (2) is obtained by adding a saturated aqueous solution of sodium fluoride to the reaction system, stirring the mixture for 1 to 24 hours, removing insoluble matters by suction filtration, separating an aqueous phase, and washing an organic phase with a saturated aqueous solution of sodium chloride.
  5. 5. The process according to claim 1, wherein the crude product of 1- (2-amino-6-thiomethyl purine) -2-deoxy-3, 5-di-O-p-methylbenzoyl-D-ribose in the step (3) is isolated by removing acetonitrile in the reaction system, adding ethyl acetate and water to the reaction system, separating the organic phase, washing the organic phase with saturated brine, and drying with anhydrous sodium sulfate to obtain the crude product.
  6. 6. The preparation method according to claim 1, wherein the 6-methylthioethylpurine-2 '-deoxynucleoside in the step (4) is separated by dispersing the system after the reaction is completed in ethyl acetate, subjecting the mixture to ultrasonic treatment for 1 to 3 hours, suction-filtering the mixture to obtain a crude product, and crystallizing the crude product in isopropyl ether to obtain 6-methylthioethylpurine-2' -deoxynucleoside.
  7. 7. 6-methylthioethylpurine-2' -deoxynucleoside produced by the production process according to any one of claims 1 to 6.
  8. Application of 6-methylthioethyl purine-2' -deoxynucleoside in preparation of functional nucleic acid-based material, preparation of nucleic acid medicine or DNA encryption.
  9. 9. A phosphoramidite unit for preparing cross-linked DNA is characterized in that 6-methylthioethyl purine-2' -deoxynucleoside is prepared by the protection reaction of 3-hydroxyl, 5-hydroxyl and amino, and the structure is shown as formula II:
    Figure FDA0003156058390000021
  10. 10. the method for preparing phosphoramidite units for use in preparing crosslinked DNA according to claim 9, wherein the synthetic route is as follows:
    Figure FDA0003156058390000022
    wherein the reaction conditions of the steps in the synthetic route are set forth as follows: a. chlorotrimethylsilane, pyridine, phenoxyacetyl chloride, 25 ℃; 4,4' -dimethoxytriphenylchloromethane, pyridine, 25 ℃; 2-O-cyanoethyl N, N-diisopropyl chlorophosphite amide, anhydrous tetrahydrofuran, triethylamine at 0 ℃.
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