CN118271378A

CN118271378A - Sugar ring modified nucleoside phosphoramidite monomer and preparation method and application thereof

Info

Publication number: CN118271378A
Application number: CN202211714135.5A
Authority: CN
Inventors: 梅辉; 熊成鹤; 温骏林; 何雷; 赵颖
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2024-07-02
Also published as: WO2024139896A1

Abstract

The invention discloses a sugar ring modified nucleoside phosphoramidite monomer, a preparation method and application thereof. The sugar ring modified nucleoside phosphoramidite monomer comprises any one of a compound with a structure shown as a formula (1), a salt of the compound or an isomer of the compound. The invention designs a brand new modification strategy, takes 3 '-deoxyapiose as a framework to design nucleoside phosphoramidite monomer, and the oligonucleotide prepared by the method has universality and obviously improved nuclease stability, has potential medical application value for preparing nucleic acid medicines, diagnostic reagents and the like, and develops a new route for synthesizing the nucleoside monomer taking 3' -deoxyapiose as the framework, thereby obviously simplifying the process, improving the efficiency and being beneficial to wide popularization and application.

Description

Sugar ring modified nucleoside phosphoramidite monomer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a sugar ring modified nucleoside phosphoramidite monomer, and a preparation method and application thereof.

Background

Nucleic acid medicaments comprise antisense nucleic acid, small interfering RNA, messenger RNA (mRNA) aptamer, ribozyme and the like, and are new-generation medicaments subsequent to small molecule medicaments, protein medicaments and antibody medicaments. However, nucleic acid drugs based on natural DNA/RNA structures have some drawbacks that are difficult to overcome, such as being easily degraded by endogenous nucleases, low target binding capacity, insufficient recognition specificity, poor bioavailability, etc. Currently, the antisense nucleic acid drug eteplirsen for treating duchenne muscular dystrophy, for example, can treat Fomivirsen of cytomegalovirus retinitis (CMV) all with non-natural modification mainly by chemically modifying three structural units (phosphate, base and sugar rings) of natural nucleic acid and even completely replacing to form non-natural nucleic acid, thereby improving and solving the above problems.

Research shows that non-natural nucleic acid based on sugar ring modification becomes one of the best solutions to the problems of nuclease degradation, target binding specificity and the like. In recent years, there have been few sugar ring-modified nucleic acids effective in improving enzyme resistance, such as 2 '-fluoro-arabinonucleic acid (2' -fluoroarabino nucleic acids, FANA), hexose nucleic acid (hexose nucleic acid, HNA), lock nucleic acid (Locked nucleic acid, LNA), threose nucleic acid (Threose nucleic acid, TNA) and the like, which have been developed and applied to nucleic acid drug development, such as Wang Jing and the like (see: synthesis and property studies of 2 '-deoxy-2' -fluoro-4 '-substituted arabino-diabetic modified antisense oligonucleotides [ D ], wang Jing, zhengzhou university, 2016) starting from 2' -deoxy-2 '-fluoro-arabino-glucoside (2' -F-araU), and by introducing different substituents at the sugar group 4 'position, a novel 2' -deoxy-2 '-fluoro-4' -substituted arabino-glucoside (i.e., 2'-F-4' -X-araU) modification strategy has been proposed, which is expected to increase the stability of antisense oligonucleotides to nucleic acids as well as affinity and specificity to target genes and improve the biological activity and side effect occurrence properties thereof.

However, in general, the existing non-natural nucleic acid types are still rare, the synthesis difficulty is high, a novel natural nucleic acid modification strategy is designed, and the novel non-natural nucleic acid is developed, so that the method has important significance in the field of research and development of nucleic acid medicines.

Disclosure of Invention

Aiming at the problems that the existing non-natural nucleic acid is still rare and the synthesis difficulty is high, the invention provides a sugar ring modified nucleoside phosphoramidite monomer, and a preparation method and application thereof, so as to realize efficient preparation of novel non-natural nucleic acid and provide a novel method and a novel idea for developing novel nucleic acid medicines.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a sugar ring-modified nucleoside phosphoramidite monomer comprising any one of a compound having the structure of formula (1), a salt of the compound, or an isomer of the compound;

Wherein:

r ₁ is selected from any one of natural base, non-natural base, modified base or salt thereof;

R ₂、R₃ is independently selected from alkyl groups with 1-4 carbon atoms;

DMTr is 4,4' -dimethoxytrityl.

In the invention, a brand new modification strategy is designed, a nucleoside phosphoramidite monomer is designed by taking 3' -deoxyapiose as a framework, and the stability of the oligonucleotide modified by the nucleoside phosphoramidite monomer is obviously improved, so that the oligonucleotide has potential medical application value in preparing nucleic acid medicines, diagnostic reagents and the like.

In a second aspect, the present invention provides a method for preparing the sugar ring modified nucleoside phosphoramidite monomer according to the first aspect, the method comprising the steps of:

(1) Reacting a reaction substrate (I) with glyoxylate in the presence of a catalyst, and carrying out a reduction reaction on the obtained product in the presence of a reducing agent to obtain a compound (II) and/or an isomer thereof;

(2) Removing the protecting group of the compound (II) and/or the isomer thereof, and carrying out intramolecular cyclization reaction under an acidic condition to obtain the compound (III) and/or the isomer thereof;

(3) Carrying out reduction reaction on the compound (III) and/or an isomer thereof in the presence of a reducing agent to obtain a compound (IV) and/or an isomer thereof;

(4) Reacting compound (IV) and/or an isomer thereof with R ₁ in the presence of a protecting agent to obtain compound (V) and/or an isomer thereof;

(5) Reacting the compound (V) and/or an isomer thereof with 4,4' -dimethoxy triphenylchloride under the catalysis of a catalyst;

(6) And (3) carrying out phosphoramidition reaction on the product of the step (5) to obtain the sugar ring modified nucleoside phosphoramidite monomer.

Wherein the structural formulas of the compounds (I) to (V) are shown below, and W ₁、W₂、W₃、W₄ and W ₅ are each independently selected from hydrogen or a protecting group:

In the invention, a new route for synthesizing nucleoside monomers with 3 '-deoxyapiose as a framework is developed, the process is obviously simplified, the efficiency is improved, and the method is suitable for preparing 3' -deoxyapiose nucleosides in a scale of 10-100 g.

Optionally, the catalyst of step (1) is selected from L-proline and/or derivatives thereof.

Optionally, the reducing agent of step (1) is selected from sodium borohydride and/or lithium aluminum hydride.

Optionally, the reducing agent of step (3) is selected from Dibal-H and/or RED-Al.

Optionally, the protecting reagent of step (4) comprises N, O-bis (trimethylsilyl) acetamide (BSA).

Optionally, the catalyst in step (5) comprises 4-dimethylaminopyridine and/or triethylamine.

Optionally, the solvent of the reaction of step (5) comprises dichloromethane or pyridine.

Optionally, the reaction of step (5) is carried out under the protection of an inert gas, wherein the inert gas comprises nitrogen or/and argon.

Alternatively, the reaction in step (5) may be carried out at a temperature of 10 to 50 ℃ (for example, 11 ℃, 12 ℃, 13 ℃, 15 ℃, 16 ℃,18 ℃, 20 ℃, 25 ℃, 30 ℃, 32 ℃, 35 ℃, 36 ℃, 38 ℃, 40 ℃, 45 ℃, 46 ℃, 47 ℃,48 ℃ or 49 ℃) for a time of 2 to 12 hours (for example, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or 11 hours).

Optionally, the catalyst of the phosphoramidition reaction of step (6) comprises triethylamine and/or N, N-diisopropylethylamine.

Alternatively, the phosphoramiditing reagent of the phosphoramidition reaction comprises 2-cyanoethyl N, N-diisopropylchlorophosphamide.

Alternatively, the phosphoramidite reaction is carried out at a temperature of-10 to 25 ℃ (including, but not limited to, -9 ℃, -8 ℃, -5 ℃,0 ℃,5 ℃, 10 ℃, 15 ℃,20 ℃, 21 ℃, 22 ℃, 23 ℃ or 24 ℃) for a time of 1 to 6 hours (which may be, for example, 2 hours, 3 hours, 4 hours or 5 hours).

In a third aspect, the present invention provides the use of a sugar ring modified nucleoside phosphoramidite monomer according to the first aspect in the preparation of an oligonucleotide.

In a fourth aspect, the present invention provides an oligonucleotide, the starting material for the preparation of which comprises the sugar ring modified nucleoside phosphoramidite monomer of the first aspect.

In a fifth aspect, the present invention provides a method of preparing an oligonucleotide, the method comprising synthesizing an oligonucleotide using a starting material comprising a sugar ring modified nucleoside phosphoramidite monomer according to the first aspect.

It will be appreciated that methods of nucleic acid synthesis common in the art are applicable to the present invention.

Alternatively, the method of synthesis may comprise a solid phase phosphoramidite method.

In a sixth aspect, the present invention provides the use of a sugar ring modified nucleoside phosphoramidite monomer according to the first aspect or an oligonucleotide according to the fourth aspect in the preparation of a nucleic acid drug.

The nucleoside phosphoramidite monomer with 3' -deoxyapiose as a framework can be used for preparing a functional oligonucleotide, and can be inserted into one or more positions of the oligonucleotide at fixed points to prepare the functional oligonucleotide, wherein the functional oligonucleotide is a 3' -deoxyapiose-framework nucleoside fixed-point modified or fully modified oligonucleotide, and the 3' -deoxyapiose-framework nucleoside phosphoramidite monomer can be inserted into any position of an oligonucleotide chain.

Alternatively, the nucleic acid drug includes antisense nucleic acids, small interfering RNAs, messenger RNA (mRNA) aptamers, ribozymes, and the like.

In a seventh aspect, the present invention provides a nucleic acid pharmaceutical composition comprising a sugar ring modified nucleoside phosphoramidite monomer according to the first aspect or an oligonucleotide according to the fourth aspect.

Optionally, the nucleic acid pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention designs a brand new modification strategy, takes 3 '-deoxyapiose as a framework to design a nucleoside phosphoramidite monomer, and the oligonucleotide prepared by the 3' -deoxyapiose has universality and obviously improved stability, and has potential medical application values for preparing nucleic acid medicines, diagnostic reagents and the like;

(2) The invention designs and develops a new route for synthesizing nucleoside monomers with 3 '-deoxyapiose as a framework, which remarkably simplifies the process, improves the efficiency and is suitable for preparing the 3' -deoxyapiose nucleoside in a scale of 10-100 g.

Drawings

FIG. 1 is a ¹ HNMR spectrum of Compound 1 in example 1;

FIG. 2 is a ¹³ C NMR spectrum of compound 1 in example 1;

FIG. 3 is a ¹ HNMR spectrum of Compound 2 in example 1;

FIG. 4 is a ¹³ C NMR spectrum of compound 2 in example 1;

FIG. 5 is a ¹ HNMR spectrum of Compound 3 in example 1;

FIG. 6 is a ¹³ C NMR spectrum of compound 3 in example 1;

FIG. 7 is a ¹ HNMR spectrum of Compound 4 in example 1;

FIG. 8 is a ¹³ C NMR spectrum of compound 4 in example 1;

FIG. 9 is a ¹ HNMR spectrum of Compound 5 in example 1;

FIG. 10 is a ¹³ C NMR spectrum of compound 5 in example 1;

FIG. 11 is a ¹ HNMR spectrum of Compound 6 in example 1;

FIG. 12 is a ¹³ C NMR spectrum of compound 6 in example 1;

FIG. 13 is a ³¹ PNMR spectrum of compound 7 in example 1;

FIG. 14 is a ¹ HNMR spectrum of Compound 8 in example 1;

FIG. 15 is a ¹³ C NMR spectrum of compound 8 in example 1;

FIG. 16 is a ¹ HNMR spectrum of Compound 9 in example 1;

FIG. 17 is a ¹³ C NMR spectrum of compound 9 in example 1;

FIG. 18 is a ³¹ PNMR spectrum of compound 10 of example 1;

FIG. 19 is a ¹ HNMR spectrum of Compound 11 in example 1;

FIG. 20 is a ¹³ C NMR spectrum of compound 11 in example 1;

FIG. 21 is a ¹ HNMR spectrum of Compound 12 in example 1;

FIG. 22 is a ¹³ C NMR spectrum of compound 12 in example 1;

FIG. 23 is a ³¹ PNMR spectrum of compound 13 in example 1;

FIG. 24 is a ¹ H NMR spectrum of compound 14 from example 1;

FIG. 25 is a ¹³ C NMR spectrum of compound 14 in example 1;

FIG. 26 is a ¹ H NMR spectrum of compound 15 in example 1;

FIG. 27 is a ¹³ C NMR spectrum of compound 15 in example 1;

FIG. 28 is a ³¹ PNMR spectrum of compound 16 of example 1;

FIG. 29 is a graph showing the molecular weight of the fully modified oligonucleotide ON-1 of example 2;

FIG. 30 is an electrophoresis chart of the experiment of serum resistance of the oligonucleotide in example 3;

FIG. 31 is an experimental electrophoresis of the snake venom phosphodiesterase resistance of the oligonucleotide of example 3.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

The invention designs a nucleoside phosphoramidite monomer by taking 3' -deoxyapiose as a framework, wherein the nucleoside phosphoramidite monomer can be used for preparing a functional oligonucleotide by a chemical method, for example, a solid phase phosphoramidite method is adopted, the functional oligonucleotide can be a nucleoside phosphoramidite monomer with 3' -deoxyapiose as a framework, which is subjected to site-directed modification or full modification, and the nucleoside phosphoramidite monomer with 3' -deoxyapiose as a framework can be inserted into any position of an oligonucleotide chain.

Example 1

In the embodiment, 3-deoxyapiose is taken as a framework, and R ₁ is four natural bases: the synthesis of nucleoside phosphoramidite monomers of thymine, cytosine, adenine and guanine exemplifies the preparation method of the sugar ring modified nucleoside phosphoramidite monomer of the present invention, and the specific routes are shown below.

The reaction conditions of each step are as follows, and nuclear magnetic resonance is adopted to characterize the structure of the compound:

a: 15g of 3-benzyloxypropionaldehyde was dissolved in 45mL of acetonitrile and 5mL of water, 28g of ethyl glyoxylate was added at 25℃and 1g L-proline derivative (catalyst) was added at 0℃followed by reaction at 5℃for 60 hours. All solvent was removed under reduced pressure and the resulting slurry was dissolved in 100mL of methanol. 6g of sodium borohydride was added at 0℃and after 30 minutes the reaction solution was adjusted to pH about 7 with concentrated hydrochloric acid, all solvents were removed under reduced pressure, the organic phase was extracted with methylene chloride, washed with water and saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to give 14.7g of colorless syrup 1, yield 60%, and the structure of compound 1 was confirmed as follows (FIGS. 1 and 2):

¹H NMR(400MHz,CDCl₃)δ7.41–7.29(m,6H),4.56(s,2H),4.46–4.40(m,1H),4.26(dt,J＝13.8,6.8Hz,2H),3.80(d,J＝5.0Hz,2H),3.70(dd,J＝6.6,2.9Hz,2H),3.22(d,J＝4.8Hz,1H),2.49–2.40(m,1H),2.09(d,J＝15.1Hz,1H),1.31(t,J＝7.1Hz,3H);

¹³C NMR(101MHz,CDCl₃)δ174.70,137.93,128.46,127.79,127.73,73.50,70.40,69.21,61.85,61.12,44.40,14.15;

b: 22.2g of Compound 1 was dissolved in 200mL of dry dichloromethane at 25℃followed by addition of 14.4mL of triethylamine, 14.34g of t-butyldimethylchlorosilane followed by addition of 200mg of 4-dimethylaminopyridine, stirring overnight, extraction with dichloromethane, washing with water and washing the organic phase with saturated brine, collecting the organic phase, drying over anhydrous sodium sulfate, filtration, concentration, purification by silica gel column chromatography (Petroleum ether/ethyl acetate=10/1) to give 25.4g of colorless syrup 2 in 80% yield, the structure confirmation result of Compound 2 was as follows (FIGS. 3 and 4):

¹H NMR(400MHz,CDCl₃)δ7.40–7.30(m,5H),4.61–4.50(m,2H),4.41(dd,J＝6.8,2.9Hz,1H),4.29–4.15(m,2H),3.79(dd,J＝10.1,6.0Hz,1H),3.73(dd,J＝10.2,5.2Hz,1H),3.65(d,J＝6.6Hz,2H),3.46(d,J＝6.8Hz,1H),2.46–2.37(m,1H),1.29(t,J＝7.1Hz,3H),0.94–0.88(m,9H),0.10–0.01(m,6H);

¹³C NMR(101MHz,CDCl₃)δ174.41,138.16,128.39,127.71,127.66,73.40,70.61,68.49,61.29,60.83,44.42,25.87,18.27,14.19,-5.63,-5.67;

c: 200mL of tetrahydrofuran was used to dissolve 28g of Compound 2, 0.5g of 20% palladium hydroxide was further added under nitrogen atmosphere, the reaction solution was stirred under hydrogen atmosphere for 48 hours, after the reaction was completed, a pad layer of celite was filtered, 630mg of p-toluenesulfonic acid was added to the filtrate, stirring was carried out at 25℃overnight, the reaction solution was adjusted to pH of about 7 with triethylamine, and concentrated, the obtained oil was dissolved with 150mL of methylene chloride, 12.2mL of triethylamine and 9.3mL of benzoyl chloride were added to the solution, respectively, the reaction was continued for 3 hours, extraction with methylene chloride, washing with water and saturated brine, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, concentrated, purified by silica gel column chromatography (Petroleum ether/ethyl acetate=10/1), 20.5g of colorless syrup 3 was obtained, the yield was 80%, and the structure of Compound 3 was confirmed as follows (FIG. 5 and FIG. 6):

¹H NMR(400MHz,CDCl₃)δ8.10(d,J＝7.2Hz,2H),7.62(t,J＝7.4Hz,1H),7.48(t,J＝7.7Hz,2H),5.71(d,J＝9.0Hz,1H),4.56(t,J＝8.8Hz,1H),4.30(t,J＝9.0Hz,1H),3.89(dd,J＝10.6,4.8Hz,1H),3.81(dd,J＝10.6,3.5Hz,1H),2.96(qdd,J＝8.7,4.7,3.7Hz,1H),0.91(s,9H),0.10(d,J＝7.8Hz,6H);

¹³C NMR(101MHz,CDCl₃)δ172.68,165.38,133.70,130.04,128.81,128.52,69.36,67.32,60.24,43.75,25.77,18.20,-5.58,-5.60;

d: 4.71g of Compound 3 was dissolved in 45mL of tetrahydrofuran, then cooled to-78℃and, after stirring for 10min, 16.8mLDIBAL-H hexane solution (1M) was slowly added dropwise to the flask. The reaction was kept at-78 ℃ for 10h with stirring at-78 ℃ and 5mL of methanol was slowly dropped into the reaction flask, followed by heating to 25 ℃ and 45mL of saturated aqueous potassium sodium tartrate and 90mL of ethyl acetate were sequentially added to the reaction solution, stirred overnight, extracted with ethyl acetate, the organic phase was washed with water and saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, the resulting slurry was dissolved in 40mL of methylene chloride, then 5.6mL of triethylamine was added, 1.5mL of acetic anhydride, a small amount of 4-dimethylaminopyridine was added to the reaction solution under ice bath conditions, stirring at 25 ℃ for 4h, extracted with methylene chloride, the organic phase was washed with water and saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl acetate=10/1), to give 5g of colorless slurry 4, yield 94%, the structure confirmation result of the compound 4 was as follows (fig. 7 and 8):

¹H NMR(400MHz,CDCl₃)δ8.03(dd,J＝5.2,3.3Hz,2H),7.61–7.55(m,1H),7.49–7.41(m,2H),6.47(d,J＝4.3Hz,0.2H),6.30(s,0.8H),5.34(d,J＝2.9Hz,0.8H),5.26(dd,J＝8.2,4.4Hz,0.2H),4.36–4.25(m,1H),4.00(dd,J＝8.9,7.2Hz,1H),3.95–3.86(m,1H),3.86–3.71(m,1H),2.84–2.70(m,0.2H),2.72–2.61(m,0.8H),2.10(s,2.4H),2.00(s,0.6H),0.95–0.82(m,9H),0.09–0.04(m,6H);

¹³C NMR(101MHz,CDCl₃)δ169.61,165.74,133.40,129.77,128.46,100.97,80.16,71.12,61.67,47.51,25.81,21.14,18.23,-5.44,-5.46;

e: 383mg of thymine was weighed into a50 mL reaction flask, 8mL of acetonitrile was added under nitrogen, followed by 1.5mL of N, O-bis-trimethylsilylacetamide, and the solution was stirred at 25℃until the solution became clear. Acetonitrile solution (4 mL) containing 1.14g of compound 4 was added to the reaction solution, followed by addition of 0.85mL of trimethylsilicone triflate, stirring at 25 ℃ for 1.5 hours, neutralization reaction with saturated aqueous sodium bicarbonate until no bubble was generated, extraction with ethyl acetate, washing with water and washing with saturated brine, collecting the organic phase, drying with anhydrous sodium sulfate, filtration, concentration, dissolution of the resulting slurry with 10mL of tetrahydrofuran, followed by addition of 1.4mL of triethylamine hydrogen fluoride solution, stirring at 25 ℃ overnight, concentration, silica gel column chromatography purification (dichloromethane/methanol=30/1) to give 0.85g of colorless slurry 5 in 85% yield, confirmation of structure of compound 5 being as follows (fig. 9 and 10):

¹H NMR(400MHz,CDCl₃)δ9.27(s,1H),8.09–8.01(m,2H),7.61(t,J＝7.4Hz,1H),7.47(t,J＝7.7Hz,2H),7.23(s,1H),5.99(d,J＝4.6Hz,1H),5.62(t,J＝5.1Hz,1H),4.32(t,J＝8.6Hz,1H),4.28–4.20(m,1H),3.96(dd,J＝11.0,5.1Hz,1H),3.89(dd,J＝11.0,5.2Hz,1H),2.96(s,1H),2.85–2.75(m,1H),1.94(s,3H);

¹³C NMR(101MHz,CDCl₃)δ166.67,163.98,150.81,136.81,133.74,129.92,128.88,128.58,111.37,92.01,78.84,70.65,60.95,47.25,12.54;

f: 1.2g of Compound 5 was weighed into a 50mL reaction flask, dissolved in 10mL of pyridine, 1.53g of DMTrCl was added under ice bath conditions, stirring was continued for 10 minutes, the ice bath was removed, stirring was performed at 25℃for 1 hour, and the reaction was completed. To the reaction solution was added 1mL of methanol, followed by removal of all solvents under reduced pressure, the residue was dissolved with ethyl acetate, the organic phase was washed with water and saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The brown slurry obtained was dissolved in 10mL of methanol and 10mL of tetrahydrofuran, 5.4mL of 1mol/L aqueous sodium hydroxide solution was added to the reaction solution under ice bath conditions, after stirring for thirty minutes, 20mL of water was added, the organic solvent was removed under reduced pressure, the remaining aqueous solution was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol 15/1) to give 1.5g of colorless foam-like solid 6 in 80% yield. The structure of compound 6 was confirmed as follows (fig. 11 and 12):

¹H NMR(400MHz,CDCl₃)δ8.98(s,1H),7.42–7.36(m,2H),7.36–7.18(m,10H),6.87–6.79(m,4H),5.56(d,J＝3.9Hz,1H),4.42–4.33(m,1H),4.19–4.09(m,1H),3.97(t,J＝8.6Hz,1H),3.81(s,6H),3.76(d,J＝2.3Hz,1H),3.30(dd,J＝9.3,5.6Hz,1H),3.15(dd,J＝9.2,7.3Hz,1H),2.80–2.67(m,1H),1.90(d,J＝0.9Hz,3H);

¹³C NMR(101MHz,CDCl₃)δ164.09,158.54,151.48,144.65,135.81,135.73,134.55,129.95,128.04,127.87,126.91,113.17,110.59,94.28,86.19,79.33,71.65,61.93,55.22,46.57,12.53;

g: 365mg of Compound 6 was weighed into a 25mL reaction flask, dissolved in 8mL of methylene chloride, followed by the addition of 0.47mLN, N-diisopropylethylamine, 0.45mL of 2-cyanoethyl-N, N-diisopropylchlorophosphamide under ice bath conditions, continued stirring for 10 minutes, and after removing the ice bath, stirring at 25℃for 1 hour, the reaction was completed. Extracting with dichloromethane, washing the organic phase with saturated aqueous sodium bicarbonate, collecting the organic phase, drying over anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography (petroleum ether/ethyl acetate/triethylamine)

=60/40/1), To give 450mg of white foam-like solid compound 7 in 90% yield, the phosphorus spectrum of compound 7 characterizes the following (fig. 13):

³¹PNMR(162MHz,CDCl₃)δ150.56,150.51。

1.05g of N ⁴ -Bz cytosine are weighed into a 50mL reaction flask, 15mL of acetonitrile are added under nitrogen, followed by 4.77mL of N, O-bis-trimethylsilyl acetamide, and the solution is stirred at 25℃until clear. An acetonitrile solution (10 mL) containing 3.5g of compound 4 was added to the reaction solution, followed by 2.56mL of trimethylsilyl triflate, and stirred at 25℃for 1.5 hours. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting slurry was dissolved in 20mL of tetrahydrofuran, followed by the addition of 2.86mL of a solution of hydrogen fluoride in triethylamine, and stirring overnight at 25 ℃. Concentration and purification by column chromatography on silica gel (dichloromethane/methanol 30/1) gave 2.95g of colourless syrup 8 in 76% yield. The structure of compound 8 was confirmed as follows (fig. 14 and 15):

¹H NMR(400MHz,CDCl₃)δ9.31(s,1H),8.00–7.85(m,5H),7.53(dt,J＝15.5,7.4Hz,3H),7.44–7.34(m,4H),6.08(d,J＝2.5Hz,1H),5.86–5.77(m,1H),4.44(t,J＝8.7Hz,1H),4.36(dd,J＝8.8,6.4Hz,1H),4.07–3.90(m,2H),3.74(dd,J＝10.8,4.6Hz,1H),2.72(qt,J＝8.5,4.4Hz,1H);

¹³C NMR(101MHz,CDCl₃)δ167.01,166.21,162.95,155.69,145.29,133.38,133.04,132.85,129.82,129.24,128.69,128.39,127.87,96.73,94.31,81.59,72.49,60.80,48.18;

i: 2.6g of Compound 8 was weighed into a 50mL reaction flask, dissolved in 15mL of pyridine, added with 2.63g of DMTrCl under ice bath conditions, and after stirring for 10 minutes, the ice bath was removed, and after stirring for 1 hour at 25℃the reaction was completed. To the reaction solution was added 2mL of methanol, followed by removal of all solvents under reduced pressure, the residue was dissolved with ethyl acetate, the organic phase was washed with water and saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The brown slurry obtained was dissolved in 20mL of methanol and 20mL of tetrahydrofuran, 5.9mL of 1mol/L aqueous sodium hydroxide solution was added to the reaction solution under ice-bath conditions, and after stirring for fifteen minutes, 5.9mL of 1mol/L aqueous sodium hydroxide solution was added thereto, and stirring was continued for fifteen minutes. To the reaction solution was added saturated ammonium chloride solution to neutral pH, the organic solvent was removed under reduced pressure, the remaining aqueous solution was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol 15/1) to give 2.5g of a colorless foam-like solid 9 in 66% yield. The structure of compound 9 was confirmed as follows (fig. 16 and 17):

¹H NMR(400MHz,CDCl₃)δ8.68(s,1H),7.93(dd,J＝12.5,7.6Hz,3H),7.72–7.50(m,4H),7.38(d,J＝7.5Hz,2H),7.32–7.19(m,10H),6.83(d,J＝8.8Hz,4H),5.62(d,J＝3.6Hz,1H),4.51–4.43(m,1H),4.39(s,1H),4.12–4.04(m,1H),3.99(t,J＝8.8Hz,1H),3.80(s,6H),3.36(dd,J＝9.2,4.7Hz,1H),3.14–3.04(m,1H),2.80(dq,J＝14.8,7.4Hz,1H);

¹³C NMR(101MHz,CDCl₃)δ166.53,162.60,158.52,156.50,144.66,143.10,135.85,135.78,133.21,133.01,129.94,129.02,128.05,127.88,127.65,126.88,113.18,113.17,96.47,86.13,80.12,72.14,61.70,55.22,47.02;

0.5g of compound 9 is weighed into a 50mL reaction bottle, dissolved in 8mL of dichloromethane, then 0.55mLN, N-diisopropylethylamine and 0.53mL 2-cyanoethyl-N, N-diisopropylchlorophosphamide are added under ice bath condition, the ice bath is removed after stirring for 10 minutes, and the reaction is finished after stirring for 1 hour at 25 ℃. The organic phase was washed with a saturated aqueous sodium hydrogencarbonate solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl acetate/triethylamine 60/40/1) to give 528mg of compound 10 as a yellow foam-like solid in 80% yield. The phosphorus spectrum of compound 10 is characterized as follows (fig. 18):

³¹PNMR(162MHz,CDCl₃)δ151.79,149.47；

3.03g of N ⁶ -Bz adenine is weighed into a 100mL reaction flask, 30mL of toluene is added under nitrogen, followed by 6.35mL of N, O-bis-trimethylsilyl acetamide, and the solution is stirred at 95℃until clear. A toluene solution (15 mL) containing 5.0g of Compound 4 was added to the reaction solution, followed by addition of 3.44mL of trimethylsilyl triflate and stirring at 95℃for 2 hours. After the reaction solution was cooled to room temperature, it was then subjected to neutralization reaction with a saturated aqueous sodium hydrogencarbonate solution until no bubbles were generated, extraction was performed with ethyl acetate, the organic phase was washed with water and saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting slurry was dissolved in 30mL of tetrahydrofuran, followed by the addition of 4.1mL of a solution of hydrogen fluoride in triethylamine, and stirred overnight at 25 ℃. Concentration and purification by column chromatography on silica gel (dichloromethane/methanol 30/1) gave 3.5g of colorless syrup 11 in 60% yield. The structure of compound 11 was confirmed as follows (fig. 19 and 20):

¹H NMR(400MHz,CDCl₃)δ9.23(s,1H),8.81(s,1H),8.28(s,1H),8.10–7.99(m,4H),7.66–7.58(m,2H),7.50(dt,J＝23.2,7.7Hz,4H),6.28(d,J＝3.5Hz,1H),6.23–6.15(m,1H),4.41(d,J＝7.6Hz,2H),4.13(dd,J＝11.3,4.6Hz,1H),4.00(dd,J＝11.3,5.2Hz,1H),3.80(s,1H),2.99–2.88(m,1H);

¹³C NMR(101MHz,CDCl₃)δ166.34,164.80,152.63,151.36,149.83,142.36,133.81,133.64,132.79,129.83,128.82,128.74,128.59,127.96,123.33,90.55,79.95,70.73,60.70,48.05;

l: 3.1g of Compound 11 was weighed into a 50mL reaction flask, dissolved in 20mL of pyridine, 2.97g of DMTrCl was added under ice bath conditions, stirring was continued for 10 minutes, the ice bath was removed, stirring was performed at 25℃for 1 hour, and the reaction was completed. To the reaction solution was added 2mL of methanol, followed by removal of all solvents under reduced pressure, the residue was dissolved with ethyl acetate, the organic phase was washed with water and saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The brown slurry obtained was dissolved in 20mL of methanol and 20mL of tetrahydrofuran, 6.8mL of 1mol/L aqueous sodium hydroxide solution was added to the reaction solution under ice-bath conditions, and after stirring for fifteen minutes, 6.8mL of 1mol/L aqueous sodium hydroxide solution was added thereto, and stirring was continued for fifteen minutes. To the reaction solution was added saturated ammonium chloride solution to neutral pH, the organic solvent was removed under reduced pressure, the remaining aqueous solution was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol 15/1) to give 3.2g of a colorless foam-like solid 12 in a yield of 72%. The structure of compound 12 was confirmed as follows (fig. 21 and 22):

¹H NMR(400MHz,CDCl₃)δ8.67(s,1H),8.08(s,1H),8.02(d,J＝7.4Hz,2H),7.60(t,J＝7.4Hz,1H),7.51(t,J＝7.6Hz,2H),7.41(d,J＝7.6Hz,2H),7.29(t,J＝7.7Hz,7H),7.21(t,J＝7.2Hz,1H),6.82(d,J＝8.8Hz,4H),5.88(d,J＝5.4Hz,1H),5.09(s,1H),4.69(dd,J＝8.2,5.5Hz,1H),4.43(t,J＝8.3Hz,1H),4.15(t,J＝9.1Hz,1H),3.79(s,6H),3.44(dd,J＝9.2,5.2Hz,1H),3.27(dd,J＝9.0,7.3Hz,1H),2.94–2.80(m,1H);

¹³C NMR(101MHz,CDCl₃)δ164.69,158.53,152.24,151.12,149.50,144.67,141.12,135.81,135.76,133.55,132.86,129.97,128.87,128.05,127.90,126.89,123.28,113.18,92.62,86.27,77.87,71.71,62.02,55.23,46.27.

m.0.7 g of Compound 12 is weighed into a 50mL reaction flask, dissolved in 8mL of dichloromethane, then 1.12mLN, N-diisopropylethylamine is added, 0.95mL 2-cyanoethyl-N, N-diisopropylchlorophosphamide is added under ice bath condition, stirring is continued for 10 minutes, ice bath is removed, stirring is carried out at 25 ℃ for 1 hour, and the reaction is completed. The organic phase was extracted with methylene chloride, washed with saturated aqueous sodium hydrogencarbonate, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl acetate/triethylamine 60/40/1) to give 0.75g of compound 13 as a white foam-like solid in 82% yield. The phosphorus spectrum of compound 13 is characterized as follows (fig. 23):

³¹PNMR(162MHz,CDCl₃)δ150.42,150.37；

n.into a 100mL reaction flask, 5.32g O ⁶-DPC-N² -Ac guanine was weighed, 30mL of 1, 2-dichloroethane was added under nitrogen atmosphere, followed by 8.37mL of N, O-bis-trimethylsilyl acetamide, stirred at 70℃until the solution was clear, and 1, 2-dichloroethane was removed under reduced pressure. The resulting slurry was dissolved in 20mL of toluene, and a toluene solution (15 mL) containing 4.5g of Compound 4 was added to the reaction solution, followed by addition of 4.33mL of trimethylsilyl triflate and stirring at 70℃for 2 hours. After the reaction solution was cooled to room temperature, it was then subjected to neutralization reaction with a saturated aqueous sodium hydrogencarbonate solution until no bubbles were generated, extraction was performed with ethyl acetate, the organic phase was washed with water and saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting slurry was dissolved in 30mL of tetrahydrofuran, followed by the addition of 3.7mL of a solution of hydrogen fluoride in triethylamine, and stirred overnight at 25 ℃. Concentration and purification by column chromatography on silica gel (dichloromethane/methanol 30/1) gave 5.2g of colorless syrup 14 in 75% yield. The structure of compound 14 was confirmed as follows (fig. 24 and 25):

¹H NMR(400MHz,CD₃OD_SPE)δ8.50(s,1H),8.02–7.95(m,2H),7.59(t,J＝7.4Hz,1H),7.43(ddd,J＝22.5,13.3,7.2Hz,10H),7.27(t,J＝7.3Hz,2H),6.33(d,J＝3.9Hz,1H),6.02(dd,J＝6.3,4.0Hz,1H),4.50(t,J＝8.8Hz,1H),4.37(t,J＝8.5Hz,1H),3.98–3.86(m,2H),3.01–2.89(m,1H),2.22(s,3H);

¹³C NMR(101MHz,CDCl₃)δ174.58,170.14,159.57,158.31,156.16,154.74,148.34,148.31,145.75,137.25,133.33,132.94,132.89,132.19,130.91,124.69,94.25,83.38,74.85,63.60,51.62,27.29;

o: 4.3g of compound 14 was weighed into a 50mL reaction flask, dissolved in 20mL of pyridine, 3.11g of DMTrCl was added under ice bath conditions, stirring was continued for 10 minutes, the ice bath was removed, stirring was performed at 25℃for 1 hour, and the reaction was completed. To the reaction solution was added 2mL of methanol, followed by removal of all solvents under reduced pressure, the residue was dissolved with ethyl acetate, the organic phase was washed with water and saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated. The brown slurry obtained was dissolved in 40mL 7N ammonia-methanol, stirred at 25℃for 2 days, all solvents were removed under reduced pressure, and then the residue was dissolved in 30m methanol, 1.9mL DMF-DMA was added to the reaction solution, stirred at 25℃overnight, all solvents were removed under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol 15/1) to give 3.5g of a colorless foam-like solid 15 in 79% yield. The structure of compound 15 was confirmed as follows (fig. 26 and 27):

¹H NMR(400MHz,CDCl₃)δ9.26(s,1H),8.39(s,1H),7.41(d,J＝7.5Hz,2H),7.38(s,1H),7.34–7.23(m,7H),7.18(t,J＝7.3Hz,1H),6.81(dd,J＝8.8,1.3Hz,4H),5.76(d,J＝5.8Hz,1H),4.67(dd,J＝8.3,5.9Hz,1H),4.34(t,J＝8.3Hz,1H),4.02(t,J＝9.0Hz,1H),3.77(s,6H),3.45(dd,J＝9.2,5.0Hz,1H),3.25(t,J＝8.4Hz,1H),2.96(s,3H),2.94(s,3H),2.83(dq,J＝12.1,6.7Hz,1H);

¹³C NMR(101MHz,CDCl₃)δ158.83,158.38,158.00,156.83,150.35,145.25,136.45,136.28,136.20,130.37,130.35,128.40,128.20,127.14,120.28,113.51,91.26,86.47,77.25,71.36,62.91,55.54,46.48,41.55,35.40.

0.8g of compound 15 is weighed into a 50mL reaction bottle, dissolved in 8mL of dichloromethane, then 0.9mLN, N-diisopropylethylamine and 0.85mL 2-cyanoethyl-N, N-diisopropylchlorophosphamide are added under ice bath condition, the ice bath is removed after stirring for 10 minutes, and the reaction is finished after stirring for 1 hour at 25 ℃. The organic phase was extracted with methylene chloride, washed with saturated aqueous sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether/ethyl acetate/triethylamine 60/40/1) to give 0.9g of compound 16 as a white foam-like solid in 85% yield. The phosphorus spectrum of compound 16 is characterized as follows (fig. 28):

³¹PNMR(162MHz,CDCl₃)δ150.61,150.41。

In conclusion, the invention can rapidly and efficiently prepare the sugar ring modified nucleoside phosphoramidite monomer, and different nucleoside phosphoramidite monomers can be prepared by selecting different bases for R ₁.

Example 2

This example demonstrates the synthesis of fully modified oligonucleotides with 3-deoxy apiose as a backbone.

Based ON the phosphoramidite prepared in example 1, the phosphoramidite monomer prepared in example 1 was inserted into the corresponding oligonucleotide sequence using a standard phosphoramidite method ON an automatic DNA synthesizer to obtain the following fully modified oligonucleotide ON-1, the molecular weight diagram of which is shown in FIG. 29. As a control, the natural DNA oligonucleotide ON-2 was prepared from commercial DNA phosphoramidite by a synthesizer.

ON-1:3’-apio(CGTAGTGAC CTG)-2’(apioNA)

ON-2:5’-CGTAGTGAC CTG-3’(DNA)

Example 3

This example performs nuclease resistance experiments.

Serum and snake venom phosphodiesterase tests were performed on the basis of the fully modified oligonucleotides with 3-deoxyapiose as a backbone obtained in example 2.

(1) Human serum resistance test

The above ON-1 was incubated with DMEM containing 50% human serum at 37℃for 15min, 30min, 1h, 2h, 5h, 24h and 48h, respectively, and the reaction was stopped by adding 10. Mu.L of stop buffer (containing 8M urea, 5mM Tris-HCl, 20mM EDTA, pH 7.5), and the control group was subjected to electrophoresis with 20% denaturing PAGE gel using DNAON-2 (FIG. 30), stained with SYBR GOLD, and the fluorescence intensity of the band was extracted with Imagr J to calculate the human serum resistance of OD-1.

(2) Venom-resistant phosphodiesterase

The reaction was stopped by incubating ON-1 with snake venom phosphodiesterase (0.4 mU/. Mu.L) at 37℃for 0.5h,1h,3h,5h,7h, and 10. Mu.L stop buffer (containing 8M urea, 5mM Tris-HCl, 20mM EDTA, pH 7.5), electrophoresed ON a DNAON-2 control, 20% denaturing PAGE gel, stained with SYBR GOLD (FIG. 31), and the fluorescence intensity of the bands was extracted with Imagr J to calculate the snake venom phosphodiesterase resistance of OD-1.

Enzyme resistance test results: the stability of ON-1 in human serum (half-life of about 58 hours) is 29 times that of the common DNA oligonucleotide ON-2, and the resistance to snake venom phosphodiesterase is 29 times as high.

In summary, the invention designs a brand-new modification strategy, designs a nucleoside phosphoramidite monomer by taking 3' -deoxyapiose as a framework, the oligonucleotide prepared by the nucleoside phosphoramidite monomer has universality and obviously improves the stability of nuclease, has potential medical application values for preparing nucleic acid medicines, diagnostic reagents and the like, and in addition, designs and develops a new route for synthesizing the nucleoside phosphoramidite monomer by taking 3' -deoxyapiose as the framework, thereby obviously simplifying the process, improving the efficiency and being applicable to the preparation of 3' -deoxyapiose nucleoside with the scale of 10-100 g.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A sugar ring-modified nucleoside phosphoramidite monomer, characterized in that the sugar ring-modified nucleoside phosphoramidite monomer comprises any one of a compound having a structure represented by formula (1), a salt of the compound, or an isomer of the compound;

Wherein:

R ₂、R₃ is independently selected from alkyl groups with 1-4 carbon atoms;

DMTr is 4,4' -dimethoxytrityl.

2. A method for preparing the sugar ring modified nucleoside phosphoramidite monomer of claim 1, comprising the steps of:

(6) Carrying out phosphoramidition reaction on the product of the step (5) to obtain the sugar ring modified nucleoside phosphoramidite monomer;

3. The process for the preparation of a sugar ring modified nucleoside phosphoramidite monomer according to claim 2, wherein the catalyst of step (1) is selected from L-proline and/or derivatives thereof;

preferably, the reducing agent of step (1) is selected from sodium borohydride and/or lithium aluminum hydride;

Preferably, the reducing agent of step (3) is selected from Dibal-H and/or RED-Al;

preferably, the protecting agent of step (4) comprises N, O-bis (trimethylsilyl) acetamide (BSA).

4. A process for the preparation of sugar ring modified nucleoside phosphoramidite monomers according to claim 2 or 3 wherein the catalyst in step (5) comprises 4-dimethylaminopyridine and/or triethylamine;

Preferably, the solvent of the reaction of step (5) comprises dichloromethane or pyridine;

preferably, the reaction of step (5) is carried out under the protection of an inert gas comprising nitrogen or/and argon;

preferably, the temperature of the reaction in the step (5) is 10-50 ℃ and the time is 2-12 h.

5. The method for producing a sugar ring-modified nucleoside phosphoramidite monomer according to any one of claims 2 to 4, wherein the catalyst for phosphoramidite reaction of step (6) comprises triethylamine and/or N, N-diisopropylethylamine;

Preferably, the phosphoramiditing reagent of the phosphoramidition reaction comprises 2-cyanoethyl N, N-diisopropylchlorophosphamide;

Preferably, the temperature of the phosphoramidite reaction is-10-25 ℃ and the time is 1-6 h.

6. Use of the sugar ring modified nucleoside phosphoramidite monomer of claim 1 in the preparation of an oligonucleotide.

7. An oligonucleotide, wherein the oligonucleotide is prepared from a starting material comprising the sugar ring-modified nucleoside phosphoramidite monomer of claim 1.

8. A method for preparing an oligonucleotide, comprising synthesizing an oligonucleotide using a starting material comprising the sugar ring-modified nucleoside phosphoramidite monomer of claim 1;

preferably, the method of synthesis comprises a solid phase phosphoramidite method.

9. Use of the sugar ring modified nucleoside phosphoramidite monomer of claim 1 or the oligonucleotide of claim 7 in the preparation of a nucleic acid drug.

10. A nucleic acid pharmaceutical composition comprising the sugar ring modified nucleoside phosphoramidite monomer of claim 1 or the oligonucleotide of claim 7.