Bio-orthogonal group-containing phosphoramidite monomer as well as preparation method and application thereof

Abstract

The invention discloses a phosphoramidite monomer containing a biological orthogonal group as shown in a formula I and a preparation method and application thereof, and belongs to the field of chemical modification of oligonucleotides and development of functional nucleic acid. The monomer can be used for preparing modified oligonucleotides through a DNA / RNA synthesizer, the biological orthogonal group is introduced into any position of the oligonucleotides, and the modified oligonucleotides can be prepared through a DNA / RNA synthesis instrument. The types of phosphoramidite monomer modules containing biological orthogonal groups are enriched, and a nucleic acid chemical modification raw material system is perfected. The obtained modified oligonucleotide can adapt to a biological orthogonal reaction, is used for preparing nucleic acid drugs and oligonucleotide probes, and supports diversified application of functional nucleic acid in the fields of chemistry, biology and medicine.

Description

Technical Field

[0001] This invention belongs to the field of chemical modification of oligonucleotides and development of functional nucleic acids, and relates to phosphoramidite monomers containing bioorthogonal groups, their preparation methods and applications, specifically to phosphoramidite monomers with trans-4-hydroxymethylpiperidin-3-ol as the backbone containing bioorthogonal groups such as norbornene, syldione, or cyclooctyne, and their application in the chemical modification of oligonucleotides. Background Technology

[0002] Bioorthogonal reactions are chemical reactions that occur within cells or tissues of an organism without interfering with the organism's own biochemical reactions. These reactions can achieve chemical labeling and imaging in living cells without disrupting the biological system. The [4+2] cycloaddition reaction between electron-deficient dienes and electron-rich diephiles is called the "reverse electron demand Diels-Alder reaction (IEDDA)". IEDDA reactions have good biocompatibility and high specificity, and are the fastest bioorthogonal reactions, commonly used in nucleic acid labeling and coupling. Norbornene is a structural unit formed by the fusion of a cyclohexene ring with a methylene bridge; its intracyclic double bond endows the molecule with high reactivity and stability. It can undergo rapid bioorthogonal reactions with tetrazine (Tz).

[0003] With the development of nucleic acid chemical synthesis technology, nucleic acids and their modified analogs have been widely used in chemistry, biology, and medicine. Through DNA / RNA solid-phase synthesis and phosphoramide synthesis, comprehensive chemical modification or partial substitution of oligonucleotides can be achieved. Chemically modified oligonucleotides can not only be used as biomimetic drugs but also have wide applications in the preparation of nanoprobe materials. Chemical modification of oligonucleotides includes modification of sugar rings, base sites, phosphodiester backbones, and substitution of nucleosides with non-natural monomers. In nucleic acid modification, bioorthogonal reactions can be used to introduce specific functional groups, achieving post-synthetic modification of DNA / RNA, which can then be further used for nucleic acid labeling, imaging, or drug delivery.

[0004] In recent years, chemists have designed and synthesized a series of bioorthogonal group-modified oligonucleotide probes for applications such as nucleic acid imaging and targeted drug delivery. The most common approach is to introduce bioorthogonal groups as coupling handles onto natural nucleosides, but this method is complex. While polymerase chain reaction (PCR) can introduce nucleoside triphosphates with coupling handles, it does not achieve selective site-specific introduction into oligonucleotides. Furthermore, the strategy of introducing coupling handles onto natural nucleosides in the form of phosphorous amide monomers has limited reported applications, requires stringent and complex solid-phase synthesis conditions, and may affect the function of the natural nucleosides. Most reported bioorthogonal-modified oligonucleotides are derived from phosphorous amide monomers modified at base sites; reports on introducing bioorthogonal groups using nucleoside substitutes are extremely rare. Therefore, to enrich the variety of phosphorous amide monomer modules containing bioorthogonal groups, it is essential to design and synthesize novel phosphorous amide monomers with simple and practical synthesis methods. This invention designs and synthesizes phosphoramidide monomers containing bioorthogonal groups using trans-4-hydroxymethylpiperidin-3-ol as the backbone, and prepares oligonucleotides modified with bioorthogonal groups using DNA / RNA solid-phase synthesis technology, providing usable molecular tools for the subsequent development of functional nucleic acids. Summary of the Invention

[0005] The purpose of this invention is to provide a phosphoramidite monomer containing a bioorthogonal group and an oligonucleotide modified with the bioorthogonal group.

[0006] This invention first discloses a phosphorus amide monomer containing a bioorthogonal group, which is a compound with the structure shown in Formula I:

[0007] Where n is an integer between 1 and 8.

[0008] This invention provides a method for preparing a phosphorus amide monomer containing a bioorthogonal group as shown in Formula I, comprising the following steps:

[0009] (a) trans-4-hydroxymethylpiperidine-3-ol and N -Benzyloxyformylaminobromoamine undergoes a nucleophilic reaction; (b) The product obtained in step (a) was reacted with 4,4′-dimethoxytriphenylmethyl chloride under anhydrous, oxygen-free and inert gas protection and catalyzed by 4-dimethylaminopyridine; (c) The product obtained in step (b) reacts with palladium on carbon and ammonium formate to remove the benzyloxycarbonyl protecting group; (d) The product obtained in step (c) was reacted with a carboxylic acid containing a bioorthogonal group under anhydrous, oxygen-free and inert gas protection, catalyzed by 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. (e) The product obtained in step (d) is subjected to anhydrous, oxygen-free, and inert gas protection, in N,N -Reaction of 2-cyanoethyl with diisopropylethylamine catalyzed by diisopropylethylamine N,N - The reaction of diisopropylchlorophosphamide yields a phosphorus amide monomer containing a bioorthogonal group.

[0010] This invention utilizes phosphoramidite monomers containing bioorthogonal groups to prepare bioorthogonal-modified oligonucleotides using a DNA / RNA synthesizer. The bioorthogonal groups can be introduced into any position on the oligonucleotide, providing a usable molecular tool for the subsequent development of functional nucleic acids. Simultaneously, it enriches the types of phosphoramidite monomer modules containing bioorthogonal groups, solving problems such as complex synthesis steps and limited coverage of nucleosides linked to the coupling handle in existing similar monomers. This provides support for post-synthetic modification of nucleic acids and related chemical biology and nucleic acid drug development research.

[0011] Oligonucleotides modified with bioorthogonal groups can be applied to nucleic acid-related research and detection. Leveraging the properties of bioorthogonal reactions, chemical labeling and imaging of nucleic acids in living cells can be achieved without interfering with the organism's own biochemical reactions. After nucleic acid synthesis, modification and the introduction of specific functional groups facilitate precise labeling and conjugation. Simultaneously, they can be used as nucleic acid-based biomimetic drugs for targeted disease therapy. Furthermore, bioorthogonal reactions can also produce oligonucleotide nanoprobes labeled with fluorescent or quencher groups for use in biological detection and disease diagnosis. Attached Figure Description

[0012] Figure 1 Synthetic routes for phosphorous amide monomers containing norbornene; Figure 2 1H NMR spectrum of phosphoramide monomers containing norbornene; Figure 3 Mass spectrometry of phosphoramide monomers containing norbornene; Figure 4 Mass spectrometry of norbornene-modified DNA. Detailed Implementation

[0013] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0014] This invention uses trans-4-hydroxymethylpiperidine-3-ol as the backbone and starting material, and its piperidine N and N Following a nucleophilic reaction, 3-benzyloxycarbonyl-3-bromoethylamine is protected by DMTr (4,4'-dimethoxytriphenylmethyl), deprotected by the benzyloxycarbonyl group, and then amidated and phosphoramidized with 5-norbornene-2-carboxylic acid to finally yield a phosphoramidite monomer containing norbornene. The above synthetic route is as follows: Figure 1 As shown.

[0015] Example 1 Synthesis of benzyl (2-(trans-3-hydroxy-4-hydroxymethylpiperidin-1-yl)ethyl)carbamate (compound 2): Take 530 mg of 4-hydroxymethyl-piperidine-3-ol into a 100 mL round-bottom flask, add 15 mL of anhydrous... N,N Dissolve dimethylformamide and add 970 mg. N -Benzyloxycarbonyl-3-bromoethylamine and 2.08 g of anhydrous potassium carbonate were reacted in an oil bath at 70 °C for 12 h. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with water and extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous NaSO4, and the solvent was evaporated. The residue was subjected to column chromatography (eluent: dichloromethane / methanol = 100 / 1-20 / 1). V / V Compound 2 was purified to obtain 360 mg of a pale yellow oily liquid, with a yield of 31.12%. 1 H NMR (400 MHz, Chloroform-d) δ: 7.33–7.21 (m, 5H), 5.02 (s, 2H), 3.68 (dd, J = 10.7, 3.7 Hz, 1H), 3.62–3.53 (m,2H), 3.22 (q, J = 5.8 Hz, 2H), 2.97–2.88 (m, 1H), 2.73 (d, J = 11.3 Hz, 1H), 2.47–2.36 (m, 2H), 1.96–1.75 (m, 2H), 1.54–1.41 (m, 2H), 1.22–1.11 (m, 1H).

[0016] Example 2 Synthesis of benzyl (2-(trans-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxypiperidin-1-yl)ethyl)carbamate (compound 3): Take 1.07 g of compound 2 into a 100 mL three-necked flask, add 42 mg of [unclear text - possibly a specific ingredient or ingredient]. N,N -Diisopropylethylamine, 1.9 mL anhydrousN, N - Diisopropylethylamine was dissolved in 20 mL of anhydrous dichloromethane, and after argon protection, the solution was transferred to a 0°C cold trap. 1.41 g of DMTr-Cl was dissolved in 5 mL of anhydrous dichloromethane and added dropwise to a three-necked flask. After the addition was complete, the mixture was allowed to react at room temperature for 4 h. The solution was extracted three times with dichloromethane, and the organic phase was collected. Column chromatography (eluent: dichloromethane / methanol = 100 / 1-20 / 1) was then performed. V / V The purified compound 3 was obtained as 910 mg of a pale yellow oily liquid, with a yield of 42.97%. 1 H NMR (400 MHz, Chloroform- d ) δ: 7.21(s, 14H), 6.76 (ddd, J = 8.9, 3.5, 1.4 Hz, 4H), 5.01 (s, 1H), 3.71 (d, J =2.1 Hz, 7H), 3.44 (td, J = 9.5, 4.4 Hz, 1H), 3.27–3.18 (m, 2H), 3.12–2.70 (m,4H), 2.67 (d, J = 10.4 Hz, 1H), 2.40 (t, J = 5.9 Hz, 2H), 1.79 (t, J = 10.1Hz, 2H), 1.58 (ddt, J = 13.3, 9.1, 4.3 Hz, 1H), 1.50–1.35 (m, 1H), 1.18 (s, 1H).

[0017] Example 3 Synthesis of trans-1-(2-aminoethyl)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidine-3-ol (compound 4): 910 mg of compound 3 and 564 mg of ammonium formate were placed in a 100 mL single-necked flask, dissolved in 15 mL of methanol, and 25 mg of palladium on carbon were added. The mixture was heated to 70 °C and refluxed for 3 h. After standing and returning to room temperature, the mixture was filtered through diatomaceous earth and distilled under reduced pressure to remove methanol and excess ammonium formate, yielding compound 4, a colorless oily liquid, 420 mg, yield: 59.00%. 1H NMR (400 MHz, Chloroform-d) δ: 7.94 (s, 1H), 7.36–7.28 (m, 2H), 7.21 (dd, J = 8.1, 5.6 Hz, 7H), 6.77–6.70 (m, 4H), 5.22 (s, 1H), 3.69 (s, 6H), 2.98 (s, 2H), 2.88 (s,8H), 2.47 (d, J = 38.8 Hz, 4H), 2.10 (d, J = 9.7 Hz, 1H), 1.68 (s, 2H).

[0018] Example 4 Synthesis of (1R,2S,4R)-N-(2-(trans-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxypiperidin-1-yl)ethyl)bicyclo[2,2,1]hept-5-en-2-carboxamide (compound 5): Take 94 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 60 mg of 4-dimethylaminopyridine into a 100 mL double-necked flask, and add 20 mL of anhydrous... N,N Dissolve dimethylformamide under argon protection and stir at 0°C for 5 min. Dissolve 50 mg of 5-norbornene-2-carboxylic acid and 20 mg of compound 4 separately in anhydrous dichloromethane and add them dropwise to a two-necked flask. After the addition is complete, stir for 30 min, raise the temperature to room temperature, and continue the reaction for 12 h. Evaporate the solvent, dissolve the residue in 10 mL of water, extract three times with ethyl acetate, wash once with saturated NaCl solution, collect the organic phase, and perform column chromatography (eluent: dichloromethane / methanol = 100 / 1-20 / 1). V / V After purification, compound 5 was obtained, 127 mg of colorless oily liquid, with a yield of 74.02%. 1 H NMR (400 MHz, Chloroform- d) δ: 7.37–7.29 (m, 2H), 7.27–7.20 (m, 6H), 7.18–7.11 (m, 1H), 6.82–6.71 (m, 4H), 6.12 (dd, J = 5.7, 3.1 Hz, 1H), 3.72 (s, 6H), 3.48 (td, J = 9.4, 4.4 Hz, 1H), 3.28 (dt, J = 9.3, 3.4 Hz, 1H), 3.20 (q, J = 5.7 Hz, 1H), 3.06–2.97 (m, 2H), 2.93 (dd, J = 10.7, 4.6 Hz, 1H), 2.85–2.73 (m, 2H), 2.69(d, J = 11.2 Hz, 1H), 2.39 (t, J = 6.0 Hz, 2H), 1.94–1.79 (m, 3H), 1.62 (q, J= 6.1 Hz, 1H), 1.45 (d, J = 12.8 Hz, 1H), 1.37 (dq, J = 8.3, 2.0 Hz, 1H), 1.21 (dd, J = 15.0, 6.1 Hz, 4H).

[0019] Example 5 Synthesis of trans-1-(2-((1R,2S,4R)-bicyclo[2,2,1]hept-5-en-2-carbamoyl)ethyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidin-3-ylbut-3-yn-1-yl diisopropylphosphonamide monomer (compound 6): Take 127 mg of compound 5 into a 100 mL double-necked flask, add 20 mL of anhydrous dichloromethane to dissolve it, and then add 0.07 mL of... N,N Diisopropylethylamine was slowly added dropwise with 0.07 mL of 2-cyanoethyl- at 0 °C under argon protection. N,N -Diisopropylphosphonamide, heated to room temperature and reacted for another 6 hours. After the reaction, the mixture was washed once with saturated NaHCO3 solution and once with saturated NaCl solution. The organic phase was dried over anhydrous NaSO4, the solvent was evaporated, and the residue was subjected to column chromatography (eluent: petroleum ether / ethyl acetate = 100 / 1-10 / 1). V / V Compound 6 was purified to obtain 94 mg of a pale yellow oily liquid, with a yield of 57.14%. 1H NMR (400 MHz, Chloroform-d)) δ 7.25–7.17 (m, 5H), 7.13–7.06 (m, 4H), 6.79–6.73 (m, 4H), 3.72 (s, 6H), 2.88 (s, 1H), 2.81 (s, 1H), 2.71 (d, J = 3.4 Hz, 1H), 1.38–1.28(m, 1H), 1.18 (s, 8H), 0.85–0.73 (m, 2H) ( Figure 2 ). HRMS (ESI):calcd.forC 46 H 61 N4O6P: 796.3429; found: 819.4206 [M+Na] + ( Figure 3 ).

[0020] Example 6 Synthesis of norbornene-modified oligonucleotides: Compound 6 was introduced into the desired position in the DNA sequence using a DNA / RNA solid-phase synthesis system. After ammonolysis and HPLC reverse-phase column purification, norbornene-modified DNA was obtained. The final product was confirmed by ESI-MS, with a theoretical value of 2965.08 and a measured value of 2765.4. Figure 4 ).

[0021] The oligonucleotide sequences used in this invention are as follows: DNA: 5'-GCAT- NBE -GATC-3' ( NBE Norbornene).

Claims

1. A phosphorus amide monomer containing a bioorthogonal group, characterized in that, It is a compound with the structure shown in Formula I: Where n is an integer between 1 and 8.

2. A method for preparing a phosphorus amide monomer containing a bioorthogonal group as described in claim 1, characterized in that, Includes the following steps: Wherein, n and R1 are as described in claim 1; Step a: trans-4-hydroxymethylpiperidine-3-ol and N -Benzyloxyformylaminobromoamine undergoes a nucleophilic reaction; Step b: The product obtained in step a reacts with 4,4′-dimethoxytriphenylmethyl chloride under the protection of anhydrous and oxygen-free inert gas and catalysis. Step c: The product obtained in step b reacts with palladium on carbon and ammonium formate to remove the benzyloxycarbonyl protecting group; Step d: The product obtained in step c reacts with a carboxylic acid containing a bio-orthogonal group under the protection of anhydrous and oxygen-free inert gas and catalysis. Step e: The product obtained in step d is catalyzed by a catalyst under anhydrous and oxygen-free inert gas protection, using 2-cyanoethyl... N, N -Diisopropylchlorophosphamide undergoes a phosphoramidation reaction to obtain a phosphoramid monomer containing a bioorthogonal group.

3. The application of the phosphoramidite monomer containing a bioorthogonal group as described in claim 1 in the preparation of bioorthogonal group-modified oligonucleotides, characterized in that, Biological orthogonal groups can be introduced into any position of oligonucleotides using DNA / RNA solid-phase synthesis technology.

4. A bioorthogonally modified oligonucleotide, characterized in that, The oligonucleotide was prepared by introducing the phosphoramidite monomer containing the bioorthogonal group described in claim 1 into any position using DNA / RNA solid-phase synthesis technology.

5. The application of the bioorthogonally modified oligonucleotides as described in claim 4 in nucleic acid-related research and detection.

6. The application of the bio-orthogonally modified oligonucleotides of claim 4 in the preparation of biomimetic drugs in medical treatment-related research.

7. The application of the bioorthogonally modified oligonucleotides of claim 4 in the preparation of nanoprobe materials.

Images