CN110590886B - Modified nucleoside, nucleotide and nucleic acid polymer as well as preparation method and application thereof - Google Patents
Modified nucleoside, nucleotide and nucleic acid polymer as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to modified nucleosides, nucleotides and nucleic acid polymers, and preparation methods and applications thereof. The modified nucleoside is selected from a compound with a structure shown in a formula (I), a salt or an isomer thereof; wherein R is1Selected from substituted or unsubstituted bases or salts thereof; x represents O or S; r2Selected from hydrogen or substituted or unsubstituted: c1～C6Alkyl radical, C1～C6Heteroalkyl group, C2～C6Alkenyl radical, C2～C6Alkynyl, aryl or heteroaryl; r3And R4Independently selected from hydrogen or substituted or unsubstituted: c1～C6Alkyl radical, C1～C6A heteroalkyl group; w1And W2Independently selected from H or a protecting group. The nitrile group is introduced into the 6' position of the modified nucleoside, and the modified nucleotide and the nucleic acid polymer are further obtained on the basis, so that the ribozyme tolerance of the modified nucleoside is greatly improved, and the modified nucleoside has high selectivity and strong binding affinity to target RNA.
Description
Technical Field
The invention relates to the fields of chemical modification of nucleotides and nucleic acid medicines, in particular to modified nucleosides, nucleotides and nucleic acid polymers as well as preparation methods and applications thereof.
Background
Compared with the traditional micromolecule and protein drugs, the nucleic acid drug has the advantages of rapid design, universal target, high specificity, capability of playing a role in cells, relatively rapid synthesis and preparation and the like, can break through the treatment of serious diseases of which the protein target is difficult to prepare, and has unique value particularly in emergency research and development of special cases, new emergent infectious diseases and the like.
However, natural oligonucleotide molecules also present a number of problems that must be overcome in order to be effective as therapeutic agents, such as: (1) the in vivo stability is poor, and the natural phosphodiester-linked oligonucleotides are easily and rapidly degraded by various ribozymes which are widely present in blood and cells in vivo; (2) low binding affinity and specificity with target genes, susceptibility to off-target effects, immunostimulation and toxic side effects, especially hepatotoxicity; (3) the bioavailability is low, oligonucleotides are usually polyvalent anionic macromolecules, and thus, target organs and tissues are difficult to enter, and lipophilic cell membranes are not easy to permeate into cells.
Chemical modification of nucleic acids is a key technology and bottleneck for the development of nucleic acid drugs. In view of the above problems, since 1970s, many successful oligonucleotide structure modification strategies have been developed by modifying phosphate backbone, sugar group, and base, such as phosphorothioate, 2 '-OMe modified at 2' -position, 2 '-OMOE, 2' -F, Morpholino, PNA, etc., which greatly improves the nuclease resistance of oligonucleotides, the affinity and specificity to target genes, reduces immune stimulation and toxic side effects, and promotes the development of nucleic acid drugs.
However, the currently approved nucleic acid drugs are all focused on the rare disease field, and further breakthrough is still expected in terms of major diseases, the problem of drug formation of the nucleic acid drug ADME-Tox and the like is still outstanding, and the need for a novel modified structure with high efficiency and low toxicity is very urgent, and particularly for the second-generation antisense nucleic acid drug gapmer, the development of C6' substituted LNA with high nuclease resistance and target RNA affinity is an important development direction of the new-generation nucleic acid chemical modification, and has important theoretical and practical significance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a modified nucleoside, wherein a nitrile group is introduced into the position of a bridge ring C6' to improve the ribozyme tolerance of the modified nucleoside when the modified nucleoside is used for nucleic acid drugs.
Meanwhile, the invention further provides modified nucleotides and nucleic acid polymers on the basis of the modified nucleosides, wherein the modified nucleotides and the nucleic acid polymers have good ribozyme tolerance, high selectivity and strong binding affinity for target RNA, and are beneficial to reducing off-target effect.
The invention also provides the application of the nucleic acid polymer in preparing nucleic acid diagnostic agents and/or nucleic acid therapeutic agents.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a modified nucleoside selected from a compound having a structure represented by formula (I), a salt thereof, or an isomer thereof:
wherein R is1Selected from substituted or unsubstituted bases or salts thereof; x represents O or S;
R2selected from hydrogen, or R2Selected from substituted or unsubstituted: c1～C6Alkyl radical, C1～C6Heteroalkyl group, C2～C6Alkenyl radical, C2～C6Alkynyl, aryl or heteroaryl; said C is1～C6The heteroatom of heteroalkyl or said heteroaryl is selected from O, S, N, P or Si;
R3and R4Independently selected from hydrogen, or R3And R4Independently selected from substituted or unsubstituted: c1～C6Alkyl radical, C1～C6A heteroalkyl group;
W1and W2Independently selected from H or a protecting group.
Alternatively, the protecting group may be selected from hydroxyl protecting groups conventional in the art, such as benzyl (Bn), 4' -dimethoxytrityl (DMTr), silicon protecting groups (e.g., t-butyldiphenylsilane, etc.), and the like.
The modified nucleoside provided by the invention is modified by introducing a nitrile group (-CN) at the site of bridge ring C6' to obtain the modified nucleoside with a novel structure, and can effectively enhance the tolerance of the modified nucleoside to nuclease.
After a polar group CN is introduced to the 6' position, the steric hindrance of a methylene bridge in a nucleoside structure can be further increased, and higher nuclease tolerance is facilitated to be obtained; the hydration of the polar group CN is stronger, the antisense activity and the tissue distribution can be further improved, more structures are provided, and the optimization space of modification is enlarged; the introduction of the polar group CN can greatly improve the cutting specificity of RNaseH enzyme on target RNA; meanwhile, the length of the antisense gapmer sequence is shortened, and the binding specificity to the target RNA is greatly improved, so that the off-target effect of antisense nucleic acid is greatly reduced, and the hepatotoxicity is reduced.
Alternatively, the R is1Selected from substituted or unsubstituted: purine, pyrimidine or their respective salts.
Alternatively, the R is1Selected from substituted or unsubstituted: adenine (a), guanine (G), thymine (T), cytosine (C), uracil (U), or their respective salts.
Alternatively, when said R is1When selected from substituted cytosines, said R1Is 5' -alkyl substituted cytosine.
Alternatively, the R is1Is 5 '-methyl-substituted cytosine (5' -mC).
Alternatively, when R1、R2、R3And R4When substituted, the substituents may be independently selected from halogen, alkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, mercapto, amino, amide, carbonyl, carboxyl, sulfonic, ester groups, and the like.
Alternatively, the R is2Is hydrogen.
Alternatively, the R is3And R4Independently selected from hydrogen.
On the basis of any of the above nucleosides, the present invention also provides a nucleotide including a 3' -phosphoramidite derivative of any of the above modified nucleosides or a salt thereof.
Alternatively, the nucleotide is selected from a compound having a structure shown in formula (II), a salt thereof or an isomer thereof:
optionally, W in the formula (II)2Is 4, 4' -dimethoxytrityl (DMTr).
Modified oligonucleotides can be obtained by incorporating the above nucleotides into oligonucleotide sequences using standard synthetic methods.
As an embodiment, the modified oligonucleotide is obtained by inserting the above-mentioned nucleotide into an oligonucleotide sequence by an automated synthesizer using a standard phosphoramidite method.
According to another aspect of the present invention, there is also provided a nucleic acid polymer.
The nucleic acid polymer comprises at least one nucleotide structure as described above.
Alternatively, the nucleic acid polymer contains a condensation product structure of the above-mentioned nucleotide.
Optionally, the nucleic acid polymer comprises ribonucleic acid, deoxyribonucleic acid, or a copolymer of ribonucleotides and deoxyribonucleotides.
The invention also provides a preparation method of the nucleoside. When R is3And R4Independently of one another is hydrogen, R1In the case of thymine, the method comprises:
reacting the reaction substrate (I) with thymine in the presence of a protective agent to produce a compound (II);
in the presence of a catalyst A, carrying out intramolecular cyclization reaction on the compound (II) to generate a compound (III); protecting group W of Compound (III)2' removing and oxidizing in the presence of an oxidizing agent to form compound (IV);
in the presence of a catalyst B, carrying out nucleophilic substitution reaction on the compound (IV) and a nitrile source to obtain a compound (V) and/or an isomer thereof;
converting compound (V) and/or its isomer into compound (VI) and/or its isomer under basic conditions;
wherein the structural formula of each compound is shown as the following formula, W1、W2And W2' is hydrogen or a protecting group:
optionally, the protecting reagent is N, O-bis (trimethylsilyl) acetamide (BSA).
Alternatively, the catalyst A is selected from 4-Dimethylaminopyridine (DMAP) and/or trifluoromethanesulfonic anhydride (Tf)2O)。
Optionally, the oxidizing agent is selected from at least one of 2-iodoxybenzoic acid, dess-martin reagent, dimethyl sulfoxide-oxalyl chloride, preferably 2-iodoxybenzoic acid.
Optionally, the catalyst B is selected from AlCl3、CeCl3、ZnCl2、TiCl4Preferably AlCl3。
Optionally, the nitrile source is selected from at least one of Trimethylnitrilosilane (TMSCN), sodium cyanide, potassium ferricyanate, preferably trimethylnitrilosilane.
The invention also provides the application of the nucleic acid polymer in preparing nucleic acid diagnostic agents and/or nucleic acid therapeutic agents.
Compared with the prior art, the invention has the beneficial effects that:
according to the modified nucleoside provided by the invention, a novel 6'-CN modified structure is obtained by introducing-CN at the position of C6', and a nucleotide and a nucleic acid polymer are obtained by further modification, so that the ribozyme tolerance is greatly improved, the affinity to target RNA is high, the modified nucleoside has high RNA selectivity, good antisense property is shown, and the hepatotoxicity is low.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the results of the hydrolysis tolerance of Snake Venom Phosphodiesterase (SVPDE) comprising a sequence having a nitrile group at the 6' position substituted for the nucleotide structure and other alignment sequences in accordance with one embodiment of the present invention;
FIG. 2 shows the mass spectrum of the nucleotide sequence ON1 according to one embodiment of the present invention;
FIG. 3 shows the mass spectrum of the nucleotide sequence ON2 according to one embodiment of the present invention;
FIG. 4 shows the result of mass spectrometry of nucleotide sequence ON3 according to one embodiment of the present invention;
FIG. 5 shows the mass spectrum of the nucleotide sequence ON4 according to one embodiment of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
As used herein, the term "C1～C6"means having 1 in the main chain of the groupAny integer value of carbon atoms in the range of to 6, for example 1, 2, 3, 4,5, 6 carbon atoms. Similarly, the term "C2～C6"refers to a group having any integer value of carbon atoms in the backbone ranging from 2 to 6, for example 2, 3, 4,5, 6 carbon atoms.
As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having a straight chain or a branched chain; non-limiting examples thereof include methyl, ethyl, propyl, n-butyl, t-butyl, pentyl, hexyl and the like.
As used herein, the term "alkenyl" refers to a hydrocarbyl group having at least one carbon-carbon double bond at one or more positions along the carbon chain of the alkyl group; non-limiting examples thereof include ethenyl, propenyl, butenyl and the like.
As used herein, the term "alkynyl" refers to a hydrocarbon group having at least one carbon-carbon triple bond at one or more positions along the carbon chain of the alkyl group; non-limiting examples thereof include ethynyl, propynyl, and the like.
As used herein, the term "aryl" refers to a group comprising a carbocyclic aromatic system; non-limiting examples thereof include phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, and the like; when the aryl group includes a plurality of rings, the respective rings may be fused to each other.
As used herein, the term "heteroaryl" refers to a group having a carbocyclic aromatic system containing at least one heteroatom selected from N, O, Si, P and S as a ring-forming atom; non-limiting examples thereof include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolyl, isoquinolyl and the like; when the heteroaryl group includes a plurality of rings, the respective rings may be fused to each other.
As used herein, the terms "heteroalkyl," "heteroaryl" refer to an alkyl group containing at least one heteroatom selected from N, O, Si, P and S.
The term "salt" as used herein refers to a corresponding salt, e.g., a pharmaceutically acceptable salt, of a modified nucleoside compound (or nucleotide compound) of the present invention which can be conveniently or desirably prepared, purified and/or processed. Unless otherwise indicated, reference to a particular compound in the present invention also includes its salt form.
As used herein, the term "nucleic acid polymer" may refer to any nucleic acid molecule, including but not limited to DNA, RNA, and hybrids thereof, including but not limited to single stranded, double stranded, and the like. The number of nucleotides polymerized to form the nucleic acid is 2, 3 or more, and may be an oligonucleotide having a number of nucleotides of 20 or less, or a polymer having a number of nucleotides of 20 or more.
As used herein, the portion between the wavy lines in the nucleic acid polymer is a structure embedded in the nucleic acid polymer sequence, and the portion outside the wavy lines represents other sequences in the nucleic acid polymer.
In one embodiment, the modified nucleoside of the present invention is selected from a compound having a structure represented by the following formula:
wherein R is1Selected from substituted or unsubstituted: a purine, pyrimidine or their respective salts; r2、R3And R4Are all hydrogen.
In one embodiment, the modified nucleoside of the present invention is selected from a compound having a structure represented by the following formula:
wherein R is1Selected from substituted or unsubstituted: a purine, pyrimidine or their respective salts; r2、R3And R4Are all hydrogen.
As an embodiment, the nucleotide can be obtained by subjecting the hydroxyl group at the 3' -position or the protecting group structure to a phosphating treatment on the basis of the modified nucleoside in the present invention, and the nucleotide is selected from a compound having a structure represented by the following formula:
in one embodiment, the nucleic acid polymer is obtained by condensing a compound containing the nucleotide to obtain a condensate.
As an embodiment, a polymer or a sequence comprising a condensate of the above-mentioned nucleotide also falls within the scope of the nucleic acid polymer of the present invention.
In one embodiment, the structure of the nucleic acid polymer of the present invention includes a structure in which at least one nucleotide is modified by being incorporated into an oligonucleotide sequence.
EXAMPLE 1 preparation of modified nucleosides
In this example, the synthetic route for the modified nucleoside is as follows:
wherein the reaction conditions of the steps are as follows, and the nuclear magnetic resonance and mass spectrometry are adopted to characterize the structure of the compound:
a: in a solvent DMF, taking a compound 1 as a substrate, taking tert-butyldiphenylchlorosilane (TBDPSCl) as a protective reagent, and carrying out quantitative reaction for 12h at room temperature in the presence of imidazole to complete hydroxyl protection to obtain a compound 2. The method comprises the following specific steps:
TBDPSCl (10mL,37.5mmol) was added to a solution of compound 1(10g,25mmol) and imidazole (3.86g,56mmol) in 100mL of anhydrous DMF under ice-bath, reacted at room temperature for 12h, quenched with excess methanol, extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by flash column (petroleum ether/ethyl acetate: 9/1) to give 16.5g of anhydrous oil 2, yield 97%. The structure confirmation results of compound 2 are as follows:
1H-NMR(400MHz,CDCl3)δ7.70-7.20(m,20H,ArH),5.77(d,J＝3.6Hz,1H,H-1),4.68-4.44(m,5H,5-OBnH,H-2,6-CH2),4.21(d,J＝4.2Hz,1H,H-3),4.10-4.30(m,2H,3-OBnH),3.75(d,J＝10.4Hz,1H,5-Ha),3.63(d,J＝10.0Hz,5-Hb)；
13C-NMR(100MHz,CDCl3)δ135.51,135.29,133.23,133.08,130.90,130.57,126.84,125.67,125.60,125.08,124.97,124.92,110.66,101.55,85.02,77.00,75.49,71.03,69.78,69.34,62.05,24.22,23.95,23.69,16.66；
ESI-MS(m/z)656.32[M+NH4]+。
b: in a solvent of ethyl acetate, firstly, reacting a compound 2 at room temperature for 12 hours in the presence of acetic anhydride, acetic acid and sulfuric acid; then taking acetonitrile as a solvent, and carrying out reflux reaction with thymine for 18h in the presence of N, O-bis (trimethylsilyl) acetamide (BSA) and trimethylsilyl trifluoromethanesulfonate to obtain a compound 3.
The method comprises the following specific steps:
adding 20mL of acetic acid, 35mL of acetic anhydride and a catalytic amount of concentrated sulfuric acid (160 μ L,2.6mmol) in sequence to a 40mL ethyl acetate solution of compound 2(18.0g,28.2mmol) at room temperature, reacting at room temperature for 3h, detecting completion of the reaction by TLC (petroleum ether/ethyl acetate: 9/1), extracting with ethyl acetate, washing with water, washing with saturated sodium bicarbonate, drying with anhydrous sodium sulfate, filtering, concentrating, vacuum drying, dissolving the obtained residue in 200mL acetonitrile, adding thymine (10.6g,84mmol), adding N, O-bis (trimethylsilyl) acetamide (34.0mL,140mmol), slightly heating to completely dissolve the thymine, cooling to room temperature, adding TMSOTf (7.7mL,42mmol), refluxing for 18h, stopping heating, slowly pouring the reaction solution into 1L after the reaction solution is cooled to room temperature, a large amount of white solid was precipitated, and the precipitate was allowed to stand, filtered, dissolved in methylene chloride, filtered through celite, washed with water, washed with a saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain 18.8g of 3 as a white solid in a yield of 89%. The structure confirmation results of compound 3 are as follows:
1H-NMR(400MHz,CDCl3)δ8.45(s,1H,NH),7.64-7.20(m,21H,ArH,6-H),6.17(d,J＝6.0Hz,1’-H),5.39(t,J＝5.6Hz,1H,H-2),4.59-4.52(m,4H,ArC 2H×2),4.21(d,J＝5.6Hz,1H,H-3),3.96-3.67(m,4H,6’-C 2H,5’-C 2H),1.96(s,3H,Ac),1.52(s,3H,5-CH3),1.06(s,9H,TBDPS-H)；
13C-NMR(100MHz,CDCl3)δ172.42,165.67,152.55,139.72,139.41,137.96,137.93,137.74,135.14,134.79,130.88,130.59,130.34,130.08,129.97,129.94,129.92,129.88,113.53,90.10,87.66,79.93,77.15,76.85,75.96,74.44,66.05,29.09,22.86,21.41,14.24；
ESI-MS(m/z)771.33[M+Na]+。
c: with K2CO3Basic conditions were provided and Compound 3(20g,26.7mmol) was added to 70mL of 2N NH3-CH3To the OH solution, the reaction was carried out at room temperature for 48h, concentrated under reduced pressure, and then dissolved with DCM, followed by flash column purification (dichloromethane/methanol-10/1) to obtain 18g of product 4 as a white solid with a yield of 90%. The structure of compound 4 was confirmed as follows:
1H-NMR(400MHz,DMSO-d6)δ11.38(s,1H,NH),7.62-7.16(m,21H,ArH,6-H),5.91(d,J＝6.8Hz,1H,H-1’),5.58(d,J＝6.4Hz,1H,2-OH),4.83-4.87(m,5H,ArC 2H×2,H-2’),4.15-3.75(m,5H,3’-H,6’-C 2H,5’-C 2H),1.57(s,3H,5-CH3),0.96(s,9H,TBDPS-H)；
13C-NMR(100MHz,DMSO-d6)δ163.81,151.20,138.57,138.20,136.02,135.39,135.29,132.90,132.72,130.08,130.01,128.61,128.32,128.10,127.86,127.70,127.60,127.55,110.02,86.47,86.19,79.32,73.40,73.12,73.03,71.60,64.30,26.78,19.01,12.15；
ESI-MS(m/z)707.32[M+H]+,729.30[M+Na]+。
d: in DMAP and Tf2In the presence of O, with CH2Cl2As a solvent, the compound 4 reacts at 0 ℃ for 2h to undergo intramolecular cyclization reaction to obtain a compound 5. The method comprises the following specific steps:
to a solution of compound 4(17g,24mmol) and DMAP (11.7g,96mmol) in 100mL of dichloromethane was slowly added dropwise a solution of trifluoromethanesulfonic anhydride (12mL,72mmol) in 50mL of dichloromethane under ice bath, after dropwise addition, the reaction was carried out at room temperature for 2 hours, quenched with ice water, extracted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and flash column purified (dichloromethane/methanol ═ 10/1) to give 12.3g of product 5 as a white solid in 74% yield. The structure confirmation results of compound 5 are as follows:
1H-NMR(400MHz,CDCl3)δ7.66-7.21(m,21H,ArH,H-6),6.25(d,J＝6.0Hz,1H,H-1’),4.81(d,J＝11.6Hz,1H,5’-OCHa-Ar),4.62(d,J＝11.2Hz,1H,5’-OCHb-Ar),4.36-4.31(m,2H,5’-OC 2H-Ar),3.84(d,J＝10.8Hz,1H,6’-Ha),3.70(d,J＝10.4Hz,1H,6’-Hb),3.36(dd,J＝10.8,15.2Hz,2H,5’-CH2),1.98(s,3H,5-CH3),1.03(s,9H,(C 3 3H)C-)；
13C-NMR(100MHz,CDCl3)δ172.29,159.23,137.06,136.46,135.59,135.43,132.35,131.95,130.15,129.93,129.88,128.54,128.37,128.24,127.89,127.83,127.73,127.53,119.21,90.02,88.79,87.21,84.06,73.50,69.48,64.12,26.75,18.89。
e: THF is used as a solvent, and the compound 5 reacts for 2 days at room temperature in the presence of tetrabutylammonium fluoride (TBAF), and the TBDPS-protective group is removed to obtain a compound 6. The method comprises the following specific steps:
to a solution of compound 5(15g,21.8mmol) in 200mL of tetrahydrofuran at room temperature was added 5mL of water, followed by addition of TBAF.3H2O (10.3g,32.7mmol), reacted at room temperature for 2 days, extracted with ethyl acetate, washed with water to precipitate a large amount of solid, filtered and dried to give 9.3g of product 6 as a white solid in 95% yield. The structure confirmation results of compound 6 are as follows:
1H-NMR(400MHz,DMSO-d6)δ7.76(d,J＝1.2Hz,1H,H-6),7.38-7.13(m,10H,ArH),6.34(d,J＝6.4Hz,1H,H-1’),5.51(dd,J＝6.0,2.8Hz,1H,H-2’),5.08(t,J＝5.2Hz,1H,6’-OH),4.78(d,J＝12.0Hz,1H,3’-OC aH-Ar),4.62(d,J＝12.0Hz,1H,3’-OC bH-Ar),4.36(s,2H,5’-OC 2H-Ar),4.27(d,J＝3.2Hz,1H,H-3’),3.65(dd,J＝11.2,5.2Hz,1H,6’-Ha),3.54(dd,J＝11.2,4.8Hz,1H,6’-Hb),3.40(s,2H,5’-CH2),1.80(d,J＝1.2Hz,1H,5-CH3)；
13C-NMR(100MHz,DMSO-d6)δ171.16,158.86,137.41,137.03,131.80,127.90,127.79,127.35,127.27,127.05,126.80,116.37,82.29,88.26,86.23,83.11,72.10,71.49,69.24,60.47,13.10。
f: performing reflux reaction for 6h by using IBX as an oxidant and acetonitrile as a solvent, oxidizing hydroxyl without a protective group in the compound 6 into carbonyl to obtain an aldehyde intermediate, filtering and pumping to dry;
TMSCN is used as nitrile source, AlCl is used as3As a catalyst, an aldehyde intermediate in CH2Cl2Reacting for 12 hours at a medium temperature, and then reacting for 1 hour at the room temperature by taking THF as a solvent in the presence of tetrabutylammonium fluoride (TBAF) to obtain a mixture 7 with an R/S configuration; the structure confirmation results of compound 7 are as follows:
1H-NMR(400MHz,CD3OD)δ7.71(d,J＝1.2Hz,1H,H-6),5.69(s,1H,H-1’),4.93(s,1H,H-6’),4.46(s,1H,H-2’),4.28(s,1H,H-3’),3.97(s,2H,5’-CH2),1.89(d,J＝1.2Hz,1H,5-CH3)；
13C-NMR(100MHz,CD3OD)δ166.42,151.79,136.28,117.52,111.13,90.91,88.08,82.13,71.36,70.78,56.63,12.67；
ESI-MS(m/z)296.10[M+H]+。
g: at K2CO3In the presence of acetonitrile as a solvent, carrying out reflux reaction on the compound 7 for 4h to obtain a compound S-8 with an S configuration and a compound R-8 with an R configuration.
In the final product, compound S-8 (about 25 wt.%) was an amorphous solid and compound R-8 (about 75 wt.%) was needle-like crystals. The structural characterization results of compound S-8 and compound R-8 are as follows:
compound S-8:
1H-NMR(400MHz,DMSO-d6)δ11.47(s,1H,NH),7.37-7.24(m,11H,H-6,ArH),5.59(s,1H,H-1’),5.11(s,1H,H-6’),4.84(s,1H,H-2’),4.75-4.61(m,4H,ArC 2H×2),4.24-4.07(m,3H,H-3’,5’-C 2H),1.64(s,3H,5-C 3H)；
13C-NMR(100MHz,CDCl3)δ163.74,149.81,137.63,137.29,134.11,128.33,128.15,127.71,127.58,127.31,115.30,108.87,87.98,86.14,77.95,76.28,73.00,71.27,68.72,64.46,12.15；
compound R-8:
1H-NMR(400MHz,DMSO-d6)δ11.45(s,1H,NH),7.35-7.31(m,11H,H-6,ArH),5.67(s,1H,H-1’),5.19(s,1H,H-6’),4.78(s,1H,H-2’),4.67-4.60(m,4H,ArC 2H×2),4.25(s,1H,H-3’),4.05(d,J＝11.2Hz,1H,H-5a’),3.93(d,J＝11.2Hz,1H,H-5b’),1.59(d,J＝1.2Hz,1H,H-5)；
13C-NMR(100MHz,CDCl3)δ163.74,149.81 137.70,137.18,133.94,128.30,128.30,128.23,127.65,127.42,116.81,108.87,87.20,86.28,79.98,76.18,72.87,71.23,70.65,63.90,12.15；
the mass spectrum results for compound S-8 and compound R-8 are: ESI-MS (M/z)476.19[ M + H ]]+。
EXAMPLE 2 preparation of nucleotides
In this example, nucleotides were obtained by further modification based on the modified nucleoside R-8 obtained in example 1, and the synthetic route was as follows:
wherein the reaction conditions of the steps are as follows:
h: with CH2Cl2As a solvent in anhydrous FeCl3In the presence, the compound R-8 reacts for 2h at room temperature to obtain a compound 9.
i: and (3) reacting the compound 9 at room temperature for 5 hours in the presence of pyridine by using DMTrCl as a protective reagent to obtain a compound 10. The structure confirmation results for compound 10 are as follows:
1H-NMR(400MHz,DMSO-d6)δ11.48(s,1H,NH),7.47-6.91(m,14H,DMTrH),6.27(d,J＝4.0Hz,1H,H-1’),5.63(s,1H,H-6’),5.01(s,1H,H-2’),4.52(s,1H,H-3’),3.42(d,J＝4.4Hz,1H,3’-OH),3.74(s,6H,OC 3H×2),3.65(d,J＝11.2Hz,1H,5’-Ha),3.36(d,J＝11.2Hz,1H,5’-Hb),1.60(d,J＝0.8Hz,1H,5-CH3)；
13C-NMR(100MHz,DMSO-d6)δ163.81,158.27,149.91,144.50,135.07,134.84,134.00,129.91,129.84,128.00,127.71,126.94,117.41,113.34,109.01,87.99,86.38,86.35,80.37,70.52,70.33,58.47,55.09,12.35；
ESI-MS(m/z)620.20[M+Na]+。
j: compound 10 was reacted in acetonitrile solvent with 2-cyanoethyl N, N '-tetraisopropylphosphorodiamidite (2-cyanoethyl N, N' -tetraisopropylphosphorodiamidite, bis (diisopropylamino) (2-cyanoethoxy) phosphine) as a phosphating agent in the presence of 4, 5-dicyanoimidazole at room temperature for 8h to give compound 11. The results of the phosphorus and mass spectrometric characterization of compound 11 are as follows:
31P-NMR(152MHz,DMSO-d6)δ149.35,149.29；
ESI-HRMS(m/z)798.3264[M+H]+,820.3077[M+Na]+。
EXAMPLE 3 preparation of oligonucleotide sequences
Based ON the nucleotides prepared in example 2, the phosphoramidite monomers obtained in the above examples were incorporated into the corresponding oligonucleotide sequences using the standard phosphoramidite method above for DNA automated synthesis to obtain the following oligonucleotides ON 1-ON 4, as shown in Table 1. The mass spectrometry results of ON 1-ON 4 are shown in FIG. 2, FIG. 3, FIG. 4, and FIG. 5, respectively.
In Table 1, underlined bases are sites modified by substitution, i.e.TTo replace the modified nucleotide with a nucleotide according to the invention.
TABLE 1 oligonucleotide sequences ON 1-ON 4 and their corresponding mass spectral data
Experimental example 1 Properties of oligonucleotides
To illustrate the properties of the modified nucleosides and nucleotides obtained in the different examples of the present invention, the properties of the different modified oligonucleotides obtained in example 3 were tested.
Nuclease resistance test
Based ON the ON4 sequence obtained in example 3, one of the ON4 sequencesTThe sequence obtained by the replacement with LNA is denoted ON 5;
will be in the ON4 sequenceTThe sequence obtained by replacing Ts (3' -phosphothioate-T) with Ts was designated ON 6;
will be in the ON4 sequenceTThe sequence obtained by substitution with the natural base T was designated ON 7;
native sequence GCGTTTTTTGCT was designated as ON 8.
The experimental method is as follows:
carrying out enzymolysis on the oligonucleotide under the physiological condition of 37 ℃; taking out the incubation liquid at different time points (0min, 2min, 5min, 10min, 20min, 30min and 40min), and quantifying by using HPLC to obtain corresponding content-time curves;
a buffer system: 50mM Tris-HCl, 10mM MgCl2，pH 8.0；
Conditions for HPLC analysis: waters model HPLC, flow rate: 1 mL/min; sample introduction amount: 10 mu L of the solution; mobile phase A: water, mobile phase B: methanol; the gradient is set from A: B98: 2(v/v) to A: B92: 8(v/v) within 0-8 min; wavelength of ultraviolet detector: 260 nm;
and (3) enzyme stability determination: mu.L (about 7nmol based on molecular weight-3000) of 1. mu.g/. mu.L sample was dissolved in 375. mu.L buffer, while 0.02. mu.g/. mu.L of SVPDE 5. mu.L or 5. mu.L of high purity water was added as a blank (total volume of 400. mu.L), and incubated at 37 ℃; taking out 50 μ L of the incubation liquid at 0min, 2min, 5min, 10min, 20min, 30min and 40min respectively, precipitating protein with 150 μ L of methanol, centrifuging at 10000rpm for 10min, taking 150 μ L of supernatant, draining, adding 150 μ L of water, and detecting the content of the sample by HPLC method. Based on the measured percent content of the undegraded sample, a sample content-time curve is prepared.
The results of nuclease resistance experiments are shown in FIG. 1, under the condition of 1.0 μ g/mL SVPDE, the sequence ON4 in the invention is degraded slowly, more than 40% of the sequence is not degraded at 40min, and the LNA modified ON5 sequence is only remained less than 10%, which shows that the 6' position-CN substituted LNA in the invention can greatly improve the nuclease resistance of the oligonucleotide and is better than LNA.
Thermal denaturation test
Based ON the ON1, ON2 and ON3 sequences obtained in example 3, one of the ON1, ON2 and ON3 sequencesTThe resulting sequences were designated Y-ON1, Y-ON2, and Y-ON3, respectively, with Y (LNA-T) replaced.
The experimental method comprises the following steps:
annealing buffer solution: 10mM Na3PO4100mM NaCl, pH 7.4 (in the case of using phosphate buffer solution of pH 7.4 as it is, it should be noted that MgCl is not contained2)；
The annealing method comprises the following steps: diluting the two oligonucleotide single strands with annealing buffer solution to make the final concentration of the two oligonucleotide single strands be 2 μ M and the volume be 3.5mL (800 μ L × 3 times can be measured, and CD spectrum is 1mL, each strand needs 7nmol), heating in water bath at 95 ℃ for 5min (taking care to cover tightly and prevent from being blown open by heat), slowly cooling to room temperature, and placing in a refrigerator at 4 ℃ overnight;
Tmthe determination method comprises the following steps: add 800. mu.L of the sample to be tested to the cuvette and cover it firmly with a heat-isolating lid. Selecting 15 ℃ as initial measurement temperature, 85 ℃ as termination temperature, the temperature rise rate is 1 ℃/min, the A260 reading rate is 2 times/DEG C, and finally, giving out T by an instrumentmA value; the assay was repeated 3 times for each sample and the average was taken as the final result.
TABLE 2 melting temperature T of DNA/RNA duplexes formed by the oligonucleotide sequencesmValue (. degree. C.)
In table 2, Y ═ LNA-T, corresponding TmValues were derived from literature (chem. Commun.,2015,51, 9737-9740); delta Tm＝Tm(modified)-Tm(ON8)。
As a result of heat denaturation experiments, it was found that R-6' -CN-T in the present invention is superior to the natural sequence ON8LModification sequences ON 1-ON 3 and targetsSignificantly enhanced RNA binding affinity,. DELTA.TmAt a temperature of 3.3 ℃ to 3.6 ℃ in relation to the Mod, although lower than the LNA modification, the RNA selectivity of ON1 to ON3 is comparable to that of LNA, and the binding affinity to the corresponding DNA is hardly changed (by less than 1 ℃) since R-6' -CN-TLThe 100% 3 '-endo configuration of glycosyl locking and hydration enhance the stability of double chains, therefore, the reason that 6' -CN-LNA has weaker binding affinity to target RNA than LNA is inferred that strong electronegative CN can enhance electrostatic repulsion between two anionic single chains, which is comprehensively expressed as high selectivity and stronger binding affinity to target RNA.
In conclusion, the preliminary property evaluation result shows that the 6 '-CN-LNA modified sequence obtained by introducing-CN at the C6' position can obviously improve the nuclease tolerance and the binding affinity with the target RNA, and the unique affinity and selectivity to the target RNA provide a new probe for further clarifying the action mechanism of RNaseH to antisense gapmer-RNA and provide a new structure-activity relationship for developing a novel efficient and low-toxicity nucleic acid chemical modification structure.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> modified nucleoside, nucleotide and nucleic acid polymer, and preparation method and application thereof
<130> PA19029815
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> DNA
<213> Artificial sequence ON1
<400> 1
gcgttttttg ct 12
<210> 2
<211> 12
<212> DNA
<213> Artificial sequence ON2
<400> 2
gcgttttttg ct 12
<210> 3
<211> 12
<212> DNA
<213> Artificial sequence ON3
<400> 3
gcgttttttg ct 12
<210> 4
<211> 10
<212> DNA
<213> Artificial sequence ON4
<400> 4
<210> 5
<211> 12
<212> DNA
<213> Artificial sequence ON8
<400> 5
gcgttttttg ct 12
<210> 6
<211> 12
<212> DNA
<213> Artificial sequence Y-ON1
<400> 6
gcgttytttg ct 12
<210> 7
<211> 12
<212> DNA
<213> Artificial sequence Y-ON2
<400> 7
gcgttytytg ct 12
<210> 8
<211> 12
<212> DNA
<213> Artificial sequence Y-ON3
<400> 8
gcgytytytg ct 12
Claims (13)
1. A modified nucleoside selected from compounds having a structure represented by formula (I), salts thereof or isomers thereof:
wherein R is1Selected from substituted or unsubstituted: adenine, guanine, thymine, cytosine, uracil, or their respective salts; x represents O;
R2selected from hydrogen, or R2Selected from substituted or unsubstituted: c1～C6Alkyl radical, C1～C6Heteroalkyl group, C2～C6Alkenyl radical, C2～C6Alkynyl, aryl or heteroaryl;
the aryl is selected from phenyl, naphthyl, anthryl, phenanthryl or pyrenyl; the heteroaryl group is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolinyl, or isoquinolinyl;
said C is1～C6The heteroatom of the heteroalkyl group is selected from O, S, N, P or Si;
when R is1And R2When substituted, the substituents are selected from halogen, methyl, ethyl, propyl, n-butyl, tert-butyl, pentyl, hexyl, hydroxy, mercapto, amino, carboxy or sulfonic acid;
R3and R4Independently selected from hydrogen;
W1and W2Independently selected from protecting groups.
2. The modified nucleoside of claim 1, wherein when R is1Is selected fromWhen substituted cytosine, said R1Is 5' -methyl-substituted cytosine.
3. A nucleotide, wherein the nucleotide comprises a 3' -phosphoramidite derivative of a modified nucleoside of claim 1 or 2, or a salt thereof.
4. The nucleotide according to claim 3, wherein the nucleotide is selected from a compound having a structure represented by formula (II), a salt thereof, or an isomer thereof:
5. the nucleotide of claim 4, wherein W in the formula (II)2Is 4, 4' -dimethoxytrityl.
6. A nucleic acid polymer, characterized in that it comprises at least one nucleotide according to any one of claims 3 to 5.
7. The nucleic acid polymer of claim 6, wherein the nucleic acid polymer is a ribonucleic acid, a deoxyribonucleic acid, or a copolymer of a ribonucleotide and a deoxyribonucleotide.
8. A method of preparing a modified nucleoside according to claim 1 or 2, comprising the steps of:
when R is3And R4Independently of one another is hydrogen, R1In the case of thymine, the method comprises:
reacting the reaction substrate (I) with thymine in the presence of a protective agent to produce a compound (II); in the presence of a catalyst A, carrying out intramolecular cyclization reaction on the compound (II) to generate a compound (III); protecting group W of Compound (III)2' removal from, and in, oxidizing agentsIn the presence of oxygen to produce compound (IV); in the presence of a catalyst B, carrying out nucleophilic substitution reaction on the compound (IV) and a nitrile source to obtain a compound (V) and/or an isomer thereof; converting compound (V) and/or its isomer into compound (VI) and/or its isomer under basic conditions;
wherein the structural formula of each compound is shown as the following formula, W1、W2And W2' is hydrogen or a protecting group:
9. the process according to claim 8, characterized in that the catalyst A is selected from 4-dimethylaminopyridine and/or trifluoromethanesulfonic anhydride.
10. The method of claim 8, wherein the oxidizing agent is selected from at least one of 2-iodoxybenzoic acid, dess-martin reagent, dimethyl sulfoxide-oxalyl chloride.
11. The process of claim 8 wherein the catalyst B is selected from AlCl3、CeCl3、ZnCl2、TiCl4At least one of (1).
12. The method of claim 8, wherein the nitrile source is selected from at least one of trimethylnitrilosilane, sodium cyanide, potassium ferricyanate.
13. Use of the nucleic acid polymer according to claim 6 or 7 for the preparation of a nucleic acid diagnostic and/or therapeutic agent.
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