WO2021197371A1 - Biological conjugate containing rigid linker - Google Patents

Biological conjugate containing rigid linker Download PDF

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WO2021197371A1
WO2021197371A1 PCT/CN2021/084341 CN2021084341W WO2021197371A1 WO 2021197371 A1 WO2021197371 A1 WO 2021197371A1 CN 2021084341 W CN2021084341 W CN 2021084341W WO 2021197371 A1 WO2021197371 A1 WO 2021197371A1
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bond
bioconjugate
linker
biotin
oligonucleotide
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PCT/CN2021/084341
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French (fr)
Chinese (zh)
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陈锦森
黄静
王建鹏
李竑
吴政宪
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南京金斯瑞生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention belongs to the field of bioconjugate coupling, and specifically relates to the application field of a fixed-length rigid linker between conjugated biomolecules.
  • Bioconjugation refers to the joining of two molecules, at least one of which is a biomolecule, by a covalent bond through chemical means.
  • Biomolecules synthesized and modified through biological coupling can have multiple functions, such as tracking cellular events, revealing enzyme functions, determining protein biodistribution, imaging specific biomarkers, and delivering drugs to target cells.
  • a linker spacer
  • Linkers are divided into two types, flexible and rigid in molecular structure.
  • the substructure of flexible linker unit is generally composed of carbon-carbon single bonds, carbon-oxygen single bonds, and carbon-nitrogen single bonds.
  • Rigid linkers are generally composed of benzene in structure. Groups, pyridine groups, conjugated heterocycles, alkenes, alkynes, and three-dimensional rigid structures (such as ⁇ helices and ⁇ sheets) formed by hydrogen bonds. This difference in molecular structure makes them different in aqueous solutions in that the flexible linker unit forms a worm-like coiled chain due to the free rotation of the covalent bond; while the rigid linker is restricted due to the limited rotation of the covalent bond. Make it into a rod-like linear chain.
  • oligoproline was used as a rigid linker in the early days, but because the peptide bond belongs to the sp 2 hybrid property , Due to the type of solvent and the difference of the substituents, the normal/trans isomerism will occur, which will cause the spacer linkers composed of the same unit number to have different lengths, resulting in poor data reproducibility.
  • the biological coupling modification of oligonucleotides is helpful to solve the problem of how to improve the delivery of oligonucleotides to the cytosol or nucleus.
  • This method has enabled many such drug precursors to successfully enter the clinic in the past 5 years.
  • Experimental research stage At present, the market can provide spacer linkers for oligonucleotide coupling modification with linear alkanes with different carbon numbers, linear PEG structures, and dSpacers with tetrahydrofuran five-membered sugar ring structures. They all It is a flexible linker; for bioconjugates containing rigid linkers, there is no product on the market, and there is no similar research in the literature.
  • the present invention provides a formula
  • n 1 and n 2 are each independently selected from O, S or N;
  • X is selected from molecules containing fluorescent dyes, biotin, N-acetylgalactosamine, cholesterol, enzymes or antibodies;
  • Y is selected from nucleic acid sequences containing any base of 1-200 nt;
  • P 1 and P 2 are each independently selected from a phosphodiester bond, a phosphorothioate bond, a phosphorothioate bond, an alkyl phosphonate bond,
  • R1 and R2 each independently have the structure shown in formula (II),
  • Z is O, NH or CONH, and n is selected from any integer value of 1-10.
  • both n 1 and n 2 are oxygen.
  • the rigid linker structure is Length is
  • the X is selected from molecules containing fluorescent dyes, biotin, N-acetylgalactosamine or cholesterol. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes, biotin, or cholesterol. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes or biotin. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes. In some embodiments of the invention, a biotin molecule. In some embodiments of the invention, the cholesterol molecule.
  • the X further includes a flexible linker structure.
  • Flexible linkers refer to covalent bonds that can rotate freely to form a worm-like coiled chain structure.
  • Common flexible linkers include, but are not limited to, linear alkanes, linear PEG, branched alkanes, and tetrahydrofuran five-membered rings Structure.
  • the flexible linker is selected from alkanes, linear PEG, branched alkanes, tetrahydrofuran five-membered rings, or combinations thereof.
  • the flexible connector structure is or
  • the Y is selected from a nucleic acid sequence of any base of 5-150 nt, preferably a nucleic acid sequence of any base of 10-100 nt, and more preferably a nucleic acid sequence of any base of 15-50 nt.
  • the sequence is most preferably a nucleic acid sequence of any base of 21 nt.
  • the P 1 and P 2 are both phosphodiester bonds.
  • the Z is oxygen, and n is any integer value of 2-8. In some embodiments of the present invention, the Z is oxygen, and n is 2, 3, 4, 5, 6, 7, or 8. In some specific embodiments of the present invention, said Z is oxygen and n is 2.
  • the X is selected from fluorescent dyes containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -. In a specific embodiment of the present invention, said X is comprising For the fluorescent dye of the structure, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the X is selected from biotin containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -.
  • said X is selected from the group comprising In the structure of biotin, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the X is selected from N-acetylgalactosamine containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-( CH 2 ) 2 -.
  • said X is selected from the group comprising In the structure of N-acetylgalactosamine, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the X is selected from cholesterol containing a flexible linker structure, P 1 and P 2 are selected from phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2- .
  • said X is selected from the group comprising In the structure of cholesterol, P1 and P2 are selected from phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the biological conjugate of the present invention is used in nucleic acid diagnosis, sequencing, genome analysis, detection of genetic variation, identification of single nucleotide polymorphism, oligonucleotide enrichment and purification, solid-phase difference display scheme, and protein interaction process , Peptide modification, drug delivery system or mass spectrometry analysis and detection applications.
  • the present invention in order to obtain a rigid linker that can be used for oligonucleotide modification, the present invention selects 4,4'-biphenol with a rigid structure as the main body, and the two ends are connected with ethylene glycol to form L-1, this structure can improve the water solubility of the rigid linker.
  • the rigid linker phosphoramidite monomer can be used for solid-phase synthesis.
  • the fluorescent dyes of the present invention include many common commercial dyes, such as Rhodamine series, BODIPY series, fluorescein (FITC) series, Coumarin series, cyanine series, Oxazine, ATTO series, AleaxFluor, LightCycler series, etc.
  • the fluorescent dye includes, but is not limited to, 4-acetylamino-4'-isothiocyanatostilbene (isothiocyanatostilbene)- 2,2'Disulfonic acid, acridine, derivatives of acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino- N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide Amine, anthranilamide, Brilliant Yellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-trifluoromethyl Coumaran 151, cyanosine; 4',6-d
  • rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123 , Rhodamine isothiocyanate X, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and Lanthanide chelate derivatives, cyanine, pyrelium dyes,
  • ROX 6-carboxy
  • nucleic acid refers to a polymer of nucleotides (such as ribonucleotides, deoxyribonucleotides, nucleotide analogs, etc.), and includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA-RNA Hybrids, oligonucleotides, polynucleotides, aptamers, peptide nucleic acids (PNA), PNA-DNA conjugates, PNA-RNA conjugates, locked nucleic acids (LNA), etc., which include linear or branched co- Nucleotides linked together.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA-RNA Hybrids DNA-RNA Hybrids
  • oligonucleotides polynucleotides
  • PNA peptide nucleic acids
  • PNA PNA-DNA conjugates
  • PNA-RNA conjugates locked nucleic acids
  • Nucleic acids are usually single-stranded or double-stranded, and generally contain phosphodiester bonds, although in some cases, nucleic acid analogs that may have alternative backbones are included, including, for example, phosphoramidites (Beaucage et al. (1993) Tetrahedron 49 (10 ): 1925) phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc.
  • nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al. (1995) Chem. Soc. Rev. pages 169-176), and analogs such as Rawls, C&E News Jun. 2 , 1997, described on page 35.
  • the nucleic acid is deoxyribonucleic acid or ribonucleic acid.
  • the nucleic acid is a primer or a probe. In some specific embodiments of the invention, the nucleic acid is a primer.
  • the bioconjugate with a rigid linker of fixed length provided by the present invention can fix the spacing between the biomolecules connected at the two ends of the rigid linker, avoid the mutual interference between the two biomolecules, and thus can better Complete the coupling process of biomolecules, and in the process of using coupled molecules, better performance of the connected biomolecules.
  • the synthesis efficiency of the coupling structure is higher, and the fluorescence intensity of the dye is basically The same; in addition, the present invention also found that when the two ends of the rigid linker are connected to biotin and oligonucleotide chains, the rigid linker can extend the biotin, making it easier to be coupled by streptavidin Obtained; and the use of rigid linkers to connect cholesterol is more flexible than flexible linkers (such as C6), which can better improve the lipophilicity of oligonucleotides.
  • Figure 1 Synthetic route diagram of the rigid linker phosphoramidite monomer L.
  • Figure 2 Structure diagram of oligonucleotide modified by rigid linker L'.
  • Figure 3 Structure diagram of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 4 Structure diagram of oligonucleotide modified by rigid linker L'and Biotin.
  • Figure 5 Structure diagram of oligonucleotide modified by rigid linker L'and GalNAc.
  • Figure 6 Structure diagram of oligonucleotide modified with rigid linker L'and cholesterol.
  • Figure 7 LC-MS image of crude oligonucleotide structure modified by rigid linker L'.
  • Figure 8 The crude quality spectrum of the oligonucleotide structure modified by the rigid linker L'.
  • Figure 9 Crude HPLC chart of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 10 HPLC chart after purification of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 11 Mass spectrum of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 12 Fluorescence spectrum of the dye FAM after the rigid linker L'and the flexible linkers C6 and C18 are connected.
  • Figure 13 HPLC chart of crude oligonucleotide modified by rigid linker L'and Biotion.
  • Figure 14 HPLC chart after purification of oligonucleotide modified with rigid linker L'and Biotin.
  • Figure 15 Mass spectrum of oligonucleotide modified with rigid linker L'and Biotin.
  • Figure 16 HPLC chart of crude oligonucleotide modified with rigid linker L'and GalNAc.
  • Figure 17 HPLC chart after purification of oligonucleotide modified with rigid linker L'and GalNAc.
  • Figure 18 Mass spectrum of oligonucleotide modified with rigid linker L'and GalNAc.
  • Figure 19 HPLC chart of crude oligonucleotide modified with rigid linker L'and cholesterol.
  • Figure 20 HPLC chart after purification of oligonucleotide modified with rigid linker L'and cholesterol.
  • Figure 21 Mass spectrum of oligonucleotide modified with rigid linker L'and cholesterol.
  • the present invention selects four widely used biomolecules, namely fluorescent dyes, biotin, cholesterol and N-acetylgalactosamine, all of which can perform efficient biological coupling reactions with linkers.
  • Fluorescent labeling of oligonucleotides is the main detection method in DNA diagnosis, sequencing and genome analysis. Fluorescent probes have a wide range of applications in detecting genetic variation and identifying single nucleotide polymorphisms. Biotin-modified oligonucleotides have been widely used in enrichment, purification, solid-phase difference display schemes, and solid-phase genome and plasmid sequencing schemes.
  • GalNAc N-acetylgalactosamine
  • ASGPR ASialoGlycoProtein receptor
  • Cholesterol-modified oligonucleotides are recognized by high-density lipoproteins and low-density lipoproteins (HDL and LDL) in vivo, and are internalized through cholesterol-binding receptors. After intravenous or intraperitoneal injection, these oligonucleotide conjugates escape renal clearance, thereby greatly affecting their pharmacokinetics by extending the retention time of nucleic acids in plasma.
  • the rigid linker phosphoramidite monomer L was used as a raw material to connect to the 5 of an oligonucleotide with a length of 21 bases.
  • 'End (5'-HO-CTCTATGGGCAGTCGGTGAAT-OH-3', the structure is shown in Figure 2).
  • CTCTATGGGCAGTCGGTGAAT After editing the position information containing the rigid linker phosphoramidite monomer L and the oligonucleotide sequence (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer.
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, respectively placed in the corresponding synthesis channel of the synthesizer, solid-phase synthesis and
  • the method of cleavage and deprotection is the same as that of conventional primer solid-phase synthesis, see (Beaucage et al., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), where the coupling time of compound L is 320s ⁇ 4 times, and the coupling time is 3min. After obtaining the primer containing the rigid linker L', it was separated, purified and recovered by HPLC.
  • the product was analyzed by HPLC.
  • the HPLC gradient elution condition for crude product analysis was 0-6min, and the concentration of acetonitrile was 5%-20%; 6.01-8min, The acetonitrile concentration is 88%; 8.01-10min, the acetonitrile concentration is 5%.
  • the pure product analysis HPLC gradient elution conditions are 0-6min, the acetonitrile concentration is 5%-30%, 6.01-8min, the acetonitrile concentration is 88%, 8.01 10min, the concentration of acetonitrile was 5%.
  • the terminal hydroxyl group of the linker can be further modified, for example, by coupling with the phosphoramidite monomer of the dye (Carboxyfluorescein FAM) to obtain the dye and the oligonucleotide by the rigid linker L'
  • the spaced-apart structure is shown as Probe 1 in Figure 3.
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, dye FAM phosphoramidite monomers with a concentration of 0.057M; respectively placed in the synthesis After the corresponding synthesis channel of the instrument alone, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis.
  • the coupling time is 320s ⁇ 4 times, and the dye coupling time is 3min. After the primer containing the dye donor and linker is obtained, it is separated and purified by HPLC. The product is analyzed by HPLC.
  • the crude product is analyzed by HPLC gradient elution conditions of 0-6min , Acetonitrile concentration is 5%-20%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • the main peak with retention time of 6.065min is the pure product further modified by dye
  • Analytical HPLC gradient elution conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • the purity is 95.4%.
  • the yield after synthesis and purification is shown in Table 1. It can be seen that the synthesis yield from the rigid linker is 76.9% and 72.6% higher than the yield of the flexible linker, respectively.
  • the terminal hydroxyl group of the linker can also be modified by biotin, which is a biomolecule that is an important part of an enzyme that affects cell growth and affects amino acids involved in protein synthesis, as shown in Probe A in Figure 4.
  • biotin phosphoramidite monomer is coupled to obtain a structure in which the biotin and the oligonucleotide are separated by the L'between the rigid linkers.
  • sequence position information including Biotin (5'-Biotin phosphoramidite monomer), rigid linker phosphoramidite monomer L and oligonucleotide (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA Synthesizer.
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, and the concentration of biotin monomer was 0.067M; they were placed in separate synthesizers. After the corresponding synthesis channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981), where the coupling time of compound L is 320s ⁇ 4 The coupling time of Biotin is 3min.
  • the primer containing the biotin monomer and the linker After obtaining the primer containing the biotin monomer and the linker, it is separated, purified and recovered by HPLC.
  • the product was analyzed by HPLC.
  • the crude product analysis conditions are 0-6min, acetonitrile concentration is 5%-20%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • the main peak with a retention time of 6.047 min is a product further modified by biotin, which has a high coupling efficiency.
  • the pure product analysis conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • Oligonucleic acid chains connected to biotin are respectively marked as Probe B (5'-Biotin-C6-CTCTATGGGCAGTCGGTGAAT-OH-3'), Probe C (5'-Biotin-C18-CTCTATGGGCAGTCGGTGAAT-OH-3'); in the same amount
  • Probe B (5'-Biotin-C6-CTCTATGGGCAGTCGGTGAAT-OH-3')
  • Probe C (5'-Biotin-C18-CTCTATGGGCAGTCGGTGAAT-OH-3')
  • the amount of remaining oligo-nucleic acid strands in the supernatant solution after the magnetic bead grabbing is detected to obtain a comparison of grabbing efficiency.
  • the Dynabeads TM magnetic beads used in this experiment have a maximum adsorption value of 200 pmol for single-stranded oligonucleotides. Take 20 ⁇ L of Dynabeads TM magnetic beads, which can adsorb single-stranded oligonucleotides with a maximum adsorption value of 40pmol. Take 0.95 times of the maximum adsorption value (experiment 1, 38pmol single-stranded oligonucleotide), 1.30 times (experiment 2, 52pmol single-stranded oligonucleotide) and 1.70 times (experiment three, 68pmol single-stranded oligonucleotide).
  • Glycidyl oligonucleotides for experiments.
  • the supernatant was taken for concentration test using a Nanodrop instrument, and the measured results are shown in Table 2. From the concentration of oligonucleotides in the supernatant, the ratio of the remaining oligonucleotides in the supernatant to the total primers can be calculated.
  • the volume of the supernatant is 80 ⁇ L
  • the relative molecular mass of the oligonucleotide is 7218
  • the results obtained by referring to the above calculation method are shown in Table 3 (where the relative molecular mass of Probe B oligonucleotide is 7062.8, and Probe C oligonucleotide The relative molecular mass of nucleotides is 7227).
  • Probe B 0.0ng/ ⁇ L 0.6ng/ ⁇ L 1.9ng/ ⁇ L
  • Probe C 0.0ng/ ⁇ L 0.5ng/ ⁇ L 2.1ng/ ⁇ L
  • GalNAc N-Acetylgalactosamine
  • sequence information including N-acetylgalactosamine (5'-GalNAc C3 phosphoramidite monomer), rigid linker phosphoramidite monomer L, and oligonucleotide sequence position information (CTCTATGGGCAGTCGGTGAAT), upload In the automated Dr. Oligo48 DNA synthesizer.
  • sequence information including N-acetylgalactosamine (5'-GalNAc C3 phosphoramidite monomer), rigid linker phosphoramidite monomer L, and oligonucleotide sequence position information (CTCTATGGGCAGTCGGTGAAT)
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, and the concentration of GalNAc monomer was 0.054M; respectively placed in a separate corresponding synthesizer After synthesizing the channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981), where the coupling time of compound L is 320s ⁇ 4 times , 5'-GalNAcC3 phosphoramidite monomer coupling time is 12min.
  • the primer containing 5'-GalNAc C3 and the linker After obtaining the primer containing 5'-GalNAc C3 and the linker, it is separated, purified and recovered by HPLC.
  • the product was analyzed by HPLC.
  • the analysis conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • 16 the main peak with a retention time of 3.405 min is the product further modified by N-acetylgalactosamine, and the coupling efficiency is high.
  • the yield was 65.0%, and the purity of the obtained modification was 98.9%.
  • the addition of lipophilic groups such as cholesterol molecules to oligonucleotides is expected to enhance its cellular uptake and membrane permeability.
  • the L linker can also connect cholesterol molecules to oligonucleotides by solid-phase synthesis, as shown in Probe 4 in Figure 6. After editing the sequence information including cholesterol (5'-Cholesteryl-TEG phosphoramidite monomer) and linker sequence position information (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer.
  • Monomers A, C, G, T phosphoramidite monomers are dissolved in acetonitrile with a concentration of 0.06M; compound L is dissolved in acetonitrile with a concentration of 0.10M, and the concentration of cholesterol monomer is 0.050M; they are placed in separate corresponding synthesizers After synthesizing the channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see (Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), where the coupling time of compound L is 320s ⁇ 4 The coupling time of cholesterol monomer is 3min.
  • the primer containing cholesterol and linker After obtaining the primer containing cholesterol and linker, it is separated, purified and recovered by HPLC. The product is analyzed by HPLC. The analysis conditions are 0-6min, the concentration of acetonitrile is 40%-80%, 6.01 -8min, the acetonitrile concentration is 88%. 8.01-10min, the acetonitrile concentration is 5%. As shown in Figure 19, the main peak with a retention time of 3.0157min is the product further modified by N-acetylgalactosamine, and the coupling efficiency is high. 20, after to give a clean product, retention time 3.189min; ESI mass spectrum having a molecular weight by a confirmation (FIG.
  • the partition coefficient (abbreviated as P) is defined as the specific ratio of the solute concentration between two solvents (liquid phase two phases), especially for unionized solutes, so the ratio
  • the logarithm of is logP.
  • the logP value is a measure of lipophilicity or hydrophobicity, and the theoretically calculated partition coefficient is recorded as clogP.

Abstract

A biological conjugate containing a rigid linker in the field of biological conjugate coupling. The provided biological conjugate can fix an interval between biomolecules connected at two ends of the rigid linker, and avoid mutual interference between two biomolecules, thereby better completing the coupling of the biomolecules; in the process of using the conjugate molecule, the properties between connected biomolecules are better exhibited.

Description

一种含有刚性连接子的生物偶联物A biological conjugate containing rigid linker 技术领域Technical field
本发明属于生物共轭偶联领域，具体涉及固定长度刚性连接子在共轭偶联的生物分子之间的应用领域。The invention belongs to the field of bioconjugate coupling, and specifically relates to the application field of a fixed-length rigid linker between conjugated biomolecules.
背景技术Background technique
生物共轭偶联(Bioconjugation)是指通过化学手段将至少一个是生物分子的两分子通过共价键相连。通过生物偶联合成修饰的生物分子可以具有多种功能，例如跟踪细胞事件，揭示酶功能，确定蛋白质生物分布，成像特定生物标记物以及将药物递送至靶细胞等。而当需要将两个或者多个不同或者相同生物分子偶联，例如，将酶或者抗体偶联于寡核苷酸上时，需要加入连接子(spacer)便可以降低反应物间的空间位阻作用从而提高偶联的效率。连接子从分子结构上分有柔性与刚性两种，柔性连接子单元子结构上一般由碳-碳单键、碳-氧单键、碳-氮单键构成，刚性连接子结构上一般由苯基、吡啶基团、共轭杂环，烯烃、炔烃、由氢键形成的三维刚性结构(如α螺旋和β折叠)构成。这种分子结构上的差别使他们在水溶液中的区别在于，柔性连接子单元子由于共价键的自由旋转，使其形成如蠕虫状卷曲链；而刚性连接子由于共价键旋转受限，使其形成如棒状直链。例如，在应用于蛋白质的融合时，采用自由摆动的柔性连接子单元相连时，导致两个蛋白相互干扰，从而造成低表达或者生物活性的丧失的现象。当将小分子染料偶联至生物大分子，利用荧光共振能量传递用于测定生物分子间的距离时，早期将寡聚脯氨酸作为刚性连接子使用，但由于肽键属于sp 2杂化属性，会由于溶剂的种类以及取代基的不同而发生正/反异构从而导致由相同单位数组成的间隔连接子其长短不一，造成数据的重复性差。生物固相纯化领域涉及到共轭偶联修饰时，如商业化产品Thermo Scientific的Pierce NHS活化的磁珠在磁珠表面与反应位点NHS官能团间有43埃
Figure PCTCN2021084341-appb-000001
BcMag TM的胺基修饰的磁珠表面与反应位点间有18个原子的间隔连接子，这些都是为了降低共轭偶联反应的位阻，然而这些间隔连接子同样使用了上述的柔性连接子，造成间隔空间不定的问题。 Bioconjugation refers to the joining of two molecules, at least one of which is a biomolecule, by a covalent bond through chemical means. Biomolecules synthesized and modified through biological coupling can have multiple functions, such as tracking cellular events, revealing enzyme functions, determining protein biodistribution, imaging specific biomarkers, and delivering drugs to target cells. When two or more different or the same biomolecules need to be coupled, for example, when an enzyme or antibody is coupled to an oligonucleotide, a linker (spacer) needs to be added to reduce the steric hindrance between the reactants To improve the efficiency of coupling. Linkers are divided into two types, flexible and rigid in molecular structure. The substructure of flexible linker unit is generally composed of carbon-carbon single bonds, carbon-oxygen single bonds, and carbon-nitrogen single bonds. Rigid linkers are generally composed of benzene in structure. Groups, pyridine groups, conjugated heterocycles, alkenes, alkynes, and three-dimensional rigid structures (such as α helices and β sheets) formed by hydrogen bonds. This difference in molecular structure makes them different in aqueous solutions in that the flexible linker unit forms a worm-like coiled chain due to the free rotation of the covalent bond; while the rigid linker is restricted due to the limited rotation of the covalent bond. Make it into a rod-like linear chain. For example, when applied to protein fusion, when a free swinging flexible linker unit is used to connect, the two proteins interfere with each other, resulting in low expression or loss of biological activity. When coupling small molecule dyes to biological macromolecules and using fluorescence resonance energy transfer to measure the distance between biomolecules, oligoproline was used as a rigid linker in the early days, but because the peptide bond belongs to the sp 2 hybrid property , Due to the type of solvent and the difference of the substituents, the normal/trans isomerism will occur, which will cause the spacer linkers composed of the same unit number to have different lengths, resulting in poor data reproducibility. When the field of biological solid-phase purification involves conjugated coupling modification, such as the Pierce NHS activated magnetic beads of the commercial product Thermo Scientific, there is 43 angstroms between the surface of the magnetic beads and the NHS functional group of the reaction site
Figure PCTCN2021084341-appb-000001
There are 18-atom spacer linkers between the surface of the amine-modified magnetic beads of BcMag TM and the reaction site. These spacer linkers are designed to reduce the steric hindrance of the conjugation coupling reaction. However, these spacer linkers also use the above-mentioned flexible linkers. Sub, causing the problem of indeterminate space.
对寡核苷酸的生物偶联修饰有利于解决如何改善将寡核苷酸向胞质溶胶或细胞核的传递难题，这种方法在过去的5年中使许多此类药物前体成功进入了临床实验研究阶段。而目前市场中能够提供用于寡核苷酸偶联修饰的间隔连接子有不同碳原子数的直链烷烃类，有直链PEG结构类，以及四氢呋喃五元环糖环结构的dSpacer，他们都属于柔性连接子；而对于含有刚性连接子的生物偶联物，市面上没有产品，文献报道中也没有相类似的研究。The biological coupling modification of oligonucleotides is helpful to solve the problem of how to improve the delivery of oligonucleotides to the cytosol or nucleus. This method has enabled many such drug precursors to successfully enter the clinic in the past 5 years. Experimental research stage. At present, the market can provide spacer linkers for oligonucleotide coupling modification with linear alkanes with different carbon numbers, linear PEG structures, and dSpacers with tetrahydrofuran five-membered sugar ring structures. They all It is a flexible linker; for bioconjugates containing rigid linkers, there is no product on the market, and there is no similar research in the literature.
发明内容Summary of the invention
针对市场及研究中的缺少含有刚性连接子的生物偶联物现状，本发明提供了一种具有式In view of the lack of bioconjugates containing rigid linkers in the market and research, the present invention provides a formula
(Ⅰ)所示结构的生物偶联物(Ⅰ) Biological conjugate of the structure shown
X-P 1-R 1-L’-R 2-P 2-Y， XP 1 -R 1 -L'-R 2 -P 2 -Y,
(Ⅰ)(Ⅰ)
其中，in,
L’为刚性连接子，结构为
Figure PCTCN2021084341-appb-000002
所述n 1、n 2各自独立地选自O、S或者N； L'is a rigid linker, the structure is
Figure PCTCN2021084341-appb-000002
The n 1 and n 2 are each independently selected from O, S or N;
X选自包含荧光染料、生物素、N-乙酰半乳糖胺、胆固醇、酶或者抗体的分子；X is selected from molecules containing fluorescent dyes, biotin, N-acetylgalactosamine, cholesterol, enzymes or antibodies;
Y选自包含1-200nt的任意碱基的核酸序列；Y is selected from nucleic acid sequences containing any base of 1-200 nt;
P 1，P 2各自独立地选自磷酸二酯键、硫代磷酸酯键、二硫代磷酸酯键、烷基膦酸酯键、 P 1 and P 2 are each independently selected from a phosphodiester bond, a phosphorothioate bond, a phosphorothioate bond, an alkyl phosphonate bond,
烷基硫代磷酸酯键、磷酸三酯键、磷醢胺酸酯键、硅氧烷基、碳酸酯键、烷氧基甲醢基、Alkyl phosphorothioate linkages, phosphotriester linkages, phosphamate linkages, siloxane groups, carbonate linkages, alkoxymethanyl groups,
乙醢胺酸酯键、胺基甲酸酯键、吗啉基、硼烷基、硫醚键、桥接磷醢胺酸酯键、桥接亚Acetate bond, urethane bond, morpholino, boryl group, thioether bond, bridged phosphamate bond, bridging sub
甲基膦酸酯键、桥接硫代磷酸酯键或砜核苷键；Methyl phosphonate bond, bridging phosphorothioate bond or sulfone nucleoside bond;
R1，R2各自独立地具有式(Ⅱ)所示的结构，R1 and R2 each independently have the structure shown in formula (II),
-Z-(CH 2) n-， -Z-(CH 2 ) n -,
(Ⅱ)(Ⅱ)
其中，Z为O、NH或者CONH，n选自1-10任一整数值。Wherein, Z is O, NH or CONH, and n is selected from any integer value of 1-10.
在本发明的一些实施方案中，所述n 1，n 2均为氧。在本发明的一个具体实施方案中，刚性连接子结构为
Figure PCTCN2021084341-appb-000003
长度为
Figure PCTCN2021084341-appb-000004
In some embodiments of the present invention, both n 1 and n 2 are oxygen. In a specific embodiment of the present invention, the rigid linker structure is
Figure PCTCN2021084341-appb-000003
Length is
Figure PCTCN2021084341-appb-000004
在本发明的一些实施方案中，所述X选自包含荧光染料、生物素、N-乙酰半乳糖胺或者胆固醇的分子。在本发明的一些实施方案中，所述X选自包含荧光染料、生物素或者胆固醇的分子。在本发明的一些实施方案中，所述X选自包含荧光染料或者生物素的分子。在本发明的一些实施方案中，所述X选自包含荧光染料的分子。在本发明的一些实施方案中，生物素的分子。在本发明的一些实施方案中，胆固醇的分子。In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes, biotin, N-acetylgalactosamine or cholesterol. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes, biotin, or cholesterol. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes or biotin. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes. In some embodiments of the invention, a biotin molecule. In some embodiments of the invention, the cholesterol molecule.
在本发明的一些实施方案中，所述X还包含柔性连接子结构。柔性连接子是指共价键可以自由旋转，使其形成如蠕虫状卷曲链的结构，常见的柔性连接子包括，但不限于直链烷烃、直链PEG、支链烷烃、以及四氢呋喃五元环结构，在本发明的一些实施方案中，所述柔性连接子选自烷烃、直链PEG、支链烷烃、四氢呋喃五元环或其组合。在本发明的一些具体的实施例中，柔性连接子结构为
Figure PCTCN2021084341-appb-000005
或者
Figure PCTCN2021084341-appb-000006
In some embodiments of the present invention, the X further includes a flexible linker structure. Flexible linkers refer to covalent bonds that can rotate freely to form a worm-like coiled chain structure. Common flexible linkers include, but are not limited to, linear alkanes, linear PEG, branched alkanes, and tetrahydrofuran five-membered rings Structure. In some embodiments of the present invention, the flexible linker is selected from alkanes, linear PEG, branched alkanes, tetrahydrofuran five-membered rings, or combinations thereof. In some specific embodiments of the present invention, the flexible connector structure is
Figure PCTCN2021084341-appb-000005
or
Figure PCTCN2021084341-appb-000006
在本发明的一些实施方案中，所述Y选自5-150nt的任意碱基的核酸序列，优选为10-100nt的任意碱基的核酸序列，更优选为15-50nt的任意碱基的核酸序列，最优选为21nt的任意碱基的核酸序列。In some embodiments of the present invention, the Y is selected from a nucleic acid sequence of any base of 5-150 nt, preferably a nucleic acid sequence of any base of 10-100 nt, and more preferably a nucleic acid sequence of any base of 15-50 nt. The sequence is most preferably a nucleic acid sequence of any base of 21 nt.
在本发明的一些实施方案中，所述P 1，P 2均为磷酸二酯键。 In some embodiments of the present invention, the P 1 and P 2 are both phosphodiester bonds.
在本发明的一些实施方案中，所述Z为氧，n为2-8任一整数值。在本发明的一些实施方案中，所述Z为氧，n为2、3、4、5、6、7或者8。在本发明的一些具体的实施方案中，所述Z为氧，n为2。In some embodiments of the present invention, the Z is oxygen, and n is any integer value of 2-8. In some embodiments of the present invention, the Z is oxygen, and n is 2, 3, 4, 5, 6, 7, or 8. In some specific embodiments of the present invention, said Z is oxygen and n is 2.
在本发明的一个具体实施方案中，所述X选自包含柔性连接子结构的荧光染料，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。在本发明的一个具体的实施方案中，所述X为包含
Figure PCTCN2021084341-appb-000007
结构的荧光染料，P1，P2均为磷酸二酯键，R1，R2均为-O-(CH 2) 2-。 In a specific embodiment of the present invention, the X is selected from fluorescent dyes containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -. In a specific embodiment of the present invention, said X is comprising
Figure PCTCN2021084341-appb-000007
For the fluorescent dye of the structure, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
在本发明的一些实施方案中，所述X选自包含柔性连接子结构的生物素，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。在本发明的一个具体的实施方案中，所述X选自包含
Figure PCTCN2021084341-appb-000008
结构的生物素，P1，P2均为磷酸二酯键，R1，R2均为-O-(CH 2) 2-。 In some embodiments of the present invention, the X is selected from biotin containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -. In a specific embodiment of the present invention, said X is selected from the group comprising
Figure PCTCN2021084341-appb-000008
In the structure of biotin, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
在本发明的一些实施方案中，所述X选自包含柔性连接子结构的N-乙酰半乳糖胺，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。在本发明的一个具体的实施方案中，所述X选自包含
Figure PCTCN2021084341-appb-000009
结构的N-乙酰半乳糖胺，P1，P2均为磷酸二酯键，R1，R2均为-O-(CH 2) 2-。 In some embodiments of the present invention, the X is selected from N-acetylgalactosamine containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-( CH 2 ) 2 -. In a specific embodiment of the present invention, said X is selected from the group comprising
Figure PCTCN2021084341-appb-000009
In the structure of N-acetylgalactosamine, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
在本发明的一些实施方案中，所述X选自包含柔性连接子结构的胆固醇，P 1，P 2选自磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。在本发明的一个具体的实施方案中，所述X选自包含
Figure PCTCN2021084341-appb-000010
结构的胆固醇，P1，P2选自磷酸二酯键，R1，R2均为-O-(CH 2) 2-。 In some embodiments of the present invention, the X is selected from cholesterol containing a flexible linker structure, P 1 and P 2 are selected from phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2- . In a specific embodiment of the present invention, said X is selected from the group comprising
Figure PCTCN2021084341-appb-000010
In the structure of cholesterol, P1 and P2 are selected from phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
本发明所述的生物偶联物在核酸诊断、测序、基因组分析、检测遗传变异、鉴定单核苷酸多态性、寡核苷酸富集和纯化、固相差异显示方案、蛋白质相互作用过程、多肽修饰、药物递送***或质谱分析检测中的应用。The biological conjugate of the present invention is used in nucleic acid diagnosis, sequencing, genome analysis, detection of genetic variation, identification of single nucleotide polymorphism, oligonucleotide enrichment and purification, solid-phase difference display scheme, and protein interaction process , Peptide modification, drug delivery system or mass spectrometry analysis and detection applications.
在本发明的一些实施方案中，为了获得可用于寡核苷酸修饰的刚性连接子，本发明选择了具有刚性结构的4,4’-联苯二酚为主体，两端连接乙二醇形成L-1，该结构可以提高刚性连接子的水溶性，通过亚磷酰胺化学合成可用于固相合成的刚性连接子亚磷酰胺单体。在本发明的一个具体实施方案中，在乙醇中，在碱性条件下，由4,4’-联苯二酚与2-溴乙醇反应得到L-1，再由4,4'-二甲氧基三苯基氯甲烷与L-1在吡啶中合成L-2，最后L-2与2-氰乙基N,N,N',N'-四异丙基亚磷酰二胺在BTT的催化下偶联得到刚性连接子亚磷酰胺单体L，其中刚性连接子的长度为
Figure PCTCN2021084341-appb-000011
如图1。 In some embodiments of the present invention, in order to obtain a rigid linker that can be used for oligonucleotide modification, the present invention selects 4,4'-biphenol with a rigid structure as the main body, and the two ends are connected with ethylene glycol to form L-1, this structure can improve the water solubility of the rigid linker. Through phosphoramidite chemical synthesis, the rigid linker phosphoramidite monomer can be used for solid-phase synthesis. In a specific embodiment of the present invention, in ethanol, under alkaline conditions, 4,4'-biphenol and 2-bromoethanol are reacted to obtain L-1, and then 4,4'-dimethyl Synthesize L-2 with oxytriphenylchloromethane and L-1 in pyridine, and finally L-2 with 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite in BTT Coupling under the catalysis of the rigid linker phosphoramidite monomer L, wherein the length of the rigid linker is
Figure PCTCN2021084341-appb-000011
Figure 1.
本发明所述的荧光染料包括诸多常见的商业化染料，如罗丹明(Rhodamine)系列、BODIPY系列、荧光素(FITC)系列、香豆素(Coumarin)系列、花青素(cyanine)系列、恶嗪(oxazine)、ATTO系列、AleaxFluor、LightCycler系列等，本发明的一些实施方案中，所述的荧光染料包括，但不限于，4-乙酰氨基-4'-异硫氰酸芪(isothiocyanatostilbene)-2,2'二磺酸、吖啶、吖啶和吖啶异硫氰酸酯的衍生物、5-(2'-氨基乙基)氨基萘-1-磺酸(EDANS)、4-氨基-N-[3-乙烯基磺酰基)苯基]萘二甲酰亚胺-3,5二磺酸盐(Lucifer Yellow VS)、N-(4-苯胺基-1-萘基)马来酰亚胺、邻氨基苯甲酰胺、亮黄(Brilliant Yellow)、香豆素、香豆素衍生物、7-氨基-4-甲基香豆素(AMC、Coumarin 120)、7-氨基-三氟甲基香豆素(Coumaran 151)、焰红染料(cyanosine)；4',6-二脒基(diaminidino)-2-苯基吲哚(DAPI)、5',5”-二溴邻苯三酚(dibromopyrogallol)-磺酞(溴邻苯三酚红)、7-二乙基氨基-3-(4'-异硫氰酸基苯基)-4-甲基香豆 素、4,4'-二异硫氰酸基二氢-芪-2,2'-二磺酸、4,4'-二异硫氰酸芪-2,2'-二磺酸、5-[二甲基氨基]萘-1-磺酰氯(DNS、丹磺酰氯)、荧光素和衍生物例如5-羧基荧光素(FAM)、5-(4,6-二氯三嗪-2-基)氨基荧光素(DTAF)、2'7'-二甲氧基-4'5'-二氯-6-羧基荧光素(JOE)、荧光素、异硫氰酸荧光素(FITC)、QFITC(XRITC)；荧光胺衍生物(荧光与胺反应时)；IR144；IR1446；异硫氰酸孔雀绿；4-甲基伞形酮；邻甲酚酞；硝基酪氨酸；副品红；酚红、B-藻红蛋白；邻苯二甲醛衍生物(荧光与胺反应时)；芘和衍生物例如芘、丁酸芘和1-芘丁酸琥珀酰亚胺基酯；活性红4(Cibacron.RTM.亮红3B-A)、罗丹明和衍生物例如6-羧基-X-罗丹明(ROX)、6-羧基罗丹明(R6G)、丽丝胺罗丹明B磺酰氯、罗丹明(Rhod)、罗丹明B、罗丹明123、异硫氰酸罗丹明X、磺基罗丹明B、磺基罗丹明101和磺基罗丹明101的磺酰氯衍生物(得克萨斯红)；N,N,N',N'-四甲基-6-羧基罗丹明(TAMRA)；四甲基罗丹明、异硫氰酸四甲基罗丹明(TRITC)；核黄素；玫红酸和镧系元素螯合物衍生物、花青、pyrelium染料、方酸箐30(squaraines)、1,3-二氯-7-羟基-9,9-二甲基-2(9H)-吖啶酮(DDAO)和二甲基吖啶酮(DAO)。The fluorescent dyes of the present invention include many common commercial dyes, such as Rhodamine series, BODIPY series, fluorescein (FITC) series, Coumarin series, cyanine series, Oxazine, ATTO series, AleaxFluor, LightCycler series, etc. In some embodiments of the present invention, the fluorescent dye includes, but is not limited to, 4-acetylamino-4'-isothiocyanatostilbene (isothiocyanatostilbene)- 2,2'Disulfonic acid, acridine, derivatives of acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino- N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide Amine, anthranilamide, Brilliant Yellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-trifluoromethyl Coumaran 151, cyanosine; 4',6-diaminidino-2-phenylindole (DAPI), 5',5”-dibromopyrogallol (dibromopyrogallol)-sulfophthalein (bromopyrogallol red), 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, 4,4'- Diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 5-[dimethylamino]naphthalene -1-sulfonyl chloride (DNS, dansyl chloride), fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) , 2'7'-Dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC); fluorescein derivatives (When fluorescence reacts with amine); IR144; IR1446; malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; para-fuchsin; phenol red, B-phycoerythrin; Phthalaldehyde derivatives (when fluorescence reacts with amines); pyrene and derivatives such as pyrene, pyrene butyrate and 1-pyrene butyrate succinimidyl ester; Reactive Red 4 (Cibacron.RTM. Brilliant Red 3B-A ), rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123 , Rhodamine isothiocyanate X, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and Lanthanide chelate derivatives, cyanine, pyrelium dyes, squaraines 30 (squaraines), 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone (DDAO) and dimethylacridone (DAO).
术语“核酸”指核苷酸(例如核糖核苷酸、脱氧核糖核苷酸、核苷酸类似物等)的聚合物，并且包含脱氧核糖核酸(DNA)、核糖核酸(RNA)、DNA-RNA杂交体、寡核苷酸、多核苷酸、适体、肽核酸(PNA)、PNA-DNA缀合物、PNA-RNA缀合物、锁核酸(LNA)等，其包含以线性或分支方式共价连接在一起的核苷酸。核酸通常是单链或双链的，并且一般含有磷酸二酯键，尽管在一些情况下，包括可以具有替代主链的核酸类似物，包括例如磷酰胺(Beaucage等人(1993)Tetrahedron 49(10):1925)硫代磷酸酯(Mag等人(1991)Nucleic Acids Res.19:1437；和美国专利号5,644,048)，二硫代磷酸酯(Briu等人(1989)J.Am.Chem.Soc.111:2321)，O-甲基亚磷酰胺键(参见Eckstein，Oligonucleotides and Analogues:APractical Approach，Oxford University Press(1992))，以及肽核酸主链和键(参见Egholm(1992)J.Am.Chem.Soc.114:1895)。其他类似物核酸包括具有带正电的主链(Denpcy等(1995)Proc.Natl.Acad.Sci.USA 92:6097)；非离子主链(美国专利号5386023，5637684，5602240，5216141和4469863)和非核糖主链的那些，包括在美国专利号5,235,033和5,034,506中描述的那些。含有一种或多种碳环糖的核酸也包括在核酸的定义内(参见Jenkins等人(1995)Chem.Soc.Rev.第169-176页)，并且类似物例如Rawls,C&E News Jun.2,1997第35页中描述。本发明的一些实施方案中，所述核酸是脱氧核糖核酸或核糖核酸。本发明的一些实施方案中，所述核酸是引物或探针。在发明的一些具体的实施方案中，所述核酸是引物。The term "nucleic acid" refers to a polymer of nucleotides (such as ribonucleotides, deoxyribonucleotides, nucleotide analogs, etc.), and includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA-RNA Hybrids, oligonucleotides, polynucleotides, aptamers, peptide nucleic acids (PNA), PNA-DNA conjugates, PNA-RNA conjugates, locked nucleic acids (LNA), etc., which include linear or branched co- Nucleotides linked together. Nucleic acids are usually single-stranded or double-stranded, and generally contain phosphodiester bonds, although in some cases, nucleic acid analogs that may have alternative backbones are included, including, for example, phosphoramidites (Beaucage et al. (1993) Tetrahedron 49 (10 ): 1925) phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111:2321), O-methyl phosphoramidite bond (see Eckstein, Oligonucleotides and Analogues: APractical Approach, Oxford University Press (1992)), and peptide nucleic acid backbone and bonds (see Egholm (1992) J. Am. Chem .Soc.114:1895). Other analog nucleic acids include a positively charged backbone (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097); non-ionic backbone (U.S. Patent Nos. 5386023, 5637684, 5602240, 5216141 and 4469863) And non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al. (1995) Chem. Soc. Rev. pages 169-176), and analogs such as Rawls, C&E News Jun. 2 , 1997, described on page 35. In some embodiments of the invention, the nucleic acid is deoxyribonucleic acid or ribonucleic acid. In some embodiments of the invention, the nucleic acid is a primer or a probe. In some specific embodiments of the invention, the nucleic acid is a primer.
发明有益效果：本发明提供的具有固定长度刚性连接子的生物偶联物，能够固定刚性连接子两端连接的生物分子之间的间隔，避免两生物分子间的相互干扰，从而能够更好地完成生物分子的偶联过程，并且在偶联分子的使用过程中，更好的表现所连接的各生物分子之间的性能。如与市面上常见的含有柔性连接子(C6和C18)相比，将刚性连接子两端分别连接染料和寡核苷酸链，该偶联结构的合成效率更高，且染料的荧光强度基本相同；除此之外，本发明还发现，当刚性连接子两端分别连接生物素和寡核苷酸链时，刚性连接子可以使生物素伸出，更容易被链霉亲和素偶联获得；且利用刚性连接子连接胆固醇较柔性连接子(如C6),可以更好的提高寡核苷酸的亲脂性。Advantageous effects of the invention: The bioconjugate with a rigid linker of fixed length provided by the present invention can fix the spacing between the biomolecules connected at the two ends of the rigid linker, avoid the mutual interference between the two biomolecules, and thus can better Complete the coupling process of biomolecules, and in the process of using coupled molecules, better performance of the connected biomolecules. For example, compared with the common flexible linkers (C6 and C18) on the market, connecting the two ends of the rigid linker to the dye and oligonucleotide chain respectively, the synthesis efficiency of the coupling structure is higher, and the fluorescence intensity of the dye is basically The same; in addition, the present invention also found that when the two ends of the rigid linker are connected to biotin and oligonucleotide chains, the rigid linker can extend the biotin, making it easier to be coupled by streptavidin Obtained; and the use of rigid linkers to connect cholesterol is more flexible than flexible linkers (such as C6), which can better improve the lipophilicity of oligonucleotides.
附图说明Description of the drawings
图1：刚性连接子亚磷酰胺单体L的合成路线图。Figure 1: Synthetic route diagram of the rigid linker phosphoramidite monomer L.
图2：经刚性连接子L’修饰后的寡核苷酸结构图。Figure 2: Structure diagram of oligonucleotide modified by rigid linker L'.
图3：经刚性连接子L’与染料FAM修饰后的寡核苷酸结构图。Figure 3: Structure diagram of oligonucleotide modified with rigid linker L'and dye FAM.
图4：经刚性连接子L’与Biotin修饰后的寡核苷酸结构图。Figure 4: Structure diagram of oligonucleotide modified by rigid linker L'and Biotin.
图5：经刚性连接子L’与GalNAc修饰后的寡核苷酸结构图。Figure 5: Structure diagram of oligonucleotide modified by rigid linker L'and GalNAc.
图6：经刚性连接子L’与胆固醇修饰后的寡核苷酸结构图。Figure 6: Structure diagram of oligonucleotide modified with rigid linker L'and cholesterol.
图7：经刚性连接子L’修饰后的寡核苷酸结构粗品的LC-MS图。Figure 7: LC-MS image of crude oligonucleotide structure modified by rigid linker L'.
图8：经刚性连接子L’修饰后的寡核苷酸结构粗品质谱图。Figure 8: The crude quality spectrum of the oligonucleotide structure modified by the rigid linker L'.
图9：经刚性连接子L’与染料FAM修饰后的寡核苷酸的粗品HPLC图。Figure 9: Crude HPLC chart of oligonucleotide modified with rigid linker L'and dye FAM.
图10：经刚性连接子L’与染料FAM修饰后的寡核苷酸的纯化后的HPLC图。Figure 10: HPLC chart after purification of oligonucleotide modified with rigid linker L'and dye FAM.
图11：经刚性连接子L’与染料FAM修饰后的寡核苷酸的质谱图。Figure 11: Mass spectrum of oligonucleotide modified with rigid linker L'and dye FAM.
图12：刚性连接子L’与柔性连接子C6和C18连接染料FAM后的荧光图谱。Figure 12: Fluorescence spectrum of the dye FAM after the rigid linker L'and the flexible linkers C6 and C18 are connected.
图13：经刚性连接子L’与Biotion修饰后的寡核苷酸粗品的HPLC图。Figure 13: HPLC chart of crude oligonucleotide modified by rigid linker L'and Biotion.
图14：经刚性连接子L’与Biotin修饰后的寡核苷酸纯化后的HPLC图。Figure 14: HPLC chart after purification of oligonucleotide modified with rigid linker L'and Biotin.
图15：经刚性连接子L’与Biotin修饰后的寡核苷酸的质谱图。Figure 15: Mass spectrum of oligonucleotide modified with rigid linker L'and Biotin.
图16：经刚性连接子L’与GalNAc修饰后的寡核苷酸粗品的HPLC图。Figure 16: HPLC chart of crude oligonucleotide modified with rigid linker L'and GalNAc.
图17：经刚性连接子L’与GalNAc修饰后的寡核苷酸纯化后的HPLC图。Figure 17: HPLC chart after purification of oligonucleotide modified with rigid linker L'and GalNAc.
图18：经刚性连接子L’与GalNAc修饰后的寡核苷酸的质谱图。Figure 18: Mass spectrum of oligonucleotide modified with rigid linker L'and GalNAc.
图19：经刚性连接子L’与胆固醇修饰后的寡核苷酸粗品的HPLC图。Figure 19: HPLC chart of crude oligonucleotide modified with rigid linker L'and cholesterol.
图20：经刚性连接子L’与胆固醇修饰后的寡核苷酸纯化后的HPLC图。Figure 20: HPLC chart after purification of oligonucleotide modified with rigid linker L'and cholesterol.
图21：经刚性连接子L’与胆固醇修饰后的寡核苷酸的质谱图。Figure 21: Mass spectrum of oligonucleotide modified with rigid linker L'and cholesterol.
具体实施方式Detailed ways
下面通过实施例，并结合附图，对本发明的技术方案作进一步详细的说明，但本发明不限于下面的实施例。除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。The technical solutions of the present invention will be further described in detail below through embodiments and in conjunction with the drawings, but the present invention is not limited to the following embodiments. Unless otherwise specified, the technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.
为了验证刚性连接子的有效性，本发明选取了四种应用广泛的生物分子，即荧光染料、生物素、胆固醇及N-乙酰半乳糖胺，他们与连接子都可以进行高效生物偶联反应。寡核酸的荧光标记是DNA诊断，测序和基因组分析中的主要检测方法。荧光探针在检测遗传变异和鉴定单核苷酸多态性方面具有广泛的应用。生物素修饰的寡核苷酸已经广泛应用于富集、纯化、固相差异显示方案以及固相基因组和质粒测序方案。N-乙酰半乳糖胺(GalNAc)已经被证明可以与ASialoGlycoProtein受体(ASGPR)结合从而提高寡核苷酸在肝细胞的内吞作用实现在肝部的富集。胆固醇修饰的寡核苷酸在体内被高密度脂蛋白和低密度脂蛋白(HDL和LDL)识别，并通过胆固醇结合受体被内化。在静脉内或腹膜内注射后，这些寡核苷酸结合物逃脱了肾脏清除，从而通过延长核酸在血浆中的保留时间而极大地影响了它们的药代动力学。In order to verify the effectiveness of rigid linkers, the present invention selects four widely used biomolecules, namely fluorescent dyes, biotin, cholesterol and N-acetylgalactosamine, all of which can perform efficient biological coupling reactions with linkers. Fluorescent labeling of oligonucleotides is the main detection method in DNA diagnosis, sequencing and genome analysis. Fluorescent probes have a wide range of applications in detecting genetic variation and identifying single nucleotide polymorphisms. Biotin-modified oligonucleotides have been widely used in enrichment, purification, solid-phase difference display schemes, and solid-phase genome and plasmid sequencing schemes. N-acetylgalactosamine (GalNAc) has been shown to bind to ASialoGlycoProtein receptor (ASGPR) to increase the endocytosis of oligonucleotides in hepatocytes to achieve liver enrichment. Cholesterol-modified oligonucleotides are recognized by high-density lipoproteins and low-density lipoproteins (HDL and LDL) in vivo, and are internalized through cholesterol-binding receptors. After intravenous or intraperitoneal injection, these oligonucleotide conjugates escape renal clearance, thereby greatly affecting their pharmacokinetics by extending the retention time of nucleic acids in plasma.
实施例1 刚性连接子亚磷酰胺单体L的合成Example 1 Synthesis of Rigid Linker Phosphoramidite Monomer L
L-1的合成Synthesis of L-1
将4,4'-二羟基联苯(1.86g，10mmol)溶解于20mL的乙醇在圆底反应瓶中，加入氢氧化钠(1.6g，40mmol)，将反应液升温至回流。回流反应二十分钟后，滴加入2-溴乙醇(2.80mL，40mmol)，回流24小时后。将反应液倒入水中，产物析出后，用抽滤得到L-1产物1.73g，产率64％。MS(ESI)m/z理论值C 16H 19O 4[M+H] +为275.13；测量值，275.20。 4,4'-Dihydroxybiphenyl (1.86g, 10mmol) was dissolved in 20mL of ethanol in a round bottom reaction flask, sodium hydroxide (1.6g, 40mmol) was added, and the reaction solution was heated to reflux. After refluxing the reaction for 20 minutes, 2-bromoethanol (2.80 mL, 40 mmol) was added dropwise, and after refluxing for 24 hours. The reaction solution was poured into water, and after the product precipitated, 1.73 g of L-1 product was obtained by suction filtration, with a yield of 64%. MS (ESI) m/z theoretical value C 16 H 19 O 4 [M+H] + is 275.13; measured value, 275.20.
L-2的合成Synthesis of L-2
将得到的L-1(1.36g，5mmol)溶于50mL的无水吡啶中，加入4-二甲氨基吡啶(DMAP，610mg，5mmol)与4,4'-二甲氧基三苯基氯甲烷(1.69g，5mmol),反应液在室温下搅拌24小时。将反应液中的吡啶蒸出后，加入饱合碳酸氢钠水溶液与乙酸乙酯后，分液，上层有机相经过饱合氯化钠水溶液洗涤后，用无水硫酸钠干燥。产物经柱层析分离，展开剂(乙醇乙酯/石油醚＝1：1)，得到产物L-2 1.85g，产率65％。MS(ESI)m/z理论值C 37H 36NaO 6[M+Na] +为599.25；测量值，599.60。 Dissolve the obtained L-1 (1.36g, 5mmol) in 50mL of anhydrous pyridine, add 4-dimethylaminopyridine (DMAP, 610mg, 5mmol) and 4,4'-dimethoxytriphenylchloromethane (1.69g, 5mmol), the reaction solution was stirred at room temperature for 24 hours. After the pyridine in the reaction solution was distilled off, a saturated aqueous sodium bicarbonate solution and ethyl acetate were added, and then the layers were separated. The upper organic phase was washed with a saturated sodium chloride aqueous solution and dried with anhydrous sodium sulfate. The product was separated by column chromatography, and the developing solvent (ethyl ethyl alcohol/petroleum ether = 1:1), to obtain 1.85 g of product L-2, with a yield of 65%. MS (ESI) m/z theoretical value C 37 H 36 NaO 6 [M+Na] + is 599.25; measured value, 599.60.
L的合成Synthesis of L
将L-2(60mg，0.1mmol)溶解于2mL无水乙腈中，加入5-苄硫基四氮(BTT，30mg，0.15mmol)与2-氰乙基N,N,N',N'-四异丙基亚磷酰二胺(50μL，0.15mmol)。反应在室温下搅拌1小时后，蒸去乙腈后，产物由柱层析分离，展开剂(乙酸乙酯/石油醚/三乙胺＝25：75：2)。产物72mg，产率95％。MS(ESI)m/z理论值C 46H 53N 2NaO 7P[M+Na] +为799.35；测量值，799.67。 Dissolve L-2 (60mg, 0.1mmol) in 2mL anhydrous acetonitrile, add 5-benzylthio tetrazolium (BTT, 30mg, 0.15mmol) and 2-cyanoethyl N,N,N',N'- Tetraisopropyl phosphorodiamidite (50 μL, 0.15 mmol). After the reaction was stirred at room temperature for 1 hour, after evaporating the acetonitrile, the product was separated by column chromatography with a developing solvent (ethyl acetate/petroleum ether/triethylamine=25:75:2). The product was 72 mg, and the yield was 95%. MS (ESI) m/z theoretical value C 46 H 53 N 2 NaO 7 P[M+Na] + is 799.35; measured value, 799.67.
为了验证连接子L’在寡核苷酸固相合成中的应用，以刚性连接子亚磷酰胺单体L为原料，被用于连接在一个长度为21个碱基的寡核苷酸的5’端(5’-HO-CTCTATGGGCAGTCGGTGAAT-OH-3’，结构为图2)。将包含刚性连接子亚磷酰胺单体L和寡核苷酸序列(CTCTATGGGCAGTCGGTGAAT)的位置信息编辑完毕后，上载于自动化Dr.Oligo48DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；化合物L溶解于乙腈，浓度为0.10M，分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成参见(Beaucage et al.,J.Tetrahedron Letters.22.20:1859-1862(1981)，其中，化合物L偶联时间为320s×4次，偶联时间为3min。在得到包含刚性连接子L’的引物后，经过HPLC分离纯化回收。产物经HPLC分析。粗品分析HPLC梯度洗脱条件为0-6min，乙腈浓度为5％-20％；6.01-8min，乙腈浓度为88％；8.01-10min，乙腈浓度为5％。纯品分析HPLC梯度洗脱条件为0-6min，乙腈浓度为5％-30％，6.01-8min，乙腈浓度为88％，8.01-10min，乙腈浓度为5％。经过切割脱除碱基保护基团后的样品，经过LC-MS分析确认(图7为LC-MS谱图，图8为质谱)，保留时间为4.07分钟处的主峰为连接子连接于寡核苷酸上的产物，产率为95％，说明刚性连接子在固相合成时，刚性连接子亚磷酰胺单体与 寡核苷酸的5’端羟基的偶联效率高，连接子的另一端的DMTr在固相合成完成后，可以被顺利脱除，同时说明了该连接子在寡核苷酸合成后的切割及脱保护步骤强碱性条件下可以稳定存在。In order to verify the application of the linker L'in the solid-phase synthesis of oligonucleotides, the rigid linker phosphoramidite monomer L was used as a raw material to connect to the 5 of an oligonucleotide with a length of 21 bases. 'End (5'-HO-CTCTATGGGCAGTCGGTGAAT-OH-3', the structure is shown in Figure 2). After editing the position information containing the rigid linker phosphoramidite monomer L and the oligonucleotide sequence (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer. Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, respectively placed in the corresponding synthesis channel of the synthesizer, solid-phase synthesis and The method of cleavage and deprotection is the same as that of conventional primer solid-phase synthesis, see (Beaucage et al., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), where the coupling time of compound L is 320s×4 times, and the coupling time is 3min. After obtaining the primer containing the rigid linker L', it was separated, purified and recovered by HPLC. The product was analyzed by HPLC. The HPLC gradient elution condition for crude product analysis was 0-6min, and the concentration of acetonitrile was 5%-20%; 6.01-8min, The acetonitrile concentration is 88%; 8.01-10min, the acetonitrile concentration is 5%. The pure product analysis HPLC gradient elution conditions are 0-6min, the acetonitrile concentration is 5%-30%, 6.01-8min, the acetonitrile concentration is 88%, 8.01 10min, the concentration of acetonitrile was 5%. The sample after cleavage to remove the base protecting group was confirmed by LC-MS analysis (Figure 7 is the LC-MS spectrum, Figure 8 is the mass spectrum), and the retention time is 4.07 minutes. The main peak is the product of the linker connected to the oligonucleotide, and the yield is 95%, indicating that the rigid linker phosphoramidite monomer and the oligonucleotide 5'hydroxyl The linking efficiency is high. The DMTr at the other end of the linker can be removed smoothly after the solid phase synthesis is completed. It also shows that the linker can be stable under strong alkaline conditions during the cleavage and deprotection steps after oligonucleotide synthesis. exist.
实施例2 刚性连接子L’用于染料修饰Example 2 Rigid linker L’ is used for dye modification
将L’连接在寡核酸末端后，连接子的末端羟基可以被利用进一步修饰，比如通过与染料(羧基荧光素FAM)的亚磷酰胺单体偶联得到染料与寡核酸被刚性连接子L’间隔开的结构，如图3中Probe 1。将包含染料(6-FAM亚磷酰胺单体)、刚性连接子亚磷酰胺单体L和寡核苷酸序列(CTCTATGGGCAGTCGGTGAAT)的位置信息编辑完毕后，上载于自动化Dr.Oligo48DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；化合物L溶解于乙腈，浓度为0.10M，染料FAM亚磷酰胺单体浓度为0.057M；分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成参见(Beaucage et al.,J.Tetrahedron Letters.22.20:1859-1862(1981)，其中，化合物L偶联时间为320s×4次，染料偶联时间为3min。在得到包含染料供体及连接子的引物后，经过HPLC分离纯化回收。产物经HPLC分析。粗品分析HPLC梯度洗脱条件为0-6min，乙腈浓度为5％-20％，6.01-8min，乙腈浓度为88％。8.01-10min，乙腈浓度为5％。如图9，保留时间为6.065min的主峰为进一步被染料修饰的产品纯品分析HPLC梯度洗脱条件为0-6min，乙腈浓度为5％-30％，6.01-8min，乙腈浓度为88％。8.01-10min，乙腈浓度为5％。如图10，经过纯化后得到干净的产品，保留时间为3.987min；其分子量由ESI质谱确认无误(图11)。MS(ESI)m/z理论值7348.2，测量值[M-H] -为7349.6。产率为57.3％，所得的修饰物纯度为95.4％。 After connecting L'to the end of the oligonucleotide, the terminal hydroxyl group of the linker can be further modified, for example, by coupling with the phosphoramidite monomer of the dye (Carboxyfluorescein FAM) to obtain the dye and the oligonucleotide by the rigid linker L' The spaced-apart structure is shown as Probe 1 in Figure 3. After editing the position information containing the dye (6-FAM phosphoramidite monomer), rigid linker phosphoramidite monomer L and oligonucleotide sequence (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer. Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, dye FAM phosphoramidite monomers with a concentration of 0.057M; respectively placed in the synthesis After the corresponding synthesis channel of the instrument alone, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis. The coupling time is 320s×4 times, and the dye coupling time is 3min. After the primer containing the dye donor and linker is obtained, it is separated and purified by HPLC. The product is analyzed by HPLC. The crude product is analyzed by HPLC gradient elution conditions of 0-6min , Acetonitrile concentration is 5%-20%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%. As shown in Figure 9, the main peak with retention time of 6.065min is the pure product further modified by dye Analytical HPLC gradient elution conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%. As shown in Figure 10, after purification, a clean product retention time 3.987min; ESI mass spectrum having a molecular weight by a confirmation (FIG. 11) .MS (ESI) m / z 7348.2 theoretical value, the measured value [MH] - is 7349.6 57.3% yield, the resulting modifications. The purity is 95.4%.
为了验证刚性连接子作用，本实验将市面上商业化已有的柔性连接子(C6和C18)换成刚性连接子后，用于染料的连接与利用刚性连接子连接染料后作了对比。由刚性连接子连接染料的寡核苷酸链记为Probe 1(5’-FAM-Linker-CTCTATGGGCAGTCGGTGAAT-OH-3’)，由柔性连接子(C6、C18)连接染料FAM的寡核苷酸链分别记为Probe 2(5’-FAM-C6-CTCTATGGGCAGTCGGTGAAT-OH-3’)、Probe 3(5’-FAM-C18-CTCTATGGGCAGTCGGTGAAT-OH-3’)。合成纯化后的产率如表1，可以看到由刚性连接子合成产率较柔性连接子的产率分别提高76.9％和72.6％。取200μL浓度为2μM的寡核苷酸链用酶标仪进行荧光测试。选取发射波长为470nm-700nm，激发波长为430nm，所得图谱相比(图12)，荧光全谱扫描性质没有发性变化，考虑浓度测量误差可以认为荧光强度基本相同。In order to verify the role of rigid linkers, in this experiment, the commercially available flexible linkers (C6 and C18) were replaced with rigid linkers, and the connection of dyes was compared with the use of rigid linkers to connect dyes. The oligonucleotide chain connected to the dye by the rigid linker is marked as Probe 1 (5'-FAM-Linker-CTCTATGGGCAGTCGGTGAAT-OH-3'), and the oligonucleotide chain of the dye FAM is connected by the flexible linker (C6, C18) They are respectively marked as Probe 2 (5'-FAM-C6-CTCTATGGGCAGTCGGTGAAT-OH-3') and Probe 3 (5'-FAM-C18-CTCTATGGGCAGTCGGTGAAT-OH-3'). The yield after synthesis and purification is shown in Table 1. It can be seen that the synthesis yield from the rigid linker is 76.9% and 72.6% higher than the yield of the flexible linker, respectively. Take 200μL of the oligonucleotide chain with a concentration of 2μM for fluorescence test with a microplate reader. The emission wavelength is 470nm-700nm, and the excitation wavelength is 430nm. Compared with the obtained spectrum (Figure 12), there is no change in the fluorescence full-spectrum scanning properties. Considering the concentration measurement error, it can be considered that the fluorescence intensity is basically the same.
表1利用不同连接子合成寡核苷酸链的产率Table 1 The yields of synthesizing oligonucleotide chains with different linkers
寡核苷酸链Oligonucleotide chain 产率Yield
Probe 1 Probe 1 57.3％57.3%
Probe 2Probe 2 32.4％32.4%
Probe 3Probe 3 33.2％33.2%
实施例3 连接子L’用于生物素修饰Example 3 The linker L’ is used for biotin modification
连接子的末端羟基也可以被生物素修饰，它是一种作为影响细胞生长并影响参与蛋白质合成的氨基酸的酶的重要组成部分的生物分子，如图4中的Probe A。将生物素亚磷酰胺单体，偶联得到生物素与寡核苷酸被刚性连接子间L’隔开的结构。将包含生物素(Biotin)(5’-Biotin亚磷酰胺单体)、刚性连接子亚磷酰胺单体L和寡核苷酸(CTCTATGGGCAGTCGGTGAAT)的序列位置信息编辑完毕后，上载于自动化Dr.Oligo48DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；化合物L溶解于乙腈，浓度为0.10M，生物素单体浓度为0.067M；分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成参见Beaucageetal.,J.Tetrahedron Letters.22.20:1859-1862(1981)，其中，化合物L偶联时间为320s×4次，Biotin偶联时间为3min。在得到包含生物素单体及连接子的引物后，经过HPLC分离纯化回收。产物经HPLC分析。粗品分析条件为0-6min，乙腈浓度为5％-20％,6.01-8min，乙腈浓度为88％。8.01-10min，乙腈浓度为5％。如图13，保留时间为6.047min的主峰为进一步被生物素修饰的产品，偶联效率高。纯品分析条件为0-6min，乙腈浓度为5％-30％，6.01-8min，乙腈浓度为88％。8.01-10min，乙腈浓度为5％。如图14，经过纯化后得到干净的产品，保留时间为4.104min；其分子量由ESI质谱确认无误(图15)MS(ESI)m/z理论值7218.6，测量值[M-H] -为7218.0。产率为56.2％，所得的修饰物纯度为97.5％。 The terminal hydroxyl group of the linker can also be modified by biotin, which is a biomolecule that is an important part of an enzyme that affects cell growth and affects amino acids involved in protein synthesis, as shown in Probe A in Figure 4. The biotin phosphoramidite monomer is coupled to obtain a structure in which the biotin and the oligonucleotide are separated by the L'between the rigid linkers. After editing the sequence position information including Biotin (5'-Biotin phosphoramidite monomer), rigid linker phosphoramidite monomer L and oligonucleotide (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA Synthesizer. Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, and the concentration of biotin monomer was 0.067M; they were placed in separate synthesizers. After the corresponding synthesis channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981), where the coupling time of compound L is 320s×4 The coupling time of Biotin is 3min. After obtaining the primer containing the biotin monomer and the linker, it is separated, purified and recovered by HPLC. The product was analyzed by HPLC. The crude product analysis conditions are 0-6min, acetonitrile concentration is 5%-20%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%. As shown in Figure 13, the main peak with a retention time of 6.047 min is a product further modified by biotin, which has a high coupling efficiency. The pure product analysis conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%. 14, after to give a clean product, retention time 4.104min; ESI mass spectrum having a molecular weight by a confirmation (FIG. 15) MS (ESI) m / z 7218.6 theoretical value, the measured value [MH] - is 7218.0. The yield was 56.2%, and the purity of the obtained modification was 97.5%.
为了验证将生物素通过刚性连接子连接后是否可以提高生物素的寡核酸链性能，链霉亲和素偶联的磁珠(DynabeadsM-270Streptavidin)对生物素的寡核酸链的抓取实验被用来验证该目的。实验设计的逻辑是，利用刚性连接子连接的寡核酸链记为Probe A(5'-Biotin-Linker-CTCTATGGGCAGTCGGTGAAT-OH-3’),按照类似的步骤合成两条利用柔性连接子(C6,C18)连接生物素的寡核酸链分别记为Probe B(5'-Biotin-C6-CTCTATGGGCAGTCGGTGAAT-OH-3’)、Probe C(5'-Biotin-C18-CTCTATGGGCAGTCGGTGAAT-OH-3’)；在相同量的寡核酸链溶液与磁珠溶液的情况下，检测磁珠抓取后上清溶液剩余寡核酸链的量来获得抓取效率对比。In order to verify whether the performance of biotin oligo-nucleic acid chain can be improved by connecting biotin through a rigid linker, streptavidin-coupled magnetic beads (DynabeadsM-270Streptavidin) were used to grasp the biotin oligo-nucleic acid chain. To verify the purpose. The logic of the experimental design is that the oligonucleotide strands connected by rigid linkers are recorded as Probe A (5'-Biotin-Linker-CTCTATGGGCAGTCGGTGAAT-OH-3'), and two flexible linkers (C6, C18) are synthesized according to similar steps. ) Oligonucleic acid chains connected to biotin are respectively marked as Probe B (5'-Biotin-C6-CTCTATGGGCAGTCGGTGAAT-OH-3'), Probe C (5'-Biotin-C18-CTCTATGGGCAGTCGGTGAAT-OH-3'); in the same amount In the case of the oligo-nucleic acid strand solution and the magnetic bead solution, the amount of remaining oligo-nucleic acid strands in the supernatant solution after the magnetic bead grabbing is detected to obtain a comparison of grabbing efficiency.
本实验所用的Dynabeads TM磁珠对于单链寡核苷酸的最大吸附值为200pmol。取20μL的Dynabeads TM磁珠，可吸附的最大吸附值为40pmol的单链寡核苷酸。分别取最大吸附值的0.95倍(实验一，38pmol的单链寡核苷酸)、1.30倍(实验二，52pmol的单链寡核苷酸) 以及1.70倍(实验三，68pmol的单链寡核苷酸)的寡核苷酸进行实验。所取的上清液使用Nanodrop仪器进行浓度测试，测得的结果如下表2。由上清液寡核苷酸浓度可以计算出上清液中残留的寡核苷酸占总引物的比例，计算过程如下：以实验二Probe A用磁珠吸附后的上清液寡核苷酸浓度计算为例，计算方式为：0.5ng/μL×80μL÷7218×1000＝5.54pmol，(5.54pmol÷52pmol)×100％＝10.65％。其中上清液的体积为80μL，寡核苷酸的相对分子质量为7218，参考上面的计算方法所得到的结果如表3(其中Probe B寡核苷酸的相对分子质量为7062.8，Probe C寡核苷酸的相对分子质量为7227)。从表中可以看出，在投入与寡核苷酸量(0.95当量)相当的磁珠的情况下，三种连接子修饰的寡核苷酸(Probe A、B和C)的剩余比例当为零说明都可以被抓取完全；当增加寡核苷酸的量，使其过量(130％当量)条件下，刚性连接子Probe A剩余比短柔性连接子Probe B少，与长柔性连接子Probe C剩余相当；当进一步增加寡核苷酸一(170％当量)刚性连接子Probe A剩余明显比柔性连接子Probe B、C少。这说明利用刚性连接子可以使生物素伸出从而更容易被链霉亲和素偶联获得。以下为具体的实验过程： The Dynabeads TM magnetic beads used in this experiment have a maximum adsorption value of 200 pmol for single-stranded oligonucleotides. Take 20μL of Dynabeads TM magnetic beads, which can adsorb single-stranded oligonucleotides with a maximum adsorption value of 40pmol. Take 0.95 times of the maximum adsorption value (experiment 1, 38pmol single-stranded oligonucleotide), 1.30 times (experiment 2, 52pmol single-stranded oligonucleotide) and 1.70 times (experiment three, 68pmol single-stranded oligonucleotide). Glycidyl) oligonucleotides for experiments. The supernatant was taken for concentration test using a Nanodrop instrument, and the measured results are shown in Table 2. From the concentration of oligonucleotides in the supernatant, the ratio of the remaining oligonucleotides in the supernatant to the total primers can be calculated. The calculation process is as follows: Take the oligonucleotides in the supernatant after the probe A is adsorbed with magnetic beads in Experiment 2 Take the concentration calculation as an example, the calculation method is: 0.5ng/μL×80μL÷7218×1000=5.54pmol, (5.54pmol÷52pmol)×100%=10.65%. The volume of the supernatant is 80μL, the relative molecular mass of the oligonucleotide is 7218, and the results obtained by referring to the above calculation method are shown in Table 3 (where the relative molecular mass of Probe B oligonucleotide is 7062.8, and Probe C oligonucleotide The relative molecular mass of nucleotides is 7227). As can be seen from the table, when the magnetic beads equivalent to the amount of oligonucleotides (0.95 equivalent) are put in, the remaining proportions of the three linker-modified oligonucleotides (Probe A, B, and C) are regarded as Zero means that all can be grasped completely; when the amount of oligonucleotide is increased to make it excessive (130% equivalent), the remaining rigid linker Probe A is less than the short flexible linker Probe B, and the long flexible linker Probe B The remaining C is equivalent; when the oligonucleotide-one (170% equivalent) is further increased, the remaining rigid linker Probe A is significantly less than the flexible linker Probe B and C. This shows that the use of rigid linkers can make biotin stretch out so that it can be more easily obtained by coupling with streptavidin. The following is the specific experimental process:
清洗Dynabeads TM磁珠： To clean Dynabeads TM magnetic beads:
1、振荡30秒，复苏磁珠。1. Vibrate for 30 seconds to recover the magnetic beads.
2、将所需体积的磁珠转移到离心管中。2. Transfer the required volume of magnetic beads to a centrifuge tube.
3、加入600μL的缓冲液洗涤。3. Add 600μL of buffer to wash.
4、将离心管放在磁力架上1分钟，弃去上清液。4. Place the centrifuge tube on the magnetic stand for 1 minute, and discard the supernatant.
5、重复步骤3和4，共洗涤3次。5. Repeat steps 3 and 4 for a total of 3 washes.
吸附生物素寡核苷酸实验步骤：Experimental steps for adsorbing biotin oligonucleotides:
1、将磁珠重置于2X B&W缓冲液中，使其最终浓度为5μg/μL。1. Reset the magnetic beads in 2X B&W buffer to make the final concentration 5μg/μL.
2、在蒸馏水中加入等量的生物素化的核酸来稀释2X B&W缓冲液中的NaCl浓度从2M到1M。2. Add the same amount of biotinylated nucleic acid to distilled water to dilute the NaCl concentration in the 2X B&W buffer from 2M to 1M.
3、在1200rpm的涡流下培养15分钟。3. Incubate for 15 minutes under a vortex at 1200 rpm.
4、用磁铁将涂有生物素核酸的磁珠分离2-3分钟，取上清液。4. Use a magnet to separate the magnetic beads coated with biotin nucleic acid for 2-3 minutes, and take the supernatant.
表2经过磁珠抓取后上清液寡核苷酸浓度Table 2 Oligonucleotide concentration in supernatant after magnetic beads grabbing
 To 实验一experiment one 实验二Experiment two 实验三Experiment Three
Probe AProbe A 0.0ng/μL0.0ng/μL 0.5ng/μL0.5ng/μL 1.9ng/μL1.9ng/μL
Probe BProbe B 0.0ng/μL0.0ng/μL 0.6ng/μL0.6ng/μL 1.9ng/μL1.9ng/μL
Probe CProbe C 0.0ng/μL0.0ng/μL 0.5ng/μL0.5ng/μL 2.1ng/μL2.1ng/μL
表3上清液寡核苷酸剩余比例Table 3 The remaining proportion of oligonucleotides in the supernatant
 To 实验一experiment one 实验二Experiment two 实验三Experiment Three
Probe AProbe A 00 10.65％10.65% 30.96％30.96%
Probe BProbe B 00 13.07％13.07% 31.64％31.64%
Probe CProbe C 00 10.64％10.64% 34.18％34.18%
实施例4 连接子L’用于N-乙酰半乳糖胺修饰Example 4 Linker L’ is used for N-acetylgalactosamine modification
N-乙酰半乳糖胺(GalNAc)是一种个半乳糖的氨基糖衍生物，将其生物偶联于寡核苷酸上可以提高靶向作用，被成功应用于世界第一种(patisiran，商品名Onpattro，2018)及第二种(givosiran，商品名Givlaari，2019)RNA干扰药物中。连接子L’也可以用于GalNAc的修饰，如图5。将包含N-乙酰半乳糖胺(5'-GalNAc C3亚磷酰胺单体)、刚性连接子亚磷酰胺单体L和寡核苷酸的序列位置信息(CTCTATGGGCAGTCGGTGAAT)的序列信息编辑完毕后，上载于自动化Dr.Oligo48DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；化合物L溶解于乙腈，浓度为0.10M，GalNAc单体浓度为0.054M；分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成参见Beaucageetal.,J.Tetrahedron Letters.22.20:1859-1862(1981)，其中，化合物L偶联时间为320s×4次，5'-GalNAcC3亚磷酰胺单体偶联时间为12min。在得到包含5'-GalNAc C3及连接子的引物后，经过HPLC分离纯化回收。产物经HPLC分析。分析条件为0-6min，乙腈浓度为5％-30％，6.01-8min，乙腈浓度为88％。8.01-10min，乙腈浓度为5％。如16，保留时间为3.405min的主峰为进一步被N-乙酰半乳糖胺修饰的产品，偶联效率高。如图17，经过纯化后得到干净的产品，保留时间为3.460min；其分子量由ESI质谱确认无误(图18)，MS(ESI)m/z理论值7422.6，测量值[M-H] -为7421.8。产率为65.0％，所得的修饰物纯度为98.9％。 N-Acetylgalactosamine (GalNAc) is an amino sugar derivative of galactose, which can be biologically coupled to oligonucleotides to improve targeting. It has been successfully applied to the world's first (patisiran, commercial product). Name Onpattro, 2018) and the second (givosiran, trade name Givlaari, 2019) RNA interference drugs. The linker L'can also be used to modify GalNAc, as shown in Figure 5. After editing the sequence information including N-acetylgalactosamine (5'-GalNAc C3 phosphoramidite monomer), rigid linker phosphoramidite monomer L, and oligonucleotide sequence position information (CTCTATGGGCAGTCGGTGAAT), upload In the automated Dr. Oligo48 DNA synthesizer. Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, and the concentration of GalNAc monomer was 0.054M; respectively placed in a separate corresponding synthesizer After synthesizing the channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981), where the coupling time of compound L is 320s×4 times , 5'-GalNAcC3 phosphoramidite monomer coupling time is 12min. After obtaining the primer containing 5'-GalNAc C3 and the linker, it is separated, purified and recovered by HPLC. The product was analyzed by HPLC. The analysis conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%. For example, 16, the main peak with a retention time of 3.405 min is the product further modified by N-acetylgalactosamine, and the coupling efficiency is high. 17, after to give a clean product, retention time 3.460min; ESI mass spectrum having a molecular weight by a confirmation (FIG. 18), MS (ESI) m / z 7422.6 theoretical value, the measured value [MH] - is 7421.8. The yield was 65.0%, and the purity of the obtained modification was 98.9%.
实施例5 连接子L’用于胆固醇分子修饰Example 5 Linker L’ is used for cholesterol molecular modification
将亲脂性基团如胆固醇分子添加至寡核苷酸可望增强其细胞摄取和膜渗透性能。L连接子也可以将胆固醇分子通过固相合成连接于寡核苷酸上，如图6中的Probe 4。将包含胆固 醇(5'-Cholesteryl-TEG亚磷酰胺单体)和连接子的序列位置信息(CTCTATGGGCAGTCGGTGAAT)的序列信息编辑完毕后，上载于自动化Dr.Oligo48DNA合成仪。单体A、C、G、T亚磷酰胺单体溶解于乙腈，浓度为0.06M；化合物L溶解于乙腈，浓度为0.10M，胆固醇单体浓度为0.050M；分别置于合成仪单独的对应合成通道后，固相合成及切割脱保护的方法同常规的引物固相合成参见(Beaucageetal.,J.Tetrahedron Letters.22.20:1859-1862(1981)，其中，化合物L偶联时间为320s×4次，胆固醇单体偶联时间为3min。在得到包含胆固醇及连接子的引物后，经过HPLC分离纯化回收。产物经HPLC分析。分析条件为0-6min，乙腈浓度为40％-80％，6.01-8min，乙腈浓度为88％。8.01-10min，乙腈浓度为5％。如图19，保留时间为3.0157min的主峰为进一步被N-乙酰半乳糖胺修饰的产品，偶联效率高。如图20，经过纯化后得到干净的产品，保留时间为3.189min；其分子量由ESI质谱确认无误(图21)，MS(ESI)m/z理论值7494.8，测量值[M-H] -为7493.9。产率为41.9％，所得的修饰物纯度为95.4％。分配系数(缩写为P)定义为两种溶剂(液相两相)之间的溶质浓度的特定比率，特别是对于未电离的溶质，因此比率的对数为logP。当一种溶剂是水而另一种是非极性溶剂时，logP值是亲脂性或疏水性的量度，而理论计算的分配系数记为clogP。经过MarvinSketch软件计算，利用刚性连接子的修饰后含胆固醇的clogP＝-6.10，而利用柔性连接子如C6修饰后(如图6中Probe 5)含胆固醇的clogP＝-7.77，因此利用刚性连接子连接胆固醇较柔性连接子如C6可以更好的提高寡苷酸的亲脂性。 The addition of lipophilic groups such as cholesterol molecules to oligonucleotides is expected to enhance its cellular uptake and membrane permeability. The L linker can also connect cholesterol molecules to oligonucleotides by solid-phase synthesis, as shown in Probe 4 in Figure 6. After editing the sequence information including cholesterol (5'-Cholesteryl-TEG phosphoramidite monomer) and linker sequence position information (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer. Monomers A, C, G, T phosphoramidite monomers are dissolved in acetonitrile with a concentration of 0.06M; compound L is dissolved in acetonitrile with a concentration of 0.10M, and the concentration of cholesterol monomer is 0.050M; they are placed in separate corresponding synthesizers After synthesizing the channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see (Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), where the coupling time of compound L is 320s×4 The coupling time of cholesterol monomer is 3min. After obtaining the primer containing cholesterol and linker, it is separated, purified and recovered by HPLC. The product is analyzed by HPLC. The analysis conditions are 0-6min, the concentration of acetonitrile is 40%-80%, 6.01 -8min, the acetonitrile concentration is 88%. 8.01-10min, the acetonitrile concentration is 5%. As shown in Figure 19, the main peak with a retention time of 3.0157min is the product further modified by N-acetylgalactosamine, and the coupling efficiency is high. 20, after to give a clean product, retention time 3.189min; ESI mass spectrum having a molecular weight by a confirmation (FIG. 21), MS (ESI) m / z 7494.8 theoretical value, the measured value [MH] - is 7493.9 yield. Is 41.9%, and the purity of the resulting modification is 95.4%. The partition coefficient (abbreviated as P) is defined as the specific ratio of the solute concentration between two solvents (liquid phase two phases), especially for unionized solutes, so the ratio The logarithm of is logP. When one solvent is water and the other is a non-polar solvent, the logP value is a measure of lipophilicity or hydrophobicity, and the theoretically calculated partition coefficient is recorded as clogP. Calculated by MarvinSketch software, using rigidity After the linker is modified, the cholesterol-containing clogP=-6.10, and the flexible linker such as C6 (as shown in Probe 5) contains cholesterol-containing clogP=-7.77. Therefore, using a rigid linker to connect cholesterol is more flexible than a flexible linker such as C6 can better improve the lipophilicity of oligonucleotides.
实施例中所用化学品的来源Sources of chemicals used in the examples
Figure PCTCN2021084341-appb-000012
Figure PCTCN2021084341-appb-000012
Figure PCTCN2021084341-appb-000013
Figure PCTCN2021084341-appb-000013

Claims (13)

  1. 一种具有式(Ⅰ)所示结构的生物偶联物A biological conjugate with the structure shown in formula (Ⅰ)
    X-P 1-R 1-L’-R 2-P 2-Y， XP 1 -R 1 -L'-R 2 -P 2 -Y,
    (Ⅰ)(Ⅰ)
    其中，in,
    L’为刚性连接子，结构为
    Figure PCTCN2021084341-appb-100001
    所述n 1、n 2各自独立地选自O、S或者N；     L'is a rigid linker, the structure is
    Figure PCTCN2021084341-appb-100001
    The n 1 and n 2 are each independently selected from O, S or N;
    X为包含荧光染料、生物素、N-乙酰半乳糖胺、胆固醇、酶或者抗体的分子；X is a molecule containing fluorescent dye, biotin, N-acetylgalactosamine, cholesterol, enzyme or antibody;
    Y为包含1-200nt的任意碱基的核酸序列；Y is a nucleic acid sequence containing any base of 1-200 nt;
    P 1，P 2各自独立地选自磷酸二酯键、硫代磷酸酯键、二硫代磷酸酯键、烷基膦酸酯键、烷基硫代磷酸酯键、磷酸三酯键、磷醢胺酸酯键、硅氧烷基、碳酸酯键、烷氧基甲醢基、乙醢胺酸酯键、胺基甲酸酯键、吗啉基、硼烷基、硫醚键、桥接磷醢胺酸酯键、桥接亚甲基膦酸酯键、桥接硫代磷酸酯键或砜核苷键； P 1 and P 2 are each independently selected from a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, an alkyl phosphonate bond, an alkyl phosphorothioate bond, a phosphotriester bond, and a phosphoric acid ester bond. Amino acid ester bond, siloxane group, carbonate bond, alkoxy methanoate group, acetamino acid ester bond, urethane bond, morpholino group, boryl group, thioether bond, bridging phosphate Amino acid ester linkage, bridging methylene phosphonate linkage, bridging phosphorothioate linkage or sulfone nucleoside linkage;
    R 1，R 2各自独立地具有式(Ⅱ)所示的结构， R 1 and R 2 each independently have a structure represented by formula (II),
    -Z-(CH 2) n-， -Z-(CH 2 ) n -,
    (Ⅱ)(Ⅱ)
    其中，Z为O、NH或者CONH，n为1-10任一整数值。Among them, Z is O, NH or CONH, and n is any integer value of 1-10.
  2. 权利要求1所述的生物偶联物，所述n 1，n 2均为O。 The bioconjugate of claim 1, wherein both n 1 and n 2 are O.
  3. 权利要求1所述的生物偶联物，所述X选自荧光染料、生物素、N-乙酰半乳糖胺或者胆固醇。The bioconjugate of claim 1, wherein the X is selected from a fluorescent dye, biotin, N-acetylgalactosamine or cholesterol.
  4. 权利要求1-3中任一项所述的生物偶联物，所述X还包含柔性连接子。The bioconjugate of any one of claims 1 to 3, wherein the X further comprises a flexible linker.
  5. 权利要求4所述的生物偶联物，所述柔性连接子结构为
    Figure PCTCN2021084341-appb-100002
    Figure PCTCN2021084341-appb-100003
    或者
    Figure PCTCN2021084341-appb-100004
    The bioconjugate of claim 4, the flexible linker structure is
    Figure PCTCN2021084341-appb-100002
    Figure PCTCN2021084341-appb-100003
    or
    Figure PCTCN2021084341-appb-100004
  6. 权利要求1所述的生物偶联物，所述Y选自5-150nt的任意碱基的核酸序列，优选为10-100nt的任意碱基的核酸序列。The biological conjugate according to claim 1, wherein the Y is selected from a nucleic acid sequence of any base from 5 to 150 nt, preferably a nucleic acid sequence of any base from 10 to 100 nt.
  7. 权利要求1所述的生物偶联物，所述P 1，P 2均为磷酸二酯键。 The bioconjugate of claim 1, wherein the P 1 and P 2 are both phosphodiester bonds.
  8. 权利要求1所述的生物偶联物，所述Z为O，n为2-8任一整数值。The bioconjugate of claim 1, wherein Z is O, and n is any integer value of 2-8.
  9. 权利要求1-8中任一项所述的生物偶联物，所述X选自包含柔性连接子结构的荧光染料，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。 The bioconjugate according to any one of claims 1-8, wherein X is selected from fluorescent dyes containing flexible linker structures, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -.
  10. 权利要求1-8中任一项所述的生物偶联物，所述X选自包含柔性连接子结构的生物素，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。 The bioconjugate according to any one of claims 1-8, wherein X is selected from biotin containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -.
  11. 权利要求1-8中任一项所述的生物偶联物，所述X选自包含柔性连接子结构的N-乙酰半乳糖胺，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。 The bioconjugate according to any one of claims 1-8, wherein X is selected from N-acetylgalactosamine containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, R 1 , R 2 is all -O-(CH 2 ) 2 -.
  12. 权利要求1-8中任一项所述的生物偶联物，所述X选自包含柔性连接子结构的胆固醇，P 1，P 2均为磷酸二酯键，R 1，R 2均为-O-(CH 2) 2-。 The bioconjugate of any one of claims 1-8, wherein X is selected from cholesterol containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both- O-(CH 2 ) 2 -.
  13. 权利要求1-12中任一项所述的生物偶联物在核酸诊断、测序、基因组分析、检测遗传变异、鉴定单核苷酸多态性、寡核苷酸的富集和纯化、固相差异显示方案、蛋白质相互作用过程、多肽修饰、药物递送***或质谱分析检测中的应用。The biological conjugate of any one of claims 1-12 is used in nucleic acid diagnosis, sequencing, genome analysis, detection of genetic variation, identification of single nucleotide polymorphisms, enrichment and purification of oligonucleotides, solid phase Application in difference display scheme, protein interaction process, peptide modification, drug delivery system or mass spectrometry analysis.
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Citations (5)

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WO2003097104A1 (en) * 2002-05-15 2003-11-27 Pentraxin Therapeutics Limited Treatment and prevention of tissue damage
CN104244987A (en) * 2011-12-15 2014-12-24 株式会社百奥尼 Novel oligonucleotide conjugates and use thereof
CN106795200A (en) * 2014-10-10 2017-05-31 豪夫迈·罗氏有限公司 GALNAC phosphoramidites, its nucleic acid conjugate and application thereof
WO2017158136A1 (en) * 2016-03-16 2017-09-21 Oryzon Genomics, S.A. Methods to determine kdm1a target engagement and chemoprobes useful therefor
CN109957566A (en) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 The compound of the oligonucleotides of modification and the oligonucleotides that can be used for synthetic modification

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003097104A1 (en) * 2002-05-15 2003-11-27 Pentraxin Therapeutics Limited Treatment and prevention of tissue damage
CN104244987A (en) * 2011-12-15 2014-12-24 株式会社百奥尼 Novel oligonucleotide conjugates and use thereof
CN106795200A (en) * 2014-10-10 2017-05-31 豪夫迈·罗氏有限公司 GALNAC phosphoramidites, its nucleic acid conjugate and application thereof
WO2017158136A1 (en) * 2016-03-16 2017-09-21 Oryzon Genomics, S.A. Methods to determine kdm1a target engagement and chemoprobes useful therefor
CN109957566A (en) * 2017-12-26 2019-07-02 广州市锐博生物科技有限公司 The compound of the oligonucleotides of modification and the oligonucleotides that can be used for synthetic modification

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