Azobenzene quenching units, solid phase synthesis supports and methods of making same, nucleic acid probes

Abstract

The application relates to an azobenzene quencher unit, a preparation method thereof, a solid-phase synthesis carrier, a preparation method thereof, and a nucleic acid probe. The structural general formula of the azobenzene quencher unit is shown in the following formula: wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12 and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso or cyano, n=0-6, and m=2-12. The azobenzene quencher unit contains a polyfluorocarboxylic acid connecting structure. The azobenzene quencher unit is connected to the solid-phase synthesis carrier through the formation of an amide bond between the carboxylic acid of the polyfluorocarboxylic acid connecting structure and the amino group on the surface of the solid-phase synthesis carrier, and is used for synthesizing nucleic acids. The synthesis yield of 3' azobenzene quencher-labeled nucleic acids can be effectively improved.

Description

Technical Field

[0001] This application relates to the field of molecular biology, specifically to azobenzene quenching units, solid-phase synthetic supports and their preparation methods, and nucleic acid probes. Background Technology

[0002] Nucleic acid probes have important applications in various industries such as clinical testing, disease control, environmental monitoring, food safety, and agriculture, forestry, animal husbandry, and fisheries, serving as a highly efficient and rapid detection tool. Fluorescent probes are one of the most widely used types of nucleic acid probes. Their structure consists of natural or non-natural nucleic acids containing one or more fluorescent molecules. By binding to target sequences or molecules to form double-stranded or other secondary structures, they control fluorescence generation and alter the detection wavelength, enabling the detection of the target sequence or molecule. Complementing fluorescent probes are nucleic acid sequences containing quenching functional groups, also known as quenching sequences. These sequences quench the luminescence of fluorescent units by quenching the functional groups, thereby improving detection efficiency.

[0003] Currently, the quenching units used for nucleic acid probes are mainly azobenzene dyes. By connecting the quenching units to porous glass microspheres through linking groups with different structures, a solid-phase synthetic support that can be modified at the 3' end of nucleic acid can be obtained.

[0004] However, current azobenzene quenching units, solid-phase synthesis supports and their preparation methods, and nucleic acid probes still need improvement. Summary of the Invention

[0005] This application provides an azobenzene-based quenching unit and its preparation method, a solid-phase synthesis support and its preparation method, and a nucleic acid probe. The azobenzene-based quenching unit of this application contains a polyfluorocarboxylic acid linkage structure. The azobenzene-based quenching unit is linked to the solid-phase synthesis support by forming an amide bond between the carboxylic acid in the polyfluorocarboxylic acid linkage structure and an amino group on the surface of the solid-phase synthesis support, thereby achieving a high nucleic acid synthesis yield.

[0006] Firstly, this application proposes an azobenzene-based quenching unit. The general structural formula of the azobenzene-based quenching unit is as follows:

[0007]

[0008] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano, n = 0-6, and m = 2-12.

[0009] Furthermore, the structural formula of the azobenzene quenching unit is as follows:

[0010] ,or

[0011] .

[0012] Secondly, this application proposes a solid-phase synthesis support modified with an azobenzene quenching unit. The general structural formula of the solid-phase synthesis support is:

[0013]

[0014] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano, n = 0-6, and m = 2-12.

[0015] Furthermore, the structural formula of the solid-phase synthesis support is as follows:

[0016] ,or

[0017] .

[0018] Furthermore, the solid-phase synthesis support satisfies at least one of the following conditions: the particle size of the porous glass microspheres is 10-100 micrometers; the pore size of the porous glass microspheres is 800-1200 angstroms; and the loading of azobenzene quenching units modified on the surface of the solid-phase synthesis support is 20-120 micromoles / gram.

[0019] Thirdly, this application proposes a nucleic acid probe. The nucleic acid probe is obtained through a solid-phase synthesis support modified with an azobenzene quenching unit as described above, and the general structural formula of the nucleic acid probe is:

[0020]

[0021] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano.

[0022] Furthermore, the structural formula of the nucleic acid probe is as follows:

[0023] ,or

[0024] .

[0025] Further, the nucleic acid probe includes any one or more of the following: natural and non-natural nucleic acids modified on the sugar ring, natural and non-natural nucleic acids modified on the base, natural and non-natural nucleic acids modified on the linker unit, and other non-natural structurally modified nucleic acids linked by nucleosides and phosphates. The natural and non-natural nucleic acids modified on the sugar ring include: natural DNA, natural RNA, 2'-methoxy-substituted deoxyribose, 2'-fluorine-substituted deoxyribose, 2'-methoxyethyl-substituted deoxyribose, 2'-methylamino-2-oxyethyl-substituted deoxyribose, and 2'-propargyl... Oxyl-substituted deoxyribose, 2'-butoxy-substituted deoxyribose, 2'-hexyloxy-substituted deoxyribose, 2'-octyloxy-substituted deoxyribose, 2'-decyloxy-substituted deoxyribose, 2'-dodecyloxy-substituted deoxyribose, 2'-tetradecyloxy-substituted deoxyribose, 2'-hexadecyloxy-substituted deoxyribose, 2'-octadecyloxy-substituted deoxyribose, 2'-eicosyloxy-substituted deoxyribose, 2'-oxy-4'-carbonylmethylene-locked deoxyribose, 2'-oxy-4'-carbonylvinyl-locked deoxyribose The bases in the natural and non-natural nucleic acids modified with the bases include: 2'-oxy-4'-carbon-S-configuration ethyl-locked deoxyribose, 2'-oxy-4'-carbon-R-configuration ethyl-locked deoxyribose, 2'-fluorinated arabinose, 2'-methoxylated arabinose, L-configuration deoxyribose, L-configuration ribose, 3' and 5' linked inverted deoxyribose, and 3' and 5' linked inverted ribose; the bases in the modified natural and non-natural nucleic acids include: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, and 6-methyladenine. The linking units of the natural and non-natural nucleic acids modified on the linking units include: racemic phosphothioester bonds, R-configured phosphothioester bonds, S-configured phosphothioester bonds, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, and monooctyl hypophosphite; the other non-natural structurally modified nucleic acids linking nucleosides and phosphates include: 2',3'-Dideoxy-β-D-glucopyranosyl, wherein the base is at the 6-β position; 2-hydroxymethylmorpholine, wherein the base is at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, wherein the base is at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, wherein the base is at the 2-position; unlocked nucleic acid unit, having an RNA-like structure with no chemical bond between the 2' and 3' ends, wherein the base is at the 1'-β position; the length of the nucleic acid is 2-240 nucleotides.

[0026] Fourthly, this application proposes a method for preparing the aforementioned azobenzene quenching unit. The method includes: mixing an azobenzene compound with 4,4'-dimethoxytriphenylchloromethane to undergo an etherification reaction to obtain azobenzene-O-DMTr; and mixing the azobenzene-O-DMTr with a fluorocarboxylic acid to undergo an esterification reaction to obtain the azobenzene quenching unit.

[0027] Fifthly, this application proposes a method for preparing the aforementioned solid-phase synthetic support modified with azobenzene quenching units. The method includes: mixing porous glass microspheres with amino-modified surfaces with fluorocarboxylic acids to undergo an amidation reaction, obtaining porous glass microspheres with fluorocarboxylic acids modified on the surface; and performing an esterification reaction between a polyazobenzene compound and the porous glass microspheres with the fluorocarboxylic acids to obtain the solid-phase synthetic support. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0029] Figure 1 This application provides embodiments of azobenzene quenching units containing polyfluorocarboxylic acid linkages that are connected to a solid-phase synthesis support for nucleic acid synthesis cycles.

[0030] Figure 2 This is a schematic diagram of the synthetic route of compound 2 in Example 1;

[0031] Figure 3 This is a schematic diagram of the synthetic route of compound 4 in Example 2;

[0032] Figure 4 The molecular weight diagram of the T20 sequence synthesized from the solid-phase synthesis support of Example 1;

[0033] Figure 5 The high-performance liquid chromatography (HPLC) spectrum of the T20 sequence synthesized from the solid-phase synthesis support of Example 1;

[0034] Figure 6 The high-performance liquid chromatography (HPLC) spectrum of the T20 sequence synthesized from the solid-phase synthesis support of Comparative Example 1 is shown.

[0035] Figure 7 The molecular weight diagram of the T20 sequence synthesized from the solid-phase synthesis support of Example 2;

[0036] Figure 8The high-performance liquid chromatography (HPLC) spectrum of the T20 sequence synthesized from the solid-phase synthesis support of Example 2;

[0037] Figure 9 The image shows the high-performance liquid chromatography (HPLC) spectrum of the T20 sequence synthesized from the solid-phase synthesis support of Comparative Example 2. Detailed Implementation

[0038] The embodiments of this application are hereby disclosed in detail with appropriate reference to the accompanying drawings. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of actually identical structures may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.

[0039] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a specific parameter, it is also expected that ranges of 60 to 110 and 80 to 120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise stated, the numerical range "a to b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0 to 5" means that all real numbers between "0 and 5" have been listed in this article; "0 to 5" is just a shortened representation of these numerical combinations. In addition, when a parameter is stated as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0040] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0041] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0042] Unless otherwise specified, all steps of this application may be performed sequentially or randomly, preferably sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0043] In one aspect of this application, an azobenzene-based quenching unit is proposed. In some embodiments of this application, the general structural formula of the azobenzene-based quenching unit is shown in formula (1):

[0044] (1)

[0045] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano, n = 0-6, and m = 2-12.

[0046] The azobenzene quenching unit of this application contains a polyfluorocarboxylic acid (PFCA) linker, while currently reported azobenzene quenching units do not contain PFCA linkers. Compared to azobenzene quenching units without PFCA linkers, the azobenzene quenching unit with PFCA linkers in this application results in a higher synthesis yield for nucleic acid probes.

[0047] Among them, R1 to R13 can be the same or different, or some of R1 to R13 are the same, while the other part is different from other substituents.

[0048] In some specific embodiments of this application, the structural formula of the azobenzene quenching unit is shown in formula (2):

[0049] (2)

[0050] Alternatively, in some other specific embodiments of this application, the structural formula of the azobenzene quenching unit is shown in formula (3):

[0051] (3)

[0052] The aforementioned azobenzene quenching units all contribute to higher synthesis yields of nucleic acid probes.

[0053] In another aspect of this application, a solid-phase synthesis support modified with an azobenzene quenching unit is proposed. In some embodiments of this application, the general structural formula of the solid-phase synthesis support modified with the azobenzene quenching unit is shown in formula (4):

[0054] (4)

[0055] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano, n = 0-6, and m = 2-12.

[0056] This application utilizes molecular design to link azobenzene-based quenching functional groups to a porous glass microsphere support via amide bonds of polyfluorocarboxylic acids, resulting in porous glass microspheres loaded with azobenzene-based quenching functional groups. These porous glass microspheres are suitable for the chemical synthesis of natural and non-natural nucleic acids, including DNA, RNA, synthetic nucleotides containing non-natural nucleotide units, and synthetic nucleotides containing both natural and non-natural nucleotide units, modified with azobenzene-based quenching functional groups at their 3' ends. Nucleic acid probes synthesized using this solid-phase synthetic support exhibit high synthesis yields.

[0057] In this application, the azobenzene quenching unit containing the polyfluorocarboxylic acid linkage structure forms an amide bond by condensing the carboxyl group in the polyfluorocarboxylic acid linkage structure with the primary amino group on the surface of the porous glass microsphere, thereby connecting the azobenzene quenching unit with the porous glass microsphere.

[0058] In some specific embodiments of this application, the structural formula of the solid-phase synthesis support is shown in formula (5):

[0059] (5)

[0060] The solid-phase synthesis support of formula (5) is prepared by the azobenzene quenching unit of formula (2) through the condensation reaction of the carboxyl group with the primary amino group on the surface of the porous glass microsphere to form an amide bond.

[0061] Alternatively, in some other specific embodiments of this application, the structural formula of the solid-phase synthesis support is shown in formula (6):

[0062] (6)

[0063] The solid-phase synthesis support of formula (6) is prepared by the azobenzene quenching unit of formula (3) through the condensation reaction of the carboxyl group with the primary amino group on the surface of the porous glass microsphere to form an amide bond.

[0064] In some embodiments of this application, the particle size of porous glass microspheres (CPG) can be 10-100 micrometers, for example, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, and 100 micrometers. If the particle size of the porous glass microspheres is too small, it will result in poor flowability; if the particle size of the porous glass microspheres is too large, it will result in a small specific surface area, affecting the synthesis efficiency. Setting the particle size of the porous glass microspheres within the above-mentioned range can balance the synthesis efficiency and hydrodynamic properties, achieving both high synthesis efficiency and good flowability.

[0065] In some embodiments of this application, the pore size of the porous glass microspheres can be 800-1200 angstroms, for example, 800 angstroms, 900 angstroms, 1000 angstroms, 1100 angstroms, and 1200 angstroms. This allows for the synthesis of nucleic acids of different lengths while ensuring the free diffusion of reagents and products, enabling the reaction to proceed fully and rapidly. Those skilled in the art can adjust the pore size of the porous glass microspheres according to actual needs during the design process.

[0066] In some embodiments of this application, the loading of azobenzene quenching units modified on the surface of the solid-phase synthesis support can be 20-120 μmol / g, for example, 20 μmol / g, 50 μmol / g, 80 μmol / g, 100 μmol / g, and 120 μmol / g. This balances signal intensity and synthesis efficiency, achieving high quenching efficiency without causing steric hindrance or side reactions.

[0067] The following is a brief explanation of the principle of synthesizing nucleic acids using this solid-phase synthesis carrier:

[0068] refer to Figure 1The first step, deprotection, is carried out in a dichloromethane solution of trichloroacetic acid to remove the 5'-terminal dimethoxytriphenylmethyl (DMT) protecting group of the nucleotide attached to the solid-phase synthesis support, exposing the free 5'-hydroxyl group (5'-OH) to prepare for the attachment of the next nucleotide. The second step, condensation, involves the newly added nucleotide monomer (with a DMT-protected 5' end and an active phosphorous amide group at the 3' end) being activated by 5-ethylthiotetrazole. The activated monomer undergoes a nucleophilic substitution reaction with the free 5'-OH of the nucleotide on the solid-phase synthesis support, forming a phosphite trimer bond, thus attaching the phosphite-protected nucleotide monomer to the exposed 5'-OH. The third step, end-capping, uses acetic anhydride-methylimidazole to rapidly acetylate the unreacted 5'-OH, rendering it inactive and blocking the very few 5'-OHs that failed to participate in the condensation reaction. This prevents these unreacted chains from being extended in subsequent cycles, thereby reducing the generation of failed sequences with missing bases and simplifying the purification of the final product. The fourth step is oxidation, which takes place in iodine-water-pyridine-tetrahydrofuran. This oxidation converts the unstable phosphite-triester bonds formed in the condensation step into stable phosphotriester bonds. These four steps constitute one cycle, which is repeated for each base extension until the desired nucleotide chain length is obtained. After synthesis, a fifth step, ammonolysis, is performed in ammonia at 60°C. This cleaves the nucleotide chain from the solid-phase synthesis support, specifically from the polyfluorocarboxylic acid linker, to obtain the nucleic acid probe.

[0069] The porous glass microsphere carrier of this application can be directly used in solid-phase synthesis equipment for nucleic acid molecules. The usage method is similar to that of general solid-phase synthesis carriers, and no changes to the synthesis operation or procedure are required.

[0070] In another aspect of this application, a nucleic acid probe is proposed. In some embodiments of this application, the nucleic acid probe is obtained through a solid-phase synthetic support modified with an azobenzene quenching unit as described above, and the general structural formula of the nucleic acid probe is shown in formula (7):

[0071] (7)

[0072] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano.

[0073] This nucleic acid probe has a high synthesis yield.

[0074] In some specific embodiments of this application, the structural formula of the nucleic acid probe is shown in formula (8):

[0075] (8)

[0076] The nucleic acid probe of formula (8) is prepared by solid-phase synthesis carrier of formula (5).

[0077] Alternatively, in some other specific embodiments of this application, the structure of the nucleic acid probe is shown in formula (9):

[0078] (9)

[0079] The nucleic acid probe of formula (9) is prepared by solid-phase synthesis carrier of formula (6).

[0080] In some embodiments of this application, the nucleic acid probe includes any one or more of the following: natural and non-natural nucleic acids modified on the sugar ring, natural and non-natural nucleic acids modified on the base, natural and non-natural nucleic acids modified on the linker unit, and other non-natural structurally modified nucleic acids linked by nucleosides and phosphates. Specifically, it can be polymerized from any one or more of the following: natural and non-natural nucleic acids modified on the sugar ring, natural and non-natural nucleic acids modified on the base, natural and non-natural nucleic acids modified on the linker unit, and other non-natural structurally modified nucleic acids linked by nucleosides and phosphates.

[0081] Among them, the natural and non-natural nucleic acids modified on the sugar ring include: natural DNA, natural RNA, 2'-methoxy-substituted deoxyribose, 2'-fluorine-substituted deoxyribose, 2'-methoxyethyl-substituted deoxyribose, 2'-methylamino-2-oxoethyl-substituted deoxyribose, 2'-propoxy-substituted deoxyribose, 2'-butoxy-substituted deoxyribose, 2'-hexyloxy-substituted deoxyribose, 2'-octyloxy-substituted deoxyribose, 2'-decyloxy-substituted deoxyribose, 2'-dodecyloxy-substituted deoxyribose, 2'-tetradecyloxy-substituted deoxyribose, and 2'-hexadecyloxy-substituted deoxyribose. Substituted deoxyribose, 2'-octadecyloxy-substituted deoxyribose, 2'-eicosyloxy-substituted deoxyribose, 2'-oxy-4'-carbonylmethylene-locked deoxyribose, 2'-oxy-4'-carbonylvinyl-locked deoxyribose, 2'-oxy-4'-carbonyl-S-configuration ethyl-locked deoxyribose, 2'-oxy-4'-carbonyl-R-configuration ethyl-locked deoxyribose, 2'-fluorinated arabinose, 2'-methoxy-substituted arabinose, L-configuration deoxyribose, L-configuration ribose, 3' and 5'-linked inverted deoxyribose, 3' and 5'-linked inverted ribose;

[0082] The bases in natural and non-natural nucleic acids modified with bases include: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine, and N-methylpseudouridine.

[0083] The linker units in natural and non-natural nucleic acids modified on the linker unit include: racemic phosphothioester bonds, R-configured phosphothioester bonds, S-configured phosphothioester bonds, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, and monooctyl hypophosphite.

[0084] Other non-natural structural modifications of nucleic acids linking nucleosides and phosphates include: 2',3'-dideoxy-β-D-glucopyranosyl, with the base at the 6-β position; 2-hydroxymethylmorpholine, with the base at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, with the base at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, with the base at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, with the base at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, with the base at the 2-position; and unlocked nucleic acid units, which are RNA-like structures with no chemical bond between the 2' and 3' ends, with the base at the 1'-β position.

[0085] The length of the nucleic acid is 2-240 nucleotides, preferably 20-160 nucleotides.

[0086] In another aspect of this application, a method for preparing the solid-phase synthetic support modified with azobenzene quenching units as described above is proposed. In some embodiments of this application, the method includes:

[0087] S100:

[0088] Amidation of porous glass microspheres with amino groups on their surface and an excess of polyfluorodicarboxylic acid in the presence of a condensing agent yields porous glass microspheres with carboxyl groups on their surface. Then, an azobenzene compound modified with a 4,4'-dimethoxytriphenyl (DMTr) functional group is linked to the porous glass microspheres with carboxyl groups in the presence of a condensing agent to obtain porous glass microspheres supported on azo compounds.

[0089] In some embodiments of this application, the general structural formula of a polyfluorodicarboxylic acid benzene compound is shown in formula (10):

[0090] (10)

[0091] In the formula, n = 0-6, m = 2-12.

[0092] In some embodiments of this application, the general structural formula of an azobenzene compound containing a 4,4'-dimethoxytriphenyl (4,4'-dimethoxytrityl, DMTr) functional group is shown in formula (11):

[0093] (11)

[0094] In the formula, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently hydrogen, methyl, ethyl, propyl, isopropyl, phenyl, biphenyl, 2-furanyl, 2-thienyl, methoxy, ethoxy, isopropoxy, nitro, nitroso, or cyano.

[0095] In this step, amino-modified porous glass microspheres are dispersed in dichloromethane, and excess fluorocarboxylic acid, an activator (such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC·HCl), and a catalyst (such as 4,4-dimethylaminopyridine, DMAP) are added. The activator reacts with one carboxyl group of the fluorocarboxylic acid to form an activated O-acylisourea intermediate. The catalyst nucleophilically attacks this intermediate to form an acyl-DMAP salt, which then attacks the amino group on the amino-modified porous glass microspheres to form an amide bond, releasing DMAP. Subsequently, the organic phase is separated by filtration and subjected to washing, drying, and elution steps to obtain porous glass microspheres containing a polyfluorocarboxylic acid linkage structure.

[0096] In this step, porous glass microspheres modified with polyfluorocarboxyl groups are dispersed in dichloromethane, and azobenzene-O-DMTr, an activator (such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC·HCl), and a catalyst (such as 4,4-dimethylaminopyridine, DMAP) are added. The activator reacts with one carboxyl group of the fluorocarboxylic acid to form an activated O-acyl isourea intermediate. The catalyst nucleophilically attacks this intermediate to form an acyl-DMAP salt, which then attacks the amino group on the amino-modified porous glass microspheres to form an amide bond and release DMAP. Subsequently, the organic phase is separated by filtration and subjected to washing, drying, and elution steps to obtain porous glass microspheres containing azobenzene quenching units with polyfluorocarboxylic acid linkage structures.

[0097] In another aspect of this application, a method for preparing the solid-phase synthetic support modified with azobenzene quenching units as described above is proposed. In some embodiments of this application, the method includes:

[0098] In this step, porous glass microspheres modified with polyfluorocarboxyl groups are dispersed in dichloromethane, and azobenzene-O-DMTr, an activator (such as dicyclohexylcarbodiimide, DCC), and a catalyst (such as 4,4-dimethylaminopyridine, DMAP) are added. The activator reacts with one carboxyl group of the fluorocarboxylic acid to form an activated O-acylisourea intermediate. The catalyst nucleophilically attacks this intermediate to form an acyl-DMAP salt, which then attacks the amino group on the amino-modified porous glass microspheres to form an amide bond, releasing DMAP. Subsequently, the organic phase is separated by filtration and subjected to washing, drying, and elution steps to obtain porous glass microspheres containing azobenzene quenching units with polyfluorocarboxylic acid linkage structures.

[0099] The azobenzene quenching units described above were mixed with porous glass microspheres with amino-modified surfaces and subjected to a condensation reaction to obtain a solid-phase synthetic support modified with azobenzene quenching units. Example

[0100] The following embodiments describe the contents disclosed in this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of the embodiments of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0101] Example 1

[0102] The preparation process of the solid-phase synthesis support with the structural formula (2) is as follows:

[0103] 1) Synthesis of the azobenzene quenching unit (i.e., compound 2) with the structural formula (2) (see reference) Figure 2 )

[0104] Weigh 3.45 g of 3,3,4,4,5,5,6,6-octafluorooctanoic acid (0.01 mol), dissolve it in 50 mL of anhydrous dichloromethane, add 5 g of amino-modified porous glass microspheres with a pore size of 1000 Å, add 2.06 g of dicyclohexylcarbodiimide (0.01 mol) and 0.1 g of 4,4-dimethylaminopyridine, and stir at room temperature for 24 hours. Filter, and wash the filter cake three times with 50 mL of dimethyl sulfoxide each time, then wash five times with 50 mL of anhydrous ethanol each time. Collect the solid, dry it under vacuum to obtain the product.

[0105] 7.95 g of 4'-(2-nitro-4-tolylazo)-2'-methoxy-5'-methyl-azophenyl-4''-(N--2-O-(4,4'-dimethoxytriphenylmethyl))-N-hydroxyethanol (0.01 mol) was dissolved in 50 mL of anhydrous dichloromethane. 5 g of amino-modified porous glass microspheres with a pore size of 1000 Å were added, along with 2.06 g of dicyclohexylcarbodiimide (0.01 mol) and 0.1 g of 4,4-dimethylaminopyridine. The mixture was stirred at room temperature for 24 hours. The mixture was filtered, and the filter cake was washed three times with 50 mL of dimethyl sulfoxide each time, followed by five times with 50 mL of anhydrous ethanol each time. The solid was collected and dried under vacuum to obtain the product.

[0106] Example 2

[0107] The preparation process of the solid-phase synthesis support with the structural formula (3) is as follows:

[0108] Weigh 3.45 g of 3,3,4,4,5,5,6,6-octafluorooctanoic acid (0.01 mol), dissolve it in 50 mL of anhydrous dichloromethane, add 5 g of amino-modified porous glass microspheres with a pore size of 1000 Å, add 2.06 g of dicyclohexylcarbodiimide (0.01 mol) and 0.1 g of 4,4-dimethylaminopyridine, and stir at room temperature for 24 hours. Filter, and wash the filter cake three times with 50 mL of dimethyl sulfoxide each time, then wash five times with 50 mL of anhydrous ethanol each time. Collect the solid, dry it under vacuum to obtain the product.

[0109] 7.95 g of 4'-(4-nitrophenylazobenzene)-2',5'-dimethoxyazobenzene-4''-(N-2-O-(4,4'-dimethoxytriphenylmethyl)-N-hydroxyethanol (0.01 mol) was dissolved in 50 mL of anhydrous dichloromethane. 5 g of amino-modified porous glass microspheres with a pore size of 1000 Å were added, along with 2.06 g of dicyclohexylcarbodiimide (0.01 mol) and 0.1 g of 4,4-dimethylaminopyridine. The mixture was stirred at room temperature for 24 hours. The mixture was filtered, and the filter cake was washed three times with 50 mL of dimethyl sulfoxide each time, followed by five times with 50 mL of anhydrous ethanol each time. The solid was collected and dried under vacuum to obtain the product.

[0110] Comparative Example 1

[0111] The azobenzene quenching unit modified on the surface of the solid-phase synthesis support does not contain polyfluorocarboxylic acid linkage structures, and its structural formula is shown in formula (12):

[0112] (12)

[0113] Comparative Example 2

[0114] The azobenzene quenching unit modified on the surface of the solid-phase synthesis support does not contain polyfluorocarboxylic acid linkage structures, and its structural formula is shown in formula (13):

[0115] (13)

[0116] Nucleic acids were synthesized using the solid-phase synthesis vectors of Examples 1, 2, 1, and 2, respectively. The structural formulas of the nucleic acids synthesized using the solid-phase synthesis vectors of Examples 1 and 1 are shown in formula (8), and the structural formulas of the nucleic acids synthesized using the solid-phase synthesis vectors of Examples 2 and 2 are shown in formula (9).

[0117] (8)

[0118] (9)

[0119] The process of nucleic acid synthesis:

[0120] The experimental environment was 25±1℃ and the humidity was 10%~30%. The nucleic acid structure was DNA, with a sequence of 20 thymine deoxynucleotides, T20, and a sequence of 5'-TTTTTTTTTTTTTTTTTTTX-3', where X represents azadiphenylcyclooctyne.

[0121] The specific synthesis process was as follows: the amount of nucleic acid synthesized was 200 nanomoles; the synthesis column was a porous glass microsphere powder-packed column modified with aziridine cyclooctyne; the concentration of deoxythymidine phosphoramidide monomer was 0.05 mmol / L; the water content of the eluting acetonitrile was 15 ppm; the deprotecting reagent was a 3% (w / v) trichloroacetic acid solution in dichloromethane; the activator was a 0.45 mol / L acetonitrile solution of 5-ethylthiotetrazole; the oxidant was a 0.05 mol / L tetrahydrofuran-water-pyridine mixed solution (where the volume ratio of tetrahydrofuran-water-pyridine was 90:5:5); the end-capping reagent A was a 10% acetic anhydride solution in tetrahydrofuran; and the end-capping reagent B was a 5% 1-methylimidazole solution in tetrahydrofuran.

[0122] Nucleic acid synthesis was performed using the DMT-off method. After synthesis, the porous glass microsphere carrier was removed and placed in 1 mL of 28% ammonia solution for ammonolysis at 60°C for 2 hours. The ammonolysis solution was dried and analyzed using high-performance liquid chromatography (HPLC).

[0123] Figure 4 The molecular weight diagram of the T20 sequence synthesized from the solid-phase synthesis support of Example 1 is shown. The theoretical molecular weight of the T20 sequence is 6618.2, and the detection result is 6618.9, which proves that the azobenzene quenching group has been successfully modified to the 3' end of the T20 sequence (as shown in structural formula (8)).

[0124] Figure 5 Table 1 shows the high-performance liquid chromatography (HPLC) chromatograms of the T20 sequence synthesized from the solid-phase synthesis support of Example 1. Table 1 also shows the HPLC analysis data of the T20 sequence synthesized from the solid-phase synthesis support of Example 1.

[0125] Table 1

[0126]

[0127] Depend on Figure 5 As shown in Table 1, the retention time for the largest peak area and peak ratio is 19.066 min, and the signal at this point is a DNA sequence modified with an azobenzene quencher group.

[0128] Figure 6 Table 2 shows the high-performance liquid chromatography (HPLC) chromatogram of the T20 sequence synthesized from the solid-phase synthesis support of Comparative Example 1. Table 2 shows the HPLC analysis data of the T20 sequence synthesized from the solid-phase synthesis support of Comparative Example 1.

[0129] Table 2

[0130]

[0131] Depend on Figure 6 As shown in Table 2, the retention time for the largest peak area and peak ratio is 18.981 min, and the signal at this point is a DNA sequence modified with an azobenzene quencher group.

[0132] Figure 7 The molecular weight diagram of the T20 sequence synthesized from the solid-phase synthesis support of Example 2 is shown. The theoretical molecular weight of the T20 sequence is 6577.6, and the test result is 6577.6, which proves that the azobenzene quenching group has been successfully modified to the 3' end of the T20 sequence (as shown in structural formula (9)).

[0133] Figure 8 Table 3 shows the high-performance liquid chromatography (HPLC) chromatogram of the T20 sequence synthesized from the solid-phase synthesis support of Example 2. Table 3 shows the HPLC analysis data of the T20 sequence synthesized from the solid-phase synthesis support of Example 2.

[0134] Table 3

[0135]

[0136] Depend on Figure 8 As shown in Table 3, the retention time for the largest peak area and peak ratio is 14.819 min, and the signal at this point is a DNA sequence modified with an azobenzene quencher group.

[0137] Figure 9Table 4 shows the high-performance liquid chromatography (HPLC) chromatogram of the T20 sequence synthesized from the solid-phase synthesis support of Comparative Example 2. Table 4 shows the HPLC analysis data of the T20 sequence synthesized from the solid-phase synthesis support of Comparative Example 2.

[0138] Table 4

[0139]

[0140] Depend on Figure 9 As shown in Table 4, the retention time for the largest peak area and peak ratio is 14.849 min, and the signal at this point is a DNA sequence modified with an azobenzene quencher group.

[0141] The synthesis yields of nucleic acids synthesized from the solid-phase synthesis vectors of Examples 1, 2, 1, and 2 are listed in Table 5.

[0142] Table 5

[0143]

[0144] Experimental results show that the target product yield of porous glass microspheres containing polyfluorocarboxylic acid (PFCA) linking structures is higher than that of porous glass microspheres without PFCA linking structures. Compared to Comparative Example 1, the target product yield of porous glass microspheres containing PFCA linking structures in Example 1 was 77.4%, while the target product yield of porous glass microspheres without PFCA linking structures in Comparative Example 1 was 67.2%. Compared to Comparative Example 2, the target product yield of porous glass microspheres containing PFCA linking structures in Example 2 was 85.2%, while the target product yield of porous glass microspheres without PFCA linking structures in Comparative Example 2 was 57.2%.

[0145] High performance liquid chromatography test conditions:

[0146] Mobile phase A was chromatographically pure acetonitrile, mobile phase B was a 10% deionized aqueous solution of triethylamine acetate, the analytical column was a C18 Waters X-Bridge with a particle size of 5 μm and dimensions of 250 mm × 4.6 mm, the flow rate was 0.2 mL / min, and the column temperature was 25℃.

[0147] Method for measuring load:

[0148] Weigh 15 mg of the solid-phase synthesis support with surface-modified azobenzene quenching units and place it into a 15 mL test tube;

[0149] Weigh 3.55 g of p-toluenesulfonic acid and add it to a clean 250 mL Erlenmeyer flask. Dissolve it in anhydrous acetonitrile to a final volume of 200 mL to obtain a 0.1 mol / L p-toluenesulfonic acid acetonitrile solution.

[0150] Add 1 mL of 0.1 mol / L p-toluenesulfonic acid acetonitrile solution as eluent to a test tube containing a solid-phase synthesis support with azobenzene quenching units on its surface, mix well, let stand for 5 minutes, centrifuge for 10 seconds, and take out the supernatant.

[0151] The detection wavelength of the UV-Vis spectrophotometer was determined to be 498 nm. The eluent was used as a reference solution, and the absorbance coefficient of the supernatant was measured.

[0152] The formula for calculating load is as follows:

[0153] Loading capacity (μmol / g) = OD λ498 × Dilution factor × 12 ÷ CPG powder mass (mg)

[0154] Although illustrative embodiments have been demonstrated and described, those skilled in the art should understand that the above embodiments should not be construed as limiting the present application, and that changes, substitutions and modifications can be made to the embodiments without departing from the spirit, principles and scope of the present application.

Claims

1. An azobenzene-based quenching unit, characterized in that, Its general structural formula is: ， In the formula, R3 is methyl or nitro, R5 is H or nitro, R7 is methyl or methoxy, R9 is methoxy, and the remaining R groups are all hydrogen, n = 2, m = 4.

2. The azobenzene quenching unit according to claim 1, characterized in that, Its structural formula is: ,or 。 3. A solid-phase synthesis support modified with azobenzene quenching units, characterized in that, Its general structural formula is: ， In the formula, R3 is methyl or nitro, R5 is H or nitro, R7 is methyl or methoxy, R9 is methoxy, and the remaining R groups are all hydrogen, n = 2, m = 4.

4. The solid-phase synthesis support modified with azobenzene quenching units according to claim 3, characterized in that, Its structural formula is: ,or 。 5. The solid-phase synthesis support modified with azobenzene quenching units according to claim 3, characterized in that, It meets at least one of the following conditions: The porous glass microspheres have a particle size of 10-100 micrometers; the pore size of the porous glass microspheres is 800-1200 angstroms; and the loading of the azobenzene quenching unit modified on the surface of the solid-phase synthesis support is 20-120 micromoles / gram.

6. A nucleic acid probe, characterized in that, The nucleic acid probe is obtained using a solid-phase synthesis support modified with an azobenzene quenching unit as described in any one of claims 3-5, and the general structural formula of the nucleic acid probe is: ， In the formula, R3 is methyl or nitro, R5 is H or nitro, R7 is methyl or methoxy, R9 is methoxy, and the remaining substituent R groups are all hydrogen.

7. The nucleic acid probe according to claim 6, characterized in that, Its structural formula is: ,or 。 8. The nucleic acid probe according to claim 6, characterized in that, The nucleic acid probe is any one or more of the following: natural and non-natural nucleic acids modified on the sugar ring, natural and non-natural nucleic acids modified on the base, natural and non-natural nucleic acids modified on the linker unit, and other non-natural structurally modified nucleic acids linked by nucleosides and phosphates. The natural and non-natural nucleic acids modified on the sugar ring include: natural DNA, natural RNA, 2'-methoxy-substituted deoxyribose, 2'-fluorine-substituted deoxyribose, 2'-methoxyethyl-substituted deoxyribose, 2'-methylamino-2-oxoethyl-substituted deoxyribose, 2'-propoxy-substituted deoxyribose, 2'-butoxy-substituted deoxyribose, 2'-hexoxy-substituted deoxyribose, 2'-octoxy-substituted deoxyribose, and 2'-decoxy-substituted deoxyribose. 2'-Dodecyloxy-substituted deoxyribose, 2'-Tetradecyloxy-substituted deoxyribose, 2'-Hexadecyloxy-substituted deoxyribose, 2'-Octadecyloxy-substituted deoxyribose, 2'-Eicosyloxy-substituted deoxyribose, 2'-Oxy-4'Carbylmethylene-locked deoxyribose, 2'-Oxy-4'Carbylvinyl-locked deoxyribose, 2'-Oxy-4'Carbyl-S-configuration ethyl-locked deoxyribose, 2'-Oxy-4'Carbyl-R-configuration ethyl-locked deoxyribose, 2'-Fluoro-substituted arabinose, 2'-Methoxy-substituted arabinose, L-configuration deoxyribose, L-configuration ribose, 3' and 5'-linked inverted deoxyribose, 3' and 5'-linked inverted ribose; The bases in the natural and non-natural nucleic acids modified on the bases are: uracil, thymine, cytosine, adenine, guanine, hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, dihydrouracil, tetrahydrouracil, pseudouridine, and N-methylpseudouridine; the linking units in the natural and non-natural nucleic acids modified on the linking units include: racemic phosphothioester bonds, R-configured phosphothioester bonds, S-configured phosphothioester bonds, monomethyl hypophosphite, monoethyl hypophosphite, monopropyl hypophosphite, monobutyl hypophosphite, monopentyl hypophosphite, monohexyl hypophosphite, monoheptyl hypophosphite, and monooctyl hypophosphite. The other non-natural structural modifications of the nucleic acids linking nucleosides and phosphates are: 2',3'-dideoxy-β-D-glucopyranosyl, with the base at the 6-β position; 2-hydroxymethylmorpholine, with the base at the 6-position; 1-hydroxy-S-2-hydroxy-3-methylene unit, with the base at the 3-position; 1-hydroxy-R-2-hydroxy-3-methylene unit, with the base at the 3-position; S-1,3-dihydroxymethyl-2-methylene unit, with the base at the 2-position; R-1,3-dihydroxymethyl-2-methylene unit, with the base at the 2-position; and an unlocked nucleic acid unit, which is an RNA-like structure with no chemical bond between the 2' and 3' ends, with the base at the 1'-β position; The nucleic acid is 2-240 nucleotides in length.

9. A method for preparing a solid-phase synthetic support modified with azobenzene quenching units as described in any one of claims 3-5, characterized in that, include: Amidation reaction was carried out on porous glass microspheres with amino-modified surfaces and fluorocarboxylic acids to obtain porous glass microspheres with fluorocarboxylic acids. An esterification reaction was carried out on the porous glass microspheres with fluorocarboxylic acids to obtain the solid-phase synthetic support.

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