Compounds, compositions and methods for isolating nucleic acids

Abstract

Provided herein are compounds, compositions and methods for the rapid isolation of nucleic acids from a sample.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 333,813, filed April 22, 2022, which is incorporated by reference in its entirety.

[0002] Provided herein are compounds, compositions and methods for the rapid isolation of nucleic acids from a sample. [Background technology]

[0003] Isolation of nucleic acids from a sample is a critical step in many biochemical and diagnostic procedures, such as those that rely on nucleic acid amplification or sequencing reactions. Although compositions and methods for isolating nucleic acids from a sample are available, many are time consuming and / or involve the use of components that are not compatible with downstream applications (e.g., sequencing reactions), such as chaotropic agents, salts, detergents, and organic solvents. Summary of the Invention

[0004] In one aspect, disclosed herein is a composition comprising: solid surface; A linker that is covalently attached to the solid surface; and The moiety of formula (I) that is covalently attached to the linker: [ka] [During the ceremony, X is selected from O, S, S(O) and S(O); R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a , -SR b , -C(O)OR c , -C(O)NR d R e , -OC(O)NR f R g , -NR h C(O)NR i R j , and -NR h C(S)NR i R j Each R a , R b , R c , R d , R e , R f , R g , R h , R i and R j are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and aryl. The composition comprises:

[0005] In some embodiments, the solid surface comprises a material selected from silica, glass, polymers, and metals. In some embodiments, the solid surface comprises a polymer selected from cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethylene, polyethersulfone, polyolefin, polyvinylidene fluoride, polyacrylate, polystyrene, or any combination thereof. In some embodiments, the solid surface is in the form of a bead, resin, magnetic particle, membrane, vial, plate, film, tube, syringe, cartridge, cassette, pipette tip, microfluidic cartridge, or cuvette.

[0006] In some embodiments, the linker comprises one or more methylene, ether, ester, amide, carbamate, carbonate, urea, thioether, thioester, thioamide, thiocarbamate, thiocarbonate, thiourea, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof. In some embodiments, the linker comprises one or more -CH2-, -O-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -OC(O)O-, -NHC(O)NH-, -S-, -C(O)S-, -C(S)NH-, -NHC(S)O-, -OC(S)O-, -NHC(S)NH-, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof.

[0007] In some embodiments, the linker is [ka] The compound includes a moiety selected from

[0008] In some embodiments, the linker has the structure: -(CH2) n1 -Z-(CH2) n2 - wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8; and Z is selected from -NHC(O)NH-, -C(O)NH-, -OC(O)NH-, -OC(O)O-, -NHC(O)S-, -NHC(S)NH-, -O-, -C(O)O-, and a bond. has.

[0009] In some embodiments, the linker is [ka] is selected from.

[0010] In some embodiments, the linker has the structure: [ka] has.

[0011] In some embodiments, X is O. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each hydrogen.

[0012] In some embodiments, the solid surface further comprises one or more additional moieties covalently attached to the surface, hi some embodiments, the solid surface further comprises at least one polyethylene glycol (PEG) moiety covalently attached to the surface.

[0013] In another aspect, disclosed herein is a method for isolating nucleic acid from a sample, the method comprising: (a) providing a sample containing nucleic acid; (b) contacting the sample with a composition disclosed herein (e.g., a composition disclosed herein, e.g., a composition comprising a solid surface, a linker covalently attached to the solid surface, and a moiety of Formula (I) covalently attached to the linker) at a pH of less than about 8.0, whereby the nucleic acid binds to the composition to provide a nucleic acid binding composition; and (c) isolating the nucleic acid binding composition from the sample. The method includes:

[0014] In some embodiments, step (b) is performed at a pH of about 2.0 to less than about 8.0. In some embodiments, step (b) is carried out at a pH of about 6.0 to about 7.5. In some embodiments, step (b) comprises contacting the sample with the composition for about 10 seconds to about 10 minutes. In some embodiments, step (b) comprises contacting the sample with the composition for about 1 minute to about 5 minutes.

[0015] In some embodiments, the method further comprises one or more steps of washing the nucleic acid binding composition after step (c) by contacting the composition with a wash solution having a pH of less than about 8.0.

[0016] In some embodiments, the method further comprises contacting the nucleic acid binding composition with an elution solution at a pH of about 8.5 or greater to release the nucleic acid from the composition. In some embodiments, the elution solution comprises an aqueous buffer. In some embodiments, the method further comprises contacting the nucleic acid binding composition with an elution solution at a pH of about 8.5 to about 9.5. In some embodiments, the method further comprises contacting the nucleic acid binding composition with an elution solution at a pH of about 8.5 to about 9.0. In some embodiments, the method further comprises contacting the nucleic acid binding composition with the elution solution for about 10 seconds to about 10 minutes. In some embodiments, the method further comprises contacting the nucleic acid binding composition with the elution solution for about 1 minute to about 5 minutes.

[0017] In some embodiments, the method further comprises performing a polymerase chain reaction on the nucleic acid binding composition.

[0018] In some embodiments, the sample comprises a bodily fluid. In some embodiments, the bodily fluid is selected from blood, saliva, lymph, breast milk, mucus, urine, sweat, amniotic fluid, cerebrospinal fluid, feces, vaginal fluid, and semen. In some embodiments, the bodily fluid comprises cultured cells, tissue cells, or cells from a bodily fluid (e.g., from blood, saliva, lymph, breast milk, mucus, urine, sweat, amniotic fluid, cerebrospinal fluid, feces, vaginal fluid, or semen). In some embodiments, the sample is a blood sample selected from whole blood, plasma, and serum. In some embodiments, the sample is an environmental sample selected from soil and water. In some embodiments, the sample is a food sample (e.g., a meat sample). In some embodiments, the sample is a plant (e.g., a vegetable). In some embodiments, the sample comprises a virus, a bacteria, a fungus (e.g., a yeast or mold), or any combination thereof. In some embodiments, the sample comprises culture medium from cultured cells, a cell lysate, a cell supernatant, or a purified fraction of cells (e.g., a subcellular fraction).

[0019] In some embodiments, the method comprises a step of lysing the sample prior to step (b).

[0020] In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is selected from total DNA, mtDNA (mitochondrial DNA), gDNA (genomic DNA), cfDNA (cell-free DNA), ccfDNA (circulating cell-free DNA), cffDNA (cell-free fetal DNA), bacterial DNA, viral DNA, and ctDNA (circulating tumor DNA). In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is selected from total RNA, miRNA, mRNA, tRNA, rRNA, siRNA, ctRNA (circulating tumor RNA), and viral RNA. In some embodiments, the nucleic acid is total nucleic acid from the sample.

[0021] In one aspect, disclosed herein is a compound of formula (IIa): [ka] [During the ceremony, X is selected from O, S, S(O) and S(O); L is a linker; Each R a is independently selected from hydroxy, C1-C6 alkoxy, and halo; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a , -SR b , -C(O)OR c , -C(O)NR d R e , -OC(O)NR f R g , N.R. h C(O)NR i R j , and -NR h C(S)NR i R j Each R a , R b , R c , R d , R e , R f , R g , R h , R i and R j are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and aryl. or a salt thereof.

[0022] In some embodiments, X is O. In some embodiments, each R a is C1-C6 alkoxy. In some embodiments, R 1 , R 2 , R 3 , R4 , R 5 , R 6 , R 7 and R 8 are each hydrogen.

[0023] In some embodiments, the linker comprises one or more methylene, ether, ester, amide, carbamate, carbonate, urea, thioether, thioester, thioamide, thiocarbamate, thiocarbonate, thiourea, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof. In some embodiments, the linker comprises one or more -CH2-, -O-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -OC(O)O-, -NHC(O)NH-, -S-, -C(O)S-, -C(S)NH-, -NHC(S)O-, -OC(S)O-, -NHC(S)NH-, or any combination thereof.

[0024] In some embodiments, L is [ka] The compound includes a moiety selected from

[0025] In some embodiments, L is of the formula: -(CH2) n1 -Z-(CH2) n2 - wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8; and Z is selected from -NHC(O)NH-, -C(O)NH-, -OC(O)NH-, -OC(O)O-, -NHC(O)S-, -NHC(S)NH-, -O-, -C(O)O-, and a bond. has.

[0026] In some embodiments, L is [ka] The structure is selected from:

[0027] In some embodiments, L has the structure: [ka] has. [Brief description of the drawings]

[0028] [Figure 1] 1 shows the structures of various amine-containing silane ligands. [Diagram 2] 1 shows a reaction scheme for functionalizing magnetic silica beads with exemplary silane ligands. [Diagram 3] 2 shows the percentage binding and elution of DNA by various amine-functionalized magnetic silica particles when bound at pH 6.5 and eluted at pH 9.0. [Figure 4] 2 shows the percentage binding and elution of DNA by various amine-functionalized magnetic silica particles when bound at pH 7.4 and eluted at pH 9.0. [Diagram 5] The percentage of DNA binding and elution by morpholine-functionalized magnetic silica particles is shown at various pH values ​​(5.8-8.4) and salt concentrations (0-600 mM NaCl) upon binding, and upon elution at pH 9.0. [Figure 6] The percentage of DNA binding and elution by morpholine-functionalized magnetic silica particles using various DNA input concentrations is shown. [Figure 7] The percentage binding and elution of RNA by morpholine-functionalized magnetic silica particles using various RNA input concentrations is shown. [Figure 8] FIG. 1 shows the isolation and concentration of ccfDNA (circulating cell-free DNA) using morpholine-functionalized beads. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Provided herein are compounds, compositions and methods for the rapid isolation of nucleic acids from a sample. The compounds and compositions allow for rapid capture of nucleic acids at low (e.g., less than about 8) pH. Upon shifting to a higher pH (e.g., about 8.5 or higher), the nucleic acids are rapidly released into solution. In some embodiments, the entire process can be performed in less than 10 minutes, and the nucleic acids can be released under conditions compatible with downstream applications, such as polymerase chain reaction (PCR), including reverse transcription PCR (RT-PCR).

[0030] I. Definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, devices and materials are described herein. However, prior to describing the materials and methods of the present invention, it is to be understood that the disclosure is not limited to the specific molecules, compositions, methodologies or implementations described herein, as these may vary according to routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular variations or embodiments only, and is not intended to be limiting on the scope of the embodiments described herein.

[0031] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization techniques described herein is well known and commonly used in the art. The meaning and scope of the terms should be clear, but nevertheless, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide" refers to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0033] As used herein, the term "and / or" includes any and all combinations of the listed items, including any of the listed items individually. For example, "A, B and / or C" includes A, B, C, AB, AC, BC and ABC, each of which should be considered as being individually listed by the explicit reference "A, B and / or C."

[0034] As used herein, the term "comprising" and its linguistic variants refer to the presence of the recited feature(s), element(s), method step(s), etc., but do not exclude the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and its linguistic variants refer to the presence of the recited feature(s), element(s), method step(s), etc., but excludes any feature(s), element(s), method step(s), etc. that are not recited, apart from normally accompanying impurities. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., plus any additional feature(s), element(s), method step(s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended "comprising" phrase. Such embodiments include the limiting "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language.

[0035] For the recitation of numerical ranges herein, each intervening number of equal precision is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0036] Definitions of certain functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (endpaper), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry and specific functional moieties and reactivity are described in Sorrell, Organic Chemistry, 2001, 14th Ed., 1999, 1999-2002, 1999-2002, 1999-2002, 1999-2003, 1999-2004, 1999-2005, 1999-2006, 1999-2007, 1999-2008, 1999-2000, 1999-2001, 1999-2002, 1999-2002, 1999-2003, 1999-2004, 1999-2005, 1999-2006, nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley & Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the contents of each of which are incorporated herein by reference in their entirety.

[0037] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon chain. An alkyl chain can be, for example, 1 to 30 carbon atoms (C1-C 30 Alkyl), 1 to 24 carbon atoms (C1-C 24 alkyl), e.g., alkyl having 1 to 16 carbon atoms (C1-C 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10 The alkyl group may contain 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0038] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) may be located at any position within the hydrocarbon chain. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0039] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond(s) may be located at any position within the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0040] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic), including fused ring systems, and zero heteroatoms. As used herein, aryl refers to an aromatic carbocyclic ring system having 6 to 20 carbon atoms (C6-C 20 Aryl), 6 to 14 ring carbon atoms (C6-C 14 Aryl), 6 to 12 ring carbon atoms (C6-C 12 aryl), or 6 to 10 ring carbon atoms (C6-C 10 Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl.

[0041] As used herein, the term "cyano" refers to the group --CN.

[0042] As used herein, the term "cycloalkyl" refers to a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls can be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0043] As used herein, the term "halogen" or "halo" means F, Cl, Br or I.

[0044] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, in which at least one hydrogen atom (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms) is replaced with a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced with a halogen. Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0045] As used herein, the term "heteroaryl" refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) with one or more ring heteroatoms independently selected from O, N, and S. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from O, N, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, N, and S). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. Bicyclic heteroaryl groups are exemplified by monocyclic aryl groups as defined herein or monocyclic heteroaryl groups as defined herein fused with an additional monocyclic heteroaryl ring. Tricyclic heteroaryl groups are exemplified by monocyclic heteroaryl rings fused with two rings independently selected from monocyclic aryl groups as defined herein and monocyclic heteroaryl groups as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic and tricyclic heteroaryls are linked to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0046] As used herein, the term "heterocycle" or "heterocyclic" refers to a saturated or partially unsaturated non-aromatic ring group having one or more ring heteroatoms independently selected from O, N, and S, meaning a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S. The 7- and 8-membered rings contain 0, 1, 2 or 3 double bonds and 1, 2 or 3 heteroatoms selected from O, N and S. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl. , pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are joined by an alkylene bridge of 1, 2, 3 or 4 carbon atoms or an alkenylene bridge of 2, 3 or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Examples of tricyclic heterocycles include bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are connected by an alkylene bridge having 1, 2, 3, or 4 carbon atoms or an alkenylene bridge having 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane). The monocyclic, bicyclic and tricyclic heterocycles are linked to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring.

[0047] As used herein, the term "nitro" refers to the group --NO.

[0048] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and can include fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum, and the like. Samples can also refer to cells or cell lysates. Cell lysates can include cells lysed with a lysing agent, or lysates, such as rabbit reticulocytes or wheat germ lysates. Environmental samples include environmental materials, such as surface materials, soil, water (e.g., wastewater), crystals, and industrial samples. Samples can also include purified samples, such as purified protein samples. However, such examples should not be construed as limiting the sample types applicable to the present disclosure.

[0049] Where a group or moiety can be substituted, the term "substituted" refers to one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) hydrogens on the group indicated with the phrase "substituted" can be replaced with a selection of the enumerated and indicated groups, or suitable substituents known to those of skill in the art (e.g., one or more of the groups listed below), provided that the normal valence of the designated atom is not exceeded. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0050] As used herein, in a chemical structure: [ka] The designation "-" represents the point at which one moiety is attached to another moiety (eg, a substituent to the remainder of a compound).

[0051] With respect to the compounds described herein, groups or substituents thereof may be selected for the permissible valences of atoms and substituents, e.g., such that the selection and substitution result in stable compounds, e.g., compounds that do not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like.

[0052] Where substituents are specified by their conventional chemical formula written from left to right, this optionally includes the substituents resulting from writing the structure from right to left, e.g., -CHO- optionally also recites -OCH-, -OC(O)NH- optionally also recites -NHC(O)O-.

[0053] I. Compositions and Compounds Provided herein are compounds and compositions that can be used to rapidly isolate nucleic acids from a sample. The compounds and compositions include a morpholino moiety or a derivative thereof. At lower pH (e.g., less than about 8), the morpholino moiety is protonated and positively charged, and therefore can rapidly bind to negatively charged nucleic acids. Upon shifting to a higher pH (e.g., greater than or equal to about 8.5), the morpholino moiety is deprotonated, resulting in a form that rapidly releases nucleic acids into solution. The compositions of the present disclosure include a morpholino moiety or a derivative thereof (e.g., morpholine, thiomorpholine, thiomorpholine 1-oxide, thiomorpholine 1,1-dioxide, or a salt thereof) that is attached to a solid surface via a linker. Although similar materials with other ligands, such as imidazole, methylpiperazine, and other primary and secondary amines, can also bind nucleic acids, compositions containing morpholine groups (or derivatives thereof) can rapidly release a large amount of nucleic acid (e.g., in some embodiments, at least 80% of the bound nucleic acid can be released in less than 5 minutes or less than 1 minute). This provides a significant advantage to the materials of the present disclosure. Moreover, nucleic acids can be released using elution solutions that do not require components (e.g., chaotropic agents) that can interfere with downstream applications, such as sequencing reactions. Thus, the compounds and compositions of the present disclosure can be used for rapid and efficient isolation of nucleic acids, resulting in solutions that can be used directly for downstream applications without further sample processing.

[0054] Thus, disclosed herein is a composition comprising a solid surface; a linker covalently attached to the solid surface; and a moiety of formula (I) covalently attached to the linker: [ka] [During the ceremony, X is selected from O, S, S(O) and S(O); R 1 , R 2 , R 3 , R 4 , R 5 , R 6, R 7 and R 8 are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a , -SR b , -C(O)OR c , -C(O)NR d R e , -OC(O)NR f R g , -NR h C(O)NR i R j , and -NR h C(S)NR i R j Each R a , R b , R c , R d , R e , R f , R g , R h , R i and R j are independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, and aryl. The composition comprises:

[0055] In the moiety of formula (I), X is selected from O, S, S(O) and S(O). In some embodiments, X is O. In some embodiments, X is S. In some embodiments, X is S(O). In some embodiments, X is S(O).

[0056] In the moiety of formula (I), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a , -SR b , -C(O)OR c , -C(O)NR d R e , -OC(O)NR f R g , -NR h C(O)NR i R j , and -NR h C(S)NR i R j Each R a , R b , R c , R d , R e , R f , R g , R h , R i and R j is independently selected from hydrogen, C-C alkyl, C-C alkenyl, C-C alkynyl, C-C haloalkyl, C-C cycloalkyl, and aryl. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently hydrogen, C1-C3 alkyl, halo, and -OR a Selected from R a is selected from hydrogen and C1-C3 alkyl. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8one or two of are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a , -SR b , -C(O)OR c , -C(O)NR d R e , -OC(O)NR f R g , -NR h C(O)NR i R j , and -NR h C(S)NR i R j and the remainder are hydrogen. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 one or two of are each independently selected from hydrogen, C1-C3 alkyl, halo, and -OR a Selected from R a is selected from hydrogen and C1-C3 alkyl, and the remainder are hydrogen. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each hydrogen.

[0057] Any solid surface suitable for isolating nucleic acids can be used. For example, in some embodiments, the solid surface comprises a material selected from silica, glass, polymers, and metals. In some embodiments, the solid surface is a glass surface. In some embodiments, the solid surface comprises silica. In some embodiments, the solid surface comprises a polymer, such as a polymer selected from cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethylene, polyethersulfone, polyolefin, polyvinylidene fluoride, polyacrylate, polystyrene, or any combination thereof. The solid surface can be in the form of, for example, beads, resins, magnetic particles, membranes, vials, plates, films, tubes, syringes, cartridges, cassettes, pipette tips, microfluidic cartridges, or cuvettes. In some embodiments, the solid surface is in the form of beads. In some embodiments, the solid surface is in the form of a resin. In some embodiments, the solid surface is in the form of a membrane. In some embodiments, the solid surface is in the form of a plate. In some embodiments, the solid surface is in the form of a pipette tip. In some embodiments, the solid surface is in the form of a microfluidic cartridge.

[0058] In the composition, the moiety of formula (I) is attached to the solid surface via a linker. In some embodiments, the linker is a direct bond. In other embodiments, the linker separates the moiety of formula (I) from the solid surface by about 5 Å, about 10 Å, about 20 Å, about 50 Å, about 100 Å, about 150 Å, about 200 Å, about 300 Å, about 400 Å, about 500 Å, about 600 Å, about 700 Å, about 800 Å, about 900 Å, about 1000 Å, or any suitable range therebetween (e.g., about 5-100 Å, about 50-500 Å, about 150-700 Å, etc.). In some embodiments, the linker separates the functional element from the remainder of the compound of Formula (I) by about 1-200 atoms (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, or any suitable range therebetween (e.g., about 2-20, about 10-50, etc.).

[0059] The linker may include one or more groups independently selected from methylene (-CH-), ethylene (-CH=CH-), ethynylene (-C≡C-), ether (-O-), amine (-NR-, where R is hydrogen or an alkyl group), thioether (-S-), carbonyl (-C(O)-), thiocarbonyl (-C(S)-), sulfonyl (-S(O)-), arylene, heteroarylene, and heterocyclylene moieties, or any combination thereof. For example, the above moieties can be combined to form additional groups that can be included in a linker, e.g., a carbonyl group and an ether group can together provide an ester moiety (-C(O)O-), a carbonyl group and two ether groups can together provide a carbonate moiety (-OC(O)O-), a carbonyl group and an unsubstituted amine group can together provide an unsubstituted amide moiety (-C(O)NH-), a carbonyl group and two unsubstituted amine groups can together provide an unsubstituted urea moiety (-NHC(O)NH-), a carbonyl group can together with an unsubstituted amine group and an ester group to provide an unsubstituted carbamate moiety (-OC(O)NH-), a carbonyl group can together with a thioether and an unsubstituted amine group to provide an S-thiocarbamate moiety, a thiocarbonyl group can together with an ether and an unsubstituted amine group to provide an O-thiocarbamate moiety, multiple methylene groups can together form an alkylene chain, and so forth.

[0060] In some embodiments, the linker comprises one or more methylene, ether, ester, amide, carbamate, carbonate, urea, thioether, thioester, thioamide, thiocarbamate, thiocarbonate, thiourea, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof. In some embodiments, the linker comprises one or more -CH2-, -O-, -C(O)O-, -C(O)NH-, -NHC(O)O-, -OC(O)O-, -NHC(O)NH-, -S-, -C(O)S-, -C(S)NH-, -NHC(S)O-, -OC(S)O-, -NHC(S)NH-, arylene, heteroarylene, or heterocyclylene moieties, or any combination thereof.

[0061] In some embodiments, the linker comprises one or more alkylene groups (e.g., -(CH) n -wherein n is 1 to 12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween. In some embodiments, the linker comprises one or more branched alkylene groups.

[0062] In some embodiments, the linker has the formula -(CH) n1 -Z-(CH2) n2 wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8; and Z is selected from -NHC(O)NH-, -C(O)NH-, -OC(O)NH-, -OC(O)O-, -NHC(O)S-, -NHC(S)NH-, -O-, -C(O)O-, and a bond.

[0063] In some embodiments, the linker has the formula -(CH) n1 -NH-C(O)-NH-(CH2) n2 wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0064] In some embodiments, the linker is [ka] having a formula selected from:

[0065] In some embodiments, the linker has the formula: [ka] has.

[0066] In some embodiments, the linker may include one or more moieties that are obtained by the reaction of two reactive groups, such as reactive groups known in various biomolecular conjugation reactions. For example, the linker may include a triazole group, which is formed by the reaction of an azide with an alkyne. Exemplary moieties that may be obtained from such a reaction include: [ka] These include, but are not limited to:

[0067] In some embodiments, the solid surface comprises one or more additional moieties in addition to the moiety of formula (I). For example, the solid surface may be functionalized with additional ligands. In some embodiments, the solid surface further comprises one or more PEG ligands.

[0068] The composition can be prepared by reacting a solid surface with a compound comprising a moiety of formula (I). Typically, the compound comprising a moiety of formula (I) will have a reactive group that is complementary to the reactive group present on the solid surface to allow for covalent bonding. For example, a suitable solid surface can be reacted with a compound of formula (II): [ka] wherein RM is a reactive moiety, L is a linker, and X, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are as defined and described herein. In some embodiments, the reactive moiety comprises a group selected from a silane, an alkyne, an azide, an alkene, a thiol, a maleimide, an amine, a succinimidyl ester, a -COOH group, a tetrazole, an arylphosphine, or the like. For example, in some embodiments, RM is [ka] [In the formula, each R a are independently selected from hydroxy, C1-C6 alkoxy, and halo (e.g., fluoro, chloro, or bromo). The skilled artisan will recognize that when the compound of formula (II) contains any of the above groups, the solid surface will be functionalized with a complementary reactive group. For example, when the compound of formula (II) contains an alkyne-containing group, the solid surface may be functionalized with an azide such that the compound of formula (II) can be attached to the solid surface via a click chemistry reaction (e.g., to form a triazole). Similarly, when the compound of formula (II) contains a succinimidyl ester ... [ka] In the latter case, the solid surface may be functionalized with a primary amine (-NH2) such that the compound of formula (II) can be attached to the solid surface via an amide bond.

[0069] In the compound of formula (II), the group L can be any suitable linking group. For example, in some embodiments, L comprises one or more groups independently selected from methylene (-CH2-), ethylene (-CH=CH-), ethynylene (-C≡C-), ether (-O-), amine (-NR-), thioether (-S-), carbonyl (-C(O)-), thiocarbonyl (-C(S)-), sulfonyl (-S(O)2-), arylene, heteroarylene, and heterocyclylene moieties, or any combination thereof. For example, the moieties can be combined to form further groups, such as esters, carbonates, amides, ureas, thioureas, carbamates, alkylenes, and the like. In some embodiments, L comprises one or more alkylene groups (e.g., -(CH2) n -, where n is 1 to 12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween. In some embodiments, L comprises one or more branched alkylene groups.

[0070] In some embodiments, L is a group of the formula -(CH2) n1 -Z-(CH2) n2 wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8; and Z is selected from -NHC(O)NH-, -C(O)NH-, -OC(O)NH-, -OC(O)O-, -NHC(O)S-, -NHC(S)NH-, -O-, -C(O)O-, and a bond.

[0071] In some embodiments, L is a group of the formula -(CH2) n1 -NH-C(O)-NH-(CH2) n2 wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8.

[0072] In some embodiments, L is [ka] having a formula selected from:

[0073] In some embodiments, L is of the formula: [ka] has.

[0074] In some embodiments, provided herein is a compound of formula (IIa): [ka] [In the formula, L, X, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R a are as defined and described herein. It is. In one embodiment, provided herein is a compound having the formula: [ka] It is a compound of the formula:

[0075] III.How to use The compositions disclosed herein can be used to rapidly isolate nucleic acids (e.g., DNA and RNA) from a sample, e.g., a biological sample. In one aspect, the disclosure provides a method for isolating nucleic acids from a sample, comprising: (a) providing a sample containing nucleic acid; (b) contacting the sample with a composition disclosed herein (e.g., a composition comprising a solid surface, a linker covalently attached to the solid surface, and a moiety of Formula (I) covalently attached to the linker) at a pH of less than about 8.0, whereby the nucleic acid binds to the composition to provide a nucleic acid binding composition; and (c) isolating the nucleic acid binding composition from the sample. The method includes:

[0076] In some embodiments, the nucleic acid isolated from the sample is selected from DNA and RNA. In some embodiments, the nucleic acid isolated from the sample is DNA (e.g., genomic DNA (gDNA), plasmid DNA, cDNA, mitochondrial DNA (mtDNA), cosmid DNA, cell-free DNA (cfDNA), circulating cell-free DNA (ccfDNA), cell-free fetal DNA (cffDNA), bacterial DNA, viral DNA, circulating tumor DNA (ctDNA), total DNA, or the like). In some embodiments, the nucleic acid isolated from the sample is RNA, e.g., messenger RNA (mRNA), microRNA (miRNA), precursor miRNA, ribosomal RNA (rRNA), mitochondrial RNA, non-coding RNA, circulating RNA, small interfering RNA (siRNA), guide RNA, total RNA, or the like. In some embodiments, the nucleic acid isolated from the sample is total nucleic acid.

[0077] Any suitable sample containing nucleic acid can be used in the disclosed method. In some embodiments, the sample comprises a bodily fluid, such as blood, saliva, lymph, breast milk, mucus, urine, sweat, amniotic fluid, cerebrospinal fluid, feces, vaginal fluid, or semen. In some embodiments, the sample comprises cultured cells, tissue (e.g., fresh tissue, or formalin-fixed paraffin-embedded (FFPE) tissue), or cells from a bodily fluid. In some embodiments, the sample comprises a virus, a bacteria, a fungus (e.g., yeast or mold), or any combination thereof. In some embodiments, the sample is a food sample, such as a meat sample. In some embodiments, the sample is a plant, such as a vegetable. In certain embodiments, the sample is a blood sample selected from whole blood, plasma, and serum. In some embodiments, the sample is an environmental sample, such as soil or water (e.g., wastewater). The method can also be used to isolate nucleic acids from other environments, such as agar or polyacrylamide gels, or solutions where amplification of the target nucleic acid has already been performed.

[0078] In some embodiments, the method may further comprise a step of lysing the sample before contacting the sample with the composition disclosed herein. Any suitable method of lysing the sample may be used, such as osmotic (i.e., using a hypotonic solution), enzymatic (e.g., using lysozyme), chemical (e.g., using detergents, such as non-ionic detergents or cationic detergents, with or without other ingredients such as sodium hydroxide), physical (e.g., mechanical lysis, e.g., by mixing with glass beads, shaking, using a homogenizer or French press, using an ultrasonicator, freeze-thaw cycles, or mechanical grinding), or heat lysis, or any combination thereof.

[0079] The method includes contacting a sample (e.g., a lysed sample) with a composition disclosed herein (e.g., a composition comprising a moiety of Formula (I) disclosed herein) such that nucleic acids in the sample bind to the composition to provide a nucleic acid binding composition. During this contacting step, the pH of the sample is less than about 8.0 to ensure that the moiety of Formula (I) in the composition is positively charged. In some embodiments, the contacting step is performed at a pH of about 2.0 to about 8.0, about 2.5 to about 8.0, about 3.0 to about 8.0, about 3.5 to about 8.0, about 4.0 to about 8.0, about 4.5 to about 8.0, about 5.5 to about 8.0, or about 6.0 to about 7.5, e.g., about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about The method is carried out at a pH of about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or any range therebetween.

[0080] Nucleic acids rapidly bind to the compositions disclosed herein. For example, in some embodiments, more than about 50% (e.g., about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the nucleic acids in a sample bind rapidly to the compositions disclosed herein. , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater) bind to the composition in less than about 10 minutes (e.g., less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes or less than about 1 minute). In some embodiments, greater than 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater than 99%) of the nucleic acids in the sample bind to the composition in less than about 10 minutes (e.g., less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes or less than about 1 minute). In some embodiments, greater than 75% (e.g., greater than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the nucleic acids in the sample bind to the composition in less than about 5 minutes (e.g., less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes or less than about 1 minute). In some embodiments, greater than 75% (e.g., greater than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the nucleic acids in the sample bind to the composition in less than about 1 minute.Thus, although the step of contacting the sample with the composition can be performed for a longer period of time as appropriate or desired, in some embodiments, the contacting step is performed for less than about 10 minutes (e.g., less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute). Because this period is sufficient to allow binding of the nucleic acid to the composition. In some embodiments, the contacting step is performed for about 10 seconds to about 10 minutes, or about 1 minute to about 5 minutes. The contacting step results in the production of a nucleic acid binding composition.

[0081] The method optionally includes one or more washing steps after the contacting step. In some embodiments, the method further includes contacting the nucleic acid binding composition with at least one washing solution. Typically, the washing solution is an aqueous solution (e.g., an aqueous buffer) having a pH of about 2.0 to about 8.0, about 2.5 to about 8.0, about 3.0 to about 8.0, about 3.5 to about 8.0, about 4.0 to about 8.0, about 4.5 to about 8.0, about 5.5 to about 8.0, or about 6.0 to about 7.5, optionally containing chemical additives such as detergents, salts (e.g., NaCl) or organic solvents (e.g., methanol, ethanol, isopropanol, acetonitrile, DMSO, or DMF), such that the composition remains protonated and the nucleic acids remain bound or water-soluble while other contaminants are washed away. In some embodiments, the wash solution has a pH of about 2.0 to about 8.0, about 2.5 to about 8.0, about 3.0 to about 8.0, about 3.5 to about 8.0, about 4.0 to about 8.0, about 4.5 to about 8.0, about 5.5 to about 8.0, or about 6.0 to about 7.5, e.g., about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 6.8, about 6.9, about 6.9, about 6.1, about 6.2 ... 2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or any range therebetween.

[0082] After the contacting step and optional washing step(s), the nucleic acid binding composition can be used for various applications. In some embodiments, the nucleic acid is released from the nucleic acid binding composition by contacting the nucleic acid binding composition with an elution solution having a pH of, for example, about 8.5 or higher, for example, a pH of about 8.5 to about 9.5, for example, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.4 or about 9.5, or any range therebetween. In some embodiments, the elution solution comprises a buffer, for example, tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS) or the like. The elution solution may contain other components, such as salts, provided that they are present at concentrations that do not interfere with downstream applications such as PCR. For example, in some embodiments, the elution solution contains salt (e.g., NaCl) at a concentration of less than about 25 nM.

[0083] A significant advantage of the disclosed composition is that it releases nucleic acids at a very fast rate upon shifting to a higher pH. Although other compositions have the ability to effect nucleic acid binding and release, similar to the compositions described herein, including compositions having moieties other than the moiety of formula (I), such as imidazole, methylpiperazine, amines (e.g., primary amines (-NH2) or substituted amines (e.g., -N(CH3)2 or -N(CH2CH2OH)2), the compositions disclosed herein are capable of liberating large amounts of nucleic acid in a very short time frame. In some embodiments, contacting the nucleic acid binding composition with an elution solution having a pH of about 8.5 or greater results in a nucleic acid-binding cleavage. and, whereby greater than 50% (e.g., greater than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the bound nucleic acid is removed in less than about 5 minutes (e.g., In some embodiments, by contacting the nucleic acid binding composition with an elution solution having a pH of about 8.5 or greater, greater than 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99%) of the bound nucleic acid is released in less than about 5 minutes (e.g., less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, The nucleic acid binding composition is eluted for less than about 2 minutes or less than about 1 minute. This significantly reduces the processing time required to isolate nucleic acid from the sample. Therefore, although the step of contacting the nucleic acid binding composition with the elution solution can be carried out for a longer period of time as appropriate or desired, in some embodiments, this step is carried out for less than about 5 minutes (e.g., less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute), because this period is sufficient to allow efficient release of the bound nucleic acid.In some embodiments, the step of contacting the nucleic acid binding composition with the elution solution occurs for a period of from about 10 seconds to about 10 minutes, or from about 1 minute to about 5 minutes.

[0084] In embodiments where an elution solution is used, the elution solution does not contain chaotropic agents (e.g., guanidine hydrochloride or guanidine isothiocyanate), organic solvents (e.g., alcohols, e.g., ethanol or methanol, or other organic solvents, e.g., dimethyl sulfoxide), or detergents (e.g., sodium dodecyl sulfate). Even trace amounts of such components in a solution of target nucleic acid can significantly limit the usefulness of the nucleic acid in downstream processing or analysis. This is another major advantage of the compositions of the present disclosure - the nucleic acid can be rapidly released from the composition using aqueous buffers that do not contain components that would interfere with downstream applications.

[0085] If specific pH conditions are required in any of the steps of the method (e.g., the contacting step, the optional washing step, and the elution step), an acidifying or basifying agent can be used as necessary to adjust the pH of the sample or solution to the specified pH. Suitable acidifying agents include inorganic acids, such as HCl, HBr, HClO4, HClO3, H2SO4, H2SO3, H3PO4, or H3PO3; organic acids, such as 2-acetoxyoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalene acid, hydroxyphenyl ether ... Suitable basifying agents include, but are not limited to, carboxylic acids, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, tetrafluoroboric acid, toluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, and valeric acid. Suitable basifying agents include, but are not limited to, inorganic bases, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium bicarbonate, sodium carbonate, ammonia, diethanolamine, meglumine, lysine, arginine, ethanolamine, piperazine, trometamol, triethanolamine, and the like.

[0086] In some embodiments, the nucleic acid binding composition can be used directly in downstream applications without an elution step, for example, in some embodiments, the nucleic acid binding composition can be used directly in an on-bead polymerase chain reaction.

[0087] In some embodiments, the isolated nucleic acid may be used in any number of downstream applications, such as methods to analyze or further process the nucleic acid. For example, in some embodiments, the methods of the disclosure further comprise analyzing or processing the nucleic acid using, for example, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), quantitative PCR, quantitative reverse transcription PCR (RT-qPCR), real-time PCR, hot-start PCR, single-cell PCR, nested PCR, in situ colony PCR, digital PCR (dPCR), droplet digital™ PCR (ddPCR), emulsion PCR, ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), hyper-branched RCA (HRCA), isothermal amplification, gel electrophoresis, capillary electrophoresis, mass spectrometry, fluorescence detection, ultraviolet spectroscopy, hybridization assays, DNA or RNA sequencing, reverse transcription, next generation sequencing (NGS), or the like. EXAMPLES

[0088] IV. Working Examples Abbreviations used in the examples include the following: DCM is dichloromethane, DMF is dimethylformamide, MeOH is methanol, MS is mass spectrometry, NMR is nuclear magnetic resonance, RT is room temperature, and THE is tetrahydrofuran.

[0089] Example 1 Synthesis of 1-(3-morpholinopropyl)-3-(3-(triethoxysilyl)propyl)urea 6.41 g of 3-morpholinopropylamine was added to 40 mL of anhydrous THF, followed by slow addition of 10 mL of 3-(triethoxysilyl)propyl isocyanate. The solution was heated to reflux overnight. The solution was cooled to room temperature and purified by chromatography using a Teledyne ISCO system. The solvent system was DCM / MeOH, with a gradient of 0-20% MeOH in DCM. The compound was eluted with DCM containing approximately 15% MeOH. The combined solution was rotary evaporated to remove the solvent to produce 9.5 g of a viscous colorless liquid (60% yield). MS: 392.6 (M+H); 1 H NMR (400MHz, methanol-d4)3.88-3.79(m,6H),3.70(d,J=5.1Hz,4H),3.17(t,J=7.0Hz,2H),3.10(t,J=7.0Hz,2H),2.48(s,4H), 2.41(t,J=7.6Hz,2H),1.69(p,J=7.2Hz,2H),1.57(p,J=7.4Hz,2H),1.22(td,J=7.1,2.1Hz,9H),0.62(dd,J=9.8,6.9Hz,2H).

[0090] Example 2 Synthesis of morpholine-functionalized magnetic silica beads The synthesis procedure is outlined in Figure 2. 1 g of Grace MP-50 magnetic silica particles (6 μm) was added to 50 mL of toluene. The suspension was stirred using an overhead mechanical stirrer at 200 rpm. 1-(3-morpholinopropyl)-3-(3-(triethoxysilyl)propyl)urea (783 mg) was dissolved in 2 mL of DMF. The solution was added to the toluene suspension and stirred at room temperature for 30 min. The suspension was heated to 75° C. overnight. The particles were washed 3 times with DMF and 3 times with water. The particles were stored in nanopure water. CHN elemental analysis: C (1.53%), H (0.40%), and N (0.38%).

[0091] Example 3 Synthesis of functionalized magnetic silica beads 1 g of Grace MP-50 magnetic silica particles (6 μm) was added to 50 mL of toluene. The suspension was stirred using an overhead mechanical stirrer at 200 rpm. 1-(3-morpholinopropyl)-3-(3-(triethoxysilyl)propyl)urea (783 mg) was dissolved in 2 mL of DMF. The solution was added into the toluene suspension. 500 μL of acetic acid was added as an additive to improve the ligand coupling efficiency. The suspension was stirred for 30 min at room temperature. The suspension was heated to 75 °C overnight. The particles were washed 3 times with DMF and 3 times with water. The particles were stored in nanopure water. CHN elemental analysis: C (6.48%), H (1.2%), and N (1.9%).

[0092] Following a similar procedure, other amine-containing silane ligands were used instead of 1-(3-morpholinopropyl)-3-(3-(triethoxysilyl)propyl)urea (i.e., 1-(3-(1H-imidazol-1-yl)propyl)-3-(3-(triethoxysilyl)propyl)urea, 1-(3-(4-methylpiperazin-1-yl)propyl)-3-(3-(triethoxysilyl)propyl)urea, 3-(trimethoxysilyl)propyl)urea, and 1-(3-(4-methylpiperazin-1-yl)propyl)-3-(3-(triethoxysilyl)propyl)urea. Additional magnetic silica beads were prepared using propan-1-amine, 1-(3-(dimethylamino)propyl)-3-(3-(triethoxysilyl)propyl)urea, 2,2'-((3-(triethoxysilyl)propyl)azanediyl)bis(ethan-1-ol), and 1-(2-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)ethyl)-3-(3-(triethoxysilyl)propyl)urea, respectively.

[0093] Example 4 DNA binding and elution assays 1 mg of various amine-functionalized magnetic beads was added into 1 mL of a solution of 200 bp DNA (100 ng / mL, 10 mM PBS, pH 6.5). The suspension was mixed for 40 seconds. The particles were collected with a magnetic bar for 1 minute. The supernatant was collected to measure the DNA concentration remaining in the solution and calculate the percentage of binding. The DNA concentration was quantified using the Quantifluor® dsDNA system (Promega). 50 μL of Tris buffer (10 mM, pH 9.0) was added into the magnetic particles and the suspension was vortexed for 1 minute. The particles were collected with a magnetic bar. The elution solution was collected and the DNA concentration in the elution solution was quantified to calculate the percentage of elution.

[0094] The results are shown in Figure 3. All particles rapidly bind DNA, but after a 1 min elution step, the morpholine-functionalized magnetic beads released much higher amounts of DNA than the other materials.

[0095] Example 5 DNA binding and elution assays 1 mg of various amine-functionalized magnetic beads was added into 1 mL of a solution of 200 bp DNA (100 ng / mL, 10 mM PBS, pH 7.4). The suspension was mixed for 40 seconds. The particles were collected with a magnetic bar for 1 minute. The supernatant was collected to measure the DNA concentration remaining in the solution and calculate the percentage of binding. The DNA concentration was quantified using the Quantifluor® dsDNA system (Promega). 50 μL of Tris buffer (10 mM, pH 9.0) was added into the magnetic particles and the suspension was vortexed for 1 minute. The particles were collected with a magnetic bar. The elution solution was collected and the DNA concentration in the elution solution was quantified to calculate the percentage of elution.

[0096] The results are shown in Figure 4. All particles rapidly bind DNA, but after a 1 min elution step, the morpholine-functionalized magnetic beads released much higher amounts of DNA than the other materials.

[0097] Example 6 DNA binding and elution assays 1 mg of morpholine-functionalized magnetic beads was added to 1 mL of 200 bp DNA solutions with various pH and salt concentrations (pH 5.8-pH 8.4, 0-600 mM NaCl). The suspension was mixed for 40 seconds. The particles were collected with a magnetic bar for 1 minute. The supernatant was collected to measure the DNA concentration remaining in the solution and calculate the percentage of binding. The DNA concentration was quantified using the Quantifluor® dsDNA system (Promega). 50 μL of Tris buffer (10 mM, pH 9.0) was added to the magnetic particles and the suspension was vortexed for 1 minute. The particles were collected with a magnetic bar. The elution solution was collected and the DNA concentration in the elution solution was quantified to calculate the percentage of elution.

[0098] The results are shown in Figure 5 and demonstrate that the morpholine-functionalized beads can function over a wide range of initial binding pH and NaCl concentrations.

[0099] Example 7 DNA binding and elution assays 1 mg of morpholine-functionalized magnetic beads was added into 1 ml of total human DNA (100, 10 or 1 ng) solution in phosphate-buffered saline (PBS) pH 7.0. The suspension was mixed for 60 seconds. The particles were collected with a magnetic bar for 1 minute. The supernatant was removed and 50 μL of Tris buffer (10 mM, pH 9.0) was added into the magnetic particles and the suspension was vortexed for 1 minute. The particles were collected with a magnetic bar. The elution solution was collected and the DNA concentration in the elution solution was quantified using a probe-based quantitative real-time PCR (qPCR) to calculate the elution concentration. The DNA solution input and elution concentration were compared using a beta-2-microglobulin probe and primers.

[0100] The results are shown in Figure 6. Morpholine-functionalized beads can concentrate total DNA, which can then be utilized in downstream qPCR reactions.

[0101] Example 8 RNA binding and elution assays One milligram of morpholine-functionalized magnetic beads was added to 1 mL of total RNA (1000, 100, or 10 ng) in phosphate-buffered saline (PBS) pH 7.0. The total RNA used in this assay was isolated from human embryonic kidney 293 cells (commonly referred to as HEK293, HEK-293, or 293 cells). The suspension was mixed for 60 seconds. The particles were collected with a magnetic bar for 1 minute. The supernatant was removed, and 50 μL of Tris buffer (10 mM, pH 9.0) was added to the magnetic particles, and the suspension was vortexed for 1 minute. The particles were collected with a magnetic bar. The elution solution was collected and the DNA concentration in the elution solution was quantified using a probe-based one-step quantitative real-time PCR (RT-qPCR) to calculate the elution concentration. The RNA solution input and elution concentration were compared using a beta-2-microglobulin probe and primers.

[0102] The results are shown in Figure 7. Morpholine-functionalized beads can enrich total RNA, and the zRNA can then be utilized in a downstream one-step RT-qPCR reaction.

[0103] Example 9 CcfDNA (cell-free circulating DNA) was isolated from 1 mL of human plasma using a Promega Maxwell Instrument. Method 1 is the current Promega off-the-shelf product (#AS1480). Method 2 uses the morpholine-functionalized magnetic beads of the present disclosure. Method 2 also included a salt wash at low pH after the binding step. Recovered ccfDNA was measured by qPCR.

[0104] The results are shown in Figure 8. The rapid sample preparation chemistry using morpholine-functionalized beads of the present disclosure in Method 2 can isolate and enrich ccfDNA quickly and with high recovery yields. In all three plasma samples, the rapid sample preparation chemistry using morpholine-functionalized beads provided higher yields compared to the commercially available Promega Maxwell ccfDNA kit.

Claims

1. A composition, solid surface; Linkers covalently bonded to the solid surface; Here, the linker has the following structure: -(CH 2 ) n1 -Z-(CH 2 ) n2 - [wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8, and Z is selected from -NHC(O)NH-, -C(O)NH-, -OC(O)NH-, -OC(O)O-, -NHC(O)S-, -NHC(S)NH-, and -O-]; and The part of equation (I) that is covalently bonded to the aforementioned linker: 【Chemistry 1】 [During the ceremony, X is O, S, S(O) and S(O) 2 Selected from; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 and R 8 are each independently hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a 、-SR b 、-C(O)OR c 、-C(O)NR d R e 、-OC(O)NR f R g 、-NR h C(O)NR i R j 、and -NR h C(S)NR i R j selected from, and each R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i and R j is independently hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 3 -C 6 cycloalkyl, and aryl selected therefrom] The composition comprising the above.

2. The composition according to claim 1, wherein the solid surface comprises a material selected from silica, glass, polymer, and metal.

3. The aforementioned solid surface Polymers selected from cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethylene, polyethersulfone, polyolefin, polyvinylidene fluoride, polyacrylate, polystyrene, or any combination thereof. The composition according to claim 1, comprising:

4. The aforementioned solid surface Beads, resins, magnetic particles, membranes, vials, plates, films, tubes, syringes, cartridges, cassettes, pipette tips, microfluidic cartridges, or cuvettes. The composition according to claim 1, in the form of...

5. The aforementioned linker, 【Transformation 3】 A composition according to claim 1, selected from the following.

6. The aforementioned linker, 【Chemistry 4】 The composition according to claim 5.

7. The composition according to claim 1, wherein X is O.

8. R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 The composition according to claim 1, wherein each of them is hydrogen.

9. The composition according to claim 1, wherein the solid surface further comprises one or more additional portions covalently bonded to the surface.

10. The composition according to claim 1, wherein the solid surface further comprises at least one polyethylene glycol (PEG) portion covalently bonded to the surface.

11. A method for isolating nucleic acids from a sample, (a) To provide a sample containing nucleic acid; (b) Contacting the sample with the composition according to any one of claims 1 to 10 at a pH of less than approximately 8.0, wherein the nucleic acid binds to the composition to provide a nucleic acid-binding composition; and (c) Separating the nucleic acid binding composition from the sample. The method, including the method described above.

12. The method according to claim 11, wherein step (b) is performed at a pH of approximately 2.0 to less than approximately 8.

0.

13. The method according to claim 11, wherein step (b) is performed at a pH of approximately 6.0 to approximately 7.

5.

14. The method according to claim 11, wherein step (b) includes contacting the sample with the composition for about 10 seconds to about 10 minutes.

15. One or more steps, after step (c), to wash the nucleic acid binding composition by contacting the composition with a washing solution with a pH of less than approximately 8.

0. The method according to claim 11, further comprising:

16. The method according to claim 11, further comprising the step of contacting the nucleic acid binding composition with an eluent at a pH of approximately 8.5 or higher to release the nucleic acid from the composition.

17. The method according to claim 16, wherein the eluent solution comprises an aqueous buffer.

18. The method according to claim 16, comprising contacting the nucleic acid binding composition with the eluent at a pH of approximately 8.5 to approximately 9.

5.

19. The method according to claim 16, comprising contacting the nucleic acid binding composition with the eluent for approximately 10 seconds to approximately 10 minutes.

20. The method according to claim 11, comprising carrying out a polymerase chain reaction with respect to the nucleic acid binding composition.

21. The method according to claim 11, wherein the sample comprises body fluid, cultured cells, tissue cells, cells derived from body fluid, environmental samples, food samples, plant samples, or any combination thereof.

22. The method according to claim 21, wherein the sample is a blood sample selected from whole blood, plasma, and serum.

23. The method according to claim 11, wherein the sample comprises a cell lysate, a cell supernatant, or a purified fraction of cells.

24. The method according to claim 11, wherein the method includes a step of dissolving the sample before step (b).

25. The method according to claim 11, wherein the nucleic acid is DNA.

26. The method according to claim 25, wherein the DNA is selected from total DNA, mtDNA (mitochondrial DNA), gDNA (genomic DNA), cfDNA (cell-free DNA), ccfDNA (circulating cell-free DNA), cffDNA (cell-free fetal DNA), bacterial DNA, viral DNA, and ctDNA (circulating tumor DNA).

27. The method according to claim 11, wherein the nucleic acid is RNA.

28. The method according to claim 27, wherein the RNA is selected from total RNA, miRNA, mRNA, tRNA, rRNA, siRNA, ctRNA (circulating tumor RNA), and viral RNA.

29. The method according to claim 11, wherein the nucleic acid is the total nucleic acid from the sample.

30. Compound of formula (IIa): 【Transformation 5】 [During the ceremony, X is O, S, S(O) and S(O) 2 Selected from; L is in the formula: -(CH 2 ) n1 -Z-(CH 2 ) n2 - [wherein n1 and n2 are each independently selected from 1, 2, 3, 4, 5, 6, 7, and 8, and Z is a linker selected from -NHC(O)NH-, -C(O)NH-, -OC(O)NH-, -OC(O)O-, -NHC(O)S-, -NHC(S)NH-, -O-, and -C(O)O-]; Each R a These are independently hydroxy, C 1 -C 6 Selected from alkoxys and halos; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 及びR 8 are each independently hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, -OR a 、-SR b 、-C(O)OR c 、-C(O)NR d R<000s087>、-OC(O)NR f R g 、-NR h C(O)NR i R j 、及び-NR h C(S)NR i R j selected from, and each R a 、R b 、R c 、R d 、R e 、R f 、R g 、R h 、R i 及びR j is independently hydrogen, C 1 -C 6 alkyl, C 2 -C<00s0109>alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 haloalkyl, C 3 -C 6 cycloalkyl, and aryl selected from〕 or its salt.

31. The compound according to claim 30, or a salt thereof, wherein X is O.

32. Each R a However, C 1 -C 6 The compound according to claim 30, or a salt thereof, which is an alkoxy.

33. R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 The compound according to claim 30, or a salt thereof, wherein each of these is hydrogen.

34. L, 【Transformation 7】 A compound according to any one of claims 30 to 33, or a salt thereof, having a structure selected from the above.

35. L is structure: 【Transformation 8】 A compound according to claim 34, or a salt thereof, having the properties of the compound according to claim 34.

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