Oligonucleotide synthesis on solid support

Abstract

Functionalized solid supports are useful for the synthesis of oligonucleotides. The functionalized solid supports contain a terminal functional group or an extended linker to the first nucleotide or nucleoside moiety. The extended linker allows oligonucleotide synthesis to be performed at a greater distance from the solid support more efficiently.

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 980,880, filed February 24, 2020, the entire contents of which are incorporated herein by reference.

[0002]

[0002] The present invention relates to functionalized solid supports for oligonucleotide synthesis. [Background technology]

[0003] Solid-phase oligonucleotide synthesis is typically performed on frits composed of controlled-pore glass (CPG) particles. CPG consists of a porous network that facilitates rapid mass transfer during synthesis, with pore diameters ranging from 500 to 2000 Å. For oligonucleotide synthesis, CPG particles are uniformly coated with a substrate, such as (3-aminopropyl)triethoxysilane, containing reactive amino groups that serve as attachment points for non-nucleoside linkers or nucleoside succinates, and oligonucleotides during the synthesis process. The oligonucleotides are covalently attached to the linker via the hydroxyl group at their 3' ends and remain attached throughout the assembly of the chain, after which they are cleaved and deprotected. In high-throughput, parallel, or small-scale oligonucleotide synthesis, the solid support is contained within the wells of a multiwell plate.

[0004] To maximize the scale of synthesis, the molecular structure of the substrate must have a high density of evenly distributed attachment points for optimal oligo synthesis. However, such a high density can cause crowding and steric hindrance of the oligos during the synthesis process, especially for long oligos, which can lead to errors in the final product.

[0005] One way to avoid steric hindrance is to use larger pore sizes. However, because the surface area of ​​CPGs is inversely proportional to pore size and the density of silanol-bonding groups is a function of surface area, large-pore CPGs have low ligand loading capacity. Furthermore, not all silanol-bonding sites are uniformly distributed; some are too densely packed to be useful for full-length oligonucleotide synthesis.

[0006] Another way to avoid steric hindrance is to use a spacer to which the oligo is tethered, which serves to position the oligo and the synthesis process farther from the substrate, which would allow more flexibility and more space for synthesis.

[0007] One commonly used spacer is a long-chain alkylamine (LCAA; Figures 1A and 1B). The LCAA is attached to an aminopropyl CPG (Figure 1A). In some cases, the LCAA is attached via a linker, such as acetylglycerol (Figure 1B). The amino group of the attached LCAA is then used as an anchor point for a linker suitable for oligonucleotide synthesis, such as a succinyl spacer. Summary of the Invention

[0008] The present invention relates to solid-phase oligonucleotide synthesis in which synthesis is performed at a greater distance from the surface of a solid support (e.g., CPG particles) using length-extended linkers comprising one or more polyalkylene glycol phosphate / phosphonate units. The length-extended linkers serve as starting points for oligonucleotide synthesis. The polyalkylene glycol phosphate / phosphonate units can be attached via phosphoramidite chemistry. The extended linkers described herein are compatible with universal linkers and conventional linker arms used in oligonucleotide synthesis.

[0009] In one embodiment, there is provided a functionalized solid support of formula (I), or a salt thereof, [ka] A is a solid support material; L 1 is a divalent chemical linker consisting of atoms selected from the group consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, and halogens; where (a) the atoms of the divalent linker are arranged to form an optionally substituted chain or an optionally substituted chain interrupted by an optionally substituted ring; (b) the chain or the chain and ring together comprise a continuous linear array of 12 to 40 members of atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon; L 2 may, at each occurrence independently, be -(CH2CH2O) m -, -(CHCH3CH2O) m - or (CH2CHCH3O) m - and G 1 But hydrogen, PG 1 , or P(O)(R 1 )-L 3 -R 2 and R 1 However, at each occurrence, independently, R 1a OR 1a and R 1a But, at each occurrence, independently, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-10 Cycloalkyl, or C 5-10 cycloalkenyl, where R 1a But halogen, cyano, oxo, OH, -OC 1-4 Alkyl, -OC 1-4 Haloalkyl or C 3-6 optionally substituted with 1 to 6 substituents independently selected from the group consisting of cycloalkyl, C 3-6 Cycloalkyl is halogen or C 1-4 optionally substituted with alkyl; L 3But -(OCH2CH2) p -, -(OCHCH3CH2) p -, -(OCH2CHCH3) p -, -OC 2-10 Alkylene-, -OC 0-4 Alkylene-C 3-10 Cycloalkylene-C 0-4 Alkylene- or OC 0-4 Alkylene-phenylene-C 0-4 alkylene-, where L 3 The alkylene in the alkylene group is optionally substituted with 1 to 6 halogens, and the cycloalkylene and phenylene groups are optionally substituted with halogens, C 1-4 Alkyl, C 1-4 Haloalkyl, cyano, -OC 1-4 Alkyl and OC 1-4 optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl; R 2 Ga-NR 2a R 2b and R 2a But hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 1-3 Alkylene-C 3-6 is cycloalkyl, R 2b But hydrogen, PG 2 , C(O)-L 4 -OH, C(O)-L 4 -OPG 3 , C(O)-L 4 -G 1a , C(O)-L 5 -H, C(O)-L 5 -PG 1 , C(O)-L 5 -P(O)(R 1 )-G 1a , C(O)-L 5 -P(O)(OH)-G 1a , or C(O)-L 6 -G 1a or R 2a and R 2btogether with the nitrogen to which they are attached form a protected nitrogen atom, L 4 is C(O) or L 7 -C(O), L 5 But, L 7 -O, L 6 But, L 7 -Si(C 1-4 alkyl)2, L 7 is a divalent chemical linker consisting of atoms selected from the group consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, and halogens; where (a) the atoms of the divalent linker are arranged to form an optionally substituted chain, an optionally substituted ring, or a combination of optionally substituted chain(s) and ring(s); (b) the chain(s), ring(s), or combinations thereof comprise a 1- to 20-membered continuous linear array of atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon; G 1a is a nucleotide or nucleoside moiety, PG 1 is a hydroxy protecting group, PG 2 is an amino protecting group, PG 3 is a carboxylic acid protecting group, m and p are independently an integer of 3 to 10; n is an integer from 1 to 20; Disclosed is a functionalized solid support of formula (I), or a salt thereof:

[0010] In another aspect, the present invention provides a method for producing a compound comprising: 1 is hydrogen or PG 1 1. A method for preparing a functionalized solid support of formula (I), wherein: a) reacting a functionalized solid support of formula (II) with R 20 and R 21 independently C 1-6 Formula (III) [ka] to form a compound of formula (IV) [ka] providing a functionalized solid support of b) oxidizing the functionalized solid support of formula (IV) to form G 1 is PG 1 providing a functionalized solid support of formula (I) wherein n is 1; and c) optionally a protecting group PG 1 Remove and G 1 providing a functionalized solid support of formula (I) wherein is hydrogen and n is 1; The present invention provides a method comprising:

[0011] In another aspect, the present invention provides a method for producing a compound comprising: 1 -P(O)(R 1 )-L 3 -R 2 A method for preparing a functionalized solid support of formula (I), which comprises the steps of: 1 is hydrogen, the functionalized solid support of formula (I) is 20 and R 21 independently C 1-6 alkyl, formula (V) [ka] By reacting with the compound Formula (VI) [ka] providing a functionalized solid support of The functionalized solid support of formula (VI) is oxidized to form G 1 -P(O)(R 1 )-L 3 -R 2 and R 2b is C(O)-L 4 -G 1a providing a functionalized solid support of formula (I), The present invention provides a method comprising:

[0012] In another aspect, the present invention provides a method for producing a compound comprising: 1 -P(O)(R 1 )-L 3 -R 2 1. A method for preparing a functionalized solid support of formula (I), wherein R 2b is C(O)-L 7 The functionalized solid support of formula (I), which is —C(O)OH, is reacted with a nucleoside or nucleotide at the 3′ or 5′ OH to form a compound of formula (IX-a): [ka] providing a functionalized solid support of formula (I) having: The present invention provides a method comprising:

[0013] In another aspect, the present invention provides a method for producing a compound comprising: 1 -P(O)(R 1 )-L 3 -R 2 1. A method for preparing a functionalized solid support of formula (I), wherein R 2b is C(O)-L 7 The functionalized solid support of formula (I) is -OH is reacted with R 20 and R 21 independently C 1-6 is alkyl, G 1a Formula (VII) in which [ka] to form a compound of formula (VIII-a) [ka] providing a functionalized solid support of The functionalized solid support of formula (VIII-a) is oxidized to form a compound of formula (Xa) [ka] providing a functionalized solid support of formula (I) having: The present invention provides a method comprising:

[0014] In another aspect, the present invention provides a method for synthesizing an oligonucleotide. [Brief explanation of the drawings]

[0015] [Figure 1A] FIG. 1 shows a controlled pore glass (CPG) functionalized with chains having a first moiety 1 derived from 3-aminopropylsilane and a second moiety 2 called a long chain alkylamine (LCAA). [Figure 1B]

[0016] FIG. 1 shows a CPG functionalized with a chain having moiety 3 derived from acetylglycerol. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1.Definition

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0017]

[0018] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0018]

[0019] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses a range of "2 to 4." The term "about" can refer to plus or minus 10% of the indicated numerical value. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" may be apparent from context, such as rounding, so for example, "about 1" can also mean 0.5 to 1.4.

[0019]

[0020] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described herein. Furthermore, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0020]

[0021] As used herein, the term "alkyl" means a straight or branched saturated hydrocarbon chain. The terms "lower alkyl" or "C 1-6 "Alkyl" means a straight or branched chain hydrocarbon containing 1 to 6 carbon atoms. 1-4 "Alkyl" means a straight or branched chain saturated hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0021]

[0022] The term "alkenyl" as used herein means a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond.

[0022]

[0023] The term "alkylene," as used herein, refers to a divalent group derived from a straight or branched saturated chain hydrocarbon, e.g., of 1 to 6 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and CH2CH2CH2CH2CH2-.

[0023]

[0024] As used herein, the term "alkenylene" refers to a divalent group derived from a straight or branched chain hydrocarbon having at least one carbon-carbon double bond.

[0024]

[0025] The term "alkoxy" as used herein refers to the group -O-alkyl. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0025]

[0026] The term "alkoxyalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkylene group.

[0026]

[0027] As used herein, the term "aryl" refers to phenyl or phenyl attached to a parent molecular moiety and fused to a cycloalkane group (e.g., the aryl can be indan-4-yl), a six-membered arene group (i.e., the aryl is naphthyl), or a non-aromatic heterocycle (e.g., the aryl can be benzo[d][1,3]dioxol-5-yl). The term "phenyl" is used to refer to the substituent, and the term "six-membered arene" is used to refer to the fused ring. Six-membered arenes are monocyclic (e.g., benzene or benzo). Aryl can be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic ring).

[0027]

[0028] As used herein, the term "cycloalkyl" or "cycloalkane" refers to a saturated ring system containing all carbon atoms as ring members and zero double bonds. The term "cycloalkyl" is used herein to refer to a cycloalkane when it is present as a substituent. A cycloalkyl may be a monocyclic cycloalkyl (e.g., cyclopropyl), a fused bicyclic cycloalkyl (e.g., decahydronaphthalenyl), a spirocyclic ring, or a bridged cycloalkyl (e.g., bicyclo[2.2.1]heptanyl) in which two non-adjacent atoms of the ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, and bicyclo[1.1.1]pentanyl.

[0028]

[0029] As used herein, the term "cycloalkenyl" or "cycloalkene" refers to a non-aromatic monocyclic or polycyclic ring system containing all carbon atoms and at least one carbon-carbon double bond as ring members, preferably having 5 to 10 carbon atoms per ring. The term "cycloalkenyl" is used herein to refer to a cycloalkene when present as a substituent. A cycloalkenyl may be a monocyclic cycloalkenyl (e.g., cyclopentenyl), a fused bicyclic cycloalkenyl (e.g., octahydronaphthalenyl), or a bridged cycloalkenyl (e.g., bicyclo[2.2.1]heptenyl) in which two non-adjacent atoms of the ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0029]

[0030] The term "fluoroalkyl" as used herein means an alkyl group, as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced with fluorine. Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, such as 3,3,3-trifluoropropyl.

[0030]

[0031] The term "halogen" or "halo" as used herein means Cl, Br, I, or F.

[0031]

[0032] The term "haloalkyl" as used herein means an alkyl group, as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced with halogen.

[0032]

[0033] As used herein, the term "heteroaryl" refers to an aromatic monocyclic heteroatom-containing ring (monocyclic heteroaryl) or a bicyclic ring system containing at least one monocyclic heteroaromatic ring (bicyclic heteroaryl). The term "heteroaryl," when present as a substituent, is used herein to refer to a heteroarene. A monocyclic heteroaryl is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A five-membered aromatic monocycle has two double bonds, and a six-membered aromatic monocycle has three double bonds. Bicyclic heteroaryls are 8- to 12-membered ring systems and include fused bicyclic heterocyclic ring systems (i.e., 10π-electron systems), such as monocyclic heteroaryl rings fused to 6-membered arenes (e.g., quinolin-4-yl, indol-1-yl), monocyclic heteroaryl rings fused to monocyclic heteroarene rings (e.g., naphthyridinyl), and phenyl rings fused to monocyclic heteroarene rings (e.g., quinolin-5-yl, indol-4-yl). Bicyclic heteroaryl / heteroarene groups include 9-membered fused bicyclic heteroaromatic ring systems with four double bonds and at least one heteroatom contributing a lone pair to a fully aromatic 10π-electron system, such as ring systems with a nitrogen atom at the ring junction (e.g., imidazopyridine) or benzoxadiazolyl. Bicyclic heteroaryl also includes fused bicyclic systems consisting of one heteroaromatic ring and one non-aromatic ring, such as a monocyclic heteroaryl ring fused to a monocyclic carbocyclic ring (e.g., 6,7-dihydro-5H-cyclopenta[b]pyridinyl) or a monocyclic heteroaryl ring fused to a monocyclic heterocyclic ring (e.g., 2,3-dihydrofuro[3,2-b]pyridinyl). A bicyclic heteroaryl is attached to the parent molecular moiety through an aromatic ring atom.Other representative examples of heteroaryl include, but are not limited to, indolyl (e.g., indol-1-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl (e.g., pyrazol-4-yl), pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl (e.g., triazol-4-yl), 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl (e.g., thiazol-4-yl), isothiazolyl, thienyl, benzimidazolyl (e.g., Examples include benzimidazol-5-yl), benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl (e.g., indazol-4-yl, indazol-5-yl), quinazolinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, isoquinolinyl, quinolinyl, imidazo[1,2-a]pyridinyl (e.g., imidazo[1,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.

[0033]

[0034] As used herein, the term "heterocycle" or "heterocyclic ring" refers to a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The term "heterocyclyl" is used herein to refer to a heterocycle when it is present as a substituent. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 7- and 8-membered ring contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, Includes oxetanyl, oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a 6-membered arene, or a monocyclic heterocycle fused to a monocyclic cycloalkane, or a monocyclic heterocycle fused to a monocyclic cycloalkene, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a monocyclic heterocycle fused to a monocyclic heteroarene, or a spiroheterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. A bicyclic heterocyclyl is attached to the parent molecular moiety at a non-aromatic ring atom (e.g., indolin-1-yl). Representative examples of bicyclic heterocyclyl include, but are not limited to, chroman-4-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien-2-yl, 1,2,3,4-tetrahydroisoquinolin-2-yl, 2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan-6-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), azabicyclo[3.1]heptyl, and 2-azabicyclo[2.2.1]heptyl. 0]hexanyl (including 3-azabicyclo[3.1.0]hexan-3-yl), 2,3-dihydro-1H-indol-1-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7-oxabicyclo[2.2.1]heptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, and 3-oxaspiro[5.5]undecanyl. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a 6-membered arene, or a bicyclic heterocycle fused to a monocyclic cycloalkane, or a bicyclic heterocycle fused to a monocyclic cycloalkene, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic 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.Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, azaadamantane (1-azatricyclo[3.3.1.13,7]decane), and oxaadamantane (2-oxatricyclo[3.3.1.13,7]decane). Monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through a non-aromatic ring atom.

[0034]

[0035] The terms "hydroxyl" or "hydroxy" as used herein refer to an --OH group.

[0035]

[0036] The term "hydroxyalkyl," as used herein, means that at least one --OH group is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0036]

[0037] The terms cycloalkylene, arylene, phenylene, heterocyclylene, etc. refer to divalent groups derived from a base ring, e.g., a cycloalkane, a heterocycle, etc. For illustrative purposes, examples of cycloalkylene and phenylene are, respectively: [ka] Cycloalkylene includes 1,1-cycloalkylene (e.g., [ka] ), for example, 1,1-cyclopropylene (i.e., [ka] ) is included.

[0037]

[0038] The terms "alkyl," "cycloalkyl," "alkylene," and the like, can in certain cases be preceded by a symbol indicating the number of atoms present in the group (e.g., "C 1-4 Alkyl," "C 3-6cycloalkyl," "C 1-4 These symbols are used as commonly understood by those skilled in the art. For example, the expression "C" followed by a subscript number indicates the number of carbon atoms present in the group that follows. Thus, a "C alkyl" is an alkyl group having 3 carbon atoms (i.e., n-propyl, isopropyl). 1-4 When a range is specified, such as ", the group portion that follows can have any number of carbon atoms that falls within the recited range. For example, "C 1-4 "Alkyl" is an alkyl group having from 1 to 4 carbon atoms in any arrangement (ie, straight or branched).

[0038]

[0039] For the compounds described herein, groups and substituents thereof may be chosen according to the allowed valences of atoms and substituents so that selection and substitution result in stable compounds, e.g., those that do not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like.

[0039]

[0040] When reciting numerical ranges herein, each intervening number to the same degree of precision is expressly contemplated. For example, for a range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for a range of 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.

[0040] 2.Functionalized solid support

[0041] An embodiment of the present invention provides a functionalized solid support of formula (I), wherein A, L 1 , L 2 , R 1 , G 1 , and n is as defined herein.

[0041]

[0042] In the solid support of formula (I), A is a solid support material suitable for oligonucleotide synthesis. Suitable solid support materials include controlled pore glass (CPG), silica gel, macroporous cross-linked polystyrene, polymethacrylate vinyl alcohol copolymer, silicon chips, glass, polystyrene beads, polypropylene sheets, nonporous silica beads, polyacrylamide, or polyacrylate. Solid supports are well known in the art, as described in Current Protocols in Nucleic Acid Chemistry 2000, Chapter 3, Section 3.1, "Solid-Phase Supports for Oligonucleotide Synthesis." CPG can have a pore size (e.g., 300-2000 Å) suitable for preparing oligonucleotide synthesis, such as 1000 or 2000 Å. CPG supports have a particle size of 40-500 mesh, a bulk density of 0.1-1.0 g / cc, and a molecular weight of 10-200 M. 2 / g and a pore volume of 0.2 to 3.050 cc / g.

[0042]

[0043] In formula (I), L 1 is a divalent chemical linker composed of atoms selected from the group consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, and halogen, wherein (a) the atoms of the divalent linker are arranged to form an optionally substituted chain, or an optionally substituted chain interrupted by an optionally substituted ring, and (b) the chain, or the chain and ring together, comprise a 12-40 member contiguous linear array of atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon.

[0043]

[0044] A 12-40 membered continuous linear array of atoms refers to an unbroken string of atoms, where at least some of the atoms are chain atoms or non-cyclic atoms, and optionally some of the atoms are cyclic atoms. A 12-40 membered continuous linear array of atoms is illustrated by the following example, where the atoms in the continuous linear array of atoms are shown in bold: [ka]

[0044]

[0045] In continuous linear array (a), all atoms in the array are chain atoms. Continuous linear arrays (b) through (f) contain a mixture of chain atoms and ring atoms, with (b) having one ring atom, (c) having two ring atoms, (d) having three ring atoms, and (e) and (f) having four ring atoms.

[0045]

[0046] The continuous linear array is [ka] or (f), may have unsaturated bonds between adjacent atoms.

[0046]

[0047] L 1 The optional substituents on the above are halogen (e.g., fluoro), C 1-6 Alkyl (e.g., methyl), C 1-6 Haloalkyl (e.g., trifluoromethyl), oxo (i.e., ═O), thione (i.e., ═S), OH, -OC 1-6 Alkyl (e.g., methoxy), -OC 3-6 Cycloalkyl, -OCH2C 3-6 Cycloalkyl, -OC(O)C 1-6 Alkyl (e.g., -OC(O)CH3), -OC(O)C 3-6 Cycloalkyl, -OC(O)CH2C 3-6 Cycloalkyl, C 1-6 Hydroxyalkyl and C 1-6 Alkoxy C 1-6 Contains alkyl (e.g., -CH2CH2OCH3). 1 Any permutation on L 1 This allows the compound to have a branched structure.

[0047]

[0048] L 1 may contain various functional groups, such as amines (e.g., -NH-, -N(C 1-6 alkyl)-), amides (e.g., -NHC(O)-, -N(C 1-6alkyl)C(O)-), thioamides (e.g., -NHC(S)-, -N(C 1-6 alkyl)C(S)-), ether (-O-), ester (-OC(O)-), thioester (-OC(S)-), sulfide (-S-), sulfoxide (-S(O)-), sulfone (-S(O)2-), sulfonamide (e.g., -NHS(O)2-, -N(C 1-6 alkyl)S(O)2-), silyl, alkyl orthosilicates (e.g., -OSi(OC 1-6 alkyl)O-), carbamates (e.g., -NHC(O)O-, -N(C 1-6 alkyl)C(O)O-), urea (e.g., -NHC(O)NH-, -N(C 1-6 alkyl)C(O)NH-, -N(C 1-6 alkyl)C(O)N(C 1-6 alkyl)-), hydrazides (e.g., -NHNHC(O)-), guanidines (e.g., -NHC(NH)=N-), and phosphate esters (e.g., -OP(O)(OC 1-6 alkyl)O-). Thus, L 1 may contain one or more covalently bonded moieties, including heteroatom-containing functional groups, alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene (e.g., phenylene), and heteroarylene groups. For example, L 1 is C 2-10 Alkylene, -NH-, -NHC(O)-, silyl, and OCHCH(OC(O)C 1-4 L may contain one or more covalently attached moieties independently selected from the group consisting of: 1 is a C bonded to a silyl group 11-39 alkylene, and the silyl group is attached to a solid support material, wherein C 11-39 2 to 6 methylene units of the alkylene are replaced by -NH-, -O- or NHC(O)-, C 11-39 The alkylene is optionally substituted with -OC(O)CH3. 1 L 1a -L 1b where L 1a is attached to a solid support material.1a -silyl-C 2-4 Alkylene-NH-C 2-10 It may be alkylene-NH-. 1a -silyl-C 2-4 Alkylene-NH-C 3-10 It may be cycloalkylene-NH-. 1a -silyl-C 2-4 Alkylene-C(O)NH-C 2-10 It may be alkylene-NH-. 1a -silyl-C 2-4 Alkylene-NH-C(O)C 2-10 Alkylene-C(O)NH-C 2-10 It may be alkylene-NH-. 1a -silyl-C 2-4 Alkylene-NHC(O)NH-C 2-10 It may be alkylene-NH-. 1b is -C(O)C 1-10 It may be alkylene-. 1 -silyl-C 2-4 Alkylene-NH-C 2-10 Alkylene-NHC(O)-C 2-10 Alkylene- or silyl-C 2-4 Alkylene-OCH2CH(OC(O)CH3)CH2OC(O)NH-C 2-10 Alkylene-NHC(O)-C 2-10 It may be alkylene-. 1 may be -silyl-(CH2)3-NH-(CH2)6-NHC(O)-(CH2)5- or silyl-(CH2)3-OCH2CH(OC(O)CH3)CH2OC(O)NH-(CH2)6-NHC(O)-(CH2)5-.

[0048]

[0049] L 1 When contains a terminal silyl group, the silyl group is Si-L 1* L 1 (i.e., L 1* But, L 1excluding one member (i.e., silyl) of a 12-40 member continuous linear array of such atoms), can be attached to an oxygen atom on a solid support such as CPG, shown in the formula: [ka] Other attachment arrangements of the silyl to the solid support material are possible, for example: [ka] is.

[0049]

[0050] L 2 represents, independently at each occurrence, -(CH2CH2O) m -, -(CHCH3CH2O) m - or (CH2CHCH3O) m -, where m is 3, 4, 5, 6, 7, 8, 9, or 10, i.e. the solid support of formula (I) is [ka] Preferably, L 2 is -(CH2CH2O) m Preferably, m is 6.

[0050]

[0051] To further illustrate, the functionalized solid support of formula (I) can be, for example, [ka] Includes:

[0051]

[0052] R 1 is independently calculated for each occurrence by R 1a OR 1a where R 1a is independently C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-10 Cycloalkyl, or C 5-10 cycloalkenyl, where R 1ais halogen (e.g., fluoro), cyano, oxo, OH, -OC 1-4 Alkyl, -OC 1-4 Haloalkyl, or C 3-6 optionally substituted with 1 to 6 substituents independently selected from the group consisting of cycloalkyl, C 3-6 Cycloalkyl can be halogen (e.g., fluoro) or C 1-4 Optionally substituted with alkyl. R 1 is, independently at each occurrence, C 1-4 Alkyl, -OC 1-4 It may be alkyl, or OCH2CH2CN. R 1 may, at each occurrence, independently be —CH 3 , —CH 2 CH 3 , —OCH 3 , —OCH 2 CH 3 , —OCH(CH 3 ) 2 , or OCH 2 CH 2 CN.

[0052]

[0053] G 1 may be hydrogen, i.e., formula (I) terminates on the right side with a hydroxy group.

[0053]

[0054] G 1 is PG 1 It may be here PG 1 is a hydroxy-protecting group. Hydroxy-protecting groups are well known in the art, as described in P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis," 4th ed., John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Suitable hydroxy-protecting groups include, for example, trityl groups (e.g., trityl, dimethoxytrityl, methoxytrityl), acetyl, benzoyl, benzyl, p-methoxybenzyl, β-methoxyethoxymethyl (MEM), methoxymethyl (MOM), methylthiomethyl, pivaloyl, tetrahydropyranyl (THP), tetrahydrofuranyl (THF), silyl (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), methyl, and ethoxyethyl.

[0054]

[0055] G 1 is -P(O)(R 1 )-L 3 -R 2 where R 1 , R 2 , and L 3 is as described herein.

[0055]

[0056] L 3 is -(OCH2CH2) p -, -(OCHCH3CH2) p -, -(OCH2CHCH3) p -, -OC 2-10 Alkylene-, -OC 0-4 Alkylene-C 3-10 Cycloalkylene-C 0-4 Alkylene- or OC 0-4 Alkylene-phenylene-C 0-4 alkylene-, where L 3 The alkylene in is optionally substituted with 1 to 6 halogens (e.g., fluoro), and the cycloalkylene and phenylene are optionally substituted with halogens, C 1-4 Alkyl, C 1-4 Haloalkyl, cyano, -OC 1-4 Alkyl and OC 1-4 and optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl, and p is 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, p is 6.

[0056]

[0057] L 3 is -(OCH2CH2) p -, for example, -(OCH2CH2)6-.

[0057]

[0058] L 3 -OC 2-10 Alkylene-, e.g. -OC 4-8 It may be alkylene-, -OC6 alkylene-, or O(CH2)6-.

[0058]

[0059] R2 Ha-NR 2a R 2b where R 2a is hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 1-3 Alkylene-C 3-6 is cycloalkyl, and R 2b is hydrogen, PG 2 , C(O)-L 4 -OH, C(O)-L 4 -OPG 3 , C(O)-L 4 -G 1a , C(O)-L 5 -H, C(O)-L 5 -PG 1 , C(O)-L 5 -P(O)(R 1 )-G 1a , C(O)-L 5 -P(O)(OH)-G 1a , or C(O)-L 6 -G 1a or R 2a and R 2b together with the nitrogen to which they are attached form a protecting nitrogen atom (e.g., phthalimide).

[0059]

[0060] R 2a and R 2b may each be hydrogen (i.e., R 2 is NH2).

[0060]

[0061] R 2a is hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 1-3 Alkylene-C 3-6 cycloalkyl, where R 2b PG 2 and PG 2is an amino-protecting group. Amino-protecting groups are well known in the art, as described in P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis," 4th ed., John Wiley & Sons, NY (2006). Suitable amino-protecting groups include, for example, carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl, and trichloroethylcarbonyl (Troc). R 2a may be hydrogen, where R 2b is PG 2 and PG 2 is preferably BOC.

[0061]

[0062] R 2a is hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 1-3 Alkylene-C 3-6 may be cycloalkyl, R 2b is C(O)-L 4 -OH, C(O)-L 4 -OPG 3 or C(O)-L 4 -G 1a and L 4 is C(O) or L 7 -C(O). Therefore, L 4 is a C(O) moiety, OH, OPG 3 , and G 1a Binds to R 2b is C(O)-C(O)-OH, C(O)-C(O)-OPG 3 , or C(O)-C(O)-G 1a Alternatively, L 4 L 7 -C(O), then R 2bis C(O)-L 7 -C(O)-OH, C(O)-L 7 -C(O)-OPG 3 , or C(O)-L 7 -C(O)-G 1a R 2a may be hydrogen, where R 2b is C(O)-L 4 -OH, C(O)-L 4 -OPG 3 , or C(O)-L 4 -G 1a where L 4 is as shown or described herein.

[0062]

[0063] L 7 is a divalent chemical linker comprised of atoms selected from the group consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, and halogen, wherein (a) the atoms of the divalent linker are arranged to form an optionally substituted chain, an optionally substituted ring, or a combination of optionally substituted chain(s) and ring(s), and (b) the chain(s), ring(s), or combination thereof comprises a 1-20 member contiguous linear array of atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon. 7 A continuous linear array of 1 to 20 atoms of L 1 It refers to an unbroken string of atoms similar to

[0063]

[0064] L 7 The optional substituents on the above are halogen (e.g., fluoro), C 1-6 Alkyl (e.g., methyl), C 1-6 Haloalkyl (e.g., trifluoromethyl), oxo (i.e., ═O), thione (i.e., ═S), OH, -OC 1-6 Alkyl (e.g., methoxy), -OC 3-6 Cycloalkyl, -OCH2C 3-6 Cycloalkyl, -OC(O)C 1-6 Alkyl (e.g., -OC(O)CH3), -OC(O)C 3-6 Cycloalkyl, -OC(O)CH2C 3-6Cycloalkyl, C 1-6 Hydroxyalkyl and C 1-6 Alkoxy C 1-6 Contains alkyl (e.g., -CH2CH2OCH3). 7 Any permutation on L 7 This allows the compound to have a branched structure.

[0064]

[0065] L 7 may contain, together with its optional substituents, various functional groups, such as amines (e.g., -NH-, -N(C 1-6 alkyl)-), amides (e.g., -NHC(O)-, -N(C 1-6 alkyl)C(O)-), thioamides (e.g., -NHC(S)-, -N(C 1-6 alkyl)C(S)-), ether (-O-), ester (-OC(O)-), thioester (-OC(S)-), sulfide (-S-), sulfoxide (-S(O)-), sulfone (-S(O)2-), sulfonamide (e.g., -NHS(O)2-, -N(C 1-6 alkyl)S(O)2-), silyl, alkyl orthosilicates (e.g., -OSi(OC 1-6 alkyl)O-), carbamates (e.g., -NHC(O)O-, -N(C 1-6 alkyl)C(O)O-), urea (e.g., -NHC(O)NH-, -N(C 1-6 alkyl)C(O)NH-, -N(C 1-6 alkyl)C(O)N(C 1-6 alkyl)-), hydrazides (e.g., -NHNHC(O)-), guanidines (e.g., -NHC(NH)=N-), and phosphate esters (e.g., -OP(O)(OC 1-6 alkyl)O-). Thus, L 7 may contain one or more covalently linked moieties, including heteroatom-containing functional groups, alkylene, alkenylene, alkynylene, cycloalkylene, heterocyclylene, arylene (eg, phenylene), and heteroarylene groups.

[0065]

[0066] L 4 -C 0-4 Alkylene-C(O)-, -C1-3 Alkylene-OC 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3 Alkylene-C(O)-, -phenylene-C(O)-, -C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-, -phenylene-C 1-3 Alkylene-OC(O)-, -C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-OC(O)-, -C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3 alkylene-OC(O)-, or [ka] wherein phenylene may be, for example, NO2, -OC 1-4 Alkyl, halogen, C 1-4 Alkyl, and C 1-4 L is optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl. 4 Each of the above choices for -C 0-4 L except for -C0-C(O)- in alkylene-C(O)- 4 L 7 -C(O). Therefore, L in these examples 7 -C 1-4 Alkylene-, -C 1-3 Alkylene-OC 1-3 Alkylene-, -C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3 Alkylene-, -phenylene-, -C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-, -phenylene-C 1-3 Alkylene -O-, -C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene -O-, -C 1-3 Alkylene-C(O)NH-Fluorene-C1-3 alkylene-O-, or [ka] L 4 Further explaining the bonding arrangement of C(O)-L 4 -G 1a is C(O)-C 0-4 Alkylene-C(O)-G 1a , C(O)-C 1-3 Alkylene-OC 1-3 Alkylene-C(O)-G 1a , C(O)-C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3 Alkylene-C(O)-G 1a , C(O)-phenylene-C(O)-G 1a , C(O)-C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-G 1a , C(O)-phenylene-C 1-3 Alkylene-OC(O)-G 1a , C(O)-C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-OC(O)-G 1a , C(O)-C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3 Alkylene-OC(O)-G 1a , or [ka] may be.

[0066]

[0067] L 4 may be -C(O)-, or L 4 -CH2-C(O)-, -(CH2)2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O)-, [ka] That is, L7 -CH2-, -(CH2)2-, -CH2-O-CH2-, -CH2-N(CH3)C(O)-(CH2)2-, [ka] where L 7 The continuous linear atomic arrangement of the group is shown in bold.

[0067]

[0068] R 2b is C(O)-L 4 -OH or C(O)-L 4 -OPG 3 where L 4 is -C(O)- or L 4 -CH2-C(O)-, -(CH2)2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O)-, [ka] is.

[0068]

[0069] PG 3 is a carboxylic acid protecting group. Carboxylic acid protecting groups are well known in the art, as described in P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis," 4th ed., John Wiley & Sons, NY (2006). Suitable carboxylic acid protecting groups include, for example, methyl, benzyl, tert-butyl, 2,6-disubstituted phenols (e.g., 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), and silyl.

[0069]

[0070] G 1 -P(O)(R 1 )-L 3 -R 2 and R 2 Ga-NR 2a R 2b and R 2bis C(O)-L 4 -OH, C(O)-L 4 -OPG 3 , or C(O)-L 4 -G 1a The functionalized solid supports of formula (I) have formulas (IA), (IB) and (IC), respectively, where A, L 1 , L 2 , L 3 , L 4 , R 1 , R 2a , P.G. 3 , n and G 1a is as defined herein. [ka]

[0070]

[0071] For example, in the functionalized solid supports of formulas (IA), (IB), and (IC), L 1 is -silyl-(CH2)3-NH-(CH2)6-NHC(O)-(CH2)5- or silyl-(CH2)3-OCH2CH(OC(O)CH3)CH2OC(O)NH-(CH2)6-NHC(O)-(CH2)5-, and L 2 is -(CH2CH2O)6-, n is 4 to 7, and L 3 is -O(CH2)3- and R 1 is, independently in each occurrence, -CH, -CHCH, -OCH, -OCHCH, -OCH(CH), or OCHCHCN; R 2a is hydrogen, and L 4 is as defined herein, for example, —C(O)—, —CH—C(O)—, —(CH)—C(O)—, —CH—O—CH—C(O)—, —CH—N(CH)C(O)—(CH)—C(O)—, [ka] and PG 3 and G 1a is as defined herein.

[0071]

[0072] Further, for example, in the functionalized solid supports of formulas (IA), (IB), and (IC), L 1 is -silyl-(CH2)3-NH-(CH2)6-NHC(O)-(CH2)5- or silyl-(CH2)3-OCH2CH(OC(O)CH3)CH2OC(O)NH-(CH2)6-NHC(O)-(CH2)5-, and L 2 is -(CH2CH2O)6-, n is 4 to 7, and L 3 is -(OCH2CH2)6-, and R 1 is, independently in each occurrence, -CH, -CHCH, -OCH, -OCHCH, -OCH(CH), or OCHCHCN; R 2a is hydrogen, and L 4 is as defined herein, for example, —C(O)—, —CH—C(O)—, —(CH)—C(O)—, —CH—O—CH—C(O)—, —CH—N(CH)C(O)—(CH)—C(O)—, [ka] and PG 3 and G 1a is as defined herein.

[0072]

[0073] R 2a is hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 1-3 Alkylene-C 3-6 cycloalkyl, where R 2b is C(O)-L 5 -H, C(O)-L 5 -PG 1 , C(O)-L 5 -P(O)(R 1 )-G 1a , or C(O)-L 5 -P(O)(OH)-G 1a and L 5 L 7 -O, where L 7is as defined herein. 5 (L 7 -O) is L 5 The oxygen atom on the right side of 1 , P(O)(R 1 )-G 1a , and P(O)(OH)-G 1a Combine with.

[0073]

[0074] L 5 (i.e., L 7 -O) is L 5b or L 5a -L 5b where L 5a -C 0-4 Alkylene-C(O)-, -C 1-3 Alkylene-OC 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3 Alkylene-C(O)-, -phenylene-C(O)-, -C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-, -phenylene-C 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-OC(O)- or C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3 alkylene -OC(O)-, and L 5b teeth, [ka] , -(CH2) 1-10 -O-, or (CH2CH2O) 2-6 - and R 10 is phenyl, C 1-10 Alkyl or C 1-10 haloalkyl, where phenylene and phenyl are HBr, -OC 1-4 Alkyl, halogen, C 1-4 Alkyl and C 1-4L is optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl. 5 Further explaining the bonding arrangement of C(O)-L 5 -P(O)(R 1 )-G 1a is C(O)-L 5a -L 5b -P(O)(R 1 )-G 1a which in turn may be C(O)-C 0-4 Alkylene-C(O)-L 5b -P(O)(R 1 )-G 1a , C(O)-C 1-3 Alkylene-OC 1-3 Alkylene-C(O)-L 5b -P(O)(R 1 )-G 1a , C(O)-C 1-3 Alkylene-N(C 1-3 alkyl)-C(O)-C 1-3 Alkylene-C(O)-L 5b -P(O)(R 1 )-G 1a , C(O)-phenylene-C(O)-L 5b -P(O)(R 1 )-G 1a , C(O)-C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-L 5b -P(O)(R 1 )-G 1a , C(O)-phenylene-C 1-3 Alkylene-OC(O)-L 5b -P(O)(R 1 )-G 1a , C(O)-C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-OC(O)-L 5b -P(O)(R 1 )-G 1a , or C(O)-C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3 Alkylene-OC(O)-L 5b -P(O)(R 1)-G 1a where -L 5b -P(O)(R 1 )-G 1a teeth, [ka] , -(CH2) 1-10 -OP(O)(R 1 )-G 1a , or (CH2CH2O) 2-6 -P(O)(R 1 )-G 1a and R 10 is phenyl, C 1-10 Alkyl or C 1-10 It is haloalkyl.

[0074]

[0075] L 5b base [ka] is useful for the preparation of oligonucleotides bearing a 3'-terminal amino group following removal of the phthalimide group.

[0075]

[0076] L 5 (i.e., L 7 -O) is L 5b or L 5a -L 5b where L 5a -C(O)-, -CH2-C(O)-, -(CH2)2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O), [ka] and L 5b teeth [ka] -CH2-O-, -(CH2)3-O-, or (CH2CH2O)4-.

[0076]

[0077] L5 (i.e., L 7 -O) is L 5b where L 5b teeth, [ka] , -(CH2) 1-10 -O-, or (CH2CH2O) 2-6 - and phenyl is NO2, -OC 1-4 Alkyl, halogen, C 1-4 Alkyl, and C 1-4 L is optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl. 5 (i.e., L 7 -O) is L 5b where L 5b teeth, [ka] , —CH2—O—, —(CH2)3—O—, or (CH2CH2O)4—.

[0077]

[0078] L 5 (i.e., L 7 -O) is L 5a -L 5b where L 5a -C 0-4 Alkylene-C(O)-, -C 1-3 Alkylene-OC 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3 Alkylene-C(O)-, -phenylene-C(O)-, -C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-, -phenylene-C 1-3 Alkylene-OC(O)-, -C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-OC(O)- or C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3alkylene -OC(O)-, and L 5b teeth, [ka] and R 10 is phenyl, C 1-10 Alkyl or C 1-10 haloalkyl, where phenylene and phenyl are H, NO, -OC 1-4 Alkyl, halogen, C 1-4 Alkyl and C 1-4 L is optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl. 5 (i.e., L 7 -O) is L 5a -L 5b where L 5a -C(O)-, -CH2-C(O)-, -(CH2)2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O)-, [ka] and L 5b teeth, [ka] is.

[0078]

[0079] Thus, for example, L 5 (i.e., L 7 -O) is [ka] , —CH—O—, —(CH)—O—, or (CHCHO)—, where L 5 L 7 The continuous linear atomic arrangement of the moiety is shown in bold.

[0079]

[0080] R 2b is C(O)-L 5 -H or C(O)-L 5-PG 1 where PG 1 is as defined herein, and L 5 teeth, [ka] , —CH2—O—, —(CH2)3—O—, or (CH2CH2O)4—.

[0080]

[0081] R 2b is C(O)-L 6 -G 1a where G 1a is as defined herein, and L 6 L 7 -Si(C 1-4 alkyl)2, and L 7 is as defined herein. For example, L 6 teeth, [ka] That is, L 7 teeth, [ka] For example, L 6 teeth, [ka] It may be. 6 To explain the bond, C(O)-L 6 -G 1a teeth, [ka] may be.

[0081]

[0082] G 1ais a nucleotide or nucleoside moiety, which corresponds to the respective nucleotide or nucleoside minus the hydrogen at the attachment point. Nucleotide refers to both mononucleotides and oligonucleotides (i.e., polynucleotides). Nucleoside, by definition, is limited to one nucleoside residue. Nucleotides and nucleosides include deoxyribonucleotides / deoxyribonucleosides and ribonucleotides / ribonucleosides.

[0082]

[0083] The oligonucleotide may be a DNA oligonucleotide, an RNA oligonucleotide, or an antisense oligonucleotide (eg, antisense RNA).

[0083]

[0084] Nucleotides and nucleosides include free (unprotected), modified nucleotides and nucleosides, and protected versions of nucleotides and nucleosides.

[0084]

[0085] Modified nucleotides or nucleosides, in the case of polynucleotides, include molecules having chemical modifications or substitutions on the nucleobase, sugar, or internucleotide linkage, as described, for example, in Ann. Rev. Biochem. Rev. Biochem. 1998, 67:99-134; Int. J. Mol. Sci. 2017, 18(8), 1683; Curr. Opin. Biotechnol. 1995, 6:12-19; ​​and Tetrahedron 1993, 49(28), 6123-6194, which are incorporated herein by reference. For example, modifications of internucleotide linkages can include phosphorothioates, N3'-O5' phosphoramidates, O3'-N5' phosphoramidates, 3-phosphorothiolates, 5'-phosphorothiolates, phosphorodithioates, methylphosphonates, and triazoles.

[0085]

[0086] Nucleotides and nucleosides include not only the known purine and pyrimidine bases, but also modified purine and pyrimidine bases and other heterocyclic base-containing moieties (these moieties are sometimes collectively referred to as "purine and pyrimidine bases and their analogs"). Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, etc. Nucleobase modifications can include, for example, deazapurines, N-1-methylguanosine, isoguanine, 2-aminopurine, 1,3-diaza-2-oxophenothiazine, 1,3-diaza-2-oxophenoxazine, 7-nitro-1,3-diaza-2-oxophenothiazine, 2,6-diaminopurine, purine, 6-thioguanine, hypoxanthine, 2-pyrimidinone, 2-pyridone, 4-thiouridine, imidazole-4-carboxamide, N-substituted 5-(carboxyamido)uridines, e.g., 5-(N-benzylcarboxyamido)-uridine, 5-fluoro-deoxyuridine.

[0086]

[0087] Sugar modifications include, for example, abasic nucleotides / nucleosides, 4'-carbocyclo analogs, 4'-thio analogs, 4' amino analogs, and 2'-substituted sugars such as fluoro, amino, methoxy, allyloxy, e.g., [ka] may include:

[0087]

[0088] Further modified nucleotides or nucleosides include substitution with non-radioactive labels, such as fluorescent labels or fluorescence quenchers, which can be attached via a spacer unit. Fluorophore classes include xanthenes (e.g., fluorescein, rhodamine), cyanines (e.g., cyanines 3, 3.5, 5, 5.5), and boron dipyrromethenes (BODIPY™ dyes). Fluorescence quencher classes include azo quenchers (e.g., dabcyl, IOWA BLACK® FQ) and anthraquinone quenchers (e.g., IOWA BLACK® RQ). Fluorescent labels and quenchers can be attached away from the 3'- or 5'-position of the nucleobase or sugar. For example, [ka]

[0088]

[0089] Additional nucleotide / nucleoside modifications are well known to those skilled in the art and include, for example, substitution with polyethylene glycol groups, cholesterol groups, biotin groups, lipid groups, and various functional groups such as amino, aldehyde, carboxylic acid, and alkyne.

[0089]

[0090] A protecting group may be present on an oxygen or nitrogen atom of the sugar or nucleobase portion of a nucleotide or nucleoside. A protecting group may also be present on the oxygen of the phosphate portion of a nucleotide. Protecting groups for nucleotides and nucleosides are well known in the art.

[0090]

[0091] Representative protecting groups are disclosed by Beaucage, S.L.; Uyer, R.P., "Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach," Tetrahedron, 1992, 48, pp. 2223-2311, which is incorporated by reference in its entirety. Exemplary protecting groups that can be removed under acidic or neutral conditions include trityl (Tr), dimethoxytrityl (DMTr), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), 9-(p-methoxyphenyl)xanthen-9-yl (Mox), and 4,4',4"-tris-tert-butyltrityl (TTTr). Exemplary protecting groups that can be removed under neutral conditions are base-labile protecting groups, such as acyl or aroyl groups.

[0091]

[0092] The exocyclic amino groups on nucleobases can be protected by acylation. Benzoyl groups are used to protect both adenine and cytosine, and isobutyryl and dimethylformamidyl groups can be used to protect guanine. Other alternative protecting groups are known, such as phenoxyacetyl, phthaloyl, or di-N-butylaminomethylene for the amino group of adenine, acetyl for cytosine, and isopropylphenoxyacetyl for guanine.

[0092]

[0093] As those skilled in the art will recognize, 5'-hydroxyl or 3'-hydroxyl groups are typically protected with triphenylmethyl (trityl), 4-methoxytriphenylmethyl (methoxytrityl), or 4,4'-dimethoxytriphenylmethyl (dimethoxytrityl). The dimethoxytrityl protecting group is generally preferred. 2'-Hydroxy protecting groups include silyl (e.g., t-butyldimethylsilyl), acetal, and the like, as described in "Current Protocols in Nucleic Acid Chemistry 2000, Chapter 3.5, 'Strategies for Oligoribonucleotide Synthesis According to the Phosphharamidite Method.' Phosphate protecting groups include 2-cyanoethyl and methyl groups.

[0093]

[0094] G 1a The nucleotide or nucleoside moiety of formula (I) is linked to the parent molecular moiety of formula (I) through the 3' or 5' oxygen atom in the nucleotide or nucleoside moiety.

[0094]

[0095] For illustrative purposes, R 2b is C(O)-L 7 -C(O)-G 1a G 1a An example of a functionalized solid support of formula (I) attached to the 3' oxygen of [ka] where A, L 1 , L 2 , L3 , L 7 , R 1 , R 2a and n is as defined herein.

[0095]

[0096] R 2b is C(O)-L 5a -L 5b -P(O)(R 1 )-G 1a G 1a Further examples of functionalized solid supports of formula (I) attached to the 3' oxygen of [ka] where A, L 1 , L 2 , L 3 , L 5a , L 5b , R 1 , R 2a , and n are as defined herein.

[0096]

[0097] R 2b is C(O)-L 6 -G 1a and L 6 L 7 -Si(C 1-4 Further examples of functionalized solid supports of formula (I) attached to the 3' oxygen are: [ka] where A, L 1 , L 2 , L 3 , L 7 , R 1 , R 2a , and n is as defined herein.

[0097]

[0098] G 1a A typical example of [ka] Includes:

[0098]

[0099] Throughout the embodiments and descriptions of the compounds of the present invention, all examples of haloalkyl may also be fluoroalkyl (e.g., any C 1-4 Haloalkyl is C 1-4 It may be fluoroalkyl).

[0099]

[0100] Compounds may exist as stereoisomers where asymmetric or chiral centers are present. Stereoisomers are "R" or "S" depending on the configuration of substituents around the chiral carbon atom. As used herein, the terms "R" and "S" refer to the configurations as defined in the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem., 1976, 45:13-30. This disclosure contemplates various stereoisomers and mixtures thereof, which are specifically included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials containing asymmetric or chiral centers, or by preparation of racemic mixtures followed by separation methods well known to those skilled in the art. These separation methods are exemplified by (1) coupling the mixture of enantiomers to a chiral auxiliary and separating the resulting mixture of diastereomers by recrystallization or chromatography, and optionally liberating the optically pure product from the auxiliary, as described in Furniss, Hannaford, Smith, and Tatchell, "Vogel's Textbook of Practical Organic Chemistry," 5th Edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England; (2) direct separation of a mixture of optical enantiomers on a chiral chromatographic column; or (3) fractional recrystallization techniques.

[0100]

[0101] It is understood that the compounds may have tautomeric forms, as well as geometric isomers, which also form an aspect of the present invention.

[0101]

[0102] The present disclosure also includes isotopically labeled compounds in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in compounds of the invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, including, but not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively. 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Cl. Deuterium, i.e. 2 Substitution with heavier isotopes, such as H, can confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and may therefore be preferred under certain circumstances. Compounds may incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated into compounds of formula (I) include: 11 C. 13 N, 15 O, and 18 F. Isotopically labeled compounds of formula (I) may generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples using an appropriate isotopically labeled reagent in place of the non-isotopically labeled reagent.

[0102]

[0103] The disclosed compounds can exist as salts. The salts can be prepared during the final isolation and purification of the compounds, or separately by reacting the amino group of the compound with a suitable acid. For example, the compound can be dissolved in a suitable solvent, such as, but not limited to, methanol and water, and treated with at least one equivalent of an acid, such as hydrochloric acid. The resulting salt can precipitate, be isolated by filtration, and dried under reduced pressure. Alternatively, the solvent and excess acid can be removed under reduced pressure to provide the salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, paratoluenesulfonate, undecanoate, hydrochloride, hydrobromide, sulfate, phosphate, and the like. The amino groups of the compounds may also be quaternized with alkyl chlorides, bromides, and iodides, such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl, and the like.

[0103]

[0104] Base addition salts can be prepared during the final isolation and purification of the disclosed compounds by reacting the carboxyl group with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a metal cation, such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0104] B. Method of synthesis

[0105] An embodiment of the present invention provides a method for preparing a functionalized solid support of formula (I). Functionalized supports of formula (I) include supports substituted with nucleotide or nucleoside moieties and supports that are chemical intermediates for preparing supports substituted with nucleotide or nucleoside moieties. Certain chemical intermediate supports can be reacted to prepare other chemical intermediate supports. Functionalized supports bearing nucleotide or nucleoside moieties can also be reacted to prepare oligonucleotides using well-known oligonucleotide synthesis techniques, such as those described in Biochem. Soc. Trans. Soc. Trans. 2011, 39, 575-580; and "Synthesis of Unmodified Oligoribonucleotides," Current Protocols in Nucleic Acid Chemistry 2000, Chapter 3 (each of which sections is incorporated herein by reference). Oligonucleotides can be cleaved from the solid supports of the invention using established protocols.

[0105]

[0106] One aspect of the present invention is G 1 is PG 1 and n is 1, comprising: (a) reacting a functionalized solid support of formula (II) with R 20 and R 21 independently C 1-6 Formula (III) [ka] to form a compound of formula (IV) [ka] providing a functionalized solid support of (b) oxidizing the functionalized solid support of formula (IV) to form A, L 1 , L 2 , R 1 , and P.G. 1 is as defined herein: [ka] providing a functionalized solid support of Steps (a) and (b) can be carried out using standard phosphoramidite coupling and oxidation procedures.

[0106]

[0107] The functionalized support of formula (Ia) is provided with a protecting group PG 1 with a suitable reagent in step (c) to remove [ka] The functionalized solid support may be provided as follows:

[0107]

[0108] The functionalized support of formula (Ib) can be converted to an additional chemical intermediate by reaction with a compound of formula (III) in step (d) to form a compound of formula (IV-A): [ka] (e) oxidizing the functionalized solid support of formula (IV-A) to form A, L 1 , L 2 , R 1 , and P.G. 1 is as defined herein, [ka] Steps (d) and (e) may be carried out using standard phosphoramidite coupling and oxidation procedures.

[0108]

[0109] Formula (Ic) is prepared by the addition of a protecting group PG in step (f). 1 to provide formula (I-c1), and the resulting alcohol may be further reacted according to steps (d) and (e), or (d), (e) and (f) to provide a functionalized solid support of formula (I), wherein G 1 is hydrogen or PG 1 and n is an integer from 3 to 20. PG at the end of a specific sequence 1 The removal of formula (I-c2) [ka] to provide. [ka]

[0109]

[0110] Shown in Scheme 1 are specific A, L 1 , L 2 , and G 1 1 is a representative synthesis showing the implementation of steps (a) through (f) with a group.

[0110]

[0111] Another aspect of the present invention is G 1 is hydrogen, in step (g), R 20 and R 21 independently C 1-6 alkyl, formula (Va) [ka] Further intermediates of the invention of formula (I) may be prepared by reaction with a compound of Formula (VI-a) [ka] providing a functionalized solid support of (h) oxidizing the functionalized solid support of formula (VI-a) to form A, L 1 , L 2 , L 3 , R 1 , R 2a , n, and PG 2 is as defined herein (i.e., in formula (I), G 1 -P(O)(R 1 )-L 3 -R 2 and R 2 NR 2a R 2b and R 2b is PG 2 ), formula (Id) [ka] In some embodiments, R 2a is hydrogen. Steps (g) and (h) can be carried out using standard phosphoramidite coupling and oxidation procedures.

[0111]

[0112] The functionalized support of formula (Id) is prepared by the addition of a protecting group PG 2 with a suitable reagent to remove the compound of formula (Ie) [ka] In some embodiments, a functionalized solid support of R 2a is hydrogen. [ka]

[0112]

[0113] Shown in Scheme 2 are specific A, L 2 , L 3 , and R 2b 1 is a representative synthesis showing the implementation of steps (g) through (i) with a group.

[0113]

[0114] A functionalized solid support of formula (Ie) (e.g., R 2a is hydrogen) can be further treated to provide additional intermediates of the invention by reaction with succinic anhydride to give R 2b is C(O)-CH2CH2COOH, the functionalized solid support of formula (I) is [ka] It may be provided in the form of

[0114]

[0115] A functionalized solid support of formula (Ie) (e.g., R 2a is hydrogen) has the formula HO-C(O)-L 7 -C(O)OH or HO-C(O)-L 7 -C(O)OPG 3 and further treating to provide additional intermediates of the invention by reaction with a compound of formula 2b is C(O)-L 7 -C(O)OH or C(O)-L 7 -C(O)OPG 3 a functionalized solid support of formula (I) of formula (Ig) or (Ih) [ka] Formula (Ih) can be prepared by removing the protecting group PG using standard conditions for removing carboxylic acid protecting groups well known in the art. 3 can be converted to formula (Ig) by removing [ka]

[0115]

[0116] Shown in Scheme 3 are specific A, L 2 , L 3 , R2a , and R 2b

[0049] Figure 1 is a representative synthesis showing the coupling of a compound of formula (Ie), representative (i-e2), with succinic anhydride to produce (i-f1).

[0116]

[0117] A functionalized solid support of formula (Ie) (e.g., R 2a is hydrogen) has the formula HO-C(O)-L 7 -OH or HO-C(O)-L 7 -OPG 1 may be further processed to provide additional intermediates of the invention by reaction with a compound of formula 2b is C(O)-L 7 -OPG 1 or C(O)-L 7 -OH (i.e., R 2b is C(O)-L 5 -PG 1 or C(O)-L 5 -H) to a functionalized solid support of formula (I) with a compound of formula (Ii) and (Ij) [ka] In formula (Ii) and (Ij), L 7 L 7 -O-PG 1 is the ether moiety, and L 7 Formula (Ii) can be prepared by removing the protecting group PG using standard conditions for removing alcohol protecting groups well known in the art. 1 can be converted to formula (Ij) by removing [ka]

[0117]

[0118] Scheme 4 shows a representative synthesis of a compound of formula (ii) via amide coupling with a representative universal linker, followed by alcohol deprotection to provide a compound of formula (ij). Universal linkers are well known in the art and are described in Org. Process Res. Dev. (2008) 12(3):399-410; Nucleosides, Nucleotides, and Nucl. Acids (2010) 29(11):867-878; "Solid-Phase Supports for Oligonucleotide Synthesis," Current Protocols in Nucleic Acid Chemistry 2000, Chapter 3, Section 3.1; U.S. Patent No. 6,770,754; U.S. Patent No. 7,202,264; U.S. Patent No. 7,491,817; and International Publication No. WO 2018 / 177881.

[0118]

[0119] The intermediates of formula (Ib), (I-c1), (I-c2), (Ie), (If), (Ig), and (Ij) may be further reacted to form the nucleotide or nucleoside moiety G 1a A functionalized solid support of formula (I) may be prepared having the formula:

[0119]

[0120] Collectively the expression (I-c3) [ka] Formulas (Ib), (I-c1), and (I-c2) can be prepared by coupling R 20 and R 21 independently C 1-6 Formula (Vb) which is alkyl [ka] to react with a compound of formula (VI-b) [ka] a functionalized solid support of The functionalized solid support of formula (VI-b) is oxidized to form A, L 1 , L 2 , L 3 , L 4 , R 1 , R 2a , and G 1a is as defined herein (i.e., in formula (I), G 1 is -P(O)(R 1 )-L 3 -R 2 and R 2 is NR 2a R 2b and R 2b is C(O)-L 4 -G 1a ), formula (Ik) [ka] In some embodiments, R 2a is hydrogen.

[0120]

[0121] Formula (Ie) can be prepared using standard amide bond forming conditions to give the compound of formula HO-C(O)-L 4 -G 1a reacts with the compound R 2b is C(O)-L 4 -G 1a The functionalized solid support of formula (I), wherein: may be provided in the form of formula (Ik): [ka]

[0121]

[0122] Shown in Scheme 5 is a compound of formula (I) in which the nucleotide or nucleoside moiety G 1a , and certain A, L 3 , R 2a , and R 2b 1 is a representative synthesis of a functionalized solid support of formula (ik) bearing a group.

[0122]

[0123] Formula (Ig) reacts with a nucleoside or nucleotide at the 3' or 5' OH under ester forming conditions to give formula (IX-a) [ka] The reaction of formula (If) can provide a functionalized solid support of formula (I) having: 7 is —CH2CH2—.

[0123]

[0124] Formula (Ij) is R 20 and R 21 independently C 1-6 is alkyl, G 1a is a nucleotide or nucleoside moiety, [ka] to react with a compound of formula (VIII-a) [ka] A functionalized solid support can be provided, The functionalized solid support of formula (VIII-a) is oxidized to form a compound of formula (Xa) [ka] A functionalized solid support having formula (I) is provided: [ka]

[0124]

[0125] Shown in Scheme 6 is a compound of formula (I) in which the nucleotide or nucleoside moiety G 1a , and certain A, L 5 , R 2a , and R 2b 1 is a representative synthesis of a functionalized solid support of formula (Xa) bearing a group.

[0125]

[0126] Another aspect of the present invention is a method for the preparation of a nucleotide-containing solid support of formula (I) comprising the steps of: (a) subjecting the functionalized solid support of formula (I) to one or more cycles of nucleotide addition chemistry, wherein G 1 But -P(O)(R 1 )-L 3-R 2 and R 2 But, -NR 2a R 2b and R 2b But C(O)-L 4 -G 1a , C(O)-L 5 -P(O)(R 1 )-G 1a , or C(O)-L 6 -G 1a and G 1a is a nucleotide or nucleoside moiety, the nucleotide and nucleoside moieties comprising a single nucleotide or nucleoside residue; and (b) obtaining a functionalized solid support of formula (I), 1 But -P(O)(R 1 )-L 3 -R 2 and R 2 But, -NR 2a R 2b and R 2b But C(O)-L 4 -G 1a , C(O)-L 5 -P(O)(R 1 )-G 1a , or C(O)-L 6 -G 1a and G 1a is an oligonucleotide moiety, The present invention provides a method for synthesizing an oligonucleotide comprising:

[0126]

[0127] The one or more cycles of nucleotide addition chemistry may be one or more cycles of nucleotide addition via phosphoramidites.

[0127]

[0128] Using the functionalized solid supports of the present invention, oligonucleotides can be prepared with an average error and / or deletion rate of less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 per 10,000 base pairs.

[0128]

[0129] The oligonucleotide synthesis method may further include cleaving the oligonucleotide portion from the functionalized solid support. The method may further include removing one or more protecting groups from the oligonucleotide portion before cleavage. The method may further include removing one or more protecting groups from the oligonucleotide after cleavage.

[0129]

[0130] The functionalized solid supports of the present invention can be synthesized using a number of techniques and synthetic protocols known to those skilled in the art.

[0130]

[0131] Phosphoramidite coupling can be carried out by activation of the appropriate phosphoramidite with an acidic azole catalyst in a solvent such as acetonitrile, followed by reaction of an excess of the activated phosphite with an alcohol reaction partner and oxidation of the intermediate phosphite triester. Azole catalysts include 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or numerous similar compounds, as described in "Coupling activators for the oligonucleotide synthesis via phosphoramidite approach," Tetrahedron (2013) 69(18), pp. 3615-3637. The unstable trivalent phosphite triester can be oxidized to a stable pentavalent phosphate triester using either 0.1 M or 0.02 M iodine in THF / pyridine / water.

[0131]

[0132] Amide coupling can be carried out using well-known conditions, for example, by reacting an amine with a carboxylic acid using a coupling reagent such as HATU, EDC, PyBOP, DCC, HBTU, or TBTU in a solvent such as THF, DMF, dichloromethane, ethyl acetate, or DMSO, optionally with the addition of an amine base such as N-methylmorpholine, Hunig's base, pyridine, 2,6-lutidine, triethylamine, etc., to provide the product, for example, by reacting an amine (1 equivalent) with an acid (2.5 equivalents) and HATU (2.5 equivalents) in the presence of diisopropylethylamine (3 equivalents) in DMSO at about room temperature.

[0132]

[0133] Conditions for removing alcohol and amine protecting groups are well known in the art, as described in P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis," 4th ed., John Wiley & Sons, NY (2006), incorporated herein by reference. For example, the DMTr group can be removed by reaction with 3% trichloroacetic acid (TCA) in dichloromethane (DCM), 3% dichloroacetic acid (DCA) in DCM, or 5% DCA in toluene. The Boc protecting group on the amine can be removed using trifluoroacetic acid (TFA) in DCM or HCl in a dioxane / water mixture.

[0133]

[0134] Oligonucleotide synthesis can be carried out by the stepwise addition of nucleotide residues to the 5'-end of a growing chain until the desired sequence is assembled. Each addition, called a synthesis cycle, generally consists of four chemical reactions: (1) deblocking, (2) coupling, (3) capping, and (4) oxidation. Deblocking can involve removal of the 5'-DMTr group, providing a solid-support-bound precursor with a free 5'-terminal hydroxy group. Typical conditions for DMTr removal include reaction with a solution of an acid, such as 2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in an inert solvent (dichloromethane or toluene). The free 5'-hydroxy can be coupled to the phosphoramide of a nucleoside in acetonitrile activated with an acidic azole catalyst, 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, or many similar compounds, to form a phosphite triester linkage. Any unreacted 5'-OH residues are then capped by acetylation. Finally, the phosphite triester bond is typically oxidized to a phosphate triester using iodine and water in the presence of a weak base (e.g., pyridine, lutidine, choline). Alternatively, oligonucleotide synthesis can be performed in the 3' direction, as described in Appl. Sci. 2019, 9, 1357.

[0134]

[0135] After synthesis, oligonucleotides can be cleaved from the support and deprotected. Deprotection conditions are generally determined by the type of modification incorporated into the oligonucleotide. They can also be determined by the protection of the nucleobases used to synthesize the oligonucleotide backbone. N-acyl base protection and 2-cyanoethyl phosphate protection can be simultaneously removed by treatment with an inorganic base or amine. Solid support-bound oligonucleotides can be treated with a solution of base in an organic solvent, such as 50% triethylamine in acetonitrile or 10% diethylamine in acetonitrile. Standard DNA bases protected with conventional groups (Bz-dA, Bz-dC, and iBu-dG) can be deprotected using ammonium hydroxide solution. However, this deprotection is generally slow and not compatible with all modifications. Ac-dC and dmf-dG, as well as the corresponding RNA bases, can be deprotected much more quickly using a 1:1 mixture of 28% (or more) ammonium hydroxide and 40% aqueous methylamine. If the oligonucleotide contains any 2'-O-protected ribonucleotide residues, the deprotection protocol includes a second step in which the 2'-O-protected silyl group is removed by treatment with fluoride ion using a variety of methods. The fully deprotected product can be used directly, or the desired oligonucleotide can be purified by a number of methods. Most commonly, the crude product is desalted using ethanol precipitation, size-exclusion chromatography, or reverse-phase HPLC. To remove unwanted cleavage products, the oligonucleotide can be purified by polyacrylamide gel electrophoresis or anion-exchange HPLC, followed by desalting. Oligonucleotides can be synthesized with "DMT ON," and the final dimethoxytrityl group can be removed during or after purification. To achieve this, no deblocking treatment is performed after the addition of the final phosphoramidite. The oligo is cleaved and deprotected as needed. If the DMTr group remains upon purification, it can be removed by treatment with acid (80% acetic acid in water). To completely remove the universal linker, one of the following methods can be used:(1) aqueous ammonium hydroxide at 80° C. for 17 hours; (2) (a 1:1 mixture of 28% (or more) ammonium hydroxide and 40% aqueous methylamine) at 80° C. for 5 hours; or (3) (a 1:1 mixture of 28% (or more) ammonium hydroxide and 40% aqueous methylamine) overnight at 55° C. The Q linker can be removed during deprotection by reaction with a 1:1 mixture of 28% (or more) ammonium hydroxide and 40% aqueous methylamine at 70° C. for 2.5 hours.

[0135]

[0136] The compounds of the present disclosure can be prepared using the exemplary reactions and techniques described in this section. Reactions are carried out in solvents appropriate to the reagents and materials used and suitable for efficient transformation. It will also be understood that in the description of the synthetic methods below, all proposed reaction conditions, including solvents, reaction atmospheres, reaction temperatures, experimental times, and workup procedures, are selected as standard conditions for the reactions and would be readily apparent to one of ordinary skill in the art. One of ordinary skill in the art can adjust one or more of the conditions described herein. Those familiar with the art of organic synthesis will understand that the functionality present on various portions of the edict molecule must be compatible with the proposed reagents and reactions. Not all compounds of the present disclosure falling within a given class may be compatible with some of the reaction conditions required in some of the methods described. Such limitations on substituents that are compatible with the reaction conditions will be readily apparent to one of ordinary skill in the art, and alternative methods may be used.

[0136] Abbreviation Ac-dC = N-acetyl-2'-deoxycytidine Ac2O = acetic anhydride aq. = aqueous Bz-dA = benzoyl-2'-deoxyadenosine Bz-dC = benzoyl-2'-deoxycytidine conc.=concentration DCC=N,N'-dicyclohexylcarbodiimide DCM = dichloromethane, CH2Cl2 DIPEA = di-isopropylethylamine dmf-dG = N-dimethylformamidine-2'-deoxyguanosine DMSO = dimethyl sulfoxide EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide eq=equivalent EtOH = ethanol EtOAc = ethyl acetate Et3N = triethylamine h=time HOAc = acetic acid HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HBTU = (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate iBu-dG = isobutyl-2'-deoxyguanosine MeCN = acetonitrile MeOH = methanol min=minutes PyBop = bromotripyrrolidinophosphonium hexafluorophosphate Q Linker = [ka] rt=room temperature rt=room temperature sat.=saturated TBDMS = tert-butyldimethylsilyl TBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate TFA = trifluoroacetic acid THF = tetrahydrofuran TMS = trimethylsilyl wt.=weight XantPhos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene [Example]

[0137] Example 1. Synthesis of extended linker CPG support Scheme 7 shows the synthesis of an exemplary precursor molecule 8. LCAA CPG beads with a pore size of 1000 Å were reacted with excess ε-caprolactone 7 in toluene at reflux for 24 hours. The resulting structure 8 has an alcohol-based support that is conducive to subsequent phosphoramidite chemistry. [ka]

[0138] As shown in Scheme 8, one to four spacer 18 phosphoramidites were then coupled to the cyanoethyl protecting group 9 using up to four cycles of standard phosphoramidite chemistry to generate the extended linker CPG10. [ka]

[0139]

[0139] As shown in Scheme 9, after deprotection of extended linker 10, a cleavable modified dT phosphoramidite 11 is then attached to 10 by standard phosphoramidite chemistry to generate 12, to which subsequent nucleotides are attached by standard phosphoramidite chemistry to generate the final oligonucleotide product. [ka]

[0140] Example 2. Coupling efficiency of oligo synthesis using extended linker CPG support structures Using a functionalized support of the general type as compound 12, oligonucleotides were produced with an error and deletion rate of less than 3 per 10,000 bp. Example 3. Synthesis of extended linker CPG support

[0141] Further embodiments of extended linker CPGs include modifications of spacer 18 to increase shelf life and reduce branching and side reactions. For example, spacer 18 amidites can be modified to replace the cyanoethyl protecting group with non-cleavable or more stable groups such as methyl, ethyl, methoxy, ethoxy, and isopropoxy.

[0141]

[0142] To prepare these modified structures, spacer 18, protected at one end with DMTr attached to the free OH at its terminus 14, was reacted with ethyl N,N,N',N'-tetraisopropylphosphorodiamidite 15, rather than 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, in the presence of pyridine trifluoroacetic acid (Py-TFA) in a Mixer Mill 400 ball mill homogenizer. This produced the spacer 18 amidite, also protected with DMTr and bearing an ethyl protecting group on phosphoramidite 16. [ka]

[0142]

[0143] The spacer 18 amidite 16 was then coupled to an alcohol-based CPG bearing an LCAA derivatized with ε-caprolactone 8 and oxidized, and the coupling-oxidation process was repeated six times to provide 18 . [ka]

[0143]

[0144] To add an amine group to the terminus of the extended linker, another spacer 18 19 was modified so that one free OH at one end was replaced with a free amine protected with a tert-butyloxycarbonyl (Boc) group 20. Ethyl-protected amidite 15 was then coupled as described above to generate 21. [ka]

[0144]

[0145] Compound 21 was then coupled to extended linker 18 to generate product 22, which contains eight spacers 18, is protected via the ethyl group on the phosphate, and has a free amine at the terminus. [ka]

[0145]

[0146] Similar chemistry can be used to prepare functionalized solid supports 27 and 28. [ka]

[0146]

[0147] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations on the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0147]

[0148] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation, with respect to the chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof. The claims as filed are set forth below. [Claim 1] Formula (I) [ka] or a salt thereof, A is a solid support material; L 1 is a divalent chemical linker consisting of atoms selected from the group consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, and halogens; where (a) the atoms of the divalent linker are arranged to form an optionally substituted chain or an optionally substituted chain interrupted by an optionally substituted ring; (b) the chain, or the chain and ring together, comprises a continuous linear array of 12 to 40 atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon; L 2 may, at each occurrence independently, be -(CH2CH2O) m -, -(CHCH3CH2O) m - or (CH2CHCH3O) m - and G 1 But -P(O)(R 1 )-L 3 -R 2 , hydrogen, or PG 1 and R 1 but, independently at each occurrence, -OR 1a or R 1a and R 1a But, at each occurrence, independently, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-10 Cycloalkyl, or C 5-10 cycloalkenyl, where R 1a Cyano, halogen, oxo, OH, -OC 1-4 Alkyl, -OC 1-4 Haloalkyl or C 3-6 cycloalkyl, wherein the C 3-6 Cycloalkyl is halogen or C 1-4 optionally substituted with alkyl; L 3 But -(OCH2CH2) p -, -(OCHCH3CH2) p -, -(OCH2CHCH3) p -, -OC 2-10 Alkylene-, -OC 0-4 Alkylene-C 3-10 Cycloalkylene-C 0-4 Alkylene- or OC 0-4 Alkylene-phenylene-C 0-4 alkylene-, where L 3wherein the alkylene is optionally substituted with 1 to 6 halogens, and the cycloalkylene and phenylene are optionally substituted with halogens, C 1-4 Alkyl, C 1-4 Haloalkyl, cyano, -OC 1-4 Alkyl and OC 1-4 optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl; R 2 But, -NR 2a R 2b and R 2a But hydrogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 1-3 Alkylene-C 3-6 is cycloalkyl, R 2b But C(O)-L 4 -G 1a , hydrogen, PG 2 , C(O)-L 4 -OH, C(O)-L 4 -OPG 3 , C(O)-L 5 -H, C(O)-L 5 -PG 1 , C(O)-L 5 -P(O)(R 1 )-G 1a , C(O)-L 5 -P(O)(OH)-G 1a , or C(O)-L 6 -G 1a or R 2a and R 2b together with the nitrogen to which they are attached form a protected nitrogen atom, L 4 But, L 7 -C(O) or C(O), L 5 But, L 7 -O, L 6 But, L 7 -Si(C 1-4 alkyl)2, L 7is a divalent chemical linker consisting of atoms selected from the group consisting of carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, silicon, and halogens; where (a) the atoms of the divalent linker are arranged to form an optionally substituted chain, an optionally substituted ring, or a combination of optionally substituted chain(s) and ring(s); (b) the chain(s), ring(s), or combinations thereof comprise a 1- to 20-membered contiguous linear array of atoms selected from the group consisting of carbon, oxygen, nitrogen, sulfur, phosphorus, and silicon; G 1a is a nucleotide or nucleoside moiety, PG 1 is a hydroxy protecting group, PG 2 is an amino protecting group, PG 3 is a carboxylic acid protecting group, m and p are independently an integer of 3 to 10; n is an integer from 1 to 20; A functionalized solid support of formula (I), or a salt thereof. [Claim 2] G 1 10. The functionalized solid support of claim 1, wherein is hydrogen. [Claim 3] G 1 is PG 1 2. The functionalized solid support of claim 1, wherein: [Claim 4] G 1 is P(O)(R 1 )-L 3 -R 2 2. The functionalized solid support of claim 1, wherein: [Claim 5] L 3 -(OCH2CH2) p 5. The functionalized solid support of claim 1, wherein [Claim 6] 6. The functionalized solid support of any one of claims 1, 4 and 5, wherein p is 6. [Claim 7] L 3 Ga-OC 2-10 10. The functionalized solid support of claim 1 or 4, which is alkylene-. [Claim 8] L 3 Ga-OC 4-8 8. The functionalized solid support of any one of claims 1, 4 and 7, which is alkylene-. [Claim 9] L 3 9. The functionalized solid support of any one of claims 1, 4, 7 and 8, wherein is -OC6 alkylene-. [Claim 10] L 3 9. The functionalized solid support of any one of claims 1, 4, 7 and 8, wherein is -O(CH2)6-. [Claim 11] R 2a 11. The functionalized solid support according to claim 1, wherein is hydrogen. [Claim 12] R 2b 12. The functionalized solid support according to claim 1, wherein is hydrogen. [Claim 13] R 2b is PG 2 12. The functionalized solid support according to claim 1, wherein [Claim 14] R 2b is C(O)-L 4 -G 1a , C(O)-L 4 -OH or C(O)-L 4 -OPG 3 12. The functionalized solid support according to claim 1, wherein [Claim 15] L 4 But -C 0-4 Alkylene-C(O)-, -C 1-3 Alkylene-OC 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3Alkylene-C(O)-, -phenylene-C(O)-, -C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-, -phenylene-OC 1-3 Alkylene-OC(O)-, -C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3 alkylene-OC(O)-, or [ka] wherein the phenylene is selected from the group consisting of NO2, -OC 1-4 Alkyl, halogen, C 1-4 Alkyl, and C 1-4 15. The functionalized solid support of claim 14, optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl. [Claim 16] L 4 is -(CH2)2-C(O)-, -C(O)-, -CH2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O)-, [ka] 16. The functionalized solid support of claim 15, wherein: [Claim 17] R 2b But C(O)-L 5 -H, C(O)-L 5 -PG 1 , C(O)-L 5 -P(O)(R 1 )-G 1a , or C(O)-L 5 -P(O)(OH)-G 1a 12. The functionalized solid support according to claim 1, wherein [Claim 18] L 5 But, L 5b or L 5a -L 5band L 5a But -C 0-4 Alkylene-C(O)-, -C 1-3 Alkylene-OC 1-3 Alkylene-C(O)-, -C 1-3 Alkylene-N(C 1-3 alkyl)C(O)-C 1-3 Alkylene-C(O)-, -phenylene-C(O)-, -C 1-3 Alkylene-O-phenylene-OC 1-3 Alkylene-C(O)-, -phenylene-C 1-3 Alkylene-OC(O)-, -C 1-3 Alkylene-O-phenylene-C 1-3 Alkylene-OC(O)- or C 1-3 Alkylene-C(O)NH-Fluorene-C 1-3 alkylene -OC(O)-, L 5b but, [ka] , -(CH2) 1-10 -O-, or (CH2CH2O) 2-6 - and R 10 But phenyl, C 1-10 Alkyl or C 1-10 is haloalkyl, wherein the phenylene and phenyl are selected from NO2, -OC 1-4 Alkyl, halogen, C 1-4 Alkyl, and C 1-4 18. The functionalized solid support of claim 17, optionally substituted with 1 to 4 substituents independently selected from the group consisting of haloalkyl. [Claim 19] L 5a is -C(O)-, -CH2-C(O)-, -(CH2)2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O)-, [ka] and L 5b but, [ka] 19. The functionalized solid support of claim 18, wherein the functionalized solid support is -CH2-O-, -(CH2)3-O-, or (CH2CH2O)4-. [Claim 20] L 5 but, [ka] 20. The functionalized solid support of claim 19, wherein the functionalized solid support is CH2-O-, -(CH2)3-O-, or (CH2CH2O)4-. [Claim 21] R 2b But C(O)-L 4 -OH or C(O)-L 4 -OPG 3 and L 4 is -C(O)-, -CH2-C(O)-, -(CH2)2-C(O)-, -CH2-O-CH2-C(O)-, -CH2-N(CH3)C(O)-(CH2)2-C(O)-, [ka] 15. The functionalized solid support of claim 14, wherein: [Claim 22] R 2b But C(O)-L 5 -H or C(O)-L 5 -PG 1 and L 5 but, [ka] 18. The functionalized solid support of claim 17, wherein the functionalized solid support is CH2-O-, -(CH2)3-O-, or (CH2CH2O)4-. [Claim 23] R 2b But C(O)-L 6 -G 1a and L 6 but [ka] 12. The functionalized solid support according to claim 1, wherein [Claim 24] L 6 but, [ka] 24. The functionalized solid support of claim 23, wherein: [Claim 25] L 1 comprises one or more covalently attached moieties, and said one or more moieties are C 2-10 Alkylene, -NH-, -NHC(O)-, silyl, and OCHCH(OC(O)C 1-4 25. The functionalized solid support of any one of claims 1 to 24, wherein the alkyl groups are independently selected from the group consisting of: alkyl)CH2O-; [Claim 26] L 1 C bonded to a silyl group 11-39 alkylene, wherein the silyl group is attached to a solid support material, and 11-39 2 to 6 methylene units of the alkylene are replaced by -NH-, -O-, or NHC(O)-, and 11-39 25. The functionalized solid support of any one of claims 1 to 24, wherein alkylene is optionally substituted with -OC(O)CH3. [Claim 27] L 1 But, -silyl-C 2-4 Alkylene-NH-C 2-10 Alkylene-NHC(O)-C 2-10 Alkylene- or silyl-C 2-4 Alkylene-OCH2CH(OC(O)CH3)CH2OC(O)NH-C 2-10 Alkylene-NHC(O)-C 2-10 25. The functionalized solid support of any one of claims 1 to 24, which is alkylene-. [Claim 28] L 125. The functionalized solid support of any one of claims 1 to 24, wherein is -silyl-(CH2)3-NH-(CH2)6-NHC(O)-(CH2)5- or silyl-(CH2)3-OCH2CH(OC(O)CH3)CH2OC(O)NH-(CH2)6-NHC(O)-(CH2)5-. [Claim 29] L 2 may, at each occurrence independently, be -(CH2CH2O) m 29. The functionalized solid support according to any one of claims 1 to 28, wherein - [Claim 30] 30. The functionalized solid support of claim 1, wherein m is 6. [Claim 31] 31. The functionalized solid support of claim 1, wherein n is 4 to 7. [Claim 32] R 1 32. The functionalized solid support of claim 1, wherein each occurrence is independently -OCH2CH2CN, -CH3, -CH2CH3, -OCH3, -OCH2CH3, or OCH(CH3)2. [Claim 33] 33. The functionalized solid support of any one of claims 1 to 32, wherein the solid support material is controlled pore glass (CPG), silica gel, macroporous cross-linked polystyrene, polymethacrylate vinyl alcohol copolymer, silicon chip, glass, polystyrene beads, polypropylene sheet, non-porous silica beads, polyacrylamide, or polyacrylate. [Claim 34] G 1 is hydrogen or PG 1 34. A method for preparing a functionalized solid support according to any one of claims 1 to 3 and 25 to 33, wherein: (a) A functionalized solid support of formula (II) is prepared by reacting a 20 and R 21 independently C 1-6 Formula (III) [ka] to form a compound of formula (IV) [ka] providing a functionalized solid support of (b) oxidizing the functionalized solid support of formula (IV) to form G 1 is PG 1 providing a functionalized solid support of formula (I) wherein n is 1; and (c) optionally the protecting group PG 1 Remove and G 1 providing a functionalized solid support of formula (I) wherein is hydrogen and n is 1; A method comprising: [Claim 35] (d)G 1 is hydrogen and n is 1, with a compound of formula (III) to form a compound of formula (IV-A): [ka] providing a functionalized solid support of (e) oxidizing the functionalized solid support of formula (IV-A) to form G 1 is PG 1 providing a functionalized solid support of formula (I) wherein n is 2; (f) optionally the protecting group PG 1 Remove and G 1 providing a functionalized solid support of formula (I) wherein is hydrogen and n is 2; and Optionally, steps (d) and (e), or (d), (e) and (f) may be repeated to give G 1 is hydrogen or PG 1 providing a functionalized solid support of formula (I) wherein n is an integer between 3 and 20; 35. The method of claim 34, further comprising: [Claim 36] G 1 -P(O)(R 1 )-L 3 -R 234. A method for preparing a functionalized solid support according to any one of claims 1 and 4 to 33, wherein: (g)G 1 is hydrogen, the functionalized solid support of formula (I) is 20 and R 21 independently C 1-6 alkyl, formula (V) [ka] to form a compound of formula (VI) [ka] providing a functionalized solid support of (h) oxidizing the functionalized solid support of formula (VI) to form G 1 -P(O)(R 1 )-L 3 -R 2 providing a functionalized solid support of formula (I), A method comprising: [Claim 37] R 2a is hydrogen, R 2b But C(O)-L 4 -G 1a That is, 37. The method of claim 36. [Claim 38] R 2a is hydrogen, R 2b But, PG 2 That is, 37. The method of claim 36. [Claim 39] the protecting group PG 2 Remove and R 2b 39. The method of claim 38, further comprising providing a functionalized solid support of formula (I), wherein is hydrogen. [Claim 40] R 2 is -NH2, by the addition of a functionalized solid support of formula (I) 4 -G 1a By reacting with the compound of R2b is C(O)-L 4 -G 1a 40. The method of claim 39, further comprising providing a functionalized solid support of formula (I), wherein: [Claim 41] R 2 A functionalized solid support of formula (I) where R is -NH is reacted with succinic anhydride to form a 2b 40. The method of claim 39, further comprising providing a functionalized solid support of formula (I), wherein is C(O)-CH2CH2COOH. [Claim 42] R 2 is -NH2, by the addition of a functionalized solid support of formula (I) 7 -C(O)OH or HO-C(O)-L 7 -C(O)OPG 3 By reacting with the compound of R 2b is C(O)-L 7 -C(O)OH or C(O)-L 7 -C(O)OPG 3 40. The method of claim 39, further comprising providing a functionalized solid support of formula (I), wherein: [Claim 43] R 2b is C(O)-L 7 -C(O)OPG 3 wherein the protecting group PG 3 43. The method of claim 42, further comprising removing: [Claim 44] R 2b is C(O)-L 7 The functionalized solid support of formula (I), which is —C(O)OH, is reacted with a nucleoside or nucleotide at the 3′ or 5′ OH to form a compound of formula (IX): [ka] 44. The method of any one of claims 41 to 43, further comprising providing a functionalized solid support of formula (I) having: [Claim 45] R 2 is -NH2, by the addition of a functionalized solid support of formula (I)7 -OH or HO-C(O)-L 7 -OPG 1 By reacting with the compound of R 2b is C(O)-L 7 -OH or C(O)-L 7 -OPG 1 40. The method of claim 39, further comprising providing a functionalized solid support of formula (I), wherein: [Claim 46] R 2b is C(O)-L 7 -OPG 1 wherein the protecting group PG 1 46. ​​The method of claim 45, further comprising removing: [Claim 47] R 2b is C(O)-L 7 The functionalized solid support of formula (I) is -OH is reacted with R 20 and R 21 independently C 1-6 is alkyl, G 1a Formula (VII) in which [ka] to form a compound of formula (VIII) [ka] providing a functionalized solid support; and The functionalized solid support of formula (VIII) is oxidized to form a compound of formula (X) [ka] providing a functionalized solid support of formula (I) having: 47. The process of claim 45 or 46, further comprising: [Claim 48] (a) subjecting a functionalized solid support of formula (I) to one or more cycles of nucleotide addition chemistry, [ka] G 1 But -P(O)(R 1 )-L 3 -R 2 and R 2 But, -NR 2a R 2b and R 2b But C(O)-L 4 -G 1a , C(O)-L 5 -P(O)(R 1 )-G 1a , or C(O)-L 6 -G 1a and G 1a is a nucleotide or nucleoside moiety, and the nucleotide and nucleoside moieties contain a single nucleotide or nucleoside residue; A, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , R 1 , R 2a and n is as defined in any one of claims 1 to 33. Steps, and (b) obtaining a functionalized solid support of formula (I), G 1 But -P(O)(R 1 )-L 3 -R 2 and R 2 But, -NR 2a R 2b and R 2b But C(O)-L 4 -G 1a , C(O)-L 5 -P(O)(R 1 )-G 1a , or C(O)-L 6 -G 1a and G 1a is an oligonucleotide moiety, Step, A method for synthesizing an oligonucleotide comprising: [Claim 49] 49. The method of Claim 48, further comprising cleaving the oligonucleotide portion from the functionalized solid support to provide the oligonucleotide. [Claim 50] 50. The method of claim 48 or 49, further comprising removing one or more protecting groups from said oligonucleotide moiety prior to cleavage. [Claim 51] 51. The method of any one of claims 48 to 50, further comprising the step of removing one or more protecting groups from the oligonucleotide after cleavage.

Claims

1. Formula (I) 【Chemistry 1】 or a salt thereof, A is a solid support material; L １ But -silyl- (CH ２ ) ２－４ -NH-(CH ２ ) ２－１０ -NHC(O)-(CH ２ ) ２－１０ - and L ２ But -(CH ２ CH ２ O) ｍ - and G １ But -P(O)(R １ )-L ３ -R ２ , hydrogen, or PG １ and R １ may, at each occurrence, independently be -OCH ２ CH ２ CN, -CH ３ , -CH ２ CH ３ , -OCH ３ , -OCH ２ CH ３ , or -OCH(CH ３ ) ２ and L ３ But -(OCH ２ CH ２ ) ｐ - or -OC ２－１０ alkylene-, R ２ But, -NR ２ａ R ２ｂ and R ２ａ is hydrogen, R ２ｂ But C(O)-L ４ -G １ａ , C(O)-L 4 -OH, C(O)-L 4 -OPG 3 , C(O)-L 7 -OPG 1 , hydrogen, or PG ２ and L ４ But, -C ０－４ alkylene-C(O)-; L 7 is —C 1-4 alkylene; G １ａ is a nucleotide or nucleoside moiety, PG １ is a hydroxy protecting group, wherein the hydroxy protecting group is selected from the group consisting of trityl, dimethoxytrityl, methoxytrityl, acetyl, benzoyl, benzyl, p-methoxybenzyl, β-methoxyethoxymethyl (MEM), methoxymethyl (MOM), methylthiomethyl, pivaloyl, tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), methyl, and ethoxyethyl; PG ２ is an amino protecting group, wherein the amino protecting group is selected from the group consisting of carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl, and trichloroethylcarbonyl (Troc); PG 3 is a carboxylic acid protecting group, wherein said carboxylic acid protecting group is selected from the group consisting of methyl, benzyl, tert-butyl, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol, and silyl; m and p are independently integers from 3 to 10; n is an integer from 1 to 20; A functionalized solid support of formula (I), or a salt thereof:

2. G １ 10. The functionalized solid support of claim 1, wherein is hydrogen.

3. G １ is PG １ 2. The functionalized solid support of claim 1, wherein:

4. G １ is P(O)(R １ )-L ３ -R ２ 2. The functionalized solid support of claim 1, wherein:

5. 5. The functionalized solid support of claim 1 or 4, wherein p is 6.

6. L ３ Ga-OC ４－８ The functionalized solid support of claim 1 or 4, which is alkylene-.

7. L ３ Ga-OC ６ The functionalized solid support of any one of claims 1, 4, and 6, which is alkylene-.

8. L ３ -O(CH ２ ) ６ The functionalized solid support according to any one of claims 1, 4, 6 and 7, wherein

9. L ４ But -(CH ２ ) ２ —C(O)—, —C(O)—, or —CH ２ 2. The functionalized solid support of claim 1, wherein the functionalized solid support is -C(O)-.

10. The functionalized solid support of any one of claims 1 to 9, wherein m is 6.

11. The functionalized solid support of any one of claims 1 to 10, wherein n is 4 to 7.

12. 12. The functionalized solid support of any one of claims 1 to 11, wherein the solid support material is controlled pore glass (CPG), silica gel, macroporous cross-linked polystyrene, polymethacrylate vinyl alcohol copolymer, silicon chip, glass, polystyrene beads, polypropylene sheet, non-porous silica beads, polyacrylamide, or polyacrylate.

13. G １ is hydrogen or PG １ A method for preparing a functionalized solid support according to any one of claims 1 to 3, wherein (a) A functionalized solid support of formula (II) is treated with R ２０ and R ２１ became independent and became C １－６ Formula (III) 【Chemistry 2】 to react a compound of formula (IV) 【Transformation 3】 providing a functionalized solid support of (b) oxidizing the functionalized solid support of formula (IV) to form G １ is PG １ providing a functionalized solid support of formula (I) wherein n is 1; and (c) optionally the protecting group PG １ By removing １ providing a functionalized solid support of formula (I) wherein is hydrogen and n is 1; A method comprising:

14. (d) G １ is hydrogen and n is 1, with a compound of formula (III) to form a compound of formula (IV-A): 【Chemistry 4】 providing a functionalized solid support of (e) oxidizing the functionalized solid support of formula (IV-A) to form G １ is PG １ providing a functionalized solid support of formula (I) wherein n is 2; (f) optionally the protecting group PG １ By removing １ providing a functionalized solid support of formula (I) wherein is hydrogen and n is 2; and Optionally, steps (d) and (e), or (d), (e) and (f) may be repeated to form G １ is hydrogen or PG １ providing a functionalized solid support of formula (I) wherein n is an integer from 3 to 20; 14. The method of claim 13, further comprising:

15. G １ -P(O)(R １ )-L ３ -R ２ 13. A method for preparing a functionalized solid support according to any one of claims 1 or 4 to 12, wherein (g) G １ is hydrogen, the functionalized solid support of formula (I) is ２０ and R ２１ became independent and became C １－６ alkyl, formula (V) 【Transformation 5】 to react a compound of formula (VI) 【Transformation 6】 providing a functionalized solid support of (h) oxidizing the functionalized solid support of formula (VI) to form G １ -P(O)(R １ )-L ３ -R ２ providing a functionalized solid support of formula (I), A method comprising:

16. R ２ａ is hydrogen, R ２ｂ But C(O)-L ４ -G １ａ That is, 16. The method of claim 15.

17. R ２ａ is hydrogen, R ２ｂ But, PG ２ That is, 16. The method of claim 15.

18. The protecting group PG ２ By removing ２ｂ 18. The method of claim 17, further comprising providing a functionalized solid support of formula (I), wherein is hydrogen.

19. R ２ Ga-NH ２ a functionalized solid support of formula (I) of formula HO—C(O)—L ４ -G １ａ by reacting with a compound of formula R ２ｂ is C(O)-L ４ -G １ａ 20. The method of claim 18, further comprising providing a functionalized solid support of formula (I), wherein:

20. R ２ Ga-NH ２ A functionalized solid support of formula (I), wherein R ２ｂ is C(O)-CH ２ CH ２ 20. The method of claim 18, further comprising providing a functionalized solid support of formula (I) where:

21. R ２ Ga-NH ２ a functionalized solid support of formula (I) of formula HO—C(O)—L ７ -C(O)OH or HO-C(O)-L ７ -C(O)OPG ３ by reacting with a compound of formula R ２ｂ is C(O)-L ７ -C(O)OH or C(O)-L ７ -C(O)OPG ３ providing a functionalized solid support of formula (I), Here, P.G. ３ is a carboxylic acid protecting group, wherein said carboxylic acid protecting group is selected from the group consisting of methyl, benzyl, tert-butyl, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol, and silyl; L ７ But, -C 1－４ is alkylene, 20. The method of claim 18.

22. R ２ｂ is C(O)-L ７ -C(O)OPG ３ wherein the protecting group PG ３ 22. The method of claim 21, further comprising the step of removing:

23. R ２ｂ is C(O)-L ７ The functionalized solid support of formula (I), which is —C(O)OH, is reacted with a nucleoside or nucleotide at the 3′ or 5′ OH to form a compound of formula (IX): 【Transformation 7】 23. The method of any one of claims 20 to 22, further comprising providing a functionalized solid support of formula (I) having:

24. R ２ Ga-NH ２ a functionalized solid support of formula (I) of formula HO—C(O)—L ７ -OH or HO-C(O)-L ７ -OPG １ by reacting with a compound of formula R ２ｂ is C(O)-L ７ —OH or C(O)-L ７ -OPG １ 20. The method of claim 18, further comprising providing a functionalized solid support of formula (I), wherein:

25. R ２ｂ is C(O)-L ７ -OPG １ wherein the protecting group PG １ 25. The method of claim 24, further comprising the step of removing:

26. (a) subjecting a functionalized solid support of formula (I) to one or more cycles of nucleotide addition chemistry, 【Transformation 8】 G １ But -P(O)(R １ )-L ３ -R ２ and R ２ But, -NR ２ａ R ２ｂ and R ２ｂ But C(O)-L ４ -G １ａ , C(O)-L ５ -P(O)(R １ )-G １ａ , or C(O)-L ６ -G １ａ and G １ａ is a nucleotide or nucleoside moiety, and the nucleotide and nucleoside moieties contain a single nucleotide or nucleoside residue; A, L １ , L ２ , L ３ , L ４ , R １ , R ２ａ and n is as defined in any one of claims 1 to 12. Steps, and (b) obtaining a functionalized solid support of formula (I), G １ But -P(O)(R １ )-L ３ -R ２ and R ２ But, -NR ２ａ R ２ｂ and R ２ｂ But C(O)-L ４ -G １ａ , C(O)-L ５ -P(O)(R １ )-G １ａ , or C(O)-L ６ -G １ａ and L ５ But, L ７ -O and L ６ But, L ７ -Si(C １－４ alkyl) ２ and L 7 is —C 1-4 alkylene; G １ａ is an oligonucleotide moiety, Step, A method for synthesizing an oligonucleotide comprising:

27. 27. The method of claim 26, further comprising cleaving the oligonucleotide portion from the functionalized solid support to provide the oligonucleotide.

28. 28. The method of claim 26 or 27, further comprising removing one or more protecting groups from said oligonucleotide moiety prior to cleavage.

29. The method of any one of claims 26 to 28, further comprising removing one or more protecting groups from the oligonucleotide after cleavage.

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