Chiral Design

Abstract

Chiral controlled oligonucleotides of carefully selected designs, compositions of chiral controlled oligonucleotides, and methods for their purification and use are provided. [Solution] A chirally controlled oligonucleotide composition, the composition being chirally controlled to be enriched for oligonucleotides of a particular oligonucleotide type compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length, wherein the common pattern of backbone chiral centers comprises 5' to 3' Rp(Sp)2, each oligonucleotide in the plurality has five or more chirally modified phosphate linkages, and at least 50% of the chirally modified phosphate linkages in each oligonucleotide in the plurality have the Sp structure.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 61 / 928,405, filed January 16, 2014, and 62 / 063,359, filed October 13, 2014, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Oligonucleotides are useful in therapeutic and diagnostic research and nanomaterial applications. Naturally occurring nucleic acids (e.g., unmodified DNA or RNA) have limited therapeutic use due to their instability to extracellular and intracellular nucleases and / or poor cell penetration and distribution. Furthermore, in vitro studies have demonstrated that the binding affinity, sequence-specific binding to complementary RNA, and nuclease stability of antisense oligonucleotides (Cosstick and Eckstein, 1985; LaPlanche et al., 1986; Latimer et al., 1989; Hacia et al., 1994; Mesmaeker et al., 1995) are sensitive to the absolute stereochemical configuration of the phosphorus atom. Therefore, new and improved oligonucleotides and oligonucleotide compositions, such as novel antisense oligonucleotides, siRNA oligonucleotides, and oligonucleotide compositions, are needed. Summary of the Invention

[0003] Among other things, the present invention encompasses the recognition that a stereorandom oligonucleotide preparation contains multiple unique chemical entities that differ from one another in the stereochemical configuration of individual backbone chiral centers within the oligonucleotide chain. Furthermore, the present invention encompasses the insight that a stereorandom oligonucleotide preparation is generally unlikely to contain every possible stereoisomer of a related oligonucleotide. Thus, among other things, the present invention provides novel chemical entities that are specific stereoisomers of an oligonucleotide of interest. That is, the present invention provides substantially pure preparations of single oligonucleotide compounds, where a particular oligonucleotide compound may be defined by its base sequence, its length, its backbone bond pattern, and its backbone chiral center pattern.

[0004] The present invention demonstrates, inter alia, that individual stereoisomers of a particular oligonucleotide may exhibit different stabilities and / or activities. Furthermore, the present disclosure demonstrates that the stability improvements achieved by the inclusion and / or location of particular chiral structures within an oligonucleotide can be comparable to or even better than the stability improvements achieved by the use of particular modified backbone linkages, bases, and / or sugars (e.g., by the use of particular types of modified phosphates, 2'-modifications, base modifications, etc.).

[0005] In particular, the present invention recognizes that the properties and activity of an oligonucleotide can be tuned by optimizing the pattern of its backbone chiral centers. In some embodiments, the present invention provides compositions of oligonucleotides, wherein the oligonucleotides have a common pattern of backbone chiral centers that unexpectedly and significantly improves the stability and / or biological activity of the oligonucleotide. In some embodiments, the pattern of backbone chiral centers results in increased stability. In some embodiments, the pattern of backbone chiral centers surprisingly results in increased activity. In some embodiments, the pattern of backbone chiral centers results in increased stability and activity. In some embodiments, when the oligonucleotide is utilized to cleave a nucleic acid polymer, the pattern of backbone chiral centers of the oligonucleotide surprisingly alone alters the cleavage pattern of the target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers effectively prevents cleavage at a second site. In some embodiments, the pattern of backbone chiral centers creates new cleavage sites. In some embodiments, the pattern of backbone chiral centers minimizes the number of cleavage sites. In some embodiments, the pattern of backbone chiral centers minimizes the number of cleavage sites such that the target nucleic acid polymer is cleaved at only one site within the sequence of the target nucleic acid polymer that is complementary to the oligonucleotide. In some embodiments, the pattern of backbone chiral centers improves cleavage efficiency at the cleavage site. In some embodiments, the pattern of backbone chiral centers of an oligonucleotide improves cleavage of a target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers increases selectivity. In some embodiments, the pattern of backbone chiral centers minimizes off-target effects. In some embodiments, the pattern of backbone chiral centers increases selectivity, for example, cleavage selectivity between two target sequences that differ only by a single nucleotide polymorphism (SNP).

[0006] All publications and patent documents cited in this application are incorporated herein by reference in their entirety.

[0007] definition Aliphatic: As used herein, the terms "aliphatic" or "aliphatic group" refer to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or polycyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocycle," "alicyclic," or "cycloalkyl"). In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. Unless otherwise specified, an aliphatic group contains 1-10 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-4 aliphatic carbon atoms. In still other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic or bicyclic C3-C6 ring that is fully saturated or contains one or more unsaturated units, but is not aromatic, with one point of attachment to another molecule. 10 refers to a hydrocarbon. In some embodiments, "alicyclic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, with one point of attachment to another molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, and composites thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0008] Alkylene: The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n-, where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0009] Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene group is a polymethylene group containing at least one double bond in which one or more hydrogen atoms have been replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0010] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development. In some embodiments, "animal" refers to a non-human animal, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0011] Approximately: As used herein, the word "approximately" or "about" when describing a number is generally intended to include numbers within a range of 5%, 10%, 15%, or 20% in either direction (greater or less) of that number, unless otherwise stated or otherwise apparent from the context (unless such number is less than 0% or more than 100% of its possible values). In some embodiments, the use of the word "about" when describing dosage means ±5 mg / kg / day.

[0012] Aryl: The term "aryl," used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring structures in which at least one ring of the structure is aromatic and each ring of the structure contains 3 to 7 ring members, with a total of 5 to 14 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to aromatic ring structures, including but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, are also included within the scope of the term "aryl" herein.

[0013] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide includes a stretch of amino acids, or a set of stretches of amino acids, that, taken together, are characteristic of the protein or polypeptide. Each such stretch will generally contain at least two amino acids. Moreover, one of skill in the art will recognize that at least 5, 10, 15, 20, or more amino acids are usually required to be characteristic of a protein. Generally, a characteristic portion shares at least one functional characteristic with the related intact protein, in addition to the specific sequence homology described above.

[0014] Signature Sequence: A "signature sequence" is a sequence that is found in all members of a family of polypeptides or nucleic acids and therefore can be used by those of skill in the art to define the members of that family.

[0015] Characteristic structural element: The term "characteristic structural element" refers to a distinct structural element (e.g., backbone structure, collection of pendant moieties, sequence element, etc.) found in all members of a family of polypeptides, small molecules, or nucleic acids, and thus can be used by those skilled in the art to define the members of that family.

[0016] Equivalent: The term "equivalent" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit a comparison of the results obtained or the phenomena observed. In some embodiments, equivalent sets of conditions or circumstances are characterized by a plurality of substantially the same characteristics and one or a few altered characteristics. Those skilled in the art will recognize that sets of conditions are equivalent to one another when they are characterized by a sufficient number and type of substantially the same characteristics such that the differences in the results obtained or the phenomena observed under the different sets of conditions or circumstances can justify a reasonable conclusion that the sets of conditions are caused by or exhibit differences in those altered characteristics.

[0017] Dosage regimen: As used herein, a "dosage regimen" or "treatment regimen" refers to a set of unit doses (usually one or more) administered individually to a subject, usually separated by a period of time. In some embodiments, a given therapeutic agent has a required dosing regimen that may include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated from the other by a period of equal length; in some embodiments, a dosing regimen includes multiple doses and at least two different periods that separate the separate doses. In some embodiments, all doses within a dosing regimen are the same unit dosage. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is the same as the first dosage amount.

[0018] Equivalent Agents: Upon reading this disclosure, those skilled in the art will recognize that the scope of agents useful in the context of the present invention is not limited to those specifically mentioned or exemplified herein. Specifically, those skilled in the art will recognize that active agents typically have a structure consisting of a backbone and attached pendant moieties, and will therefore understand that simple modifications of such backbone and / or pendant moieties do not significantly alter the activity of the agent. For example, in some embodiments, replacement of one or more pendant moieties with groups of equivalent three-dimensional structure and / or chemical reactivity properties can produce substituted compounds or moieties equivalent to the parent reference compound or moiety. In some embodiments, addition or removal of one or more pendant moieties can produce substituted compounds equivalent to the parent reference compound. In some embodiments, for example, alteration of the backbone structure by addition or removal of a small number of bonds (usually no more than 5, 4, 3, 2, or 1 bond, and often only a single bond) can produce substituted compounds equivalent to the parent reference compound. In many embodiments, equivalent compounds can be synthesized, for example, by the methods shown in the following general reaction schemes, or modifications thereof, using readily available materials, reagents, and conventional or provided synthetic procedures. Modifications to these reactions that are known per se but not mentioned here can also be employed.

[0019] Equivalent Dosage: The term "equivalent dosage" is used herein to compare dosages of different pharmaceutically active agents that produce the same biological result. Doses of two different agents are considered "equivalent" to one another according to the present invention if they achieve the same level or degree of biological result. In some embodiments, equivalent dosages of different pharmaceuticals used according to the present invention are determined using the in vitro and / or in vivo assays described herein. In some embodiments, one or more lysosomotropic agents used according to the present invention are utilized at a dose equivalent to that of a reference lysosomotropic agent; in some embodiments, such a reference lysosomotropic agent is selected from the group consisting of small molecule allosteric activators (e.g., pyrazolpyrimidines), iminosugars (e.g., isofagomine), antioxidants (e.g., n-acetylcysteine), and regulators of cellular trafficking (e.g., Rab1a polypeptides).

[0020] Heteroaliphatic: The term "heteroaliphatic" refers to an aliphatic group in which one or more units selected from C, CH, CH, or CH are independently replaced by a heteroatom. In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0021] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 pi electrons shared in the cyclic arrangement; and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and the quaternized form of any basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the aromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "aromatic heterocycle," any of which includes optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently may be optionally substituted.

[0022] Heteroatom: The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, boron, selenium, or silicon (any oxidized form of nitrogen, boron, selenium, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or a heterocycle, such as N (in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR + (including the substitutable nitrogen of) in N-substituted pyrrolidinyl).

[0023] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1 to 4, heteroatoms as defined above. When used to refer to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, wherein the nitrogen is N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + It can be NR (as in N-substituted pyrrolidinyl).

[0024] A heterocycle can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl groups, heteroaryl groups, or aliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. The heterocyclyl ring can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions are independently optionally substituted.

[0025] Intraperitoneal: As used herein, the phrases "intraperitoneal administration" and "administered intraperitoneally" have their art-recognized meaning to refer to administration of a compound or composition into the peritoneal membrane of a subject.

[0026] In vitro: As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, and / or microorganism) but in an artificial environment, e.g., in a test tube or reactor, in cell culture, etc.

[0027] In vivo: As used herein, the term "in vivo" refers to events that occur within a living organism (e.g., an animal, a plant, and / or a microorganism).

[0028] Lower alkyl: The term "lower alkyl" refers to a C 1~4It represents a straight or branched chain alkyl group. Illustrative lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0029] Lower haloalkyl: The term "lower haloalkyl" refers to a C alkyl group substituted with one or more halogen atoms. 1~4 It represents a straight or branched chain alkyl group.

[0030] Optionally substituted: As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," with or without the word "may," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, where one or more positions in any given structure may be the same or different at each position. Combinations of substituents contemplated by the present invention are preferably those that result in the production of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are substantially unchanged when in a condition that allows for their preparation, detection, and, in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0031] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently: halogen; -(CH) 0~4 R ○ ;-(CH2) 0~4 OR ○ ;-O(CH2) 0~4 R ○ , -O-(CH2) 0~4 C(O)OR ○ ;-(CH2) 0~4 CH(OR ○ )2;-(CH2) 0~4 SR ○ ;R ○ may be substituted with (CH2) 0~4 Ph;R ○ may be substituted with (CH2) 0~4O(CH2) 0~1 Ph;R ○ CH=CHPh optionally substituted with R ○ may be substituted with (CH2) 0~4 O(CH2) 0~1 -pyridyl; -NO2; -CN; -N3; ​​(CH2) 0~4 N(R ○ )2;-(CH2) 0~4 N(R ○ )C(O)R ○ ;-N(R ○ )C(S)R ○ ;-(CH2) 0~4 N(R ○ )C(O)NR ○ 2;N(R ○ )C(S)NR ○ 2;-(CH2) 0~4 N(R ○ )C(O)OR ○ ;-N(R ○ )N(R ○ )C(O)R ○ ;N(R ○ )N(R ○ )C(O)NR ○ 2;N(R ○ )N(R ○ )C(O)OR ○ ;-(CH2) 0~4 C(O)R ○ ;-C(S)R ○ ;-(CH2) 0~4 C(O)OR ○ ;-(CH2) 0~4 C(O)SR ○ ;(CH2) 0~4 C(O)OSiR ○ 3;-(CH2) 0~4 OC(O)R ○ ;-OC(O)(CH2) 0~4 SR-, SC(S)SR ○ ;-(CH2) 0~4 SC(O)R ○ ;-(CH2) 0~4 C(O)NR ○ 2;-C(S)NR ○ 2;-C(S)SR ○ ;-SC(S)SR ○ , (CH2)0~4 OC(O)NR ○ 2;C(O)N(OR ○ )R ○ ;-C(O)C(O)R○;-C(O)CH2C(O)R○;-C(NOR○)R ○ ;(CH2) 0~4 SSR ○ ;-(CH2) 0~4 S(O)2R ○ ;-(CH2) 0~4 S(O)2OR ○ ;-(CH2) 0~4 OS(O)2R ○ ;-S(O)2NR ○ 2;(CH2) 0~4 S(O)R ○ ;N(R ○ )S(O)NR ○ 2;-N(R ○ )S(O)2R ○ ;-N(OR ○ )R ○ ;-C(NH)NR ○ 2;-P(O)2R ○ ;P(O)R ○ 2;OP(O)R ○ 2;-OP(O)(OR ○ )2;-SiR○3;-(C 1~4 Linear or branched alkylene)ON(R ○ )2; or -(C 1~4 Linear or branched alkylene)C(O)ON(R ○ )2(in the formula, each R ○ are optionally substituted as described below and independently represent hydrogen, C 1~6 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2- (5- to 6-membered heteroaryl ring) or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independently occurring R ○taken together with their intervening atoms form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as described below.

[0032] R ○ The appropriate monovalent substituent (or two independently occurring R ○ together with the intervening atoms to form a ring) are independently halogen, -(CH2) 0~2 R ● ,-(Halo R ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2;O(Halo R ● ), -CN, -N3, -(CH2) 0~2 C(O)R ● , -(CH2) 0~2 C(O)OH, -(CH2) 0~2 C(O)OR ● , -(CH2) 0~2 SR ● , -(CH2) 0~2 SH, -(CH2) 0~2 NH2, -(CH2) 0~2 NHR ● , -(CH2) 0~2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. C(O)SR ● , -(C 1~4 Straight or branched chain alkylene)C(O)OR ● , or -SSR ● (In the formula, each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and, independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. ○ Suitable divalent substituents on a saturated carbon atom of include ═O and ═S.

[0033] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2~3 O-, or -S(C(R * 2)) 2~3 S-(wherein each R * can be substituted with hydrogen, 1~6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to adjacent substitutable carbon atoms of an "optionally substituted" group include: -O(CR * 2) 2~3 O-(wherein each R * can be substituted with hydrogen, 1~6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0034] Suitable substituents on the aliphatic group of R* include halogen, -R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ●2, or -NO2 (wherein each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and, independently, C 1~4 Aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0035] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † ;(In the formula, each R † are independently selected from halogen, and the following may be substituted C 1~6 an aliphatic, unsubstituted -OPh, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, notwithstanding the above definition, two independently occurring R † together with their intervening atoms form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0036] Suitable substituents on an aliphatic group of R† are independently halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2 (wherein each R● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and is independently a C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0037] Oral: As used herein, the phrases "oral administration" and "orally administered" refer to administration of a compound or composition by mouth and have their art-recognized meaning.

[0038] Parenteral: As used herein, the phrases "parenteral administration" and "parenterally administered" have their art-recognized meaning to refer to modes of administration that are not enteral or topical, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0039] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0040] As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., for buccal, sublingual, and systemic absorption, boluses, powders, granules, and pastes to be applied to the tongue; parenteral administration, e.g., as a sterile solution or suspension, or sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray to be applied to the skin, lungs, or oral cavity; intravaginally or rectally, e.g., as a pessary, cream, or foam; sublingually; ophthalmically; transdermally; or intranasally to the lungs and other mucosal surfaces.

[0041] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that, within sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0042] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the transport or transfer of a substance from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0043] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to a salt of such a compound that is suitable for use in a pharmaceutical context, i.e., a salt that is suitable for use in contact with the tissues of humans and lower animals, within the scope of sound medical judgment, at a reasonable benefit / risk ratio, and without undue harmful effects, irritation, allergic response, etc. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxypropyl methylcellulose ... Examples of suitable salts include, but are not limited to, sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates and arylsulfonates having 1 to 6 carbon atoms, as appropriate.

[0044] Prodrug: Generally, a "prodrug," as the term is used herein, and as understood in the art, is an entity that, when administered to an organism, is metabolized in the body to deliver an active (e.g., therapeutic or diagnostic) agent of interest. Typically, such metabolism results in the removal of at least one "prodrug moiety," so that the active agent is produced. Various forms of "prodrugs" are well known in the art. Examples of such prodrug moieties include: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42:309-396, edited by K. Widder, et al. (Academic Press, 1985); b)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011); c)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011); d)A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen; e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H. Bundgaard, p. 113-191 (1991); f) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992); g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and h)Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984) reference.

[0045] Like other compounds described herein, prodrugs can be provided in any of a variety of forms, e.g., crystalline forms, salt forms, etc. In some embodiments, the prodrug is provided as a pharmaceutically acceptable salt thereof.

[0046] Protecting group: As used herein, the term "protecting group" is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd edition, John Wiley & Sons, 1999 (the entire text of which is incorporated herein by reference). It also includes protecting groups specifically adapted to nucleotides and nucleotide chemistry described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012 (the entire text of Chapter 2 is incorporated herein by reference). Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenanthyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2- Phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitro p-Toluenesulfonylmethylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 2-methylthioethylcarbamate, 2-methylsulfonylethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthio Phenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acryloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-Dimethoxycarbonylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methyl 1-methyl-1-cyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamates, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenoxy)acetamide o-(methyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-Tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, Quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np- Methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylideneamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenyl Carbolic acid derivatives, N-[phenyl(pentacarbonylchromium or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4- Methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0047] Suitable protected carboxylic acids include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0048] Suitable hydroxyl protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (ME), and the like. M), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl] -4-Methoxypiperazin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trimethyl- methylethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazolinone) (4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthiazolyl Silyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate ester, benzoylformate ester, acetate ester, chloroacetate ester, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenoxyacetate ester, p-chlorophenoxyacetate Acid esters, 3-phenylpropionate esters, 4-oxopentanoate esters (levulinate esters), 4,4-(ethylenedithio)pentanoate esters (levulinoyl dithioacetal), pivalate esters, adamantate, crotonate esters, 4-methoxycrotonate esters, benzoate esters, p-phenylbenzoate esters, 2,4,6-trimethylbenzoate esters (mesitoate), alkyl methyl carbonates, 9-fluorenylmethyl carbonates (Fmoc), alkyl ethyl carbonates, alkyl 2,2,2-Trichloroethyl (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoic acid ester, 4-azidobutyric acid ester, 4-nitro-4-methylpentanoic acid ester, o-(dibromomethyl)benzoic acid ester, 2-formylbenzenesulfonic acid ester, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyric acid Acid esters, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoic acid, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothionyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,Examples of suitable boronic acid derivatives include 4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl borate, and phenyl borate.

[0049] In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl, ( DMTr) and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0050] In some embodiments, the phosphite protecting group is a group attached to an internucleotide phosphite bond throughout oligonucleotide synthesis. In some embodiments, the phosphite protecting group is attached to the sulfur atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is attached to the oxygen atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group is attached to the oxygen atom of an internucleotide phosphate bond. In some embodiments, the phosphite protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0051] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked to one another by peptide bonds). In some embodiments, a protein contains only naturally occurring amino acids. In some embodiments, a protein contains one or more non-naturally occurring amino acids (e.g., a moiety that forms one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues of a protein chain contain a non-amino acid moiety (e.g., a glycan, etc.). In some embodiments, a protein contains more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means. In some embodiments, a protein contains L-amino acids, D-amino acids, or both; in some embodiments, a protein contains one or more amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0052] Sample: As used herein, a "sample" refers to a specific organism or material obtained from. In some embodiments, the sample is a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample includes biological tissue or biological fluid. In some embodiments, the biological sample is or includes bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; suspended nucleic acid; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural fluid; feces; lymphatic fluid; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washings or lavage fluids, such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimen; tissue biopsy specimen; surgical specimen; feces, other body fluids, secretions, and / or cells therefrom, etc. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), etc. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), for example, by filtration through a semipermeable membrane. Such a "processed sample" can include, for example, nucleic acids or proteins obtained by extraction from a sample or processing a primary sample by techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components, etc. In some embodiments, the sample is an organism. In some embodiments, the sample is a plant. In some embodiments, the sample is an animal. In some embodiments, the sample is a human. In some embodiments, the sample is a non-human organism.

[0053] Stereochemical isomers: As used herein, the phrase "stereochemical isomers" refers to different compounds constructed of the same atoms connected by the same series of bonds but having different, incompatible three-dimensional structures. In some embodiments of the present invention, the provided chemical compositions may be or contain a pure synthesis of an individual stereochemical isomer of a compound; in some embodiments, the provided chemical compositions may be or contain a mixture of two or more stereochemical isomers of the compound. In certain embodiments, such mixtures contain equal amounts of different stereochemical isomers; in certain embodiments, such mixtures contain unequal amounts of at least two different stereochemical isomers. In some embodiments, the chemical composition may contain all diastereomers and / or enantiomers of the compound. In some embodiments, the chemical composition may contain less than all diastereomers and / or enantiomers of the compound. In some embodiments, if a specific enantiomer of a compound of the present invention is desired, it may be synthesized, for example, by asymmetric synthesis or derivatization with a chiral auxiliary, and the resulting diastereomeric mixture separated and the auxiliary cleaved to yield the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino, diastereomeric salts can be formed with a suitable optically active acid and resolved, for example, by fractional crystallization.

[0054] Subject: As used herein, the term "subject" or "subject" refers to any organism to which provided compounds or compositions are administered in accordance with the present invention, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject is suffering from and / or susceptible to a disease, disorder, and / or condition.

[0055] Substantial: As used herein, the term "substantial" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property under consideration. Those skilled in the biological arts will understand that biological and chemical phenomena rarely eschew completeness and / or completion or accomplishment or absolute results. Thus, the term "substantial" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0056] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0057] Susceptible (to a disease): An individual who is "susceptible to" a disease, disorder, and / or condition is one who is at a higher risk of developing the disease, disorder, and / or condition than members of the general community. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not be diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0058] Systemic: As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" have their art-recognized meaning of referring to administering a compound or composition so that it enters the recipient's entire body.

[0059] Tautomers: As used herein, the phrase "tautomers" is used to describe different isomers of an organic compound that are readily interchangeable. Tautomers can be characterized by the formal migration of a hydrogen atom or a proton accompanied by the shifting of a single bond and an adjacent double bond. In some embodiments, tautomers can result from proton tautomerism (i.e., relocation of a proton). In some embodiments, tautomers can result from valence tautomerism (i.e., rapid relocation of bonding electrons). All such tautomers are intended to be included within the scope of the present invention. In some embodiments, tautomers of a compound exist in mobile equilibrium with one another such that attempts to synthesize the separate substances would result in mixtures. In some embodiments, tautomers of a compound are separable and isolatable compounds. In some embodiments of the present invention, chemical compositions can be provided that are or contain a pure composition of a single tautomer of a compound. In some embodiments of the present invention, chemical compositions can be provided as mixtures of two or more tautomers of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomers; in certain embodiments, such mixtures contain different amounts of at least two tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain all tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain fewer than all tautomers of a compound. In some embodiments of the present invention, a chemical composition may contain one or more tautomers of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the present invention, the tautomer is keto-enol tautomerism. One skilled in the chemical arts can "capture" (i.e., chemically modify to retain the "enol" form) keto-enol tautomerism using any suitable reagent known in the chemical arts to obtain an enol derivative that can be subsequently isolated using one or more suitable techniques known in the art. Unless otherwise indicated, the present invention encompasses all tautomers of the relevant compounds, whether in pure form or in mixtures with each other.

[0060] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any agent that, when administered to a subject, induces a therapeutic effect and / or a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0061] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and others. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, suppresses, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0062] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, suppress, prevent, delay onset, reduce severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0063] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0064] Unit dose: As used herein, the phrase "unit dose" refers to an amount administered as a single dose of a pharmaceutical composition and / or in a physically discrete unit. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, one or more unit doses are administered to achieve the entire single dose. In some embodiments, administration of multiple unit doses is necessary or expected to be necessary to achieve the intended effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of a solid form, sustained-release formulation, or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose can be in a formulation containing any variety of ingredients in addition to the therapeutic agent. For example, an acceptable carrier (e.g., a pharmaceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, etc., as described below, can be included. It will be understood by those skilled in the art that in many embodiments, an appropriate total daily dosage of a particular therapeutic agent may comprise a fraction or multiple unit doses, and may be determined, for example, by an attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dosage level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active compound used; the specific composition used; the age, weight, health, sex, and diet of the subject; the number of administrations and the excretion rate of the specific compound used; the duration of treatment; drugs and / or additional therapies used in combination or simultaneously with the specific compound used, and similar factors well known in the medical arts.

[0065] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning of referring to an entity having a structure and / or activity actually found in a "normal" (as opposed to mutant, diseased, altered, etc.) state or context. Those of skill in the art will recognize that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0066] Nucleic Acid: The term "nucleic acid" includes any nucleotide, its analogs, and polymers thereof. As used herein, the term "polynucleotide" refers to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, by way of equivalents, analogs of either RNA or DNA synthesized from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. The term encompasses poly- or oligoribonucleotides (RNA) and poly- or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N- or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages"). The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. The prefix poly refers to nucleic acids containing from 2 to about 10,000 nucleotide monomer units, and the prefix oligo refers to nucleic acids containing from 2 to about 200 nucleotide monomer units.

[0067] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate or phosphorus-containing internucleotide linkages. The natural bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are purine or pyrimidine derivatives, although it should be understood that natural and unnatural base analogs are also included. The natural sugars are the pentoses (five-carbon sugars) deoxyribose (forming DNA) or ribose (forming RNA), although it should be understood that natural and unnatural base analogs are also included. Nucleotides are linked via internucleotide linkages to form nucleic acids, or polynucleotides. Many internucleotide linkages are well known in the art, including, but not limited to, phosphate, phosphorothioate, and boranophosphate. Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothioates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of natural nucleic acids such as those described herein.

[0068] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar.

[0069] Sugar: The term "sugar" refers to a monosaccharide in closed and / or open form. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term also encompasses structural analogs used in place of normal sugar molecules, such as glycols, whose polymers form the backbone of nucleic acid analogs, glycol nucleic acids ("GNAs").

[0070] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar, such that the modified sugar mimics the spatial arrangement, electronic state, or some other physicochemical property of a sugar.

[0071] Nucleobase: The term "nucleobase" refers to a nucleic acid moiety that participates in hydrogen bonds to bind one nucleic acid strand to another complementary strand in a sequence-specific manner. Most naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase," e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobases mimic the spatial arrangement, electronic state, or some other physicochemical property of nucleobases and retain the hydrogen-bonding properties that allow one nucleic acid strand to bind to another complementary strand in a sequence-specific manner. In some embodiments, the modified nucleobases can pair with all five natural bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex.

[0072] Chiral Ligand: The term "chiral ligand" or "chiral auxiliary" refers to a moiety that is chiral and can be incorporated into a reactant so that a reaction can be carried out with a particular stereoselectivity.

[0073] Condensing Reagent: In a condensation reaction, the term "condensing reagent" refers to a reagent that activates a less reactive site, making it more susceptible to action with another reagent. In some embodiments, such another reagent is a nucleophile.

[0074] Blocking Group: The term "blocking group" refers to a group that masks the reactivity of a functional group that can be subsequently unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

[0075] Moiety: The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity incorporated into or added to a molecule.

[0076] Solid support: The term "solid support" refers to any support that allows for the synthesis of nucleic acids. In some embodiments, the term refers to glass or polymer that is insoluble in the medium used in the reaction step to carry out nucleic acid synthesis and derivatize to introduce reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid support is controlled pore glass (CPG). In some embodiments, the solid support is a composite support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP).

[0077] Linking moiety: The term "linking moiety" refers to any moiety that may be located between the terminal nucleotide and the solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0078] DNA molecule: The term "DNA molecule" refers to either its single-stranded form or the polymeric form of double-helical deoxyribonucleotides (adenine, guanine, thymine, or cytosine). The term refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found in, inter alia, linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing specific double-stranded DNA molecule structures, the sequence may be described herein according to the convention of providing only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA).

[0079] Coding sequence: A DNA "coding sequence" or "coding region" is a double-stranded DNA that is transcribed and translated into an in vivo polypeptide when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence ("open reading frame" or "ORF") are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxylic) terminus. Coding sequences include, but are not limited to, prokaryotic sequences, cDNA from prokaryotic mRNA, genomic DNA sequences from prokaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence. The terms "non-coding sequence" or "non-coding region" refer to regions of a polynucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0080] Reading Frame: The term "reading frame" refers to one of six possible reading frames, three in each direction of a double-stranded DNA molecule. The reading frame used determines which codons are used to code for amino acids within the coding sequence of the DNA molecule.

[0081] Antisense: As used herein, an "antisense" nucleic acid molecule comprises a nucleic acid sequence that is complementary to, for example, the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence, or complementary to a "sense" nucleic acid encoding a protein that is complementary to the coding strand of a gene. Thus, an antisense nucleic acid molecule can associate with a sense nucleic acid molecule through hydrogen bonding.

[0082] Wobble position: As used herein, "wobble position" refers to the third position of a codon. In some embodiments, a mutation in a DNA molecule within the wobble position of a codon results in a silent or conservative mutation at the amino acid level. For example, there are four codons that code for glycine, namely, GGU, GGC, GGA, and GGG; therefore, mutation of any nucleotide at any wobble position to another nucleotide selected from A, U, C, and G will not result in a change at the amino acid level of the encoded protein, and is therefore a silent substitution.

[0083] Silent Substitution: A "silent substitution" or "silent mutation" is one in which a nucleotide in a codon is changed but does not result in a change in the amino acid residue encoded by the codon. Examples include mutations in the first position of a particular codon, such as the codon "CGG," which when mutated to AGG still encodes Arg, as well as mutations in the third position of the codon.

[0084] Gene: As used herein, the terms "gene," "recombinant gene," and "gene construct" refer to a DNA molecule, or portion of a DNA molecule, that encodes a protein or portion thereof. The DNA molecule may include an open reading frame that encodes the protein (as an exon sequence) and may further include intron sequences. As used herein, the term "intron" refers to a DNA sequence present in a given gene that is not translated into protein, and in some, but not all, cases, found between exons. As is well known in the art, it may be desirable for a gene to be operably associated with (or may include) one or more promoters, enhancers, repressors, and / or other control sequences that regulate the activity or expression of the gene.

[0085] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes recombinant polynucleotides synthesized by reverse transcription of mRNA, from which intervening sequences (introns) have been removed.

[0086] Homology: "Homology" or "identity" or "similarity" refers to the sequence similarity between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence that can be positioned for comparison purposes. When the same position in the compared sequences is occupied by the same base, then the molecules are identical at that position; when the same site is occupied by the same or similar nucleic acid residue (e.g., similar in steric and / or electronic state), then the molecules can be said to be homologous (similar) at that position. Expression of percentage homology / similarity or identity represents a function of the number of identical or similar nucleic acids at a position shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with a sequence described herein. When comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of extra residues also reduces the identity and homology / similarity.

[0087] In some embodiments, the term "homology" describes a mathematically based comparison of sequence similarity used for identical genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" to perform searches against public databases, for example, to identify other family members, related sequences, or homologs. In some embodiments, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments, BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. In some embodiments, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, to obtain gapped alignments for comparison purposes. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

[0088] Identity: As used herein, "identity" means the percentage of identical nucleotide residues at corresponding positions in two or more sequences when the sequences are aligned for maximum sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to maximize the match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith-Waterman algorithm may also be used to determine identity.

[0089] Heterologous: A "heterologous" region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found in relation to the larger sequence. Thus, when a heterologous region encodes a mammalian gene, the gene may be located on a side of DNA that is not normally adjacent to the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a sequence in which the coding sequence itself is not found at all (e.g., a cDNA containing introns or synthetic sequences in which the genomic coding sequence has codons or motifs that differ from the native gene). Allelic variation or natural mutational events do not give rise to a non-homologous region of DNA as defined herein.

[0090] Transition Mutation: The term "transition mutation" refers to a base change in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T)) is replaced by another pyrimidine, or a purine (adenosine (A) or guanosine (G)) is replaced by another purine.

[0091] Transversion Mutation: The term "transversion mutation" refers to a base change in a DNA sequence in which a pyrimidine (cytidine (C) or thymidine (T)) is replaced by a purine, or a purine (adenosine (A) or guanosine (G)) is replaced by a pyrimidine.

[0092] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages," as further defined herein).

[0093] Oligonucleotides can be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" encompasses single-stranded oligonucleotides. Single-stranded oligonucleotides can have double-stranded regions, and double-stranded oligonucleotides can have single-stranded regions. Illustrative oligonucleotides include, but are not limited to, structural genes, genes including regulatory and terminal regions, viral or plasmid DNA, self-replicating systems such as single-stranded and double-stranded siRNAs and other RNA interference agents (RNAi agents or iRNA agents), shRNAs, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0094] The double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as siRNA, RNAi agent or iRNA agent.In some embodiments, these RNA interference-inducing oligonucleotides are associated with the cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC).In many embodiments, single-stranded and double-stranded RNAi agents are long enough that they can be cut by endogenous molecules, such as Dicer, to produce smaller oligonucleotides that can enter RISC mechanism and participate in the RISC-mediated cleavage of target sequence, such as target mRNA.

[0095] Oligonucleotides of the present invention can vary in length. In certain embodiments, oligonucleotides can range from about 2 to about 200 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides can range in length from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides. In some embodiments, the oligonucleotides are about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotides are at least 4 nucleotides in length. In some embodiments, the oligonucleotides are at least 5 nucleotides in length. In some embodiments, the oligonucleotides are at least 6 nucleotides in length. In some embodiments, the oligonucleotides are at least 7 nucleotides in length. In some embodiments, the oligonucleotides are at least 8 nucleotides in length. In some embodiments, the oligonucleotides are at least 9 nucleotides in length. In some embodiments, the oligonucleotides are at least 10 nucleotides in length. In some embodiments, the oligonucleotides are at least 11 nucleotides in length. In some embodiments, the oligonucleotides are at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand at least 21 nucleotides in length.

[0096] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a phosphorus-containing bond between nucleotide units of an oligonucleotide, and is synonymous above and herein with "intersugar linkage" and "phosphorus atom bridge." In some embodiments, the internucleotide linkage is a phosphodiester linkage found in natural DNA and RNA molecules. In some embodiments, the internucleotide linkage is a "modified internucleotide linkage" in which each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but not limited to, =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se-, and -N(R')- (wherein each R' is independently as defined and described below). In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a phosphodiester diester linkage. [ka] or a modified phosphorothioate triester bond. Those skilled in the art will appreciate that the internucleotide linkage may exist as an anion or cation at a given pH depending on the presence of an acid or base moiety in the linkage.

[0097] Unless otherwise specified, when used with an oligonucleotide sequence, each s, s1, s2, s3, s4, s5, s6 and s7 independently represents the following modified internucleotide linkages shown in Table 1 below:

[0098] Table 1. Illustrative modified internucleotide linkages [Table 1] TIFF0007662687000003.tif224166TIFF0007662687000004.tif96166

[0099] For example, (Rp,Sp)-ATsCs1GA has 1) a phosphorothioate internucleotide bond between T and C; ( [ka] ) and 2) between C and G [ka] Unless otherwise specified, the Rp / Sp designation preceding an oligonucleotide sequence refers to the configuration of the chiral phosphorus atoms of the internucleotide linkages sequentially from 5' to 3' of the oligonucleotide sequence. For example, in (Rp,Sp)-ATsCs1GA, the phosphorus of the "s" bond between T and C has the Rp configuration, and the phosphorus of the "s1" bond between C and G has the Sp configuration. In some embodiments, "all (Rp)" or "all (Sp)" is used to indicate that all chiral phosphorus atoms of the oligonucleotide have the same Rp or Sp configuration, respectively. For example, all (Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linking phosphorus atoms of the oligonucleotide have the Rp configuration; all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linking phosphorus atoms of the oligonucleotide have the Sp configuration.

[0100] Oligonucleotide Type: As used herein, the phrase "oligonucleotide type" refers to an oligonucleotide having a particular base sequence, backbone linkage pattern (i.e., internucleotide linkage pattern, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linked phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I). 1 "Type" is used to define oligonucleotides having a pattern of "groups." Oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0101] Those skilled in the art will recognize that the synthesis methods of the present invention provide a degree of control during the synthesis of an oligonucleotide chain, such that each nucleotide unit of the oligonucleotide chain can be designed and / or preselected to have a specific stereochemistry at the linking phosphorus and / or a specific modification at the linking phosphorus and / or a specific base and / or a specific sugar. In some embodiments, the oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linking phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. The present invention provides compositions (e.g., chiral-controlled oligonucleotide compositions) comprising or consisting of a plurality of oligonucleotide molecules. In some embodiments, all such molecules are of the same type (i.e., structurally identical to each other). However, in many embodiments, the provided compositions typically contain multiple oligonucleotides of different types in predetermined relative amounts.

[0102] Chiral control: As used herein, "chiral control" refers to the ability to control the stereochemical orientation of each chiral linkage phosphorus within an oligonucleotide chain. The phrase "chiral controlled oligonucleotide" refers to an oligonucleotide that exists in a single diastereoisomer about the chiral linkage phosphorus.

[0103] Chiral control oligonucleotide composition: As used herein, the phrase "chiral control oligonucleotide composition" refers to an oligonucleotide composition that contains a predetermined level of an individual oligonucleotide type. For example, in some embodiments, the chiral control oligonucleotide composition contains one oligonucleotide type. In some embodiments, the chiral control oligonucleotide composition contains a mixture of multiple oligonucleotide types. Illustrative chiral control oligonucleotide compositions are further described herein.

[0104] Chiral pure: The phrase "chiral pure" is used herein to describe chiral controlled oligonucleotide compositions in which the entire oligonucleotide exists in a single diastereoisomer with respect to the attached phosphorus.

[0105] Chiral homogeneous: As used herein, the phrase "chiral homogeneous" is used to describe an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the junction phosphorus. For example, an oligonucleotide in which all nucleotide units have Rp stereochemistry at the junction phosphorus is chiral homogeneous. Similarly, an oligonucleotide in which all nucleotide units have Sp stereochemistry at the junction phosphorus is chiral homogeneous.

[0106] Predetermined: Predetermined means deliberately selected, e.g., as opposed to randomly occurring or achieved. Those skilled in the art will understand upon reading this specification that the present invention provides new and surprising technology that allows for the selection of specific oligonucleotide types for formulation and / or inclusion in provided compositions, and further allows for the controlled formulation of the selected specific types, optionally in selected specific relative amounts, precisely so that provided compositions are formulated. Such provided compositions are "predetermined" as described herein. Compositions that may contain specific individual oligonucleotide types are not "predetermined" compositions because they were created through a process that, by chance, does not control the intentional creation of specific oligonucleotide types. In some embodiments, a predetermined composition is one that can be intentionally replicated (e.g., through the repetition of a controlled process).

[0107] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom depicted is present in an internucleotide linkage, and that the phosphorus atom corresponds to the phosphorus atom of the phosphodiester internucleotide linkage that occurs in natural DNA and RNA. In some embodiments, the bound phosphorus atom is in a modified internucleotide linkage, where each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the bound phosphorus atom is represented by P * In some embodiments, the linking phosphorus atom is chiral. In some embodiments, the chiral linking phosphorus atom is P of Formula I * is.

[0108] P modification: As used herein, the term "P modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus. In some embodiments, the "P modification" ... 1 (Wherein X, L and R 1 are independently as defined and described herein and below).

[0109] Blockmir: As used herein, the term "blockmir" refers to an oligonucleotide chain in which the pattern of structural features that characterize each individual nucleotide unit is characterized by the presence of at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus. In some embodiments, the at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus linkage are referred to as a "block."

[0110] In some embodiments, the blockmirror is a "stereoblockmirror," e.g., at least two consecutive nucleotide units have the same stereochemistry at the linked phosphorus. Such at least two consecutive nucleotide units form a "stereoblockmirror." For example, (Rp,Sp)-ATsCs1GA is a stereoblockmirror because at least two consecutive nucleotide units, Ts and Cs1, have the same stereochemistry at the linked phosphorus (both Sp). In the same oligonucleotide, (Rp,Sp)-ATsCs1 forms a block, which is a stereoblock.

[0111] In some embodiments, a blockmir is a "P-modified blockmir," e.g., at least two consecutive nucleotide units have the same modification at the linked phosphorus. Such at least two consecutive nucleotide units form a "P-modified block." For example, (Rp,Sp)-ATsCsGA is a P-modified blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same P modification (i.e., both are phosphorothioate diester). In the same oligonucleotide of (Rp,Sp)-ATsCsGA, Ts and Cs form a block, which is a P-modified block.

[0112] In some embodiments, a blockmir is a "linked blockmir," e.g., at least two consecutive nucleotide units have the same stereochemistry and the same modification at the linking phosphorus. At least two consecutive nucleotide units form a "linked block." For example, (Rp,Rp)-ATsCsGA is a linked blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P modification (both phosphorothioate). In the same oligonucleotide of (Rp,Rp)-ATsCsGA, TsCs forms a block and is a linked block.

[0113] In some embodiments, the blockmir comprises one or more blocks independently selected from a stereoblock, a P-modified block, and a linking block, hi some embodiments, the blockmir is a stereoblockmir to one block, and / or a P-modified blockmir to another block, and / or a linking blockmir to yet another block. For example, (Rp,Rp,Rp,Rp,Rp,Sp,Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG is a stereoblockmir with respect to the stereoblock AsTsCsGsAs1 (all Rp's at the bound phosphorus) or Ts1Cs1Gs1 (all Sp's at the bound phosphorus), a P-modified blockmir with respect to the P-modified block AsTsCsGs (all s-bonds) or As1Ts1Cs1Gs1 (all s1-bonds), or a bonded blockmir with respect to the bonded block AsTsCsGs (all Rp's and all s-bonds at the bound phosphorus) or Ts1Cs1Gs1 (all Sp's and all s1-bonds at the bound phosphorus).

[0114] Altmer: As used herein, the term "altmer" refers to an oligonucleotide chain whose structural feature pattern characterizing each individual nucleotide unit is characterized by the absence of two consecutive nucleotide units in the oligonucleotide chain that share a particular structural feature in the internucleotide phosphorus bond. In some embodiments, an altmer is designed so that it contains a repeating pattern. In some embodiments, an altmer is designed so that it does not contain a repeating pattern.

[0115] In some embodiments, the altomer is a "stereoaltomer," e.g., no two consecutive nucleotide units have the same stereochemistry at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

[0116] In some embodiments, the altmer is a "P-modified altmer," e.g., no two consecutive nucleotide units have the same modification at the linked phosphorus, e.g., all (Sp)CAs1GsT, where each linked phosphorus has a different P modification than the others.

[0117] In some embodiments, the altmer is a "linked altmer," e.g., no two consecutive nucleotide units have the same stereochemistry or the same modification at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsTs2CsGs3CsAs4CsC.

[0118] Unimer: As used herein, the term "unimer" refers to an oligonucleotide chain whose structural feature pattern characterizes each individual nucleotide unit, in which all nucleotide units in the chain share at least one common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus.

[0119] In some embodiments, the unimer is a "stereounimer," e.g., all nucleotide units have the same stereochemistry at the linkage phosphorus, e.g., all (Sp)-CsAs1GsT, where all the linkages have Sp phosphorus.

[0120] In some embodiments, the unimer is a "P-modified unimer," e.g., all nucleotide units have the same modification at the linked phosphorus, e.g., (Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp,Sp,Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, where all the internucleotide linkages are phosphorothioate diesters.

[0121] In some embodiments, the unimer is a "linked unimer," e.g., all nucleotide units have the same stereochemistry and the same modification at the linked phosphorus, e.g., all (Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC, where all the internucleotide linkages are phosphorothioate diesters with an Sp linked phosphorus.

[0122] Gapmer: As used herein, the term "gapmer" refers to an oligonucleotide chain characterized in that at least one internucleotide phosphorus bond of the oligonucleotide chain is a phosphodiester bond, such as that found in natural DNA or RNA. In some embodiments, one or more internucleotide phosphorus bonds of the oligonucleotide chain are phosphodiester bonds, such as those found in natural DNA or RNA. For example, all (Sp)-CAs1GsT, in which the internucleotide bond between C and A is a phosphodiester bond.

[0123] Skipmer: As used herein, the term "skipmer" refers to a type of gapmer in which every other internucleotide phosphorus bond of the oligonucleotide strand is a phosphodiester bond, such as found in natural DNA or RNA, and every other internucleotide phosphorus bond of the oligonucleotide strand is a modified internucleotide bond, e.g., all (Sp)-AsTCs1GAs2TCs3G.

[0124] For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, edited by CAS, Handbook of Chemistry and Physics, 67th Edition, 1986-87, inside cover.

[0125] The methods and structures described herein relating to the compounds and compositions of the present invention also apply to pharmaceutically acceptable acid or base addition salts and all stereoisomers of these compounds and compositions. [Brief explanation of the drawings]

[0126] [Figure 1]This figure shows reverse-phase HPLC analysis of rat liver homogenate after incubation. The total amount of remaining oligonucleotide was measured when rat whole liver homogenate was incubated at 37°C for different days. The in vitro metabolic stability of ONT-154 was found to be similar to that of ONT-87, which has a 2'-MOE wing, but both have significantly better stability than the stereorandom 2'-MOE gapmer (ONT-41, mipomersen). The amount of remaining full-length oligomer was measured by reverse-phase HPLC, and the peak area of ​​the peak of interest was normalized to the internal standard.

[0127] [Figure 2] Degradation of various chirally pure analogs of mipomersen (ONT-41) in rat whole liver homogenate. The total amount of remaining oligonucleotide was measured when rat whole liver homogenate was incubated at 37°C for different days. Increased Sp internucleotide linkages were found to increase the in vitro metabolic stability of chirally pure diastereomers of the human ApoB sequence ONT-41 (mipomersen). The amount of remaining full-length oligomer was measured by reverse-phase HPLC, and the peak area of ​​the peak of interest was normalized to an internal standard.

[0128] [Figure 3] Degradation of various chirally pure analogs of the mouse ApoB sequence (ISIS 147764, ONT-83) in rat whole liver homogenate was measured. The total amount of remaining oligonucleotide was measured after incubation of rat whole liver homogenate at 37°C for different days. The in vitro metabolic stability of chirally pure diastereomers of the mouse ApoB sequence (ONT-83, 2'-MOE gapmer, stereorandom phosphorothioate) was found to increase with increasing Sp internucleotide linkage. The amount of remaining full-length oligomer was measured by reverse-phase HPLC, and the peak area of ​​the peak of interest was normalized to the internal standard.

[0129] [Figure 4]1 shows the degradation of the mipomersen analog ONT-75 in rat whole liver homogenate over 24 hours. This figure shows the stability of ONT-75 in rat whole liver homogenate.

[0130] [Figure 5] 1 shows the degradation of the mipomersen analog ONT-81 in rat whole liver homogenate over a 24 hour period. This figure shows the stability of ONT-81 in rat whole liver homogenate.

[0131] [Figure 6] This figure shows the duration of knockdown in ONT-87, ONT-88, and ONT-89. Stereoisomers can exhibit significantly different durations of knockdown. ONT-87 produces significantly more durable inhibition than other stereoisomers. The long duration of action of ONT-87 was observed in multiple in vivo studies. ONT-88 demonstrated a similar efficacy and recovery profile to ONT-41 (mipomersen) in certain in vivo studies. Hu ApoB transgenic mice (n=4) were administered a 10 mpk IP bolus (2x / week for 3 weeks). Mice were randomized into groups and administered 10 mg / kg intraperitoneally (IP) based on individual mouse weights measured before dosing on each dosing day (days 1, 4, 8, 11, 15, 18, and 22). Blood was collected by submandibular (cheek) bleeds on days 0, 17, 24, 31, 38, 45, and 52, followed by sacrifice by cardiac puncture and serum processing on day 52. ​​ApoB was measured by ELISA. Highlighted: sustained knockdown after 3 weeks of dosing (72% vs. 35%).

[0132] [Figure 7] FIG. 1 shows HPLC profiles measured in human serum demonstrating differences in metabolic stability of siRNA duplexes with several Rp, Sp, or stereorandom phosphorothioate linkages.

[0133] [Figure 8] Effect of stereochemistry on RNase H activity. Oligonucleotides were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1x RNase H buffer. From top to bottom (120 min): ONT-89, ONT-77, ONT-81, ONT-80, ONT-75, ONT-41, ONT-88, ONT-154, ONT-87. ONT-77 / 154 are very close to each other.

[0134] [Figure 9] Analysis of human RNase H1 cleavage of a 20-mer RNA when hybridized with different preparations of stereoisomers of phosphorothioate oligonucleotides targeting the same region of human ApoB mRNA. The specific site of cleavage is strongly influenced by the differential stereochemistry. The arrow indicates the position of cleavage (cleavage site). Products were analyzed by UPLC / MS. The length of the arrow represents the amount of product present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment (the longer the arrow, the more cleavage product was detected). (A) explains the cleavage map symbols. (B) and (C) show the cleavage maps of the oligonucleotides.

[0135] [Figure 10]Cleavage maps of different oligonucleotide compositions are shown ((A)-((C))). These three sequences target different regions in the FOXO1 mRNA. Each sequence was probed with five different chemistries. Cleavage maps were generated from the reaction mixture obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer at 37°C for 30 minutes. Arrows indicate the cleavage site. (┬) indicates that both fragments, the 5'-phosphate species and the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH species was detected by mass spectrometry. The length of the arrow represents the amount of product present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment (the longer the arrow, the more detectable cleavage product). The 5'-phosphate species peak was used for quantification only when 5'-OH and 3'-OH were not detected in the reaction mixture. The cleavage rate was determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the designated time points, the reaction was stopped with 30 mM Na2EDTA.

[0136] [Figure 11]Figures (A-B) show cleavage maps of oligonucleotide compositions with different consensus base sequences and lengths. The cleavage maps show a comparison of a stereorandom DNA composition (top panel) with three stereochemically pure, unique oligonucleotide compositions. The data compare the results of a chirality-controlled oligonucleotide composition with two stereorandom phosphorothioate oligonucleotide compositions (ONT-366 and ONT-367) targeting different regions in the FOXO1 mRNA. Each panel shows a comparison of stereorandom DNA (top panel) with three stereochemically pure, unique oligonucleotide preparations. Cleavage maps were generated from the reaction mixtures obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer for 30 minutes at 37°C. Arrows indicate the cleavage sites. (┬) indicates that both fragments, the 5'-phosphate species and the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment (the longer the arrow, the more detectable cleavage product). The 5'-phosphate species peak was used for quantification only if the 5'-OH 3'-OH species was not detected in the reaction mixture.

[0137] [Figure 12]Effect of stereochemistry on RNase H activity. In two independent experiments, antisense oligonucleotides targeting the same region of FOXO1 mRNA were hybridized to RNA and then incubated with RNase H in the presence of 1x RNase H buffer at 37°C. The disappearance of full-length RNA was measured by RP-HPLC from its peak area at 254 nm. (A) shows the remaining RNA substrate (from top to bottom (60 min): ONT-355, ONT-316, ONT-367, ONT-392, ONT-393, and ONT-394 (at 60 min, ONT-393 and ONT-394 were similar; at 5 min, ONT-393 had a higher remaining RNA substrate (%)). (B) shows the remaining RNA substrate (%) (from top to bottom (60 min): ONT-315, ONT-354, ONT-366, ONT-391, ONT-389, and ONT-390). Cleavage rates were determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the indicated time points, the reaction was stopped with 30 mM Na2EDTA.

[0138] [Figure 13] Figure 1 shows the turnover of antisense oligonucleotides. Duplexes were formed with a concentration of each DNA strand equal to 6 μM and 100 μM of RNA. These duplexes were incubated with 0.02 μM RNase H enzyme, and the disappearance of full-length RNA was measured by RP-HPLC from its peak area at 254 nm. The cleavage rate was determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the specified time points, the reaction was stopped with 30 mM Na2EDTA. From top to bottom (40 min): ONT-316, ONT-367, and ONT-392.

[0139] [Figure 14] FIG. 1 shows a cleavage map comparing a stereorandom phosphorothioate oligonucleotide with six unique stereochemically pure oligonucleotide preparations targeting the same FOXO1 mRNA region.

[0140] [Figure 15] Effect of stereochemistry on RNase H activity. Antisense oligonucleotides were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1x RNase H buffer. A stereochemical dependence on RNase H activity was observed. Similarly, a comparison of ONT-367 (stereorandom DNA) and ONT-316 (5-10-5 2'-MOE gapmer) reveals a strong dependence of compositional chemistry on RNase H activity. From top to bottom (40 min): ONT-316, ONT-421, ONT-367, ONT-392, ONT-394, ONT-415, and ONT-422 (at 40 min, ONT-394 / 415 / 422 are at equivalent levels; at 5 min, the percentage of RNA remaining in the DNA / RNA duplex is ONT-422>ONT-394>ONT-415).

[0141] [Figure 16] Effect of stereochemistry on RNase H activity. Antisense oligonucleotides targeting the same region of FOXO1 mRNA were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1x RNase H buffer. A stereochemical dependence on RNase H activity was observed. From top to bottom (40 minutes): ONT-396, ONT-409, ONT-414, ONT-408 (at 40 minutes, ONT-396 / 409 / 414 / 408 are at the same level), ONT-404, ONT-410, ONT-402 (at 40 minutes, ONT-404 / 410 / 408 are at the same level), ONT-403, ONT-407, ONT-405, ONT-401, ONT-406 and ONT-400 (at 40 minutes, ONT-401 / 405 / 406 / 400 are at the same level).

[0142] [Figure 17]Effect of stereochemistry on RNase H activity. Antisense oligonucleotides targeting the same region of FOXO1 mRNA were hybridized to RNA and then incubated with RNase H at 37°C in the presence of 1× RNase H buffer. A stereochemical dependence on RNase H activity was observed. ONT-406 was observed to induce cleavage of double-stranded RNA at a rate slightly greater than that of the phosphodiester oligonucleotide ONT-415. From top to bottom (40 min): ONT-396, ONT-421, ONT-392, ONT-394, ONT-415 ONT-406, and ONT-422 (at 40 min, ONT-394 / 415 / 406 are at equivalent levels; at 5 min, the percentage of RNA remaining in the DNA / RNA duplex is ONT-394>ONT-415>ONT-406).

[0143] [Figure 18] Figure 1 shows exemplary UV chromatograms of RNA cleavage products obtained when RNA (ONT-388) was duplexed with stereorandom DNA (ONT-367) (top) and when stereopure DNA was duplexed with repeating triplet motif-3'-SSR-5' (ONT-394) (bottom). 2.35 min: 7mer; 3.16 min: 8mer and P-6mer; 4.48 min: P-7mer; 5.83 min: P-8mer; 6.88 min: 12mer; 9.32 min: 13mer; 10.13 min: P-11mer; 11.0 min: P-12mer and 14mer; 11.93 min: P-13mer; 13.13 min: P-14mer. Peak assignments for ONT-394 (bottom): 4.55 min: p-7mer; 4.97 min: 10mer; 9.53 min: 13mer.

[0144] [Figure 19]Electrospray ionization spectra of RNA cleavage products: RNA fragments obtained from the duplexes ONT-387, RNA / ONT-354, (7-6-7, DNA-2'-OMe-DNA) (top) and ONT-387, RNA / ONT-315, (5-10-5,2'-MOE gapmer) (bottom) when the duplexes were incubated with RNase H in the presence of 1x RNse H buffer for 30 min.

[0145] [Figure 20] Figure 1 shows UV chromatograms and TICs of ONT-406 and ONT-388 duplexes after 30 min incubation with RNase H.

[0146] [Figure 21] 1 shows exemplary proposed cleavages. The provided chiral controlled oligonucleotide compositions are capable of cleaving targets as shown.

[0147] [Figure 22]Figure 1 shows exemplary allele-specific cleavage targeting mutant huntingtin mRNA. (A) and (B): Exemplary oligonucleotides. (C)-(E): Cleavage maps. (F)-(H): RNA cleavage. Stereorandom and chiral-controlled oligonucleotide compositions were prepared to target single nucleotide polymorphisms for allele-selective suppression of mutant huntingtin. ONT-450 (stereorandom), targeting ONT-453 (muHTT) and ONT-454 (wtHTT), showed minor differences in RNA cleavage and their cleavage maps. Chiral-controlled ONT-451, targeting ONT-453 (muHTT) and ONT-454 (wtHTT) and possessing a selective placement of a 3'-SSR-5' motif within the RNase H recognition site, showed significant differences in RNA cleavage rates. It is noteworthy that in the cleavage maps, the 3'-SSR-5' motif is positioned to direct cleavage between positions 8 and 9, after the mismatch, when read from the 5' end of the RNA. ONT-452, which targets ONT-453 (muHTT) and ONT-454 (wtHTT) and has an alternative placement of the 3'-SSR-5' motif within the RNase H recognition site, showed moderate differences in RNA cleavage rates. The 3'-SSR-5' motif was positioned to direct cleavage at positions 7 and 8, before the mismatch, when read from the 5' end of the RNA. The exemplary data demonstrate that the position of the 3'-SSR-5' motif is important for achieving high discrimination for allele-specific cleavage. All cleavage maps were generated from reaction mixtures obtained after incubating each duplex with RNase H1C for 5 minutes at 37°C in the presence of 1x PBS buffer. Arrows indicate the cleavage sites. (┬) indicates that both fragments, the 5'-phosphate species as well as the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined from the ratio of the UV peak area to the theoretical extinction coefficient of that fragment.The 5'-phosphate species peak was used for quantification only if 5'-OH 3'-OH was not detected in the reaction mixture.

[0148] [Figure 23] (A)-(C) show exemplary allele-specific cleavage targeting FOXO1 mRNA.

[0149] [Figure 24] Figure 1 shows in vitro dose response silencing of ApoB mRNA after treatment with ApoB oligonucleotides. Stereochemically pure diastereomers with and without the 2'-MOE wing show similar efficacy to ONT-41 (mipomersen).

[0150] [Figure 25]Figure 1 shows a comparison of RNase H cleavage maps (A) and RNA cleavage rates (B) for stereorandom (ONT-367) and chiral-controlled oligonucleotide compositions (ONT-421 (all Sp) and ONT-455 (all Rp)) and DNA (ONT-415). These sequences target the same region in the FOXO1 mRNA. Cleavage maps were generated from the reaction mixture obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer at 37°C for 5 minutes. Arrows indicate the cleavage site. (┬) indicates that both fragments, the 5'-phosphate species and the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only when 5'-OH and 3'-OH were not detected in the reaction mixture. The cleavage rate was determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. At the designated time points, the reaction was stopped with 30 mM Na2EDTA.

[0151] [Figure 26]This figure shows a comparison of cleavage maps of sequences containing one Rp with a change in position starting from the 3'-end of the DNA. These sequences target the same region in the FOXO1 mRNA. The cleavage maps were generated from the reaction mixture obtained after incubating each duplex with RNase H1C for 5 minutes at 37°C in the presence of 1x PBS buffer. Arrows indicate the cleavage site. (┬) indicates that both fragments, the 5'-phosphate species and the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only if the 5'-OH 3'-OH species was not detected in the reaction mixture.

[0152] [Figure 27](A) Comparison of RNase H cleavage rates of stereopure oligonucleotides (ONT-406), (ONT-401), (ONT-404), and (ONT-408). All four sequences are stereopure phosphorothioates with one Rp linkage. These sequences target the same region in the FOXO1 mRNA. All duplexes were incubated with RNase H1C in the presence of 1x PBS buffer at 37°C. At designated time points, the reaction was stopped with 30 mM Na2EDTA. Cleavage rates were determined by measuring the amount of full-length RNA remaining in the reaction mixture by reverse-phase HPLC. ONT-406 and ONT-401 were found to have superior cleavage rates. (B) Correlation between % RNA cleaved in the RNase H assay (10 μM oligonucleotide) and % mRNA knockdown in the in vitro assay (20 nM oligonucleotide). All sequences target the same region of the FOXO1 mRNA. The amount of remaining RNA was determined by the UV peak area of ​​the RNA normalized to DNA in the same reaction mixture. All cleavage maps described above were generated from the reaction mixture obtained after incubating each duplex with RNase H1C for 5 minutes at 37°C in the presence of 1x PBS buffer. All sequences from ONT-396 to ONT-414 contain a single Rp phosphorothioate, and these sequences differ in the position of the Rp. ONT-421 (all Sp) phosphorothioates were inactive in in vitro assays. This is related to the slow rate of RNA cleavage in RNase H assays when ONT-421 is duplexed with complementary RNA.

[0153] [Figure 28]Figure 1 shows a 2-day serum stability assay in rat serum of single Rp walk PS DNA (ONT-396-ONT-414), stereorandom PS DNA (ONT-367), all Sp PS DNA (ONT-421), and all Rp PS DNA (ONT-455). Note that ONT-396 and ONT-455 degraded at the time tested.

[0154] [Figure 29] Figures 1A and 1B show exemplary oligonucleotides containing hemimers. (A): Cleavage map. (B): RNA cleavage assay. (C): FOXO1 mRNA knockdown. In some embodiments, the introduction of 2'-modifications at the 5'-end of the sequence increases the stability of binding to target RNA while maintaining RNase H activity. ONT-367 (stereorandom phosphorothioate DNA) and ONT-440 (5-15, 2'-F-DNA) have similar cleavage maps and similar RNA cleavage rates in RNase H assays (10 μM oligonucleotide). In some embodiments, the ONT-440 (5-11, 2'-F-DNA) sequence may have better cell permeability. In some embodiments, asymmetric 2'-modifications provide a Tm advantage while maintaining RNase H activity. The introduction of RSS motifs in hemimers may further improve the efficiency of RNase H. Cleavage maps were generated from the reaction mixture obtained after incubating each duplex with RNase H1C in the presence of 1x PBS buffer at 37°C for 5 minutes. Arrows indicate the cleavage site. (┬) indicates that both fragments, the 5'-phosphate species and the 5'-OH 3'-OH species, were identified in the reaction mixture. (┌) indicates that only the 5'-phosphate species was detected, and (┐) indicates that the 5'-OH 3'-OH component was detected by mass spectrometry. The length of the arrow represents the amount of metabolite present in the reaction mixture, which was determined by the ratio of the UV peak area to the theoretical extinction coefficient of that fragment. The 5'-phosphate species peak was used for quantification only if the 5'-OH 3'-OH species was not detected in the reaction mixture.

[0155] [Figure 30] Figure 1 shows exemplary mass spectrometry data for a cleavage assay. Top: Data for ONT-367: 2.35 min: 7-mer; 3.16 min: 8-mer and P-6-mer; 4.58 min: P-7-mer; 5.91 min: P-8-mer; 7.19 min: 12-mer; 9.55 min: 13-mer; 10.13 min: P-11-mer; 11.14 min: P-12-mer and 14-mer; 12.11 min: P-13-mer; 13.29 min: P-14-mer; 14.80 min: full-length RNA (ONT-388) and 18.33 min: stereorandom DNA (ONT-367). Bottom: Data for ONT-406: 4.72 min: p-rArUrGrGrCrUrA, 5'-phosphorylated 7-mer RNA; 9.46 min: 5'-rGrUrGrArGrCrArGrCrUrGrCrA, 5'-OH 3'-OH 13-mer RNA; 16.45 min: full-length RNA (ONT-388); 19.48 min and 19.49 min: stereopure DNA (ONT-406). DETAILED DESCRIPTION OF THE INVENTION

[0156] Synthetic oligonucleotides provide useful molecular tools for a wide variety of applications. For example, oligonucleotides are useful in therapeutics, diagnostics, research, and novel nanomaterial applications. The use of natural nucleic acids (e.g., unmodified DNA or RNA) is limited by their susceptibility to endo- and exonucleases. Therefore, to circumvent these shortcomings, various synthetic equivalents have been developed. These include synthetic oligonucleotides containing backbone modifications that make these molecules less susceptible to degradation. From a structural perspective, such modifications to internucleotide phosphate linkages introduce chirality. It has been shown that certain properties of oligonucleotides can be influenced by the arrangement of phosphorus atoms forming the oligonucleotide backbone. For example, in vitro studies have shown that properties of antisense nucleotides, such as binding affinity, specific binding sequences with complementary RNA, and stability against nucleases, are particularly influenced by backbone chirality (e.g., the arrangement of phosphorus atoms).

[0157] Among other things, the present invention encompasses the recognition that a stereorandom oligonucleotide preparation contains multiple unique chemical entities that differ from one another in the stereochemical configuration of individual backbone chiral centers within the oligonucleotide chain. Furthermore, the present invention encompasses the insight that a stereorandom oligonucleotide preparation is generally unlikely to contain every possible stereoisomer of a related oligonucleotide. Thus, among other things, the present invention provides novel chemical entities that are specific stereoisomers of an oligonucleotide of interest. That is, the present invention provides substantially pure preparations of single oligonucleotide compounds, where a particular oligonucleotide compound may be defined by its base sequence, its length, its backbone bond pattern, and its backbone chiral center pattern.

[0158] The present invention demonstrates, inter alia, that individual stereoisomers of a particular oligonucleotide can exhibit different stability and / or activity from one another. Furthermore, the present disclosure demonstrates that the improved stability achieved by the inclusion and / or location of specific chiral structures within an oligonucleotide can be comparable to or even better than the improved stability achieved by the use of modified backbone linkages, bases, and / or sugars (e.g., by the use of specific types of modified phosphate esters, 2'-modifications, base modifications, etc.). In some embodiments, the present disclosure also demonstrates that the improved activity achieved by the inclusion and / or location of specific chiral structures within an oligonucleotide can be comparable to or even better than the improved activity achieved by the use of modified backbone linkages, bases, and / or sugars (e.g., by the use of specific types of modified phosphate esters, 2'-modifications, base modifications, etc.). In some embodiments, the inclusion and / or location of specific chiral linkages within an oligonucleotide can surprisingly alter the cleavage pattern of a nucleic acid polymer when such an oligonucleotide is utilized to cleave the nucleic acid polymer. For example, in some embodiments, the pattern of backbone chiral centers results in unexpectedly high cleavage efficiency of a target nucleic acid polymer. In some embodiments, the pattern of backbone chiral centers results in new cleavage sites. In some embodiments, the pattern of backbone chiral centers results in fewer cleavage sites, for example, by blocking certain existing cleavage sites. Even more unexpectedly, in some embodiments, the pattern of backbone chiral centers results in cleavage at only one site in the target nucleic acid polymer within the sequence complementary to the oligonucleotide utilized for cleavage. In some embodiments, even higher cleavage efficiency is achieved by selecting a pattern of backbone chiral centers that minimizes the number of cleavage sites.

[0159] In some embodiments, the present invention provides 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides chirally controlled (and / or stereochemically pure) oligonucleotide compositions comprising oligonucleotides defined by having: wherein the compositions are substantially pure preparations of single oligonucleotides, in which at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. The pattern of backbone chiral centers of an oligonucleotide can be specified by the combination of 5' to 3' bond phosphorus stereochemistry (Rp / Sp). For example, as illustrated below, ONT-154 has a pattern of 5S-(SSR)3-5S, and ONT-80 has a pattern of S 19 It has.

[0160] In some embodiments, the invention provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein the compositions are enriched for oligonucleotides of a single oligonucleotide type, relative to substantially racemic preparations of the same oligonucleotide. 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides chiral controlled oligonucleotide compositions of oligonucleotides in which the compositions are enriched for oligonucleotides of a single oligonucleotide type that share a common nucleotide sequence. In some embodiments, in a substantially racemic (or non-chiral controlled) preparation of an oligonucleotide, all or most of the coupling steps are not chiral controlled, in that the coupling step is not specifically performed to enhance stereoselectivity. An exemplary substantially racemic preparation of an oligonucleotide is a phosphorothioate oligonucleotide prepared by a process known in the art, in which a phosphite triester is sulfurized with either tetraethylthiuram disulfide (TETD) or 3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD). In some embodiments, a substantially racemic preparation of an oligonucleotide results in a substantially racemic oligonucleotide composition (or a non-chiral controlled oligonucleotide composition).

[0161] In some embodiments, the present invention provides 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides a chirality-controlled oligonucleotide composition comprising an oligonucleotide of a particular oligonucleotide type, characterized by: wherein the composition is chirality-controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length. In some embodiments, the chirality-controlled oligonucleotide composition is a substantially pure preparation of an oligonucleotide type, wherein oligonucleotides in the composition that are not of the oligonucleotide type are impurities from the preparation process of said oligonucleotide type (optionally after specific purification procedures).

[0162] In some embodiments, at least about 20% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 25% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 30% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 35% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 40% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 45% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 50% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 55% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 60% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 65% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 70% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, at least about 75% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 80% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 85% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 90% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 92% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 94% of the oligonucleotides in the composition share a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers. In some embodiments, at least about 95% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, more than about 99% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.In some embodiments, the purity of the chiral-controlled oligonucleotide composition of oligonucleotides can be expressed as the percentage of oligonucleotides in the composition that have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0163] In some embodiments, the purity of a chiral controlled oligonucleotide composition of an oligonucleotide type is expressed as the percentage of oligonucleotides in the composition that are of that oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type, hi some embodiments, at least about 94% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type.In some embodiments, at least about 95% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in a chiral controlled oligonucleotide composition are of the same oligonucleotide type.

[0164] In some embodiments, the purity of a chiral controlled oligonucleotide composition can be controlled by the stereoselectivity of each coupling step in its preparation process. In some embodiments, the coupling step has 60% stereoselectivity (e.g., diastereoselectivity) (60% of the new internucleotide linkages formed by the coupling step have the desired stereochemistry). After such a coupling step, the new internucleotide linkages formed may be said to have 60% purity. In some embodiments, each coupling step has at least 60% stereoselectivity. In some embodiments, each coupling step has at least 70% stereoselectivity. In some embodiments, each coupling step has at least 80% stereoselectivity. In some embodiments, each coupling step has at least 85% stereoselectivity. In some embodiments, each coupling step has at least 90% stereoselectivity. In some embodiments, each coupling step has at least 91% stereoselectivity. In some embodiments, each coupling step has at least 92% stereoselectivity. In some embodiments, each coupling step has at least 93% stereoselectivity. In some embodiments, each coupling step has at least 94% stereoselectivity. In some embodiments, each coupling step has at least 95% stereoselectivity. In some embodiments, each coupling step has at least 96% stereoselectivity. In some embodiments, each coupling step has at least 97% stereoselectivity. In some embodiments, each coupling step has at least 98% stereoselectivity. In some embodiments, each coupling step has at least 99% stereoselectivity. In some embodiments, each coupling step has substantially 100% stereoselectivity. In some embodiments, the coupling step has substantially 100% stereoselectivity, wherein all products of the coupling step, detectable by analytical methods (e.g., NMR, HPLC, etc.), have the desired stereoselectivity.

[0165] In some embodiments, the chirality-controlled (and / or stereochemically pure) preparations provided are antisense oligonucleotides (e.g., chiromersen). In some embodiments, the chirality-controlled (and / or stereochemically pure) preparations provided are siRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions provided are of oligonucleotides that may be antisense oligonucleotides, antagomirs, microRNAs, pre-microRNs, anti-mirs, supermirs, ribozymes, Ul adapters, RNA activators, RNAi agents, decoy oligonucleotides, triplex-forming oligonucleotides, aptamers, or adjuvants. In some embodiments, the chirality-controlled oligonucleotide compositions are of antisense oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of antagomir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of microRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of pre-microRNA oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of anti-mir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide compositions are of supermir oligonucleotides. In some embodiments, the chirality-controlled oligonucleotide composition is a ribozyme oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is a Ul adaptor oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an RNA activator oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an RNAi agent oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is a decoy oligonucleotide.In some embodiments, the chirality-controlled oligonucleotide composition is a triplex-forming oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an aptamer oligonucleotide. In some embodiments, the chirality-controlled oligonucleotide composition is an adjuvant oligonucleotide.

[0166] In some embodiments, chirally controlled (and / or stereochemically pure) preparations provided are of oligonucleotides containing one or more modified backbone linkages, bases, and / or sugars.

[0167] In some embodiments, provided oligonucleotides contain one or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain two or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain three or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain four or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain five or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain five or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain six or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 7 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 8 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 9 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 10 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 11 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 12 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 13 or more chiral modified phosphate linkages. In some embodiments, provided oligonucleotide types contain 14 or more chiral modified phosphate linkages.In some embodiments, the oligonucleotide types provided contain 15 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 16 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 17 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 18 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 19 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 20 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 21 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 22 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 23 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 24 or more chiral modified phosphate linkages. In some embodiments, the oligonucleotide types provided contain 25 or more chiral modified phosphate linkages.

[0168] In some embodiments, provided oligonucleotides contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral modified phosphate linkages. Exemplary such chiral modified phosphate linkages are described above and herein. In some embodiments, provided oligonucleotides contain at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral modified phosphate linkages in the Sp configuration.

[0169] In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 80% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 85% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 90% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 91% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 92% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 93% stereochemical purity. In some embodiments, provided chiral controlled (and / or stereochemically pure) preparations are of greater than about 94% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 95% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 96% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 97% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 98% stereochemical purity. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of greater than about 99% stereochemical purity.

[0170] In some embodiments, the chiral modified phosphate linkage is a chiral phosphorothioate linkage, i.e., a phosphorothioate internucleotide linkage. In some embodiments, the provided oligonucleotide contains at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% chiral phosphorothioate internucleotide linkages. In some embodiments, all chiral modified phosphate linkages are chiral phosphorothioate internucleotide linkages. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages of the provided oligonucleotide are in the Sp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Sp configuration.In some embodiments, at least about 90% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Sp configuration. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are in the Rp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 90% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate internucleotide linkages in the provided oligonucleotides are of the Rp configuration.In some embodiments, less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 10% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 20% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 30% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 40% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 50% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 60% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 70% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 80% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 90% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 95% of the chiral phosphorothioate internucleotide linkages in a provided oligonucleotide are of the Rp configuration. In some embodiments, a provided oligonucleotide has only one Rp chiral phosphorothioate internucleotide linkage. In some embodiments, a provided oligonucleotide has only one Rp chiral phosphorothioate internucleotide linkage, where all internucleotide linkages are chiral phosphorothioate internucleotide linkages.In some embodiments, the chiral phosphorothioate internucleotide linkages are chiral phosphorothioate diester linkages. In some embodiments, the chiral phosphorothioate internucleotide linkages are each independently a chiral phosphorothioate diester linkage. In some embodiments, the internucleotide linkages are each independently a chiral phosphorothioate diester linkage. In some embodiments, the internucleotide linkages are each independently a chiral phosphorothioate diester linkage and only one linkage is Rp.

[0171] In some embodiments, the chiral (and / or stereochemically pure) preparations provided are of oligonucleotides containing one or more modified bases. In some embodiments, the chiral (and / or stereochemically pure) preparations provided are of oligonucleotides that do not contain modified bases. Exemplary such modified bases are described above and herein.

[0172] In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 8 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 9 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 10 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 11 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 12 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 13 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 14 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 15 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 16 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 17 bases. In some embodiments, chirally controlled (and / or stereochemically pure) preparations are provided of oligonucleotides having a common base sequence of at least 18 bases.In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 19 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 20 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 21 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 22 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 23 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 24 bases. In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided for oligonucleotides having a common base sequence of at least 25 bases. In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are of oligonucleotides having a common base sequence of at least 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 bases.

[0173] In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides containing one or more residues modified at the sugar moiety. In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides containing one or more residues modified at the 2'-position of the sugar moiety (referred to herein as "2'-modified"). Exemplary such modifications are described above and herein and include, but are not limited to, 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, etc. In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides containing one or more residues that are 2'-modified. For example, in some embodiments, provided oligonucleotides contain one or more residues that are 2'-O-methoxyethyl (2'-MOE)-modified residues. In some embodiments, provided chirally controlled (and / or stereochemically pure) preparations include oligonucleotides that do not contain any 2'-modifications. In some embodiments, the chirally controlled (and / or stereochemically pure) preparations provided are oligonucleotides that do not contain any 2'-MOE residues, i.e., in some embodiments, the oligonucleotides provided are not MOE-modified.

[0174] In some embodiments, provided chirally controlled (and / or stereochemically pure) oligonucleotides are of the general motif of wing-core-wing (also generally represented herein as XYX). ​​In some embodiments, each wing contains one or more residues with a particular modification that is absent from the core "Y" portion. In some embodiments, each wing contains one or more residues with a 2' modification that is absent from the core portion. For example, in some embodiments, provided chirally controlled (and / or stereochemically pure) oligonucleotides have a wing-core-wing motif represented as XYX, where the residue in each "X" portion is a 2'-modified residue of a particular type, and the residue in the core "Y" portion is not a 2'-modified residue of the same particular type. For example, in some embodiments, provided chirally controlled (and / or stereochemically pure) oligonucleotides have a wing-core-wing motif represented as XYX, where the residue in each "X" portion is a 2'-MOE-modified residue, and the residue in the core "Y" portion is not a 2'-MOE-modified residue. In some embodiments, provided chirally controlled (and / or stereochemically pure) oligonucleotides have a wing-core-wing motif designated XYX, where the residues of each "X" portion are 2'-MOE-modified residues and the residues of the core "Y" portion are 2'-deoxyribonucleotides. Those skilled in the art will understand that in the context of such an XYX motif, all such 2'-modifications described above and herein are contemplated.

[0175] In some embodiments, each wing region independently has a length of 1 base or more. In some embodiments, each wing region independently has a length of 2 bases or more. In some embodiments, each wing region independently has a length of 3 bases or more. In some embodiments, each wing region independently has a length of 4 bases or more. In some embodiments, each wing region independently has a length of 5 bases or more. In some embodiments, each wing region independently has a length of 6 bases or more. In some embodiments, each wing region independently has a length of 7 bases or more. In some embodiments, each wing region independently has a length of 8 bases or more. In some embodiments, each wing region independently has a length of 9 bases or more. In some embodiments, each wing region independently has a length of 10 bases or more. In certain embodiments, each wing region has a length of 1 base. In certain embodiments, each wing region has a length of 2 bases. In certain embodiments, each wing region has a length of 3 bases. In certain embodiments, each wing region has a length of 4 bases. In certain embodiments, each wing region has a length of 5 bases.

[0176] In some embodiments, the core region is 1 base or more in length. In some embodiments, the core region is 2 bases or more in length. In some embodiments, the core region is 3 bases or more in length. In some embodiments, the core region is 4 bases or more in length. In some embodiments, the core region is 5 bases or more in length. In some embodiments, the core region is 6 bases or more in length. In some embodiments, the core region is 7 bases or more in length. In some embodiments, the core region is 8 bases or more in length. In some embodiments, the core region is 9 bases or more in length. In some embodiments, the core region is 10 bases or more in length. In some embodiments, the core region is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 bases or more in length. In certain embodiments, the core region is 10 bases in length. In certain embodiments, the core region is 3 bases in length. In certain embodiments, the core region is 4 bases in length. In certain embodiments, the core region is 5 bases in length. In certain embodiments, the core region is 6 bases in length. In certain embodiments, the core region has a length of 7 bases. In certain embodiments, the core region has a length of 8 bases. In certain embodiments, the core region has a length of 9 bases. In certain embodiments, the core region has a length of 10 bases. In certain embodiments, the core region has a length of 11 bases. In certain embodiments, the core region has a length of 12 bases. In certain embodiments, the core region has a length of 13 bases. In certain embodiments, the core region has a length of 14 bases. In certain embodiments, the core region has a length of 15 bases. In certain embodiments, the core region has a length of 16 bases. In certain embodiments, the core region has a length of 17 bases. In certain embodiments, the core region has a length of 18 bases. In certain embodiments, the core region has a length of 19 bases. In certain embodiments, the core region has a length of 11 bases or more. In certain embodiments, the core region has a length of 12 bases or more. In certain embodiments, the core region has a length of 13 bases or more.In certain embodiments, the core region has a length of 14 bases or more. In certain embodiments, the core region has a length of 15 bases or more. In certain embodiments, the core region has a length of 16 bases or more. In certain embodiments, the core region has a length of 17 bases or more. In certain embodiments, the core region has a length of 18 bases or more. In certain embodiments, the core region has a length of 19 bases or more. In certain embodiments, the core region has a length of 20 bases or more. In certain embodiments, the core region has a length of 20 bases or more.

[0177] In some embodiments, the wing-core-wing (i.e., XYX) motif of the provided oligonucleotides is represented numerically, e.g., 5-10-5, meaning that each wing region of the oligonucleotide is 5 bases long and the core region of the oligonucleotide is 10 bases long. In some embodiments, the wing-core-wing motif is, for example, 2-16-2, 3-14-3, 4-12-4, 5-10-5, etc. In certain embodiments, the wing-core-wing motif is 5-10-5.

[0178] In some embodiments, the internucleoside linkages of provided oligonucleotides having such a wing-core-wing (i.e., XYX) motif are all chiral modified phosphate linkages. In some embodiments, the internucleoside linkages of provided oligonucleotides having such a wing-core-wing (i.e., XYX) motif are all chiral phosphorothioate internucleotide linkages. In some embodiments, the chiral internucleotide linkages of provided oligonucleotides having such a wing-core-wing motif are at least about 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, or 90% chiral modified phosphate internucleotide linkages. In some embodiments, the chiral internucleotide linkages of provided oligonucleotides having such a wing-core-wing motif are at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% chiral phosphorothioate internucleotide linkages. In some embodiments, the chiral internucleotide linkages of provided oligonucleotides of such wing-core-wing motifs are at least about 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, or 90% chiral phosphorothioate internucleotide linkages in the Sp configuration.

[0179] In some embodiments, each wing region of a wing-core-wing motif may comprise a chiral modified phosphate internucleotide linkage. In some embodiments, each wing region of a wing-core-wing motif may comprise a chiral phosphorothioate internucleotide linkage. In some embodiments, each wing region of a wing-core-wing motif comprises a chiral phosphorothioate internucleotide linkage. In some embodiments, the two wing regions of a wing-core-wing motif have the same internucleotide bond stereochemistry. In some embodiments, the two wing regions have different internucleotide bond stereochemistry. In some embodiments, each internucleotide linkage within a wing is independently a chiral internucleotide linkage.

[0180] In some embodiments, the core region of the wing-core-wing motif may comprise chiral modified phosphate internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif may comprise chiral phosphorothioate internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises a repeating pattern of internucleotide linkage stereochemistry. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide linkage stereochemistry. In some embodiments, the core region of the wing-core-wing motif comprises a repeating pattern of internucleotide linkage stereochemistry, where the repeating pattern is (Sp)mRp or Rp(Sp)m, where m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide linkage stereochemistry, where the repeating pattern is (Sp)mRp or Rp(Sp)m, where m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide linkage stereochemistry, where the repeating pattern is (Sp)mRp, where m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide linkage stereochemistry, where the repeating pattern is Rp(Sp)m, where m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide linkage stereochemistry, where the repeating pattern is (Sp)mRp or Rp(Sp)m, where m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide linkage stereochemistry, where the repeating pattern is a motif that includes at least 33% of the internucleotide linkages in the S configuration.In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide bond stereochemistry, wherein the repeating pattern is a motif with at least 50% of the internucleotide bond in an S configuration. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide bond stereochemistry, wherein the repeating pattern is a motif with at least 66% of the internucleotide bond in an S configuration. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide bond stereochemistry, wherein the repeating pattern is a repeating triplet motif selected from RpRpSp and SpSpRp. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide bond stereochemistry, wherein the repeating pattern is a repeating RpRpSp. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of internucleotide bond stereochemistry, wherein the repeating pattern is a repeating SpSpRp.

[0181] In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises (Sp)mRp or Rp(Sp)m. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises Rp(Sp)m. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises (Sp)mRp. In some embodiments, m is 2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises Rp(Sp)2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises (Sp)2Rp(Sp)2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises (Rp)2Rp(Sp)2. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises RpSpRp(Sp)2. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises SpRpRp(Sp)2. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of an oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises (Sp)2Rp.

[0182] In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising (Sp)mRp or Rp(Sp)m. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising Rp(Sp)m. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising (Sp)mRp. In some embodiments, m is 2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising Rp(Sp)2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising (Sp)2Rp(Sp)2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising (Rp)2Rp(Sp)2. In some embodiments, the present invention provides chirality-controlled oligonucleotide compositions of an oligonucleotide type having a pattern of backbone chiral centers comprising RpSpRp(Sp)2. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises SpRpRp(Sp)2. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises (Sp)2Rp.

[0183] As defined herein, m is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, m is 3, 4, 5, 6, 7, or 8. In some embodiments, m is 4, 5, 6, 7, or 8. In some embodiments, m is 5, 6, 7, or 8. In some embodiments, m is 6, 7, or 8. In some embodiments, m is 7 or 8. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8.

[0184] In some embodiments, the repeat pattern is (Sp)m(Rp)n, where n is independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is independently as defined above and described herein. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises (Sp)m(Rp)n. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers in the core region comprises (Sp)m(Rp)n. In some embodiments, the repeat pattern is (Rp)n(Sp)m, where n is independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is independently as defined above and described herein. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises (Rp)n(Sp)m. In some embodiments, (Rp)n(Sp)m is (Rp)(Sp)2. In some embodiments, (Sp)n(Rp)m is (Sp)2(Rp).

[0185] In some embodiments, the repeat pattern is (Sp)m(Rp)n(Sp)t, where n and t are each independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is as defined above and described herein. In some embodiments, the invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises (Sp)m(Rp)n(Sp)t. In some embodiments, the invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers in the core region comprises (Sp)m(Rp)n(Sp)t. In some embodiments, the repeat pattern is (Sp)m(Rp)n(Sp)t, where n and t are each independently 1, 2, 3, 4, 5, 6, 7, or 8, and m is as defined above and described herein. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type in which the pattern of backbone chiral centers comprises (Sp)t(Rp)n(Sp)m. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type in which the pattern of backbone chiral centers in the core region comprises (Sp)t(Rp)n(Sp)m.

[0186] In some embodiments, the repeat pattern is (Np)t(Rp)n(Sp)m, where n and t are each independently 1, 2, 3, 4, 5, 6, 7, or 8, Np is independently Rp or Sp, and m is as defined above and described herein. In some embodiments, the invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises (Np)t(Rp)n(Sp)m. In some embodiments, the invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers in the core region comprises (Np)t(Rp)n(Sp)m. In some embodiments, the repeat pattern is (Np)m(Rp)n(Sp)t, where n and t are each independently 1, 2, 3, 4, 5, 6, 7, or 8, Np is independently Rp or Sp, and m is as defined above and described herein. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers comprises (Np)m(Rp)n(Sp)t. In some embodiments, the present invention provides chiral controlled oligonucleotide compositions of the oligonucleotide type, in which the pattern of backbone chiral centers in the core region comprises (Np)m(Rp)n(Sp)t. In some embodiments, Np is Rp. In some embodiments, Np is Sp. In some embodiments, all Np are the same. In some embodiments, all Np are Sp. In some embodiments, at least one Np is different from the other Np. In some embodiments, t is 2.

[0187] As defined herein, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 3, 4, 5, 6, 7, or 8. In some embodiments, n is 4, 5, 6, 7, or 8. In some embodiments, n is 5, 6, 7, or 8. In some embodiments, n is 6, 7, or 8. In some embodiments, n is 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8.

[0188] As defined herein, t is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, t is 2, 3, 4, 5, 6, 7, or 8. In some embodiments, t is 3, 4, 5, 6, 7, or 8. In some embodiments, t is 4, 5, 6, 7, or 8. In some embodiments, t is 5, 6, 7, or 8. In some embodiments, t is 6, 7, or 8. In some embodiments, t is 7 or 8. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8.

[0189] In some embodiments, at least one of m and t is greater than 2. In some embodiments, at least one of m and t is greater than 3. In some embodiments, at least one of m and t is greater than 4. In some embodiments, at least one of m and t is greater than 5. In some embodiments, at least one of m and t is greater than 6. In some embodiments, at least one of m and t is greater than 7.

[0190] In some embodiments, n is 1 and at least one of m and t is greater than 1. In some embodiments, n is 1 and m and t are each independently greater than 1. In some embodiments, m>n and t>n. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)2Rp(Sp)2. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)2Rp(Sp)2. In some embodiments, (Sp)t(Rp)n(Sp)m is SpRp(Sp)2. In some embodiments, (Np)t(Rp)n(Sp)m is (Np)tRp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is (Np)2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is (Rp)2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is (Rp)2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is (Sp)2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is RpSpRp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is SpRpRp(Sp)m.

[0191] In some embodiments, (Sp)t(Rp)n(Sp)m is SpRpSpSp. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)2Rp(Sp)2. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)3Rp(Sp)3. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)4Rp(Sp)4. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)tRp(Sp)5. In some embodiments, (Sp)t(Rp)n(Sp)m is SpRp(Sp)5. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)2Rp(Sp)5. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)3Rp(Sp)5. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)4Rp(Sp)5. In some embodiments, (Sp)t(Rp)n(Sp)m is (Sp)5Rp(Sp)5.

[0192] In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)2Rp(Sp)2. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)3Rp(Sp)3. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)4Rp(Sp)4. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)mRp(Sp)5. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)2Rp(Sp)5. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)3Rp(Sp)5. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)4Rp(Sp)5. In some embodiments, (Sp)m(Rp)n(Sp)t is (Sp)5Rp(Sp)5.

[0193] In some embodiments, the core region of the wing-core-wing motif comprises at least one Rp internucleotide linkage. In some embodiments, the core region of the wing-core-wing motif comprises at least one Rp phosphorothioate internucleotide linkage. In some embodiments, the core region of the wing-core-wing motif comprises at least two Rp internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least two Rp phosphorothioate internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least three Rp internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least three Rp phosphorothioate internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least 4, 5, 6, 7, 8, 9, or 10 Rp internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least 4, 5, 6, 7, 8, 9, or 10 Rp phosphorothioate internucleotide linkages.

[0194] In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which the residues in each "X" wing region are 2'-MOE-modified residues. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which the residues in the core "Y" region are 2'-deoxyribonucleotide residues. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which all internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which all internucleoside linkages are chiral phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which the residues in each "X" wing region are 2'-MOE-modified residues, the residues in the core "Y" region are 2'-deoxyribonucleotides, and all internucleoside linkages are chiral phosphorothioate internucleoside linkages.

[0195] In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which the residues in each "X" wing region are not 2'-MOE-modified residues. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which the residues in the core "Y" region are 2'-deoxyribonucleotide residues. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which all internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which all internucleoside linkages are chiral phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif in which the residues in each "X" wing region are not 2'-MOE-modified residues, the residues in the core "Y" region are 2'-deoxyribonucleotides, and all internucleoside linkages are chiral phosphorothioate internucleoside linkages.

[0196] In certain embodiments, the chirally controlled (and / or stereochemically pure) preparations provided comprise an oligonucleotide of the base sequence GCCTCAGTCTGCTTCGCACC.

[0197] In some embodiments, the present invention provides stereochemical design parameters for oligonucleotides. That is, among other things, the present disclosure demonstrates the effect of the stereochemical structure at different positions along the oligonucleotide chain on the stability and / or activity of the oligonucleotide, including, for example, the effect on the interaction of the oligonucleotide with its cognate ligand and / or processing enzyme. The present invention specifically provides oligonucleotides whose structures incorporate or reflect the design parameters. Such oligonucleotides are novel chemical entities compared to stereorandom preparations of the same base sequence and length.

[0198] In some embodiments, the present invention provides stereochemical design parameters for antisense oligonucleotides. In some embodiments, the present invention provides design parameters for oligonucleotides that may be specifically bound and / or cleaved by RNase H. In one embodiment, the present invention provides stereochemical design parameters for siRNA oligonucleotides. In some embodiments, the present invention provides design parameters for oligonucleotides that may be specifically bound and / or cleaved by, for example, DICER, Argonaute proteins (e.g., Argonaute-1 and Argonaute-2), etc.

[0199] In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least one of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages is chiral. In some embodiments, at least two of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least three of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least four of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least five of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least six of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least seven of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least eight of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least nine of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the region comprises one of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th internucleotide linkages is chiral.In some embodiments, the nucleic acid sequence includes a region in which two of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which three of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which nine of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which ten of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral.

[0200] In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least one of the 1st, 2nd, 3rd, 5th, 7th, 18th, 19th, and 20th internucleotide linkages is chiral. In some embodiments, at least two of the 1st, 2nd, 3rd, 5th, 7th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least three of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least four of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least five of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least six of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which at least seven of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which one of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages is chiral. In some embodiments, the nucleic acid sequence includes a region in which two of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which three of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages are chiral. In some embodiments, the region comprises four of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth and twentieth internucleotide linkages are chiral.In some embodiments, the nucleic acid sequence includes a region in which five of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which six of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which seven of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral. In some embodiments, the nucleic acid sequence includes a region in which eight of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages are chiral.

[0201] In some embodiments, a single oligonucleotide of a provided composition comprises a region in which at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages is chiral and at least one internucleotide linkage is achiral. In some embodiments, a single oligonucleotide of a provided composition comprises a region in which at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleotide linkages is chiral and at least one internucleotide linkage is achiral. In some embodiments, at least two internucleotide linkages are achiral. In some embodiments, at least two internucleotide linkages are achiral. In some embodiments, at least three internucleotide linkages are achiral. In some embodiments, at least four internucleotide linkages are achiral. In some embodiments, at least five internucleotide linkages are achiral. In some embodiments, at least six internucleotide linkages are achiral. In some embodiments, at least seven internucleotide linkages are achiral. In some embodiments, at least 8 internucleotide linkages are achiral. In some embodiments, at least 9 internucleotide linkages are achiral. In some embodiments, at least 10 internucleotide linkages are achiral. In some embodiments, at least 11 internucleotide linkages are achiral. In some embodiments, at least 12 internucleotide linkages are achiral. In some embodiments, at least 13 internucleotide linkages are achiral. In some embodiments, at least 14 internucleotide linkages are achiral. In some embodiments, at least 15 internucleotide linkages are achiral. In some embodiments, at least 16 internucleotide linkages are achiral. In some embodiments, at least 17 internucleotide linkages are achiral.In some embodiments, at least 18 internucleotide linkages are achiral. In some embodiments, at least 19 internucleotide linkages are achiral. In some embodiments, at least 20 internucleotide linkages are achiral. In some embodiments, 1 internucleotide linkage is achiral. In some embodiments, 2 internucleotide linkages are achiral. In some embodiments, 3 internucleotide linkages are achiral. In some embodiments, 4 internucleotide linkages are achiral. In some embodiments, 5 internucleotide linkages are achiral. In some embodiments, 6 internucleotide linkages are achiral. In some embodiments, 7 internucleotide linkages are achiral. In some embodiments, 8 internucleotide linkages are achiral. In some embodiments, 9 internucleotide linkages are achiral. In some embodiments, 10 internucleotide linkages are achiral. In some embodiments, 11 internucleotide linkages are achiral. In some embodiments, 12 internucleotide linkages are achiral. In some embodiments, 13 internucleotide linkages are achiral. In some embodiments, 14 internucleotide linkages are achiral. In some embodiments, 15 internucleotide linkages are achiral. In some embodiments, 16 internucleotide linkages are achiral. In some embodiments, 17 internucleotide linkages are achiral. In some embodiments, 18 internucleotide linkages are achiral. In some embodiments, 19 internucleotide linkages are achiral. In some embodiments, 20 internucleotide linkages are achiral. In some embodiments, 1 internucleotide linkage is achiral. In some embodiments, 2 internucleotide linkages are achiral.In some embodiments, a single oligonucleotide of a provided composition comprises a region in which all internucleotide linkages are achiral except for at least one of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages, which are chiral.

[0202] In some embodiments, a single oligonucleotide of a provided composition comprises a region in which at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages is chiral and at least one internucleotide linkage is a phosphate ester. In some embodiments, a single oligonucleotide of a provided composition comprises a region in which at least one of the first, second, third, fifth, seventh, eighteenth, nineteenth, and twentieth internucleotide linkages is chiral and at least one internucleotide linkage is a phosphate ester. In some embodiments, at least two internucleotide linkages are phosphate esters. In some embodiments, at least three internucleotide linkages are phosphate esters. In some embodiments, at least four internucleotide linkages are phosphate esters. In some embodiments, at least five internucleotide linkages are phosphate esters. In some embodiments, at least six internucleotide linkages are phosphate esters. In some embodiments, at least seven internucleotide linkages are phosphate esters. In some embodiments, at least eight internucleotide linkages are phosphate esters. In some embodiments, at least 9 internucleotide linkages are phosphate esters. In some embodiments, at least 10 internucleotide linkages are phosphate esters. In some embodiments, at least 11 internucleotide linkages are phosphate esters. In some embodiments, at least 12 internucleotide linkages are phosphate esters. In some embodiments, at least 13 internucleotide linkages are phosphate esters. In some embodiments, at least 14 internucleotide linkages are phosphate esters. In some embodiments, at least 15 internucleotide linkages are phosphate esters. In some embodiments, at least 16 internucleotide linkages are phosphate esters. In some embodiments, at least 17 internucleotide linkages are phosphate esters.In some embodiments, at least 18 internucleotide linkages are phosphate esters. In some embodiments, at least 19 internucleotide linkages are phosphate esters. In some embodiments, at least 20 internucleotide linkages are phosphate esters. In some embodiments, 1 internucleotide linkage is phosphate ester. In some embodiments, 2 internucleotide linkages are phosphate esters. In some embodiments, 3 internucleotide linkages are phosphate esters. In some embodiments, 4 internucleotide linkages are phosphate esters. In some embodiments, 5 internucleotide linkages are phosphate esters. In some embodiments, 6 internucleotide linkages are phosphate esters. In some embodiments, 7 internucleotide linkages are phosphate esters. In some embodiments, 8 internucleotide linkages are phosphate esters. In some embodiments, 9 internucleotide linkages are phosphate esters. In some embodiments, 10 internucleotide linkages are phosphate esters. In some embodiments, 11 internucleotide linkages are phosphate esters. In some embodiments, 12 internucleotide linkages are phosphate esters. In some embodiments, 13 internucleotide linkages are phosphate esters. In some embodiments, 14 internucleotide linkages are phosphate esters. In some embodiments, 15 internucleotide linkages are phosphate esters. In some embodiments, 16 internucleotide linkages are phosphate esters. In some embodiments, 17 internucleotide linkages are phosphate esters. In some embodiments, 18 internucleotide linkages are phosphate esters. In some embodiments, 19 internucleotide linkages are phosphate esters. In some embodiments, 20 internucleotide linkages are phosphate esters. In some embodiments, 1 internucleotide linkage is a phosphate ester.In some embodiments, two internucleotide linkages are phosphate esters. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which all internucleotide linkages are phosphate esters, except for at least one of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages, which are chiral.

[0203] In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least one of the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th internucleotide linkages is chiral, and at least 10% of the internucleotide linkages in that region are achiral. In some embodiments, a single oligonucleotide of the provided compositions comprises a region in which at least one of the 1st, 2nd, 3rd, 5th, 7th, 18th, 19th, and 20th internucleotide linkages is chiral, and at least 10% of the internucleotide linkages in that region are achiral. In some embodiments, at least 20% of the internucleotide linkages in that region are achiral. In some embodiments, at least 30% of the internucleotide linkages in that region are achiral. In some embodiments, at least 40% of the internucleotide linkages in that region are achiral. In some embodiments, at least 50% of the internucleotide linkages in that region are achiral. In some embodiments, at least 60% of the internucleotide linkages in the region are achiral. In some embodiments, at least 70% of the internucleotide linkages in the region are achiral. In some embodiments, at least 80% of the internucleotide linkages in the region are achiral. In some embodiments, at least 90% of the internucleotide linkages in the region are achiral. In some embodiments, at least 50% of the internucleotide linkages in the region are achiral. In some embodiments, a single achiral internucleotide linkage is a phosphate linkage. In some embodiments, each achiral internucleotide linkage is a phosphate linkage.

[0204] In some embodiments, the first internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the first internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the second internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the second internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the third internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the third internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the fifth internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the seventh internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the seventh internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the eighth internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the eighth internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the fifth internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the 9th internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the 9th internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the 18th internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the 18th internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the 19th internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the 19th internucleotide linkage in the region is an Rp modified internucleotide linkage. In some embodiments, the 20th internucleotide linkage in the region is an Sp modified internucleotide linkage. In some embodiments, the 20th internucleotide linkage in the region is an Rp modified internucleotide linkage.

[0205] In some embodiments, the region has a length of at least 21 bases. In some embodiments, the region has a length of 21 bases. In some embodiments, a single oligonucleotide of the provided compositions has a length of at least 21 bases. In some embodiments, a single oligonucleotide of the provided compositions has a length of 21 bases.

[0206] In some embodiments, the chiral internucleotide linkage has the structure of Formula I. In some embodiments, the chiral internucleotide linkage is phosphorothioate. In some embodiments, each chiral internucleotide linkage of a single oligonucleotide of a provided composition independently has the structure of Formula I. In some embodiments, each chiral internucleotide linkage of a single oligonucleotide of a provided composition is phosphorothioate.

[0207] As known to those skilled in the art and as described in this disclosure, various modifications can be introduced at the 2'-position of the sugar moiety. Commonly used 2'-modifications include 2'-OR 1 (In the formula, R 1is not hydrogen.) In some embodiments, the modification is 2'-OR (wherein R can be a substituted aliphatic). In some embodiments, the modification is 2'-OMe. In some embodiments, the modification is 2'-O-MOE. In some embodiments, the present invention demonstrates that the inclusion and / or location of specific chirally pure internucleotide linkages can result in stability improvements that are comparable to or better than those achieved by the use of modified backbone linkages, bases, and / or sugars. In some embodiments, a single oligonucleotide of a provided composition does not have a modification on the sugar. In some embodiments, a single oligonucleotide of a provided composition does not have a modification at the 2'-position of the sugar (i.e., the two groups at the 2'-position are either -H / -H or -H / -OH). In some embodiments, a single oligonucleotide of a provided composition does not have any 2'-MOE modifications.

[0208] In some embodiments, the single oligonucleotides of the provided compositions are better substrates for Argonaute proteins (e.g., hAgo-1 and hAgo-2) than stereorandom oligonucleotide compositions. The selection and / or location of chirally pure linkages described in the present closure are useful design parameters for oligonucleotides, such as siRNAs, that interact with such proteins.

[0209] In some embodiments, a single oligonucleotide of a provided composition has at least about 25% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 30% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 35% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 40% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 45% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 50% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 55% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 60% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 65% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 70% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 75% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 80% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 85% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of a provided composition has at least about 90% of its internucleotide linkages in the Sp configuration.

[0210] In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: TIFF0007662687000007.tif34166 The underlined nucleotides are 2'-modified.

[0211] In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: TIFF0007662687000008.tif33166 The underlined nucleotides are 2'-O-MOE modified.

[0212] In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: TIFF0007662687000009.tif65166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-OH RNA residues; bold and "s" indicate phosphorothioate moieties; TIFF0007662687000010.tif63166In the table, lowercase letters represent 2'-OMe RNA residues; capital letters represent RNA residues; d = 2'-deoxy residue; "s" indicates a phosphorothioate moiety; TIFF0007662687000011.tif21166TIFF0007662687000012.tif96166In the table, lowercase letters represent 2'-OMe RNA residues; capital letters represent RNA residues; d = 2'-deoxy residue; "s" indicates a phosphorothioate moiety; TIFF0007662687000013.tif62166In the table, lowercase letters represent 2'-OMe RNA residues; capital letters represent 2'-F RNA residues; d = 2'-deoxy residue; "s" indicates a phosphorothioate moiety; TIFF0007662687000014.tif45166TIFF0007662687000015.tif40166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-F RNA residues; d = 2'-deoxy residue; "s" indicates a phosphorothioate moiety.

[0213] In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: d[A R C S A R C S A R C S A R C S A R C], d[C S C S C S C R C R C S C S C S C S C], d[C S C S C S C S C S C S C R C R C S C] and d[C S C S C S C S C S C R C R C S C S C] (where R is an Rp phosphorothioate linkage and S is an Sp phosphorothioate linkage).

[0214] In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: GGA R T S G R T S T Rm C S TCGA, GGA R T R G S T S T R m C R TCGA, GGA S T S G R T R T S m C S TCGA (wherein R is an Rp phosphorothioate linkage, S is an Sp phosphorothioate linkage, and all other linkages are PO, each m C is a 5-methylcytosine modified nucleoside).

[0215] In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: k T k m C k AGT m CATGA m CT k T m C k m C k where each nucleoside followed by a subscript "k" indicates an (S)-cEt modification, R is an Rp phosphorothioate linkage, S is an Sp phosphorothioate linkage, and each mC is a 5-methylcytosine modified nucleoside and all internucleoside linkages are phosphorothioate (PS) containing a stereochemical pattern selected from RSSSRSRRRS, RSSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, SSRSSSRRRS, RSSRSRSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRRSR, RRRRRRRRRR and SSSSSSSSSS). In some embodiments, the single oligonucleotide of the provided compositions is not an oligonucleotide selected from: k T k m C k AGT m CATGA m CTT k m C k m C k where each nucleoside followed by a subscript "k" indicates an (S)-cEt modification, R is an Rp phosphorothioate linkage, S is an Sp phosphorothioate linkage, and each mC is a 5-methylcytosine modified nucleoside and all internucleoside linkages are phosphorothioate (PS) containing a stereochemical pattern selected from RSSSRSRRRS, RSSSSSSSSSS, SRRSRSSSSR, SRSRSSRSSR, RRRSSSRSSS, RRRSRSSRSR, RRSSSRSRSR, SRSSSRSSSS, SSRRSSRSRS, SSSSSSRRSS, RRRSSRRRSR, RRRRSSSSRS, SRRSRRRRRR, RSSRSSRRRR, RSRRSRRSRR, RRSRSSRSRS, SSRRRRRSRR, RSRRSRSSSR, RRSSRSRRRR, RRSRSRRSSS, RRSRSSSRRR, RSRRRRSRSR, SSRSSSRRRS, RSSRSRSRSR, SSRSSSRRRS, RSSRSRSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRRSR, RRRRRRRRRR and SSSSSSSSSS).

[0216] Modified Oligonucleotide Structures As noted above, in view of the usefulness of oligonucleotide compositions in various applications and conditions, those skilled in the art have endeavored to develop modifications of oligonucleotide structures, as compared to native oligonucleotide molecules, that may, for example, have preferred or desirable properties or characteristics for use in particular applications and conditions. Exemplary such modifications are described below.

[0217] WO 2010 / 141471 (hereinafter "Traversa I") teaches the modification of different types of nucleic acid structures modified to have reduced net polyanionic charge. WO 2010 / 039543 (hereinafter "Traversa II") teaches compositions and methods for making neutral polynucleotides (NNs) with reduced polyanionic charge. WO 2008 / 008476 (hereinafter "Traversa III") describes the synthesis of SATE (Imbach-type) phosphate prodrugs. Traversa I, II, and III do not disclose the chiral-controlled oligonucleotides, compositions thereof, and methods for making them described in the present invention.

[0218] International Publication No. 2010 / 072831 (hereinafter "Girindus et al.") also teaches the modification of oligonucleotides. In particular, Girindus et al. disclose the use of sulfurizing agents to generate phosphorothioate triesters as prodrugs. Girindus et al. does not disclose the chiral controlled oligonucleotides, compositions thereof, and methods of making them described in the present invention.

[0219] Similarly, International Publication No. 2004 / 085454 (hereinafter "Avecia I") teaches, for example, the preparation of phosphorothioate oligonucleotides through transient silylation of poly-H-phosphonate diesters. International Publication No. 2001 / 027126 (hereinafter "Avecia II") teaches a process for the solid-phase synthesis of phosphotriester oligonucleotides in which an H-phosphonate monomer is coupled to a solid-supported 5'-hydroxyl oligonucleotide and the resulting H-phosphonate diester is further sulfurized to a phosphorothioate triester. International Publication No. 2001 / 064702 (hereinafter "Avecia III") is similar to Avecia II, but describes solid-phase synthesis on a different solid support. Avecia I, II, and III do not disclose the chiral-controlled oligonucleotides, compositions thereof, and methods of making them according to the present invention.

[0220] WO 1997 / 006183 (hereinafter "Chiron") teaches oligonucleotides containing asymmetric phosphorus, such as stereopure amidated oligonucleotides, having cationic internucleotide bonds. Chiron teaches mixtures of diastereomers or stereopure oligonucleotides obtained by crystallization using a solution such as column chromatography. Chiron does not teach the chiral controlled oligonucleotides, their compositions, and methods of preparation using the same described in the present invention.

[0221] WO 2009 / 146123 (hereinafter "Spring Bank I") discloses compositions and methods for treating viral infections using substituted phosphate oligonucleotides and phosphorothioate triesters. WO 2007 / 070598 (hereinafter "Spring Bank II") teaches the synthesis of phosphotriester and phosphorothioate prodrugs as antiviral nucleic acids. Spring Bank I and II do not disclose the chiral-controlled oligonucleotides, compositions thereof, and methods of making them described in the present invention.

[0222] European Patent No. 0779893 (hereinafter "Hybridon") teaches lipid-soluble prodrugs of antisense oligonucleotides that have high cellular uptake, and identifies Rp and Sp phosphorothioate and phosphorothioate triester dimers with different enzymatic stabilities. Hybridon does not disclose the chiral controlled oligonucleotides, their compositions, and methods of making them described in the present invention.

[0223] International Publication No. WO 1997 / 047637 (hereinafter "Imbach I") generally teaches Imbach "SATE" (S-acylthioethyl) prodrug oligonucleotide compositions and methods. Imbach I describes, for example, the preparation of certain prodrug oligonucleotides using bioreversible phosphotriester prodrugs and phosphoramidites containing post-synthetic alkylation or prodrug groups. U.S. Patent No. 6,124,445 (hereinafter "Imbach II") teaches modified antisense and chimeric prodrug oligonucleotides. Imbach I and II do not disclose the chiral-controlled oligonucleotides, compositions thereof, and methods of making them according to the present invention.

[0224] WO 2006 / 065751 (hereinafter "Beaucage") teaches CpG oligonucleotide phosphorothioate prodrugs containing thermally labile substituents (substituents introduced via phosphoramidite monomers) and their uses. Beaucage does not disclose the chiral controlled oligonucleotides, their compositions, and methods of making them described in the present invention.

[0225] Takeshi Wada et al. have developed a novel method for the stereocontrolled synthesis of P-chiral nucleic acids using amidite chiral auxiliaries (Japanese Patent No. 4348077, International Publication Nos. 2005 / 014609, 2005 / 92909, and 2010 / 064146, collectively referred to herein as "Wada I"). In particular, International Publication No. 2010 / 064146 (referred to herein as "Wada II") discloses a method for the synthesis of phosphate-modified nucleic acids with controlled stereochemical configuration at phosphorus. However, the Wada II method is limited in that it does not provide for the individual P-modification of each chiral linked phosphate in a controlled and designed manner. That is, the Wada II P-modification attachment method provides for the production of a condensation intermediate poly H-phosphonate oligonucleotide chain that, once created to the desired length, is mass-modified at the attached phosphate to provide, for example, the desired phosphorothioate diester, phosphoramidate, or boranophosphate, or another such phosphorus-atom-modified nucleic acid (referred to in the literature as Route B, Scheme 6, p. 36). Furthermore, the Wada II H-phosphonate oligonucleotide chains are shorter in length (e.g., dimers, trimers, or tetramers). Combined with the fact that no capping step is included in Route B, which generally exhibits lower purity as a result of the accumulation of "n-1" by-products, the Wada II pathway has limitations with regard to the synthesis of longer oligonucleotides. While Wada II generally contemplates that a particular oligonucleotide may contain different modifications at each attached phosphate, Wada II does not describe or suggest the method described herein for repeatedly incorporating such controlled modifications.To the extent that Wada II describes a synthesis cycle that does not require complete incorporation of the H-phosphonate intermediate oligonucleotide prior to modification with the attached phosphate (referred to in the literature as Route A, p. 35, Scheme 5, "Synthesis of Nucleic Acids Containing Chiral X-Phosphonate Moieties of Formula 1 via Route A"), this general disclosure does not teach the critical steps required to incorporate certain P-modifications provided by the present invention, nor does it provide the efficiency or versatility to make this cycle useful in the synthesis of chiral controlled P-modified oligonucleotides, particularly for the synthesis of longer oligonucleotides.

[0226] At least one such inefficiency in Wada II is described by Wada et al. in International Publication No. 2012 / 039448 (hereinafter "Wada III"). Wada III discloses the use of novel chiral auxiliaries in the Wada II method to generate and subsequently modify H-phosphonate oligonucleotides, once assembled, to yield, among other things, phosphorothioates. Wada et al. confirmed in Wada III that the four types of chiral auxiliaries disclosed in Wada II cannot be efficiently removed because they form strong bonds with the phosphorus in the attached phosphate. Wada III noted that harsh conditions are required to remove the Wada II chiral auxiliaries, which tend to compromise the integrity of the oligonucleotide product. Wada III confirmed this is particularly problematic when synthesizing long oligonucleotides, at least because as the degradation reaction proceeds, additional by-products are generated that further react with and degrade the oligonucleotide product. Thus, Wada III demonstrates the use of S N The mechanism of 1 has been used to provide chiral auxiliaries that can be efficiently cleaved from oligonucleotides via a β-elimination pathway under mildly acidic conditions (route B) or via a β-elimination pathway under relatively mildly basic conditions.

[0227] Those skilled in the chemical and synthetic arts will readily appreciate the complexities associated with producing, for example, the chiral controlled oligonucleotides provided by the present invention. For example, to synthesize and isolate a chiral controlled oligonucleotide, the conditions for each monomer addition must be designed so that: (1) the chemistry is compatible with each portion of the incremental oligonucleotide; (2) by-products generated during each monomer addition do not compromise the structural and stereochemical integrity of the incremental oligonucleotide; and (3) the composition of the final crude product is such that the desired chiral controlled oligonucleotide product is isolated.

[0228] Oligonucleotide phosphorothioates have shown therapeutic potential (Stein et al., Science (1993), 261:1004-12; Agrawal et al., Antisense Res. and Dev. (1992), 2:261-66; Bayever et al., Antisense Res. and Dev. (1993), 3:383-390). Oligonucleotide phosphorothioates prepared without regard to the stereochemistry of the phosphorothioate have been shown to be 2 nThey exist as a mixture of diastereomers, where n is the number of internucleotide phosphorothioate linkages. The chemical and biological properties of these diastereomeric phosphorothioates can be distinctly different from one another. For example, Wada et al. (Nucleic Acids Symposium Series No. 51, p. 119-120; doi:10.1093 / nass / nrm060) found that the stereodefined -(Rp)-(Ups)U / (Ap)A duplex exhibited higher Tm values ​​than the normal -(Up)U / (Ap)A and the stereodefined, non-duplex -(Sp)-(Ups)U. In another example, a study by Tang et al. (Nucleosides Nucleotides (1995), 14:985-990) found that stereo-pure Rp oligodeoxyribonucleoside phosphorothioates have lower stability to nucleases present in human serum than the parent oligodeoxyribonucleoside phosphorothioates with undefined phosphoryl chirality.

[0229] Chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions The present invention provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high crude purity and high diastereomeric purity. In some embodiments, the present invention provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high crude purity. In some embodiments, the present invention provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high diastereomeric purity.

[0230] In some embodiments, the present invention provides 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers wherein the composition is a substantially pure preparation of a single oligonucleotide, wherein at least about 10% of the oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0231] In some embodiments, the invention provides chiral controlled oligonucleotide compositions of oligonucleotides, wherein the compositions are enriched for oligonucleotides of a single oligonucleotide type, relative to substantially racemic preparations of the same oligonucleotide. 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers The present invention provides chiral controlled oligonucleotide compositions of oligonucleotides in which the compositions are enriched for oligonucleotides of a single oligonucleotide type that share a common nucleotide sequence.

[0232] In some embodiments, the present invention provides 1) consensus sequence and length; 2) common patterns of skeletal bonding; and 3) Common patterns of skeletal chiral centers wherein the composition is chirally controlled in that it is enriched for oligonucleotides of a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0233] In some embodiments, as will be understood by those skilled in the art, in a substantially racemic (or non-chiral controlled) preparation of oligonucleotides, all or most of the conjugation steps are not chiral controlled, in that the conjugation step is not specifically performed to enhance stereoselectivity. An exemplary substantially racemic preparation of oligonucleotides is the preparation of phosphorothioate oligonucleotides by a process well known in the art of sulfurizing a phosphite triester with either tetraethylthiuram disulfide (TETD) or 3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD). In some embodiments, a substantially racemic preparation of oligonucleotides results in a substantially racemic oligonucleotide composition (or a non-chiral controlled oligonucleotide composition).

[0234] In some embodiments, the chiral controlled oligonucleotide composition is a substantially pure preparation of an oligonucleotide type, wherein any oligonucleotides in the composition that are not of the oligonucleotide type are impurities form the preparation process of said oligonucleotide type (optionally after certain purification procedures).

[0235] In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with respect to the chirally linked phosphate. In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages having the structure of Formula I. In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with respect to the chirally linked phosphate and one or more phosphodiester linkages. In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with the structure of Formula I and one or more phosphodiester linkages. In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with the structure of Formula Ic and one or more phosphodiester linkages. In some embodiments, such oligonucleotides are prepared using stereoselective oligonucleotide synthesis, as described herein, to form pre-designed diastereomerically pure internucleotide linkages with respect to the chirally linked phosphate. For example, in one exemplary oligonucleotide designated (Rp / Sp,Rp / Sp,Rp / Sp,Rp,Rp,Sp,Sp,Sp,Sp,Sp,Sp,Sp,Sp,Rp,Rp,Rp,Rp)-d[GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGs1Cs1As1CsC], the first three internucleotide linkages are prepared using conventional oligonucleotide synthesis methods, and diastereomerically pure internucleotide linkages are prepared with stereochemical control as described herein. Exemplary internucleotide linkages include those having the structure of Formula I, further described below.

[0236] In some embodiments, the present invention provides chirality-controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P-modifications. In certain embodiments, the present invention provides chirality-controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P-modifications from each other. In certain embodiments, the present invention provides chirality-controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P-modifications from each other, wherein the chirality-controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage. In certain embodiments, the present invention provides chirality-controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P-modifications from each other, wherein the chirality-controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one phosphorothioate diester internucleotide linkage. In certain embodiments, the present invention provides chirality-controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P-modifications from each other, wherein the chirality-controlled oligonucleotide comprises at least one phosphorothioate triester internucleotide linkage. In certain embodiments, the present invention provides chirality-controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different P-modifications, and the chirality-controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least one phosphorothioate triester internucleotide linkage.

[0237] In certain embodiments, the present invention provides compounds independently comprising Formula I: [ka] wherein each variable is defined and described as follows. In some embodiments, the present invention provides chiral controlled oligonucleotides comprising one or more modified internucleotide linkages of Formula I, wherein each internucleotide linkage of Formula I within the oligonucleotide has a P-modification that is different from each other ... 1 In some embodiments, the present invention provides chirality-controlled oligonucleotides comprising one or more modified internucleotide linkages of Formula I, wherein each internucleotide linkage of Formula I within the oligonucleotide has a different X from each other. In some embodiments, the present invention provides chirality-controlled oligonucleotides comprising one or more modified internucleotide linkages of Formula I, wherein each internucleotide linkage of Formula I within the oligonucleotide has a different -LR 1 It has.

[0238] In some embodiments, the present invention provides chiral controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P-modifications from one another. In some embodiments, the present invention provides chiral controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry from one another, and at least a portion of the structure of the chiral controlled oligonucleotide is characterized by a repeating pattern of alternating stereochemistry.

[0239] In some embodiments, the present invention provides chiral controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide are -XLR 1with different X atoms in the moiety, and / or -XLR 1 with different L groups in the moiety and / or -XLR 1 Different R in different parts 1 They have different P-modifications from each other in the sense that they have different atoms.

[0240] In some embodiments, the present invention provides chiral controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P-modifications from one another, and the oligonucleotide has the following formula: [S B n1R B n2S B n3R B n4...S B nxR B ny] and having a structure represented by Here, each R B independently represents a block of nucleotide units having the R configuration at the attached phosphate; Each S B independently represents a block of nucleotide units having an S configuration at the attached phosphate; each of n1 through ny is zero or an integer, with the requirement that at least one odd number n and at least one even number n is non-zero, such that the oligonucleotide contains at least two individual internucleotide linkages of different stereochemistry; and wherein the sum of n1 through ny is 2 to 200, and in some embodiments, the lower limit is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more, and the upper limit is selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 or more, and the upper limit is greater than the lower limit.

[0241] In some such embodiments, each n is the same value; in some embodiments, each even n is the same value as each other even n; in some embodiments, each odd n is the same value as each other odd n; in some embodiments, at least two even n are different values ​​from each other; in some embodiments, at least two odd n are different values ​​from each other.

[0242] In some embodiments, at least two adjacent n's are equal to each other, and the provided oligonucleotides comprise adjacent blocks of equal length S and R stereochemical bonds. In some embodiments, the provided oligonucleotides comprise repeating blocks of equal length S and R stereochemical bonds. In some embodiments, the provided oligonucleotides comprise repeating blocks of S and R stereochemical bonds, and at least two such blocks are different lengths from each other; in some such embodiments, each S stereochemical block is the same length and different from the length of each R stereochemical, and the lengths of each R stereochemical may optionally be the same length as each other.

[0243] In some embodiments, at least two adjacent n's, skipping one other than n, are equal to each other, and the provided oligonucleotide comprises at least two blocks of bonds of a first stereochemistry that are equal in length, separated by blocks of bonds of a different stereochemistry, which may be the same length as or different from the blocks of the first stereochemistry.

[0244] In some embodiments, the n's associated with the linking blocks at the ends of a provided oligonucleotide are the same length. In some embodiments, a provided oligonucleotide has endblocks of the same linking stereochemistry. In some such embodiments, the endblocks are separated from one another by midblocks of different linking stereochemistry.

[0245] In some embodiments, the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], provided oligonucleotides are stereoblockmers. B n1R B n2S B n3R B n4...S B nxR B ny] and provided oligonucleotides are stereoskipmers. B n1R B n2S B n3R B n4...S B nxR B ny], provided that the oligonucleotide is a stereoisomer. B n1R B n2S B n3R B n4...S B nxR B ny] and provided oligonucleotides are gapmers.

[0246] In some embodiments, the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], provided oligonucleotides can have any of the patterns described above, and further include patterns of P-modifications. For example, in some embodiments, oligonucleotides of the formula [S B n1R B n2S B n3R B n4...S B nxR B ny] and provided oligonucleotides are stereoskipmers and P-modified skippmers.B n1R B n2S B n3R B n4...S B nxR B ny], provided oligonucleotides are stereoblockmers and P-modified altmers. In some embodiments, oligonucleotides of formula [S B n1R B n2S B n3R B n4...S B nxR B ny] and provided oligonucleotides are stereoaltomers and P-modified blockmirs.

[0247] In some embodiments, the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], independently represent the formula I: [ka] a chiral controlled oligonucleotide containing one or more modified internucleotide linkages having the structure: where P * is an asymmetric phosphorus atom, either Rp or Sp; W is O, S or Se; Each of X, Y and Z is independently -O-, -S-, -N(-LR 1 )-, or L; L is a covalent bond or an optionally substituted, straight-chain or branched C1-C 10alkylene, wherein one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R') optionally and independently substituted by -C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 1 is halogen, R, or optionally substituted C1-C 50 aliphatic, wherein one or more methylene units are optionally and independently replaced by optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; Each R' is independently -R, -C(O)R, -COR, or -SOR, or: two R' on the same nitrogen together with their intervening atoms form an optionally substituted heterocyclic or heteroaryl ring, or two R' on the same carbon together with their intervening atoms form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring; -Cy- is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene; each R is independently hydrogen or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each TIFF0007662687000018.tif10166 represents the bond to the nucleoside independently.

[0248] In some embodiments, the chiral controlled oligonucleotide comprises one or more modified internucleotide phosphate linkages. In some embodiments, the chiral controlled oligonucleotide comprises, for example, phosphorothioate or phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide comprises phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide comprises at least two phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide comprises at least three phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide comprises at least four phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide comprises at least five phosphorothioate triester linkages. Such exemplary modified internucleotide phosphate linkages are further described herein.

[0249] In some embodiments, the chiral controlled oligonucleotide contains different internucleotide phosphate linkages. In some embodiments, the chiral controlled oligonucleotide contains at least one phosphodiester internucleotide linkage and at least one modified internucleotide linkage. In some embodiments, the chiral controlled oligonucleotide contains at least one phosphodiester internucleotide linkage and at least one phosphorothioate triester linkage. In some embodiments, the chiral controlled oligonucleotide contains at least one phosphodiester internucleotide linkage and at least two phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide contains at least one phosphodiester internucleotide linkage and at least three phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide contains at least one phosphodiester internucleotide linkage and at least four phosphorothioate triester linkages. In some embodiments, the chiral controlled oligonucleotide contains at least one phosphodiester internucleotide linkage and at least five phosphorothioate triester linkages. Such exemplary modified internucleotide phosphate linkages are further described herein.

[0250] In some embodiments, the phosphorothioate triester linkage includes a chiral auxiliary used, for example, to control the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester linkage does not include a chiral auxiliary. In some embodiments, the phosphorothioate triester linkage is intentionally maintained until and / or throughout administration to a subject.

[0251] In some embodiments, the chiral controlled oligonucleotide is attached to a solid support. In some embodiments, the chiral controlled oligonucleotide is cleaved from the solid support.

[0252] In some embodiments, the chiral controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive modified internucleotide linkages, hi some embodiments, the chiral controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive phosphorothioate triester internucleotide linkages.

[0253] In some embodiments, the chiral controlled oligonucleotide is a blockmir. In some embodiments, the chiral controlled oligonucleotide is a stereoblockmir. In some embodiments, the chiral controlled oligonucleotide is a P-modified blockmir. In some embodiments, the chiral controlled oligonucleotide is a linked blockmir.

[0254] In some embodiments, the chiral controlled oligonucleotide is an altomer. In some embodiments, the chiral controlled oligonucleotide is a stereoaltomer. In some embodiments, the chiral controlled oligonucleotide is a P-modified altomer. In some embodiments, the chiral controlled oligonucleotide is a linked altomer.

[0255] In some embodiments, the chiral controlled oligonucleotide is a unimer. In some embodiments, the chiral controlled oligonucleotide is a stereounimer. In some embodiments, the chiral controlled oligonucleotide is a P-modified unimer. In some embodiments, the chiral controlled oligonucleotide is a linked unimer.

[0256] In some embodiments, the chiral controlled oligonucleotide is a gapmer.

[0257] In some embodiments, the chiral controlled oligonucleotide is a skipmer.

[0258] In some embodiments, the present invention provides a compound of formula I: [ka] and a chiral controlled oligonucleotide comprising one or more modified internucleotide linkages independently having the structure: where P * is an asymmetric phosphorus atom, either Rp or Sp; W is O, S or Se; Each of X, Y and Z is independently -O-, -S-, -N(-LR 1 )-, or L; L is a covalent bond or an optionally substituted, straight-chain or branched C1-C 10 alkylene, wherein one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R') optionally and independently substituted by -C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 1 is halogen, R, or optionally substituted C1-C 50aliphatic, wherein one or more methylene units are optionally and independently replaced by optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; Each R' is independently -R, -C(O)R, -COR, or -SOR, or: two R' on the same nitrogen together with their intervening atoms form an optionally substituted heterocyclic or heteroaryl ring, or two R' on the same carbon together with their intervening atoms form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring; -Cy- is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene; each R is independently hydrogen or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each TIFF0007662687000020.tif10166 represents the bond to the nucleoside independently.

[0259] As generally described above and herein, P * is an asymmetric phosphorus atom and is either Rp or Sp. In some embodiments, P * is Rp. In another embodiment, P * In some embodiments, the oligonucleotide comprises each P *In some embodiments, the oligonucleotide comprises one or more internucleotide linkages of Formula I, each P being independently Rp or Sp. * In some embodiments, the oligonucleotide comprises one or more internucleotide linkages of Formula I, where each P * In some embodiments, the oligonucleotide comprises one or more internucleotide linkages of Formula I, where P * In some embodiments, the oligonucleotide comprises at least one internucleotide linkage of Formula I, where P * In some embodiments, the oligonucleotide comprises at least one internucleotide linkage of Formula I, where P * contains at least one internucleotide linkage of formula I, where Rp, and P * contains at least one internucleotide linkage of formula I, where Sp.

[0260] As generally described above and herein, W is O, S, or Se. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, where W is O. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, where W is S. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, where W is Se.

[0261] As generally described above and herein, each R is independently hydrogen or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

[0262] In some embodiments, R is hydrogen. In some embodiments, R is an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

[0263] In some embodiments, R is an optionally substituted C1-C6 aliphatic. In some embodiments, R is an optionally substituted C1-C6 alkyl. In some embodiments, R is an optionally substituted straight-chained or branched hexyl. In some embodiments, R is an optionally substituted straight-chained or branched pentyl. In some embodiments, R is an optionally substituted straight-chained or branched butyl. In some embodiments, R is an optionally substituted straight-chained or branched propyl. In some embodiments, R is an optionally substituted ethyl. In some embodiments, R is an optionally substituted methyl.

[0264] In some embodiments, R is an optionally substituted phenyl. In some embodiments, R is a substituted phenyl. In some embodiments, R is phenyl.

[0265] In some embodiments, R is an optionally substituted carbocyclyl. In some embodiments, R is an optionally substituted C3-C 10 In some embodiments, R is an optionally substituted monocyclic carbocyclyl. In some embodiments, R is an optionally substituted cycloheptyl. In some embodiments, R is an optionally substituted cyclohexyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is an optionally substituted cyclobutyl. In some embodiments, R is an optionally substituted cyclopropyl. In some embodiments, R is an optionally substituted bicyclic carbocyclyl.

[0266] In some embodiments, R is an optionally substituted aryl. In some embodiments, R is an optionally substituted bicyclic aryl ring.

[0267] In some embodiments, R is an optionally substituted heteroaryl. In some embodiments, R is an optionally substituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, sulfur, or oxygen. In some embodiments, R is a substituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an unsubstituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, sulfur, or oxygen.

[0268] In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0269] In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R is selected from pyrrolyl, furanyl, or thienyl.

[0270] In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom, and the additional heteroatom is selected from sulfur or oxygen. Exemplary R groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl.

[0271] In some embodiments, R is a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms. In other embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1 to 2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In certain embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1 nitrogen. Exemplary R groups are optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

[0272] In certain embodiments, R is an optionally substituted 8- to 10-membered bicyclic heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted indolyl. In some embodiments, R is an optionally substituted azabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0273] In certain embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted quinolinyl. In some embodiments, R is an optionally substituted isoquinolinyl. In one aspect, R is an optionally substituted 6,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is quinazoline or quinoxaline.

[0274] In some embodiments, R is an optionally substituted heterocyclyl. In some embodiments, R is an optionally substituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is a substituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an unsubstituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0275] In some embodiments, R is an optionally substituted heterocyclyl. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 oxygen atoms.

[0276] In certain embodiments, R is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl, tetra ... hydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxthiolinonyl, oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0277] In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

[0278] In some embodiments, R is an optionally substituted 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted indolinyl. In some embodiments, R is an optionally substituted isoindolinyl. In some embodiments, R is an optionally substituted 1,2,3,4-tetrahydroquinoline. In some embodiments, R is an optionally substituted 1,2,3,4-tetrahydroisoquinoline.

[0279] As generally described above and herein, each R' is independently -R, -C(O)R, -COR, or -SOR, or: two R' on the same nitrogen together with their intervening atoms form an optionally substituted heterocyclic or heteroaryl ring, or two R' on the same carbon together with their intervening atoms form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;

[0280] In some embodiments, R' is -R, -C(O)R, -CO2R, or -SO2R, where R is as defined above and described herein.

[0281] In some embodiments, R' is -R, where R is as defined and described above and herein. In some embodiments, R' is hydrogen.

[0282] In some embodiments, R' is -C(O)R, where R is as defined above and as described herein. In some embodiments, R' is -COR, where R is as defined above and as described herein. In some embodiments, R' is -SOR, where R is as defined above and as described herein.

[0283] In some embodiments, two R' on the same nitrogen, together with their intervening atoms, form an optionally substituted heterocyclic or heteroaryl ring. In some embodiments, two R' on the same carbon, together with their intervening atoms, form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring.

[0284] As generally described above and herein, -Cy- is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene.

[0285] In some embodiments, -Cy- is optionally substituted phenylene. In some embodiments, -Cy- is optionally substituted carbocyclylene. In some embodiments, -Cy- is optionally substituted arylene. In some embodiments, -Cy- is optionally substituted heteroarylene. In some embodiments, -Cy- is optionally substituted heterocyclylene.

[0286] As generally described above and herein, each of X, Y and Z independently represents -O-, -S-, -N(-LR 1 )-, or L, and L and R 1 each of which is independently as defined above and as described below.

[0287] In some embodiments, X is -O-. In some embodiments, X is -S-. In some embodiments, X is -O- or -S-. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, and X is -O-. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, and X is -S-. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, and X is -O-, and at least one internucleotide linkage of Formula I, and X is -S-. In some embodiments, an oligonucleotide comprises at least one internucleotide linkage of Formula I, and X is -O-, and at least one internucleotide linkage of Formula I, and X is -S-, and at least one internucleotide linkage of Formula I, and L is an optionally substituted linear or branched C1-C 10 is alkylene, and one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C( Optionally and independently substituted by -O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-.

[0288] In some embodiments, X is -N(-LR 1 In some embodiments, X is —N(R 1 )-. In some embodiments, X is -N(R')-. In some embodiments, X is -N(R)-. In some embodiments, X is -NH-.

[0289] In some embodiments, X is L. In some embodiments, X is a covalent bond. In some embodiments, X is an optionally substituted, straight-chain or branched C1-C 10 is alkylene, and one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C( In some embodiments, X is optionally and independently substituted by an optionally substituted C1-C2 alkyl group. In some embodiments, X is optionally substituted by an optionally substituted C1-C2 alkyl group. In some embodiments, X is optionally substituted by an optionally substituted C1-C2 alkyl group. In some embodiments, X is optionally substituted by an optionally substituted C1-C2 alkyl group. In some embodiments, X is optionally substituted by an optionally substituted C1-C2 alkyl group. 10 Alkylene or C1-C 10 In some embodiments, X is methylene.

[0290] In some embodiments, Y is -O-. In some embodiments, Y is -S-.

[0291] In some embodiments, Y is -N(-LR 1 In some embodiments, Y is —N(R 1 )-. In some embodiments, Y is -N(R')-. In some embodiments, Y is -N(R)-. In some embodiments, Y is -NH-.

[0292] In some embodiments, Y is L. In some embodiments, Y is a covalent bond. In some embodiments, Y is an optionally substituted, straight-chain or branched C1-C 10is alkylene, and one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C( In some embodiments, Y is optionally and independently substituted with an optionally substituted C1-C2 alkyl group. 10 Alkylene or C1-C 10 In some embodiments, Y is methylene.

[0293] In some embodiments, Z is -O-. In some embodiments, Z is -S-.

[0294] In some embodiments, Z is -N(-LR 1 In some embodiments, Z is —N(R 1 )-. In some embodiments, Z is -N(R')-. In some embodiments, Z is -N(R)-. In some embodiments, Z is -NH-.

[0295] In some embodiments, Z is L. In some embodiments, Z is a covalent bond. In some embodiments, Z is an optionally substituted, straight or branched C1-C 10is alkylene, and one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C( In some embodiments, Z is optionally and independently substituted by an optionally substituted C1-C2 alkyl group. 10 Alkylene or C1-C 10 In some embodiments, Z is methylene.

[0296] As generally described above and herein, L is a covalent bond or an optionally substituted, straight or branched C1-C 10 is alkylene, and one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C( Optionally and independently substituted by -O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-.

[0297] In some embodiments, L is a covalent bond. In some embodiments, L is an optionally substituted, straight or branched C1-C 10is alkylene, and one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C( Optionally and independently substituted by -O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-.

[0298] In some embodiments, L is -L 1 -V- structure, L 1 is an optionally substituted group: [ka] TIFF0007662687000022.tif34166TIFF0007662687000023.tif31166Selected from C1-C6 alkylene, C1-C6 alkenylene, carbocyclylene, arylene, C1-C6 heteroalkylene, heterocyclylene, and heteroarylene; V is -O-, -S-, -NR'-, C(R')2, -SS-, -BSSC-, [ka] or an optionally substituted group selected from C1-C6 alkylene, arylene, C1-C6 heteroalkylene, heterocyclylene, and heteroarylene; A is =O, =S, =NR', or =C(R')2; Each of B and C is independently -O-, -S-, -NR'-, -C(R')2-, or an optionally substituted group selected from C1-C6 alkylene, carbocyclylene, arylene, heterocyclylene, or heteroarylene; and Each R' is independently as defined above and described herein.

[0299] In some embodiments, L 1 teeth, [ka] TIFF0007662687000026.tif18166.

[0300] In some embodiments, L 1 teeth, [ka] and wherein ring Cy' is an optionally substituted arylene, carbocyclylene, heteroarylene, or heterocyclylene. 1 is optionally substituted [ka] In some embodiments, L 1 teeth, [ka] is.

[0301] In some embodiments, L 1 is bonded to X. In some embodiments, L 1 represents an optionally substituted group [ka] TIFF0007662687000031.tif30166, and the sulfur atom is bonded to V. In some embodiments, L 1 represents an optionally substituted group [ka] TIFF0007662687000033.tif30166, and the carbon atom is bonded to X.

[0302] In some embodiments, L is [ka] having the structure where: E is -O-, -S-, -NR'- or -C(R')2-; - - - is a single or double bond; The Two R's L1 taken together with the two carbon atoms to which they are attached form an optionally substituted aryl, carbocyclic, heteroaryl or heterocyclic ring; each R' is independently as defined above and as described herein.

[0303] In some embodiments, L is [ka] having the structure where: G is -O-, -S-, or -NR'; - - - is a single or double bond; and The Two R's L1 together with the two carbon atoms to which they are attached, form an optionally substituted aryl, C3-C 10 Forms a carbocyclic, heteroaryl or heterocyclic ring.

[0304] In some embodiments, L is [ka] having the structure wherein E is —O—, —S—, —NR′—, or —C(R′)2—; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and Each R' is independently as defined above and described herein.

[0305] In some embodiments, L is [ka] having the structure where: G is -O-, -S-, or -NR'; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-.

[0306] In some embodiments, L is [ka] having the structure where: E is -O-, -S-, -NR'- or -C(R')2-; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and Each R' is independently as defined above and described herein.

[0307] In some embodiments, L is [ka] having the structure where: G is -O-, -S-, or -NR'; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-.

[0308] In some embodiments, L is [ka] having the structure wherein E is —O—, —S—, —NR′—, or —C(R′)2—; - - - is a single or double bond; The Two R's L1 together with the two carbon atoms to which they are attached, form an optionally substituted aryl, C3-C 10 forming a carbocyclic, heteroaryl or heterocyclic ring; Each R' is independently as defined above and described herein.

[0309] In some embodiments, L is [ka] having the structure wherein G is -O-, -S-, or -NR'; - - - is a single or double bond; The Two R's L1 together with the two carbon atoms to which they are attached, form an optionally substituted aryl, C3-C 10 forming a carbocyclic, heteroaryl or heterocyclic ring; Each R' is independently as defined above and described herein.

[0310] In some embodiments, L is [ka] having the structure where: E is -O-, -S-, -NR'- or -C(R')2-; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and Each R' is independently as defined above and described herein.

[0311] In some embodiments, L is: [ka] (In the formula, G is -O-, -S-, or -NR'; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and each R' is independently as defined above and described herein.

[0312] In some embodiments, L is: [ka] (In the formula, E is -O-, -S-, -NR'- or -C(R')2-; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and each R' is independently as defined above and described herein.

[0313] In some embodiments, L is: [ka] (In the formula, G is -O-, -S-, or -NR'; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and each R' is independently as defined above and described herein.

[0314] In some embodiments, L is: [ka] (wherein E is —O—, —S—, —NR′—, or —C(R′)2—; - - - is a single or double bond; two R L1 together with the two carbon atoms to which they are attached, represent an optionally substituted aryl, C3-C 10 forming a carbocyclic, heteroaryl, or heterocyclic ring; and each R' is independently as defined above and described herein.

[0315] In some embodiments, L is [ka] having the structure wherein G is -O-, -S-, or -NR'; - - - is a single or double bond; The Two R's L1 together with the two carbon atoms to which they are attached, form an optionally substituted aryl, C3-C 10 forming a carbocyclic, heteroaryl or heterocyclic ring; Each R' is independently as defined above and described herein.

[0316] In some embodiments, L is [ka] having the structure wherein E is —O—, —S—, —NR′—, or —C(R′)2—; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and Each R' is independently as defined above and described herein.

[0317] In some embodiments, L is [ka] having the structure where: G is -O-, -S-, or -NR'; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and R' is as defined above and described herein.

[0318] In some embodiments, L is [ka] having the structure where: E is -O-, -S-, -NR'- or -C(R')2-; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and Each R' is independently as defined above and described herein.

[0319] In some embodiments, L is [ka] having the structure where: G is -O-, -S-, or -NR'; D is =N-, =C(F)-, =C(Cl)-, =C(Br)-, =C(I)-, =C(CN)-, =C(NO2)-, =C(CO2-(C1-C6 aliphatic))-, or =C(CF3)-; and R' is as defined above and described herein.

[0320] In some embodiments, L is [ka] having the structure wherein the phenyl ring may be substituted. In some embodiments, the phenyl ring is unsubstituted. In some embodiments, the phenyl ring is substituted.

[0321] In some embodiments, L is [ka] having the structure wherein the phenyl ring may be substituted. In some embodiments, the phenyl ring is unsubstituted. In some embodiments, the phenyl ring is substituted.

[0322] In some embodiments, L is [ka] having the structure where - - - is a single or double bond; and The Two R's L1 together with the two carbon atoms to which they are attached, form an optionally substituted aryl, C3-C 10 It forms a carbocyclic, heteroaryl or heterocyclic ring.

[0323] In some embodiments, L is [ka] having the structure where: G is -O-, -S-, or -NR'; - - - is a single or double bond; and The Two R's L1 together with the two carbon atoms to which they are attached, form an optionally substituted aryl, C3-C 10 It forms a carbocyclic, heteroaryl or heterocyclic ring.

[0324] As generally described above and herein, E is -O-, -S-, -NR'-, or -C(R')2-, where each R' is independently as defined above and described herein. In some embodiments, E is -O-, -S-, or -NR'-. In some embodiments, E is -O-, -S-, or -NH-. In some embodiments, E is -O-. In some embodiments, E is -S-. In some embodiments, E is -NH-.

[0325] As generally described above and herein, G is -O-, -S-, or -NR', where each R' is independently as defined above and described herein. In some embodiments, G is -O-, -S-, or -NH-. In some embodiments, G is -O-. In some embodiments, G is -S-. In some embodiments, G is -NH-.

[0326] In some embodiments, L is -L 3 -G-, where L 3is an optionally substituted C1-C5 alkylene or alkenylene, wherein one or more methylene units are optionally and independently -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -S(O)-, -S(O)2-, or [ka] is replaced by Each of G and R' and the ring Cy' is independently as defined above and as described herein.

[0327] In some embodiments, L is -L 3 -S- and L 3 is defined above and described herein. In some embodiments, L is -L 3 -O- and L 3 is defined above and described herein. In some embodiments, L is -L 3 -N(R')-, where L 3 and R' are each independently as defined above and as described herein. In some embodiments, L is -L 3 -NH- and L 3 and R' are each independently as defined above and described herein.

[0328] In some embodiments, L 3 is an optionally substituted C alkylene or alkenylene, wherein one or more methylene units are optionally and independently -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -S(O)-, -S(O)2-, or [ka] wherein each of R' and ring Cy' is independently as defined above and as described herein. 3is an optionally substituted C alkylene. 3 -G- is [ka] is.

[0329] In some embodiments, L 3 is an optionally substituted C alkylene or alkenylene, wherein one or more methylene units are optionally and independently —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)2—, or [ka] and each of R' and Cy' is independently as defined above and described herein.

[0330] In some embodiments, -L 3 -G- is [ka] The file is TIFF0007662687000061.tif34166.

[0331] In some embodiments, L 3 is an optionally substituted C alkylene or alkenylene, wherein one or more methylene units are optionally and independently —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)2—, or [ka] and each of R' and Cy' is independently as defined above and described herein.

[0332] In some embodiments, -L 3 -G- is [ka] The file is TIFF0007662687000064.tif29166.

[0333] In some embodiments, L is [ka] In some embodiments, L is [ka] In some embodiments, L is [ka] is.

[0334] In some embodiments, L 3 C2 is alkylene or alkenylene, and one or more methylene units are -O-, -S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -S(O)-, -S(O)2-, or [ka] wherein each of R' and Cy' is independently as defined above and as described herein.

[0335] In some embodiments, -L 3 -G- is [ka] and each of G and Cy' is independently as defined above and as described herein. In some embodiments, L is [ka] is.

[0336] In some embodiments, L is -L 4 -G- and L 4 is an optionally substituted C1-C2 alkylene; and G is as defined above and described herein. In some embodiments, L is -L 4 -G- and L 4 is an optionally substituted C1-C2 alkylene; G is as defined above and described herein; G is R 1 In some embodiments, L is bonded to -L 4 -G- and L 4 is an optionally substituted methylene; G is as defined above and described herein; and G is R 1 In some embodiments, L is linked to -L 4 -G- and L 4 is methylene; G is as defined above and described herein; and G is R 1 In some embodiments, L is linked to -L 4 -G- and L 4 is an optionally substituted —(CH)—; G is as defined above and described herein; and G is R 1 In some embodiments, L is linked to -L 4 -G- and L 4 is —(CH)—; G is as defined above and described herein; and G is R 1 is bonded to.

[0337] In some embodiments, L is [ka] or [ka] where G is as defined above and described herein, and G is R 1In some embodiments, L is attached to [ka] and G is as defined above and as described herein, and G is R 1 In some embodiments, L is attached to [ka] and G is as defined above and as described herein, and G is R 1 In some embodiments, L is attached to [ka] or [ka] where the sulfur atom is R 1 In some embodiments, L is attached to [ka] or TIFF0007662687000078.tif12166, where the oxygen atom is R 1 is connected to

[0338] In some embodiments, L is [ka] and G is as defined above and described herein.

[0339] In some embodiments, L is -SR L3 -or-SC(O)-R L3 - and R L3is an optionally substituted, straight-chain or branched C1-C9 alkylene, and one or more methylene units are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R' )C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, where each of R′ and -Cy- is independently as defined above and described herein. In some embodiments, L is selected from the group consisting of —SR L3 -or-SC(O)-R L3 - and R L3 is an optionally substituted C1-C6 alkylene. In some embodiments, L is -SR L3 -or-SC(O)-R L3 - and R L3 In some embodiments, L is -SR L3 -or-SC(O)-R L3 - and R L3 is an optionally substituted C1-C6 alkylene, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C1-C6 alkenylene, arylene, or heteroarylene. L3 is optionally substituted -S-(C1-C6 alkenylene)-, -S-(C1-C6 alkylene)-, -S-(C1-C6 alkylene)-arylene-(C1-C6 alkylene)-, -S-CO-arylene-(C1-C6 alkylene)-, or -S-CO-(C1-C6 alkylene)-arylene-(C1-C6 alkylene)-.

[0340] In some embodiments, L is [ka] The file is TIFF0007662687000081.tif30166.

[0341] In some embodiments, L is [ka] is. In some embodiments, L is [ka] is. In some embodiments, [ka] is.

[0342] In some embodiments, the sulfur atom of L in the embodiments above and described herein is bonded to X. In some embodiments, the sulfur atom of L in the embodiments above and described herein is bonded to R 1 is connected to

[0343] As generally described above and herein, R 1 is halogen, R, or optionally substituted C1-C 50an aliphatic group in which one or more methylene units are optionally substituted, such as C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, and optionally and independently substituted by -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, where each variable is independently as defined above and described herein. In some embodiments, R 1 is halogen, R, or optionally substituted C1-C 10 an aliphatic group in which one or more methylene units are optionally substituted, such as C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, and optionally and independently substituted by -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, where each variable is independently as defined above and described herein.

[0344] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is halogen. In some embodiments, R 1 is -F. In some embodiments, R 1 is -Cl. In some embodiments, R 1 is -Br. In some embodiments, R 1 is -I.

[0345] In some embodiments, R 1 is R, where R is as defined above and described herein.

[0346] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is C1~C 50 It is an optionally substituted group selected from aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

[0347] In some embodiments, R 1 is optionally substituted C1 to C 50 In some embodiments, R 1 is optionally substituted C1 to C 10 In some embodiments, R 1 is an optionally substituted C1-C6 aliphatic. In some embodiments, R 1 is an optionally substituted C1-C6 alkyl. In some embodiments, R 1 is optionally substituted, straight or branched hexyl. In some embodiments, R 1 is optionally substituted, straight or branched chain pentyl. In some embodiments, R 1 is optionally substituted, straight or branched butyl. In some embodiments, R 1 is optionally substituted, straight or branched propyl. In some embodiments, R 1 is optionally substituted ethyl. In some embodiments, R 1 is optionally substituted methyl.

[0348] In some embodiments, R 1 is optionally substituted phenyl. In some embodiments, R 1 is a substituted phenyl. In some embodiments, R 1 is phenyl.

[0349] In some embodiments, R 1 is an optionally substituted carbocyclyl. In some embodiments, R 1 is an optionally substituted C3 to C 10 In some embodiments, R 1 is an optionally substituted monocyclic carbocyclyl. In some embodiments, R 1 is optionally substituted cycloheptyl. In some embodiments, R 1 is optionally substituted cyclohexyl. In some embodiments, R 1 is optionally substituted cyclopentyl. In some embodiments, R 1 is optionally substituted cyclobutyl. In some embodiments, R 1 is optionally substituted cyclopropyl. In some embodiments, R 1 is an optionally substituted bicyclic carbocyclyl.

[0350] In some embodiments, R 1 is optionally substituted C1 to C 50 In some embodiments, R 1 is optionally substituted C1 to C 50It is a polycyclic hydrocarbon, and one or more methylene units are optionally substituted C1 to C6 alkylene, C1 to C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O) and optionally and independently substituted by -, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, where each variable is independently as defined above and described herein. 1 is optionally substituted [ka] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 is optionally substituted [ka] is.

[0351] In some embodiments, R 1 is an optionally substituted C1-C50 aliphatic group containing one or more optionally substituted polycyclic hydrocarbon moieties. 1 is an optionally substituted C1-C cyclic hydrocarbon group containing one or more optionally substituted polycyclic hydrocarbon moieties. 50an aliphatic group in which one or more methylene units are optionally substituted, such as C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, and optionally and independently substituted by -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, where each variable is independently as defined above and described herein. In some embodiments, R 1 is one or more optionally substituted C1 to C 50 aliphatic [ka] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth, [ka] In some embodiments, R 1 teeth, [ka] is.

[0352] In some embodiments, R 1 is optionally substituted aryl. In some embodiments, R 1 is an optionally substituted bicyclic aryl ring.

[0353] In some embodiments, R 1 is optionally substituted heteroaryl. In some embodiments, R 1 is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, or oxygen. In some embodiments, R 1 is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, or oxygen.

[0354] In some embodiments, R 1 is an optionally substituted 5-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0355] In some embodiments, R 1 is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is selected from pyrrolyl, furanyl, or thienyl.

[0356] In some embodiments, R 1is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. 1 is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom, and an additional heteroatom selected from sulfur or oxygen. 1 Groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

[0357] In some embodiments, R 1 is a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms. 1 is an optionally substituted 6-membered heteroaryl ring having 1 to 2 nitrogen atoms. In some embodiments, R 1 is an optionally substituted 6-membered heteroaryl ring having two nitrogen atoms. In certain embodiments, R 1 is an optionally substituted 6-membered heteroaryl ring having one nitrogen. Exemplary R groups are optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

[0358] In certain embodiments, R 1 is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted 5,6-fused heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 1 is an optionally substituted 5,6-fused heteroaryl ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 1is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted indolyl. In some embodiments, R 1 is an optionally substituted azabicyclo[3.2.1]octanyl. In certain embodiments, R 1 is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted azaindolyl. In some embodiments, R 1 is optionally substituted benzimidazolyl. In some embodiments, R 1 is optionally substituted benzothiazolyl. In some embodiments, R 1 is an optionally substituted benzoxazolyl. In some embodiments, R 1 is optionally substituted indazolyl. In certain embodiments, R 1 is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0359] In certain embodiments, R 1 is an optionally substituted 6,6-fused heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted 6,6-fused heteroaryl ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 1 is an optionally substituted 6,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1is an optionally substituted quinolinyl. In some embodiments, R 1 is optionally substituted isoquinolinyl. 1 is an optionally substituted 6,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is a quinazoline or quinoxaline.

[0360] In some embodiments, R 1 is optionally substituted heterocyclyl. In some embodiments, R 1 is an optionally substituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is a substituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an unsubstituted 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0361] In some embodiments, R 1 is optionally substituted heterocyclyl. In some embodiments, R 1 is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted, partially unsaturated 6-membered heterocyclic ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted, partially unsaturated six-membered heterocyclic ring having two oxygen atoms.

[0362] In certain embodiments, R 1 is a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 1 is oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranyl nonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxthiolinonyl, oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 1 is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0363] In certain embodiments, R 1 is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R 1 is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

[0364] In some embodiments, R 1 is an optionally substituted 8- to 10-membered bicyclic saturated or partially unsaturated heterocyclic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted indolinyl. In some embodiments, R 1 is an optionally substituted isoindolinyl. In some embodiments, R 1 is an optionally substituted 1,2,3,4-tetrahydroquinoline. In some embodiments, R is an optionally substituted 1,2,3,4-tetrahydroisoquinoline.

[0365] In some embodiments, R 1 is optionally substituted C1 to C 10 an aliphatic group in which one or more methylene units are optionally substituted, such as C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, and optionally and independently substituted by -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, where each variable is independently as defined above and described herein. In some embodiments, R 1is optionally substituted C1 to C 10 is aliphatic, wherein one or more methylene units are optionally and independently replaced by -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -OC(O)-, or -C(O)O-, each R' is independently as defined above and described herein. In some embodiments, R 1 is optionally substituted C1 to C 10 is aliphatic, wherein one or more methylene units are optionally and independently replaced by any of -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -OC(O)-, or -C(O)O-, wherein each R' is independently as defined above and described herein.

[0366] In some embodiments, R 1 teeth, [ka] The file is TIFF0007662687000095.tif184166.

[0367] In some embodiments, R 1 teeth, [ka] The file is TIFF0007662687000097.tif19166.

[0368] In some embodiments, R 1 includes a terminus having an optionally substituted —(CH)— moiety attached to L. Such exemplary R 1 The groups are as follows: [ka]

[0369] In some embodiments, R 1 is attached to L and includes an optionally substituted -(CH)- moiety. 1 The groups are as follows: [ka]

[0370] In some embodiments, R 1 -SR L2 and R L2 is an optionally substituted C1 to C9 aliphatic group, and one or more methylene units are optionally substituted C1 to C6 alkylene, C1 to C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R' )C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each of R′ and -Cy- is independently as defined above and described herein. In some embodiments, R 1 -SR L2 and the sulfur atom is bonded to the sulfur atom of the L group.

[0371] In some embodiments, R 1 is -C(O)-R L2 and R L2is an optionally substituted C1-C9 aliphatic group, and one or more methylene units are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C and optionally and independently substituted by -Cy(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, wherein each of R' and -Cy- is independently as defined above and described herein. In some embodiments, R 1 is -C(O)-R L2 and the carbonyl group is attached to the G of the L group. In some embodiments, R 1 is -C(O)-R L2 where the carbonyl group is bonded to the sulfur atom of the L group.

[0372] In some embodiments, R L2 is an optionally substituted C1-C9 aliphatic. L2 is an optionally substituted C1-C9 alkyl. In some embodiments, R L2 is an optionally substituted C1-C9 alkenyl. In some embodiments, R L2 is an optionally substituted C1-C9 alkynyl. In some embodiments, R L2 is an optionally substituted C1-C9 aliphatic, wherein one or more methylene units are optionally and independently replaced by -Cy- or -C(O)-. In some embodiments, R L2 is an optionally substituted C1-C9 aliphatic, wherein one or more methylene units are optionally and independently replaced by -Cy-. In some embodiments, R L2is an optionally substituted C1-C9 aliphatic, wherein one or more methylene units are optionally and independently replaced by an optionally substituted heterocyclohexylene. L2 is an optionally substituted C1-C9 aliphatic, wherein one or more methylene units are optionally and independently replaced by an optionally substituted arylene. L2 is an optionally substituted C1-C9 aliphatic, wherein each methylene unit is optionally and independently replaced by an optionally substituted heteroarylene. L2 is an optionally substituted C1-C9 aliphatic group, and one or more methylene units are optionally substituted C3-C 10 In some embodiments, R L2 is an optionally substituted C1-C9 aliphatic, wherein two methylene units are optionally and independently replaced by -Cy- or -C(O)-. In some embodiments, R L2 is an optionally substituted C1-C9 aliphatic, wherein two methylene units are optionally and independently replaced by -Cy- or -C(O)-. Exemplary R L2 The groups are as follows: [ka]

[0373] In some embodiments, R 1 is hydrogen or an optionally substituted group [ka] TIFF0007662687000102.tif25166-S-(C1~C 10 aliphatic), C1~C 10 In some embodiments, R is selected from aliphatic, aryl, C1-C6 heteroalkyl, heteroaryl, and heterocyclyl. 1 teeth, [ka] TIFF0007662687000104.tif24166 or -S-(C1~C 10 In some embodiments, R 1 teeth, [ka] The file is TIFF0007662687000106.tif24166.

[0374] In some embodiments, R 1 is -S-(C1~C6 aliphatic), C1~C 10 It is an optionally substituted group selected from aliphatic, C1-C6 heteroaliphatic, aryl, heterocyclyl and heteroaryl.

[0375] In some embodiments, R 1 teeth, [ka] is.

[0376] In some embodiments, R 1 In some embodiments, the sulfur atom of the R embodiment is bonded to a sulfur atom of the L embodiment described above and herein, to a G, E, or to a -C(O)- moiety. 1 The -C(O)- moiety of the embodiment of is bonded to the sulfur atom, G, E, or -C(O)- moiety of the embodiments of L described above and herein.

[0377] In some embodiments, -LR 1 is any of the embodiments of L and R described above and herein. 1 The embodiment is any one of the combinations of the above.

[0378] In some embodiments, -LR 1 -L 3-GR 1 where each variable is independently as defined above and described herein.

[0379] In some embodiments, -LR 1 -L 4 -GR 1 where each variable is independently as defined above and described herein.

[0380] In some embodiments, -LR 1 -L 3 -GSR L2 where each variable is independently as defined above and described herein.

[0381] In some embodiments, -LR 1 -L 3 -GC(O)-R L2 where each variable is independently as defined above and described herein.

[0382] In some embodiments, -LR 1 teeth, [ka] TIFF0007662687000109.tif16166, where R L2is an optionally substituted C1-C9 aliphatic group, and one or more methylene units are optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, - and optionally and independently substituted by N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-, wherein each G is independently as defined above and described herein.

[0383] In some embodiments, -LR 1 -R L3 -SSR L2 wherein each variable is independently as defined above and as described herein. In some embodiments, -LR 1 -R L3 -C(O)-SSR L2 where each variable is independently as defined above and described herein.

[0384] In some embodiments, -LR 1 teeth, [ka] having the structure wherein each variable is independently as defined above and as described herein.

[0385] In some embodiments, -LR 1 teeth, [ka] having the structure wherein each variable is independently as defined above and as described herein.

[0386] In some embodiments, -LR 1 teeth, [ka] having the structure wherein each variable is independently as defined above and as described herein.

[0387] In some embodiments, -LR 1 teeth, [ka] having the structure wherein each variable is independently as defined above and as described herein.

[0388] In some embodiments, -LR 1 teeth, [ka] having the structure wherein each variable is independently as defined above and as described herein.

[0389] In some embodiments, -LR ...

Claims

1. A compound having a structure represented by the formula: dAs dTs dTs dAs dAs dTs dAs dAs dAsdTs dTs dGs dTs dCs dAs dTs dCs dAs dCs dC, wherein the pattern of backbone chiral centers is Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Rp Sp Sp Sp Sp Sp Sp Sp Sp, Sp represents a phosphorothioate internucleotide linkage of the Sp structure; Rp indicates the phosphorothioate internucleotide linkage of the Rp structure; s represents a phosphorothioate internucleotide linkage; d represents a 2'-deoxy residue; An oligonucleotide, or a pharma- ceutically acceptable salt thereof.

2. A compound having a structure represented by the formula: dTs dAs dGs dCs dCs dAs dTs dTs dGs dCs dAs dGs dCs dTs dGs dCs dTs dCs dAs dC, wherein the pattern of backbone chiral centers is Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Rp Sp Sp Sp Sp Sp Sp, Sp represents a phosphorothioate internucleotide linkage of the Sp structure; Rp indicates the phosphorothioate internucleotide linkage of the Rp structure; s represents a phosphorothioate internucleotide linkage; d represents a 2'-deoxy residue; An oligonucleotide, or a pharma- ceutically acceptable salt thereof.

3. 3. The oligonucleotide or pharma- ceutically acceptable salt thereof according to claim 1 or 2, which is used to treat a disease by administering the oligonucleotide or pharma- ceutically acceptable salt thereof to a subject suffering from the disease.

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