Chiral design

Abstract

To provide a chirally controlled oligonucleotide, a composition thereof, and a purification method and usage thereof.SOLUTION: A chirally controlled oligonucleotide composition contains an oligonucleotide defined by having: 1) a common base sequence and length; 2) a common pattern of skeleton bond; and 3) a common pattern of a skeleton chiral center, where the composition is chirally controlled so as to be concentrated regarding a specific oligonucleotide-type oligonucleotide compared to a substantially racemic preparation of oligonucleotide having the same base sequence and length, the common pattern of the skeleton chiral center includes Rp(Sp)2 from 5' to 3', each of the plurality of oligonucleotides has 5 or more of chiral modified phosphoric acid bond, and at least 50% of chiral modified phosphoric acid bond of each of the plurality of oligonucleotides provides a composition having an Sp structure.SELECTED DRAWING: None

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 928,405, filed on January 16, 2014, and U.S. Provisional Patent Application No. 62 / 063,359, filed on October 13, 2014, the entire contents of each application being incorporated herein by reference. BACKGROUND OF THE INVENTION

[0002] Oligonucleotides are useful in therapeutic or diagnostic research and in the application of nanomaterials. Naturally occurring nucleic acids (e.g., unmodified DNA or RNA) are limited in therapeutic use due to their instability against extracellular and intracellular nucleases and / or their poor cell permeability and cell distribution properties. Furthermore, in in vitro studies, the binding affinity, sequence - specific binding to complementary RNA (Cosstick and Eckstein, 1985; LaPlanche et al., 1986; Latimer et al., 1989; Hacia et al., 1994; Mesmaeker et al., 1995), and nuclease stability, which are characteristics of antisense oligonucleotides, have been shown to be easily affected by the stereochemical absolute configuration of phosphorus atoms. Therefore, there is a need for newly improved oligonucleotides and oligonucleotide compositions, such as, for example, new antisense oligonucleotides, siRNA oligonucleotides, and oligonucleotide compositions. SUMMARY OF THE INVENTION

[0003] In particular, the present invention encompasses the recognition that a stereorandom oligonucleotide preparation contains a plurality of distinct chemical entities that differ from one another in the stereochemical structure of the individual backbone chiral centers within the oligonucleotide chain. Further, the present invention encompasses the insight that it is not usually likely that a stereorandom oligonucleotide preparation contains every possible stereoisomer of the relevant oligonucleotide. Thus, in particular, the present invention provides novel chemical entities that are specific stereoisomers of the oligonucleotide of interest. That is, the present invention provides a substantially pure preparation of a single oligonucleotide compound, which may be defined by its base sequence, its length, its pattern of backbone linkages, and its pattern of backbone chiral centers.

[0004] The present invention shows, in particular, that the individual stereoisomers of a particular oligonucleotide can exhibit different stabilities and / or activities from one another. Further, the present disclosure shows that the improvement in stability achieved by the inclusion and / or position of a particular chiral structure within an oligonucleotide can be equivalent to, or even better than, the improvement in stability 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 activities of oligonucleotides can be modulated by optimizing the pattern of backbone chiral centers. In some embodiments, the present invention provides a composition of oligonucleotides, wherein the oligonucleotides have a common pattern of backbone chiral centers that unexpectedly greatly improves the stability and / or bioactivity of the oligonucleotides. 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 an oligonucleotide is utilized to cleave a nucleic acid polymer, the pattern of backbone chiral centers of the oligonucleotide surprisingly alone changes 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 a new cleavage site. 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 the cleavage efficiency at the cleavage site. In some embodiments, the pattern of backbone chiral centers of the oligonucleotide improves the cleavage of the 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 the cleavage selectivity between two target sequences that differ only in a single nucleotide polymorphism (SNP), for example.

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

[0007] Definition Aliphatic: As used herein, the term "aliphatic" or "aliphatic group" refers to a straight-chain (i.e., unbranched) or branched-chain, substituted or unsubstituted hydrocarbon chain having one point of attachment to another molecule, which is completely saturated or contains one or more unsaturated units, or a monocyclic or polycyclic hydrocarbon that contains one or more unsaturated units but is not aromatic (also referred to herein as a "carbocyclic", "alicyclic" or "cycloalkyl"). In some embodiments, the aliphatic group contains from 1 to 50 aliphatic carbon atoms. Unless otherwise specified, the aliphatic group contains from 1 to 10 aliphatic carbon atoms. In some embodiments, the aliphatic group contains from 1 to 6 aliphatic carbon atoms. In some embodiments, the aliphatic group contains from 1 to 5 aliphatic carbon atoms. In other embodiments, the aliphatic group contains from 1 to 4 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains from 1 to 3 aliphatic carbon atoms, and in still other embodiments, the aliphatic group contains from 1 to 2 aliphatic carbon atoms. In some embodiments, "alicyclic" (or "carbocyclic" or "cycloalkyl") represents a monocyclic or bicyclic C3-C 10 hydrocarbon having one point of attachment to another molecule, which is completely saturated or contains one or more unsaturated units but is not aromatic. In some embodiments, "alicyclic" (or "carbocyclic" or "cycloalkyl") represents a monocyclic C3-C6 hydrocarbon having one point of attachment to another molecule, which is completely saturated or contains one or more unsaturated units but is not aromatic. Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl groups, alkenyl groups, alkynyl groups, and their complexes such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

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

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

[0010] Animal: As used herein, the term "animal" represents any member of the animal kingdom. In some embodiments, "animal" represents a human at any stage of development. In some embodiments, "animal" represents a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal can be a transgenic animal, a genetically modified animal, and / or a clone.

[0011] Approximately: As used herein, the words "approximately" or "about" when representing a number generally include numbers within the range of 5%, 10%, 15%, or 20% in either direction (greater or smaller) of the number, unless specifically stated or otherwise clear from the context (except when such number is a possible value less than 0% or greater than 100%). In some embodiments, the use of the word "about" when representing a dosage means ±5 mg / kg / day.

[0012] Aryl: As used alone or as part of a larger moiety as "aralkyl", "aralkoxy", or "aryloxyalkyl", the term "aryl" represents monocyclic and bicyclic ring structures having a total of 5 to 14 ring members, wherein at least one ring of the structure is aromatic and each ring of the structure contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". In certain embodiments of the present invention, "aryl" represents an aromatic ring structure including, but not limited to, phenyl, biphenyl, naphthyl, anthracyl, etc., which may have 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 moiety: As used herein, the phrase "characteristic moiety" of a protein or polypeptide generally includes a continuous series of amino acids, or a collection of continuous series of amino acids, which are characteristic of the protein or polypeptide as a whole. Each such continuous chain will generally contain at least two amino acids. Further, one of ordinary skill in the art will generally recognize that at least 5, 10, 15, 20 or more amino acids are necessary for a portion to be characteristic of a protein. Generally, a characteristic moiety shares at least one functional characteristic with a related intact protein in addition to the specific sequence homology described above.

[0014] Characteristic sequence: A "characteristic sequence" is a sequence found among all members of a family of polypeptides or nucleic acids and can thus be used by one of ordinary skill in the art to define the members of that family.

[0015] Characteristic structural element: The term "characteristic structural element" represents a clearly distinguishable structural element (e.g., backbone structure, collection of pendant moieties, sequence elements, etc.) found among all members of a family of polypeptides, small molecules, or nucleic acids and can thus be used by one of ordinary skill 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 environments that are sufficiently similar to each other to enable a comparison of the resulting outcomes or observed phenomena. In some embodiments, equivalent sets of conditions or environments are characterized by a plurality of substantially identical features and one or a few modified features. One of ordinary skill in the art will recognize that when two sets of conditions or environments are characterized by a sufficient number and type of substantially identical features, and the differences in the resulting outcomes or observed phenomena obtained under the different sets of conditions or environments can justify the reasonable conclusion that the differences in those modified features between the sets of conditions are causative or indicative, then the two sets are equivalent to each other.

[0017] Dosing regimen: As used herein, "dosing regimen" or "treatment regimen" typically refers to a set of unit doses (usually one or more) that are administered individually to a subject over 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, the dosing regimen includes multiple doses that are each separated from one another by the same length of time; in some embodiments, the dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all of the doses within the dosing regimen are the same unit dose. In some embodiments, the different doses within the dosing regimen are different amounts. In some embodiments, the dosing regimen includes a first dose at a first dosage, followed by one or more additional doses at a second dosage that is different from the first dosage. In some embodiments, the dosing regimen includes a first dose at a first dosage, followed by one or more additional doses at a second dosage that is the same as the first dosage.

[0018] Equivalent agents: Reading this disclosure, those skilled in the art will recognize that the scope of useful agents 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 an active agent typically has a structure consisting of a backbone and attached pendant moieties, and thus will understand that simple modifications of such a backbone and / or pendant moieties will not significantly change the activity of the agent. For example, in some embodiments, substitution of one or more pendant moieties having equivalent three-dimensional structures and / or chemical reactivity characteristics may produce a substituted compound or moiety equivalent to the parent reference compound or moiety. In some embodiments, addition or removal of one or more pendant moieties may produce a compound substituted equivalently to the parent reference compound. In some embodiments, for example, modification of the backbone structure by addition or removal of a small number of bonds (usually 5, 4, 3, 2 or fewer, or 1 bond, and often single bonds only) may produce a compound substituted equivalently to the parent reference compound. In many embodiments, equivalent compounds may be synthesized, for example, using readily available materials, reagents and conventional or provided synthetic procedures, by the methods shown in the following general reaction schemes, or variations thereof. Modifications that are known per se but not mentioned here are also available in these reactions.

[0019] Equivalent dosage: The term "equivalent dosage" is used herein to compare the dosages of different pharmaceutically active agents that produce the same biological result. The dosages of two different agents are considered "equivalent" to each other according to the present invention if they achieve equivalent levels or degrees of biological result. In some embodiments, the 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 lysosomal activators used according to the present invention are utilized at a dosage equivalent to the dosage of a reference lysosomal activator; in some embodiments, a reference lysosomal activator for such purposes is selected from the group consisting of small molecule allosteric activators (e.g., pyrazolpyrimidines), imminosugars (e.g., isofagomine), antioxidants (e.g., n-acetylcysteine), and regulators of cellular trafficking (e.g., Rab1a polypeptide).

[0020] Heteroaliphatic: The term "heteroaliphatic" represents an aliphatic group in which one or more units selected from C, CH, CH2, or CH3 are independently replaced by a heteroatom. In some embodiments, the heteroaliphatic group is heteroalkyl. In some embodiments, the heteroaliphatic group is heteroalkenyl.

[0021] Heteroaryl: The terms "heteroaryl" and "heteroal-" used alone or as part of a larger moiety, such as "heteroalkyl", or "heteroalkoxy", represent a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" represents nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of any basic nitrogen. Examples of 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 "heteroal-" also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, 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. A heteroaryl group can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring", "heteroaryl group", or "aromatic heterocycle", and any of these terms includes rings that may be substituted. The term "heteroalkyl" represents an alkyl group substituted by heteroaryl, and the alkyl and heteroaryl moieties are independently optionally substituted.

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

[0023] Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical", and "heterocyclic ring" are used interchangeably and represent stable 3- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moieties that are saturated or partially unsaturated and have, in addition to carbon atoms, one or more, preferably 1 to 4, of the above heteroatoms. When used to represent the ring atoms 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 can be N (such as in 3,4-dihydro-2H-pyrrolyl), NH (such as in pyrrolidinyl) or + NR (such as in N-substituted pyrrolidinyl).

[0024] The complex ring can be attached to any heteroatom or side group on the carbon atom that provides a stable structure, and any ring atom may 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 "heterocyclic ring", "heterocyclyl", "heterocyclic ring", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclic ring is fused to one or more aryl groups, heteroaryl groups, or aliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, and the radical or point of attachment is on the heterocyclic ring. The heterocyclic ring can be monocyclic or bicyclic. The term "heterocyclylalkyl" represents an alkyl group substituted by heterocyclyl, and the alkyl and heterocyclyl moieties may be independently substituted.

[0025] Intraperitoneal: As used herein, the phrases "intraperitoneal administration" and "administered intraperitoneally" have the meaning understood in the art to represent the administration of a compound or composition into the peritoneum 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, a plant, and / or a microorganism), but in an artificial environment, such as in a test tube or reactor, in cell culture medium, or otherwise.

[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 C 1~4It represents a linear or branched alkyl group. As an example, lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

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

[0030] Optionally substituted: When described herein, the compounds of the present invention may contain a portion that is "optionally substituted". In general, the term "substituted", whether or not the term "optionally" is present, means that one or more hydrogens of the designated portion are substituted with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group when one or more positions in any given structure may be the same or different for each position. Combinations of substituents contemplated by the present invention preferably result in the formation of stable or chemically possible compounds. As used herein, the term "stable" refers to a compound that does not substantially change when in a state that allows for their manufacture, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0031] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH2) 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 ○ optionally substituted (CH2) 0~4 Ph; R ○ optionally substituted (CH2) 0~4O(CH2) 0~1 Ph; R ○ CH=CHPh; R which may be substituted ○ (CH2) which may be substituted 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)2NR ○ 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)O-N(R ○ )2; or -(C 1~4 linear or branched alkylene)C(O)O-N(R ○ )2(wherein each R ○ may be substituted as follows and is independently hydrogen, C 1~6 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, -CH2-(5-6 membered heteroaryl ring) or a 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 Rs that appear independently ○which, together with the intervening atoms thereof, may be substituted as follows and independently form a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms selected from nitrogen, oxygen, or sulfur).

[0032] R ○ The above suitable monovalent substituents (or the rings formed together by two independently occurring Rs ○ are independently halogen, -(CH2) 0~2 R ● , -(haloR ● ), -(CH2) 0~2 OH, -(CH2) 0~2 OR ● , -(CH2) 0~2 CH(OR ● )2; O(haloR ● ), -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 linear or branched alkylene)C(O)OR ● , or -SSR ● (wherein each R ● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens and, independently, C 1~4 aliphatic, -CH2Ph, -O(CH2) 0~1(selected from Ph, or an aryl ring having 0 to 4 heteroatoms independently selected from 5- or 6-membered saturated, partially unsaturated, or nitrogen, oxygen, or sulfur). R ○ Suitable divalent substituents on the saturated carbon atoms of R include =O and =S.

[0033] Suitable divalent substituents on the saturated carbon atoms of the "optionally substituted" group include the following: =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 independently appearing * is hydrogen, a C that can be substituted as described below 1~6 aliphatic, or selected from unsubstituted 5- or 6-membered saturated, partially unsaturated, or aryl rings having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur). Suitable divalent substituents bonded to the adjacent substitutable carbon of the "optionally substituted" group: -O(CR * 2) 2~3 O- (wherein each R independently appearing * is hydrogen, a C that can be substituted as described below 1~6 aliphatic, or selected from unsubstituted 5- or 6-membered saturated, partially unsaturated, or aryl rings having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0034] Suitable substituents on the aliphatic group of R* include 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", is substituted only by one or more halogens, and, independently, C 1~4 aliphatic, -CH2Ph, -O(CH2) 0~1 Ph, or an aryl ring having 5 to 6 members, saturated, partially unsaturated, or having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur).

[0035] Suitable substituents on the replaceable nitrogen of the "optionally substituted" group include -R † , -NR † 2, -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH2C(O)R † , -S(O)2R † , S(O)2NR † 2, -C(S)NR † 2, -C(NH)NR † 2, or -N(R † )S(O)2R † ; (wherein each R † is independently halogen, a C 1~6 aliphatic which may be substituted as described below, unsubstituted -OPh, or an aryl ring having 5 to 6 members, saturated, partially unsaturated, or having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independently occurring R † together with the intervening atoms form, independently, an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms selected from nitrogen, oxygen, or sulfur).

[0036] Suitable substituents on the 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, independently, is C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and being saturated, partially unsaturated, or 5-6 membered).

[0037] Oral: As used herein, the terms "oral administration" and "administered orally" refer to the oral administration of a compound or composition and have the meaning understood in the art.

[0038] Parenteral: As used herein, the terms "parenteral administration" and "administered parenterally" refer to a method of administration other than enteral and topical administration, usually by injection, and have the meaning understood in the art and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injection and infusion.

[0039] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple unsaturated portions 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 amount appropriate for administration in a therapeutic regimen that, when administered to a suitable population, exhibits a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be specifically formulated for administration in solid or liquid form, for the following uses: oral administration, e.g., aqueous or non-aqueous solutions or suspensions, tablets, e.g., for buccal, sublingual, and systemic absorption uses, boluses, powders, granules, pastes applied to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, as a sterile solution or suspension, or as a sustained release formulation; topical administration, e.g., as creams, ointments, or sustained release patches or sprays applied to the skin, lung, or mouth; intravaginally or rectally, e.g., as pessaries, creams, or foams; sublingually; ophthalmically; transdermally; or nasally, the lung, and other mucosal surfaces are included.

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

[0042] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or medium such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material involved in the conveyance or transport from one organ, or part of the body, to another organ, or part of the body. 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 are: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; celluloses and their derivatives such as sodium carboxymethyl cellulose, 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; buffering agents 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 innocuous compatible substances used in pharmaceutical formulations.

[0043] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in a pharmaceutical context, i.e., within the sound medical judgment, commensurate with a reasonable benefit / risk ratio, without undue toxicity, irritation, allergic response, etc., and are suitable for use in contact with the tissues of humans and lower animals. Pharmaceutically acceptable salts are well known in the art. For example, S. M. 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, 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, etc., but are not limited thereto. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. In some embodiments, pharmaceutically acceptable salts include, where appropriate, harmless ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates and arylsulfonates having 1 to 6 carbon atoms.

[0044] Prodrug: Generally, as the term is used herein, a “prodrug” is, as understood in the art, an entity that, when administered to an organism, is metabolized in the body to deliver an agent of interest (e.g., a therapeutic or diagnostic) agent. Typically, such metabolism causes the removal of at least one “prodrug moiety” such that the active agent is generated. 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) See references.

[0045] Like the other compounds described herein, prodrugs can be provided in various forms, such as crystalline forms, salt forms, and others. In some embodiments, the prodrug is provided as its pharmaceutically acceptable salt.

[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 (which is hereby incorporated by reference in its entirety). Also included are protecting groups particularly adapted to nucleotides and nucleotide chemistry as described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012 (the entire text of Chapter 2 is hereby incorporated 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-methoxyphenanthryl 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-dicyclohexylcarboxamide)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl 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 derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamide)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamide)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-methylcyclohexyl 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 carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyl-oxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiosuccinimide (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-dinitropyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberlylamine, 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, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylideneamine, N-p-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-diphenylboric acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosoamines, amine N-oxides, diphenylphosphine amide (Dpp), dimethylthiophosphine amide (Mpt), diphenylthiophosphine amide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (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 are included.,

[0047] Suitably 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, 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-pentenyl oxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 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-trichloroethyl, 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-(imidazol-1-yl)bis(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), dimethyltexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivalate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethyl carbonate, 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-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 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-naphthoate, nitrate ester, alkyl N,N,N’,N’-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate ester, dimethylphosphinothionyl, alkyl 2,4-dinitrophenylsulfenate, sulfate ester, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). To protect 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, 3,4-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 derivative, α-(N,N'-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanilidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronic esters, ethyl boronate, and phenyl boronate are included.,

[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, benzoylglycolate, 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 group is 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 the internucleotide phosphite linkage throughout oligonucleotide synthesis. In some embodiments, the phosphite protecting group binds to the sulfur atom of an internucleotide phosphorothioate linkage. In some embodiments, the phosphite protecting group binds to the oxygen atom of an internucleotide phosphorothioate linkage. In some embodiments, the phosphite protecting group binds to the oxygen atom of an internucleotide phosphate linkage. 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 joined to another by peptide bonds). In some embodiments, the protein comprises only naturally occurring amino acids. In some embodiments, the protein comprises one or more non-naturally occurring amino acids (e.g., moieties that form one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues of the protein chain comprise non-amino acid moieties (e.g., glycans, others). In some embodiments, the protein comprises more than one polypeptide chain joined, for example, by one or more disulfide bonds or associated by other means. In some embodiments, the protein comprises L-amino acids, D-amino acids, or both; in some embodiments, the protein comprises one or more amino acid modifications or analogs well known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, others. The term "peptide" is generally used to refer to a polypeptide 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" is a specific organism or material from which it is obtained. In some embodiments, the sample is a biological sample obtained from or derived from a subject source as described herein. In some embodiments, the subject source 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 includes bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; cell-free nucleic acid; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural effusion; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; wash or lavage fluid such as catheter wash or bronchoalveolar lavage fluid; aspirate; scraping; bone marrow specimen; tissue biopsy specimen; surgical specimen; feces, other body fluids, secretions, and / or cells therefrom, among others, or includes the same. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" directly obtained from a subject source by any suitable means. For example, in some embodiments, the 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 body fluid (e.g., blood, lymph, feces, etc.). In some embodiments, as is clear from the context, the term "sample" refers to a preparation obtained by treating a primary sample (e.g., by removal of one or more of its components and / or addition of one or more agents thereto). For example, filtration using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins obtained by extraction from a sample or treatment of a primary sample by techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of specific components, among others. In some embodiments, the sample is a biological entity. 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 term "stereochemical isomers" refers to different compounds that are assembled from the same atoms bonded by the same series of bonds but have non-interchangeable different three-dimensional structures. In some embodiments of the present invention, the provided chemical composition can be, or can include, a pure composition of the individual stereochemical isomers of the compound; in some embodiments, the provided chemical composition can be, or can include, a mixture of two or more stereochemical isomers of the compound. In certain embodiments, such a mixture contains equal amounts of different stereochemical isomers; in certain embodiments, such a mixture contains different amounts of at least two different stereochemical isomers. In some embodiments, the chemical composition can include all of the diastereomers and / or enantiomers of the compound. In some embodiments, the chemical composition can include fewer diastereomers and / or enantiomers than all of the compound. In some embodiments, if a particular enantiomer of a compound of the present invention is desired, it can be synthesized, for example, by asymmetric synthesis or by derivation using a chiral auxiliary, and the resulting mixture of diastereomers is separated and the auxiliary is cleaved to obtain the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group such as an amino group, diastereomeric salts are formed using an appropriate optically active acid and resolved, for example, by fractional recrystallization.

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

[0055] Substantially: As used herein, the term "substantially" represents a qualitative state indicating all or almost all of the range or degree of a targeted feature or characteristic. One of ordinary skill in the art of biotechnology will understand that biological and chemical phenomena rarely avoid being complete and / or finished or achieved or absolute results. Thus, the term "substantially" is used herein to account for the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0056] Affected: An individual "affected" with a disease, disorder, and / or symptom has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or symptom.

[0057] Prone to (a disease): An individual "prone to" a disease, disorder, and / or symptom has a higher risk of developing the disease, disorder, and / or symptom than a member of the general population. In some embodiments, an individual prone to a disease, disorder, and / or symptom may not be diagnosed with the disease, disorder, and / or symptom. In some embodiments, an individual prone to a disease, disorder, and / or symptom may exhibit symptoms of the disease, disorder, and / or symptom. In some embodiments, an individual prone to a disease, disorder, and / or symptom may not exhibit symptoms of the disease, disorder, and / or symptom. In some embodiments, an individual prone to a disease, disorder, and / or symptom will develop the disease, disorder, and / or symptom. In some embodiments, an individual prone to a disease, disorder, and / or symptom will not develop the disease, disorder, and / or symptom.

[0058] Systemic: As used herein, the phrases "systemic administration", "systemically administered", "peripheral administration", and "peripherally administered" have the meaning understood in the art to represent administering a compound or composition such that it enters the entire body of the recipient.

[0059] Tautomers: As used herein, the term "tautomer" is used to denote an organic compound of different isomers that are readily convertible. Tautomers can be characterized by the formal migration of a hydrogen atom or proton, which occurs simultaneously with the conversion of a single bond and an adjacent double bond. In some embodiments, tautomers can result from proton tautomerism (i.e., rearrangement of protons). In some embodiments, tautomers can result from valence tautomerism (i.e., rapid rearrangement of bonding electrons). All such tautomers are intended to be included within the scope of the present invention. In some embodiments, the tautomers of a compound are present in a mobile equilibrium with each other such that attempts to synthesize the separate substances result in the formation of a mixture. In some embodiments, the tautomers of a compound are separable and isolable compounds. In some embodiments of the present invention, a chemical composition can be provided that is or includes a pure composition of a single tautomer of a compound. In some embodiments of the present invention, a chemical composition can be provided as a mixture of two or more tautomers of a compound. In certain embodiments, such a mixture contains equal amounts of the different tautomers; in certain embodiments, such a mixture contains different amounts of at least two tautomers of a compound. In some embodiments of the present invention, a chemical composition can contain all of the tautomers of a compound. In some embodiments of the present invention, a chemical composition can contain less than all of the tautomers of a compound. In some embodiments of the present invention, a chemical composition can contain one or more tautomers of a compound in amounts that change over time as a result of interconversion. In some embodiments of the present invention, the tautomerism is keto-enol tautomerism. One skilled in the art of chemistry can "capture" (i.e., chemically modify to retain the "enol" form) keto-enol tautomerism using any suitable reagent well known in the art of chemistry and can subsequently obtain an enol derivative that can be isolated using one or more suitable techniques well 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 reduce, relieve, alleviate, suppress, prevent, delay onset, reduce severity, and / or decrease 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" means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, induces a desired biological response. In some embodiments, the therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay onset of a disease, disorder, and / or condition in a subject afflicted with or susceptible to the disease, disorder, and / or condition. As will be appreciated 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 being delivered, the target cell or tissue, and others. For example, the effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that reduces, relieves, alleviates, suppresses, prevents, delays onset, reduces severity, and / or decreases incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0062] Treatment: As used herein, the terms "treating", "treatment", or "to treat" refer to any method used to partially or completely alleviate, relieve, liberate, 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. Treatment can be administered to a subject who does not exhibit symptoms of a disease, disorder, and / or condition. In some embodiments, for example, for the purpose of reducing the risk of developing a pathology associated with a disease, disorder, and / or condition, treatment can be administered to a subject who exhibits only the initial symptoms of the disease, disorder, and / or condition.

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

[0064] Unit Dose: As used herein, the expression "unit dose" refers to the single dose of a pharmaceutical composition and / or the amount administered in physically discrete units. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains the 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 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, a sustained release formulation, or a drug delivery device containing a predetermined amount of one or more therapeutic agents, among others. It will be appreciated that a unit dose can be present in a formulation containing any of a variety of additional components in addition to the therapeutic agent. For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, and the like can be included, as described below. In many embodiments, it will be understood by those skilled in the art that the appropriate total daily dosage of a particular therapeutic agent can include a portion, or multiple unit doses, and can be determined, for example, by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dosage level for any particular subject or organism can depend on a variety of factors including the disorder being treated and the severity of the disorder; the potency of the specific active compound being used; the specific composition being used; the age, weight, health status, gender, and diet of the subject; the frequency of administration, and the rate of excretion of the specific compound being used; the duration of the treatment; drugs and / or additional therapies being used in combination with or concurrently with the specific compound being used, and similar factors well known in the medical arts.

[0065] Wild-Type: As used herein, the term "wild-type" has the meaning understood in the art thereof, representing an entity having the structure and / or activity actually found in a "normal" (as contrasted with mutant, diseased, altered, etc.) state or context. Those skilled in the art will recognize that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0066] Nucleic acid: The term "nucleic acid" includes any nucleotide, its analogs, and its polymers. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, representing ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms represent the primary structure of the molecule and thus include double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. These terms include, by way of non-limiting equivalents, nucleotide analogs and modified polynucleotides such as methylated, protected, and / or capped nucleotides or polynucleotides, and analogs of either RNA or DNA synthesized from such modified polynucleotides. The term includes poly or oligoribonucleotides (RNA) and poly or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides 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 "inter-nucleotide linkages"). The term includes 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 a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both a ribose moiety and a deoxyribose moiety, and nucleic acids containing a ribose moiety and a modified ribose moiety. The prefix poly represents a nucleic acid containing 2 to about 10,000 nucleotide monomer units, and the prefix oligo represents 2 to about 200 nucleotide monomer units.

[0067] Nucleotide: As used herein, the term "nucleotide" refers to the monomeric units of polynucleotides consisting of a heterocyclic base, a sugar, and one or more phosphate groups or phosphor-containing internucleotide linkages. Natural bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are purine or pyrimidine derivatives, but it should be understood that natural and non-natural base analogs are also included. Natural sugars are pentose (five-carbon sugars) deoxyribose (forming DNA) or ribose (forming RNA), but it should be understood that natural and non-natural sugar analogs are also included. Nucleotides are joined through internucleotide linkages to produce nucleic acids, or polynucleotides. Many internucleotide linkages are well known in the art (including, but not limited to, phosphate, phosphorothioate, boranophosphate, etc.). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonate esters, amidophosphite esters, boranophosphate esters, methylphosphonothioate esters, phosphonacetate esters, thiophosphonacetate esters, and other variants of the phosphate backbone of natural nucleic acids such as those described herein.

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

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

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

[0071] Nucleobase: The term "nucleobase" refers to the nucleic acid moiety associated with the hydrogen bonds that bind one nucleic acid strand to another complementary strand in a sequence-specific manner. Most natural nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the natural nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the natural nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase", for example, 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 nucleobase mimics the configuration, electronic state, or some other physicochemical property of the nucleobase and retains the property of the hydrogen bond that binds one nucleic acid strand to another complementary strand in a sequence-specific manner. In some embodiments, the modified nucleobase can pair with the five all-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 the reaction can be carried out with a specific stereoselectivity.

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

[0074] Blocking group: The term "blocking group" refers to a group that shields the reactivity of a functional group. The functional group can subsequently have the shielding removed by removal of the blocking group. In some embodiments, the blocking group is a protecting group.

[0075] Part: The term "part" refers to a specific segment or functional group of a molecule. Chemical parts are often recognized as chemical entities incorporated into or added to a molecule.

[0076] Solid support: The term "solid support" refers to any support that enables the synthesis of nucleic acids. In some embodiments, the term refers to a glass or polymer that is insoluble in the medium used in the reaction steps for performing nucleic acid synthesis and derivatizing 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 CPG. In some embodiments, the solid support is a composite support of 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 its single-stranded form or the polymeric form of double-stranded deoxyribonucleotides (adenine, guanine, thymine, or cytosine). This term refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Thus, this term includes, inter alia, linear DNA molecules (e.g., restriction enzyme fragments), viruses, plasmids, and double-stranded DNA found in chromosomes. In discussing a particular double-stranded DNA molecule structure, sequences may be described herein according to the convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA).

[0079] Coding sequence: A DNA "coding sequence" or "coding region" is double-stranded DNA that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence (the "open reading frame" or "ORF") are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxylic acid) 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 usually located on the 3' side of the coding sequence. The term "non-coding sequence" or "non-coding region" refers to regions of 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 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 contains 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 the sense nucleic acid encoding a protein complementary to the gene coding strand. Thus, an antisense nucleic acid molecule can associate with a sense nucleic acid molecule via hydrogen bonding.

[0082] Fluctuation position: As used herein, "fluctuation position" refers to the third position of a codon. In some embodiments, mutations in a DNA molecule within the fluctuation position of a codon result in silent or conservative mutations at the amino acid level. For example, there are four codons that encode glycine, namely GGU, GGC, GGA, and GGG. Thus, mutations of the nucleotides at any fluctuation position to other nucleotides selected from A, U, C, and G do not result in a change at the amino acid level of the encoded protein and are thus silent substitutions.

[0083] Silent substitution: "Silent substitution" or "silent mutation" refers to a situation where the nucleotide within a codon is changed, but no change in the amino acid residue encoded by the codon is brought about. Examples include mutations not only at the first position of certain codons such as the codon "CGG" that still encodes Arg even when mutated to AGG, but also mutations at 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 DNA molecule portion that encodes a protein or a portion thereof. The DNA molecule may include an open reading frame that encodes the protein (as an exon sequence) and may further include an intron sequence. As used herein, the term "intron" refers to a DNA sequence that is present in a given gene that is not translated into a protein and, although not in all cases, is found in some cases between exons. As is well known in the art, it may be desirable for a gene to be operably linked to (or include) one or more promoters, enhancers, repressors, and / or other regulatory sequences that regulate the activity or expression of the gene.

[0085] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes a recombinant polynucleotide synthesized by reverse transcription of mRNA, from which intervening sequences (introns) are 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 comparison of the positions of each sequence that can be aligned for comparison purposes. When the same position of the sequences being compared 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 residues (e.g., similar in steric and / or electronic state), then the molecules can be said to be homologous (similar) at that position. Expression as a percentage of homology / similarity or identity represents a function of the number of identical or similar nucleic acids at positions shared by the sequences being compared. "Unrelated" or "non-homologous" sequences share less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with the sequences 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 genes with similar functions or motifs. The nucleic acid sequences described herein can be used, for example, as "query sequences" for performing searches against public databases 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, the BLAST nucleotide search 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, to obtain gap alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., 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 so as to maximize 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, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., 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 give the maximum match between the sequences tested. Further, methods to determine identity are encoded in publicly available computer programs.Examples of computer program methods for determining identity between two arrays 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 can also be used for identity determination.

[0089] Non-homologous: A "non-homologous" region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found at all in relation to the larger sequence. Thus, when a non-homologous region encodes a mammalian gene, the gene can typically be on the DNA side that is not adjacent to the mammalian genomic DNA in the source organism's genome. Another example of a non-homologous coding sequence is a sequence in which the coding sequence itself is not found at all (e.g., a cDNA containing an intron or synthetic sequence with codons or motifs different from the unmodified gene in the genomic coding sequence). Allelic variations or natural mutation events do not result in non-homologous regions of DNA as defined herein.

[0090] Transversion mutation: The term "transversion mutation" refers to a change in a base 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] Base conversion mutation: The term "base conversion mutation" refers to a change in the bases in a DNA sequence where 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 that includes any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate linkages, or modified phosphorus atom linkages (also referred to herein as "inter-nucleotide linkages" as further defined herein).

[0093] An oligonucleotide can be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" includes single-stranded oligonucleotides. A single-stranded oligonucleotide can have double-stranded regions, and a double-stranded oligonucleotide can have single-stranded regions. Exemplary oligonucleotides include, but are not limited to, structural genes, genes including regulatory regions and terminal regions, viral or plasmid DNA, self-replicating systems such as single-stranded and double-stranded siRNA and other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermir(supermir), aptamers, antimir, antagomir, Ul adapter, triple helix-forming oligonucleotides, guanine quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

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

[0095] The oligonucleotides of the present invention can be of various lengths. In certain embodiments, the oligonucleotide can range in length from about 2 to about 200 nucleotides. 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, about 20 to about 30 nucleotides. In some embodiments, the oligonucleotide is about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotide is at least 4 nucleotides in length. In some embodiments, the oligonucleotide is at least 5 nucleotides in length. In some embodiments, the oligonucleotide is at least 6 nucleotides in length. In some embodiments, the oligonucleotide is at least 7 nucleotides in length. In some embodiments, the oligonucleotide is at least 8 nucleotides in length. In some embodiments, the oligonucleotide is at least 9 nucleotides in length. In some embodiments, the oligonucleotide is at least 10 nucleotides in length. In some embodiments, the oligonucleotide is at least 11 nucleotides in length. In some embodiments, the oligonucleotide is 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 the complementary strand of a double-stranded at least 18 nucleotides in length. In some embodiments, the oligonucleotide is the complementary strand of a double-stranded at least 21 nucleotides in length.

[0096] Inter-nucleotide linkage: As used herein, the phrase "inter-nucleotide linkage" generally refers to a phosphorus-containing linkage between nucleotide units of an oligonucleotide and is synonymous herein and above with "sugar-sugar linkage" and "phosphorus atom bridge". In some embodiments, the inter-nucleotide linkage is a phosphodiester linkage found in natural DNA and RNA molecules. In some embodiments, the inter-nucleotide linkage is a "modified inter-nucleotide 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 inter-nucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage

Chemical formula

[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 inter-nucleotide linkages shown in Table 1 below.

[0098] Table 1. Example modified inter-nucleotide linkages

Table 1

[0099] For example, (Rp,Sp)-ATsCs1GA has 1) a phosphorothioate nucleotide internucleotide linkage between T and C (

Chem.

Chem.

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

[0101] One of ordinary skill in the art will recognize that the synthetic 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 particular stereochemistry at the linking phosphorus and / or a particular modification at the linking phosphorus and / or a particular base and / or a particular sugar. In some embodiments, the oligonucleotide chain is designed and / or determined to have a particular combination of modifications at the linking phosphorus. In some embodiments, the oligonucleotide chain is designed and / or selected to have a particular combination of bases. In some embodiments, the oligonucleotide chain is designed and / or selected to have a particular combination of one or more of the above structural features. The present invention provides a composition comprising or consisting of a plurality of oligonucleotide molecules (e.g., a chirally controlled oligonucleotide composition). In some embodiments, all such molecules are of the same type (i.e., structurally identical to each other). However, in many embodiments, the composition provided will typically contain a plurality of oligonucleotides of different types in predetermined relative amounts.

[0102] Chiral control: As used herein, "chiral control" refers to the ability to control the stereochemical designation for each chiral linking phosphorus within an oligonucleotide chain. The phrase "chirally controlled oligonucleotide" refers to an oligonucleotide that exists as a single diastereoisomer with respect to the chiral linking phosphorus.

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

[0104] Chirally Pure: As used herein, the phrase "chirally pure" is used to denote a chiral-controlled oligonucleotide composition in which all oligonucleotides are present as a single diastereoisomer with respect to the linking phosphorus.

[0105] Chirally Homogeneous: As used herein, the phrase "chirally homogeneous" is used to denote an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the linking phosphorus. For example, an oligonucleotide in which all of its nucleotide units have Rp stereochemistry at the linking phosphorus is chirally homogeneous. Similarly, an oligonucleotide in which all of its nucleotide units have Sp stereochemistry at the linking phosphorus is chirally homogeneous.

[0106] Predetermined: As used herein, "predetermined" means, for example, randomly occurring or, as the antonym of achievement, selected in a planned manner. One of ordinary skill in the art, upon reading this specification, will understand that the present invention provides novel and surprising techniques that enable the selection of a specific oligonucleotide type for the formulation and / or encapsulation of the provided composition, and further enable the precisely controlled formulation of the selected specific type in the specific relative amounts selected such that the provided composition is 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 accidentally cannot control the intentional creation of specific oligonucleotide types. In some embodiments, a predetermined composition is one that can be intentionally replicated (e.g., through repeated controlled processes).

[0107] Linking phosphorus: As defined herein, the phrase "linking phosphorus" is used to indicate that the particular phosphorus atom represented is present during the internucleotide linkage and that the phosphorus atom corresponds to the phosphorus atom of the phosphodiester of the internucleotide linkages that occur in natural DNA and RNA. In some embodiments, the linking phosphorus atom is in a modified internucleotide linkage and each oxygen atom of the phosphodiester bond is optionally and independently substituted by an organic or inorganic moiety. In some embodiments, the linking phosphorus atom is P of formula I * as set forth. In some embodiments, the linking phosphorus atom is chiral. In some embodiments, the chiral linking phosphorus atom is P of formula I * as set forth.

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

[0109] Blockmer: As used herein, the term "blockmer" refers to an oligonucleotide chain characterized by a pattern of structural features that characterize each of its individual nucleotide units, which is characterized by the presence of at least two consecutive nucleotide units that share a common structural feature in the internucleotide phosphodiester bond. The common structural feature means 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 in the internucleotide phosphodiester bond are referred to as "blocks".

[0110] In some embodiments, the blockmer is a "stereoblockmer", for example, at least two consecutive nucleotide units have the same stereochemistry at the linking phosphorus. Such at least two consecutive nucleotide units form a "stereoblockmer". For example, since at least two consecutive nucleotide units, Ts and Cs1, have the same stereochemistry at the linking phosphorus (both Sp), (Rp,Sp)-ATsCs1GA is a stereoblockmer. In the same oligonucleotide, (Rp,Sp)-ATsCs1 forms a block, which is a stereoblock.

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

[0112] In some embodiments, the blockmer is a "coupling blockmer", for example, at least two consecutive nucleotide units have the same stereochemistry and the same modification at the coupling link. At least two consecutive nucleotide units form a "coupling block". For example, since at least two consecutive nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P modification (both phosphorothioate), (Rp,Rp)-ATsCsGA is a coupling blockmer. In the same oligonucleotide of (Rp,Rp)-ATsCsGA, TsCs forms a block and is a coupling block.

[0113] In some embodiments, the blockmer independently comprises one or more blocks selected from a stereoblock, a P-modified block, and a coupling block. In some embodiments, the blockmer is a stereoblockmer for one block, and / or a P-modified blockmer for another block, and / or a coupling blockmer for yet another block. For example, (Rp,Rp,Rp,Rp,Rp,Sp,Sp,Sp)-AAsTsCsGsAs1Ts1Cs1Gs1ATCG is a stereoblockmer with respect to the stereoblock AsTsCsGsAs1 (all Rp at the coupling link) or Ts1Cs1Gs1 (all Sp at the coupling link), a P-modified blockmer with respect to the P-modified block AsTsCsGs (all s linkages) or As1Ts1Cs1Gs1 (all s1 linkages), or a coupling blockmer with respect to the coupling block AsTsCsGs (all Rp and all s linkages at the coupling link) or Ts1Cs1Gs1 (all Sp and all s1 linkages at the coupling link).

[0114] Altmer: As used herein, the term "altmer" refers to an oligonucleotide strand characterized by the absence of two consecutive nucleotide units in the oligonucleotide strand whose structural characteristic patterns characterizing each individual nucleotide unit share specific structural characteristics in the internucleotide phosphodiester bond. In some embodiments, the altmer is designed to include a repeating pattern. In some embodiments, the altmer is designed not to include a repeating pattern.

[0115] In some embodiments, the altmer is a "stereoaltmer", e.g., there are no two consecutive nucleotide units having the same stereochemistry at the phosphodiester bond. For example, (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., there are no two consecutive nucleotide units having the same modification at the phosphodiester bond. For example, all (Sp)CAs1GsT where each phosphodiester bond has a different P-modification from the others.

[0117] In some embodiments, the altmer is a "bonded altmer", e.g., there are no two consecutive nucleotide units having the same stereochemistry or the same modification at the phosphodiester bond. For example, (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 in which the structural feature pattern that characterizes each individual nucleotide unit is such that all nucleotide units in the chain share at least one common structural feature in the internucleotide linkage. The common structural feature means a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus.

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

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

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

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

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

[0124] For the purposes of the present invention, chemical elements are identified according to the periodic table of the elements on the inside cover of the 67th edition (1986 - 87) of the Handbook of Chemistry and Physics, CAS edition.

[0125] The methods and structures described herein with respect to the compounds and compositions of the present invention also apply to pharmaceutically acceptable acids or base addition salts and all stereoisomers of these compounds and compositions. BRIEF DESCRIPTION OF THE DRAWINGS

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[0156] Synthetic oligonucleotides provide useful molecular tools for a wide variety of applications. For example, oligonucleotides are useful in therapy, diagnosis, research, and novel nanomaterial applications. The use of natural nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their sensitivity to endo- and exonucleases. Thus, to avoid these drawbacks, various synthetic equivalents have been developed. Synthetic equivalents include synthetic oligonucleotides that contain backbone modifications that make these molecules less susceptible to degradation. From a structural point of view, modifications to the phosphodiester bonds of such internucleotides introduce chirality. It has been revealed that certain properties of oligonucleotides can be affected by the configuration of the phosphorus atoms that form the backbone of the oligonucleotide. For example, in in vitro studies, properties of antisense nucleotides such as binding affinity, sequences that specifically bind to complementary RNA, and stability to nucleases have been shown to be affected particularly by the chirality of the backbone (e.g., the configuration of the phosphorus atoms).

[0157] In particular, the present invention encompasses the recognition that a stereorandom oligonucleotide preparation contains a plurality of distinct chemical entities that differ from one another in the stereochemical structure of the individual backbone chiral centers within the oligonucleotide chain. Furthermore, the present invention encompasses the insight that it is not usually likely that a stereorandom oligonucleotide preparation contains every possible stereoisomer of the relevant oligonucleotide. Thus, in particular, the present invention provides novel chemical entities that are specific stereoisomers of an oligonucleotide of interest. That is, the present invention provides a substantially pure preparation of a single oligonucleotide compound, which may be defined by its base sequence, its length, its pattern of backbone linkages, and its pattern of backbone chiral centers.

[0158] The present invention shows, inter alia, that individual stereoisomers of certain oligonucleotides can exhibit different stabilities and / or activities from one another. Further, the present disclosure shows that the improvement in stability achieved by the inclusion and / or position of a specific chiral structure within an oligonucleotide can be equal to, or even better than, the improvement in 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 shows that the improvement in activity achieved by the inclusion and / or position of a specific chiral structure within an oligonucleotide can be equal to, or even better than, the improvement in 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 position of a specific chiral bond within an oligonucleotide surprisingly can alter the cleavage pattern of a nucleic acid polymer when such an oligonucleotide is utilized for cleavage of the nucleic acid polymer. For example, in some embodiments, the pattern of backbone chiral centers results in an unexpectedly high cleavage efficiency of the 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, e.g., 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 of the target nucleic acid polymer within a sequence that is 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 invention provides 1) a common base sequence and length; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers Provided is a chirally controlled (and / or stereochemically pure) oligonucleotide composition comprising an oligonucleotide defined by having, wherein at least about 10% of the oligonucleotides in the composition are a substantially pure preparation of a single oligonucleotide having 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 the oligonucleotide can be specified by the combination of 5’ to 3’ linked phosphorus stereochemistry (Rp / Sp). For example, as illustrated below, ONT-154 has a pattern of 5S-(SSR)3-5S and ONT-80 has S 19 has.

[0160] In some embodiments, the present invention provides a chirally controlled oligonucleotide composition in which the composition is enriched in oligonucleotides with respect to a single oligonucleotide type compared to a substantially racemic preparation of the same oligonucleotide. In some embodiments, the present invention provides a chirally controlled oligonucleotide composition in which the composition is enriched in oligonucleotides with respect to a single oligonucleotide type that share 1) a common base sequence and length; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers compared to a substantially racemic preparation of the same oligonucleotide. In some embodiments, in a substantially racemic (or non-chirally controlled) preparation of an oligonucleotide, all or most coupling steps are not chirally controlled in that the coupling steps are not specifically performed to improve stereoselectivity. An exemplary substantially racemic preparation of an oligonucleotide is a preparation of a phosphorothioate oligonucleotide by a process well known in the art of sulfiding a triester of phosphorous acid with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-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-chirally controlled oligonucleotide composition).

[0161] In some embodiments, the invention provides 1) a common base sequence and length; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers characterized chiral controlled oligonucleotide compositions comprising oligonucleotides of a particular oligonucleotide type, which compositions are chirally controlled in that they are enriched with respect to oligonucleotides of the particular oligonucleotide type compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length. In some embodiments, the chirally controlled oligonucleotide compositions are substantially pure preparations of the oligonucleotide type, where the oligonucleotides in compositions that are not of the oligonucleotide type are impurities from the preparation process of the oligonucleotide type (optionally after specific purification procedures).

[0162] In some embodiments, at least about 20% 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 25% 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 30% 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 35% 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 40% 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 45% 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 50% 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 55% 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 60% 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 65% 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 70% 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 75% 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 80% 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 85% 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 90% 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 92% 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 94% 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 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 a composition having 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 an oligonucleotide-type chirally controlled oligonucleotide composition is expressed as the percentage of oligonucleotides in a composition that is of the oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 94% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type.In some embodiments, at least about 95% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in the chirally controlled oligonucleotide composition are of the same oligonucleotide type.

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

[0165] In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation is an antisense oligonucleotide (e.g., chiromersen). In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation is an siRNA oligonucleotide. In some embodiments, the provided chirally controlled oligonucleotide composition is one of an antisense oligonucleotide, an antagomir, a microRNA, a pre-microRNA, an anti-mir, a supermir, a ribozyme, a U1 adapter, an RNA activator, an RNAi agent, a decoy oligonucleotide, a triple helix-forming oligonucleotide, an aptamer or an adjuvant. In some embodiments, the chirally controlled oligonucleotide composition is one of an antisense oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of an antagomir oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of a microRNA oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of a pre-microRNA oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of an anti-mir oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of a supermir oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of a ribozyme oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of a U1 adapter oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of an RNA activator oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of an RNAi agent oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is one of a decoy oligonucleotide.In some embodiments, the chirally controlled oligonucleotide composition is that of a triplex-forming oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is that of an aptamer oligonucleotide. In some embodiments, the chirally controlled oligonucleotide composition is that of an adjuvant oligonucleotide.

[0166] In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation is that of an oligonucleotide comprising one or more modified backbone linkages, bases, and / or sugars.

[0167] In some embodiments, the provided oligonucleotide comprises one or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide comprises two or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide comprises three or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide comprises four or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide comprises five or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide comprises 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, the provided oligonucleotide type comprises five or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises six or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises seven or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises eight or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises nine or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises ten or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises eleven or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises twelve or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises thirteen or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises fourteen or more chiral modified phosphate linkages.In some embodiments, the provided oligonucleotide type comprises 15 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 16 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 17 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 18 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 19 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 20 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 21 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 22 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 23 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 24 or more chiral modified phosphate linkages. In some embodiments, the provided oligonucleotide type comprises 25 or more chiral modified phosphate linkages.

[0168] In some embodiments, the provided oligonucleotide comprises 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, the provided oligonucleotide comprises 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, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 80%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 85%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 90%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 91%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 92%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 93%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 94%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 95%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 96%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 97%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 98%. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation has a stereochemical purity of greater than about 99%.

[0170] In some embodiments, the chiral modified phosphate bond is a chiral phosphorothioate bond, i.e., a phosphorothioate nucleotide internucleotide bond. In some embodiments, the provided oligonucleotide comprises 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 nucleotide internucleotide bonds. In some embodiments, all chiral modified phosphate bonds are chiral phosphorothioate nucleotide internucleotide bonds. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate nucleotide internucleotide bonds of the provided oligonucleotide have an Sp configuration.In some embodiments, at least about 90% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Sp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Sp configuration. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 10% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 20% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 30% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 40% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 50% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 60% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 70% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 80% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 90% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide are of the Rp configuration. In some embodiments, at least about 95% of the chiral phosphorothioate nucleotide linkages in the provided oligonucleotide 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 nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 10% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 20% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 30% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 40% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 50% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 60% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 70% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 80% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 90% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, less than about 95% of the chiral phosphorothioate nucleotide linkages of the provided oligonucleotide are of the Rp configuration. In some embodiments, the provided oligonucleotide has only one Rp chiral phosphorothioate nucleotide linkage. In some embodiments, the provided oligonucleotide has only one Rp chiral phosphorothioate nucleotide linkage, where all nucleotide linkages are chiral phosphorothioate nucleotide linkages.In some embodiments, the chiral phosphorothioate internucleotide linkage is a chiral phosphorothioate diester linkage. In some embodiments, each of the chiral phosphorothioate internucleotide linkages is independently a chiral phosphorothioate diester linkage. In some embodiments, each of the internucleotide linkages is independently a chiral phosphorothioate diester linkage. In some embodiments, each of the internucleotide linkages is independently a chiral phosphorothioate diester linkage, and only one linkage is Rp.

[0171] In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation is of an oligonucleotide that includes one or more modified bases. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation is an oligonucleotide that does not include modified bases. Exemplary such modified bases are described above and herein.

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

[0173] In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation comprises an oligonucleotide comprising one or more residues modified with a sugar moiety. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation comprises an oligonucleotide comprising one or more residues modified at the 2'-position of the sugar moiety (referred to herein as "2'-modification"). 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, the provided chirally controlled (and / or stereochemically pure) preparation comprises an oligonucleotide comprising one or more residues that are 2'-modified. For example, in some embodiments, the provided oligonucleotide comprises one or more residues that are 2'-O-methoxyethyl (2'-MOE)-modified residues. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation comprises an oligonucleotide that does not include any 2'-modification. In some embodiments, the provided chirally controlled (and / or stereochemically pure) preparation is an oligonucleotide that does not include any 2'-MOE residues. That is, in some embodiments, the provided oligonucleotide is not MOE-modified.

[0174] In some embodiments, the chiral-controlled (and / or stereochemically pure) oligonucleotides provided are of the wing-core-wing general motif (also generally represented herein as X-Y-X). In some embodiments, each wing comprises one or more residues having a particular modification that is not present in the core "Y" portion. In some embodiments, each wing comprises one or more residues having a 2'-modification that is not present in the core portion. For example, in some embodiments, the chiral-controlled (and / or stereochemically pure) oligonucleotides provided have a wing-core-wing motif represented as X-Y-X, where the residues of each "X" portion are of a particular type of 2'-modified residue and the residues of the core "Y" portion are not of the same particular type of 2'-modified residue. For example, in some embodiments, the chiral-controlled (and / or stereochemically pure) oligonucleotides provided have a wing-core-wing motif represented as X-Y-X, where the residues of each "X" portion are 2'-MOE-modified residues and the residues of the core "Y" portion are not 2'-MOE-modified residues. In some embodiments, the chiral-controlled (and / or stereochemically pure) oligonucleotides provided have a wing-core-wing motif represented as X-Y-X, 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 all such 2'-modifications described above and herein are intended in the context of such X-Y-X motifs.

[0175] In some embodiments, each wing region independently has a length of at least 1 base. In some embodiments, each wing region independently has a length of at least 2 bases. In some embodiments, each wing region independently has a length of at least 3 bases. In some embodiments, each wing region independently has a length of at least 4 bases. In some embodiments, each wing region independently has a length of at least 5 bases. In some embodiments, each wing region independently has a length of at least 6 bases. In some embodiments, each wing region independently has a length of at least 7 bases. In some embodiments, each wing region independently has a length of at least 8 bases. In some embodiments, each wing region independently has a length of at least 9 bases. In some embodiments, each wing region independently has a length of at least 10 bases. 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 has a length of at least 1 base. In some embodiments, the core region has a length of at least 2 bases. In some embodiments, the core region has a length of at least 3 bases. In some embodiments, the core region has a length of at least 4 bases. In some embodiments, the core region has a length of at least 5 bases. In some embodiments, the core region has a length of at least 6 bases. In some embodiments, the core region has a length of at least 7 bases. In some embodiments, the core region has a length of at least 8 bases. In some embodiments, the core region has a length of at least 9 bases. In some embodiments, the core region has a length of at least 10 bases. In some embodiments, the core region has a length of at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 bases. In certain embodiments, the core region has a length of 10 bases. In certain embodiments, the core region has a length of 3 bases. In certain embodiments, the core region has a length of 4 bases. In certain embodiments, the core region has a length of 5 bases. In certain embodiments, the core region has a length of 6 bases. 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 at least 11 bases. In certain embodiments, the core region has a length of at least 12 bases. In certain embodiments, the core region has a length of at least 13 bases.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 or more bases.

[0177] In some embodiments, the wing-core-wing (i.e., X-Y-X) motif of the provided oligonucleotide is represented numerically, for example, as 5-10-5, which means that each wing region of the oligonucleotide is 5 bases in length and the core region of the oligonucleotide is 10 bases in length. In some embodiments, the wing-core-wing motif is any of, 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 the oligonucleotides provided with such wing-core-wing (i.e., X-Y-X) motifs are all chiral modified phosphate linkages. In some embodiments, the internucleoside linkages of the oligonucleotides provided with such wing-core-wing (i.e., X-Y-X) motifs are all chiral phosphorothioate internucleoside linkages. In some embodiments, the chiral internucleotide linkages of the oligonucleotides provided with such wing-core-wing motifs are at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% chiral modified phosphate ester internucleotide linkages. In some embodiments, the chiral internucleotide linkages of the oligonucleotides provided with such wing-core-wing motifs 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 the oligonucleotides provided with such wing-core-wing motifs are at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% chiral phosphorothioate internucleotide linkages of the Sp configuration.

[0179] In some embodiments, each wing region of the wing-core-wing motif may include chiral modified phosphate ester internucleotide linkages. In some embodiments, each wing region of the wing-core-wing motif may include chiral phosphorothioate internucleotide linkages. In some embodiments, each wing region of the wing-core-wing motif includes chiral phosphorothioate internucleotide linkages. In some embodiments, the two wing regions of the wing-core-wing motif have the same internucleotide linkage stereochemistry. In some embodiments, the two wing regions have different internucleotide linkage stereochemistry. In some embodiments, the internucleotide linkages within the wing are each independently chiral internucleotide linkages.

[0180] In some embodiments, the core region of the wing-core-wing motif may include chiral modified phosphodiester nucleotide linkages. In some embodiments, the core region of the wing-core-wing motif may include chiral phosphorothioate nucleotide linkages. In some embodiments, the core region of the wing-core-wing motif includes a repeating pattern of nucleotide linkage stereochemistry. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of nucleotide linkage stereochemistry. In some embodiments, the core region of the wing-core-wing motif includes a repeating pattern of nucleotide 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 nucleotide 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 nucleotide 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 nucleotide 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 nucleotide 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 nucleotide linkage stereochemistry, where the repeating pattern is a motif that includes at least 33% of the nucleotide linkages in the S structure.In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of nucleotide bond stereochemistry, where the repeating pattern is a motif that includes at least 50% of the nucleotide bonds in an S structure. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of nucleotide bond stereochemistry, where the repeating pattern is a motif that includes at least 66% of the nucleotide bonds in an S structure. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of nucleotide bond stereochemistry, where 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 nucleotide bond stereochemistry, where the repeating pattern is the repeating RpRpSp. In some embodiments, the core region of the wing-core-wing motif has a repeating pattern of nucleotide bond stereochemistry, where the repeating pattern is the repeating SpSpRp.

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

[0182] In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Sp)mRp or Rp(Sp)m. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes Rp(Sp)m. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Sp)mRp. In some embodiments, m is 2. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes Rp(Sp)2. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Sp)2Rp(Sp)2. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Rp)2Rp(Sp)2. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes RpSpRp(Sp)2. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes SpRpRp(Sp)2. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (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 repeating pattern is (Sp)m(Rp)n, wherein n is independently 1, 2, 3, 4, 5, 6, 7 or 8, and m is independently as defined above and as described herein. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers comprises (Sp)m(Rp)n. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers in the core region comprises (Sp)m(Rp)n. In some embodiments, the repeating pattern is (Rp)n(Sp)m, wherein n is independently 1, 2, 3, 4, 5, 6, 7 or 8, and m is independently as defined above and as described herein. In some embodiments, the present invention provides an oligonucleotide-type chirally controlled oligonucleotide composition 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 repeating 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 as described herein. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Sp)m(Rp)n(Sp)t. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers in the core region includes (Sp)m(Rp)n(Sp)t. In some embodiments, the repeating 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 as described herein. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Sp)t(Rp)n(Sp)m. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers in the core region includes (Sp)t(Rp)n(Sp)m.

[0186] In some embodiments, the repeating 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 as described herein. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Np)t(Rp)n(Sp)m. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers in the core region includes (Np)t(Rp)n(Sp)m. In some embodiments, the repeating 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 as described herein. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers includes (Np)m(Rp)n(Sp)t. In some embodiments, the invention provides an oligonucleotide-type chirally controlled oligonucleotide composition in which the pattern of backbone chiral centers in the core region includes (Np)m(Rp)n(Sp)t. In some embodiments, Np is Rp. In some embodiments, Np is Sp. In some embodiments, all of the Np's are the same. In some embodiments, all of the Np's are Sp. In some embodiments, at least one Np is different from the other Np's. 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 (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 nucleotide internucleotide linkage. In some embodiments, the core region of the wing-core-wing motif comprises at least one Rp phosphorothioate nucleotide internucleotide linkage. In some embodiments, the core region of the wing-core-wing motif comprises at least two Rp nucleotide internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least two Rp phosphorothioate nucleotide internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least three Rp nucleotide internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least three Rp phosphorothioate nucleotide internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least four, five, six, seven, eight, nine, or ten Rp nucleotide internucleotide linkages. In some embodiments, the core region of the wing-core-wing motif comprises at least four, five, six, seven, eight, nine, or ten Rp phosphorothioate nucleotide internucleotide linkages.

[0194] In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where the residues of each "X" wing region are 2'-MOE-modified residues. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where the residues of the core "Y" region are 2'-deoxyribonucleotide residues. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where all internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where all internucleoside linkages are chiral phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where the residues of each "X" wing region are 2'-MOE-modified residues, the residues of 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 where 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 where 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 where all internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where all internucleoside linkages are chiral phosphorothioate internucleoside linkages. In certain embodiments, the wing-core-wing motif is a 5-10-5 motif where 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 provided chirally controlled (and / or stereochemically pure) preparation comprises an oligonucleotide having 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 shows 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 homologous ligands and / or processing enzymes. The present invention specifically provides oligonucleotides whose structure incorporates or reflects the design parameters. Such oligonucleotides are novel chemical entities compared to stereorandom preparations having 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 specifically provides design parameters for oligonucleotides that may be bound and / or cleaved by ribonuclease H. In one (ome) embodiment, the present invention provides stereochemical design parameters for siRNA oligonucleotides. In some embodiments, the present invention specifically provides design parameters for oligonucleotides that may be bound and / or cleaved by, for example, DICER, Argonaute proteins (e.g., Argonaute-1 and Argonaute-2), and the like.

[0199] In some embodiments, a single oligonucleotide of the provided composition comprises a region in which at least one of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides is chiral. In some embodiments, at least two of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least three of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least four of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least five of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least six of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least seven of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least eight of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which at least nine of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it comprises a region in which one of the internucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides is chiral.In some embodiments, it includes a region in which 2 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 3 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 4 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 5 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 6 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 7 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 8 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 9 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral. In some embodiments, it includes a region in which 10 out of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides are chiral.

[0200] In some embodiments, a single oligonucleotide of the provided composition comprises a region in which at least one of the internucleotide linkages at positions 1, 2, 3, 5, 7, 18, 19, and 20 is chiral. In some embodiments, at least two of the internucleotide linkages at positions 1, 2, 3, 5, 7, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which at least three of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which at least four of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which at least five of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which at least six of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which at least seven of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which one of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 is chiral. In some embodiments, it comprises a region in which two of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which three of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral. In some embodiments, it comprises a region in which four of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 are chiral.In some embodiments, it includes a region in which 5 of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it includes a region in which 6 of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it includes a region in which 7 of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral. In some embodiments, it includes a region in which 8 of the nucleotide linkages between the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th, and 20th nucleotides are chiral.

[0201] In some embodiments, the single oligonucleotide of the provided composition comprises a region in which at least one of the internucleotide linkages at positions 1, 2, 3, 5, 7, 8, 9, 18, 19, and 20 is chiral and at least one internucleotide linkage is achiral. In some embodiments, the single oligonucleotide of the provided composition comprises a region in which at least one of the internucleotide linkages at positions 1, 2, 3, 5, 7, 18, 19, and 20 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 eight internucleotide linkages are achiral. In some embodiments, at least nine internucleotide linkages are achiral. In some embodiments, at least ten internucleotide linkages are achiral. In some embodiments, at least eleven internucleotide linkages are achiral. In some embodiments, at least twelve internucleotide linkages are achiral. In some embodiments, at least thirteen internucleotide linkages are achiral. In some embodiments, at least fourteen internucleotide linkages are achiral. In some embodiments, at least fifteen internucleotide linkages are achiral. In some embodiments, at least sixteen internucleotide linkages are achiral. In some embodiments, at least seventeen 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 the provided composition comprises a region in which all internucleotide linkages, except for at least one of the first, second, third, fifth, seventh, eighth, ninth, eighteenth, nineteenth, and twentieth internucleotide linkages, which are chiral, are achiral.

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

[0203] In some embodiments, at least one of the internucleotide linkages at the 1st, 2nd, 3rd, 5th, 7th, 8th, 9th, 18th, 19th and 20th nucleotides of a single oligonucleotide of the provided composition contains a chiral region, and at least 10% of the total internucleotide linkages within that region are achiral. In some embodiments, at least one of the internucleotide linkages at the 1st, 2nd, 3rd, 5th, 7th, 18th, 19th and 20th nucleotides of a single oligonucleotide of the provided composition contains a chiral region, and at least 10% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 20% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 30% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 40% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 50% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 60% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 70% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 80% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 90% of the total internucleotide linkages within that region are achiral. In some embodiments, at least 50% of the total internucleotide linkages within that 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 nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the first nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the second nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the second nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the third nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the third nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the fifth nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the seventh nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the seventh nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the eighth nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the eighth nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the fifth nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the ninth nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the ninth nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the eighteenth nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the eighteenth nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the nineteenth nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the nineteenth nucleotide bond in the region is a Rp-modified nucleotide bond. In some embodiments, the twentieth nucleotide bond in the region is a Sp-modified nucleotide bond. In some embodiments, the twentieth nucleotide bond in the region is a Rp-modified nucleotide bond.

[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 composition has a length of at least 21 bases. In some embodiments, a single oligonucleotide of the provided composition 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 a phosphorothioate. In some embodiments, each of the chiral internucleotide linkages of a single oligonucleotide of the provided composition independently has the structure of Formula I. In some embodiments, the chiral internucleotide linkages of a single oligonucleotide of the provided composition are each phosphorothioate.

[0207] Known to those of skill in the art and as described in the present disclosure, various modifications can be introduced at the 2'-position of the sugar moiety. Commonly used 2'-modifications are 2'-OR 1 (wherein R 1It is not hydrogen.) and includes, but is not limited to, this. In some embodiments, the modification is 2'-OR (wherein R may 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 shows that the content and / or position of certain chirally pure nucleotide linkages can result in an improvement in stability equal to or better than that achieved by the use of modified backbone linkages, bases, and / or sugars. In some embodiments, a single oligonucleotide of the provided composition has no modification on the sugar. In some embodiments, a single oligonucleotide of the provided composition has no 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 the provided composition has no 2'-MOE modification.

[0208] In some embodiments, a single oligonucleotide of the provided composition is a better substrate for argonaute proteins (e.g., hAgo-1 and hAgo-2) compared to a stereorandom oligonucleotide composition. The selection and / or position of the chirally pure linkages described in the present disclosure are useful design parameters for oligonucleotides that interact with such proteins, such as siRNA.

[0209] In some embodiments, a single oligonucleotide of the provided composition has at least about 25% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 30% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 35% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 40% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 45% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 50% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 55% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 60% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 65% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 70% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 75% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 80% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the provided composition has at least about 85% of its internucleotide linkages in the Sp configuration. In some embodiments, a single oligonucleotide of the 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 composition is not an oligonucleotide selected from the following: TIFF2025106395000007.tif34166The underlined nucleotides are 2'-modified.

[0211] In some embodiments, the single oligonucleotide of the provided composition is not an oligonucleotide selected from the following: TIFF2025106395000008.tif33166The underlined nucleotides are 2'-O-MOE modified.

[0212] In some embodiments, the single oligonucleotide of the provided composition is not an oligonucleotide selected from the following: TIFF2025106395000009.tif65166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-OH RNA residues; bold and "s" indicate phosphorothioate moieties; TIFF2025106395000010.tif63166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent RNA residues; d = 2'-deoxy residue; "s" indicates phosphorothioate moieties; TIFF2025106395000011.tif21166TIFF2025106395000012.tif96166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent RNA residues; d = 2'-deoxy residue; "s" indicates phosphorothioate moieties; TIFF2025106395000013.tif62166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-F RNA residues; d = 2'-deoxy residue; "s" indicates phosphorothioate moieties; TIFF2025106395000014.tif45166TIFF2025106395000015.tif40166In the table, lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-F RNA residues; d represents 2'-deoxy residue; "s" indicates a phosphorothioate moiety.

[0213] In some embodiments, the single oligonucleotide of the provided composition 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] (wherein R is an Rp phosphorothioate bond and S is an Sp phosphorothioate bond).

[0214] In some embodiments, the single oligonucleotide of the provided composition 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, and each m C is a 5-methylcytosine-modified nucleoside).

[0215] In some embodiments, the single oligonucleotide of the provided composition is not an oligonucleotide selected from: T k T k m C k AGT m CATGA m CT k T m C k m C k (wherein each nucleoside followed by a subscript "k" represents 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 phosphorothioates (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, RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRRSR, RRRRRRRRRR, and SSSSSSSSSS). In some embodiments, a single oligonucleotide of the provided composition is not an oligonucleotide selected from: T k T k m C k AGT m CATGA m CTT k m C k m C k (wherein each nucleoside followed by a subscripted "k" represents 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 phosphorothioates (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, RSRSRSSRSS, RRRSSRRSRS, SRRSSRRSRS, RRRRSRSRRR, SSSSRRRRRSR, RRRRRRRRRR, and SSSSSSSSSS).

[0216] Modified oligonucleotide structure As described above, considering the usefulness of oligonucleotide compositions in various applications and conditions, those skilled in the art have made efforts to develop modifications of oligonucleotide structures that may have favorable or desirable properties and characteristics for specific applications and conditions compared to natural oligonucleotide molecules. Such exemplary modifications are described below.

[0217] International Publication No. 2010 / 141471 (hereinafter "Traversa I") teaches modifications of different types of nucleic acid constructs modified to have a reduced net polyanionic charge. International Publication No. 2010 / 039543 (hereinafter "Travera II") teaches compositions and methods of making neutral polynucleotides (NN) using a reduced polyanionic charge. International Publication No. 2008 / 008476 (herein, "Traversa III") describes the synthesis of SATE (Imbach-type) phosphate prodrugs. Traversa I, II, and III do not disclose the chirally controlled oligonucleotides, compositions thereof, and methods of making using the same 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 a sulfurizing agent to produce phosphorothioate triesters as prodrugs. Girindus et al. do not disclose the chiral-controlled oligonucleotides, compositions thereof, and methods of making using the same described in the present invention.

[0219] Similarly, International Publication No. 2004 / 085454 (hereinafter "Avecia I") teaches, for example, the preparation of phosphorothioate oligonucleotides through the temporary silylation of poly-H-phosphonate diesters. International Publication No. 2001 / 027126 (hereinafter "Avecia II") teaches a process for the solid-phase synthesis of phosphorotriester oligonucleotides by coupling H-phosphonate monomers to solid-supported 5'-hydroxyl oligonucleotides and further sulfurizing the resulting H-phosphonate diesters to phosphorothioate triesters. The disclosure of International Publication No. 2001 / 064702 (hereinafter "Avecia III") is similar to Avecia II and further describes solid-phase synthesis on different solid supports. Avecia I, II, and III do not disclose the chiral-controlled oligonucleotides, compositions thereof, and methods of making using the same described in the present invention.

[0220] International Publication No. 1997 / 006183 (hereinafter "Chiron") teaches oligonucleotides having cationic internucleotide linkages and containing asymmetric phosphorus such as stereochemically pure amidates. Chiron teaches mixtures of diastereomers or stereochemically pure oligonucleotides obtained by crystallization using resolution means such as column chromatography. Chiron does not teach the chiral-controlled oligonucleotides, compositions thereof, and methods of making using the same described in the present invention.

[0221] International Publication No. 2009 / 146123 (hereinafter "Spring Bank I") discloses compositions and methods for treating viral infections using substituted oligonucleotide phosphates and phosphorothioate triesters. International Publication No. 2007 / 070598 (hereinafter "Spring Bank II") teaches the synthesis of phosphotriester prodrugs and phosphorothioate prodrugs as antiviral nucleic acids. Spring Bank I and II do not disclose the chirally controlled oligonucleotides, their compositions, and methods of making them described in the present invention.

[0222] European Patent No. 0779893 (hereinafter "Hybridon") teaches lipophilic prodrugs that incorporate many cells of antisense oligonucleotides, and it has been confirmed that Rp and Sp phosphorothioates and phosphorothioate triester dimers have different enzyme stabilities. Hybridon does not disclose the chirally controlled oligonucleotides, their compositions, and methods of making them described in the present invention.

[0223] International Publication No. 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 bio-reversible phosphotriester prodrugs and phosphoramidites containing alkylating or prodrug groups after synthesis. 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 chirally controlled oligonucleotides, their compositions, and methods of making them described in the present invention.

[0224] International Publication No. 2006 / 065751 (hereinafter referred to as "Beaucage") teaches CpG oligonucleotide phosphorothioate prodrugs containing thermally labile substituents (substituents introduced through phosphoramidite monomers) and their uses. Beaucage does not disclose the chirally controlled oligonucleotides, compositions thereof, and preparation methods using the same as described in the present invention.

[0225] Takeshi Wada et al. developed a novel method for the stereocontrolled synthesis of P-chiral nucleic acids using amidite chiral auxiliaries (Japanese Patent No. 4348077, International Publication No. 2005 / 014609, International Publication No. 2005 / 92909, and International Publication No. 2010 / 064146, hereinafter referred to as "Wada I" in order). In particular, International Publication No. 2010 / 064146 (hereinafter referred to as "Wada II") discloses a method for synthesizing a phosphorous atom-modified nucleic acid in which the stereochemical configuration at phosphorus is controlled. However, the method of Wada II is limited in that it does not provide individual P-modifications of each chirally bound phosphate in a controlled and designed manner. That is, the method of binding P-modifications in Wada II, once made to the desired length, is mass-modified with the bound phosphate to provide, for example, a desired phosphorothioate diester, phosphoroamidate, or boranophosphate or another such phosphorous atom-modified nucleic acid, by providing the formation of a condensation intermediate poly H-phosphonate oligonucleotide chain (referred to as Route B in the literature, Scheme 6, page 36). Furthermore, the H-phosphonate oligonucleotide chains of Wada II are of shorter length (e.g., dimer, trimer, or tetramer). The capping step is not included in Route B, and when combined with the fact that it generally exhibits low purity as a result of the accumulation of "n-1" type by-products, the Wada II route includes limitations regarding the synthesis of longer oligonucleotides. Although Wada II generally contemplates that a particular oligonucleotide may contain different modifications at each bound phosphate, Wada II does not describe or suggest the methods described herein for repeatedly incorporating such controlled modifications.To the extent that Wada II describes a synthetic cycle that does not require complete incorporation of the H-phosphonate intermediate oligonucleotide before modification with the coupled phosphate (in the literature, called Route A, page 35, Scheme 5, "Synthesis of Nucleic Acids Containing the Chiral X-Phosphonate Moiety of Formula 1 via Route A"), this general disclosure does not teach the important steps required to incorporate certain P-modifications provided by the present invention, nor does this cycle have the efficiency and versatility that could be useful in the synthesis of chirally controlled P-modified oligonucleotides, particularly in the synthesis of longer oligonucleotides.

[0226] At least one such inefficiency in Wada II is described by Wada et al's International Publication No. 2012 / 039448 (hereinafter "Wada III"). Wada III discloses using a novel chiral auxiliary in the method of Wada II to generate an H-phosphonate oligonucleotide once constructed and continuously modified to obtain, 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 phosphorus in the coupled phosphate. Wada III describes that severe conditions that tend to compromise the integrity of the oligonucleotide product are required to remove the chiral auxiliary of Wada II. Wada III confirmed that this is particularly problematic when synthesizing long-chain oligonucleotides. The reason is that as at least the decomposition reaction proceeds, additional by-products are generated that further react with the oligonucleotide product and decompose the oligonucleotide product. Therefore, Wada III provides a chiral auxiliary that uses the mechanism of S N 1 to open the H-phosphonate internucleotide bond under weakly acidic conditions (Route B), or can be efficiently cleaved from the oligonucleotide through a β-elimination pathway under relatively weakly basic conditions.

[0227] Those skilled in the art of chemistry and synthesis will readily appreciate, for example, the complexity associated with the generation of chirally controlled oligonucleotides provided by the present invention. For example, in order to synthesize and isolate a chirally controlled oligonucleotide, the conditions for each monomer addition must be designed as follows: (1) the chemical action is compatible with each portion of the growing oligonucleotide; (2) the by-products generated during the addition of each monomer do not compromise the structural and stereochemical integrity of the growing oligonucleotide; and (3) the composition of the final crude product is one that allows isolation of the desired chirally controlled oligonucleotide product.

[0228] Oligonucleotide phosphorothioates have shown potential for therapy (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 2 nIt exists 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 others. For example, Wada et al. (Nucleic Acids Symposium Series No. 51 p. 119-120; doi:10.1093 / nass / nrm060) discovered that the stereodefined -(Rp)-(Ups)9U / (Ap)9A duplex exhibited a higher Tm value than the normal -(Up)9U / (Ap)9A and the -(Sp)-(Ups)9U that did not form a stereodefined duplex. In another example, a study by Tang et al. (Nucleosides Nucleotides (1995), 14:985-990) found that stereochemically pure Rp oligodeoxyribonucleoside phosphorothioates had lower stability against nucleases endogenous to human serum than the parental oligodeoxyribonucleoside phosphorothioates with undefined chirality.

[0229] Chirally Controlled Oligonucleotides and Chirally Controlled Oligonucleotide Compositions The present invention provides chirally controlled oligonucleotides with high crude purity and high diastereomeric purity, and chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides chirally controlled oligonucleotides with high crude purity, and chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides chirally controlled oligonucleotides and oligonucleotide compositions with high purity of chirally controlled diastereomers.

[0230] In some embodiments, the present invention 1) a common base sequence and length; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers Provided is a chirally controlled oligonucleotide composition comprising an oligonucleotide defined by having, wherein at least about 10% of the oligonucleotides in the composition is a substantially pure preparation of a single oligonucleotide having 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 present invention provides a chirally controlled oligonucleotide composition in which the composition is enriched in oligonucleotides with respect to a single oligonucleotide type compared to a substantially racemic preparation of the same oligonucleotide. In some embodiments, the present invention, compared to a substantially racemic preparation of the same oligonucleotide, 1) a common base sequence and length; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers Provided is a chirally controlled oligonucleotide composition in which the composition is enriched in oligonucleotides with respect to a single oligonucleotide type sharing.

[0232] In some embodiments, the present invention 1) a common base sequence and length; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers Provided is a chirally controlled oligonucleotide composition comprising an oligonucleotide of a particular oligonucleotide type characterized by, which composition is chirally controlled in that it is enriched in oligonucleotides of a particular oligonucleotide type compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length.

[0233] In some embodiments, as will be appreciated by those skilled in the art, in a substantially racemic (or chirally unregulated) preparation of oligonucleotides, all or most coupling steps are chirally unregulated in that the coupling steps are not specifically performed to improve stereoselectivity. Exemplary substantially racemic preparations of oligonucleotides are preparations of phosphorothioate oligonucleotides by processes well known in the art that sulfurize triester phosphites with either tetraethylthiuram disulfide or (TETD) or 3H-1,2-benzodithiol-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 chirally unregulated oligonucleotide composition).

[0234] In some embodiments, a chirally regulated oligonucleotide composition is a substantially pure preparation of an oligonucleotide type, where oligonucleotides in a composition that are not of the oligonucleotide type are impurities from the preparation process of the oligonucleotide type (optionally, after specific purification procedures).

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

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

[0237] In certain embodiments, the present invention independently provides Formula I:

Chemical formula

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

[0239] In some embodiments, the invention provides a chirally controlled oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide are -XLR 1Having different X atoms in part, and / or -XLR 1 Having different L groups in part, and / or -XLR 1 Differing in part 1 R atoms have different P-modifications relative to each other.

[0240] In some embodiments, the invention provides a chirally controlled oligonucleotide, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P-modifications from each other, and the oligonucleotide has the following formula: [S B n1R B n2S B n3R B n4...S B nxR B ny] has the structure represented by: where each R B independently represents a block of nucleotide units having the R configuration in the attached phosphate; each S B independently represents a block of nucleotide units having the S configuration in the attached phosphate; each of n1 to ny is zero or an integer subject to the requirement that at least one odd n and at least one even n are non-zero numbers, whereby the oligonucleotide contains at least two individual internucleotide linkages of different stereochemistry from each other; and where the sum of n1 to ny is from 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 the other even ns; in some embodiments, each odd n is the same value as the other odd ns; in some embodiments, at least two even ns are different values from each other; in some embodiments, at least two odd ns are different values from each other.

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

[0243] In some embodiments, at least two adjacent ns, skipping those other than n, are equal to each other, and the provided oligonucleotide contains at least two blocks of bonds of a first stereochemistry that are equal in length to each other, and are divided into blocks of another stereochemistry of bonds, and the divided blocks may be of the same length or different lengths from the blocks of the first stereochemistry.

[0244] In some embodiments, the ns associated with the binding blocks at the ends of the provided oligonucleotide are of the same length. In some embodiments, the provided oligonucleotide has end blocks of the same binding stereochemistry. In some such embodiments, the end blocks are separated from each other by intermediate blocks of a different binding stereochemistry.

[0245] In some embodiments, the oligonucleotide provided, represented by the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], is a stereoblockmer. In some embodiments, the oligonucleotide provided, represented by the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], is a stereoskipmer. In some embodiments, the oligonucleotide provided, represented by the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], is a stereoaltmer. In some embodiments, the oligonucleotide provided, represented by the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], is a gapmer.

[0246] In some embodiments, the oligonucleotide provided, represented by the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], may be any of the patterns described above and further includes a pattern of P-modification. For example, in some embodiments, the oligonucleotide provided, represented by the formula [S B n1R B n2S B n3R B n4...S B nxR B ny], is a stereoskipmer and a P-modified skipmer. In some embodiments, the oligonucleotide provided, represented by the formula [SB n1R B n2S B n3R B n4...S B nxR B ny] and the provided oligonucleotide is a stereoblockmer and a P-modified altmer. In some embodiments, the formula [S B n1R B n2S B n3R B n4...S B nxR B ny] and the provided oligonucleotide is a stereoaltmer and a P-modified blockmer.

[0247] In some embodiments, the formula [S B n1R B n2S B n3R B n4...S B nxR B ny] and the provided oligonucleotide independently has a structure of formula I:

Chemical formula

[0248] In some embodiments, the chirally controlled oligonucleotide comprises a phosphodiester bond of one or more modified internucleotides. In some embodiments, the chirally controlled oligonucleotide comprises, for example, phosphorothioate or phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least two phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least three phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least four phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least five phosphorothioate triester bonds. Such exemplary modified internucleotide phosphodiester bonds are further described herein.

[0249] In some embodiments, the chirally controlled oligonucleotide comprises phosphate linkages of different internucleotides. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least one modified internucleotide bond. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least one phosphorothioate triester bond. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least two phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least three phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least four phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least five phosphorothioate triester bonds. Such exemplary modified internucleotide phosphate linkages are further described herein.

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

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

[0252] In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least two consecutively modified internucleotide bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphodiester internucleotide bond and at least two consecutive phosphorothioate triester internucleotide bonds.

[0253] In some embodiments, the chirally controlled oligonucleotide is a blockmer. In some embodiments, the chirally controlled oligonucleotide is a stereoblockmer. In some embodiments, the chirally controlled oligonucleotide is a P-modified blockmer. In some embodiments, the chirally controlled oligonucleotide is a binding blockmer.

[0254] In some embodiments, the chirally controlled oligonucleotide is an altmer. In some embodiments, the chirally controlled oligonucleotide is a stereoaltmer. In some embodiments, the chirally controlled oligonucleotide is a P-modified altmer. In some embodiments, the chirally controlled oligonucleotide is a binding altmer.

[0255] In some embodiments, the chirally controlled oligonucleotide is a unimmer. In some embodiments, the chirally controlled oligonucleotide is a stereounimmer. In some embodiments, the chirally controlled oligonucleotide is a P-modified unimmer. In some embodiments, the chirally controlled oligonucleotide is a binding unimmer.

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

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

[0258] In some embodiments, the present invention provides a chirally controlled oligonucleotide comprising the linkage of one or more modified internucleotides independently having the structure of Formula I:

Chemical formula

[0259] Generally as 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 * is Sp. In some embodiments, the oligonucleotide has each P *includes one or more internucleotide linkages of Formula I that are independently Rp or Sp. In some embodiments, the oligonucleotide has each P * includes one or more internucleotide linkages of Formula I that are Rp. In some embodiments, the oligonucleotide has each P * includes one or more internucleotide linkages of Formula I that are Sp. In some embodiments, the oligonucleotide has P * includes at least one internucleotide linkage of Formula I that is Rp. In some embodiments, the oligonucleotide has, of P * includes at least one internucleotide linkage of Formula I that is Sp. In some embodiments, the oligonucleotide has P * includes at least one internucleotide linkage of Formula I that is Rp and P * includes at least one internucleotide linkage of Formula I that is Sp.

[0260] Generally as 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, the oligonucleotide includes at least one internucleotide linkage of Formula I and W is O. In some embodiments, the oligonucleotide includes at least one internucleotide linkage of Formula I and W is S. In some embodiments, the oligonucleotide includes at least one internucleotide linkage of Formula I where W is Se.

[0261] Generally as described above and herein, each R is independently hydrogen or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclic, aryl, heteroaryl, or heterocyclic.

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

[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 or branched hexyl. In some embodiments, R is an optionally substituted, straight or branched pentyl. In some embodiments, R is an optionally substituted, straight or branched butyl. In some embodiments, R is an optionally substituted, straight 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 carbocyclic. In some embodiments, R is an optionally substituted C3-C 10 carbocyclic. In some embodiments, R is an optionally substituted monocyclic carbocyclic. 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 carbocyclic.

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

[0269] In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1 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 another embodiment, 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 two nitrogen atoms. In certain embodiments, R is an optionally substituted 6-membered heteroaryl ring having one 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 from 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 from 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, R is an optionally substituted 5,6-fused heteroaryl ring having from 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 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted azaindolyl. In some embodiments, R is an optionally substituted benzimidazolyl. In some embodiments, R is an optionally substituted benzothiazolyl. In some embodiments, R is an optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0273] In certain embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having from 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 from 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, 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. From a certain perspective, 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 from 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 from 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 from 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 six-membered saturated or partially unsaturated heterocyclic ring having from 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted partially unsaturated six-membered heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted partially unsaturated six-membered heterocyclic ring having 2 oxygen atoms.

[0276] In certain embodiments, R is a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, thietanyl, 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, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, 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.

[0277] In certain embodiments, R is an optionally substituted 5- to 6-membered partially unsaturated monocyclic ring having 1 to 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] Generally as described above and herein, each R´ is independently -R, -C(O)R, -CO2R, or -SO2R, 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, and R is as defined above and as described herein.

[0281] In some embodiments, R’ is -R, and 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 defined as above and as described herein. In some embodiments, R’ is -CO2R, where R is defined as above and as described herein. In some embodiments, R’ is -SO2R, where R is defined as above and as described herein.

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

[0284] Generally as 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] Generally as described above and herein, each of X, Y and Z is independently -O-, -S-, -N(-L-R 1 )-, or L, where each of L and R 1 is independently defined as 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, the oligonucleotide comprises at least one internucleotide linkage of Formula I, and X is -O-. In some embodiments, the oligonucleotide comprises at least one internucleotide linkage of Formula I, and X is -S-. In some embodiments, the oligonucleotide comprises at least one internucleotide linkage of Formula I, X is -O-, and comprises at least one internucleotide linkage of Formula I, and X is -S-. In some embodiments, the oligonucleotide comprises at least one internucleotide linkage of Formula I, X is -O-, and comprises at least one Formula I internucleotide linkage, X is -S-, and comprises at least one internucleotide linkage of Formula I, and L is an optionally substituted, straight-chain or branched C1-C 10 alkylene, and one or more methylene units of L are optionally and independently substituted by optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-.

[0288] In some embodiments, X is -N(-L-R 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 a linear or branched C1-C 10 alkylene, and one or more methylene units of L may be optionally and independently substituted by C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-. In some embodiments, X is a substituted C1-C 10 alkylene or C1-C 10 alkenylene. 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(-L-R 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 optionally substituted or a linear or branched C1-C 10is an alkylene, and one or more methylene units of L are optionally and independently substituted by optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-. In some embodiments, Y is optionally substituted C1-C 10 alkylene or C1-C 10 is alkenylene. 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(-L-R 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 also a straight-chain or branched C1-C optionally substituted 10is an alkylene, and one or more methylene units of L are optionally and independently substituted by an optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -S-S-, -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-. In some embodiments, Z is an optionally substituted C1-C 10 alkylene or C1-C 10 alkenylene. In some embodiments, Z is methylene.

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

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

[0298] In some embodiments, L is -L 1 -V- having a structure, where L 1 is an optionally substituted group:

Chemical formula

Chemical formula

[0299] In some embodiments, L 1 is

Chemical formula

[0300] In some embodiments, L 1 is

Chemical formula

Chemical formula

Chemical formula

[0301] is bonded to X. In some embodiments, L 1 is an optionally substituted group 1 selected from

Chemical formula

Chemical formula

[0302] In some embodiments, L is

Chemical formula

[0303] In some embodiments, L is

Chemical formula

[0304] In some embodiments, L is

Chemical formula

[0305] In some embodiments, L is

Chemical formula

[0306] In some embodiments, L is

Chemical formula

[0307] In some embodiments, L is

Chemical formula

[0308] In some embodiments, L is

Chemical formula

[0309] In some embodiments, L is

Chemical formula

[0310] In some embodiments, L is

Chemical formula

[0311] In some embodiments, L is:

Chemical formula

[0312] In some embodiments, L is:

Chemical formula

[0313] In some embodiments, L is:

Chemical formula

[0314] In some embodiments, L is:

Chemical formula

[0315] In some embodiments, L is

Chemical formula

[0316] In some embodiments, L is

Chemical formula

[0317] In some embodiments, L is

Chemical formula

[0318] In some embodiments, L is

Chemical formula

[0319] In some embodiments, L is

Chemical formula

[0320] In some embodiments, L is

Chemical formula

[0321] In some embodiments, L is

Chemical formula

[0322] In some embodiments, L is

Chemical formula

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

[0324] Generally as described above and herein, E is -O-, -S-, -NR'- or -C(R')2-, each R' is independently defined as above and as 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] Generally as described above and herein, G is -O-, -S-, or -NR', each R' is independently defined as above and as 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 optionally substituted C1-C5 alkylene or alkenylene, and 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

Chemical formula

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

[0328] In some embodiments, L 3 is optionally substituted C5 alkylene or alkenylene, and 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

Chemical formula

Chemical formula

[0329] In some embodiments, L 3 is an optionally substituted C4 alkylene or alkenylene, and 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

Chemical formula

[0330] In some embodiments, -L 3 -G- is

Chemical formula

[0331] In some embodiments, L 3 is an optionally substituted C3 alkylene or alkenylene, and 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

Chemical formula

[0332] In some embodiments, -L 3 -G- is [Chem.] It is TIFF2025106395000064.tif29166.

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

[0334] In some embodiments, L 3 is optionally substituted C2 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 [Chem.] optionally and independently substituted by, and each of R’ and Cy’ is independently defined as above and as described herein.

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

[0336] In some embodiments, L is -L 4 -G-, where L 4 is optionally substituted C1-C2 alkylene; and G is as defined above and as described herein. In some embodiments, L is -L 4 -G-, where L 4 is optionally substituted C1-C2 alkylene; G is as defined above and as described herein; G is attached to R 1 . In some embodiments, L is -L 4 -G-, where L 4 is optionally substituted methylene; G is as defined above and as described herein; and G is attached to R 1 . In some embodiments, L is -L 4 -G-, where L 4 is methylene; G is as defined above and as described herein; and G is attached to R 1 . In some embodiments, L is -L 4 -G-, where L 4 is optionally substituted -(CH2)2-; G is as defined above and as described herein; and G is attached to R 1 . In some embodiments, L is -L 4 -G-, where L 4 is -(CH2)2-; G is as defined above and as described herein; and G is attached to R 1 .

[0337] In some embodiments, L is [Chemical formula] or [Chemical formula] where G is as defined above and as described herein, G is attached to R 1is attached to. In some embodiments, L is [Chemical formula] where G is defined as above and as described herein, and G is attached to R 1 is attached to. In some embodiments, L is [Chemical formula] where G is defined as above and as described herein, and G is attached to R 1 is attached to. In some embodiments, L is [Chemical formula] or [Chemical formula] where the sulfur atom is attached to R 1 is attached to. In some embodiments, L is [Chemical formula] or TIFF2025106395000078.tif12166 where the oxygen atom is attached to R 1 is attached to.

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

[0339] In some embodiments, L is -S-R L3 - or -S-C(O)-R L3 - where R L3is optionally substituted and is a straight-chain or branched C1-C9 alkylene, and one or more methylene units are optionally and independently substituted by optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-, where each of R’ and -Cy- is independently defined as above and as described herein. In some embodiments, L is -S-R L3 - or -S-C(O)-R L3 - and R L3 is optionally substituted C1-C6 alkylene. In some embodiments, L is -S-R L3 - or -S-C(O)-R L3 - and R L3 is optionally substituted C1-C6 alkenylene. In some embodiments, L is -S-R L3 - or -S-C(O)-R L3 - and R L3 is optionally substituted C1-C6 alkylene, and one or more methylene units are optionally and independently substituted by optionally substituted C1-C6 alkenylene, arylene, or heteroarylene. In some embodiments, in some embodiments, R 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

Chem.

[0341] In some embodiments, L is

Chem.

Chem.

Chem.

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

[0343] Generally as described above and herein, R 1 is halogen, R, or optionally substituted C1 - C 50It is aliphatic, and one or more methylene units are optionally and independently substituted by C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -S-S-, -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-, where each variable is independently defined as described above and as described herein. In some embodiments, R 1 is halogen, R, or optionally substituted C1-C 10 It is aliphatic, and one or more methylene units are optionally and independently substituted by C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -S-S-, -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-, where each variable is independently defined as described above and as 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 defined as above and as described herein.

[0346] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is an optionally substituted group selected from C1-C 50 aliphatic, phenyl, carbocyclic, aryl, heteroaryl, or heterocyclic.

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

[0348] In some embodiments, R 1 is an 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 carbocyclic ring. In some embodiments, R 1 is an optionally substituted C3-C 10 carbocyclic ring. In some embodiments, R 1 is an optionally substituted monocyclic carbocyclic ring. In some embodiments, R 1 is an optionally substituted cycloheptyl. In some embodiments, R 1 is an optionally substituted cyclohexyl. In some embodiments, R 1 is an optionally substituted cyclopentyl. In some embodiments, R 1 is an optionally substituted cyclobutyl. In some embodiments, R 1 is an optionally substituted cyclopropyl. In some embodiments, R 1 is an optionally substituted bicyclic carbocyclic ring.

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

Chemical formula

Chemical formula

Chemical formula

[0351] In some embodiments, R 1 is an optionally substituted C1-C50 aliphatic containing one or more optionally substituted polycyclic hydrocarbon moieties. In some embodiments, R 1 is an optionally substituted C1-C 50It is aliphatic, and one or more methylene units are optionally and independently substituted by C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-, where each variable is independently defined as described above and as set forth herein. In some embodiments, R 1 is one or more optionally substituted C1-C 50 aliphatic

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

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

[0353] In some embodiments, R 1 is an optionally substituted heteroaryl. In some embodiments, R 1 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 1 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 1 is an unsubstituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 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 1 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. In certain embodiments, R 1 is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom, and the additional heteroatom is selected from sulfur or oxygen. Exemplary R 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. In another embodiment, R 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- to 10-membered bicyclic heteroaryl ring having 1 to 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. In another embodiment, R 1 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 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 an optionally substituted benzimidazolyl. In some embodiments, R 1 is an optionally substituted benzothiazolyl. In some embodiments, R 1 is an optionally substituted benzoxazolyl. In some embodiments, R 1 is an 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 one to four heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 1 is an optionally substituted 6,6-fused heteroaryl ring having one to two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, R 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 an optionally substituted isoquinolinyl. According to one aspect, R 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 quinazoline or quinoxaline.

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

[0361] In some embodiments, R 1 is an optionally substituted heterocyclyl. In some embodiments, R 1 is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having one or two 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 6-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. In certain embodiments, R 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, dihydrothiophenyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, 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. In some embodiments, R 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 8-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 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-C 10 aliphatic, where one or more methylene units are optionally and independently substituted by optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-, where each variable is independently defined as above and as described herein. In some embodiments, R 1is optionally substituted C1-C 10 is aliphatic and one or more methylene units are optionally and independently substituted by -Cy-, -O-, -S-, -S-S-, -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-, where each R’ is independently defined as above and as described herein. In some embodiments, R 1 is optionally substituted C1-C 10 is aliphatic and one or more methylene units are optionally and independently substituted by any -Cy-, -O-, -S-, -S-S-, -N(R´)-, -C(O)-, -OC(O)-, or -C(O)O-, where each R’ is independently defined as above and as described herein.

[0366] In some embodiments, R 1 is

Chemical formula

[0367] In some embodiments, R 1 is

Chemical formula

[0368] In some embodiments, R 1 includes a terminus having an optionally substituted -(CH2)2- moiety attached to L. Such exemplary R 1 groups are as follows:

Chemical formula

[0369] In some embodiments, R 1 is attached to L and contains an optionally substituted -(CH2)- moiety. Such exemplary R 1 groups are as follows:

Chemical formula

[0370] In some embodiments, R 1 is -S-R L2 wherein R L2 is an optionally substituted C1-C9 aliphatic, and one or more methylene units are optionally and independently substituted by C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R´)2-, -Cy-, -O-, -S-, -S-S-, -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-, and each of R’ and -Cy- is independently defined as above and as described herein. In some embodiments, R 1 is -S-R L2 wherein the sulfur atom is attached to the sulfur atom of the L group.

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

Claims

【Claim 1】 1) A common base sequence and length; 2) A common pattern of backbone linkages; and 3) A common pattern of backbone chiral centers A chirally controlled oligonucleotide composition comprising an oligonucleotide defined by having, The composition is chirally controlled such that it is enriched with respect to oligonucleotides of a particular oligonucleotide type compared to a substantially racemic preparation of oligonucleotides having the same base sequence and length, The common pattern of the backbone chiral centers includes Rp (Sp) 2 from 5' to 3', Each of the plurality of oligonucleotides has 5 or more chiral modified phosphate linkages, A composition in which at least 50% of the chiral modified phosphate linkages of each of the plurality of oligonucleotides have an Sp structure.

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