Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

• Oligonucleotides may contain a variety of modifications. Certain modifications, such as phosphorothioate internucleotidic linkages, may introduce new chiral centers into
• Oligonucleotides are useful for many purposes. However, natural oligonucleotides have been found to suffer disadvantages, such as low stability, low activity, etc., that can reduce or negate their usefulness as therapeutics.
• the present Applicant appreciated that developments of technologies that could improve or facilitate production of oligonucleotide compositions, particularly stereocontrolled oligonucleotide compositions, could provide significant benefits.
• the present disclosure describes certain such developments, and provides technologies relating to oligonucleotide compositions, particularly to stereocontrolled oligonucleotide compositions. Provided technologies may be particularly useful, for example, with respect to therapeutic oligonucleotides.
• the present disclosure encompasses the recognition that certain conditions and/or sequences of steps that have been utilized in the preparation of
• oligonucleotides particularly stereocontrolled (e.g., stereopure) oligonucleotide compositions
• the present disclosure thus identifies the source of a problem with strategies that have utilized such conditions and/or steps; the present disclosure provides technologies including methods that utilize certain conditions and/or sequences of steps that are described and demonstrated to dramatically improve crude product purity and yield, in some embodiments significantly improving efficiency and/or reducing production cost.
• provided technologies can deliver chirally controlled oligonucleotide compositions with crude purity of over 70% (full-length product purity), much higher than reference technologies (crude full-length product purity of can be around 30% or lower).
• Oligonucleotide synthesis typically utilizes highly efficient chemical transformations in its steps.
• products of one or more steps often contain one or more reactive functional groups that can introduce significant impurities if uncapped, e.g., for coupling products, unreacted 5' -OH groups, and/or newly formed reactive groups (e.g., primary and/or secondary amino groups) when chiral auxiliaries are utilized for chirally controlled oligonucleotide synthesis.
• reactive functional groups are capped during oligonucleotide synthesis in order to reduce impurities from them.
• the present disclosure encompasses the recognition of a source of problem that capping steps as those typically used in traditional phosphoramidite-based oligonucleotide synthesis can lead to generation of a significant amount of byproducts, particularly for many chirally controlled (stereocontrolled, stereoselective) oligonucleotide synthesis processes.
• the present disclosure provides technologies that address the problems.
• the present disclosure provides methods comprising a post- modification capping step, e.g., after a modification step but before the next de-blocking and/or coupling step.
• a post-modification capping step is after a modification step that provides a chirally controlled internucleotidic linkage but before the next de-blocking and/or coupling step.
• the present disclosure provides methods comprising capping steps of different chemistry strategies compared to a reference capping step in traditional oligonucleotide synthesis.
• the present disclosure provides methods comprising one or more capping steps, each of which selectively caps amino groups over hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis).
• provided capping steps are selective for amidation over esterification.
• capping reagent systems for capping steps contain no or reduced levels (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis) of strong nucleophiles and/or esterification catalysts (or reagents that can provide them when contacted with a composition to be capped), e.g., no or reduced levels of DMAP, NMI, etc..
• the present disclosure provides methods comprising capping steps that can cap both amino groups and hydroxyl groups efficiently, e.g., capping steps that are comparable or identical to a reference capping step in traditional oligonucleotide synthesis.
• the present disclosure provides methods that comprise capping steps of the same or different chemistry strategies to achieve oligonucleotide synthesis and can provide various advantages, e.g., improved crude purity, improved yield, etc., particularly for chirally controlled (stereocontrolled, stereoselective) oligonucleotide synthesis.
• the present disclosure provides methods of a pre-modification capping step (after a coupling step but before the next modification step) and a post-modification capping step (after a modification step but before the next de-blocking and/or coupling step).
• a pre-modification capping step and post-modification capping step are different.
• a pre-modification capping step and post-modification capping step have different chemistry strategies.
• a pre-modification capping step caps amino groups selectively over hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis).
• a post-modification capping step can cap both amino and hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis).
• a pre- modification capping step caps amino groups selectively over hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis). In some embodiments, a pre-modification capping step can cap both amino and hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis). [0011] In some embodiments, provided methods comprise two or more capping steps in an oligonucleotide synthesis cycle.
• provided methods comprise two capping steps in an oligonucleotide synthesis cycle, wherein the two steps are separated by a modification step, e.g., oxidation, sulfurization, etc.
• provided methods comprise a step in which a chiral modified internucleotidic linkage comprising a chiral linkage phosphorus is formed with a stereoselectivity of at least 80:20, 85: 15, 90: 10, 91 :9, 92:8, 93 :7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99: 1, favoring either the Rp or Sp configuration.
• the present disclosure provides a method comprising:
• a chiral nucleoside phosphoramidite which comprises a chiral atom that is not the phosphorus atom or a sugar carbon atom
• the present disclosure provides a method comprising:
• a chiral nucleoside phosphoramidite which comprises a chiral atom that is not the phosphorus atom and is not an atom of the nucleoside unit;
• the present disclosure provides a method comprising:
• an oligonucleotide intermediate comprising a chiral linkage phosphorus atom, which is bonded to a chiral unit which does not comprise a nucleoside unit or a part thereof; and a capping step immediately following a sulfurization or oxidation step.
• the present disclosure provides a method comprising:
• oligonucleotide intermediate comprising a chiral linkage phosphorus atom, which is bonded to a chiral unit which does not comprise an atom of a nucleoside unit;
• the present disclosure provides a method for preparing a composition comprising a plurality of oligonucleotides comprising:
• a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is deblocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and coupling a partner compound with the free hydroxyl groups of a plurality of deblocked oligonucleotides or nucleosides;
• the coupling step provides a coupling product composition
• a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;
• the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides
• modification step provides a modification product composition comprising a plurality of modification product oligonucleotides
• a post-modification capping step comprising:
• post-modification capping step provides a post-modification capping product composition comprising a plurality of post-modification capping product oligonucleotides
• a de-blocking step comprising: contacting a modification product composition, or a post-modification capping product composition, with a de-blocking reagent system;
• the deblocking step provides a de-blocking product composition comprising a plurality of de-blocking product oligonucleotides, each of which
• steps b) through e) optionally repeating steps b) through e) a number of times.
• provided methods comprise one or more pre-modification capping steps. In some embodiments, provided methods comprise one or more post- modification capping steps. In some embodiments, provided methods comprise one or more pre- and post-modification capping steps. In some embodiments, provided methods comprise one or more de-blocking steps.
• the present disclosure provides a method for preparing a composition comprising a plurality of oligonucleotides comprising one or more cycles, each cycle independently comprises:
• a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is deblocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and coupling a partner compound with the free hydroxyl groups of a plurality of deblocked oligonucleotides or nucleosides;
• the coupling step provides a coupling product composition
• a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;
• the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides
• modification step provides a modification product composition comprising a plurality of modification product oligonucleotides
• a post-modification capping step comprising:
• post-modification capping step provides a post-modification capping product composition comprising a plurality of post-modification capping product oligonucleotides
• the deblocking step provides a de-blocking product composition comprising a plurality of de-blocking product oligonucleotides, each of which independently comprises a free hydroxyl group.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises the following steps:
• a cycle comprises one or more pre-modification capping steps. In some embodiments, a cycle comprises one or more post-modification capping steps. In some embodiments, a cycle comprises one or more pre- and post-modification capping steps. In some embodiments, a cycle comprises one or more de-blocking steps.
• the present disclosure encompasses the recognition that traditional capping conditions when used as in traditional oligonucleotide synthesis may be a significant source of various problems under certain circumstances, and may contribute to formation of one or more by-products (impurities) and significantly lower oligonucleotide crude purity and yield, particularly for stereoselective preparation of oligonucleotides comprising one or more chiral internucleotidic linkages.
• the present disclosure provides technologies comprising capping strategies that can deliver unexpectedly high crude impurity and yield compared to an appropriate reference technology, for example, through designed capping strategies in combination with other steps in oligonucleotide synthesis.
• a reference technology uses a traditional capping condition as in traditional phosphoramidite-based oligonucleotide synthesis, which typically is or comprises an esterification condition that acrylates hydroxyl groups, e.g., by using a mixture comprising an acylating agent (e.g., acetic anhydride), a base (e.g., 2,6-lutidine), and a catalyst (e.g., N- methylimidazole, DMAP, etc.) to contact oligonucleotides to cap hydroxyl groups (e.g., unreacted 5' -OH groups).
• an acylating agent e.g., acetic anhydride
• a base e.g., 2,6-lutidine
• a catalyst e.g., N- methylimidazole, DMAP, etc.
• acylating agent, base and catalyst for capping typically use a substantial amount of acylating agent, base and catalyst for capping, generally each independently about 5%-15% volume, and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 equivalents relative to the first nucleoside incorporated into an oligonucleotide (before any cycle forming internucleotidic linkage) or oligonucleotide loading capacity of a support (e.g., loading capacity of the support used for preparing an oligonucleotide, can be calculated by multiplying unit loading capacity of a support (e.g., umol/g) by amount of support (g)).
• unit loading capacity of a support e.g., umol/g
• each synthetic cycle of a reference technology contains a single capping step.
• a reference technology comprises no more than one capping step in each of its synthetic cycle, wherein capping is performed using an esterification condition, e.g., comprising an acylating agent (e.g., acetic anhydride), a base (e.g., 2,6-lutidine), and a catalyst (e.g., N-methylimidazole (NMI), DMAP, etc.), each independently no less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%), 13%), 14%), or 15%> by volume of the capping reagent solution, and/or the catalyst is no less than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.5, or 2 equivalents relative to the acylating agent and/or the
• the present disclosure provides technologies comprising one or more capping steps, e.g., pre-modification capping steps, post-modification capping steps, etc., each of which is independently comparable or identical to a reference capping step, e.g., of traditional oligonucleotide synthesis based on phosphoramidite chemistry.
• a reference capping step e.g., of traditional oligonucleotide synthesis based on phosphoramidite chemistry.
• the present disclosure reduces by-products that may be formed in such capping steps by strategically positioning their positions (or timing) in oligonucleotide synthesis methods and/or cycles.
• capping steps are positioned after amino groups (typically primary and secondary) are, in many instances selectively, capped (over free hydroxyl groups, particularly, 5' -OH), either as individual separate capping steps or in combination with other capping steps (e.g., capping steps capping amino groups, in many instances selectively 5'- OH).
• the present disclosure provides technologies comprising one or more capping steps that each independently comprise a condition that is selective or specific for amidation over esterification. In some embodiments, the present disclosure provides technologies comprising one or more capping steps that use an amidation condition which is not an efficient and/or typical esterification condition.
• esterification and amidation have been extensively studied, and various conditions selective or specific for amidation over esterification, and various methods for assessing selectivity and/or specificity for amidation over esterification, are widely known in the art and can be utilized in accordance with the present disclosure.
• a typical condition selective or specific for amidation over esterification is an anhydride and a base without a catalyst (e.g., Ac 2 0 and 2,6- lutidine), as a corresponding efficient esterification condition typically requires an anhydride, a base, and a catalyst (e.g., Ac 2 0, 2,6-lutidine, and NMI) as traditional capping conditions.
• the present disclosure provides technologies that comprise one or more synthetic cycles each independently comprising a coupling step, a modification step (e.g., oxidation, sulfurization, etc.), and one or more capping steps, wherein each capping step after a coupling step and before a modification step comprising an amidation condition and no estenfication condition.
• an amidation condition comprises no more than 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% by volume of a catalyst for esterification under an appropriate corresponding condition (having the same acylating agent and base), and/or no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents of a catalyst for esterification under an appropriate corresponding condition (having the same acylating agent and base), relative to an acylating agent and/or relative to oligonucleotide loading capacity of a support.
• an acylating agent is an anhydride.
• an acylating agent is Ac 2 0.
• a catalyst is MI.
• a catalyst is DMAP.
• a catalyst is a nucleophilic nitrogen base.
• the present disclosure encompasses the recognition of a source of a problem in oligonucleotide synthesis, that an nucleophilic agent, particularly when used in a capping step that is after a coupling step and before a modification step in stereoselective oligonucleotide preparation, may contribute to generation of byproducts and lower overall preparation efficiency and/or crude purity through, e.g., degradation of oligonucleotides, lowering performance of another step, etc.
• the present disclosure provides capping technologies comprising greatly reduced levels of or no strong nucleophiles, e.g., catalysts used in typical capping conditions such as DMAP, NMI, etc., in contrast to traditional capping conditions which can comprise a large amount of a nucleophilic catalyst ⁇ e.g., in some cases, 5%-15% NMI by volume of capping solutions).
• each of one or more capping steps after a coupling step and before a modification step within an oligonucleotide preparation cycle independently comprises greatly reduced levels of or no strong nucleophiles.
• a reduced level is no more than 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% by volume of a capping reagent solution. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents relative to an acylating agent. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents relative to oligonucleotide loading capacity of a support.
• a strong nucleophile is a nucleophilic base.
• a nucleophilic base is a nitrogen base.
• a nucleophilic base is a nucleophilic nitrogen base.
• a nucleophilic nitrogen base is a compound of the structure of formula B-I:
• each R N is independently R and the three R groups are taken together with the nitrogen atom to form an optionally substituted bicyclic or polycyclic ring as described in the present disclosure for R groups (and groups can be R), wherein the nitrogen of N(R N ) 3 (underlined) is a tertiary nitrogen, and there are no substitutions at any of the positions alpha to the nitrogen atom.
• a formed ring is saturated.
• a nucleophilic base is DABCO (l,4-diazabicyclo[2.2.2]octane).
• a formed ring contains one or more unsaturation.
• a nucleophilic nitrogen base is a compound of the structure of formula B-II:
• a formed ring is an optionally substituted C 5 - 3 o heteroaryl ring comprising 0-10 hetereoatoms in addition to the nitrogen atom.
• a formed ring is an optionally substituted 5-membered heteroaryl ring.
• a formed ring is a substituted 5-membered heteroaryl ring.
• a formed ring is a substituted imidazolyl ring.
• a nucleophilic base is substituted imidazole. In some embodiments, a nucleophilic nitrogen base is MI. In some embodiments, a formed ring is an optionally substituted 6- membered heteroaryl ring. In some embodiments, a formed ring is a substituted 6-membered heteroaryl ring. In some embodiments, a formed ring is a substituted pyridinyl ring. In some embodiments, a nucleophilic base is substituted pyridine. In some embodiments, a nucleophilic nitrogen base is DMAP.
• nucleophilicity e.g., of basic nitrogen atoms in bases
• nucleophilicity is related to several factors, e.g., steric hindrance, electron density, etc.
• a base that can efficiently catalyze esterification reactions e.g., a base that can be used for efficient capping of unreacted 5'-OH together with anhydride and 2,6-lutidine in traditional oligonucleotide synthesis ⁇ e.g., DMAP, NMI, etc.
• a strong nucleophilic base is a strong nucleophilic base and should be avoided or used at reduced levels for capping steps that comprise greatly reduced levels of or no strong nucleophiles, e.g., any capping step after a coupling step and before a modification step.
• a strong nucleophilic base is a base that can effectively replace DMAP or NMI in esterification. In some embodiments, a strong nucleophilic base is a base that can effectively replace DMAP or NMI in a capping step of traditional oligonucleotide synthesis (which typically uses phosphoramidite chemistry and does not use chiral auxiliaries and is considered non-stereoselective/non-stereocontrolled).
• provided methods comprise a capping step, which capping step comprises no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents of a strong nucleophilic base relative to oligonucleotides or loading capacity of a support, or no strong nucleophilic bases.
• a capping step is immediately followed by a non-capping step.
• such a capping step is immediately after a non-capping step.
• such a capping step is immediately followed by a non-capping step, and is immediately after a non-capping step.
• a non-capping step is a coupling step.
• a non-coupling step is a modification step.
• a non-capping step immediately before such a capping step is a coupling modification step.
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises no strong nucleophile, or if it comprises one or more strong nucleophiles, level of each of the one or more strong nucleophiles is independently reduced compared to an appropriate reference capping condition.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises no strong nucleophile, or if it comprises one or more strong nucleophiles, level of each of the one or more strong nucleophiles is independently no more than no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.
• the first incorporated nucleoside of the oligonucleotide is the first nucleoside loaded to a support before the first cycle that forms an internucleotidic linkage.
• equivalent of the first incorporated nucleoside of an oligonucleotide to oligonucleotide loading capacity of a support used for preparing the oligonucleotide is 1.
• a strong nucleophile is a strong nucleophile base as described in the present disclosure.
• a strong nucleophilic base is a compound of formula B-I.
• a strong nucleophilic base is a compound of formula B-I and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis.
• a strong nucleophilic base is a compound of formula B-II.
• a strong nucleophilic base is a compound of formula B-II and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis.
• a strong nucleophilic base is DMAP.
• a strong nucleophilic base in MI is DMAP.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises no catalyst that promotes capping of 5'-OH as in an appropriate reference capping condition, or if it comprises one or more such catalysts, level of each of the one or more such catalysts is independently reduced compared to an appropriate reference capping condition.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises no catalyst that promotes capping of 5'-OH as in an appropriate reference capping condition, or if it comprises one or more such catalysts, level of each of the one or more such catalysts is independently no more than 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• each capping step between the coupling step and the modification step comprises no catalyst for esterification, or if it comprises one or more catalysts for esterification, level of each of the one or more such catalysts is independently reduced compared to an appropriate reference capping condition.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises no catalyst for esterification, or if it comprises one or more catalysts for esterification, level of each of the one or more such catalysts is independently no more than 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.
• a catalyst e.g., that promotes capping of 5' -OH as in oligonucleotide synthesis, for esterification, etc.
• a catalyst is a compound of formula B-I.
• a catalyst is a compound of formula B-I and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis.
• a catalyst is a compound of formula B-II.
• a catalyst is a compound of formula B-II and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis.
• a catalyst is DMAP.
• a strong nucleophilic base in MI is e.g., a strong nucleophilic base in MI.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises a selective condition for amidation over esterification.
• the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:
• each cycle independently forms an internucleotidic linkage
• each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which
• modification step modifies the internucleotidic linkage formed in the coupling step
• each capping step between the coupling step and the modification step comprises a selective condition for amidation over esterification, and no condition identical to or comparable to an appropriate reference condition.
• selective conditions for amidation over esterification comprise reduced levels of or no catalysts for esterification, e.g., no DMAP, NMI, etc.
• a condition identical to or comparable to an appropriate reference condition can be used to replace capping conditions in traditional phosphoramidite-based oligonucleotide synthesis without significantly reducing (or with no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%), 40%), 45%), or 50%> of reduction of) efficiency, crude purity, and/or yield.
• provided methods comprise a second capping step after a modification step in one or more cycles. In some embodiments, provided methods comprise a second capping step after a modification step and before a deblocking step (which de-blocks blocked hydroxyl groups) in one or more cycles. In some embodiments, a second capping step comprises a strong nucleophile. In some embodiments, a second capping step comprises a strong nucleophile at a level comparable to a reference capping condition. In some embodiments, a second capping step comprises an esterification catalyst. In some embodiments, a second capping step comprises an esterification catalyst at a level comparable to a reference capping condition. In some embodiments, a second capping step comprises an esterification condition.
• a second capping step comprises an esterification condition that is identical or comparable with a reference capping condition, e.g., in terms of capping unreacted 5' -OH in oligonucleotide synthesis.
• a strong nucleophile is DMAP or NMI.
• a strong nucleophile is DMAP.
• a strong nucleophile is NMI.
• an esterification catalyst is DMAP or NMI.
• an esterification catalyst is DMAP.
• an esterification catalyst is NMI.
• cycle comprises steps in the order of (2)-(3)-(4);
• the present disclosure provides a method for preparing an oligonucleotide, comprising a cycle comprising steps of:
• cycle comprises steps in the order of (2)-(4)-(3);
• a first capping comprising no strong nucleophiles, or if any, of reduced levels, as described in the present disclosure.
• a first capping step comprises no esterification catalysts, of if any, of reduced levels.
• a first capping step comprises a selective condition for amidation over esterification.
• a first capping step comprises no condition identical to or comparable to an appropriate reference condition.
• a first capping step is a pre-modification capping step as described in the present disclosure.
• a first capping step utilizes a capping reagent system that is a pre-modification capping reagent system.
• a second capping step is a post-modification capping step as described in the present disclosure.
• a second capping step utilizes a capping reagent system that is a post- modification capping reagent system.
• an appropriate reference capping condition is a capping condition of traditional oligonucleotide synthesis based on phosphoramidite chemistry.
• An example cycle for traditional phosphoramidite-based oligonucleotide synthesis is described below:
• a first capping step comprises reduced levels of a strong nucleophilic base or no strong nucleophilic base. In some embodiments, a first capping step comprises a reduced level of NMI. In some embodiments, a first capping step comprises no NMI. In some embodiments, a first capping step comprises a reduced level of DMAP. In some embodiments, a first capping step comprises no DMAP.
• Capping-2 is performed after Modification.
• Capping-1 is a pre-modification capping step.
• Capping-2 is a post- modification capping step.
• cycle exit is after De-blocking before the next Coupling.
• a reduced level of the present disclosure is no more than a percentage, e.g., 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, etc., by volume of a capping reagent solution.
• a percentage is 0.01%>.
• a percentage is 0.02%.
• a percentage is 0.05%.
• a percentage is 0.1%.
• a percentage is 0.2%.
• a percentage is 0.5%.
• a percentage is 1%.
• a percentage is 2%.
• a percentage is 3%.
• a percentage is 4%.
• a percentage is 5%.
• a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents relative to a reference agent.
• a reference agent is an acylating agent.
• a reference agent is a support (by oligonucleotide loading capacity).
• a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents relative to an acylating agent.
• a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 equivalents relative to
• a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 equivalents relative to the first nucleoside incorporated to an oligonucleotide.
• equivalent of the first nucleoside incorporated into an oligonucleotide to oligonucleotide loading capacity of a support used to prepare the oligonucleotide is 1.
• a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 equivalents relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, or 10 equivalents relative to oligonucleotide loading capacity of a support.
• a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, or 5 equivalents relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalent relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01 equivalent. In some embodiments, a reduced level is no more than about 0.02 equivalent. In some embodiments, a reduced level is no more than about 0.05 equivalent.
• a reduced level is no more than about 0.1 equivalent. In some embodiments, a reduced level is no more than about 0.2 equivalent. In some embodiments, a reduced level is no more than about 0.5 equivalent. In some embodiments, a reduced level is no more than about 1 equivalent. In some embodiments, a reduced level is no more than about 1.1 equivalents. In some embodiments, a reduced level is no more than about 1.2 equivalents.
• provided technologies are particularly useful for preparing chirally controlled oligonucleotide compositions.
• provided technologies comprise formation of one or more chiral internucleotidic linkages each independently comprising a chiral linkage phosphorus, wherein each of the chiral linkage phosphorus chiral center is independently formed with a stereoselectivity as described in the present disclosure, e.g., of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
• provided technologies comprise use of one or more chiral auxiliary to stereoselectively form one or more chirally controlled internucleotidic linkages.
• provided technologies comprise providing monomeric phosphoramidites of diastereomeric purity as described in the present disclosure, e.g., of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
• phosphoramidites of provided diastereomeric purity comprise a chiral auxiliary moiety.
• phosphoramidites of traditional oligonucleotide synthesis are utilized for non-chirally controlled internucleotidic linkages, and/or non-chiral internucleotidic linkages.
• Suitable chiral auxiliaries and phosphoramidites for chirally controlled oligonucleotide synthesis that can be utilized in accordance with the present disclosure include those described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612,
• a chiral auxiliary is of formula I, I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or a salt thereof, as described in the present disclosure.
• a phosphoramidite has the structure of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V- d, V-e, VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, or Vl-e, or a salt thereof, as described in the present disclosure.
• provided technologies comprising formation of an
• provided technologies provides oligonucleotides as intermediates and/or products.
• a provided oligonucleotide is of formula VIII or a salt thereof as described in the present disclosure.
• a provided oligonucleotide is one described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, oligonucleotides of each of which are independently incorporated herein by reference.
• provided intermediates and/or products are chirally controlled oligonucleotide compositions. In some embodiments, provided intermediates and/or products are chirally controlled oligonucleotide compositions of a plurality of oligonucleotides of formula VIII or salts thereof. In some embodiments, provided intermediates and/or products are chirally controlled oligonucleotide compositions of
• WO2017/015575 WO2017/062862, WO2017/160741, WO2017/192664, WO2017/192679, WO2017/210647, WO2018/022473, WO2018/067973, WO2018/098264, oligonucleotide compositions of each of which are independently incorporated herein by reference.
• the present disclosure provides a composition comprising: the plurality of oligonucleotides is a plurality of oligonucleotides of a modification product composition;
• the reagent system is a pre-modification or post-modification capping reagent system
• the post-modification capping reagent system is in contact with the plurality of oligonucleotides.
• the present disclosure provides a composition
• a capping reagent system comprising a first compound having the structure of formula B- I or B-II,
• oligonucleotides each comprising at least one internucleotidic linkage comprising a -C(0)-N(-)- moiety or a -P-S- moiety;
• the first compound is at a level of at least 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 equivalents relative to the plurality of oligonucleotides.
• the present disclosure provides a composition
• a capping reagent system comprising a first compound having the structure of formula B- I or B-II,
• each internucleotidic linkage of oligonucleotides of the plurality is independently an internucleotidic linkage comprising a -C(0)-N(-)- moiety and a linkage phosphorus that is tetravalent;
• the first compound is at a level of at least 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 equivalents relative to the plurality of oligonucleotides.
• the plurality of oligonucleotides is a plurality of oligonucleotides of a modification product composition. In some embodiments:
• oligonucleotides of the plurality share the same base sequence
• oligonucleotides of the plurality share the same pattern of backbone linkages; and oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;
• oligonucleotides of the plurality wherein at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.
• a first compound is NMI. In some embodiments, a first compound is DMAP.
• oligonucleotides of the plurality are attached to a support, e.g., a solid support used to prepare the oligonucleotides.
• a support e.g., a solid support used to prepare the oligonucleotides.
• molar amount of the oligonucleotides of the plurality equals loading capacity of the solid support they are attached to.
• a plurality of oligonucleotides share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides share the same
• oligonucleotides share the same stereochemistry at least one internucleotidic linkage comprising a -C(0)-N(-)- moiety or a -P-S- moiety.
• about 0.1%- 100% e.g., about ⁇ %- ⁇ 00%, 5%-100%, 10%-100%, 20%-100%, 30%- 100%, 40%-100%, 50%- 100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality.
• oligonucleotides of a plurality are each of the structure of formula VIII or a salt thereof. In some embodiments, oligonucleotides of a plurality are oligonucleotides of US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399,
• a level of the present disclosure is at least 0.1 equivalent. In some embodiments, a level is at least 0.2 equivalent. In some embodiments, a level is at least 0.5 equivalent. In some embodiments, a level is at least 1 equivalent. In some embodiments, a level is at least 2 equivalents. In some embodiments, a level is at least 3 equivalents. In some embodiments, a level is at least 4 equivalents. In some embodiments, a level is at least 5 equivalents. In some embodiments, a level is at least 6 equivalents. In some embodiments, a level is at least 7 equivalents. In some embodiments, a level is at least 8 equivalents. In some embodiments, a level is at least 9 equivalents. In some embodiments, a level is at least 10 equivalents. In some embodiments, a level is at least 20 equivalents. In some embodiments, a level is at least 50 equivalents. In some embodiments, a level is at least 100 equivalents.
• a -C(0)-N(-)- is part of a capped amino group in a chiral auxiliary moiety bonded to a linkage phosphorus, wherein the corresponding chiral auxiliary (replacing bonding to -C(O)- of -C(0)-N(-)- with -H, and replacing bonding to the linkage phosphorus with -H) is a compound of formula I, I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, Il-a, II- b, III, Ill-a, Ill-b, or a salt thereof.
• the present disclosure provides oligonucleotide compositions of high crude purity.
• the present disclosure provides a crude chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein: oligonucleotides of the plurality share the same base sequence;
• oligonucleotides of the plurality share the same pattern of backbone linkages; and oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;
• oligonucleotides of the plurality wherein at least ((DS) Nc * 100)% of all oligonucleotides sharing the same base sequence in the crude composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.
• the present disclosure provides a crude chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein:
• oligonucleotides of the plurality share the same constitution
• oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;
• oligonucleotides of the plurality wherein at least 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.
• a provided crude chirally controlled oligonucleotide composition has a crude purity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.
• a crude chirally controlled oligonucleotide composition is cleaved from a support, and before any further purification.
• crude chirally controlled oligonucleotide composition is cleaved from a support, after de-salting, and before any further purification.
• crude chirally controlled oligonucleotide composition is before any chromatograph or gel purification.
• a crude purity is % full- length product.
• a crude purity is % full-length product as assessed by LC-UV monitored at UV 260 nm.
• DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, DS is at least 85%. In some embodiments, DS is at least 90%. In some embodiments, DS is at least 92%. In some embodiments, DS is at least 95%. In some embodiments, DS is at least 96%. In some embodiments, DS is at least 97%. In some embodiments, DS is at least 98%. In some embodiments, DS is at least 99%. In some embodiments, DS is diastereoselectivity at, and/or diastereopurity of, a chiral linkage phosphorus of a chirally controlled internucleotidic linkage.
• diastereoselectivity at, and/or diastereopurity of, chiral linkage phosphorus of a chiral internucleotidic linkage in an oligonucleotide may be measured or represented through a model reaction, e.g.
• the dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5'-nucleoside of the dimer is the same as the nucleoside to the 5'- end of the chiral internucleotidic linkage, and the 3 '-nucleoside of the dimer is the same as the nucleoside to the 3 '-end of the chiral internucleotidic linkage.
• diastereopurity of the underlined linkage in NNNNNNNG* SGNNNNNNN can be assessed from coupling two G moieties under the same or comparable conditions, e.g., monomers, chiral auxiliaries, solvents, activators, temperatures, etc.
• diastereopurity (and/or diastereoselectivity) of the linkage of a dimer (G*SG) is used as diastereopurity (and/or diastereoselectivity) of a corresponding linkage in an oligonucleotide (NNNNNNNG* SGNNNNNNN) .
• diastereopurity of a compound comprising multiple chiral elements is product of diastereomeric purity of all its chiral elements.
• diastereopurity ⁇ i.e., diastereomeric purity) of a provided oligonucleotide is product of diastereomeric purity of all its chiral linkage phosphorus in its chiral internucleotidic linkages.
• Nc is the number of chirally controlled internucleotidic linkage and is 1-100. In some embodiments, Nc is 1-50. In some embodiments, Nc is 1-40. In some embodiments, Nc is 1-30. In some embodiments, Nc is 1-25. In some embodiments, Nc is 1-24. In some embodiments, Nc is 1-23. In some embodiments, Nc is 1-22. In some embodiments, Nc is 1-21. In some embodiments, Nc is 1-20. In some embodiments, Nc is 1-19. In some embodiments, Nc is 1-18. In some embodiments, Nc is 1-17. In some embodiments, Nc is 1-16. In some embodiments, Nc is 1-15.
• Nc is 1-14. In some embodiments, Nc is 1-13. In some embodiments, Nc is 1-12. In some embodiments, Nc is 1-11. In some embodiments, Nc is 1-10. In some embodiments, Nc is 1-9. In some embodiments, Nc is 1-8. In some embodiments, Nc is 1-7. In some embodiments, Nc is 1-6. In some embodiments,
• Nc is 1-5. In some embodiments, Nc is 1. In some embodiments, Nc is 2. In some embodiments, Nc is 3. In some embodiments, Nc is 4. In some embodiments, Nc is 5. In some embodiments, Nc is 6. In some embodiments, Nc is 7. In some embodiments, Nc is 8. In some embodiments, Nc is 9. In some embodiments, Nc is 10. In some embodiments, Nc is 11. In some embodiments, Nc is 12. In some embodiments, Nc is 13. In some embodiments, Nc is 14. In some embodiments, Nc is 15. In some embodiments, Nc is 16. In some embodiments, Nc is 17. In some embodiments, Nc is 18. In some embodiments, Nc is 19. In some
• Nc is 20. In some embodiments, Nc is 21. In some embodiments, Nc is 22. In some embodiments, Nc is 23. In some embodiments, Nc is 24. In some embodiments, Nc is 25.
• provided technologies comprising one or more modification steps that independently comprise or are sulfurization (thiolation).
• provided intermediates and/or products comprise one or more phosphorothioate internucleotidic linkages or precursors thereof (which can be converted into phosphorothioate internucleotidic linkages upon deprotection/cleavage), optionally chirally controlled.
• provided technologies comprising one or more modification steps that independently comprise or are oxidation.
• provided intermediates and/or products comprise one or more natural phosphate linkages or precursors thereof (which can be converted into natural phosphate linkages upon deprotection/cleavage).
• provided intermediates and/or products comprise one or more natural phosphate linkages and one or more
• a support is a polymer.
• a support is a solid support.
• a solid support is a polymer, e.g., polystyrene.
• a solid support is a Primer Support (e.g., Primer Support 5G, Primer Support 200, etc.).
• a solid support is NittoPhase support (e.g., NittoPhase HL, NittoPhase UnyLinker, etc.).
• a solid support is controlled-pore glass (CPG).
• CPG controlled-pore glass
• volume of a solid support e.g., certain polystyrene based solid support, changes during oligonucleotide synthesis, e.g., at different stages of synthesis and/or when contacted with different solvent systems and/or reagents.
• volume of a solid support e.g., many CPG support, changes less than 25%, 20%, 15%, 10%, or 5%, or remains substantially the same during oligonucleotide synthesis.
• the present disclosure encompasses the recognition than volume change of solid support during synthesis may cause deviations from planned reaction conditions, e.g., solvent system, reagent concentrations, contact time, etc., and may negatively impact synthesis efficiency, crude purity and/or yield.
• solid support that does not significantly change its volume or keep substantially the same volume during oligonucleotide synthesis may provide advantages, e.g., less deviation from planned reaction conditions, higher crude purity, higher yield, etc.
• a support can have a number of chemical modifications for nucleoside loading, and may have various unit loading capacities (e.g., umol/g).
• oligonucleotide synthesis using a support typically oligonucleotides are linked to a support through a linker.
• linkers can be utilized in accordance with the present disclosure, e.g., those described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, US9403865, etc.
• the present disclosure provides designed linkers.
• a support is functionalized with amino groups.
• a support is functionalized with -CH 2 -(CH 2 )n ⁇ CH 2 -NH 2 , wherein the -CH 2 - end is connected to a support, e.g., CPG.
• a first linker is
• -CH 2 -(CH 2 )n ⁇ CH 2 -NH- wherein the -CH 2 - end is connected to a support, e.g., CPG, and the -NH- is connected to a nucleoside, e.g., 3' -OH, through a second linker, e.g.,
• n is as described in the present disclosure. In some embodiments, n is 1. In some embodiments, n is 7.
• a first linker is
• a first linker is
• a first linker is -CH 2 -(CH 2 ) n -CH 2 - H-C(0)-X-CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 -0-CH 2 -CH 2 -CH 2 - CH 2 - H-, wherein each variable is independently as described in the present disclosure.
• a first linker is
• a first linker is
• the -CH 2 - end is connected to a support, e.g., CPG, and the - H- is connected to a nucleoside, e.g. , 3 ' -OH, through a second linker, e.g., -C(0)-CH 2 -CH 2 -C(0)-.
• not all available amino moieties are loaded with nucleoside, e.g., through a second linker -C(0)-CH 2 -CH 2 -C(0)-.
• some available amino moiety can be capped with an acyl group, e.g., -C(0)-R forming
• R is -(CH 2 )o ⁇ , wherein o is 0-20.
• o is 0-12. In some embodiments, o is 0. In some
• o is 1. In some embodiments, o is 2. In some embodiments, o is 3. In some embodiments, o is 4. In some embodiments, o is 5. In some embodiments, o is 6. In some embodiments, o is 7. In some embodiments, o is 8. In some embodiments, o is 9. In some embodiments, o is 10. In some embodiments, o is 1 1. In some embodiments, o is 12. In some embodiments, o is 13. In some embodiments, o is 14. In some embodiments, o is 15.
• -0-R w is an nucleoside moiety as described in the present disclosure, is a support as described in the present disclosure, and each other variable is independently as described in the present disclosure.
• X is -0-, S ⁇ , - H-, -CH 2 -, m is 3-15, n is 1 or 7, o is 0- 12, and p is 3-15.
• X is -0-, S ⁇ , - H-, -CH 2 -, m is 0-10, n is 1 or 7, o is 0-12, and p is 3-15.
• -0-R w is a nucleoside moiety of formula IV, IV-a, IV-b, IV- c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c- 1, V-c-2, V-d, V-e, VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, or Vl-e.
• R is , wherein R s is as described in the present
• R w is ? wherein each variable is as described in the present disclosure.
• BA is a protected nucleobase selected from A, T, C, U, G and 5mC.
• BA is C(N-4-Ac or Bz), 5-Me-C(N-4-Ac or Bz),U, T, A(N-6-Bz), or G(N-2-iBu)
• R 2s is -OH, -H, -F, -OCH 3 , or -OCH 2 CH 2 OCH 3
• Z is -0-
• -S- -CH 2 - and R s is -CH 3 , -OCH 3 , or -CH 2 CH 3 .
• Oligonucleotide synthesis typically comprises a deblocking step, which de-blocks a blocked hydroxyl group for a next step, e.g., a coupling step, which keeps intact capped hydroxyl groups which should not participate in a next step, e.g., a coupling step.
• a deblocking step which de-blocks a blocked hydroxyl group for a next step, e.g., a coupling step, which keeps intact capped hydroxyl groups which should not participate in a next step, e.g., a coupling step.
• Various conditions for de-blocking can be utilized in accordance with the present disclosure, including those described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399, WO2017/015555,
• de-blocking removes DMT group from DMT-protected hydroxyl (detritylation).
• deblocking is performed by contacting oligonucleotides with an acid.
• an acid is trichloroacetic acid or dichloroacetic acid.
• a deblocking condition is 2% trichloroacetic acid (TCA) or 3%
• DCA dichloroacetic acid
• an inert solvent e.g., dichloromethane, toluene, etc.
• a coupling step forms an internucleotidic linkage, which adds a nucleoside unit to an existing oligonucleotide.
• an internucleotidic linkage formed during a coupling step is a phosphite triester linkage.
• an internucleotidic linkage can form with chirally control, e.g., as in chirally controlled oligonucleotide synthesis using diastereomerically pure phosphoramidite, typically comprising a chiral auxiliary moiety.
• a coupling reagent system comprises a nucleoside phosphoramidite and an activator.
• Various phosphoramidites may be used in provided technologies, including those described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671,
• WO2018/022473 WO2018/067973, and WO2018/098264, and those having the structure of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa- d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, or Vl-e, or a salt thereof, as described in the present disclosure.
• Example activators include those described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399, WO2017/015555,
• an activator is CMPT. In some embodiments, an activator is CMIMT. In some embodiments, an activator is ETT. In some embodiments, conditions, e.g., concentrations of phosphoramidites, concentrations of activators, contact times, solvents, etc. can be optimized for each coupling to improve, e.g., crude purity, yield, etc.
• each capping step after a coupling step and before a modification step is performed as described in the present disclosure, e.g., with reduced levels of or no strong nucleophiles, with reduced levels of or no catalysts for esterification, and/or under conditions selective or specific for amidation over esterification.
• each capping step after a coupling step and before a modification step is performed to cap one or more amino groups, e.g., one or more amino groups formed after coupling in chiral auxiliaries moieties attached to linkage phosphorus atoms.
• a modification step is performed to modify a internucleotidic linkage formed after coupling, which internucleotidic linkage, in some embodiments, comprises a linkage phosphorus atom that is trivalent (e.g., as in a phosphite linkage).
• a modification step is or comprises sulfurization.
• Example modifications and related technologies include those described in US2015100197, US9744183, US9605019, US9394333, US8859755,
• provided technologies provide more flexibility with respect to choices of modification reagents, e.g., oxidation reagents, sulfurization reagents, etc.
• modification reagents e.g., oxidation reagents, sulfurization reagents, etc.
• reagents that tended to give inferior results in previously reported chirally controlled oligonucleotide synthesis can be utilized with technologies of the present disclosure to provide significantly improved, satisfactory results.
• another capping step is performed after a modifying step.
• an after-modification capping step is performed with a substantial amount of a strong nucleophile and/or an esterification catalyst (a strong nucleophile can be the same as an esterification catalyst) under an esterification condition which is comparable or identical to a capping condition in traditional oligonucleotide synthesis.
• an after- modification capping step caps free hydroxyl groups, e.g., those residue hydroxyl groups as a result of incomplete coupling which remain intact after a modification step. After this capping step, oligonucleotides can be de-blocked to expose hydroxyl groups at sites for further chain extension, and enter another synthetic cycle.
• oligonucleotides can be fully deprotected and cleaved from support for purification and/or further uses.
• Various cleavage and/or deprotection technologies can be utilized in accordance with the present disclosure, including those described in US2015100197, US9744183, US9605019, US9394333, US8859755,
• cleavage and/or deprotection conditions can depend on the chemistry used during oligonucleotide synthesis, e.g., properties of linkers connecting oligonucleotides to a support, properties of base and/or sugar blocking groups, properties of chiral moieties etc.
• removal of chiral auxiliaries e.g., DPSE-type of chiral auxiliaries, comprises use of TEA-HF.
• TEA-HF can be successfully utilized for
• sugars and nucleobases including non-natural, modified sugars and nucleobases, can be utilized in provided technologies in accordance with the present disclosure, e.g., those sugar and nucleobases described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, WO2017/ 160741, WO2017/192664, WO2017/192679, WO2017/210647, WO2018/022473, WO2018/067973, and WO2018/098264, .
• a scale is 100 g or more. In some embodiments, a scale is 200 g or more. In some embodiments, a scale is 500 g or more. In some embodiments, a scale is at least 1000 g or more.
• a composition of another step being contacted in a step is a composition of the first step preceding the step. In some embodiments, a composition of another step being contacted in each step is a composition of the first step preceding the step.
• a de-blocked composition being contacted in a coupling step is the de-blocked composition of the first de-blocking step preceding the coupling step
• a coupling product composition being contacted in a pre-modification capping step is the coupling product composition of the first coupling step preceding the pre-modification capping step
• a coupling product composition being contacted in a modification step is the coupling product composition of the first coupling step preceding the modification step
• a pre-modification capping product composition being contacted in a modification step is the pre-modification capping product composition of the first pre-modification capping step preceding the modification step
• a modification product composition being contacted in a post-modification capping step is the modification product composition of the first modification step preceding the post-modification capping step
• a modification product composition being contacted in a de-blocking step is the modification product composition of the first modification step preceding the de-blocking step
• a method for preparing a composition comprising a plurality of oligomeric compounds comprises:
• a de-blocked composition comprising a plurality of de-blocked compounds, each independently comprising a de-blocked monomelic unit, which is deblocked in that each de-blocked monomeric unit independently comprises a free connecting group, with a coupling reagent system which comprises a partner compound comprising a monomeric unit of the oligomeric compound; and coupling a partner compound comprising a monomeric unit of the oligomeric compound with the free connecting groups of a plurality of de-blocked compounds to provide a coupling product composition comprising a plurality of coupling products, each of which independently comprises a linkage linking the connecting group of a de-blocked monomeric unit and a monomeric unit of the partner compound;
• modification product composition comprising a plurality of modification products
• a post-modification capping step comprising:
• a de-blocking reagent system to provide a de-blocked composition comprising a plurality of de-blocked products, each of which independently comprises a de-blocked monomeric unit comprising a free connecting group.
• a method optionally comprises repeating steps b) to e) a number of times, e.g., until a desired length is achieved.
• an oligomeric compound is an oligonucleotide.
• a composition comprising a plurality of oligomeric compounds is an oligonucleotide composition comprising a plurality of
• a coupling step is a coupling step as described in the present disclosure for oligonucleotide synthesis.
• a de-blocked compound is a de-blocked oligonucleotide, e.g., an oligonucleotide after de-blocking step in an
• a de-blocked monomeric unit is a deblocked 5 '-end nucleoside unit.
• a free connecting group is a free 5'- hydroxyl group.
• a coupling reagent system is a coupling reagent system in oligonucleotide synthesis cycles.
• a partner compound is a
• a coupling product is an oligonucleotide formed after coupling in oligonucleotide synthesis.
• a linkage linking the connecting group of a de-blocked monomeric unit and a monomeric unit of the partner compound is an internucleotidic linkage formed during a coupling step.
• a pre-modification capping step is a capping step in oligonucleotide synthesis as described in the present disclosure.
• a pre- modification capping reagent system is a pre-capping reagent system in oligonucleotide synthesis as described in the present disclosure.
• a pre-modification capping product composition is a composition after a pre-modification capping step in
• a pre-modification capping product is a product formed after a pre-modification capping step in oligonucleotide synthesis.
• a modification step is a modification step as used in oligonucleotide synthesis.
• a modification step as demonstrated in oligonucleotide synthesis described in the present disclosure modifies an internucleotidic linkage.
• a modification product composition is an oligonucleotide composition provided after a
• a modification product is an oligonucleotide provided after a modification step in oligonucleotide synthesis.
• a post-modification capping step is a capping step in oligonucleotide synthesis as described in the present disclosure.
• a post-modification capping reagent system is a post-capping reagent system in oligonucleotide synthesis as described in the present disclosure.
• a post-modification capping product composition is a composition after a post-modification capping step in oligonucleotide synthesis.
• a post-modification capping product is a product formed after a post-modification capping step in oligonucleotide synthesis.
• a de-blocking step is a deblocking step as described in the present disclosure for oligonucleotide synthesis.
• a non-connecting group is an amino group.
• a chirally controlled chiral center is a chirally controlled linkage phosphorus center.
• a chiral partner compound comprising a chiral atom that is not within the monomeric unit is a phosphoramidite comprising a chiral center that is not in its nucleoside unit and is not the P.
• a reagent system comprises no esterification catalyst comprises no DMAP and no MI.
• Aliphatic means a straight-chain ⁇ i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation, or combinations thereof.
• aliphatic groups contain 1- 100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms.
• aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof.
• Alkyl As used herein, the term "alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., Ci-C 2 o for straight chain, C 2 -C 20 for branched chain), and alternatively, about 1- 10.
• cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure.
• an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C 1 -C 4 for straight chain lower alkyls).
• Aryl refers to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic.
• an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members.
• an aryl group is a biaryl group.
• aryl may be used interchangeably with the term “aryl ring.”
• aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents.
• aryl is a group in which an aromatic ring is fused to one or more non- aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like, where a radical or point of attachment is on an aryl ring.
• Chiral control refers to control of the stereochemical designation of a chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide.
• a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in some embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation as exemplified in the present disclosure, which chiral auxiliaries often are part of chiral phosphoramidites used during oligonucleotide preparation.
• a person having ordinary skill in the art appreciates that conventional oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry at a chiral internucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral internucleotidic linkage.
• the stereochemical designation of each chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide is controlled.
• Chirally controlled oligonucleotide composition refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids) which share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotidic linkages (chirally controlled internucleotidic linkages), and the level of the plurality of oligonucleotides (or nucleic acids) in the composition is not random/pre-determined ⁇ e.g., through chirally controlled oligonucleotide preparation to form one or more chiral internucleotidic linkages).
• the plurality of oligonucleotides in a chirally controlled oligonucleotide composition share the same base sequence, the same, if any, nucleobase, sugar, and internucleotidic linkage modifications, and the same stereochemistry (Rp or Sp) independently at linkage phosphorus chiral centers of one or more chirally controlled internucleotidic linkages, though stereochemistry of certain linkage phosphorus chiral centers may differ.
• about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%- 100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90- 100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition are oligonucleotides of the plurality.
• about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%- 100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence are oligonucleotides of the plurality.
• about 0.1%- 100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%- 100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality.
• a predetermined level is be about 0.1%- 100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%- 100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleo
• the plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotidic linkages.
• 1-50 e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20
• the plurality of oligonucleotides share the same stereochemistry at about 0.1%-100% (e.g., about 1%-100%, 5%- 100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotidic linkages.
• 0.1%-100% e.g., about 1%-100%, 5%- 100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%,
• each chiral internucleotidic linkage is a chiral controlled internucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition.
• not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition.
• a chirally controlled oligonucleotide composition comprises predetermined levels of individual oligonucleotide or nucleic acids types. For instance, in some embodiments a chirally controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level.
• a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a predetermined level of a plurality of oligonucleotides of the oligonucleotide type.
• Cycloaliphatic refers to saturated or partially unsaturated aliphatic monocyclic, bicyclic, or poly cyclic ring systems having, e.g. , from 3 to 30, members, wherein the aliphatic ring system is optionally substituted.
• Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl.
• the cycloalkyl has 3-6 carbons.
• cycloaliphatic may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where a radical or point of attachment is on an aliphatic ring.
• a carbocyclic group is bicyclic.
• a carbocyclic group is tricyclic.
• a carbocyclic group is polycyclic.
• cycloaliphatic refers to a monocyclic C 3 -C 6 hydrocarbon, or a C 8 -Ci 0 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, or a C9-C16 tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic.
• Halogen means F, CI, Br, or I.
• Heteroaliphatic The term “heteroaliphatic” is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms are replaced with one or more heteroatoms ⁇ e.g. , oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).
• Heteroalkyl The term “heteroalkyl” is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom ⁇ e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl- substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.
• Heteroaryl and “heteroar-,” used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or poly cyclic ring systems having, for example, a total of five to thirty, ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom.
• a heteroatom is nitrogen, oxygen or sulfur.
• a heteroaryl group is a group having 5 to 10 ring atoms ⁇ i.e., monocyclic, bicyclic or poly cyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl group has 6, 10, or 14 ⁇ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
• Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
• a heteroaryl is a heterobiaryl group, such as bipyridyl and the like.
• heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroar omatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where a radical or point of attachment is on a heteroar omatic ring.
• Non- limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin- 3(4H)-one.
• heteroaryl group may be monocyclic, bicyclic or poly cyclic.
• heteroaryl may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted.
• heteroarylkyl refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.
• Heteroatom means an atom that is not carbon or hydrogen.
• a heteroatom is oxygen, sulfur, nitrogen, phosphorus, boron or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4- dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl); etc.).
• a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.
• a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus.
• a heteroatom is nitrogen, oxygen, sulfur, or phosphorus.
• a heteroatom is nitrogen, oxygen or sulfur.
• Heterocyclyl As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety ⁇ e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms.
• a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.
• a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus.
• a heteroatom is nitrogen, oxygen, sulfur, or phosphorus.
• a heteroatom is nitrogen, oxygen or sulfur.
• a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10- membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
• nitrogen includes substituted nitrogen.
• the nitrogen may be N (as in 3,4-dihydro- 2H-pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N-substituted pyrrolidinyl).
• a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
• saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
• heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
• Internucleotidic linkage refers generally to the phosphorus-containing linkage between nucleotide units of an oligonucleotide, which typically links two neighboring sugar moieties together, and is interchangeable with "inter-sugar linkage” and "phosphorus atom bridge.”
• an internucleotidic linkage is a phosphodiester linkage, as found in naturally occurring DNA and RNA molecules (a natural phosphate linkage).
• an internucleotidic linkage is a "modified internucleotidic linkage" wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety.
• an internucleotidic linkage is a phosphotriester linkage
• the internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage.
• Linkage phosphorus As defined herein, the phrase "linkage phosphorus" is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in the internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a phosphodiester of an internucleotidic linkage as occurs in naturally occurring DNA and RNA.
• a linkage phosphorus atom is in a modified internucleotidic linkage, wherein each oxygen atom of a phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety.
• a linkage phosphorus atom is the phosphorus of P L of Formula VII.
• a linkage phosphorus atom is chiral.
• a linkage phosphorus is a chiral ⁇ e.g., in natural phosphate linkage).
• Oligonucleotide type is used to define an oligonucleotide that has a particular base sequence, pattern of backbone linkages ⁇ i.e., pattern of internucleotidic linkage types, for example, phosphate, phosphorothioate, etc.), pattern of backbone chiral centers (i.e. pattern of linkage phosphorus stereochemistry (RpASp)), and pattern of backbone phosphorus modifications (e.g. , pattern of "-X-L-R 5 " groups in formula VII).
• oligonucleotides of a common designated "type" are structurally, including stereochemically, identical to one another.
• Oligonucleotide refers to a polymer or oligomer of nucleotide monomers, containing any combination of nucleobases (natural or non-natural), sugars (natural or non-natural), and internucleotidic linkages (natural or non-natural). Oligonucleotides of the present disclosure can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 200 nucleotides in length.
• oligonucleotides can range in length from about 4 to about 10 nucleotides, from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length. In some embodiments, an oligonucleotide is from about 9 to about 39 nucleotides in length. In some embodiments, an oligonucleotide is at least 4 nucleotides in length. In some embodiments, an oligonucleotide is at least 5 nucleotides in length. In some embodiments, an oligonucleotide is at least 6 nucleotides in length.
• an oligonucleotide is at least 7 nucleotides in length. In some embodiments, an oligonucleotide is at least 8 nucleotides in length. In some embodiments, an oligonucleotide is at least 9 nucleotides in length. In some embodiments, an oligonucleotide is at least 10 nucleotides in length. In some embodiments, an oligonucleotide is at least 1 1 nucleotides in length. In some embodiments, an oligonucleotide is at least 12 nucleotides in length. In some embodiments, an oligonucleotide is at least 15 nucleotides in length.
• an oligonucleotide is at least 20 nucleotides in length. In some embodiments, an oligonucleotide is at least 25 nucleotides in length. In some embodiments, an oligonucleotide is at least 30 nucleotides in length. In some embodiments, an oligonucleotide is a duplex of complementary strands of at least 18 nucleotides in length. In some embodiments, an oligonucleotide is a duplex of complementary strands of at least 21 nucleotides in length.
• Partially unsaturated refers to a moiety that includes at least one double or triple bond.
• the term “partially unsaturated” is intended to encompass groups having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties.
• composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
• active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
• compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
• oral administration for example, drenches (aqueous or non-aqueous solutions or suspension
• compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion 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.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; 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; Ring
• compositions that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
• Pharmaceutically acceptable salts are well known. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
• pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other known methods such as ion exchange.
• nontoxic acid addition salts which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other known methods such as ion exchange.
• pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palm
• pharmaceutically acceptable salts include, but are not limited to, nontoxic base addition salts, such as those formed by acidic groups of provided compounds ⁇ e.g., phosphate linkage groups of oligonucleotides, phosphorothioate linkage groups of oligonucleotides, etc.) with bases.
• Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like.
• pharmaceutically acceptable salts are ammonium salts ⁇ e.g., -N(R) 3 + ).
• pharmaceutically acceptable salts are sodium salts.
• pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
• Predetermined By predetermined (or pre-determined) is meant deliberately selected, for example as opposed to randomly occurring or achieved without control. Those of ordinary skill in the art, reading the present specification, will appreciate that the present disclosure provides technologies that permit selection of particular chemistry and/or stereochemistry features to be incorporated into oligonucleotide compositions, and further permits controlled preparation of oligonucleotide compositions having such chemistry and/or stereochemistry features. Such provided compositions are "predetermined” as described herein. A composition that may contain certain oligonucleotides because they happen to have been generated through a process that are not controlled to intentionally generate the particular chemistry and/or stereochemistry features is not a "predetermined" composition.
• a predetermined composition is one that can be intentionally reproduced ⁇ e.g., through repetition of a controlled process).
• a predetermined level of a plurality of oligonucleotides in a composition means that the absolute amount, and/or the relative amount (ratio, percentage, etc.) of the plurality of oligonucleotides in the composition is controlled.
• a predetermined level of a plurality of oligonucleotides in a composition is achieved through chirally controlled oligonucleotide preparation.
• protecting group refers to temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
• protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
• Si protecting group is a protecting group comprising a Si atom, such as Si-trialkyl ⁇ e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl ⁇ e.g., t- butyldiphenylsilyl), or Si-aryl-dialkyl ⁇ e.g., Si-phenyldialkyl).
• Si-trialkyl ⁇ e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl
• Si-triaryl Si-alkyl-diphenyl ⁇ e.g., t- butyldiphenylsilyl
• Si-aryl-dialkyl e.g., Si-phenyldialkyl
• suitably protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
• suitable esters include formates, acetates, propionates, pentanoates, crotonates, and benzoates.
• esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benzylbenzoate, 2,4,6-trimethylbenzoate.
• Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2- (trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.
• Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropyl silyl ether, and other trialkylsilyl ethers.
• alkyl ethers examples include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof.
• Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-
• arylalkyl ethers examples include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.
• Protected amines are well known in the art and include those described in detail in Greene (1999). Suitable mono-protected amines further include, but are not limited to, aralkylamines, carbamates, allyl amines, amides, and the like.
• Suitable mono- protected amino moieties include t-butyloxycarbonylamino (- HBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxocarbonylamino (- HCBZ), allylamino, benzylamino (- HBn), fluorenylmethylcarbonyl (- HFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, tri chloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, t- butyldiphenylsilyl, and the like.
• Suitable di-protected amines include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides, such as phthalimide, maleimide, succinimide, and the like. Suitable di-protected amines also include pyrroles and the like, 2,2,5,5-tetramethyl- [l,2,5]azadisilolidine and the like, and azide.
• Protected aldehydes are well known in the art and include those described in detail in Greene (1999). Suitable protected aldehydes further include, but are not limited to, acyclic acetals, cyclic acetals, hydrazones, imines, and the like. Examples of such groups include dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal, bis(2-nitrobenzyl) acetal, 1,3- dioxanes, 1,3-dioxolanes, semicarbazones, and derivatives thereof.
• Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Suitable protected carboxylic acids further include, but are not limited to, optionally substituted Ci_ 6 aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, and the like. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, wherein each group is optionally substituted. Additional suitable protected carboxylic acids include oxazolines and ortho esters.
• Protected thiols are well known in the art and include those described in detail in Greene (1999). Suitable protected thiols further include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, and thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioester, to name but a few.
• compounds of the disclosure may contain optionally substituted and/or substituted moieties.
• substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
• an "optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
• Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.
• stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, example substituents are described below.
• each R° may be substituted as defined below and is independently hydrogen, Ci_ 20 aliphatic, Ci_ 20 heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, -CH 2 -(C 6 -i4 aryl), -0(CH 2 )o-i(C6
• Suitable monovalent substituents on R° are independently halogen, -(CH 2 ) 0 _ 2 R*, -(haloR*), -(CH 2 ) 0 _ 2 OH, -(CH 2 ) 0 _ 2 OR*, -(CH 2 ) 0 _ 2 CH(OR*) 2 ; - 0(haloR*), -CN, -N 3 , -(CH 2 ) 0 _ 2 C(O)R*, -(CH 2 ) 0 _ 2 C(O)OH, -(CH 2 ) 0 _ 2 C(O)OR*, -(CH 2 ) 0 _ 2 SR*, -(CH 2 )o_ 2 SH, -(CH 2 )o_ 2 H 2 , -(CH 2 ) 0 _ 2 HR*, -(CH 2 )
• Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: -0(CR*2)2-30-, wherein each independent occurrence of R * is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• Suitable substituents on the aliphatic group of R * are halogen, -R*, -(haloR*), -OH, - OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, - H 2 , -NHR*, -NR* 2 , or -N0 2 , wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_ 4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0 _iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• suitable substituents on a substitutable nitrogen are -R ⁇ , - R ⁇ 2 , -C(0)R ⁇ , -C(0)OR ⁇ , -C(0)C(0)R ⁇ , -C(0)CH 2 C(0)R ⁇ , -S(0) 2 R ⁇ , -S(0) 2 R ⁇ 2 , - C(S) R ⁇ 2 , -C( H) R ⁇ 2 , or -N(R ⁇ )S(0) 2 R ⁇ ; wherein each R ⁇ is independently hydrogen, Ci-e aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5- 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R ⁇ , taken together with their intervening atom(s) form an unsub
• Suitable substituents on the aliphatic group of R ⁇ are independently halogen, - R*, -(haloR*), -OH, -OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, -NH 2 , -NHR*, -NR* 2 , or -N0 2 , wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_ 4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0 _iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
• Unsaturated means that a moiety has one or more units of unsaturation.
• salts such as pharmaceutically acceptable acid or base addition salts, stereoisomeric forms, and tautomeric forms, of provided compound are included.
• singular forms “a”, “an”, and “the” include the plural reference unless the context clearly indicates otherwise.
• a reference to “a compound” may include a plurality of such compounds.
• the present disclosure provides technologies for preparing oligonucleotide compositions, particularly chirally controlled oligonucleotide compositions, with unexpectedly improved crude purity and yield.
• provided technologies can dramatically reduce costs of goods, and in some embodiments, enable large production of therapeutic oligonucleotides at commercially acceptable conditions, e.g., cost, purity, yield, etc., for clinical uses and commercialization.
• provided technologies enable production of compositions of various oligonucleotides, independent of base sequences, chemical/stereochemical modifications, modes of activities, chiral auxiliaries, etc. Example embodiments of provided technologies are described herein.
• oligonucleotide compositions of provided technologies e.g., product oligonucleotide compositions of various steps, final oligonucleotide compositions, etc.
• oligonucleotides of provided technologies e.g., product oligonucleotides of various steps, final product
• oligonucleotides are oligonucleotides of formula VIII:
• R 5s is independently R' or -OR'
• each -Cy- is independently an optionally substituted bivalent group selected from a C 3-20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Cy L is independently an optionally substituted tetravalent group selected from a C 3- 2 o cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Ring A s is independently an optionally substituted 3-20 membered monocyclic, bicyclic or poly cyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R s is independently -H, halogen, -CN, -N 3 , "NO, -N0 2 , -L s -R', -L s -Si(R) 3 , -L s -OR', -L S -SR', -L S -N(R') 2 , -C-L s -R, -C-L s -Si(R) 3 , -C-L s -OR', -C-L S -SR', or -0-L s -N(R') 2 ;
• each t is independently 0-20;
• each BA is independently an optionally substituted group selected from C 3-30 cycloaliphatic, C 6- 3o aryl, C5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety;
• each L p is independently an internucleotidic linkage
• z is 1-1000
• L is— L - or— L— L— ;
• R 3E is -R', -L -R', -OR', or a support
• each R' is independently -R, -C(0)R, -C(0)OR, or -S(0) 2 R;
• each R is independently -H, or an optionally substituted group selected from Ci -30 aliphatic, Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6-3 o aryl, C 6-3 o arylaliphatic, C 6-3 o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:
• R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
• two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• an oligonucleotide composition is a chirally controlled oligonucleotide composition.
• a chirally controlled oligonucleotide composition is an oligonucleotide composition comprising a plurality of oligonucleotides which share:
• composition which composition is chirally controlled in that level of the plurality of oligonucleotides in the composition is predetermined.
• a chirally controlled oligonucleotide composition is an oligonucleotide composition comprising a plurality of oligonucleotides, wherein oligonucleotides of the plurality are of a particular oligonucleotide type defined by:
• composition which composition is chirally controlled in that level of the plurality of oligonucleotides in the composition is predetermined.
• a chirally controlled oligonucleotide composition is an oligonucleotide composition comprising a plurality of oligonucleotides which share:
• composition is a substantially pure preparation of a single oligonucleotide in that a predetermined level of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.
• a chirally controlled oligonucleotide composition is an oligonucleotide composition comprising a plurality of oligonucleotides, wherein:
• oligonucleotides of the plurality share the same base sequence
• oligonucleotides of the plurality share the same pattern of backbone linkages; and oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;
• oligonucleotides sharing the same base sequence in the composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.
• a chirally controlled oligonucleotide composition is an oligonucleotide composition comprising a plurality of oligonucleotides, wherein:
• oligonucleotides of the plurality share the same constitution
• oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;
• oligonucleotides of the plurality wherein no less than ((DS) Nc * 100)% of all oligonucleotides sharing the same base sequence in the composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.
• provided oligonucleotides comprise 1-30 non-natural internucleotidic linkages (not -0-P(0)(OH)-0- or salt forms thereof). In some embodiments, provided oligonucleotides comprise 2-30 non-natural internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 5-30 non-natural internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 10-30 non-natural internucleotidic linkages.
• provided oligonucleotides comprise 1-30 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 2-30 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 5-30 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 10-30 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 1 chirally controlled internucleotidic linkage. In some embodiments, provided oligonucleotides comprise 2 chirally controlled internucleotidic linkages.
• provided oligonucleotides comprise 3 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 4 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 5 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 6 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 7 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 8 chirally controlled internucleotidic linkages.
• provided oligonucleotides comprise 9 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 10 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 1 1 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 12 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 13 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides comprise 14 chirally controlled internucleotidic linkages.
• provided oligonucleotides have 15 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides have 16 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides have 17 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides have 18 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides have 19 chirally controlled internucleotidic linkages. In some embodiments, provided oligonucleotides have 20 chirally controlled internucleotidic linkages.
• about 1-100% of all internucleotidic linkages are chirally controlled internucleotidic linkages. In some embodiments, about 1-100% of all chiral internucleotidic linkages (comprising chiral linkage phosphorus) are chirally controlled internucleotidic linkages. In some embodiments, a percentage is about 5%-100%. In some embodiments, a percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
• each chiral internucleotidic linkage is chirally controlled.
• a portion of or all of chirally controlled internucleotidic linkages in provided oligonucleotides or one or more segments thereof are consecutive.
• all chirally controlled internucleotidic linkages in provided oligonucleotides or one or more segments thereof are consecutive.
• provided oligonucleotides comprise 1-30 natural phosphate linkages (not -0-P(0)(OH)-0- or salt forms thereof). In some embodiments, provided oligonucleotides, in addition to natural phosphate linkages, or chiral internucleotidic linkages, or chirally controlled internucleotidic linkages as described herein, further comprise 1-30 natural phosphate linkages (not -0-P(0)(OH)-0- or salt forms thereof). In some embodiments, provided oligonucleotides comprise 2-30 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 5-30 natural phosphate linkages.
• provided oligonucleotides comprise 10-30 natural phosphate linkages. In some embodiments, provided olig onucleotides comprise 1 chirally controlled internucleotidic linkage. In some embodiments, provided oligonucleotides comprise 2 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 3 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 4 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 5 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 6 natural phosphate linkages.
• provided oligonucleotides comprise 7 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 8 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 9 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 10 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 1 1 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 12 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 13 natural phosphate linkages. In some embodiments, provided oligonucleotides comprise 14 natural phosphate linkages.
• provided oligonucleotides have 15 natural phosphate linkages. In some embodiments, provided oligonucleotides have 16 natural phosphate linkages. In some embodiments, provided oligonucleotides have 17 natural phosphate linkages. In some embodiments, provided oligonucleotides have 18 natural phosphate linkages. In some embodiments, provided oligonucleotides have 19 natural phosphate linkages. In some embodiments, provided oligonucleotides have 20 natural phosphate linkages. In some embodiments, about 1-100% of all internucleotidic linkages are natural phosphate linkages.
• internucleotidic linkages are natural phosphate linkages
• about 1-99% of all internucleotidic linkages are non-natural internucleotidic linkages (e.g. , internucleotidic linkages of formula VII, VII-a-1, VII-a-2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof and are non-natural internucleotidic linkages).
• about 1- 99% of all internucleotidic linkages are natural phosphate linkages, and about 1-99% of all internucleotidic linkages are chiral internucleotidic linkages (e.g., internucleotidic linkages of formula VII, VII-a-1, VII-a-2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof and are chiral).
• about 1-99% of all internucleotidic linkages are natural phosphate linkages, and about 1-99% of all internucleotidic linkages are chirally controlled oligonucleotide composition internucleotidic linkages (e.g.
• a percentage is about 5%-100%.
• a percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
• a percentage is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
• each non- natural internucleotidic linkage independently has the structure of formula VII, VII-a-1, Vll-a- 2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof, wherein the structure of formula VII, VII-a-1, VII-a-2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof, is a non-natural internucleotidic linkage (not -0-P(0)(OH)-0- or a salt form thereof).
• each chiral linkage independently has the structure of formula VII, VII-a-1, VII-a-2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof, wherein the structure of formula VII, VII-a-1, VII-a-2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof, is not a natural phosphate linkage (not -0-P(0)(OH)-0- or a salt form thereof).
• each chirally controlled phosphate linkage independently has the structure of formula VII, VII-a-1, VII-a-2, Vll-b, VII- c, Vll-d, or Vll-e, or a salt form thereof, wherein the structure of formula VII, VII-a-1, VII-a-2, Vll-b, VII-c, Vll-d, or Vll-e, or a salt form thereof, is not a natural phosphate linkage (not -0-P(0)(OH)-0- or a salt form thereof).
• a portion of or all of natural phosphate linkages in provided oligonucleotides or one or more segments thereof are consecutive.
• all natural phosphate linkages in provided oligonucleotides or one or more segments thereof are consecutive.
• a non-natural internucleotidic linkage is a phosphorothioate linkage or a salt form thereof (-0-P(0)(SH)-0- or a salt form thereof).
• a chiral internucleotidic linkage is a phosphorothioate linkage or a salt form thereof (-0-P(0)(SH)-0- or a salt form thereof).
• a chirally controlled internucleotidic linkage is a phosphorothioate linkage or a salt form thereof (-0-P(0)(SH)-0- or a salt form thereof).
• provided oligonucleotides comprise 5-200, 5-150, 5-100, 5-50, 5-40, 5-35, 5-30, 5-25, 10-200, 10-150, 10-100, 10-50, 10-40, 10-35, 10-30, 10-25, 15-200, 15- 150, 15-100, 15-50, 15-40, 15-35, 15-30, or 15-25 nucleobases.
• provided oligonucleotides comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleobases.
• provided oligonucleotides comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleobases. In some embodiments, provided oligonucleotides comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleobases. In some embodiments, provided oligonucleotides comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleobases. In some embodiments, provided oligonucleotides comprise at least 15 nucleobases.
• provided oligonucleotides comprise at least 16 nucleobases. In some embodiments, provided oligonucleotides comprise at least 17 nucleobases. In some embodiments, provided oligonucleotides comprise at least 18 nucleobases. In some embodiments, provided oligonucleotides comprise at least 19 nucleobases. In some embodiments, provided oligonucleotides comprise at least 20 nucleobases. In some embodiments, provided oligonucleotides comprise at least 21 nucleobases. In some embodiments, provided oligonucleotides comprise at least 22 nucleobases. In some embodiments, provided oligonucleotides comprise at least 23 nucleobases.
• provided oligonucleotides comprise at least 24 nucleobases. In some embodiments, provided oligonucleotides comprise at least 25 nucleobases. In some embodiments, a nucleobase is optionally substituted adenine, cytosine, guanosine, thymine, or uracil, or a tautomer thereof.
• each chiral linkage phosphorus independently has a diastereomeric purity as described in the present disclosure.
• a provided compound has a purity, diastereomeric purity, and/or enantiomeric purity as described in the present disclosure.
• a provided compound has a purity as described in the present disclosure.
• a provided compound has a diastereomeric purity as described in the present disclosure.
• a provided compound has an enantiomeric purity as described in the present disclosure.
• a provided compound has a diastereomeric purity and an enantiomeric purity as described in the present disclosure.
• provided oligonucleotides comprise or are of a 5'-wing region-core region-3'-wing region structure. In some embodiments, provided oligonucleotides comprise or are of a 5 '-wing region-core region structure. In some embodiments, provided oligonucleotides comprise or are of a core region-3'-wing region structure. In some embodiments, provided oligonucleotides comprise of a 5 '-wing region-core region-3'-wing region structure. In some embodiments, provided oligonucleotides comprise of a 5 '-wing region-core region structure. In some embodiments, provided oligonucleotides comprise of a core region-3'-wing region structure.
• provided oligonucleotides are of a 5 '-wing region-core region-3'-wing region structure. In some embodiments, provided oligonucleotides are of a 5 '-wing region-core region structure. In some embodiments, provided oligonucleotides are of a core region-3'-wing region structure. In some embodiments, a wing- core-wing (i.e., X-Y-X) motif is represented numerically as, e.g., 5-10-4, meaning 5'-wing region is 5 bases in length, the core region is 10 bases in length, and the 3 '-wing region is 4- bases in length. In some embodiments, a wing-core-wing motif is any of, e.g.
• a wing-core-wing motif is 5-10-5.
• a wing-core-wing motif is 7-7-6.
• a wing-core-wing motif is 8-7-5.
• a wing-core motif is 5-15, 6-14, 7-13, 8-12, 9-12, etc.
• a core-wing motif is 5-15, 6-14, 7-13, 8-12, 9-12, etc.
• a wing region comprises a sugar modification absent from a core region.
• a wing region comprises a 2' -modification.
• each nucleotide unit of a wing region independently comprises a 2' -modification.
• each nucleotide unit of a wing region independently comprises the same 2' -modification.
• each nucleotide unit of a 5 '-wing region independently comprises the same 2' -modification.
• each nucleotide unit of a 3 '-wing region independently comprises the same 2' -modification.
• 2'- modifications of the 5 '-wing region are the same.
• 2' -modifications of the 5'-wing region are the different.
• a 2'-modification is 2'-OR, wherein R' is not hydrogen.
• a 2' -modification is 2' -OR, wherein R' is optionally substituted Ci -6 aliphatic.
• a 2' -modification is 2' -OR, wherein R' is optionally substituted Ci -6 alkyl.
• a 2' -modification is 2'-OMe.
• a 2' -modification is 2'-OCH 2 CH 2 OMe.
• a wing region comprises one or more natural phosphate linkages as described in the present disclosure. Additionally or alternatively, in some embodiments, a wing region comprises one or more non- natural internucleotidic linkages, e.g., phosphorothioate internucleotidic linkages.
• a core region comprises one or more natural phosphate linkages. In some embodiments, a core region comprises one or more consecutive natural phosphate linkages. In some embodiments, a core region comprises one or more chiral phosphate linkages. In some embodiments, a core region comprises one or more consecutive chiral phosphate linkages. In some embodiments, a chiral phosphate linkage is a phosphorothioate linkage. In some embodiments, a chiral phosphate linkage is a chirally controlled.
• Oligonucleotides of the present disclosure may contain a pattern of backbone chiral centers.
• a pattern of backbone chiral centers of oligonucleotides or segments thereof, e.g. , core regions provides increased stability.
• a pattern of backbone chiral centers provides surprisingly increased activity.
• a pattern of backbone chiral centers provides increased stability and activity.
• a pattern of backbone chiral centers provides surprisingly increased binding to certain proteins.
• a pattern of backbone chiral centers provides surprisingly enhanced delivery.
• a pattern of backbone chiral centers comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, (Sp)p(Rp)n(Sp)m, (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m (unless otherwise specified, from 5' to 3 '), wherein n is 1-10, and each of p and m is independently 0-50.
• a pattern of backbone chiral centers comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, (Sp)p(Rp)n(Sp)m, (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(Sp)m, or (Sp)p(Op)n(Sp)m (unless otherwise specified, from 5' to 3 '), wherein n is 1-10, and each of p and m is independently 1-50.
• a pattern of backbone chiral centers comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m (unless otherwise specified, from 5' to 3 '), wherein n is 1-10, and each of p and m is independently 1-50.
• a pattern of backbone chiral centers comprises (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m.
• a pattern of backbone chiral centers comprises (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m.
• a pattern of backbone chiral centers is (Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m.
• a pattern of backbone chiral centers is (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m.
• a pattern of backbone chiral centers comprises or is a repeating pattern comprising or being of (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(Sp)m, or (Sp)p(Rp)n(Sp)m.
• a pattern of backbone chiral centers comprises or is a repeating pattern of (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(Sp)m, or (Sp)p(Rp)n(Sp)m (from 5' to 3 ').
• a pattern of backbone chiral centers comprises or is a repeating pattern comprising or being of (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m.
• a pattern of backbone chiral centers comprises or is a repeating pattern of (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(Sp)m, or (Sp)p(Op)n(.Sp)m (from 5' to 3 ').
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m, wherein m > 2.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m, wherein n is 1, p >1, and m > 2.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, wherein m > 2.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, wherein n is 1, p >1, and m > 2.
• a pattern of backbone chiral centers of an oligonucleotide or a region thereof comprises or is two or more units independently selected from (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, (Sp)p(Rp)n(Sp)m, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, and (Sp)p(Op)n(Sp)m, wherein each variable is independently as described in the present disclosure.
• n is 1.
• n is 1 and m of each unit is independently 2 or greater as described in the present disclosure.
• a pattern of backbone chiral centers comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 such units. In some embodiments, a pattern of backbone chiral centers comprises 2 and no more than 2 such units. In some embodiments, a pattern of backbone chiral centers comprises 3 and no more than 3 such units. In some embodiments, a pattern of backbone chiral centers comprises 4 and no more than 4 such units. In some embodiments, a pattern of backbone chiral centers comprises 5 and no more than 5 such units. In some embodiments, a region of an oligonucleotide comprises such a pattern of backbone chiral centers.
• such a region comprises no 2 '-substitution at its sugar moieties (two 2'-H).
• such a region is flanked by a 5 '-region comprising a sugar modification as described in the present disclosure (e.g., a 2'-modification, e.g., 2'-OMe, 2'-MOE, 2'-F, etc., as described in the present disclosure), and/or a 5 '-region comprising a sugar modification as described in the present disclosure (e.g., a 2'-modification, e.g., 2'-OMe, 2'-MOE, 2'-F, etc., as described in the present disclosure).
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(,Sp)m, ( ⁇ ) ⁇ ( ⁇ ) ⁇ or (Sp)p(Rp)n(Sp)m.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(Sp)m, or (Sp)p(Rp)n(Sp)m, and the oligonucleotides comprise one or more 2'- modifications as described herein.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m, and the oligonucleotides comprise one or more 2'-F modifications as described herein.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(Sp)m, or (Sp)p(Rp)n(Sp)m, and the oligonucleotides comprise one or more 2' -OR modifications as described herein.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m, and the oligonucleotides comprise one or more 2' -OR modifications, wherein R is not -H.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(Sp)m, or (Sp)p(Rp)n(Sp)m, and each nucleoside unit between internucleotidic linkages having the pattern of (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m comprises no 2' -modifications.
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m, and each nucleoside unit between internucleotidic linkages having the pattern of (Rp)n(,Sp)m, (,Sp)p(Rp)n, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m comprises no 2' -substitutions (-CH 2 - at 2' -position).
• a pattern of backbone chiral centers comprises or is (Rp)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises or is (,Sp)p(Rp)n. In some embodiments, a pattern of backbone chiral centers comprises or is (Np)p(Rp)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises (Rp)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises (,Sp)p(Rp)n. In some embodiments, a pattern of backbone chiral centers comprises (Np)p(Rp)n(,Sp)m.
• a pattern of backbone chiral centers is (Rp)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers is (,Sp)p(Rp)n. In some embodiments, a pattern of backbone chiral centers is (Np)p(Rp)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises or is (Sp)p(Sp)m, optionally with n achiral phosphate diester internucleotidic linkages and/or stereorandom (non-chirally controlled) chiral internucleotidic linkages between the section having (-Sp)p and the section having (,Sp)m.
• a pattern of backbone chiral centers comprises or is (Sp)p(Rp)n(Sp)m.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, (Sp)p(Sp)m or (Sp)p(Op)n(Sp)m.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, and the oligonucleotides comprise one or more 2'- modifications as described herein.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, and the oligonucleotides comprise one or more 2'-F modifications as described herein.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, and the oligonucleotides comprise one or more 2' -OR modifications as described herein.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, and the oligonucleotides comprise one or more 2' -OR modifications, wherein R is not -H.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, and each nucleoside unit between internucleotidic linkages having the pattern of (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m comprises no 2' -modifications.
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, and each nucleoside unit between internucleotidic linkages having the pattern of (Op)n(,Sp)m, (,Sp)p(Op)n, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m comprises no 2' -substitutions (-CH 2 - at 2' -position).
• a pattern of backbone chiral centers comprises or is (Op)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises or is (,Sp)p(Op)n. In some embodiments, a pattern of backbone chiral centers comprises or is (Np)p(Op)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises (Op)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises (,Sp)p(Op)n. In some embodiments, a pattern of backbone chiral centers comprises (Np)p(Op)n(,Sp)m.
• a pattern of backbone chiral centers is (Op)n(Sp)m. In some embodiments, a pattern of backbone chiral centers is (Sp)p(Op)n. In some embodiments, a pattern of backbone chiral centers is (Np)p(Op)n(,Sp)m. In some embodiments, a pattern of backbone chiral centers comprises or is (Sp)p(Sp)m, optionally with n achiral phosphate diester internucleotidic linkages and/or stereorandom (non-chirally controlled) chiral internucleotidic linkages between the section having (-Sp)p and the section having (Sp)m.
• a pattern of backbone chiral centers comprises or is (Sp)p(Op)n(Sp)m.
• an oligonucleotide, or a region thereof comprises a pattern, or a repeating pattern, of backbone chiral centers of (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m (structurally starting from the first, and ending at the last, internucleotidic linkage of the internucleotidic linkages which have the pattern, or the repeating pattern, of (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m, respectively; a "(repeating) (,Sp)m(Rp)n region", a "(repeating) (Rp)n(,Sp)m region", a "((repeating) (Rp)
• an oligonucleotide, or a region thereof comprises a pattern, or a repeating pattern, of backbone chiral centers of (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m (structurally starting from the first, and ending at the last, internucleotidic linkage of the internucleotidic linkages which have the pattern, or the repeating pattern, of (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m, respectively; a "(repeating) (Sp)m(Op)n region", a "(repeating) (Op)n(,Sp)m region", a "(repeating) (Np)
• a (Sp)p(Rp)n(Sp)m region ((Sp)7(Rp) ⁇ (Sp)3) in WV-2555 : mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * SmU) comprises no 2' -OR sugar modifications.
• each sugar moieties in the region is -CH 2 - at the 2'-position.
• each sugar moieties in the region is an unmodified, natural, 2'-deoxyribose moiety of DNA.
• a region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m is flanked by a 5 '-wing region, which structurally ends with a nucleoside moiety (which nucleoside moiety, at its 3 '-end, is connected to the first internucleotidic linkage of the region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(Sp)m, or (Sp)p(Rp)n(Sp)m).
• flanking 5'-wing region in WV-2555 mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * SmU).
• a region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m is flanked by a 5'-wing region, which structurally ends with a nucleoside moiety (which nucleoside moiety, at its 3 '-end, is connected to the first internucleotidic linkage of the region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m).
• a region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m is flanked by a 3 '-wing region, which structurally starts with a nucleoside moiety (which nucleoside moiety, at its 5 '-end, is connected to the last internucleotidic linkage of the region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m).
• a flanking 3 '-wing region in WV-2555 mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * SmU).
• a region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m is flanked by a 3 '-wing region, which structurally starts with a nucleoside moiety (which nucleoside moiety, at its 5 '-end, is connected to the last intemucleotidic linkage of the region comprising a pattern, or a repeating partem, of backbone chiral centers which comprises or is (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m).
• a region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m is flanked by a 5'-end and a 3 '-wing regions.
• a region comprising a pattern, or a repeating pattern, of backbone chiral centers which comprises or is (Sp)m(Op)n, (Op)n(,Sp)m, (Np)p(Op)n(,Sp)m, or (Sp)p(Op)n(Sp)m is flanked by a 5'-end and a 3 '-wing regions.
• the flanking 5 '-wing region and/or the 3 '-wing region comprise a non-natural intemucleotidic linkage.
• the flanking 5'-wing region and/or the 3 '-wing region comprise a chiral intemucleotidic linkage.
• flanking 5 '-wing region and/or the 3 '-wing region comprise a chirally controlled intemucleotidic linkage. In some embodiments, the flanking 5 '-wing region and/or the 3 '-wing region comprise a modified intemucleotidic linkage comprising a Sp linkage phosphorus. In some embodiments, the flanking 5'-wing region and/or the 3 '-wing region comprise a Sp phosphorothioate linkage. In some embodiments, the flanking 5 '-wing region and/or the 3 '-wing region comprise one or more natural phosphate linkages.
• flanking 5'-wing region and/or the 3 '-wing region comprise one or more consecutive natural phosphate linkages.
• the flanking 5 '-end comprises only one modified intemucleotidic linkage which is the 5 '-end intemucleotidic linkage, and one or more consecutive natural phosphate linkages (for example, in WV-2555 : in ⁇ * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmA * SmU (SOOOSSSSSSSRSSSOOOS)).
• the flanking 3 '-end comprises only one modified intemucleotidic linkage which is the 3 '-end intemucleotidic linkage, and one or more consecutive natural phosphate linkages (for example, in WV-2555 : mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * SmU (SOOOSSSSSSSRSSSOOOS)).
• the flanking 5'-wing region and/or the 3 '-wing region comprise 2'-modified sugar units.
• each sugar unit in the 5'-wing region and/or the 3 '-wing region is independently modified.
• each sugar unit in the 5'-wing region and/or the 3 '-wing region independently comprises a 2'- modification (for example, m, 2'-OMe in WV-2555 : mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * SmU).
• each sugar unit in the 5'-wing region and/or the 3'-wing region comprises the same 2'-modification.
• a 2' -modification is 2'-OR, wherein R is optionally substituted Ci -6 aliphatic.
• a 2'-modification is 2'-OMe.
• a 2'- modification is 2'-MOE.
• a 2' -modification is a LNA modification (which comprises a type of C2-C4 bridge).
• n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 1. 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. In some embodiments, n is 9. In some embodiments, n is 10.
• n is 1. In some embodiments, m is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, p is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is at least 2. In some embodiments, p is at least 2. In some embodiments, n is 1. In some embodiments, m is at least 2, p is at least 2, n is 1. In some embodiments, p is 2, 3, 4,
• each of m and p is independently at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, each of m and p is independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, each of m and p is independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, at least one of m and p is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is at least 5,
• p is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
• p is 0-50. In some embodiments, p is 1-50. In some embodiments, p is 1. In some embodiments, p is 2-50. In some embodiments, p is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p is 3, 4, 5, 6, 7 or 8. In some embodiments, p is 4, 5, 6, 7 or 8. In some embodiments, p is 5, 6, 7 or 8. In some embodiments, p is 6, 7 or 8. In some embodiments, p is 7 or 8. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8.
• p is 9. In some embodiments, p is 10. In some embodiments, p is 11. In some embodiments, p is 12. In some embodiments, p is 13. In some embodiments, p is 14. In some embodiments, p is 15. In some embodiments, p is 16. In some embodiments, p is 17. In some embodiments, p is 18. In some embodiments, p is 19. In some embodiments, p is 20. In some embodiments, p is 21. In some embodiments, p is 22. In some embodiments, p is 23. In some embodiments, p is 24. In some embodiments, p is 25. In some embodiments, p is at least 2. In some embodiments, p is at least 3. In some embodiments, p is at least 4.
• p is at least 5. In some embodiments, p is at least 6. In some embodiments, p is at least 7. In some embodiments, p is at least 8. In some embodiments, p is at least 9. In some embodiments, p is at least 10. In some embodiments, p is at least 11. In some embodiments, p is at least 12. In some embodiments, p is at least 13. In some embodiments, p is at least 14. In some embodiments, p is at least 15. In some embodiments, p is at least 16. In some embodiments, p is at least 17. In some embodiments, p is at least 18. In some embodiments, p is at least 19. In some embodiments, p is at least 20. In some embodiments, p is at least 21. In some embodiments, p is at least 22. In some embodiments, p is at least 23. In some embodiments, p is at least 24. In some embodiments, p is at least 25.
• m is 0-50. In some embodiments, m is 1-50. In some embodiments, m is 1. In some embodiments, m is 2-50. In some embodiments, 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 0. In some embodiments, m is 1. 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.
• m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20. In some embodiments, m is 21. In some embodiments, m is 22. In some embodiments, m is 23. In some embodiments, m is 24. In some embodiments, m is 25. In some embodiments, m is at least 2.
• m is at least 3. In some embodiments, m is at least 4. In some embodiments, m is at least 5. In some embodiments, m is at least 6. In some embodiments, m is at least 7. In some embodiments, m is at least 8. In some embodiments, m is at least 9. In some embodiments, m is at least 10. In some embodiments, m is at least 11. In some embodiments, m is at least 12. In some embodiments, m is at least 13. In some embodiments, m is at least 14. In some embodiments, m is at least 15. In some embodiments, m is at least 16. In some embodiments, m is at least 17. In some embodiments, m is at least 18. In some embodiments, m is at least 19.
• m is at least 20. In some embodiments, m is at least 21. In some embodiments, m is at least 22. In some embodiments, m is at least 23. In some embodiments, m is at least 24. In some embodiments, m is at least 25. In some embodiments, m is at least greater than 25.
• At least one of m and p is greater than 2. In some embodiments, at least one of m and p is greater than 3. In some embodiments, at least one of m and p is greater than 4. In some embodiments, at least one of m and p is greater than 5. In some embodiments, at least one of m and p is greater than 6. In some embodiments, at least one of m and p is greater than 7. In some embodiments, at least one of m and p is greater than 8. In some embodiments, at least one of m and p is greater than 9. In some embodiments, at least one of m and p is greater than 10. In some embodiments, at least one of m and p is greater than 11.
• At least one of m and p is greater than 12. In some embodiments, at least one of m and p is greater than 13. In some embodiments, at least one of m and p is greater than 14. In some embodiments, at least one of m and p is greater than 15. In some embodiments, at least one of m and p is greater than 16. In some embodiments, at least one of m and p is greater than 17. In some embodiments, at least one of m and p is greater than 18. In some embodiments, at least one of m and p is greater than 19. In some embodiments, at least one of m and p is greater than 20. In some embodiments, at least one of m and p is greater than 21.
• At least one of m and p is greater than 22. In some embodiments, at least one of m and p is greater than 23. In some embodiments, at least one of m and p is greater than 24. In some embodiments, at least one of m and p is greater than 25.
• each of m and p is greater than 2. In some embodiments, each of m and p is greater than 3. In some embodiments, each of m and p is greater than 4. In some embodiments, each of m and p is greater than 5. In some embodiments, each of m and p is greater than 6. In some embodiments, each of m and p is greater than 7. In some embodiments, each of m and p is greater than 8. In some embodiments, each of m and p is greater than 9. In some embodiments, each of m and p is greater than 10. In some embodiments, each of m and p is greater than 1 1. In some embodiments, each of m and p is greater than 12.
• each of m and p is greater than 13. In some embodiments, each of m and p is greater than 14. In some embodiments, each of m and p is greater than 15. In some embodiments, each of m and p is greater than 16. In some embodiments, each of m and p is greater than 17. In some embodiments, each of m and p is greater than 18. In some embodiments, each of m and p is greater than 19. In some embodiments, each of m and p is greater than 20.
• the sum of m and p is greater than 3. In some embodiments, the sum of m and p is greater than 4. In some embodiments, the sum of m and p is greater than 5. In some embodiments, the sum of m and p is greater than 6. In some embodiments, the sum of m and p is greater than 7. In some embodiments, the sum of m and p is greater than 8. In some embodiments, the sum of m and p is greater than 9. In some embodiments, the sum of m and p is greater than 10. In some embodiments, the sum of m and p is greater than 1 1. In some embodiments, the sum of m and p is greater than 12. In some embodiments, the sum of m and p is greater than 13.
• the sum of m and p is greater than 14. In some embodiments, the sum of m and p is greater than 15. In some embodiments, the sum of m and p is greater than 16. In some embodiments, the sum of m and p is greater than 17. In some embodiments, the sum of m and p is greater than 18. In some embodiments, the sum of m and p is greater than 19. In some embodiments, the sum of m and p is greater than 20. In some embodiments, the sum of m and p is greater than 21. In some embodiments, the sum of m and p is greater than 22. In some embodiments, the sum of m and p is greater than 23. In some embodiments, the sum of m and p is greater than 24. In some embodiments, the sum of m and p is greater than 25.
• n is 1, and at least one of m and p is greater than 1. In some embodiments, n is 1 and each of m and p is independently greater than 1. In some embodiments, m>n and p>n. In some embodiments, (Sp)m(Rp)n(Sp)p is (Sp) 2 Rp(Sp) 2 . In some embodiments, (Sp)p(Rp)n(Sp)m is (Sp) 2 Rp(Sp) 2 . In some embodiments, (Sp)p(Rp)n(Sp)m is SpRp(Sp) 2 .
• (Np)p(Rp)n(Sp)m is ( Vp)tRp(Sp)m. In some embodiments, ( Vp)p(Rp)n(Sp)m is ( Vp) 2 Rp(Sp)m. In some embodiments, (Np)p(Rp)n(Sp)m is (Rp) 2 Rp(Sp)m. In some embodiments, (Np)p(Rp)n(,Sp)m is (,Sp)2Rp(-Sp)m. In some embodiments, (Np)p(Rp)n(,Sp)m is RpSpRp(Sp)m. In some embodiments, (Np)p(Rp)n(,Sp)m is SpRpRp(Sp)m.
• (Sp)p(Rp)n(Sp)m is ⁇ pRp ⁇ Sp ⁇ Sp.
• (Sp)p(Rp)n(Sp)m is (Sp) 2 Rp(Sp) 2 .
• (Sp)p(Rp)n(Sp)m is (,Sp)3Rp( ⁇ Sp)3.
• (Sp)p(Rp)n(Sp)m is (,Sp)4Rp( ⁇ Sp)4.
• (Sp)p(Rp)n(Sp)m is (Sp)tRp(Sp)5.
• (Sp)p(Rp)n(Sp)m is SpRp(Sp) 5 . In some embodiments, (Sp)p(Rp)n(Sp)m is (,Sp)2Rp( ⁇ Sp)5. In some embodiments, (Sp)p(Rp)n(Sp)m is (,Sp)3Rp( ⁇ Sp)5. In some embodiments, (Sp)p(Rp)n(Sp)m is (,Sp)4Rp( ⁇ Sp)5. In some embodiments, (Sp)p(Rp)n(Sp)m
• (Sp)m(Rp)n(Sp)p is (Sp) 2 Rp(Sp) 2 .
• (Sp)m(Rp)n(Sp)p is (,Sp)3Rp( ⁇ Sp)3.
• (Sp)m(Rp)n(Sp)p is ( ⁇ ) ⁇ 3 ⁇ 4>)4 ⁇
• (Sp)m(Rp)n(Sp)p is (Sp)mRp(Sp)5.
• (Sp)m(Rp)n(Sp)p is (,S'p)2Rp(-S'p)5.
• (Sp)m(Rp)n(Sp)p is (,Sp)3Rp( ⁇ Sp)5. In some embodiments, (Sp)m(Rp)n(Sp)p is (Sp)iRp(Sp) 5 . In some embodiments, (Sp)m(Rp)n(Sp)p is
• provided oligonucleotides are blockmers. In some embodiments, provided oligonucleotide are altmers. In some embodiments, provided oligonucleotides are altmers comprising alternating blocks. In some embodiments, a blockmer or an altmer can be defined by chemical modifications (including presence or absence), e.g. , base modifications, sugar modification, internucleotidic linkage modifications, stereochemistry, etc., or patterns thereof. Example chemical modifications, stereochemistry and patterns thereof for a block and/or an alternating unit include but are not limited to those described in this disclosure, such as those described for an oligonucleotide, etc. In some embodiments, a blockmer comprises a pattern of .. SS..RR...RR. . . . In some embodiments, an altmer comprises a pattern of SRSRSRSR.
• a provided pattern of backbone chiral centers comprises repeating (,Sp)m(Rp)n, (Rp)n(,Sp)m, (Np)p(Rp)n(,Sp)m, or (Sp)p(Rp)n(Sp)m units.
• a repeating unit is (Sp)m(Rp)n.
• a repeating unit is SpRp.
• a repeating unit is SpSpRp.
• a repeating unit is -S'pRpRp.
• a repeating unit is RpRp-S'p.
• a repeating unit is (Rp)n(Sp)m. In some embodiments, a repeating unit is (Np)p(Rp)n(Sp)m. In some embodiments, a repeating unit is (Sp)p(Rp)n(Sp)m.
• oligonucleotides of the present disclosure comprise base sequences, base modifications, sugar modifications, pattern of backbone linkages (internucleotidic linkages), and/or pattern of backbone chiral centers (e.g., of linkage phosphorus atoms) as described in US2015021 1006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, WO2017/160741, WO2017/192664, WO2017/192679, WO2017/210647, WO2018/022473, WO2018/067973, WO2018/098264, each of which is independently incorporated herein by reference.
• Product oligonucleotides and compositions thereof are useful for many purposes, e.g., those described in US2015021 1006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, WO2017/160741, WO2017/192664, WO2017/192679, WO2017/210647, WO2018/022473, WO2018/067973, WO2018/098264, etc.
• product oligonucleotides and compositions thereof prepared by provided technologies are those described in US2015021 1006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, WO2017/160741, WO2017/192664, WO2017/192679, WO2017/210647, WO2018/022473, WO2018/067973, WO2018/098264, etc.
• product oligonucleotides and compositions thereof can modulate levels and/or functions of various products of nucleic acid sequences.
• product oligonucleotides and compositions are useful for treating a variety of disease.
• the present disclosure provides pharmaceutically acceptable salts of oligonucleotides.
• oligonucleotide compositions are pharmaceutical compositions.
• a final product composition is a chirally controlled
• oligonucleotide composition of WV-887, WV-892, WV-896, WV-1714, WV-2444, WV-2445, WV-2526, WV-2527, WV-2528, WV-2530, WV-2531, WV-2578, WV-2580, WV-2587, WV- 3047, WV-3152, WV-3472, WV-3473, WV-3507, WV-3508, WV-3509, WV-3510, WV-351 1, WV-3512, WV-3513, WV-3514, WV-3515, WV-3545, or WV-3546.
• a final product composition is a chirally controlled oligonucleotide composition of WV-2603, WV-2595, WV-1510, WV- 2378, WV-2380, WV-1092, WV-1497, WV-1085, WV-1086, or WV-2623.
• provided technologies comprise labeling of oligonucleotides, e.g., using isotopes.
• provided oligonucleotides contain increased levels of one or more isotopes.
• provided oligonucleotides are labeled, e.g., by one or more isotopes of one or more elements, e.g., hydrogen, carbon, nitrogen, etc.
• provided oligonucleotides in provided compositions comprise one or more base modifications, sugar modifications, and/or internucleotidic linkage modifications, wherein the oligonucleotides contain an enriched level of an isotope.
• an isotope is deuterium.
• a hydrogen in a sugar is replaced by deuterium (e.g., at the 2' position of a 2'-deoxy).
• a hydrogen in a base is replaced by deuterium.
• a hydrogen in an internucleotidic linkage is replaced by deuterium.
• provided oligonucleotides are labeled with deuterium (replacing with - 2 H) at one or more positions.
• replacement of a hydrogen with deuterium can improve the stability, activity, bioavailability, easy of use, convenience, efficacy, and/or systemic exposure of an oligonucleotide.
• one or more 1H of an oligonucleotide or any moiety conjugated to the oligonucleotide ⁇ e.g., a targeting moiety, lipid, etc.) is substituted with 2 H.
• Such oligonucleotides can be used in any composition or method described herein.
• an oligonucleotide which targets HTT comprises one or more isotopes.
• an oligonucleotide which targets dystrophin comprises one or more isotopes.
• provided technologies are particularly useful for preparing chirally controlled oligonucleotide composition with high crude purity and/or yield.
• chiral auxiliaries are typically used to control stereochemistry of a formed linkage phosphorus chiral center.
• the present disclosure provides compounds, e.g., of formula I, I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof, that may be utilized as chiral auxiliaries for oligonucleotide synthesis.
• the present disclosure provides a compound having the structure of formula I:
• L is a covalent bond, or optionally substituted Ci- 6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L'-;
• each L' is independently a covalent bond, optionally substituted bivalent C1-3 alkylene, -C(R 3 )(R 4 )-, -C(R 3 )(R 4 )-C(R 3 )(R 4 )-, -Cy- or -C(R 3 )[C(R 4 ) 3 ]-;
• each of R 1 , R 2 , R 3 , R 4 , and R 5 is independently -H, -L s -R, halogen, -CN, -N0 2 ,
• each L s is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a Ci -3 o aliphatic group and a Ci -3 o heteroaliphatic group having 1- 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from Ci -6 alkylene, Ci -6 alkenylene, c ⁇ c , a bivalent Ci-C 6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R') 2 - -Cy-, -0-, -S- -S-S- -N(R')- -C(0)- -C(S)- -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')-, -N(R')C(0)N(R'
• each -Cy- is independently an optionally substituted bivalent group selected from a C 3-20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Cy L is independently an optionally substituted tetravalent group selected from a C 3- 20 cycloaliphatic ring, a C 6- 2o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C0 2 R, or -S0 2 R;
• R 6 is R'
• R 7 is -OH or -SH
• R 1 , R 2 , R 3 and R 4 is not -H;
• each R is independently -H, or an optionally substituted group selected from Ci-3o aliphatic, Ci -30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6-3 o aryl, C 6-3 o arylaliphatic, C 6-3 o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:
• R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
• two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• L is a covalent bond. In some embodiments, a provided
• R 5 and one or both of R 1 and R 2 , are taken together with their intervening atoms to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.
• one of R 1 and R 2 are taken together with R 5 and their intervening atoms to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having 1- 5 heteroatoms.
• a formed ring can be of various sizes, monocyclic, bicyclic or polycyclic, and contain various numbers of heteroatoms.
• a ring is a 3-membered ring. In some embodiments, a ring is a 4- membered ring. In some embodiments, a ring is a 5-membered ring. In some embodiments, a ring is a 6-membered ring. In some embodiments, a ring is monocyclic. In some embodiments, a ring contains additional ring heteroatoms other than the intervening heteroatoms. In some embodiments, a ring is a 3-membered ring containing one ring heteroatom. In some embodiments, a ring is a 3-membered ring containing two ring heteroatoms. In some embodiments, a ring is a 3-membered ring containing one carbon, one nitrogen, and one oxygen ring atom.
• L is -C(R 3 )(R 4 )-.
• a provided compound has the structure of formula I-a:
• a compound of formula I has the structure of formula I-a.
• a provided compound has the structure of or a salt thereof, wherein each variable is independently as described in the present disclosure, wherein R 4 and R 5 are not hydrogen.
• a provided compound has the structure of formula (I-a-1):
• a compound of formula I-a has the structure of formula I-a-1.
• a provided compound has the structure of formula (I-a-2):
• a compound of formula I-a has the structure of formula I-a-2.
• R 6 is -H. In some embodiments, R 6 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered heterocyclyl ring having 1-5 heteroatoms. In some embodiments, R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 4-10 membered heterocyclyl ring with the intervening nitrogen atom as the only ring heteroatom. In some embodiments, a formed ring is 3-membered. In some embodiments, a formed ring is 4-membered. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6- membered.
• a formed ring is 7-membered. In some embodiments, a formed ring is substituted. In some embodiments, a formed ring is unsubstituted. In some embodiments, a formed ring is monocyclic. In some embodiments, a formed ring is bicyclic. In some embodiments, a formed ring is polycyclic. In some embodiments, a formed ring is saturated. In some embodiments, a formed ring has no ring heteroatoms in addition to the nitrogen to which R 5 is attached.
• R 1 and R 2 are not hydrogen. In some embodiments, R 1 is hydrogen and R 2 is not hydrogen. In some embodiments, R 1 is not hydrogen and R 2 is hydrogen. In some embodiments, neither of R 1 and R 2 is hydrogen.
• one of R 1 and R 2 is -H, and the other is R, wherein R is not hydrogen. In some embodiments, one of R 1 and R 2 is -H, and the other is optionally substituted Ci-6 aliphatic. In some embodiments, one of R 1 and R 2 is -H, and the other is optionally substituted C 1-4 aliphatic. In some embodiments, one of R 1 and R 2 is -H, and the other is optionally substituted C 1-3 aliphatic. In some embodiments, one of R 1 and R 2 is -H, and the other is optionally substituted C 1-2 aliphatic.
• one of R 1 and R 2 is -H, and the other is optionally substituted Ci -6 alkenyl. In some embodiments, one of R 1 and R 2 is -H, and the other is vinyl. In some embodiments, one of R 1 and R 2 is -H, and the other is optionally substituted Ci -6 alkynyl. In some embodiments, one of R 1 and R 2 is -H, and the other is ethynyl. In some embodiments, one of R 1 and R 2 is -H, and the other is optionally substituted benzyl.
• R 1 and R 2 are -H, and the other is benzyl wherein the phenyl group of the benzyl is optionally substituted.
• R x is -H and R 2 is benzyl.
• R 1 is -H and R 2 is -R, wherein R is as described in the present disclosure and is not hydrogen.
• R 2 is optionally substituted Ci -6 aliphatic.
• R 2 is optionally substituted -CH 2 -CPh 2 Me.
• R 2 is -CH 2 -CPh 2 Me.
• R 2 is optionally substituted phenyl.
• R 2 is optionally substituted benzyl.
• HO W is Ph or a salt thereof.
• R 1 is not -H and R 2 is not -H. In some embodiments, R 1 and R 2 are independently R, wherein R is not -H. In some embodiments, R 1 is optionally substituted Ci-6 aliphatic, and R 2 is optionally substituted phenyl. In some embodiments, R 1 is methyl and R 2 is phenyl.
• R 1 and R 2 are R, wherein R comprises a ring moiety.
• R is an optionally substituted group selected from C 3-20 cycloaliphatic, C 6-2 o aryl, 5-20 membered heteroaryl having 1-5 heteroatoms, and 3-20 membered heterocyclyl having 1-5 heteroatoms.
• R is optionally substituted C 3-20 cycloaliphatic.
• R is optionally substituted C 3 . 10 cycloaliphatic.
• R is optionally substituted C 3 . 10 cycloalkyl.
• R is optionally substituted C 4 . 10 cycloalkyl.
• R is optionally substituted cyclopropyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is optionally substituted cycloheptyl. In some embodiments, R is cyclopropyl. In some embodiments, R is cyclobutyl. In some embodiments, R is cyclopentyl. In some embodiments, R is cyclohexyl. In some embodiments, R is cycloheptyl. In some embodiments, R is optionally substituted C 6- 2o aryl.
• R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is optionally substituted 5-20 membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is optionally substituted 5-membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is optionally substituted 6-membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is optionally substituted 3-20 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, the other of R 1 and R 2 is R wherein R is not hydrogen. In some embodiments, R is optionally substituted Ci -6 aliphatic. In some embodiments, R is optionally substituted Ci -6 alkyl.
• R is Ci -6 alkyl. In some embodiments, R is methyl. In some embodiments, R is substituted methyl. In some embodiments, R is ethyl. In some embodiments, R is substituted ethyl. In some embodiments, one of R 1 and R 2 is R comprising a cyclic moiety as described in the present disclosure, and the other is an alkyl group as described in the present disclosure.
• each of R 1 and R 2 is independently R, wherein R is optionally substituted Ci-2o aliphatic. In some embodiments, R is unsubstituted Ci-2o aliphatic. In some embodiments, R is optionally substituted Ci-2o alkyl. In some embodiments, R is optionally substituted Ci -6 alkyl. In some embodiments, R is linear Ci -6 alkyl. In some embodiments, one of R 1 and R 2 is optionally substituted Ci -6 alkyl, and the other is optionally substituted Ci -6 alkyl. In some embodiments, R 1 and R 2 are the same. In some embodiments, R 1 and R 2 are different.
• one of R 1 and R 2 is optionally substituted Ci -6 alkyl, and the other is optionally substituted Ci -6 alkenyl. In some embodiments, one of R 1 and R 2 is optionally substituted methyl or ethyl, and the other is vinyl. In some embodiments, one of R 1 and R 2 is methyl, and the other is vinyl.
• one of R 1 and R 2 is optionally substituted Ci -6 alkyl, and the other is optionally substituted Ci -6 alkynyl. In some embodiments, one of R 1 and R 2 is optionally substituted methyl or ethyl, and the other is ethynyl. In some embodiments, one of R 1 and R 2 is methyl, and the other is ethynyl.
• one of R 1 and R 2 is optionally substituted Ci -6 alkyl, and the other is optionally substituted Ci -6 alkyl.
• R 1 and R 2 are the same optionally substituted Ci -6 alkyl.
• R 1 and R 2 are the same optionally substituted C 1-2 alkyl, and R 1 and R 2 comprise no more than two carbon atoms.
• both R 1 and R 1 are methyl.
• both R 1 and R 1 are ethyl.
• both R 1 and R 1 are isopropyl.
• one of R 1 and R 2 is optionally substituted C 1-3 linear alkyl, and the other is optionally substituted C 3 . 10 cycloalkyl. In some embodiments, one of R 1 and R 2 is optionally substituted C 1-3 linear alkyl, and the other is optionally substituted C 5-6 cycloalkyl. In some embodiments, R 1 is methyl. In some embodiments, R 2 is cyclopentyl. In some embodiments, R 2 is cyclohexyl. In some embodiments, one of R 1 and R 2 is optionally substituted C 1-3 linear alkyl, and the other is optionally substituted benzyl. In some embodiments, R 1 is methyl and R 2 is optionally substituted benzyl.
• R 2 is benzyl. In some embodiments, R 2 is p- CH 3 O-C 6 H 4 -CH 2 -. In some embodiments, R 1 is selected from methyl, ethyl, cyclohexyl, and benzyl which is optionally substituted at the phenyl. In some embodiments, R 2 is selected from methyl, ethyl, cyclohexyl, and benzyl which is optionally substituted at the phenyl. In some embodiments, each of R 1 and R 2 is independently selected from methyl, ethyl, cyclohexyl, and benzyl which is optionally substituted at the phenyl. In some embodiments, a provided
• a provided compound or a salt thereof in some embodiments, a provided compound or a salt thereof.
• a provided compound is or a salt thereof.
• a provide mpound is or a salt thereof.
• a provide mpound is or a salt thereof.
• a provided c salt thereof in some embodiments, a provided
• a provided compound is
• a provided compound is
• a provided compound is
• a provided compound is or a salt thereof.
• one of R 1 and R 2 is optionally substituted Ci -6 alkyl, and the other is optionally substituted phenyl.
• R 1 is methyl
• R 2 is optionally substituted phenyl.
• R 1 is methyl
• 2 is phenyl.
• R 1 is methyl
• R 2 is or
• a provided compound is selected from
• a provided compound is or a salt
• a provided compound is or a salt thereof.
• a provided compound is or a salt thereof.
• R 1 and R 2 are independently R, wherein R is an optionally substituted aryl group. In some embodiments, R 1 and R 2 are independently optionally substituted phenyl. In me embodiments, R 1 and R 2 are phenyl. In some embodiments, a
• provided compound is or a salt thereof.
• R 1 and R 2 are taken together with the carbon atom they are attached on to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms. In some embodiments, R 1 and R 2 are taken together with the carbon atom they are attached on to form an optionally substituted 3-7 membered monocyclic ring having no heteroatoms.
• such a formed monocyclic ring is 3- membered: in some embodiments, 4-membered: in some embodiments, 5-membered: in some embodiments, 6-membered; in some embodiments 7-membered; in some embodiments, 8- membered; in some embodiments 9-membered; and in some embodiments 10-membered.
• a formed ring is monocyclic.
• a formed ring is bicyclic.
• a formed ring is polycyclic.
• a formed ring is aliphatic.
• a formed ring comprises no unsaturation.
• a formed ring is saturated, partially unsaturated, and/or partially aromatic, for example, a bicyclic or polycyclic ring comprising fused saturated, partially unsaturated, and/or aromatic moieties.
• such a formed ring is substituted.
• such a formed ring is not substituted.
• the carbon to which R 1 and R 2 are attached is not chiral.
• R 1 and R 2 are the same, and the carbon they are attached on is not chiral.
• the ring formed by R 1 and R 2 taken together with the carbon atom they are attached on does not introduce asymmetry, and the carbon atom R 1 and R 2 attached on is not chiral.
• R 1 and R 2 are different, and the carbon they are attached on is chiral. In some embodiments, the ring formed by R 1 and R 2 taken together with the carbon atom they are attached on introduces asymmetry, and the carbon atom R 1 and R 2 attached on is not chiral. In some embodiments a provided compound is
• a provided compound is selected from and salts thereof. In some embodiments, a provided compound is
• a prov e compoun s salt thereof in some embodiments, is or a salt thereof.
• a provided compound is salt thereof.
• R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1- 5 heteroatoms.
• a formed ring is 3-membered.
• a formed ring is 4-membered.
• a formed ring is 5-membered.
• a formed ring is 6-membered.
• a formed ring is 7- membered.
• a formed ring is 8-membered.
• a formed ring is 9-membered.
• a formed ring is 10-membered.
• R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms. In some embodiments, R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having a nitrogen atom (the one which R 5 is on). In some embodiments, R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 4-7 membered monocyclic ring having a nitrogen atom (the one which R 5 is on).
• R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 4-membered monocyclic ring having a nitrogen atom (the one which R 5 is on). In some embodiments, R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having a nitrogen atom (the one which R 5 is on). In some embodiments, R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 6-membered monocyclic ring having a nitrogen atom (the one which R 5 is on).
• R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 7-membered monocyclic ring having a nitrogen atom (the one which R 5 is on). In some embodiments, R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 8-membered monocyclic ring having a nitrogen atom (the one which R 5 is on). In some embodiments, R 3 is -H, and R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 9-membered monocyclic ring having a nitrogen atom (the one which R 5 is on).
• R 3 is -H
• R 4 and R 5 are taken together with their intervening atoms to form an optionally substituted 10-membered monocyclic ring having a nitrogen atom (the one which R 5 is on).
• a ring formed by R 4 and R 5 taken together with their intervening atoms is substituted.
• a ring formed by R 4 and R 5 taken together with their intervening atoms is unsubstituted.
• a ring formed by R 4 and R 5 taken together with their intervening atoms is monocyclic.
• a ring formed by R 4 and R 5 taken together with their intervening atoms is bicyclic.
• one of R 1 and R 2 , and one of R 3 and R 4 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or poly cyclic ring having 0-5 heteroatoms.
• a formed ring is a 3-membered ring.
• a formed ring is a 4-membered ring.
• a formed ring is a 5-membered ring.
• a formed ring is a 6-membered ring.
• a formed ring is a 7- membered ring.
• a formed ring is substituted.
• a formed ring is unsubstituted. In some embodiments, a formed ring is monocyclic. In some embodiments, a formed ring is bicyclic. In some embodiments, a formed ring is polycyclic. In some embodiments, a formed ring has no additional heteroatoms in addition to an intervening atom. In some embodiments, a formed ring has additional ring heteroatoms in addition to an intervening atom. Example rings formed are extensively described in the present disclosure. In me embodiments, a provided compound is selected from
• a provided compound is selected from thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided com ound is "° " Ph> 0 r a salt thereof. In some embodiments, a provided compound is a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is or a salt thereof. In some embodiments, a provided compound is
• one or two of R 1 and R 2 are taken together with one or more of R 3 , R 4 , and R 5 and the intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms.
• one or two of R 1 and R 2 are taken together with one or two of R 3 and R 4 and the intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0- 5 heteroatoms.
• one or two of R 1 and R 2 are taken together with R 5 and the intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms. In some embodiments, one or two of R 1 and R 2 are taken together with R 5 , one or two of R 3 and R 4 , and the intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.
• one or two of R 1 and R 2 are taken together with R 5 , one or two of R 3 and R 4 , and the intervening atoms to form an optionally substituted 6-20 membered bicyclic or polycyclic ring having 1-5 heteroatoms.
• one or two of R 1 and R 2 are taken together with R 5 , one or two of R 3 and R 4 , and the intervening atoms to form an optionally substituted 8-20 membered bicyclic or polycyclic ring having 1-5 heteroatoms.
• one of R 1 and R 2 are taken together with R 5 , one of R 3 and R 4 , and the intervening atoms to form an optionally substituted 8-20 membered bicyclic or polycyclic ring having 1-5 heteroatoms. In some embodiments, one of R 1 and R 2 are taken together with R , one of R 3 and R 4 , and the intervening atoms to form an optionally substituted 8-20 membered bicyclic ring having 1-5 heteroatoms. In some embodiments, a formed ring is 8-membered. In some embodiments, a formed ring is 9-membered.
• R 5 is taken with one of R 1 and R 2 and their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms. In some embodiments, R 5 is taken with one of R 3 and R 4 and their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.
• Example rings formed are extensively described in the present disclosure. In some embodiments, a formed ring is 3-membered. In some embodiments, a formed ring is 4-membered. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6-membered.
• R 5 is not taken with R 1 , R 2 , R 3 , or R 4 to form an optionally substituted ring.
• R 5 is optionally substituted Ci -6 aliphatic.
• R 5 is optionally substituted Ci -6 alkyl.
• R 5 is unsubstituted Ci -6 alkyl.
• R 5 is methyl.
• R 5 is ethyl.
• R 5 is isopropyl.
• a provided compound is or a salt thereof.
• a provided com or a salt thereof in some embodiments, a provided com or a salt thereof.
• a provided compound is or a salt thereof.
• L is -L'-C(R )(R )-
• a provided compound has the structure of formula I-b:
• a compound of formula I has the structure of formula I-b.
• L' is a covalent bond. In some embodiments, L' is -C(R 3 )(R 4 )-. In some embodiments, a provided compound has the structure of formula I-c:
• a compound of formula I has the structure of formula I-c.
• one or R 3 and R 4 on C2 are taken together with R 5 to form with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having a nitrogen atom (the one which R 5 is on).
• one or R 3 and R 4 on C3 are taken together with R 5 to form with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having a nitrogen atom (the one which R 5 is on).
• one of R 3 and R 4 on C2, and one of R 3 and R 4 on C3, are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms.
• R 3 and R 4 on the same carbon atom are taken together with the carbon atom to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms.
• R 3 and R 4 on C2 are taken together with C2 to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms.
• R 3 and R 4 on C3 are taken together with C3 to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-5 heteroatoms.
• Example such ring moieties e.g., formed by R 3 /R 4 and R 5 , by R 3 /R 4 and R 3 /R 4 , etc., are extensively described in the present disclosure, and can be e.g., 4-membered, 5-membered, 6-membered, 7-membered, monocyclic, bicyclic, polycyclic, substituted, unsubstituted, with additional ring heteroatoms (other than the intervening atom(s)), without additional ring hetereoatoms, combinations thereof, etc.
• R 3 on C2 is hydrogen. In some embodiments, R 4 on C2 is hydrogen. In some embodiments, R 3 on C3 is hydrogen. In some embodiments, R 4 on C3 is hydrogen. In some embodiments, both R 3 and R 4 on C2 are hydrogen. In some embodiments, both R 3 and R 4 on C3 are hydrogen. In some embodiments, both R 3 and R 4 on C2, and one of R 3 and R 4 on C3, are hydrogen. In some embodiments, both R 3 and R 4 on C3, and one of R 3 and R 4 on C2, are hydrogen.
• a provided compound is P H h > or a salt thereof.
• L is -Cy-
• a provided compound has the structure of formula I-d:
• a compound of formula I has the structure of formula I-d.
• -Cy- is 1,2-bivalent.
• -Cy- is optionally substituted
• - - is optionally substituted
• -Cy- is optionally substituted
• one of R 1 and R 2 , and one of R 3 and R 4 are R and are taken together with their intervening atoms to form an optionally substituted 3-20 membered ring having 1-10 heteroatoms as described in the present disclosure, e.g., Ring A as described herein.
• a provided compound has the structure of formula I-e:
• a compound of formula I has the structure of formula I-e.
• one of R 1 and R 2 , and R 4 are taken together with their intervening atoms to form an optionally substituted 3-20 membered ring having 1-5 heteroatoms.
• R 3 is— H
• one of R 1 and R 2 , and R 4 are taken together with their intervening atoms to form an optionally substituted 3-20 membered ring having 1-5 heteroatoms.
• R 2 and R 4 are taken together with their intervening atoms to form an optionally substituted ring (e.g., formula I-e).
• a formed ring e.g., Ring A in formula I-e, is 3, 4, 5, 6, 7, 8, 9, or 10-membered.
• a formed ring is 3- membered. In some embodiments, a formed ring is 4-membered. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6-membered. In some embodiments, a formed ring is 7-membered. In some embodiments, a formed ring is 8- membered. In some embodiments, a formed ring is 9-membered. In some embodiments, a formed ring is 10-membered. In some embodiments, a formed ring is monocyclic. In some embodiments, a formed ring is bicyclic. In some embodiments, a formed ring is polycyclic. In some embodiments, a formed ring is saturated.
• a formed ring is partially unsaturated. In some embodiments, a formed ring has no heteroatoms. In some embodiments, a formed ring is an optionally substituted 3-membered saturated aliphatic ring. In some embodiments, a formed ring is an optionally substituted 4-membered saturated aliphatic ring. In some embodiments, a formed ring is an optionally substituted 5-membered saturated aliphatic ring. In some embodiments, a formed ring is an optionally substituted 6-membered saturated aliphatic ring. In some embodiments, a formed ring is an optionally substituted 7-membered saturated aliphatic ring.
• a formed ring is an optionally substituted 8- membered saturated aliphatic ring. In some embodiments, a formed ring is an optionally substituted 9-membered saturated aliphatic ring. In some embodiments, a formed ring is an optionally substituted 10-membered saturated aliphatic ring.
• R 3 is -H
• R 1 is optionally substituted Ci -6 aliphatic or phenyl
• R 5 is optionally substituted Ci -6 aliphatic
• R 6 is -H.
• R 3 is -H
• R 1 and R 5 are taken together with their intervening atoms to form an optionally substituted ring
• R 6 is -H.
• R 3 is -H
• R 1 and R 5 are taken together with their intervening atoms to form an optionally substituted 5- or 6-membered ring
• R 6 is -H.
• R 3 is -H
• R 1 and R 5 are taken together with their intervening atoms to form an optionally substituted 5-membered saturated ring having no heteroatom in addition to the nitrogen to which R 5 is attached
• R 6 is -H
• R 3 is -H
• R 1 and R 5 are taken together with their intervening atoms to form an optionally substituted 6-membered saturated ring having no heteroatom in addition to the nitrogen to which R 5 is attached
• R 6 is -H.
• a ring formed by R 1 and R 5 taken together are unsubstituted.
• -OH and -N(R 5 )(R 6 ) are trans. In some embodiments, -OH and -N(R 5 )(R 6 ) are cis.
• the carbon to which R 1 and -OH are attached is R. In some embodiments, the carbon to which R 1 and -OH are attached is S.
• R 1 is hydrogen. In some embodiments, R 1 is not hydrogen.
• R 1 is optionally substituted Ci -6 aliphatic or phenyl. In some embodiments, R 1 is methyl. In some embodiments, R 1 is phenyl.
• R 3 is hydrogen. In some embodiments, R 5 is hydrogen.
• R 5 is not hydrogen. In some embodiments, R 5 is optionally substituted Ci -6 aliphatic or phenyl. In some embodiments, R 5 is methyl. In some embodiments, R 5 is phenyl. In some embodiments, R 6 is hydrogen. In some embodiments, R 6 is not hydrogen. In some embodiments, as demonstrated by certain example data, compounds with trans -OH and -N(R 5 )(R 6 ) can provide high yields and/or diastereoselectivity. In some embodiments, as demonstrated by certain example data, compounds with trans -OH and -N(R 5 )(R 6 ) can provide both high yields and diastereoselectivity.
• a provided compound e.g., a compound of formula I-e, is
• a a n prroovviiddeedd c coommnpoouunndd i is or a
• a provided compound is or a salt there of.
• a provided compound is or a salt there of. In some embodiments, a
• provided compound is or a salt there of. In some embodiments, a provided
• a provided compound is — / or a salt there of. In some embodiments, a provided compound is or a salt there of. In some embodiments, a provided compound is selected from , or salts thereof. In some
• provided compound is or a salt thereof. In some embodiments, a provided
• a provid mpound is or a salt thereof.
• a provided compound is or a salt thereof.
• a provided com ound has the structure of formula II:
• Ring A is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R 2 , R 3 , R 4 , and R 5 is independently -H, -L s -R, halogen, -CN, -N0 2 , -L s -Si(R) 3 , -OR, -SR, or -N(R) 2 ;
• Ci-C 6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(0)- -C(S)- -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')- -N(R')C(0)0- -S(O)- -S(0) 2 -,
• each -Cy- is independently an optionally substituted bivalent group selected from a C 3-20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Cy L is independently an optionally substituted tetravalent group selected from a C 3- 2 o cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C(0)OR, or -S(0) 2 R;
• t 0-20;
• R 6 is R' ;
• R 7 is -OH or -SH
• L is a covalent bond, or optionally substituted Ci- 6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L'-;
• L' is a covalent bond, optionally substituted bivalent Ci- 3 alkylene, -C(R 3 )(R 4 )-, -C(R 3 )(R 4 )-C(R 3 )(R 4 )-, -Cy-, or -C(R 3 )[C(R 4 ) 3 ]-;
• each R is independently -H, or an optionally substituted group selected from Ci -3 o aliphatic, Ci -30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6 -3o aryl, C 6- 3o arylaliphatic, C 6- 3o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:
• R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
• two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• a provided com ound has the structure of formula Il-a:
• a compound of formula II has the structure of formula Il-a.
• a provided compound of structure Il-a has the structure of formula Il-b:
• each variable is independently as described in the present disclosure.
• a compound of formula Il-a has the structure of formula Il-b.
• a provided compound of structure Il-a has the structure of formula II-c:
• a compound of formula Il-a has the structure of formula II-c.
• R 8 is -OH. In some embodiments, R 6 is -H. In some embodiments, R 5 is optionally substituted alkyl. In some embodiments, R 5 is methyl. In some embodiments, t is 0. In some embodiments, R 3 is optionally substituted alkyl. In some embodiments, R 3 is methyl. In some embodiments, R 3 is optionally substituted phenyl. In some embodiments, R 3 is phenyl. In some embodiments, R 3 is optionally substituted C 3 - 10 cycloalkyl. In some embodiments, R 3 is optionally substituted cyclohexyl. In some embodiments, R 3 is cyclohexyl.
• Ring A is an optionally substituted 3-20 membered monocyclic, bicyclic or poly cyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• Ring A is or comprises at least one monocyclic saturated or partially unsaturated monocyclic ring moiety, optionally as part of a bicyclic or polycyclic system.
• Ring A is monocyclic.
• Ring A is bicyclic or polycyclic comprising at least one monocyclic saturated or partially unsaturated monocyclic ring moiety, and optionally one or more aromatic monocyclic moieties.
• Ring A is or comprises at least one saturated monocyclic ring moiety.
• R 8 is connected to a sp 3 ring atom of Ring A. In some embodiments, R 8 is connected to a sp 3 carbon ring atom of Ring A. In some embodiments, R 3 is connected to a sp 3 ring atom of Ring A. In some embodiments, R 3 is connected to a sp 3 carbon ring atom of Ring A. In some embodiments, -N(R 5 )(R 6 ) is connected to a sp 3 ring atom of Ring A. In some embodiments, -N(R 5 )(R 6 ) is connected to a sp 3 carbon ring atom of Ring A.
• Ring A is optionally substituted C 3 - 10 cycloalkyl. In some embodiments, Ring A is optionally substituted cyclohexyl. In some embodiments, Ring A is cyclohexyl. In some embodiments, R 8 and -N(R 5 )(R 6 ) are cis. In some embodiments, R 8 and are trans. In some embodiments, a provided compound of formula II is selected
• a provided compound is a compound of II-c or a salt thereof.
• R 3 and R 5 are R, and are taken together to form an optionally substituted ring as described in the present disclosure.
• formula II is selected from H and salts thereof.
• a provided compound e.g. , a compound of formula I, has the structure of formula III:
• Ring A' is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein Ring A' comprises a -N(R 6 )- moiety;
• each of R 1 , R 2 , R 3 and R 4 is independently -H, -L s -R, halogen, -CN, -N0 2 , -L s -Si(R) 3 , -OR, -SR, or -N(R) 2 ;
• each -Cy- is independently an optionally substituted bivalent group selected from a C 3-20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Cy L is independently an optionally substituted tetravalent group selected from a C 3- 2 o cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C(0)OR, or -S(0) 2 R;
• t 0-20;
• R 6 is R' ;
• R 7 is -OH or -SH
• L is a covalent bond, or optionally substituted Ci- 6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L'-;
• L' is a covalent bond, optionally substituted bivalent Ci- 3 alkylene, -C(R 3 )(R 4 )-, -C(R 3 )(R 4 )-C(R 3 )(R 4 )-, -Cy-, or -C(R 3 )[C(R 4 ) 3 ]-;
• each R is independently -H, or an optionally substituted group selected from Ci -3 o aliphatic, Ci -3 o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6-3 o aryl, C 6-3 o arylaliphatic, C 6-3 o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:
• R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
• two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• a provided com ound has the structure of formula Ill-a:
• a compound of formula III has the structure of formula Ill-a.
• a provided com ound has the structure of formula Ill-b:
• a compound of formula Ill-a has the structure of formula Ill-b.
• R 8 is bonded to a carbon atom (C 2 ) next to the nitrogen atom in -N(R 6 )- (N 1 ) (e.g., formula Ill-a, formula Ill-b, etc.). In some embodiments, R 8 is bonded to
• R 8 is bonded to a carbon atom next to C that is not C 2 (C 4 ). In some embodiments, R 8 is bonded to a carbon atom next to C 4 which is not C 3 (C 5 ). In some embodiments, R 8 is bonded to a carbon atom next to C 5 which is not C 4 (C 6 ).
• R 8 is -OH. In some embodiments, R 6 is -H. In some embodiments, R 5 is optionally substituted alkyl. In some embodiments, R 5 is methyl. In some embodiments, t is 0. In some embodiments, R 3 is optionally substituted alkyl. In some embodiments, R 3 is methyl. In some embodiments, R 3 is optionally substituted phenyl. In some embodiments, R 3 is phenyl. In some embodiments, R 3 is optionally substituted C 3 - 10 cycloalkyl. In some embodiments, R 3 is optionally substituted cyclohexyl. In some embodiments, R 3 is cyclohexyl.
• Ring A' is an optionally substituted 3-20 membered monocyclic, bicyclic or poly cyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein Ring A' comprises a -N(R 6 )- moiety.
• Ring A' is Ring A as described in the present disclosure, wherein Ring A comprises a nitrogen ring atom.
• Ring A' is or comprises at least one monocyclic saturated or partially unsaturated monocyclic ring moiety, optionally as part of a bicyclic or polycyclic system. In some embodiments, Ring A' is monocyclic.
• Ring A' is bicyclic or polycyclic comprising at least one monocyclic saturated or partially unsaturated monocyclic ring moiety, and optionally one or more aromatic monocyclic moieties. In some embodiments, Ring A' is or comprises at least one saturated monocyclic ring moiety. In some embodiments, R 8 is connected to a sp 3 ring atom of Ring A'. In some embodiments, R 8 is connected to a sp 3 carbon ring atom of Ring A' . In some embodiments, R 3 is connected to a sp 3 ring atom of Ring A'. In some embodiments, R 3 is connected to a sp 3 carbon ring atom of Ring A' . In some embodiments, the nitrogen to which R 6 is attached is sp 3 .
• a provided compound of formula III is selected from
• a provided compound of formula III is selected from compounds listed in Table 4 below and salts thereof.
• a provided compound is an enantiomer of a compound selected from Table 1 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table 1 or a salt thereof.
• a provided compound is an enantiomer of a compound selected from Table 2 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table 2 or a salt thereof.
• a provided compound is an enantiomer of a compound selected from Table 3 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table 3 or a salt thereof.
• a provided compound is an enantiomer of a compound selected from Table 4 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table 4 or a salt thereof.
• provided compounds e.g., compounds of formula I, I-a, I-a-1, I-a-2, 1-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof, comprises one or more chiral elements.
• I- c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof, are chiral.
• I- c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof are of a stereopurity described in the present disclosure.
• provided chiral compounds, compounds of formula I, I-a, I-a-1, I-a-2, I-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof are of a diastereomeric purity described in the present disclosure.
• compounds of formula I, I-a, I- a-1, I-a-2, I-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof are of diastereomeric and enantiomeric purity described in the present disclosure.
• the present disclosure provides compounds, e.g., compounds of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, Vl-e, VIII, or salts thereof, that are made from compounds of formula I, I-a, I-a- 1, I-a-2, I-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof, and comprise chiral elements of compounds of formula I, I-a, I-a-1, I-a-2, I-b, I-c, I-d, I-e, II, Il-a, Il-b, III, Ill-a, or Ill-b, or salts thereof, and comprise chiral elements of
• chiral auxiliaries are utilized to prepare chirally pure phosphoramidites, which are used to stereoselectively form linkage phosphorus chiral centers compared to absence of chiral auxiliaries.
• the present disclosure provides compound, e.g., of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, or Vl-e, or salts thereof, that can be utilized as phosphoramidites for
• product oligonucleotides may contain one or more natural phosphate linkages and/or non-chirally controlled chiral internucleotidic linkages, and for such linkages, phosphoramidite for traditional oligonucleotide synthesis may be readily utilized.
• nucleobases may be blocked in
• phosphoramidite for oligonucleotide synthesis can be de-blocked, e.g., after synthesis cycles.
• Technologies for blocking and de-blocking (protecting) nucleobases are widely known in the art and can be utilized in accordance with the present disclosure.
• Technologies for preparing phosphoramidites can be utilized in accordance with the present disclosure.
• Example technologies include those described in US2015100197, US9744183, US9605019, US9394333, US8859755, US20130178612, US8470987, US8822671, US20150211006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, US9403865, WO2017/160741,
• a phosphoramidite is a DPSE-
• the present disclosure provides a compound having the structure of formula IV:
• W is O, S or Se
• L is a covalent bond, or optionally substituted Ci- 6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L'-;
• L' is a covalent bond, optionally substituted bivalent C1-3 alkylene, -C(R 3 )(R 4 )-, -C(R 3 )(R 4 )-C(R 3 )(R 4 )-, -Cy- or -C(R 3 )[C(R 4 ) 3 ]-;
• each of R 1 , R 2 , R 3 , R 4 , and R 5 is independently -H, -L s -R, halogen, -CN, -N0 2 ,
• each L s is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a Ci -3 o aliphatic group and a Ci -3 o heteroaliphatic group having 1- 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from Ci -6 alkylene, Ci -6 alkenylene, c ⁇ c , a bivalent Ci-C 6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R') 2 - -Cy-, -0-, -S- -S-S- -N(R')- -C(0)- -C(S)- -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')-, -N(R')C(0)N(R'
• each -Cy- is independently an optionally substituted bivalent group selected from a C 3-20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Cy L is independently an optionally substituted tetravalent group selected from a C 3- 20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C(0)OR, or -S(0) 2 R;
• L 7 is -O- or -S-;
• R 1 , R 2 , R 3 and R 4 is not -H;
• BA is an optionally substituted group selected from C3-30 cycloaliphatic, C 6- 3o aryl, C5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety;
• each R s is independently -H, halogen, -CN, -N 3 , " NO, -N0 2 , -L s -R', -L s -Si(R) 3 , -L s -OR', -L S -SR', -L S -N(R') 2 , -0-L s -R', -C-L s -Si(R) 3 , -0-L s -OR', -0-L s -SR', or -0-L s -N(R') 2 ;
• t 0-20;
• Ring A s is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• R 5s is R s ;
• each R' is independently -R, -C(0)R, -C(0)OR, or -S(0) 2 R;
• each R is independently -H, or an optionally substituted group selected from Ci-3o aliphatic, Ci.30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6 -3o aryl, C 6- 3o arylaliphatic, C 6- 3o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:
• R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
• two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• SU is -L s -0- In some embodiments, SU is
• SU is each of R ls , R 2s , R 3s , R 4s and R 5s is independently R s .
• SU is wherein each variable is independently
• SU is , wherein each variable is independently as described in the present disclosure.
• SU IS wherein each variable is independently as described in the present disclosure.
• SU is , wherein each variable is independently as described
• SU is , wherein each variable is independently as described in the present disclosure.
• a provided compound e.g., a compound of formula IV, has the structure of formula IV-a:
• a provided compound of formula IV has the structure of formula IV-a.
• L s -Cy- L s is an optionally substituted monocyclic or bicyclic 3-20 membered heterocyclyl ring having 1-5 heteroatoms.
• L s is an optionally substituted monocyclic or bicyclic 5-20 membered heterocyclyl ring having 1-5 heteroatoms, wherein at least one heteroatom is oxygen.
• L s is an optionally substituted bivalent tetrahydrofuran ring.
• L s is an optionally substituted furanose moiety.
• the BA in formula IV-a is bonded to CI
• the -O- in formula IV-a is bonded to C3, of the furanose moiety.
• a provided compound e.g., a compound of formula IV, has the structure of formula IV-b:
• a provided compound of formula IV has the structure of formula IV-b.
• a provided compound e.g., a compound of formula IV, has the structure of formula IV-c-1:
• a provided compound of formula IV has the structure of formula IV-c-1.
• a provided compound e.g., a compound of formula IV, has the structure of formula IV-c-2:
• a provided compound of formula IV has the structure of formula IV-c-2.
• a provided compound e.g., a compound of formula IV, has the structure of formula IV-d:
• a provided compound of formula IV has the structure of formula IV-d.
• a provided compound e.g., a compound of formula IV, has the structure of formula IV-e:
• a provided compound of formula IV has the structure of formula IV-e.
• a compound of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, or IV-e can be prepared from a compound of formula I I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, II- a, Il-b, III, Ill-a, Ill-b, etc.
• a compound of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, or IV-e has a structure such that is a compound having the structure of I, I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, or I-e, or a salt thereof.
• the present disclosure provides a compound having the structure of formula IVa:
• the present disclosure provides a compound having the structure of formula IVa-a:
• a compound of formula IVa is a compound of formula IVa-a.
• the present disclosure provides a compound having the structure of formula IVa-b:
• a compound of formula IVa is a compound of formula IVa-b.
• the present disclosure provides a compound having the structure of formula IVa- c-1:
• a compound of formula IVa is a compound of formula IVa-c-1.
• the present disclosure provides a compound having the structure of formula IVa- c-2:
• a compound of formula IVa is a compound of formula IVa-c-2.
• the present disclosure provides a com ound having the structure of formula IVa-d:
• a compound of formula IVa is a compound of formula IVa-d.
• the present disclosure provides a compound having the structure of formula IVa-e:
• a compound of formula IVa is a compound of formula IVa-e.
• L 7 is -0-.
• each of R 1 , R 5 and R 6 is independently optionally substituted Ci -6 alkyl.
• R 5 and R 6 are the same.
• P L is P.
• -I ⁇ -R 1 contains no chiral elements.
• -N(R 5 )(R 6 ) contains no chiral elements.
• -I -R 1 and -N(R 5 )(R 6 ) contains no chiral elements.
• -I -R 1 is -0-CH 2 CH 2 -CN.
• -N(R 5 )(R 6 ) is -N(/ ' -Pr) 2 .
• a compound of formula IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, or IVa-e, or a salt thereof is a phosphoramidite for non-chirally controlled oligonucleotide synthesis, e.g., oligonucleotide synthesis using traditional phosphoramidite chemistry.
• R 1 and R 5 are R and are taken together with their intervening atoms to form a ring as described in the present disclosure.
• a formed ring contain a chiral element, and a compound of formula IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, or IVa-e, or a salt thereof can be utilized for chirally controlled oligonucleotide synthesis.
• the present disclosure provides a compound having the structure of formula V:
• W is O, S or Se
• Ring A is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each of R 1 , R 2 , R 3 , R 4 , and R 5 is independently -H, -L s -R, halogen, -CN, -N0 2 , -L s -Si(R) 3 , -OR, -SR, or -N(R) 2 ;
• each L s is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a Ci -3 o aliphatic group and a Ci -3 o heteroaliphatic group having 1- 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from Ci -6 alkylene, Ci -6 alkenylene, C ⁇ C ?
• Ci-C 6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R') 2 - -Cy- -0-, -S- -S-S- -N(R')- -C(0)- -C(S)- -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')- -N(R')C(0)0- -S(O)- -S(0) 2 -,
• each -Cy- is independently an optionally substituted bivalent group selected from a C 3-20 cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each Cy L is independently an optionally substituted tetravalent group selected from a C 3- 2 o cycloaliphatic ring, a C 6-2 o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• t 0-20;
• L is a covalent bond, or optionally substituted Ci- 6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L'-;
• L' is a covalent bond, optionally substituted bivalent C 1 -3 alkylene, -C(R 3 )(R 4 )-,
• BA is an optionally substituted group selected from C3-30 cycloaliphatic, C 6- 3o aryl, C5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiet wherein SU is connected to the phosphorus atom through the oxygen atom;
• R 5s is R s ;
• each R s is independently -H, halogen, -CN, -N 3 , " NO, -N0 2 , -L s -R', -L s -Si(R) 3 , -L s -OR', -L S -SR', -L S -N(R') 2 , -0-L s -R', -0-L s -Si(R) 3 , -C-L s -OR', -0-L s -SR', or -0-L s -N(R') 2 ;
• Ring A s is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
• each R' is independently -R, -C(0)R, -C(0)OR, or -S(0) 2 R;
• each R is independently -H, or an optionally substituted group selected from Ci-3o aliphatic, Ci.30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C 6- 3o aryl, C 6- 3o arylaliphatic, C 6- 3o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:
• R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
• two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or poly cyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
• a provided compound e.g., a compound of formula V, has the structure of formula V-a:
• a provided compound of formula V has the structure of formula V-a.
• a provided compound e.g., a compound of formula V, has the structure of formula V-b:
• a provided compound of formula V has the structure of formula V-b.
• a provided compound e.g., a compound of formula V, has the structure of formula V-c-1 :
• a provided compound of formula V has the structure of formula V-c-1.
• a provided compound e.g., a compound of formula V, has the structure of formula V-c-2:
• a provided compound of formula V has the structure of formula V-c-2.
• a provided compound e.g., a compound of formula V, has the structure of formula V-d:
• a provided compound of formula V has the structure of formula V-d.
• a provided compound e.g., a compound of formula V, has the structure of formula V-e:
• a provided compound of formula V has the structure of formula V-e.
• a compound of formula V, V-a, V-b, V-c-1, V-c-2, V-d, or V- e can be prepared from a compound of formula I, I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, Il-a, II-
• a compound of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, or IV-e has a structure such that is a compound having the structure of II, Il-a, or Il-b.
• the present disclosure provides a compound having the structure of formula VI:
• each variable is independently as described in the present disclosure.
• a provided compound e.g., a compound of formula VI, has the structure of formula Vl-a:
• a provided compound of formula VI has the structure of formula Vl-a.
• a provided compound e.g., a compound of formula VI, has the structure of formula Vl-b:
• a provided compound of formula VI has the structure of formula Vl-b.
• a provided compound e.g., a compound of formula VI, has the structure of formula VI-c-1:
• a provided compound of formula VI has the structure of formula VI-c-1.
• a provided compound e.g., a compound of formula VI, has the structure of formula VI-c-2:
• a provided compound of formula VI has the structure of formula VI-c-2.
• a provided compound e.g., a compound of formula VI, has the structure of formula Vl-d:
• a provided compound of formula VI has the structure of formula Vl-d.
• a provided compound e.g., a compound of formula VI, has the structure of formula Vl-e:
• a provided compound of formula VI has the structure of formula Vl-e.
• a compound of formula VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, or Vl-e can be prepared from a compound of formul -a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, II-
• a compound of formula VI, Vl-a, Vl-b, VI-c-1, VI-c-2, Vl-d, or Vl-e has a structure such that L 8 -H _ is a compound having the structure of III, Ill-a, or Ill-b.
• a phosphoramidite has the structure of formula IV, IV-a, IV- b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a,
• P L is P.
• a phosphoramidite for non-stereocontrolled coupling is of
• a nucleobase in provided oligonucleotides is a natural nucleobase (e.g., adenine, cytosine, guanosine, thymine, or uracil) or a modified nucleobase derived from a natural nucleobase, e.g., optionally substituted adenine, cytosine, guanosine, thymine, or uracil, or tautomeric forms thereof.
• a natural nucleobase e.g., adenine, cytosine, guanosine, thymine, or uracil
• a modified nucleobase derived from a natural nucleobase e.g., optionally substituted adenine, cytosine, guanosine, thymine, or uracil, or tautomeric forms thereof.
• Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, and tautomeric forms thereof, having their respective amino groups protected by protecting groups, e.g., one or more of -R, -C(0)R, etc.
• protecting groups e.g., one or more of -R, -C(0)R, etc.
• Example protecting groups are widely known in the art and can be utilized in accordance with the present disclosure.
• a protected nucleobase and/or derivative is selected from nucleobases with one or more acyl protecting groups, 2-fluorouracil, 2- fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products).
• Example modified nucleobases are also disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al.
• a modified nucleobase is substituted uracil, thymine, adenine, cytosine, or guanine.
• a modified nucleobase is a functional replacement, e.g., in terms of hydrogen bonding and/or base pairing, of uracil, thymine, adenine, cytosine, or guanine.
• a nucleobase is optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.
• a nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.
• a modified base is optionally substituted adenine, cytosine, guanine, thymine, or uracil.
• a modified nucleobase is independently adenine, cytosine, guanine, thymine or uracil, modified by one or more modifications by which:
• a nucleobase is modified by one or more optionally substituted groups independently selected from acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof;
• one or more atoms of a nucleobase are independently replaced with a different atom selected from carbon, nitrogen or sulfur;
• one or more optionally substituted aryl or heteroaryl rings are independently inserted into a nucleobase.
• n R s an opt ona y su st tute , near or ranc e group se ecte rom a p at c, aryl, aralkyl, aryloxylalkyl, carbocyclyl, heterocyclyl and heteroaryl, each having 1 to 30 carbon atoms, and 1-10 heteroatoms if applicable, including, by way of example only, a methyl, isopropyl, phenyl, benzyl, or phenoxymethyl group; and each of R 9N and R 10N is independently an optionally substituted group selected from linear or branched aliphatic, carbocyclyl, aryl, heterocyclyl and heteroaryl, each having 1 to 30 carbon atoms, and 1-10 heteroatoms [00221]
• Modified nucleobases also include expanded-size nucleobases in which one or more aryl rings, such as phenyl rings, have been added.
• Modified nucleobases also encompass structures that are not considered typical nucleobases but are other moieties such as, but not limited to, corrin- or porphyrin-derived rings. Porphyrin-derived base replacements have been described in Morales-Rojas, H and Kool, ET, Org. Lett., 2002, 4, 4377- 4380. Shown below is an example of a porphyrin-derived ring which can be used as a base replacement:
• a modified nucleobase is fluorescent.
• fluorescent modified nucleobases include phenanthrene, pyrene, stilbene, isoxanthine, isozanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, lumazine, tethered stilbene, benzo-uracil, and na htho-uracil, as shown below:
• a modified nucleobase is unsubstituted. In some embodiments, a modified nucleobase is substituted. In some embodiments, a modified nucleobase is substituted such that it contains, e.g., heteroatoms, alkyl groups, or linking moieties connected to fluorescent moieties, biotin or avidin moieties, or other protein or peptides. In some embodiments, a modified nucleobase is a "universal base" that is not a nucleobase in the most classical sense, but that functions similarly to a nucleobase. One example of such a universal base is 3-nitropyrrole.
• nucleosides can also be used in technologies disclosed in the present disclosure and may include nucleosides that incorporate modified nucleobases, or nucleobases covalently bound to modified sugars.
• nucleosides that incorporate modified nucleobases include 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2'-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyluridine; dihydrouridine; 2'-O-methylpseudouridine; beta,D- galactosylqueosine; 2'-O-methylguanosine; A ⁇ -isopentenyladenosine; 1-methyladenosine; 1- methylpseudouridine; 1-methylguanosine; 1-methylinosine; 2,2-dimethylguanosine; 2- methyladenosine; 2-methylguanosine; N 7 -methyl
• nucleosides include 6'-modified bicyclic nucleoside analogs that have either (R) or ( ⁇ -chirality at the 6' -position and include the analogs described in US Patent No. 7,399,845.
• nucleosides include 5'-modified bicyclic nucleoside analogs that have either (R) or ( ⁇ -chirality at the 5 '-position and include the analogs described in US Patent Application Publication No. 20070287831.
• a nucleobase or modified nucleobase comprises one or more biomolecule binding moieties such as e.g., antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties.
• a nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine.
• a nucleobase or modified nucleobase is modified by substitution with a fluorescent or biomolecule binding moiety.
• the substituent on a nucleobase or modified nucleobase is a fluorescent moiety.
• the substituent on a nucleobase or modified nucleobase is biotin or avidin.
• a base e.g., BA
• a base is optionally substituted A, T, C, G or U.
• a modified base is substituted A, T, C, G or U, wherein the modified base is different than the natural A, T, C, G and U.
• a modified nucleotide or nucleotide analog is any modified nucleotide or nucleotide analog described in any of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997 Chem. Eur. J. 3 : 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res.
• Example nucleobases are also described in US 201 10294124, US 20120316224, US 20140194610, US 2015021 1006, US 20150197540, WO 2015107425, PCT/US2016/043542, and PCT/US2016/043598, nucleobases of each of which are incorporated herein by reference.
• provided compounds e.g. , oligonucleotides, comprise one or
• a sugar moiety is , wherein each variable is independently as described in the present disclosure. In some embodiments, a
• sugar moiety is wherein L s is -C(R 5s ) 2 -, wherein each R 3S is independently as described in the present disclosure.
• a sugar moiety has the structure of

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