Compositions and methods for phosphoramidite and oligonucleotide synthesis

Abstract

To provide methods for preparing phosphoramidites, which are useful for improving efficiency and / or lowering cost of oligonucleotide preparation.SOLUTION: A method for purifying a phosphoramidite comprises steps of: a) removing water from a purification medium; b) contacting the purification medium with a phosphoramidite; and c) optionally using the purification medium to purify a phosphoramidite.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 307,542, filed March 13, 2016, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Oligonucleotides are useful for many purposes, including the treatment of various diseases, and there is a need for efficient methods for the synthesis of oligonucleotides. Summary of the Invention

[0003] The present disclosure includes recognizing the source of problems with many techniques for preparing phosphoramidites. As known in the art, phosphoramidites are often utilized as monomers in oligonucleotide synthesis. Many techniques for preparing phosphoramidites suffer from low yields and / or low purity. In particular, the present disclosure provides techniques (e.g., compounds, compositions, methods, etc.) that provide significantly improved yields and / or purity for preparing phosphoramidites and / or other intermediates and products, which are beneficial, for example, for improving the efficiency and / or reducing the cost of oligonucleotide preparation.

[0004] The present disclosure encompasses several surprising findings, including that several processes for pretreating a purification medium can surprisingly improve the utility and / or effectiveness of the medium, particularly when phosphoramidites are used in the preparation. In some embodiments, such processes may be or include inactivation processes. In some embodiments, such processes may be or include removal or elimination of water from the medium. In some embodiments, such processes may be or include treatment with a hygroscopic solvent system that includes one or more hygroscopic solvents. In some embodiments, such processes may be or include treatment with a hygroscopic solvent. In some embodiments, such processes may be or include treatment with an alcohol. In some embodiments, such processes may be or include treatment with methanol. In some embodiments, such processes may be or include treatment with ethanol. In some embodiments, such processes may be or include treatment with isopropanol. In some embodiments, such processes may be or include treatment with dimethyl carbonate. In some embodiments, such processes may be or include treatment with acetonitrile. In some embodiments, such a process may be or include treatment with ethyl acetate. In some embodiments, such a process may be or include treatment with acetone. In some embodiments, the present disclosure illustrates that some such processes, including some processes previously understood or expected to degrade or otherwise damage one or more purification media, may actually improve the usefulness and / or effectiveness of such media, particularly in the preparation of phosphoramidites. In some embodiments, the teachings of the present disclosure relate to media that are or include silica media. In some embodiments, the teachings of the present disclosure relate to media that are or include silica gel.

[0005] In some embodiments, the present disclosure recognizes that one source of problems in the prior art utilizing chromatographic purification (e.g., purification using silica gel, alumina, etc.) in the preparation of phosphoramidites is that a significant amount of phosphoramidites may be lost during chromatography. Without wishing to be bound by a particular theory, the present disclosure proposes that in some cases, phosphoramidites may be lost by irreversible adsorption (e.g., not easily eluted during chromatographic purification) to the purification medium, e.g., silica gel, alumina, etc., and / or by decomposition when in contact with the purification medium. In some embodiments, the present disclosure includes the recognition that low yield and / or low purity of phosphoramidites can significantly increase the cost of oligonucleotides and / or pharmaceutical compositions thereof, especially in pharmaceutical compositions required to prepare oligonucleotide-based therapeutic pharmaceuticals on a large scale. The present disclosure provides the particular insight that, in some embodiments, treatment of the purification medium described herein can improve its performance (and thus its utility and / or efficacy) in the purification of phosphoramidites.

[0006] In some embodiments, the disclosure provides novel techniques, e.g., compounds, compositions, methods, etc., for preparing phosphoramidites and / or oligonucleotides. In some embodiments, the disclosure provides new techniques for purifying phosphoramidites. In some embodiments, the techniques provided significantly improve the efficiency of phosphoramidite synthesis and / or significantly reduce the cost of phosphoramidites and oligonucleotides, compositions and pharmaceuticals prepared therefrom.

[0007] In some embodiments, the present disclosure provides: a) pretreating a purification medium; b) contacting the purification medium with a phosphoramidite; b) optionally purifying the phosphoramidite using a purification medium.

[0008] In some embodiments, the present disclosure provides: a) pretreating a purification medium; b) purifying the phosphoramidite using a purification medium.

[0009] In some embodiments, the present disclosure provides: a) pretreating a purification medium comprising silica gel; b) contacting the purification medium with a phosphoramidite.

[0010] In some embodiments, the present disclosure provides: a) pretreating a purification medium comprising silica gel; b) purifying the phosphoramidite using the purification medium.

[0011] In some embodiments, the present disclosure provides: a) pretreating a silica gel; b) contacting the silica gel with a phosphoramidite.

[0012] In some embodiments, the present disclosure provides: a) pretreating a silica gel; b) purifying the phosphoramidite using silica gel.

[0013] In some embodiments, the pretreatment removes water from the purification medium. In some embodiments, the pretreatment inactivates the purification medium. In some embodiments, the pretreatment comprises contacting the purification medium with a first solvent system. In some embodiments, the first solvent system comprises a hygroscopic solvent. In some embodiments, the first solvent system is a hygroscopic solvent. In some embodiments, the hygroscopic solvent is an alcohol. In some embodiments, the hygroscopic solvent is methanol. In some embodiments, the hygroscopic solvent is ethanol. In some embodiments, the hygroscopic solvent is isopropanol. In some embodiments, the hygroscopic solvent is acetone. In some embodiments, the hygroscopic solvent is dimethyl carbonate. In some embodiments, the hygroscopic solvent is acetonitrile. In some embodiments, the hygroscopic solvent is ethyl acetate. In some embodiments, the first solvent system inactivates reactive sites on the purification medium. In some embodiments, the pretreatment comprises heating the purification medium.

[0014] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium.

[0015] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; and c) purifying the phosphoramidite using the purification medium.

[0016] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) purifying the phosphoramidite using the purification medium.

[0017] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) adding phosphoramidites to the purification medium; c) eluting the phosphoramidite from the purification medium with a solvent system; wherein the recovery is higher than the baseline recovery in the absence of step a).

[0018] In some embodiments, the disclosure provides a method for reducing decomposition of a phosphoramidite when the phosphoramidite contacts a purification medium comprising the step of: a) removing water from the purification medium.

[0019] In some embodiments, the present disclosure provides: a) inactivating the purification medium; b) purifying the phosphoramidite using the purification medium, wherein the inactivated purification medium provides a higher recovery than a non-inactivated purification medium.

[0020] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium.

[0021] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; and c) purifying the phosphoramidite using the purification medium.

[0022] In some embodiments, the present disclosure provides: a) removing water from the purification medium; b) purifying the phosphoramidite using the purification medium.

[0023] In some embodiments, the present disclosure provides: a) mechanically removing water from a purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium.

[0024] In some embodiments, the present disclosure provides: a) mechanically removing water from a purification medium; b) contacting the purification medium with a phosphoramidite; and c) purifying the phosphoramidite using the purification medium.

[0025] In some embodiments, the present disclosure provides: a) mechanically removing water from a purification medium; b) purifying the phosphoramidite using the purification medium.

[0026] In some embodiments, the present disclosure provides: a) treating the purification medium with a hygroscopic solvent; b) purifying the phosphoramidite using the purification medium.

[0027] In some embodiments, the present disclosure provides: a) treating the purification medium with an alcohol; b) purifying the phosphoramidite using the purification medium.

[0028] In some embodiments, the present disclosure provides: a) treating the purification medium with one or more alcohols; b) re-equilibrating the purification medium with a solvent system that is less polar than the one or more alcohols; c) purifying the phosphoramidite using silica gel, where the elution solvent system is less polar than the one or more alcohols.

[0029] In some embodiments, the present disclosure provides: a) treating the purification medium with methanol; b) re-equilibrating the purification medium with a solvent system comprising ethyl acetate and hexane; c) purifying the phosphoramidite using a purification medium, wherein the elution solvent system comprises ethyl acetate, hexane, and optionally a modifier.

[0030] In some embodiments, the present disclosure provides: a) treating the purification medium with methanol; b) re-equilibrating the purification medium with a solvent system comprising ethyl acetate and hexane; c) purifying the phosphoramidite using a purification medium, wherein the elution solvent system is a mixture of ethyl acetate and hexane.

[0031] In some embodiments, the present disclosure provides: a) treating the purification medium with methanol; b) re-equilibrating the purification medium with a solvent system comprising ethyl acetate and hexane; c) purifying the phosphoramidite using a purification medium, wherein the elution solvent system is a mixture of ethyl acetate, hexane and triethylamine.

[0032] In some embodiments, the present disclosure provides: contacting a compound with a medium, The medium is inert.

[0033] In some embodiments, the present disclosure provides: Purifying a compound using a medium, comprising: The medium is inert, and

[0034] In some embodiments, the compound is a phosphoramidite. In some embodiments, the compound is a nucleoside. In some embodiments, the compound is a nucleotide. In some embodiments, the compound is an oligonucleotide. In some embodiments, the medium is a purification medium. In some embodiments, the medium comprises silica gel. In some embodiments, the medium is silica gel. In some embodiments, the medium is mechanically inactivated as described herein. In some embodiments, the medium is inactivated with a solvent system or solvent as described herein. In some embodiments, the medium is inactivated by contact with a solvent system as described herein, or comprises a hygroscopic solvent.

[0035] In some embodiments, the method provided is a chromatographic method comprising using a medium as described herein. In some embodiments, the medium is or comprises silica gel. In some embodiments, the medium is inactivated, for example, by mechanical means, contact with a hygroscopic solvent (system), etc.

[0036] In some embodiments, the present disclosure provides compositions for preparing phosphoramidites. In some embodiments, the present disclosure provides compositions for preparing phosphoramidites, a) an inactivated purification medium; b) a phosphoramidite. In some embodiments, the phosphoramidite is contacted with an inactivated purification medium, hi some embodiments, the phosphoramidite is adsorbed to an inactivated purification medium.

[0037] In some embodiments, treating the purification medium with a solvent system or solvent is or includes contacting the purification medium with the solvent system or solvent.

[0038] In some embodiments, the phosphoramidite is a nucleoside phosphoramidite. In some embodiments, the phosphoramidite is a nucleoside phosphoramidite. In some embodiments, the phosphoramidite is a nucleoside phosphoramidite. In some embodiments, the phosphoramidite is a nucleoside phosphoramidite. In some embodiments, the phosphoramidite is a nucleoside phosphoramidite. No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178, each of which phosphoramidites are incorporated herein by reference.

[0039] In some embodiments, the phosphoramidites are stereochemically enriched. In some embodiments, the phosphoramidites are stereochemically pure. In some embodiments, the phosphoramidites are chiral controlled oligonucleotide compositions, such as those described in WO 2011 / 005761, US 2012 / 0316224, WO 2013 / 012758, US 2014 / 0194610, WO 2014 / 012081, US 2015 / 0211006, WO 2015 / 107425, US 2017 / 0037399, WO 2010 / 064146, US 201 and the chiral controlled oligonucleotide compositions used to prepare them as described in U.S. Patent Application Publication No. WO 1 / 0294124, WO 2014 / 010250, U.S. Patent Application Publication No. 2015 / 0197540, WO 2011 / 108682, U.S. Patent Application Publication No. 2013 / 0184450, WO 2012 / 039448, U.S. Patent Application Publication No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178, each of which is incorporated herein by reference.

[0040] In some embodiments, the phosphoramidite has formula I: [ka] wherein each variable is independently defined as set forth below. [Brief description of the drawings]

[0041] [Figure 1] Chromatogram: Silica without pretreatment

[0042] [Diagram 2] Chromatogram: Pretreated silica DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] 1. Definition As used herein, the following definitions apply unless otherwise indicated. In this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th In addition, the general principles of organic chemistry are identified in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5 th Ed., Ed. Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0044] Aliphatic: As used herein, "aliphatic" means a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more units of unsaturation, or a combination thereof. Unless otherwise specified, an aliphatic group contains 1-100 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 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.

[0045] Alkyl: As used herein, the term "alkyl" has its usual 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, an alkyl has 1-100 carbon atoms. In some embodiments, a straight or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C5 for straight chain). 20 , C2-C for branched chain 20 ), or about 1-10 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where the ring is monocyclic, bicyclic or polycyclic, or alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group can be a lower alkyl group, which contains 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0046] Alkenyl: The term "alkenyl" as used herein, refers to an alkyl group, as defined herein, having one or more double bonds.

[0047] Alkynyl: The term "alkynyl" as used herein, refers to an alkyl group, as defined herein, having one or more triple bonds.

[0048] Aryl: The term "aryl," used alone or as part of a larger moiety in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, with at least one ring in the system being aromatic. In some embodiments, the aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 14 ring members, with at least one ring in the system being aromatic, with each ring in the system containing 3 to 7 ring members. In some embodiments, the aryl group is a biaryl group. The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments of the present disclosure, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl, etc. In some embodiments, an aryl group has its radical or point of attachment on the aromatic ring.

[0049] Alicyclic: As used herein, the term "alicyclic" refers to a saturated or partially unsaturated aliphatic monocyclic, bicyclic, or polycyclic ring system, for example having 3-30 ring members, where the aliphatic ring system may be substituted. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a cycloalkyl has 3-6 carbons. The term "alicyclic" can also include an aliphatic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring. In some embodiments, a carbocyclic group is bicyclic. In some embodiments, a carbocyclic group is tricyclic. In some embodiments, a carbocyclic group is polycyclic. In some embodiments, "alicyclic" (or "carbocyclic" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon or C8-C6 alkyl group that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the remainder of the molecule. 10 Bicyclic hydrocarbons or C9-C rings that are fully saturated or contain one or more units of unsaturation, but are not aromatic, and have a single point of attachment to the remainder of the molecule. 16 Refers to tricyclic hydrocarbons.

[0050] Halogen: The term “halogen” means F, Cl, Br, or I.

[0051] Heteroaliphatic: The term "heteroaliphatic" has its normal meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, silicon, phosphorous, etc.).

[0052] Heteroalkyl: The term "heteroalkyl" has its normal meaning in the art and refers to an alkyl group, as described herein, in which one or more carbon atoms are replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, and the like.

[0053] Heteroaryl: The terms "heteroaryl" and "heteroaralkyl," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," refer to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, in which at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. Heteroaryl groups are groups having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, heteroaryl groups have 6, 10, or 14 pi electrons shared in the cyclic arrangement, and have 1 to 5 heteroatoms in addition to the carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl. As used herein, the terms "heteroaryl" and "heteroaralkyl" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic 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]-1,4-oxazin-3(4H)-one. Heteroaryl groups may be monocyclic, bicyclic, or polycyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "heteroaryl group" or "heteroaromatic", any of which terms include rings that are optionally substituted.The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, where the alkyl and heteroaryl portions independently may be optionally substituted.

[0054] Heteroatom: The term "heteroatom" refers to an atom that is neither carbon nor hydrogen. In some embodiments, a heteroatom is any oxidized form of nitrogen, sulfur, phosphorus, or silicon, any quaternized form of a basic nitrogen or substitutable nitrogen of a heterocyclic ring (e.g., N in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR (in N-substituted pyrrolidinyl). + etc.), oxygen, sulfur, nitrogen, phosphorus, or silicon.

[0055] 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 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, 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, having in addition to carbon atoms one or more, preferably 1-4 heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NH (as in N-substituted pyrrolidinyl). +It can be NR. The heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups may be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, where the alkyl and heterocyclyl portions may be independently substituted.

[0056] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to include groups with multiple sites of unsaturation, but is not intended to include either aryl or heteroaryl moieties.

[0057] Protecting Groups As used herein, the phrase "protecting group" refers to a temporary substituent that protects a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, acetals of aldehydes, and ketals of ketones. A "Si-protecting group" is a protecting group that contains a Si atom, such as Si-trialkyl (e.g., trimethylsilyl, tributylsilyl, t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g., t-butyldiphenylsilyl), etc., or Si-aryl-dialkyl (e.g., Si-phenyldialkyl). Generally, Si-protecting groups are attached to an oxygen atom. The field of protecting group chemistry has been reviewed (Greene, TW; Wuts, PGM Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). Such protecting groups (and related protected moieties) are described in detail below.

[0058] Protected hydroxyl groups are well known in the art and are described in Protecting Groups in Organic Synthesis, TW Greene and PG M Huts, 3 rdedition, John Wiley & Sons, 1999, the entire contents of which are incorporated herein by reference. Examples of suitable 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. Examples of suitable esters include formates, acetates, propionates, pentanoates, crotonates, and benzoates. Specific examples of suitable esters include formates, benzoyl formates, chloroacetates, trifluoroacetates, methoxyacetates, triphenylmethoxyacetates, p-chlorophenoxyacetates, 3-phenylpropionates, 4-oxopentanoates, 4,4-(ethylenedithio)pentanoates, pivalates (trimethylacetates), crotonates, 4-methoxy-crotonates, benzoates, p-benzoylbenzoates, and 2,4,6-trimethylbenzoates. Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonates. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ethers, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ethers, or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.Examples of suitable arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

[0059] 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, aralkyl amines, carbamates, allyl amines, amides, and the like. Examples of suitable mono-protected aminos include t-butyloxycarbonylamino (-NHBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxycarbonylamino (-NHCBZ), allylamino, benzylamino (-NHBn), fluorenylmethylcarbonyl (-NHFmoc) formamide, acetamide, chloroacetamide, dichloroacetamide, trichloroacetamide, phenylacetamide, trifluoroacetamide, benzamide, t-butyldiphenylsilyl, and the like. Suitable di-protected amines include amines substituted with two substituents independently selected from those listed above as mono-protected amines, and further include cyclic imides, such as phthalimides, maleimides, succinimides, etc. Suitable di-protected amines also include pyrroles, etc., 2,2,5,5-tetramethyl-[1,2,5]azadisilolidine, etc., and azides.

[0060] 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-dioxane, 1,3-dioxolane, semicarbazones, and derivatives thereof.

[0061] Protected carboxylic acids are well known in the art and include those described in detail in Greene (1999). Suitable protected carboxylic acids further include optionally substituted C 1-6 These include, but are not limited to, aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, etc. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl esters, each of which may be substituted. Further suitable protected carboxylic acids include oxazolines and orthoesters.

[0062] 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. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioesters, to name just a few.

[0063] Substituted: As described herein, the compounds of the present disclosure may include optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at every position. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is not substantially altered when exposed to conditions that allow for the production, detection, and, in some embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0064] Preferred monovalent substituents are halogen; -(CH2) 0-4 R°;-(CH2) 0-4 OR°;-O(CH2) 0-4 R o ,-O-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 CH(OR°)2; -(CH2) optionally substituted with R° 0-4 Ph; optionally substituted with R° -(CH2) 0-4 O(CH2) 0-1 Ph; optionally substituted with R° -CH=CHPh; optionally substituted with R° -(CH2) 0-4 O(CH2) 0-1 -Pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R°)2;-(CH2) 0-4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0-4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0-4N(R°)C(O)OR°;-N(R°)N(R°)C(O)R°;-N(R°)N(R°)C(O)NR°2;-N(R°)N(R°)C(O)OR°;-(CH2) 0-4 C(O)R°;-C(S)R°;-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 C(O)SR°;-(CH2) 0-4 C(O)OSiR°3;-(CH2) 0-4 OC(O)R°;-OC(O)(CH2) 0-4 SR, -SC(S)SR°;-(CH2) 0-4 SC(O)R°;-(CH2) 0-4 C(O)NR°2;-C(S)NR°2;-C(S)SR°;-SC(S)SR°, -(CH2) 0-4 OC(O)NR°2;-C(O)N(OR°)R°;-C(O)C(O)R°;-C(O)CH2C(O)R°;-C(NOR°)R°;-(CH2) 0-4 SSR°;-(CH2) 0-4 S(O)2R°;-(CH2) 0-4 S(O)2OR°;-(CH2) 0-4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0-4 S(O)R°;-N(R°)S(O)2NR°2;-N(R°)S(O)2R°;-N(OR°)R°;-C(NH)NR°2;-P(O)2R°;-P(O)R°2;-OP(O)R°2;-OP(O)(OR°)2;-SiR°3;-OSiR°3;-(C 1-4 Linear or branched alkylene)ON(R°)2; or -(C 1-4 linear or branched alkylene)C(O)ON(R°), where each R° may be substituted as defined below and is independently hydrogen, C 1-20 C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon and phosphorus 1-20 Heteroaliphatic, -CH2-(C 6-14 Aryl), -O(CH2) 0-1 (C 6-14aryl), -CH2- (5-14 membered heteroaryl ring), a 5-20 membered monocyclic, bicyclic or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the above definitions, two R° occurring independently together with their intervening atoms form a 5-20 membered monocyclic, bicyclic or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below).

[0065] Preferred monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with the intervening atoms) are independently halogen, -(CH2), 0-2 R●, -(HaloR●), -(CH2) 0-2 OH, -(CH2) 0-2 OR ●, -(CH2) 0-2 CH(OR●)2; -O(HaloR●), -CN, -N3, -(CH2) 0-2 C(O)R●, -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR●, -(CH2) 0-2 SR●, -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR●, -(CH2) 0-2 NR●2, -NO2, -SiR●3, -OSiR●3, -C(O)SR● 、 -(C 1-4 straight or branched chain alkylene)C(O)OR●, or -SSR●, where each R● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include ═O and ═S.

[0066] Preferred divalent substituents are: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S-, (wherein each R * is hydrogen, optionally substituted as defined below 1-6 A preferred divalent substituent attached to adjacent substitutable carbons of an "optionally substituted" group is -O(CR * 2) 2-3 O-, (wherein each R * is hydrogen, optionally substituted as defined below 1-6 aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0067] R * Suitable substituents on an aliphatic group are halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, where each R● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently, 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0068] In some embodiments, a suitable substituent on a substitutable nitrogen is -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH2C(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † , (where each R † are independently hydrogen, C which may be substituted as defined below 1-6 aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, regardless of the above definition, two independently occurring R † which, together with their intervening atoms, form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0069] R † Suitable substituents on an aliphatic group are independently halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, where each R● is unsubstituted or, when preceded by "halo", substituted only with one or more halogens, and independently, 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0070] Unsaturated As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0071] 2. Several modes for carrying out the invention Phosphoramidites have various uses, for example, nucleoside phosphoramidites are widely used as monomers for oligonucleotide synthesis.Many techniques for preparing phosphoramidites suffer from low yield and / or low purity.In particular, such low yield and / or low purity can result in high cost and / or low quality phosphoramidites and all downstream products thereof, such as oligonucleotides prepared therefrom.

[0072] Among other things, the present disclosure includes the recognition that in some cases, when phosphoramidites are separated from other substances, for example, via chromatography, low yields and / or low purity may result from the purification process. In some embodiments, the present disclosure recognizes that one source of problems in the prior art utilizing chromatographic purification (e.g., purification using silica gel, alumina, etc.) in the preparation of phosphoramidites is that a significant amount of phosphoramidites may be lost during chromatography. Without wishing to be bound by a particular theory, the present disclosure proposes that in some cases, phosphoramidites may be lost by irreversible absorption / adsorption onto the purification medium and / or decomposition when in contact with the purification medium during chromatography. In some embodiments, phosphoramidites may be lost by irreversible absorption / adsorption onto the purification medium in that they cannot be readily eluted. In some embodiments, phosphoramidites may decompose during chromatographic purification. For example, as described in the Examples, in some embodiments, phosphoramidites may decompose during purification using silica gel as the purification medium.

[0073] In particular, low yields and / or low purity of phosphoramidites can significantly increase the cost of compositions made therefrom, especially at large process scales, In some embodiments, when they are used in oligonucleotide synthesis, the high cost of phosphoramidites can make oligonucleotides, compositions thereof, and pharmaceutical products thereof prohibitively expensive.

[0074] The present disclosure encompasses several surprising findings, including that several processes for pretreating a purification medium can surprisingly improve the usefulness and / or effectiveness of the medium, particularly when phosphoramidites are used in the preparation. In some embodiments, such a process may be or may include an inactivation process. In some embodiments, the pretreatment is an inactivation process. In some embodiments, the pretreatment includes inactivation of the purification medium. In some embodiments, when a purification medium is inactivated, there is less irreversible absorption / adsorption and / or decomposition of phosphoramidites when such a purification medium is used to purify phosphoramidites, as compared to the same inactivated purification medium. In some embodiments, there is less irreversible absorption / adsorption. In some embodiments, there is less decomposition. In some embodiments, there is less irreversible absorption / adsorption and less decomposition. In some embodiments, the pretreated medium provides a higher yield and / or higher purity of phosphoramidites than non-pretreated medium. In some embodiments, the pretreated medium provides a higher recovery yield. In some embodiments, the pretreated medium provides a higher purity. In some embodiments, the pretreated medium provides a higher recovery yield and a higher purity.

[0075] In some embodiments, pretreatment may be or may include the removal or elimination of water from the purification medium. In some embodiments, pretreatment includes the removal or elimination of water from the purification medium. In some embodiments, pretreatment removes water from the purification medium. In some embodiments, pretreatment excludes water from the purification medium.

[0076] In some embodiments, the pretreatment is mechanical. In some embodiments, the purification medium is pretreated with heat. In some embodiments, the purification medium is pretreated with vacuum. In some embodiments, the purification medium is pretreated with heat and vacuum. In some embodiments, such mechanical pretreatment inactivates the purification medium. As one of ordinary skill in the art will recognize, heat and / or vacuum can, among other things, remove water or other solvents from the purification medium in accordance with the present disclosure. In some embodiments, the heating reaches a temperature of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, or 500° C. In some embodiments, the vacuum is less than or equal to 50 kPa, 40 kPa, 30 kPa, 20 kPa, 10 kPa, 5 kPa, 2 kPa, 1 kPa, 0.5 kPa, 0.2 kPa, 0.1 kPa, 50 Pa, 40 Pa, 30 Pa, 20 Pa, 10 Pa, 5 Pa, 2 Pa, 1 Pa, 0.5 Pa, 0.2 Pa, 0.1 Pa, 0.05 Pa, 0.02 Pa, or 0.01 Pa. In some embodiments, the treatment is for an extended period of time. In some embodiments, the treatment lasts for 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, 12 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, or more.

[0077] In some embodiments, the purification medium is pretreated with a first solvent system. In some embodiments, the first solvent system is or comprises a hygroscopic solvent. In some embodiments, the first solvent system is a hygroscopic solvent system. In some embodiments, the hygroscopic solvent system is a first solvent system described in the present disclosure. In some embodiments, the first solvent system comprises a hygroscopic solvent. In some embodiments, the first solvent is a hygroscopic solvent. In some embodiments, the purification medium is inactivated by contacting the medium with the first solvent system. In some embodiments, the first solvent system comprises a water-soluble solvent. In some embodiments, the first solvent system comprises a water-soluble organic solvent. In some embodiments, the first solvent system is a water-soluble solvent. In some embodiments, the first solvent system is a water-soluble organic solvent. In some embodiments, the first solvent system comprises two or more solvents. In some embodiments, the first solvent system comprises two or more water-soluble solvents. In some embodiments, the first solvent system comprises two or more water-soluble organic solvents. Exemplary water-soluble organic solvents include acetaldehyde, acetic acid, acetone, acetonitrile, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyric acid, diethanolamine, diethylenetriamine, dimethylformamide, dimethoxyethane, dimethylsulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol, methyldiethanolamine, methylisocyanide, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propanoic acid, propylene glycol, pyridine, tetrahydrofuran, triethylene glycol, and the like.

[0078] In some embodiments, the first solvent system comprises a hygroscopic solvent. In some embodiments, the first solvent system is a hygroscopic solvent. In some embodiments, the first solvent system comprises two or more hygroscopic solvents, for example, a hygroscopic solvent system that is a mixture of two or more hygroscopic solvents, the hygroscopic solvent system comprises two or more hygroscopic solvents and one or more dry, non-hygroscopic solvents. In some embodiments, the first hygroscopic solvent is a water-soluble solvent. In some embodiments, the hygroscopic solvent is a water-soluble organic solvent. In some embodiments, as those skilled in the art will recognize, the hygroscopic solvent system can absorb water from its environment, for example, when contacting the purification medium, the hygroscopic solvent system can absorb water, if any, from the purification medium. When passing through a column filled with the purification medium as a stationary phase, the hygroscopic solvent can remove water from the purification medium. In some embodiments, the hygroscopic solvent used for inactivation is 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.0 The hygroscopic solvent may contain less than 0.07%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% by volume of water. In some embodiments, the hygroscopic solvent is used as an anhydrous solvent purchased directly from a commercial vendor (e.g., Sigma-Aldrich, Acros Organics, etc.). Alternatively or additionally, the hygroscopic solvent can be further purified and / or dried according to protocols known in the art.

[0079] In accordance with the present disclosure, many hygroscopic solvents can be used in the provided solvent systems, e.g., the first solvent system, the hygroscopic solvent system, etc. In some embodiments, the hygroscopic solvent is an alcohol. In some embodiments, the hygroscopic solvent is methanol. In some embodiments, the hygroscopic solvent is ethanol. In some embodiments, the hygroscopic solvent is isopropyl alcohol. In some embodiments, the hygroscopic solvent is acetone. In some embodiments, the hygroscopic solvent is acetonitrile. In some embodiments, the hygroscopic solvent is dimethyl carbonate. In some embodiments, the hygroscopic solvent is ethyl acetate. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than chloroform. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than methylene chloride. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than ethyl acetate. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than acetone. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than acetonitrile. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than dimethyl carbonate. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than methanol. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than ethanol. In some embodiments, the hygroscopic solvent is a solvent that is more hygroscopic than isopropanol. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as chloroform. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as methylene chloride. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as ethyl acetate. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as acetone. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as acetonitrile. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as dimethyl carbonate. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as methanol. In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as ethanol.In some embodiments, the hygroscopic solvent is a solvent that is as hygroscopic as isopropanol.

[0080] In some embodiments, the first solvent system includes an alcohol. In some embodiments, the first solvent system is an alcohol. In some embodiments, the first solvent system includes an alcohol and another organic solvent. In some embodiments, the first solvent system includes at least 10% alcohol by volume. In some embodiments, the first solvent system includes at least 20% alcohol by volume. In some embodiments, the first solvent system includes at least 30% alcohol by volume. In some embodiments, the first solvent system includes at least 40% alcohol by volume. In some embodiments, the first solvent system includes at least 50% alcohol by volume. In some embodiments, the first solvent system includes at least 60% alcohol by volume. In some embodiments, the first solvent system includes at least 70% alcohol by volume. In some embodiments, the first solvent system includes at least 80% alcohol by volume. In some embodiments, the first solvent system includes at least 90% alcohol by volume. In some embodiments, the first solvent system includes at least 95% alcohol by volume. In some embodiments, the first solvent system includes at least 96% alcohol by volume. In some embodiments, the first solvent system comprises at least 97% alcohol by volume. In some embodiments, the first solvent system comprises at least 98% alcohol by volume. In some embodiments, the first solvent system comprises at least 99% alcohol by volume. In some embodiments, the alcohol is C 1-6 In some embodiments, the alcohol is methanol. In some embodiments, the alcohol is ethanol. In some embodiments, the alcohol is isopropanol. In some embodiments, the alcohol is 1-propanol. In some embodiments, the alcohol is butanol.

[0081] In some embodiments, the first solvent system comprises methanol. In some embodiments, the first solvent system is methanol. In some embodiments, the first solvent system comprises at least 10% by volume methanol. In some embodiments, the first solvent system comprises at least 20% by volume methanol. In some embodiments, the first solvent system comprises at least 30% by volume methanol. In some embodiments, the first solvent system comprises at least 40% by volume methanol. In some embodiments, the first solvent system comprises at least 50% by volume methanol. In some embodiments, the first solvent system comprises at least 60% by volume methanol. In some embodiments, the first solvent system comprises at least 70% by volume methanol. In some embodiments, the first solvent system comprises at least 80% by volume methanol. In some embodiments, the first solvent system comprises at least 90% by volume methanol. In some embodiments, the first solvent system comprises at least 95% by volume methanol. In some embodiments, the first solvent system comprises at least 96% by volume methanol. In some embodiments, the first solvent system comprises at least 97% by volume methanol. In some embodiments, the first solvent system comprises at least 98% by volume methanol. In some embodiments, the first solvent system comprises at least 99% by volume methanol. In some embodiments, the first solvent system is methanol.

[0082] In some embodiments, the first solvent system comprises ethanol. In some embodiments, the first solvent system is ethanol. In some embodiments, the first solvent system comprises at least 10% ethanol by volume. In some embodiments, the first solvent system comprises at least 20% ethanol by volume. In some embodiments, the first solvent system comprises at least 30% ethanol by volume. In some embodiments, the first solvent system comprises at least 40% ethanol by volume. In some embodiments, the first solvent system comprises at least 50% ethanol by volume. In some embodiments, the first solvent system comprises at least 60% ethanol by volume. In some embodiments, the first solvent system comprises at least 70% ethanol by volume. In some embodiments, the first solvent system comprises at least 80% ethanol by volume. In some embodiments, the first solvent system comprises at least 90% ethanol by volume. In some embodiments, the first solvent system comprises at least 95% ethanol by volume. In some embodiments, the first solvent system comprises at least 96% ethanol by volume. In some embodiments, the first solvent system comprises at least 97% ethanol by volume. In some embodiments, the first solvent system comprises at least 98% ethanol by volume. In some embodiments, the first solvent system comprises at least 99% ethanol by volume. In some embodiments, the first solvent system is ethanol.

[0083] In some embodiments, the first solvent system includes 1-propanol. In some embodiments, the first solvent system is 1-propanol. In some embodiments, the first solvent system includes at least 10% 1-propanol by volume. In some embodiments, the first solvent system includes at least 20% 1-propanol by volume. In some embodiments, the first solvent system includes at least 30% 1-propanol by volume. In some embodiments, the first solvent system includes at least 40% 1-propanol by volume. In some embodiments, the first solvent system includes at least 50% 1-propanol by volume. In some embodiments, the first solvent system includes at least 60% 1-propanol by volume. In some embodiments, the first solvent system includes at least 70% 1-propanol by volume. In some embodiments, the first solvent system includes at least 80% 1-propanol by volume. In some embodiments, the first solvent system includes at least 90% 1-propanol by volume. In some embodiments, the first solvent system comprises at least 95% 1-propanol by volume. In some embodiments, the first solvent system comprises at least 96% 1-propanol by volume. In some embodiments, the first solvent system comprises at least 97% 1-propanol by volume. In some embodiments, the first solvent system comprises at least 98% 1-propanol by volume. In some embodiments, the first solvent system comprises at least 99% 1-propanol by volume. In some embodiments, the first solvent system is 1-propanol.

[0084] In some embodiments, the first solvent system includes isopropanol. In some embodiments, the first solvent system is isopropanol. In some embodiments, the first solvent system includes at least 10% by volume isopropanol. In some embodiments, the first solvent system includes at least 20% by volume isopropanol. In some embodiments, the first solvent system includes at least 30% by volume isopropanol. In some embodiments, the first solvent system includes at least 40% by volume isopropanol. In some embodiments, the first solvent system includes at least 50% by volume isopropanol. In some embodiments, the first solvent system includes at least 60% by volume isopropanol. In some embodiments, the first solvent system includes at least 70% by volume isopropanol. In some embodiments, the first solvent system includes at least 80% by volume isopropanol. In some embodiments, the first solvent system includes at least 90% by volume isopropanol. In some embodiments, the first solvent system includes at least 95% by volume isopropanol. In some embodiments, the first solvent system comprises at least 96% isopropanol by volume. In some embodiments, the first solvent system comprises at least 97% isopropanol by volume. In some embodiments, the first solvent system comprises at least 98% isopropanol by volume. In some embodiments, the first solvent system comprises at least 99% isopropanol by volume. In some embodiments, the first solvent system is isopropanol.

[0085] In some embodiments, the first solvent system includes acetone. In some embodiments, the first solvent system is acetone. In some embodiments, the first solvent system includes at least 10% acetone by volume. In some embodiments, the first solvent system includes at least 20% acetone by volume. In some embodiments, the first solvent system includes at least 30% acetone by volume. In some embodiments, the first solvent system includes at least 40% acetone by volume. In some embodiments, the first solvent system includes at least 50% acetone by volume. In some embodiments, the first solvent system includes at least 60% acetone by volume. In some embodiments, the first solvent system includes at least 70% acetone by volume. In some embodiments, the first solvent system includes at least 80% acetone by volume. In some embodiments, the first solvent system includes at least 90% acetone by volume. In some embodiments, the first solvent system includes at least 95% acetone by volume. In some embodiments, the first solvent system includes at least 96% acetone by volume. In some embodiments, the first solvent system includes at least 97% acetone by volume. In some embodiments, the first solvent system comprises at least 98% acetone by volume. In some embodiments, the first solvent system comprises at least 99% acetone by volume. In some embodiments, the first solvent system is acetone.

[0086] In some embodiments, the first solvent system includes acetonitrile. In some embodiments, the first solvent system is acetonitrile. In some embodiments, the first solvent system includes at least 10% acetonitrile by volume. In some embodiments, the first solvent system includes at least 20% acetonitrile by volume. In some embodiments, the first solvent system includes at least 30% acetonitrile by volume. In some embodiments, the first solvent system includes at least 40% acetonitrile by volume. In some embodiments, the first solvent system includes at least 50% acetonitrile by volume. In some embodiments, the first solvent system includes at least 60% acetonitrile by volume. In some embodiments, the first solvent system includes at least 70% acetonitrile by volume. In some embodiments, the first solvent system includes at least 80% acetonitrile by volume. In some embodiments, the first solvent system includes at least 90% acetonitrile by volume. In some embodiments, the first solvent system includes at least 95% acetonitrile by volume. In some embodiments, the first solvent system includes at least 96% acetonitrile by volume. In some embodiments, the first solvent system comprises at least 97% acetonitrile by volume. In some embodiments, the first solvent system comprises at least 98% acetonitrile by volume. In some embodiments, the first solvent system comprises at least 99% acetonitrile by volume. In some embodiments, the first solvent system is acetonitrile.

[0087] In some embodiments, the first solvent system comprises dimethyl carbonate. In some embodiments, the first solvent system is dimethyl carbonate. In some embodiments, the first solvent system comprises at least 10% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 20% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 30% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 40% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 50% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 60% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 70% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 80% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 90% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 95% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 96% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 97% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 98% dimethyl carbonate by volume. In some embodiments, the first solvent system comprises at least 99% dimethyl carbonate by volume. In some embodiments, the first solvent system is dimethyl carbonate.

[0088] In some embodiments, the first solvent system includes ethyl acetate. In some embodiments, the first solvent system is ethyl acetate. In some embodiments, the first solvent system includes at least 10% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 20% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 30% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 40% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 50% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 60% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 70% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 80% ethyl acetate by volume. In some embodiments, the first solvent system includes at least 90% ethyl acetate by volume. In some embodiments, the first solvent system comprises at least 95% ethyl acetate by volume. In some embodiments, the first solvent system comprises at least 96% ethyl acetate by volume. In some embodiments, the first solvent system comprises at least 97% ethyl acetate by volume. In some embodiments, the first solvent system comprises at least 98% ethyl acetate by volume. In some embodiments, the first solvent system comprises at least 99% ethyl acetate by volume. In some embodiments, the first solvent system is ethyl acetate.

[0089] In some embodiments, the first solvent is hygroscopic. In some embodiments, the first solvent is hygroscopic in that it comprises a hygroscopic solvent. In some embodiments, the first solvent is hygroscopic in that it is a hygroscopic solvent. In some embodiments, the first solvent is hygroscopic in that it is a hygroscopic solution. In some embodiments, the hygroscopic solution comprises an organic solvent and a hygroscopic solute.

[0090] In some embodiments, the first solvent system optionally includes a modifier. In some embodiments, the first solvent system includes a modifier. In some embodiments, the first solvent system does not include any modifier.

[0091] Those skilled in the art will recognize that many mobile phase modifiers can be utilized in accordance with the present disclosure, for example, to modify certain properties of the solvent system as the mobile phase. In some embodiments, the modifier is a base. In some embodiments, the modifier is ammonia. In some embodiments, the modifier is triethylamine. In some embodiments, the modifier is an acid. In some embodiments, the modifier is formic acid. The modifier can be added in various amounts, for example, about 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more by volume.

[0092] In some embodiments, the first solvent system removes water from the purification medium. In some embodiments, the first solvent reacts with the reactive sites of the purification medium, inactivating the purification medium, so that the compound, e.g., phosphoramidite, is less likely to decompose when in contact with the purification medium. In some embodiments, the first solvent system can provide pretreatment by heat. For example, when methanol is contacted with silica gel, heat can be generated.

[0093] Those skilled in the art will recognize that various procedures can be used to pretreat the purification medium according to the provided methods. For example, the first solvent system can be passed through a column packed with the purification medium as the stationary phase to pretreat the stationary phase. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more column volumes of the first solvent system are passed through the column to pretreat the purification medium.

[0094] In some embodiments, the present disclosure illustrates that the provided pretreatment processes, including processes previously understood or expected to degrade or otherwise damage or wash away purification media during chromatography, can actually improve the usefulness and / or effectiveness of such media, particularly in purifying and / or preparing phosphoramidites. For example, at the time of this disclosure, it is generally taught that high percentages of certain alcohols, such as methanol, should not be passed through columns packed with certain purification media, such as silica gel. However, as exemplified by the present disclosure, pure methanol can be passed through a silica column, and silica pretreated with methanol can provide unexpectedly higher yields and / or higher purity phosphoramidites compared to non-pretreated silica.

[0095] In some embodiments, the methods provided include an equilibration process after the pretreatment process. In some embodiments, the equilibration process includes contacting the pretreated purification medium with a second solvent system. In some embodiments, the second solvent system is less polar than the first solvent system used to pretreat the purification medium.

[0096] In some embodiments, the second solvent system consists of a solvent and optionally a modifier. In some embodiments, the second solvent system comprises two or more solvents and optionally a modifier. In some embodiments, the modifier is a base. In some embodiments, the modifier is triethylamine.

[0097] In some embodiments, the second solvent system comprises an isocratic system during the equilibration process. In some embodiments, the second solvent system is isocratic during the equilibration process. In some embodiments, the second solvent system comprises a gradient system. In some embodiments, the second solvent system is a gradient system. In some embodiments, the equilibration process comprises a first isocratic process, a gradient process, and optionally a second isocratic process. In some embodiments, the gradient process begins with the first isocratic process and ends with the second isocratic process. In some embodiments, the second isocratic process uses a more polar solvent system than the first process, for example, in the case of an ethyl acetate / hexane system, with more ethyl acetate therein. In some embodiments, the second isocratic process uses a less polar solvent system than the first process, for example, in the case of an ethyl acetate / hexane system, with less ethyl acetate therein. In some embodiments, the first isocratic process uses the same solvent system as the first solvent system for equilibration.

[0098] In some embodiments, the second solvent system includes ethyl acetate. In some embodiments, the second solvent system includes ethyl acetate and triethylamine as a modifier. In some embodiments, the second solvent system includes hexane. In some embodiments, the second solvent system includes hexane and triethylamine as a modifier. In some embodiments, the second solvent system includes hexane and ethyl acetate. In some embodiments, the second solvent system is a mixture of hexane and ethyl acetate. In some embodiments, the second solvent system includes hexane, ethyl acetate, and triethylamine. In some embodiments, the second solvent system is a mixture of hexane, ethyl acetate, and triethylamine. In some embodiments, the second solvent system is ethyl acetate with 5% triethylamine / 20% ethyl acetate in hexane.

[0099] Those skilled in the art recognize that various methods can be used to equilibrate the purification medium. In some embodiments, in a column packed with the purification medium as the stationary phase, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10 or more column volumes of the second solvent system (including isocratic and / or gradient) are passed through the column to equilibrate the pretreated purification medium.

[0100] In some embodiments, after the equilibration process, the phosphoramidites are loaded onto the pretreated and equilibrated purification medium, and a third solvent system is utilized to elute, separate, and / or purify the phosphoramidites. Those skilled in the art will recognize that many suitable solvent systems can be used as the third solvent system in accordance with the present disclosure. In some embodiments, the third solvent system is less polar than the first solvent system. In some embodiments, the third solvent system is the same as the second solvent system at equilibration. In some embodiments, the third solvent system is the same as the second solvent system at the end of the equilibration process.

[0101] In some embodiments, the third solvent system consists of a solvent and optionally a modifier. In some embodiments, the third solvent system comprises two or more solvents and optionally a modifier. In some embodiments, the modifier is a base. In some embodiments, the modifier is triethylamine.

[0102] In some embodiments, the third solvent system comprises an isocratic system during the elution process. In some embodiments, the third solvent system is isocratic during the elution process. In some embodiments, the third solvent system comprises a gradient system. In some embodiments, the third solvent system is a gradient system. In some embodiments, the elution process comprises a first isocratic process, a gradient process, and optionally a second isocratic process. In some embodiments, the gradient process starts with the first isocratic process and ends with the second isocratic process. In some embodiments, the second isocratic process uses a more polar solvent system than the first process, for example, more ethyl acetate in the case of an ethyl acetate / hexane system. In some embodiments, the first isocratic elution process uses the same solvent system as the solvent system at the end of the equilibration process. In some embodiments, the elution process comprises two or more first isocratic-gradient-second isocratic processes, as illustrated in the examples.

[0103] In some embodiments, the third solvent system includes ethyl acetate. In some embodiments, the third solvent system includes ethyl acetate and a base as a modifier. In some embodiments, the third solvent system includes ethyl acetate and an amine as a modifier. In some embodiments, the third solvent system includes ethyl acetate and triethylamine as a modifier. In some embodiments, the third solvent system includes hexane. In some embodiments, the third solvent system includes hexane and triethylamine as a modifier. In some embodiments, the third solvent system includes hexane and ethyl acetate. In some embodiments, the third solvent system is a mixture of hexane and ethyl acetate. In some embodiments, the third solvent system includes hexane, ethyl acetate, and triethylamine. In some embodiments, the third solvent system is a mixture of hexane, ethyl acetate, and triethylamine. Exemplary solvent systems for elution are illustrated in the Examples.

[0104] Those skilled in the art will recognize that various methods can be used to elute, separate and / or purify phosphoramidites. In some embodiments, in a column packed with purification medium as the stationary phase, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more column volumes of a third solvent system (including isocratic and / or gradient) are passed through the column.

[0105] One of skill in the art will recognize that a variety of purification media can be used in the purification of phosphoramidites in accordance with the present disclosure and can be pretreated, equilibrated and / or eluted using the provided methods to improve the yield and / or purity of the phosphoramidites. In some embodiments, the purification media is a silica media. In some embodiments, the purification media is silica gel. In some embodiments, the purification media is alumina. In some embodiments, the purification media is basic alumina. In some embodiments, the purification media can be used for purposes other than purification.

[0106] Purification media of various particle sizes can be pretreated and utilized according to the present disclosure. In some embodiments, the particle size is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200 micrometers in diameter or more. In some embodiments, the particle size is less than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, or 200 micrometers in diameter. In some embodiments, the particle size is greater than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, or 200 micrometers in diameter. In some embodiments, the purification media is silica with a diameter of about 10 micrometers. In some embodiments, the purification medium is silica with a diameter of about 15 micrometers. In some embodiments, the purification medium is silica with a diameter of about 20 micrometers. In some embodiments, the purification medium is silica with a diameter of about 25 micrometers. In some embodiments, the purification medium is silica with a diameter of about 30 micrometers. In some embodiments, the purification medium is silica with a diameter of about 35 micrometers. In some embodiments, the purification medium is silica with a diameter of about 40 micrometers. In some embodiments, the purification medium is silica with a diameter of about 45 micrometers. In some embodiments, the purification medium is silica with a diameter of about 50 micrometers. In some embodiments, the purification medium is silica with a diameter of about 60 micrometers. In some embodiments, the purification medium is silica with a diameter of about 70 micrometers. In some embodiments, the purification medium is silica with a diameter of about 80 micrometers. In some embodiments, the purification medium is silica with a diameter of about 90 micrometers. In some embodiments, the purification medium is silica with a diameter of about 100 micrometers. In some embodiments, the purification medium is silica with a diameter greater than about 100 micrometers.

[0107] In some embodiments, the purification medium is vacuum packed into a column. In some embodiments, the purification medium is silica gel. In some embodiments, several column volumes of methanol are passed through the column to deactivate the silica gel. In some embodiments, the treated silica gel is equilibrated using several column volumes of a mixture of ethyl acetate and hexane with triethylamine as a modifier before loading the untreated phosphoramidite. In some embodiments, the phosphoramidite is eluted using a mixture of ethyl acetate and hexane with triethylamine as a modifier.

[0108] In some embodiments, the disclosure provides a composition comprising a pretreated purification medium as described herein and a phosphoramidite. In some embodiments, the disclosure provides a composition comprising a pretreated, equilibrated purification medium as described herein and a phosphoramidite. In some embodiments, the purification medium is a silica gel. In some embodiments, the silica gel is pretreated with an alcohol. In some embodiments, the silica gel is pretreated with methanol. In some embodiments, the silica gel is equilibrated with a solvent system comprising hexane. In some embodiments, the silica gel is equilibrated with a solvent system comprising ethyl acetate. In some embodiments, the silica gel is equilibrated with a solvent system comprising ethyl acetate and hexane. In some embodiments, the silica gel is equilibrated with a solvent system consisting of ethyl acetate and hexane. In some embodiments, the silica gel is equilibrated with a solvent system comprising ethyl acetate, hexane, and triethylamine. In some embodiments, the silica gel is equilibrated with a solvent system consisting of ethyl acetate, hexane, and triethylamine. In some embodiments, the silica gel is equilibrated, and the equilibration comprises contact with hexane. In some embodiments, the silica gel is equilibrated and the equilibration comprises contacting with ethyl acetate. In some embodiments, the silica gel is equilibrated and the equilibration comprises contacting with triethylamine. In some embodiments, the silica gel is equilibrated and the equilibration comprises contacting with hexane and ethyl acetate. In some embodiments, the silica gel is equilibrated and the equilibration comprises contacting with hexane and triethylamine. In some embodiments, the silica gel is equilibrated and the equilibration comprises contacting with ethyl acetate and triethylamine. In some embodiments, the silica gel is equilibrated and the equilibration comprises contacting with hexane, ethyl acetate, and triethylamine. In some embodiments, the silica gel is equilibrated and the equilibration consists of contacting with hexane, ethyl acetate, and triethylamine.

[0109] In some embodiments, the phosphoramidites are nucleoside phosphoramidites.

[0110] In some embodiments, the phosphoramidite has formula I: [ka] (In the formula, BA is R or an optionally substituted group selected from a 3-30 membered alicyclic ring, a 6-30 membered aryl ring, a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a natural nucleobase moiety, and a modified nucleobase moiety; SU is a sugar moiety, a modified sugar moiety, -LO-, or [ka] where SU is attached to the phosphorus atom in formula I via an oxygen atom; L is a covalent bond or C 1-30 C having aliphatic and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon 1-30 heteroaliphatic groups, wherein one or more methylene units are optionally substituted C 1-6 Alkylene, C 1-6 alkenylene, optionally independently replaced by -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 5s is R' or -OR'; R 2sis -F, -CN, -N3, -NO, -NO2, -R'-OR', -SR', -N(R')2, -L-R', -OL-OR', -OL-SR', or -OLN(R')2, or R 2s is L linking C2 to C1, C2, C3, C4 or C5; -Cy- is an optionally substituted divalent ring selected from 3- to 30-membered carbocyclylene, 6- to 30-membered arylene, 5- to 30-membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and 3- to 30-membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; R 1 , R 2 , and R 3 Each of is independently R′, or R 1 , R 2 and R 3 two or three of, together with their intervening atoms, form: [ka] Ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R s is independently R' or -L-R'; t is 0 to 5; each R' is independently -R, -C(O)R, -CO2R, or -SO2R, or: two or more R' together with their intervening atoms form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R is independently hydrogen or C 1-30C having 1 to 10 heteroatoms independently selected from aliphatic, oxygen, nitrogen, sulfur, phosphorus, and silicon 1-30 a heteroaliphatic ring, a 6-30 membered aryl ring, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, which may be substituted.

[0111] In some embodiments, the phosphoramidite has formula I: [ka] (In the formula, BA is C 1-30 Alicyclic, C 6-30 C having 1 to 10 heteroatoms independently selected from aryl, oxygen, nitrogen, sulfur, phosphorus, and silicon 3-30 Heterocyclyl, C having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon 5-30 an optionally substituted group selected from heteroaryl rings, natural nucleobase moieties, and modified nucleobase moieties; SU is -LO-, or [ka] wherein SU is attached to the phosphorus atom of formula I via an oxygen atom; L is a covalent bond or C 1-30 C having aliphatic and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon 1-30 heteroaliphatic groups, wherein one or more methylene units are optionally substituted C 1-6 Alkylene, C 1-6alkenylene, optionally independently replaced by -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 5s is R' or -OR'; R 2s is -F, -CN, -N3, -NO, -NO2, -R'-OR', -SR', -N(R')2, -OL-OR', -OL-SR', or -OLN(R')2, or R 2s is L linking C2 to C1, C2, C3, C4, or C5; -Cy- is an optionally substituted divalent ring selected from 3- to 30-membered carbocyclylene, 6- to 30-membered arylene, 5- to 30-membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and 3- to 30-membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; R 1 , R 2 , and R 3 Each of is independently R′, or R 1 , R 2 and R 3 two or three of, together with their intervening atoms, form: [ka] Ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R s is independently R' or -L-R'; t is 0 to 5; each R' is independently -R, -C(O)R, -CO2R, or -SO2R, or: two or more R' together with their intervening atoms form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R is independently hydrogen or C 1-30 C having 1 to 10 heteroatoms independently selected from aliphatic, oxygen, nitrogen, sulfur, phosphorus, and silicon 1-30 Heteroaliphatic, C 6-30 a 5- to 30-membered heteroaryl ring having 1 to 10 heteroatoms independently selected from aryl, oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3- to 30-membered heterocyclic ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, which may be substituted.

[0112] In some embodiments, BA is an optionally substituted group selected from a 3-30 membered alicyclic ring, a 6-30 membered aryl ring, a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. 1-30 In some embodiments, BA is an optionally substituted C 6-30 In some embodiments, BA is an optionally substituted 3-30 membered heterocyclyl. In some embodiments, BA is an optionally substituted C 3-30 In some embodiments, BA is an optionally substituted 5-30 membered heteroaryl. In some embodiments, BA is an optionally substituted C 5-30In some embodiments, BA is a heteroaryl. In some embodiments, BA is an optionally substituted natural base moiety. In some embodiments, BA is an optionally substituted modified base moiety. In some embodiments, BA is C 1-30 Alicyclic, C 6-30 Aryl, C 3-30 Heterocyclyl, and C 5-30 In some embodiments, BA is an optionally substituted group selected from the group consisting of C 1-30 Alicyclic, C 6-30 Aryl, C 3-30 Heterocyclyl, C 5-30 is an optionally substituted group selected from heteroaryl, and a natural nucleobase moiety. In some embodiments, BA is an optionally substituted A, T, C, or G. In some embodiments, BA is A, T, C, G, or 5-mC.

[0113] In some embodiments, the BA is linked to the SU via an aromatic ring. In some embodiments, the BA is linked to the SU via a heteroatom. In some embodiments, the BA is linked to the SU via a ring heteroatom of the aromatic ring. In some embodiments, the BA is linked to the SU via a ring nitrogen atom of the aromatic ring. In some embodiments, the linkage is similar to that found in naturally occurring nucleosides.

[0114] In some embodiments, BA is a natural nucleobase moiety. In some embodiments, BA is an optionally substituted natural nucleobase moiety. In some embodiments, BA is a substituted natural nucleobase moiety.

[0115] In some embodiments, BA is an optionally substituted group, which is [ka] and [ka] In some embodiments, BA is an optionally substituted group, which is formed by removing -H from [ka] and [ka] In some embodiments, BA is an optionally substituted group selected from: [ka] TIFF2025069269000014.tif25166 and [ka] In some embodiments, BA is an optionally substituted group, which is formed by removing -H from [ka] and [ka] is selected from.

[0116] In some embodiments, the BA is [ka] or [ka] In some embodiments, BA is an optionally substituted guanine residue, and O 6 The position is not protected.

[0117] Those of skill in the art will recognize that a variety of modified nucleobases are suitable for formula I in accordance with the present disclosure. Exemplary modified bases include those described in WO 2011 / 005761, U.S. Patent Application Publication No. 2012 / 0316224, WO 2013 / 012758, U.S. Patent Application Publication No. 2014 / 0194610, WO 2014 / 012081, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2015 / 0211006, WO ... Modified nucleobases include, but are not limited to, those described in WO 2011 / 0294124, WO 2014 / 010250, U.S. Patent Application Publication No. 2015 / 0197540, WO 2011 / 108682, U.S. Patent Application Publication No. 2013 / 0184450, WO 2012 / 039448, U.S. Patent Application Publication No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178. Each of these modified nucleobases is incorporated herein by reference.

[0118] In some embodiments, BA is a substituted nucleobase, and the phosphoramidite can be appropriately protected with one or more protecting groups and used in oligonucleotide synthesis. Suitable protecting groups for nucleobases are widely known in the art and include protecting groups useful in oligonucleotide synthesis and can be used in accordance with the present disclosure. In some embodiments, the protecting group is acetyl (Ac), phenylacetyl, benzoyl (Bz), isobutyryl (iBu), phenoxyacetyl (Pac), isopropyl-Pac, tertbutyl-Pac, alkyl-Pac, dimethylformamidine (DMF), or dialkylformamidine. In some embodiments, the protecting group is phthalimide, 9-fludrenylmethoxycarbonyl (FMOC), triphenylmethylsulfenyl, t-BOC, 4,4'-dimethoxytrityl (DMTr), 4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). For further suitable protecting groups, see Green and Wuts, Protective Groups in Organic Synthesis, 2nd Ed., John Wiley & Sons, New York, 1991, and International Patent Publication Nos. WO 2011 / 005761, WO 2013 / 012758, WO 2014 / 012081, WO 2015 / 107425, WO 2010 / 064146, WO 2014 / 010250, WO 2011 / 108682, WO 2012 / 039448, and WO 2012 / 073857. Each of these protecting groups is incorporated herein by reference.

[0119] In some embodiments, SU is a sugar moiety used in an oligonucleotide. In some embodiments, SU is a modified sugar moiety used in an oligonucleotide. In some embodiments, SU is a sugar moiety of a naturally occurring nucleoside. In some embodiments, SU is an optionally substituted sugar moiety found in naturally occurring nucleosides. In some embodiments, SU is a modified sugar moiety found in naturally occurring nucleosides, modified at the 2' position. In some embodiments, SU is a modified sugar moiety found in naturally occurring nucleosides, modified at the 2' position, modified with 2'-H and / or 2'-OH to R 2s In some embodiments, R 2s is not -H. In some embodiments, R 2s is not —OH. In some embodiments, R 2s is neither -H nor -OH.

[0120] In some embodiments, SU is -LO-. In some embodiments, L is -Cy-. In some embodiments, L is an optionally substituted 3-30 membered carbocyclylene. In some embodiments, L is an optionally substituted 6-30 membered arylene. In some embodiments, L is an optionally substituted 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, L is an optionally substituted 5-30 membered heteroarylene having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, L is an optionally substituted 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, L is an optionally substituted 3-30 membered heterocyclylene having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, L is an optionally substituted 5-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, L is an optionally substituted 5-30 membered heterocyclylene having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, L is an optionally substituted 5-10 membered heterocyclylene having one oxygen atom. In some embodiments, L is an optionally substituted 5-membered heterocyclylene having one oxygen atom. In some embodiments, L is an optionally substituted 6-membered heterocyclylene having one oxygen atom. In some embodiments, L is an optionally substituted 5-10 membered bicyclic heterocyclylene having one or two oxygen atoms. In some embodiments, L is an optionally substituted 7-10 membered bicyclic heterocyclylene having one or two oxygen atoms. In some embodiments, L is an optionally substituted 7-10 membered bicyclic heterocyclylene having two oxygen atoms.In some embodiments, L is an optionally substituted 7-membered bicyclic heterocyclylene having two oxygen atoms.

[0121] In some embodiments, SU is a sugar moiety used in oligonucleotide synthesis. Those skilled in the art will appreciate that phosphoramidites with various sugar moieties can benefit from improved yield and / or purity when the techniques provided are utilized to prepare phosphoramidites. In some embodiments, SU is an optionally substituted saturated monocyclic, bicyclic, or polycyclic aliphatic ring in which one or more methylene units are replaced with -O-. In some embodiments, SU is a ribose or deoxyribose moiety found in natural DNA or RNA molecules.

[0122] In some embodiments, the SU is [ka] In some embodiments, the SU has the structure: [ka] (In the formula, R 5s is -OR'; and R 2s is -F, -CN, -N3, -NO, -NO2, -R', -OR', -SR', -N(R')2, -OL-OR', -OL-SR', or -OLN(R')2; or R 2s is an L linking C2 to C1, C2, C3, C4, or C5. In some embodiments, R 2s is -H. In some embodiments, R 2s is -F. In some embodiments, R 2s In some embodiments, R 2s is -OCH2CH2OMe.

[0123] In some embodiments, the modified sugar is any of the following: -F, -CN, -N3, -NO, -NO2, -OR', -SR', or -N(R')2, where each R' is defined above and is independent as described herein; -O-(C 1-10 alkyl), -S-(C 1-10 alkyl), -NH-(C 1-10 alkyl), or -N(C 1-10 alkyl)2;-O-(C 2-10 alkenyl), -S-(C 2-10 alkenyl), -NH-(C 2-10 alkenyl), or -N(C 2-10 alkenyl)2;-O-(C 2-10 alkynyl), -S-(C 2-10 alkynyl), -NH-(C 2-10 alkynyl), or -N(C 2-10 alkynyl)2; or -O-(C 1-10 alkylene)-O-(C 1-10 alkyl), -O-(C 1-10 alkylene)-NH-(C 1-10 alkyl), or -O-(C 1-10 alkylene)-NH(C 1-10 alkyl)2, -NH-(C 1-10 alkylene)-O-(C 1-10 alkyl), or -N(C 1-10 Alkyl)-(C 1-10 alkylene)-O-(C 1-10 The 2'-position includes one or more substituents (e.g., R2s) including one of the following: -O(CH2) n OCH3 and -O(CH2) n Examples include, but are not limited to, NH2 (wherein n is from 1 to about 10), MOE, DMAOE, and DMAEOE.

[0124] Also contemplated herein are modified sugars as described in International Patent Publication No. WO 2001 / 088198, and Martin et al., Helv, Chim, Acta, 1995, 78, 486-504. In some embodiments, the modified sugar comprises one or more groups selected from a substituted silyl group, an RNA cleaving group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, a group for improving the pharmacodynamic properties of a nucleic acid, or other substituents with similar properties. In some embodiments, the modification is at one or more of the 2', 3', 4', 5', or 6' positions of the sugar or modified sugar, including the 3' position of the sugar of the 3'-terminal nucleotide or the 5' position of the 5'-terminal nucleotide.

[0125] Modified sugars include locked nucleic acids (LNAs). In some embodiments, two substituents on a sugar carbon atom together form a divalent moiety. In some embodiments, the two substituents are on two different sugar carbon atoms. In some embodiments, the divalent moiety formed has the structure -L-, as defined herein. In some embodiments, R 2s is -L-. In some embodiments, -L- is -O-CH2-, where -CH2- is optionally substituted. In some embodiments, -L- is -O-CH2-. In some embodiments, -L- is -O-CH(Et)-. In some embodiments, -L- is (S)-cEt. In some embodiments, -L- is between C2 and C4 of the sugar moiety. In some embodiments, a locked nucleic acid has the structure shown below. The structure of a locked nucleic acid is shown below. Ba represents a nucleobase or modified nucleobase as described herein, R 2s is -OCH2C4'-. [ka]

[0126] In some embodiments, the modified sugar is an ENA, e.g., as described in Seth et al., J Am Chem Soc. 2010 October 27;132(42):14942-14950. In some embodiments, the modified sugar is any of the sugars found in an XNA (xenonucleic acid), e.g., arabinose, anhydrohexitol, threose, 2'-fluoroarabinose, or cyclohexene.

[0127] Modified sugars include sugar mimetics, such as cyclobutyl or cyclopentyl moieties, in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, and 5,359,044. Some modified sugars contemplated include sugars in which the oxygen atom in the ribose ring is replaced with nitrogen, sulfur, selenium, or carbon. In some embodiments, the modified sugar is a modified ribose in which the oxygen atom in the ribose ring is replaced with nitrogen, which may be substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc.).

[0128] In some embodiments, one or more hydroxyl groups in the sugar moiety are optionally independently replaced with a halogen, -R', -N(R')2, -OR', or -SR', where each R' is defined above and independent as described herein.

[0129] In some embodiments, the SU is [ka] In some embodiments, the SU is [ka] In some embodiments, the SU is [ka] It is.

[0130] In some embodiments, L is a covalent bond. In some embodiments, L is not a covalent bond. In some embodiments, L is C 1-30 C having an aliphatic group and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 1-30 heteroaliphatic groups, wherein one or more methylene units are optionally substituted C 1-6 Alkylene, C 1-6 L is optionally independently replaced by alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-. In some embodiments, L is a divalent, optionally substituted, linear or branched C 1-30 an aliphatic group, in which one or more methylene units are optionally substituted C 1-6 Alkylene, C 1-6 L is optionally independently replaced by alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-. In some embodiments, L is a divalent, optionally substituted, linear or branched C 1-30 Heteroaliphatic groups, in which one or more methylene units are optionally substituted C 1-6 Alkylene, C1-6 L is optionally independently replaced by alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-. In some embodiments, L is optionally independently replaced by a divalent, optionally substituted, straight or branched chain C 1-30 It is a heteroaliphatic group. In some embodiments, L contains one or more Si.

[0131] In some embodiments, R 5s is R'. In some embodiments, R 5s In some embodiments, R 5s is a protected hydroxyl group suitable for oligonucleotide synthesis. In some embodiments, R 5sis DMTrO-. Exemplary protecting groups for use in accordance with the present disclosure are well known in the art. Further examples are described in Greene, TW, Wuts, PGM Protective Groups in Organic Synthesis, 2nd ed., Wiley, New York, 1991, and in WO 2011 / 005761, U.S. Patent Application Publication No. 2012 / 0316224, WO 2013 / 012758, U.S. Patent Application Publication No. 2014 / 0194610, WO 2014 / 012081, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, See U.S. Patent Application Publication No. 2011 / 0294124, WO 2014 / 010250, U.S. Patent Application Publication No. 2015 / 0197540, WO 2011 / 108682, U.S. Patent Application Publication No. 2013 / 0184450, WO 2012 / 039448, U.S. Patent Application Publication No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178, each of which protecting groups are incorporated herein by reference.

[0132] In some embodiments, R 2s is -F. In some embodiments, R 2s is -CN. In some embodiments, R 2s is -N3. In some embodiments, R 2s In some embodiments, R 2s is -NO2. In some embodiments, R 2s is R'. In some embodiments, R 2s In some embodiments, R 2s is -OR', where R' is an optionally substituted C 1-6 In some embodiments, R 2s In some embodiments, R 2s In some embodiments, R2s is -N(R'). In some embodiments, R 2s is -OL-OR'. In some embodiments, R 2s is -O-(optionally substituted C 1-6 In some embodiments, R 2s is -O-(optionally substituted C 1-6 alkylene)-OR', R' being optionally substituted C 1-6 In some embodiments, R 2s is -OCH2CH2OMe. In some embodiments, R 2s is -OL-SR'. In some embodiments, R 2s is -OLN(R'). In some embodiments, R 2s is an L connecting C2 to C1, C2, C3, C4, or C5. In some embodiments, R 2s is an L that connects C2 and C1. In some embodiments, R 2s is an L that connects C2 to C2. In some embodiments, R 2s is an L that connects C2 and C3. In some embodiments, R 2s is an L connecting C2 and C4. In some embodiments, R 2s is an L connecting C2 and C5. In some embodiments, R 2s is (C2)-O-(optionally substituted methylene)-(C4). In some embodiments, R 2s is (C2)-O-(methylene)-(C4). In some embodiments, R 2s is (C2)-O-(ethylmethylene)-(C4). In some embodiments, R 2s is (C2)-O-((R)-ethylmethylene)-(C4). In some embodiments, R 2s is (C2)-O-((S)-ethylmethylene)-(C4). In some embodiments, R 2s is not -H. In some embodiments, R 2sis not —OH. In some embodiments, R 2s is not -OMe. In some embodiments, R 2s is not -OCH2CH2OMe. 2s is not any group selected from -OH, -OMe, and OCH2CH2OMe.

[0133] In some embodiments, C1 binds to BA.

[0134] In some embodiments, -Cy- is an optionally substituted divalent 3- to 30-membered carbocyclylene. In some embodiments, -Cy- is an optionally substituted divalent 6- to 30-membered arylene. In some embodiments, -Cy- is an optionally substituted divalent 5- to 30-membered heteroarylene having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, -Cy- is an optionally substituted divalent 3- to 30-membered heterocyclylene having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, -Cy- is an optionally substituted divalent 5- to 30-membered heteroarylene having 1 to 5 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, -Cy- is an optionally substituted divalent 3- to 30-membered heterocyclylene having 1 to 5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0135] In some embodiments, R 1 is R'. In some embodiments, R 2 is R'. In some embodiments, R 3 is R'.

[0136] In some embodiments, R 1 , R 2 , and R 3 Each of is independently R′, or R 1 , R 2 , and R3 Two or three of these are formed together with their intervening atoms. [ka]

[0137] In some embodiments, the valency of ring A is t+1.

[0138] In some embodiments, R 1 is R. In some embodiments, R 1 is R and R is not hydrogen. In some embodiments, R 1 is optionally substituted C 1-10 In some embodiments, R 1 is optionally substituted C 1-10 In some embodiments, R 1 is optionally substituted methyl. In some embodiments, R 1 is optionally substituted ethyl. In some embodiments, R 1 is -O(CH2)2CN.

[0139] In some embodiments, R 2 is R. In some embodiments, R 2 is R and R is not hydrogen. In some embodiments, R 2 is optionally substituted C 1-10 In some embodiments, R 2 is optionally substituted C 1-10 In some embodiments, R 2 is optionally substituted methyl. In some embodiments, R 2 is optionally substituted ethyl. In some embodiments, R 2 is isopropyl.

[0140] In some embodiments, R 3 is R. In some embodiments, R 3is R and R is not hydrogen. In some embodiments, R 3 is optionally substituted C 1-10 In some embodiments, R 3 is optionally substituted C 1-10 In some embodiments, R 3 is optionally substituted methyl. In some embodiments, R 3 is optionally substituted ethyl. In some embodiments, R 3 is isopropyl.

[0141] In some embodiments, R 2 and R 3 is the same. In some embodiments, R 2 and R 3 It is different from.

[0142] In some embodiments, R 1 and R 2 together with their intervening atoms [ka] In some embodiments, R 1 and R 2 together with their intervening atoms [ka] In some embodiments, R 1 and R 3 together with their intervening atoms [ka] In some embodiments, R 2 and R 3 together with their intervening atoms [ka] In some embodiments, R1 , R 2 , and R 3 together with their intervening atoms [ka] Form.

[0143] In some embodiments, ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl, 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus, and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl, 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus, and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0144] In some embodiments, Ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl, 3-30 membered ring having zero heteroatoms in addition to the intervening nitrogen, phosphorus, and oxygen atoms.

[0145] In some embodiments, ring A is an optionally substituted polyvalent monocyclic, saturated, 5- to 7-membered ring having intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is an optionally substituted polyvalent monocyclic, saturated, 5-membered ring having intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is an optionally substituted polyvalent monocyclic, saturated, 6-membered ring having intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is an optionally substituted polyvalent monocyclic, saturated, 7-membered ring having intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms.

[0146] In some embodiments, ring A is an optionally substituted polyvalent bicyclic, saturated, partially unsaturated, or aryl, 5-30 membered ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon in addition to the intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, ring A is an optionally substituted polyvalent bicyclic, saturated, partially unsaturated, or aryl, 5-30 membered ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur in addition to the intervening nitrogen, phosphorus, and oxygen atoms. In some embodiments, ring A is a polyvalent bicyclic, saturated, 8-10 membered bicyclic ring having intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is a polyvalent bicyclic, saturated, 8-membered bicyclic ring having intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is a polyvalent bicyclic and saturated 9-membered bicyclic ring with intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is a polyvalent bicyclic and saturated 10-membered bicyclic ring with intervening nitrogen, phosphorus, and oxygen atoms and no additional heteroatoms. In some embodiments, ring A is bicyclic and comprises a 5-membered ring fused to a 5-membered ring. In some embodiments, ring A is bicyclic and comprises a 5-membered ring fused to a 6-membered ring. In some embodiments, the 5-membered ring comprises intervening nitrogen, phosphorus, and oxygen atoms as ring atoms. In some embodiments, ring A is [ka] In some embodiments, ring A is optionally substituted. [ka] In some embodiments, ring A is [ka] each of which independently represents 1 to 5 R s In some embodiments, ring A is substituted with [ka] each of which independently represents 1 to 5 R s where the carbon atom bonded to the oxygen atom is substituted with one or two R s In some embodiments, ring A is substituted with, for example [ka] The substituents selected from include one or more chiral centers other than the phosphorus atom and the tertiary carbon atom attached to the nitrogen. In some embodiments, for example, one or two R s As a result of the substitution with, the carbon atom attached to the oxygen atom is a chiral center. s is phenyl. In some embodiments, R s is alkyl. In some embodiments, R s is methyl. In some embodiments, one R s is phenyl and one R sIn some embodiments, Ring A is selected from the group consisting of WO 2011 / 005761, U.S. Patent Application Publication No. 2012 / 0316224, WO 2013 / 012758, U.S. Patent Application Publication No. 2014 / 0194610, WO 2014 / 012081, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, U.S. Patent Application Publication No. 2011 / 0294 124, WO 2014 / 010250, U.S. Patent Application Publication No. 2015 / 0197540, WO 2011 / 108682, U.S. Patent Application Publication No. 2013 / 0184450, WO 2012 / 039448, U.S. Patent Application Publication No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178, each of which phosphoramidites are incorporated herein by reference. In some embodiments, the compounds, e.g., phosphoramidites, are chiral and stereochemically pure. In some embodiments, provided compounds, e.g., phosphoramidites, have a purity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or greater.

[0147] In some embodiments, ring A is an optionally substituted polyvalent polycyclic, saturated, partially unsaturated, or aryl, 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus, and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, ring A is an optionally substituted polyvalent polycyclic, saturated, partially unsaturated, or aryl, 3-30 membered ring having, in addition to the intervening nitrogen, phosphorus, and oxygen atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0148] In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 5-10 membered monocyclic ring whose ring atoms include nitrogen, phosphorus, and oxygen atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 5-9 membered monocyclic ring whose ring atoms include nitrogen, phosphorus, and oxygen atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 5-8 membered monocyclic ring whose ring atoms include nitrogen, phosphorus, and oxygen atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 5-7 membered monocyclic ring whose ring atoms include nitrogen, phosphorus, and oxygen atoms. In some embodiments, Ring A is monocyclic, bicyclic, or polycyclic, including an optionally substituted 5-6 membered monocyclic ring containing nitrogen, phosphorus, and oxygen atoms interspersed therewith.

[0149] In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 5-membered monocyclic ring with nitrogen, phosphorus, and oxygen atoms interposed by its ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 6-membered monocyclic ring with nitrogen, phosphorus, and oxygen atoms interposed by its ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 7-membered monocyclic ring with nitrogen, phosphorus, and oxygen atoms interposed by its ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 8-membered monocyclic ring with nitrogen, phosphorus, and oxygen atoms interposed by its ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 9-membered monocyclic ring with nitrogen, phosphorus, and oxygen atoms interposed by its ring atoms. In some embodiments, Ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 10-membered monocyclic ring whose ring atoms include nitrogen, phosphorus, and oxygen atoms.

[0150] In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 5-membered ring consisting of nitrogen, phosphorus, and oxygen atoms and carbon atoms that are interrupted by ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 6-membered ring consisting of nitrogen, phosphorus, and oxygen atoms and carbon atoms that are interrupted by ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 7-membered ring consisting of nitrogen, phosphorus, and oxygen atoms and carbon atoms that are interrupted by ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 8-membered ring consisting of nitrogen, phosphorus, and oxygen atoms and carbon atoms that are interrupted by ring atoms. In some embodiments, ring A is monocyclic, bicyclic, or polycyclic and includes an optionally substituted 9-membered ring consisting of nitrogen, phosphorus, and oxygen atoms and carbon atoms that are interrupted by ring atoms. In some embodiments, Ring A is monocyclic, bicyclic, or polycyclic and contains an optionally substituted 10-membered ring consisting of nitrogen, phosphorus, and oxygen atoms, and carbon atoms, with the ring atoms interspersed therewith.

[0151] In some embodiments, ring A is [ka] The ring system includes a ring system having the backbone structure:

[0152] In some embodiments, R s is R'. In some embodiments, R s is R. In some embodiments, R s is an optionally substituted C 1-30 In some embodiments, R s contains one or more silicon atoms. In some embodiments, R s is -CH 2s i(Ph)2CH3.

[0153] In some embodiments, R s is -L-R'. In some embodiments, Rs is -L-R', and -L- is a divalent optionally substituted C 1-30 In some embodiments, R s is -CH2si(Ph)2CH3.

[0154] In some embodiments, t is 0. In some embodiments, t is 1-5. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5.

[0155] In some embodiments, R' is -R. In some embodiments, R' is -C(O)R. In some embodiments, R' is -CO2R. In some embodiments, R' is -SO2R. In some embodiments, two or more R', taken together with their intervening atoms, form an optionally substituted monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl, 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R' is hydrogen. In some embodiments, R' is not hydrogen.

[0156] In some embodiments, R is hydrogen. In some embodiments, R is not hydrogen. In some embodiments, R is C 1~30 C having 1 to 10 heteroatoms independently selected from aliphatic, oxygen, nitrogen, sulfur, phosphorus, and silicon 1~30 Heteroaliphatic, C 6-30 It is an optionally substituted group selected from a 5- to 30-membered heteroaryl ring having 1 to 10 heteroatoms independently selected from aryl, oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3- to 30-membered heterocyclic ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.

[0157] In some embodiments, R is hydrogen or C1-20 an optionally substituted group selected from aliphatic, phenyl, a 3- to 7-membered saturated or partially unsaturated carbocyclic ring, an 8- to 10-membered bicyclic saturated, partially unsaturated, or aryl ring, a 5- to 6-membered monocyclic heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 4- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 7- to 10-membered bicyclic saturated or partially unsaturated heterocyclic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8- to 10-membered bicyclic heteroaryl ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0158] In some embodiments, R is an optionally substituted C 1-30 In some embodiments, R is an optionally substituted C 1-20 In some embodiments, R is an optionally substituted C 1-15 In some embodiments, R is an optionally substituted C 1-10 In some embodiments, R is an optionally substituted C 1-6 In some embodiments, R is an optionally substituted C 1-6In some embodiments, R is an optionally substituted hexyl, pentyl, butyl, propyl, ethyl, or methyl. In some embodiments, R is an optionally substituted hexyl. In some embodiments, R is an optionally substituted pentyl. In some embodiments, R is an optionally substituted butyl. In some embodiments, R is an optionally substituted propyl. In some embodiments, R is an optionally substituted ethyl. In some embodiments, R is an optionally substituted methyl. In some embodiments, R is hexyl. In some embodiments, R is pentyl. In some embodiments, R is butyl. In some embodiments, R is propyl. In some embodiments, R is ethyl. In some embodiments, R is methyl. In some embodiments, R is isopropyl. In some embodiments, R is n-propyl. In some embodiments, R is tert-butyl. In some embodiments, R is sec-butyl. In some embodiments, R is n-butyl.

[0159] In some embodiments, R is an optionally substituted C 3-30 In some embodiments, R is an optionally substituted C 3-20 In some embodiments, R is an optionally substituted C 3-10 In some embodiments, R is an optionally substituted cyclohexyl. In some embodiments, R is an optionally substituted cyclohexyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is an cyclopentyl.

[0160] In some embodiments, R is an optionally substituted 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 4 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 5 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 6 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted cycloheptyl. In some embodiments, R is cycloheptyl. In some embodiments, R is an optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is an optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is an optionally substituted cyclobutyl. In some embodiments, R is a cyclobutyl. In some embodiments, R is an optionally substituted cyclopropyl. In some embodiments, R is a cyclopropyl.

[0161] In some embodiments, R is an optionally substituted C group having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. 1-30 In some embodiments, R is a heteroaliphatic group having 1 to 10 heteroatoms. 1-20 In some embodiments, R is a C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, or silicon. 1-20 In some embodiments, R is heteroaliphatic and optionally contains an oxidized form of one or more of nitrogen, sulfur, phosphorus, or selenium. [ka] , -N=, ≡N, -S-, -S(O)-, -S(O)2-, -O-, =O, [ka] C containing 1 to 10 groups independently selected from 1-30 It is heteroaliphatic.

[0162] In some embodiments, R is an optionally substituted C 6-30 In some embodiments, R is aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is substituted phenyl.

[0163] In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated, partially unsaturated, or aryl ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic partially unsaturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic aryl ring. In some embodiments, R is an optionally substituted naphthyl.

[0164] In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is an optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0165] In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0166] In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0167] In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from oxygen, nitrogen, and sulfur, In some embodiments, R is selected from optionally substituted pyrrolyl, furanyl, or thienyl.

[0168] In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom and an additional heteroatom selected from sulfur or oxygen. Exemplary R groups include, but are not limited to, optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl.

[0169] In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. Exemplary R groups include, but are not limited to, optionally substituted triazolyl, oxadiazolyl, or thiadiazolyl.

[0170] In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. Exemplary R groups include, but are not limited to, optionally substituted tetrazolyl, oxatriazolyl, and thiatriazolyl.

[0171] In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-3 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 4 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 3 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1 nitrogen atom. Exemplary R groups include, but are not limited to, optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

[0172] In some embodiments, R is a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is a 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. In some embodiments, R is a 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0173] In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 3-membered heterocyclic ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0174] In some embodiments, R is an optionally substituted 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted indolinyl. In some embodiments, R is an optionally substituted isoindolinyl. In some embodiments, R is an optionally substituted 1,2,3,4-tetrahydroquinolinyl. In some embodiments, R is an optionally substituted 1,2,3,4-tetrahydroisoquinolinyl. In some embodiments, R is an optionally substituted azabicyclo[3.2.1]octanyl.

[0175] In some embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0176] In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl, thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl, pyrrolo[2,1-b]oxazolyl, or pyrrolo[2,1-b]thiazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted dihydropyrroloimidazolyl, 1H-furomidazolyl, 1H-thinoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienoxazolyl, thienisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl, or imidazo[5,1-b]thiazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0177] In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In another embodiment, R is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted indolyl. In some embodiments, R is an optionally substituted benzofuranyl. In some embodiments, R is an optionally substituted benzo[b]thienyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted azaindolyl. In some embodiments, R is an optionally substituted benzimidazolyl. In some embodiments, R is an optionally substituted benzothiazolyl. In some embodiments, R is an optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted oxazolopyridinyl, thiazolopyridinyl, or imidazopyridinyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl, or imidazopyridazinyl.In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0178] In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted quinolinyl. In some embodiments, R is an optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted quinazolinyl, phthalazinyl, quinoxalinyl, or naphthyridinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted pyridopyrimidinyl, pyridopyridazinyl, pyridopyrazinyl, or benzotriazinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted pyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl, pyridazinopyridazinyl, pyrimidopyridazinyl, or pyrimidopyrimidinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0179] In some embodiments, R 1 , R 2 , R 3 or [ka] contains one or more chiral elements, e.g., chiral centers. In some embodiments, R 1 , R 2 , R 3 or [ka] is asymmetric. Phosphoramidites containing such asymmetric moieties can be used to prepare chiral controlled oligonucleotide compositions, such as those described in WO 2014 / 012081, WO 2015 / 107425, among others. In some embodiments, such phosphoramidites, when used in oligonucleotide synthesis, can provide greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% diastereoselectivity at the newly formed P chiral center, optionally with yields of greater than 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0180] In some embodiments, [ka] teeth [ka] (wherein one or more R s is not hydrogen. In some embodiments, [ka] teeth [ka] In some embodiments, [ka] teeth [ka] In some embodiments, [ka] teeth [ka] It is.

[0181] As one of skill in the art will recognize, phosphoramidites, including those containing amino and hydroxyl groups, can be prepared from nucleosides and chiral auxiliaries, as shown in the examples. Suitable chiral auxiliaries are described in WO 2011 / 005761, U.S. Patent Application Publication No. 2012 / 0316224, WO 2013 / 012758, U.S. Patent Application Publication No. 2014 / 0194610, WO 2014 / 012081, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, U.S. Patent Application Publication No. 2012 / 012081 ... Chiral auxiliaries include, but are not limited to, those described in WO 2011 / 0294124, WO 2014 / 010250, U.S. Patent Application Publication No. 2015 / 0197540, WO 2011 / 108682, U.S. Patent Application Publication No. 2013 / 0184450, WO 2012 / 039448, U.S. Patent Application Publication No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178, each of which is incorporated herein by reference.

[0182] In some embodiments, the phosphoramidites are those described in WO 2011 / 005761, U.S. Patent Application Publication No. 2012 / 0316224, WO 2013 / 012758, U.S. Patent Application Publication No. 2014 / 0194610, WO 2014 / 012081, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, U.S. Patent Application Publication No. 201 ... The phosphoramidites are those described in WO 2014 / 010250, U.S. Patent Application Publication No. 2015 / 0197540, WO 2011 / 108682, U.S. Patent Application Publication No. 2013 / 0184450, WO 2012 / 039448, U.S. Patent Application Publication No. 2013 / 0178612, WO 2012 / 073857, or U.S. Patent Application Publication No. 2013 / 0253178, each of which is incorporated herein by reference. In some embodiments, the disclosure provides a method that includes a method for preparing an oligonucleotide described in one of these applications, and purifying the phosphoramidites using the methods described herein.

[0183] In some embodiments, the phosphoramidite is [ka] The file is TIFF2025069269000051.tif41166. In some embodiments, the phosphoramidite is selected from Table 1 below. Table 1. Examples of phosphoramidites [Table 1] [Table 2]

[0184] In some embodiments, the disclosure provides novel techniques, e.g., compounds, compositions, methods, etc., for preparing phosphoramidites and / or oligonucleotides. In some embodiments, the disclosure provides new techniques for purifying phosphoramidites. In some embodiments, the techniques provided significantly improve the efficiency of phosphoramidite synthesis and / or significantly reduce the cost of phosphoramidites and oligonucleotides, compositions and pharmaceuticals prepared therefrom. In particular, WO 2011 / 005761, U.S. Patent Application Publication No. 2012 / 0316224, WO 2013 / 012758, U.S. Patent Application Publication No. 2014 / 0194610, WO 2014 / 012081, U.S. Patent Application Publication No. 2015 / 0211006, WO 2015 / 107425, U.S. Patent Application Publication No. 2017 / 0037399, WO 2010 / 064146, U.S. Patent Application Publication No. 2011 / 0294124, WO 2014 / 0102 Phosphoramidites for various oligonucleotide synthesis methods can be prepared using the provided techniques, including but not limited to those described in WO 2011 / 108682, WO 2013 / 0184450, WO 2012 / 039448, WO 2013 / 0178612, WO 2012 / 073857, or WO 2013 / 0253178. Each of these methods is incorporated herein by reference. In some embodiments, the methods provided herein are particularly useful for preparing chiral controlled oligonucleotide compositions, such as those described in WO 2014 / 012081, WO 2015 / 107425, etc.

[0185] As shown herein, the provided techniques can surprisingly improve the yield and / or purity of phosphoramidites. In some embodiments, the absolute improvement is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even greater. In some embodiments, the absolute improvement is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even greater. In some embodiments, the yield with the provided techniques is greater than about 80%, while the yield with the corresponding pretreatment-free techniques (corresponding to an absolute improvement of more than 20%) is less than about 60%. In some embodiments, the improvement over the corresponding technique without pre-processing is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or even greater.

[0186] In some embodiments, the techniques provided may be performed in part or in whole at temperatures above and / or below room temperature. In some embodiments, the techniques provided include procedures at temperatures above room temperature, e.g., 30, 40, 50, 60, 70, 80, 90, 100° C., or higher. In some embodiments, the techniques provided include procedures at temperatures below room temperature, e.g., 15, 10, 5, 0, −5, −10° C., or lower. In some embodiments, purification, e.g., chromatographic purification of phosphoramidites using a (often pretreated) purification medium, is performed at temperatures above room temperature. In some embodiments, purification, e.g., chromatographic purification of phosphoramidites using a (often pretreated) purification medium, is performed at temperatures below room temperature. In some embodiments, the higher and / or lower temperatures are controlled to control the temperature fluctuations within a certain range.

[0187] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the disclosure. Unless otherwise stated, all tautomers of the compounds of the disclosure are within the scope of the disclosure. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, replacement of hydrogen by deuterium or tritium, or 11 C- or 13 C- or 14 Compounds having this structure, except for the replacement of a carbon by a C-rich carbon, are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0188] As used in the specification and claims, the singular forms "a," "an," and "the" include the plural unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds.

[0189] In some embodiments, the present disclosure provides the following embodiments: 1. A method for purifying a compound, comprising: a) removing water from the purification medium; b) contacting the purification medium with a compound; c) optionally purifying the compound using a purification medium. 2. A method for purifying a compound, comprising: a) removing water from the purification medium; b) contacting the purification medium with a compound; c) purifying the compound using the purification medium. 3. A method for purifying a compound, comprising: a) removing water from the purification medium; b) purifying the compound using the purification medium. 4. A method for improving the recovery rate of a compound, comprising: a) removing water from the purification medium; b) adding a compound to the purification medium; c) eluting the compound from the purification medium with a solvent system; wherein in the absence of step a), the recovery rate is higher than the reference recovery rate. 5. A method for reducing degradation of a compound when the compound contacts a purification medium, comprising: removing water from the purification medium. 6. a) removing water from the purification medium; b) contacting the purification medium with a compound; c) optionally purifying the compound using a purification medium. 7. a) removing water from the purification medium; b) purifying the compound using the purification medium. 8. contacting the purification medium with the compound, wherein the medium is pretreated, the pretreatment comprising removing water. 9. 13. A method comprising purifying a compound using a pretreated purification medium, the medium being pretreated, the pretreatment comprising removing water. 10. a) removing water from the purification medium; b) contacting the purification medium with a compound; c) purifying the compound using the purification medium. 11. a) mechanically removing water from a purification medium; b) contacting the purification medium with a compound; c) optionally purifying the compound using a purification medium. 12. a) mechanically removing water from a purification medium; b) contacting the purification medium with a compound; c) purifying the compound using the purification medium. 13. The method of any one of embodiments 1 to 12, wherein the compound is a phosphoramidite. 14. A method for purifying phosphoramidites, comprising: a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium. 15. A method for purifying phosphoramidites, comprising: a) removing water from the purification medium; b) purifying the phosphoramidite using a purification medium. 16. A method for purifying phosphoramidites, comprising: a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; c) purifying the phosphoramidite using a purification medium. 17. A method for improving phosphoramidite recovery, comprising: a) removing water from the purification medium; b) adding phosphoramidites to the purification medium; c) eluting the phosphoramidite from the purification medium with a solvent system; wherein in the absence of step a), the recovery rate is higher than the reference recovery rate. 18. The method of embodiment 4 or 17, wherein the difference between the improved recovery rate and the reference recovery rate is at least about 10%, 20%, 30%, 40% or 50%. 19. A method for reducing decomposition of a phosphoramidite when the phosphoramidite contacts a purification medium, comprising: a) removing water from the purification medium. 20. a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium. twenty one. a) removing water from the purification medium; b) purifying the phosphoramidite using a purification medium. twenty two. a) removing water from the purification medium; b) contacting the purification medium with a phosphoramidite; c) purifying the phosphoramidite using a purification medium. twenty three. a) mechanically removing water from a purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium. twenty four. a) mechanically removing water from a purification medium; b) purifying the phosphoramidite using a purification medium. twenty five. a) mechanically removing water from a purification medium; b) contacting the purification medium with a phosphoramidite; c) purifying the phosphoramidite using a purification medium. 26. In a method for purifying phosphoramidites using a purification medium, an improvement comprising pretreatment of the purification medium by a process for removing water from the purification medium. 27. The method of any one of embodiments 1 to 26, wherein the step of removing water from the purification medium comprises heating the purification medium. 28. The method of any one of embodiments 1 to 27, wherein the step of removing water from the purification medium comprises contacting the purification medium with a hygroscopic solvent system. 29. The method of embodiment 28, wherein the hygroscopic solvent system consists of one hygroscopic solvent. 30. The method of embodiment 28, wherein the hygroscopic solvent system consists of one hygroscopic solvent and another solvent. 31. The method of embodiment 30, wherein the hygroscopic solvent system is comprised of two or more solvents, each of which is independently a hygroscopic solvent. 32. The method of any one of the preceding embodiments, wherein the hygroscopic solvent is selected from acetaldehyde, acetic acid, acetone, acetonitrile, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyric acid, diethanolamine, diethylenetriamine, dimethylcarbonate, dimethylformamide, dimethoxyethane, dimethylsulfoxide, 1,4-dioxane, ethanol, ethyl acetate, ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol, methyldiethanolamine, methylisocyanide, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propanoic acid, propylene glycol, pyridine, tetrahydrofuran, and triethylene glycol. 33. The method of any one of embodiments 1-32, wherein the hygroscopic solvent is selected from acetaldehyde, acetic acid, acetone, acetonitrile, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyric acid, diethanolamine, diethylenetriamine, dimethylformamide, dimethoxyethane, dimethylsulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol, methyldiethanolamine, methylisocyanide, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propanoic acid, propylene glycol, pyridine, tetrahydrofuran, and triethylene glycol. 34. The method of any one of the preceding embodiments, wherein the hygroscopic solvent is selected from methanol and ethanol. 35. The method of any one of embodiments 1 to 34, wherein the hygroscopic solvent is methanol. 36. The method of any one of the preceding embodiments, wherein the hygroscopic solvent is ethanol. 37. The method of any one of embodiments 1 to 35, wherein the hygroscopic solvent is isopropanol. 38. The method of any one of the preceding embodiments, wherein the hygroscopic solvent is acetone. 39. The method of any one of embodiments 1 to 35, wherein the hygroscopic solvent is acetonitrile. 40. The method of any one of the preceding embodiments, wherein the hygroscopic solvent is dimethyl carbonate. 41. The method of any one of embodiments 1-35, wherein the hygroscopic solvent is ethyl acetate. 42. a) pretreating a purification medium; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium. 43. a) pretreating a purification medium; b) contacting the purification medium with a phosphoramidite. 44. a) pretreating a purification medium; b) purifying the phosphoramidite using a purification medium. 45. a) pretreating a purification medium comprising silica; b) contacting the purification medium with a phosphoramidite; c) optionally purifying the phosphoramidite using a purification medium. 46. a) pretreating a purification medium comprising silica; b) contacting the purification medium with a phosphoramidite. 47. a) pretreating a purification medium comprising silica; b) purifying the phosphoramidite using a purification medium. 48. In a method for purifying phosphoramidites using a purification medium, an improvement comprising pretreatment of the purification medium. 49. In a method for purifying phosphoramidites using a purification medium comprising silica, an improvement comprising pretreatment of the purification medium. 50. The method of any one of embodiments 42-49, wherein pretreatment comprises heating the purification medium. 51. The method of any one of embodiments 42-50, wherein pretreatment comprises contacting the purification medium with a first solvent system. 52. The method of any one of embodiments 42-50, wherein pretreatment comprises contacting the purification medium with a first solvent system, the first solvent system comprising a hygroscopic solvent. 53. The method of any one of embodiments 42-50, wherein pretreatment comprises contacting the purification medium with a first solvent system, and the first solvent system comprises an alcohol. 54. The method of any one of embodiments 42-53, wherein the first solvent system comprises methanol. 55. The method of any one of embodiments 42-52, wherein the hygroscopic solvent is ethanol. 56. The method of any one of embodiments 42-52, wherein the hygroscopic solvent comprises isopropanol. 57. The method of any one of embodiments 42-52, wherein the hygroscopic solvent is dimethyl carbonate. 58. The method of any one of embodiments 42-52, wherein the hygroscopic solvent is acetone. 59. The method of any one of embodiments 42-52, wherein the hygroscopic solvent is acetonitrile. 60. The method of any one of embodiments 42-52, wherein the hygroscopic solvent is ethyl acetate. 61. The method of any one of embodiments 42-54, wherein the first solvent system consists of one solvent. 62. The method of any one of embodiments 42-62, wherein the first solvent system comprises a modifying agent. 63. The method of embodiment 62, wherein the modifying agent is up to 50% by volume of triethylamine. 64. The method according to any one of the preceding embodiments, further comprising the step of: a1) equilibrating the purification medium with a second solvent system before contacting the purification system with the phosphoramidite. 65. The method of any one of embodiments 1-64, wherein the second solvent system is less polar than the first solvent system. 66. The method of any one of the preceding embodiments, wherein the second solvent system comprises a lower volume percent of at least one alcohol compared to the first solvent system. 67. The method of any one of embodiments 1-66, wherein the second solvent system does not contain alcohol. 68. The method of any one of the preceding embodiments, wherein the second solvent system comprises hexane or ethyl acetate, and optionally a modifier. 69. The method of any one of embodiments 1-68, wherein the second solvent system comprises hexane, ethyl acetate, and an organic base. 70. The method of any one of embodiments 1-69, wherein the second solvent system comprises hexane, ethyl acetate, and triethylamine. 71. The method of any one of embodiments 1 to 70, wherein the purification medium is used to purify the phosphoramidites, and a third solvent system is used to elute the phosphoramidites from the purification medium. 72. The method of any one of embodiments 1-71, wherein the third solvent system is less polar than the first solvent system. 73. The method of any one of the preceding embodiments, wherein the third solvent system comprises a lower volume percent of at least one alcohol compared to the first solvent system. 74. The method of any one of embodiments 1-73, wherein the third solvent system is less polar than the second solvent system. 75. The method of any one of embodiments 1-74, wherein the third solvent system is the same as the second solvent system. 76. The method of any one of embodiments 1 to 75, wherein a gradient is used to elute the phosphoramidites from the purification medium. 77. A method according to any one of embodiments 1 to 76, wherein the solvent system at the start of the gradient elution is the same as the second solvent system at the end of the equilibration. 78. The method of any one of embodiments 1 to 77, wherein the purification medium is silica gel. 79. The method of any one of embodiments 1 to 78, wherein the purification medium is silica gel having a particle size of 50 micrometers or more. 80. The method of any one of embodiments 1 to 79, wherein the purification medium is silica gel having a particle size of 50 micrometers. 81. The method of any one of embodiments 1-80, wherein the first solvent system comprises a base. 82. The method of any one of embodiments 1-81, wherein the third solvent system comprises a base, ethyl acetate, and hexane. 83. The method of any one of embodiments 1-82, wherein the base is triethylamine. 84. The method of any one of embodiments 1 to 83, wherein the base is at most 50% by volume. 85. The method of any one of the preceding embodiments, wherein the base is 5% by volume. 86. The method of any one of embodiments 1 to 85, comprising chromatographic purification of the phosphoramidite using a purification medium. 87. The method of any one of embodiments 1 to 86, comprising chromatographic purification of the phosphoramidite using a purification medium at a temperature higher than room temperature. 88. The method of any one of embodiments 1 to 87, comprising chromatographic purification of the phosphoramidite using a purification medium at a temperature below room temperature. 89. a) a purification medium pretreated according to step a) of any one of embodiments 1 to 88; b) a phosphoramidite. 90. The composition of embodiment 89, wherein the inactivated purification medium is silica gel. 91. a) silica pretreated according to step a) of any one of embodiments 1 to 90; and b) a phosphoramidite. 92. The composition of any one of embodiments 89-91, wherein the pretreated purification medium is a silica gel pretreated with a hygroscopic solvent system. 93. The composition of any one of embodiments 92, wherein the hygroscopic solvent system is a hygroscopic solvent system or a first solvent system of any one of embodiments 1-92. 94. The composition of any one of embodiments 89-91, wherein the purification medium is equilibrated with a second solvent system. 95. The method or composition of any one of embodiments 1 to 94, wherein the phosphoramidite is a nucleoside phosphoramidite. 96. A phosphoramidite has the formula II: [ka] (In the formula, BA is R or an optionally substituted group selected from a 3-30 membered alicyclic ring, a 6-30 membered aryl ring, a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a natural nucleobase moiety, and a modified nucleobase moiety; SU is a sugar moiety, a modified sugar moiety, -LO-, or [ka] where SU is attached to the phosphorus atom in formula I via an oxygen atom; L is a covalent bond or C 1-30 C having aliphatic and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon 1-30 heteroaliphatic groups, wherein one or more methylene units are optionally substituted C 1-6 Alkylene, C 1-6Alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R ')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O )2 may be independently replaced by -, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 5s is R' or -OR'; R 2s is -F, -CN, -N3, -NO, -NO2, -R'-OR', -SR', -N(R')2, -L-R', -OL-OR', -OL-SR', or -OLN(R')2, or R 2s is L linking C2 to C1, C2, C3, C4 or C5; -Cy- is an optionally substituted divalent ring selected from 3- to 30-membered carbocyclylene, 6- to 30-membered arylene, 5- to 30-membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and 3- to 30-membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; R 1 , R 2 , and R 3 Each of is independently R′, or R 1 , R 2 and R 3 two or three of, together with their intervening atoms, form: [ka] Ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R sis independently R' or -L-R'; t is 0 to 5; each R' is independently -R, -C(O)R, -CO2R, or -SO2R, or: two or more R' together with their intervening atoms form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R is independently hydrogen or C 1-30 C having 1 to 10 heteroatoms independently selected from aliphatic, oxygen, nitrogen, sulfur, phosphorus, and silicon 1-30 The method or composition of any one of embodiments 1 to 95, wherein the heteroaryl group is an optionally substituted group selected from heteroaliphatic, 6-30 membered aryl ring, 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. 97. The method or composition of embodiment 88, wherein in some embodiments, BA is an optionally substituted group selected from a 3-30 membered alicyclic ring, a 6-30 membered aryl ring, a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. 98. A phosphoramidite has the formula II: [ka] (In the formula, BA is C 1-30 Alicyclic, C 6-30 C having 1 to 10 heteroatoms independently selected from aryl, oxygen, nitrogen, sulfur, phosphorus, and silicon 3-30Heterocyclyl, C having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon 5-30 an optionally substituted group selected from heteroaryl rings, natural nucleobase moieties, and modified nucleobase moieties; SU is -LO-, or [ka] wherein SU is attached to the phosphorus atom of formula I via an oxygen atom; L is a covalent bond or C 1-30 C having aliphatic and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon 1-30 heteroaliphatic groups, wherein one or more methylene units are optionally substituted C 1-6 Alkylene, C 1-6 alkenylene, optionally independently replaced by -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 5s is R' or -OR'; R 2s is -F, -CN, -N3, -NO, -NO2, -R'-OR', -SR', -N(R')2, -OL-OR', -OL-SR', or -OLN(R')2, or R 2s is L linking C2 to C1, C2, C3, C4, or C5; -Cy- is an optionally substituted divalent ring selected from 3- to 30-membered carbocyclylene, 6- to 30-membered arylene, 5- to 30-membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and 3- to 30-membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; R 1 , R 2 , and R 3 Each of is independently R′, or R 1 , R 2 and R 3 two or three of, together with their intervening atoms, form: [ka] Ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R s is independently R' or -L-R'; t is 0 to 5; each R' is independently -R, -C(O)R, -CO2R, or -SO2R, or: two or more R' together with their intervening atoms form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R is independently hydrogen or C 1-30 C having 1 to 10 heteroatoms independently selected from aliphatic, oxygen, nitrogen, sulfur, phosphorus, and silicon 1-30 Heteroaliphatic, C 6-30The method or composition according to any one of embodiments 1 to 97, wherein the heteroaryl group is an optionally substituted group selected from a 5-30 membered heteroaryl ring having 1 to 10 heteroatoms independently selected from aryl, oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-30 membered heterocyclic ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon. 99. In some embodiments, BA is C 1-30 Alicyclic, C 6-30 Aryl, C 3-30 Heterocyclyl, C 5-30 99. The method or composition of any one of embodiments 96-98, wherein the optionally substituted group is selected from heteroaryl, and natural nucleobase moieties. 100. BA is an optionally substituted group, which is [ka] 99. The method or composition of any one of embodiments 96 to 98, wherein the compound is formed by removing -H from TIFF2025069269000061.tif25166. 101. BA is an optionally substituted group, which is [ka] 101. The method or composition of embodiment 100, selected from TIFF2025069269000063.tif23166. 102. BA is an optionally substituted guanine residue, and its O 6 100. The method or composition of any one of embodiments 96 to 99, wherein the is unprotected. 103. The method or composition of any one of embodiments 96-99, wherein BA is a modified base. 104. The method or composition of any one of embodiments 96-103, wherein SU is a modified or unmodified sugar moiety. 105. The method or composition of any one of embodiments 96-104, wherein SU is -LO-. 106. The method or composition of any one of embodiments 96-105, wherein SU is -LO- and L is an optionally substituted 5- to 10-membered heterocyclylene having one oxygen atom. 107. The method or composition of any one of embodiments 96-106, wherein SU is -LO- and L is an optionally substituted 5-membered heterocyclylene having one oxygen atom. 108. The method or composition of any one of embodiments 96-106, wherein SU is -LO- and L is an optionally substituted 7-10 membered bicyclic heterocyclylene having two oxygen atoms. 109. SU is [ka] 107. The method or composition of any one of embodiments 96 to 106, wherein 110. R 5s The method or composition of embodiment 109, wherein is -OR'. 111. R 5s 111. The method or composition of any one of embodiments 109-110, wherein is -ODMTr. 112. R 2s 112. The method or composition of any one of embodiments 109-111, wherein 113. R 2s The method or composition of any one of embodiments 109-111, wherein is -F. 114. R 2s The method or composition of any one of embodiments 109-111, wherein is -OR'. 115. R 2s The method or composition of any one of embodiments 109-111, wherein is -OL-OR'. 116. R 2s The method or composition of any one of embodiments 109-111, wherein is -OMe. 117. R 2s The method or composition of any one of embodiments 109-111, wherein is -OCH2CH2OMe. 118. R2s 112. The method or composition of any one of embodiments 109-111, wherein is L that connects C2 to C1, C2, C3, C4, or C5. 119. R 2s 112. The method or composition of any one of embodiments 109-111, wherein is L that links C2 and C4. 120. The method or composition of embodiment 118 or 119, wherein L is (C2)-O-(optionally substituted methylene)-. 121. The method or composition of embodiment 120, wherein L is (C2)-O-(optionally substituted methylene)-, wherein the methylene group is substituted with ethylene. 122. The method or composition of embodiment 121, wherein the carbon atom of the methylene group is S. 123. The method or composition of embodiment 121, wherein the carbon atom of the methylene group is R. 124. R 2 and R 3 together with the intervening nitrogen atom [ka] 124. The method or composition of any one of embodiments 96 to 123, wherein 125. R 1 , and R 2 and R 3 with their intervening nitrogen, phosphorus and oxygen atoms. [ka] 124. The method or composition of any one of embodiments 96 to 123, wherein 126. R 1 , R 2 , and R 3 together with their intervening nitrogen, phosphorus and oxygen atoms, [ka] 124. The method or composition of any one of embodiments 96 to 123, wherein 127. Ring A is [ka] 127. The method or composition of embodiment 125 or 126, comprising a ring system having the backbone structure: 128. Ring A is [ka] 128. The method or composition of embodiment 126 or 127, comprising a ring system having the backbone structure: 129. The method or composition of any one of embodiments 96 to 128, wherein t is 0. 130. The method or composition of any one of embodiments 96-128, wherein t is 1 to 5. 131. One R s is R' or -L-R', and -L- is a divalent optionally substituted C 1-30 The method or composition of any one of embodiments 96-128, wherein the group is a heteroaliphatic group. 132. One R s The method or composition of any one of embodiments 96-128, wherein is —CH2Si(Ph)2CH3. 133. [ka] The method or composition of any one of embodiments 96-132, wherein 134. The method or composition of any one of embodiments 96 to 133, wherein the phosphoramidites, when used in oligonucleotide synthesis, can optionally provide greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% diastereoselectivity at the newly formed P chiral center, along with a yield of greater than 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. 135. The method or composition according to embodiment 134, wherein the oligonucleotide synthesis method is one described in WO / 2011 / 005761, WO / 2013 / 012758, WO / 2014 / 012081, WO / 2015 / 107425, WO / 2010 / 064146, WO / 2014 / 010250, WO / 2011 / 108682, WO / 2012 / 039448, or WO / 2012 / 073857. 136. [ka] teeth [ka] 135. The method or composition of any one of embodiments 96 to 134, wherein 137. [ka] teeth [ka] 135. The method or composition of any one of embodiments 96 to 134, wherein 138. The method or composition of any one of embodiments 1 to 137, wherein the phosphoramidite is one described in WO / 2011 / 005761, WO / 2013 / 012758, WO / 2014 / 012081, WO / 2015 / 107425, WO / 2010 / 064146, WO / 2014 / 010250, WO / 2011 / 108682, WO / 2012 / 039448, or WO / 2012 / 073857. 139. Phosphoramidites are, 137 [ka] The method or composition of any one of embodiments 1 to 137, selected from TIFF2025069269000076.tif183166. 140. The method or composition of any one of embodiments 1-137, wherein the phosphoramidite is selected from Table 1. 141. [ka] teeth The method or composition of any one of embodiments 96 to 132, wherein the nucleic acid sequence is TIFF2025069269000078.tif26166. 142. A method for preparing an oligonucleotide, comprising the method described in any one of embodiments 1 to 141. 143. A method for preparing an oligonucleotide, comprising providing a composition described in any one of embodiments 1 to 142. 144. The method of embodiment 142 or 143, wherein the method for preparing the oligonucleotide is one described in WO / 2011 / 005761, WO / 2013 / 012758, WO / 2014 / 012081, WO / 2015 / 107425, WO / 2010 / 064146, WO / 2014 / 010250, WO / 2011 / 108682, WO / 2012 / 039448, or WO / 2012 / 073857. EXAMPLES

[0190] Non-limiting examples are provided below. Those skilled in the art will recognize that other phosphoramidites can be prepared and purified in similarly greatly improved yields and / or purity as exemplified herein. [ka]

[0191] (S)-2-(methyldiphenylsilyl)-1-((S)-pyrrolidin-2-yl)ethanol 1 (28.3 g, 91.0 mmol) was dried by azeotropic evaporation with anhydrous toluene (110 mL) and left under high vacuum overnight. A solution of this dried 1 (28.3 g, 91.0 mmol) and 4-methylmorpholine (18.59 g, 184 mmol) in anhydrous toluene (150 mL) was added via cannula under argon to an ice-cold solution of trichlorophosphine (12.6 g, 8.0 mL, 92 mmol) in anhydrous toluene (80 mL) and the reaction mixture was stirred at 0° C. for 40 min. The reaction mixture was allowed to warm to room temperature (rt) and stirred for 1 h, after which the white precipitate was filtered using a Schlenk air-free filter tube under vacuum and back-flushed with argon. The solvent was removed under vacuum at 25° C. and the crude semi-solid thus obtained was dried under high vacuum for 12 h and used in the next step of the synthesis without further purification. [ka]

[0192] Nucleoside 5'-DMT-dA(N6-Bz), 3, (40 g, 60.8 mmol) was dried by azeotropic evaporation first with anhydrous pyridine (60 mL), then with anhydrous toluene (110 mL) and dried under high vacuum for 24 h. The dried compound was dissolved in THF (140 mL), followed by the addition of triethylamine (30.8 g, 42.4 mL, 304 mmol), and then cooled to -78 °C using an IPA / dry ice bath. A THF solution (80 mL) of crude compound 2 (obtained as above) was added via cannula over 15 min, and then the mixture was allowed to warm gradually to room temperature. After 1 h at rt, TLC showed complete conversion of SM to product. The mixture was cooled at 0 °C, then CHCl3 (800 mL) was added, followed by saturated NaHCO3 (400 mL). The organic layer was separated and washed with saturated NaHCO3 (2 x 400 mL). The combined aqueous layers were extracted with CHCl3 (200 mL) and washed with saturated NaHCO3 (200 mL). The combined CHCl3 extracts were dried over anhydrous Na2SO4, filtered, and the volatiles were removed on a rotary evaporator at 25 °C to give a pale yellow solid. 71 g of crude compound 4.

[0193] Purification method

[0194] The crude material was purified on a Teledyne Combiflash instrument using either pretreated or unpretreated silica gel columns, all characteristics of which were identical in both cases except for the separation method.

[0195] Purification using unpretreated silica columns

[0196] 3.0 g of crude compound 4 was dissolved in a 2:1 (vol:vol) mixture of methylene chloride:hexane containing 5% Et3N and then loaded onto a 24 g silica column equilibrated with 5 column volumes of 20% hexane / EtOAc containing 5% Et3N. After the sample was loaded onto the column, the purification process was carried out using a gradient of 20-80% EtOAc / hexane containing 5% Et3N as shown in the chromatogram (Figure 1). Fractions that were homogenous by TLC, 14-22 shown in Figure 1, were pooled together, the volatiles were removed on a rotary evaporator, and the residual solvent was removed under high vacuum for 18 hours to give a colorless white solid (yield of pure compound 4 = 1.3 g, 56%). In some embodiments, early fractions showed non-polar impurities while later fractions showed decomposition. Without intending to be bound by a particular theory, in some cases, the decomposition is due to conversion at the phosphoramidite group.

[0197] Purification by using a pretreated silica column

[0198] The exact procedure described for the present unpretreated method was followed, except that a 24 g silica gel column was first pretreated by eluting with 5 column volumes of methanol before equilibrating with 5 column volumes of 20% EtOAc / hexanes containing 5% Et3N. In this case, a colorless white solid resulting from pooling and evaporation of fractions 15-23 was also obtained (yield of pure compound 4=2.0 g, 83%). In some embodiments, the pretreated purification media provides better separation, better peak shape and / or less tailing, as shown by the examples.

[0199] Products were recorded in CDCl3 31 This was confirmed by P-NMR, and was the same for both methods. 31 P-NMR: δ 151.14.

[0200] In accordance with the techniques described in this disclosure, numerous example phosphoramidites (e.g., those listed in Table 1) having a variety of substituents have been successfully prepared and purified, providing high yields and / or purity. As described in this disclosure, the techniques provided can, among other things, improve elution efficiency and / or reduce or eliminate decomposition. As shown by the examples, O 6 Phosphoramidites containing unprotected guanine residues were successfully purified, providing high yields and purity.

[0201] As described herein, alternative hygroscopic solvents can be utilized in accordance with the present disclosure to achieve significantly improved yields and / or purity. In some embodiments, acetonitrile was utilized in place of methanol as in the examples described herein, resulting in significantly improved yields and / or purity. In some embodiments, ethyl acetate was utilized in place of methanol as in the examples described herein, resulting in significantly improved yields and / or purity.

[0202] As shown herein, the techniques provided provide surprisingly higher yields and / or purity compared to conventional techniques. In particular, the techniques provided are unexpectedly effective in suppressing decomposition of phosphoramidites during purification.

[0203] Although several embodiments of the present disclosure have been described and illustrated herein, those skilled in the art will readily envision various alternative means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein. And each such variation and / or modification is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or uses for which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Thus, the foregoing embodiments are presented by way of example only, and it is to be understood that, within the scope of the appended claims and their equivalents, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure relates to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

Claims

1. a) a pretreated purification medium; and b) Phosphoramidites A composition comprising: The phosphoramidite has the following structure as shown in Formula I: 【Chemistry 1】 BA is R or an optionally substituted group selected from a 3-30 membered alicyclic ring, a 6-30 membered aryl ring, a 3-30 membered heterocycle having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety; SU is a sugar moiety, a modified sugar moiety, -L-O-, or 【Chemistry 2】 wherein SU is attached to the phosphorus atom in formula I through an oxygen atom; L is a covalent bond or C １－３０ C having an aliphatic group and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. １－３０ heteroaliphatic groups, wherein one or more methylene units are optionally substituted C １－６ Alkylene, C １－６ Alkenylene, 【Chemistry 3】 , -C(R') ２ -, -Cy-, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, - N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O) ２ -, -S(O) ２ N(R')-, -N(R')S(O) ２ may be independently replaced by -, -SC(O)-, -C(O)S-, -OC(O)-, or -,C(O)O-; R ５ｓ is R' or -OR', R ２ｓ -F, -CN, -N ３ , -NO, -NO ２ , -R'-OR', -SR', -N(R') ２ , -L-R', -OL-OR', -OL-SR', or -OL-N(R') ２ Or, R ２ｓ is L connecting C2 to C1, C2, C3, C4 or C5, -Cy- is an optionally substituted divalent ring selected from 3- to 30-membered carbocyclylene, 6- to 30-membered arylene, 5- to 30-membered heteroarylene having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, and sulfur, and 3- to 30-membered heterocyclylene having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; R １ , R ２ , and R ３ together with their intervening atoms form 【Chemistry 4】 Ring A is an optionally substituted polyvalent monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R ｓ are independently R' or -L-R'; t is 0 to 5; Each R' is independently -R, -C(O)R, -CO ２ R or -SO ２ R, or two or more R' together with their intervening atoms form an optionally substituted monocyclic, bicyclic or polycyclic saturated, partially unsaturated or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; Each R is independently hydrogen or C １－３０ C having 1 to 10 heteroatoms independently selected from aliphatic, oxygen, nitrogen, sulfur, phosphorus, and silicon １－３０ optionally substituted groups selected from heteroaliphatic, 6-30 membered aryl ring, 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; The composition having the structure:

2. The composition of claim 1 , wherein the purification medium is silica gel.

3. 3. The composition according to claim 1 or 2, The composition, wherein BA is selected from a natural nucleobase moiety or a modified nucleobase moiety.

4. The composition according to any one of claims 1 to 3, S.U. 【Chemistry 5】 The composition.

5. The composition according to any one of claims 1 to 4, wherein R ５Ｓ The composition wherein:

6. The composition according to any one of claims 1 to 5, wherein R ２Ｓ is -H, -F, -OMe, and -OCH ２ CH ２ The composition selected from: OMe.

7. The composition according to any one of claims 1 to 6, 【Chemistry 6】 but 【Chemistry 7】 and one or more R Ｓ is not hydrogen.

8. The composition according to any one of claims 1 to 6, 【Chemistry 8】 but 【Chemistry 9】 Or, 【Chemistry 10】 The composition.

9. The composition according to any one of claims 1 to 6, 【Chemistry 11】 but 【Chemistry 12】 Wherein, one R Ｓ is hydrogen, and the other R Ｓ is -L-R', and R' is -SO ２ R and L is a divalent optionally substituted C １－３０ The composition is an aliphatic group.

10. The composition of any one of claims 1 to 9, wherein the purification medium is pretreated with a hygroscopic solvent system.

11. 11. The composition of claim 10, wherein the hygroscopic solvent system comprises a hygroscopic solvent and a modifier.

12. 12. The composition of claim 10 or 11, wherein the hygroscopic solvent system comprises methanol.

13. 12. The composition of claim 10 or 11, wherein the hygroscopic solvent system comprises acetonitrile.

14. 14. The composition of claim 13, wherein the hygroscopic solvent system comprises at least 90% (v / v) acetonitrile.

15. 14. The composition of claim 13, wherein the hygroscopic solvent system comprises 95% (v / v) acetonitrile and 5% (v / v) triethylamine.

16. 16. The composition of claim 1, wherein the phosphoramidite is selected from: 【Chemistry 13】 , where X is 【Chemistry 14】 and, R′ is H and R is H; R′ is H and R is OMe; R' is H and R is OCH ２ CH ２ Being OMe, R′ is H and R is F; R′ is Me and R is H; R′ is Me and R is OMe; R' is Me and R is OCH ２ CH ２ is OMe, or R′ is Me and R is F; 【Chemistry 15】 , where X is 【Chemistry 16】 And, R′ is H and R is H; R′ is H and R is OMe; R' is H and R is OCH ２ CH ２ Being OMe, R′ is H and R is F; R′ is Me and R is H; R′ is Me and R is OMe; R' is Me and R is OCH ２ CH ２ is OMe, or R′ is Me and R is F; 【Chemistry 17】 , where X is 【Chemistry 18】 and, R′ is H and R is H; R′ is H and R is OMe; R' is H and R is OCH ２ CH ２ Being OMe, R′ is H and R is F; R′ is Me and R is H; R′ is Me and R is OMe; R' is Me and R is OCH ２ CH ２ is OMe, or R′ is Me and R is F; 【Chemistry 19】 , where X is 【Chemistry 20】 And, R′ is H and R is H; R′ is H and R is OMe; R' is H and R is OCH ２ CH ２ Being OMe, R′ is H and R is F; R′ is Me and R is H; R′ is Me and R is OMe; R' is Me and R is OCH ２ CH ２ is OMe, or R′ is Me and R is F; 【Chemistry 21】 , where X is 【Chemical 22】 and, R′ is Ac and R is H; R′ is Ac and R is OMe; R' is Ac and R is OCH ２ CH ２ Being OMe, R′ is Ac and R is F; R′ is iBu and R is H; R′ is iBu and R is OMe; R' is iBu and R is OCH ２ CH ２ is OMe, or R′ is iBu and R is F; 【Chemistry 23】 , where X is 【Chemistry 24】 And R′ is Ac and R is H; R′ is Ac and R is OMe; R' is Ac and R is OCH ２ CH ２ Being OMe, R′ is Ac and R is F; R′ is iBu and R is H; R′ is iBu and R is OMe; R' is iBu and R is OCH ２ CH ２ is OMe, or R′ is iBu and R is F; 【Chemistry 25】 , where X is 【Chemistry 26】 and, R′ is Ac and R is H; R′ is Ac and R is OMe; R' is Ac and R is OCH ２ CH ２ Being OMe, R′ is Ac and R is F; R′ is iBu and R is H; R′ is iBu and R is OMe; R' is iBu and R is OCH ２ CH ２ is OMe, or R′ is iBu and R is F; 【Chemical 27】 , where X is 【Chemistry 28】 and, R′ is Ac and R is H; R′ is Ac and R is OMe; R' is Ac and R is OCH ２ CH ２ Being OMe, R′ is Ac and R is F; R′ is iBu and R is H; R′ is iBu and R is OMe; R' is iBu and R is OCH ２ CH ２ is OMe, or R′ is iBu and R is F; 【Chemical 29】 , where X is 【Chemistry 30】 and R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 31】 , where X is 【Chemistry 32】 and R is H; R is OMe, R is OCH ２ CH ２ is OMe, or R is F, 【Chemical 33】 , and X is 【Chemical 34】 and R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 35】 , where X is 【Chemical 36】 and R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemical 37】 , where X is 【Chemical 38】 and R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemical Formula 39】 , where X is 【Chemistry 40】 and R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 41】 , where X is 【Chemistry 42】 and R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 43】 , where X is 【Chemistry 44】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 45】 , where X is 【Chemistry 46】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 47】 , where X is 【Chemistry 48】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 49】 , where X is 【Chemistry 50】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 51】 , where X is 【Chemistry 52】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, Or, the phosphoramidite is selected from: 【Chemistry 53】 【Chemical 54】 【Chemistry 55】 【Chemistry 56】 【Chemistry 57】 【Chemistry 58】 【Chemistry 59】 【Chemistry 60】 【Chemistry 61】 and, 【Chemistry 62】 Or, the phosphoramidite is selected from: 【Chemistry 63】 , where X is 【Chemistry 64】 and, R′ is H and R is H; R′ is H and R is OMe; R' is H and R is OCH ２ CH ２ Being OMe, R′ is H and R is F; R′ is Me and R is H; R′ is Me and R is OMe; R' is Me and R is OCH ２ CH ２ is OMe, or R′ is Me and R is F; 【Chemistry 65】 , where X is 【Chemistry 66】 and, R′ is H and R is H; R′ is H and R is OMe; R' is H and R is OCH ２ CH ２ Being OMe, R′ is H and R is F; R′ is Me and R is H; R′ is Me and R is OMe; R' is Me and R is OCH ２ CH ２ is OMe, or R′ is Me and R is F; 【Chemistry 67】 , where X is 【Chemistry 68】 and, R′ is Ac and R is H; R′ is Ac and R is OMe; R' is Ac and R is OCH ２ CH ２ Being OMe, R′ is Ac and R is F; R′ is iBu and R is H; R′ is iBu and R is OMe; R' is iBu and R is OCH ２ CH ２ is OMe, or R′ is iBu and R is F; 【Chemistry 69】 , where X is 【Chemistry 70】 and, R′ is Ac and R is H; R′ is Ac and R is OMe; R' is Ac and R is OCH ２ CH ２ Being OMe, R′ is Ac and R is F; R′ is iBu and R is H; R′ is iBu and R is OMe; R' is iBu and R is OCH ２ CH ２ is OMe, or R′ is iBu and R is F; 【Chemistry 71】 , where X is 【Chemical Formula 72】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemical 73】 , where X is 【Chemical 74】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, 【Chemistry 75】 , where X is 【Chemical 76】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, and 【Chemical 77】 , where X is 【Chemical 78】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, Or, the phosphoramidite is selected from: 【Chemical 79】 , where X is 【Chemistry 80】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or R is F, and 【Chemistry 81】 , where X is 【Chemistry 82】 and, R is H; R is OMe; R is OCH ２ CH ２ is OMe, or The composition wherein R is F.

17. A purification medium, wherein the purification medium has been treated with a hygroscopic solvent system.

18. 20. The purification medium of claim 17, wherein the purification medium is silica gel.

19. 19. The purification medium of claim 17 or 18, wherein the hygroscopic solvent system comprises 50% (v / v) or more of methanol.

20. 19. The purification medium of claim 17 or 18, wherein the hygroscopic solvent system comprises 50% (v / v) or more acetonitrile.

21. The purification medium of any one of claims 17 to 20, wherein the hygroscopic solvent system comprises a base.

22. The purification medium of any one of claims 17 to 21, wherein the hygroscopic solvent system comprises triethylamine.

23. 1. A method for purifying phosphoramidites, comprising the steps of: a) contacting the pretreated purification medium with a phosphoramidite; b) purifying the phosphoramidite using said purification medium.

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