CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to United States Provisional Application No. 62/307,542, filed March 13, 2016, the entirety of each of which is incorporated herein by reference.
BACKGROUND
[0002] Oligonucleotides are useful for many purposes, including treating various diseases. There is a need for efficient synthetic methods for oligonucleotides.

SUMMARY

[0003] The present disclosure encompasses the recognition of the source of a problem with many technologies for preparation of phosphoramidites. As is known in the art, phosphoramidites are often utilized as monomers for oligonucleotide synthesis. Many technologies for preparing phosphoramidites can suffer from low yields and/or purity. Among other things, the present disclosure provides technologies (e.g., compounds, compositions, methods, etc.) that provide significantly improved yields and/or purity for preparation of phosphoramidites, and/or other intermediates and products, that are useful for e.g., improving efficiency and/or lowering cost of oligonucleotide preparation.

[0004] The present disclosure encompasses certain surprising findings, including that certain processes for pre-treating a purification medium can surprisingly improve utility and/or effectiveness of that medium, particularly for use in preparation of phosphoramidites. In some embodiments, such processes may be or comprise a deactivation process. In some embodiments, such processes may be or comprise removal or exclusion of water from the medium. In some embodiments, such processes may be or comprise treatment with a hygroscopic solvent system comprising one or more hygroscopic solvent. In some embodiments, such processes may be or comprise treatment with a hygroscopic solvent. In some embodiments, such processes may be or comprise treatment with an alcohol. In some embodiments, such processes may be or comprise treatment with methanol. In some embodiments, such processes may be or comprise treatment with ethanol. In some embodiments, such processes may be or comprise treatment with isopropanol. In some embodiments, such processes may be or comprise treatment with dimethyl carbonate. In some embodiments, such processes may be or comprise treatment with acetonitrile. In some embodiments, such processes may be or comprise treatment with ethyl acetate. In some embodiments, such processes may be or comprise treatment with acetone. In some embodiments, the present disclosure demonstrates that certain such processes, including certain processes that were previously understood or expected to degrade or otherwise damage one or more purification medium, may in fact improve utility and/or effectiveness of such a medium, particularly in the preparation of phosphoramidites. In some embodiments, teachings of the present disclosure relate to a medium that is or comprises a silica medium. In some embodiments, teachings of the present disclosure relate to a medium that is or comprises a silica gel.

[0005] In some embodiments, the present disclosure appreciates that one source of a problem with prior technologies that utilize chromatography purification (e.g., purification using silica gel, alumina, etc.) in preparation of phosphoramidites may be that a significant amount of phosphoramidite may be lost during chromatography. Without wishing to be bound by any particular theory, the present disclosure proposes that, in some instances, phosphoramidite may be lost through irreversible adsorption to (e.g., cannot be readily eluted during chromatographic purification), and/or decomposition when in contact with, a purification medium, such as silica gel, alumina, etc. In some embodiments, the present disclosure encompasses the recognition that the low yield and/or purity of phosphoramidites can greatly increase the cost of oligonucleotides and/or pharmaceutical compositions thereof, particularly at large scale such as those required for preparing oligonucleotide-based medicament for therapy. The present disclosure provides a particular insight that, in some embodiments, treatment of a purification medium as described herein may improve its performance (and therefore its utility and/or efficacy) in purification of phosphoramidites.

[0006] In some embodiments, the present disclosure provides new technologies, e.g., compounds, compositions, methods, etc., for preparing a phosphoramidite and/or an oligonucleotide. In some embodiments, the present disclosure provides new technologies for purifying phosphoramidites. In some embodiments, provided technologies greatly improve efficiency of phosphoramidite synthesis, and/or significantly decrease the cost of

phosphoramidite, and oligonucleotides, compositions and medicaments prepared therefrom.

[0007] In some embodiments, the present disclosure provides a method, comprising steps of: a) pre-treating a purification medium;

b) contacting the purification medium with a phosphoramidite; and

b) optionally using the purification medium to purify a phosphoramidite.

[0008] In some embodiments, the present disclosure provides a method, comprising steps of: a) pre-treating a purification medium; and

b) using the purification medium to purify a phosphoramidite.

[0009] In some embodiments, the present disclosure provides a method, comprising steps of: a) pre-treating a purification medium comprising silica gel; and

b) contacting the purification medium with a phosphoramidite.

[0010] In some embodiments, the present disclosure provides a method, comprising steps of: a) pre-treating a purification medium comprising silica gel; and

b) using the purification medium to purify a phosphoramidite.

[0011] In some embodiments, the present disclosure provides a method, comprising steps of: a) pre-treating silica gel; and

b) contacting the silica gel with a phosphoramidite.

[0012] In some embodiments, the present disclosure provides a method, comprising steps of: a) pre-treating silica gel; and

b) using the silica gel to purify a phosphoramidite.

[0013] In some embodiments, the pre-treatment removes water from the purification medium. In some embodiments, the pre-treatment deactivates the purification medium. In some embodiments, the pre-treatment comprises contacting the purification medium with a first solvent system. In some embodiments, a first solvent system comprises a hygroscopic solvent. In some embodiments, a first solvent system is a hygroscopic solvent. In some embodiments, a hygroscopic solvent is an alcohol. In some embodiments, a hygroscopic solvent is methanol. In some embodiments, a hygroscopic solvent is ethanol. In some embodiments, a hygroscopic solvent is isopropanol. In some embodiments, a hygroscopic solvent is acetone. In some embodiments, a hygroscopic solvent is dimethyl carbonate. In some embodiments, a hygroscopic solvent is acetonitrile. In some embodiments, a hygroscopic solvent is ethyl acetate. In some embodiments, a first solvent system deactivates a reaction site of a purification medium.

In some embodiments, the pre-treatment comprises heating the purification medium.

[0014] In some embodiments, the present disclosure provides a method, comprising 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.

[0015] In some embodiments, the present disclosure provides a method, comprising steps of: a) removing water from a purification medium;

b) contacting the purification medium with a phosphoramidite; and

c) using the purification medium to purify a phosphoramidite.

[0016] In some embodiments, the present disclosure provides a method, comprising steps of: a) removing water from a purification medium; and

b) using the purification medium to purify the phosphoramidite.

[0017] In some embodiments, the present disclosure provides a method for improving recovery rate of a phosphoramidite, , comprising steps of:

a) removing water from a purification medium;

b) adding the phosphoramidite to the purification medium; and

c) eluting the phosphoramidite from the purification medium with a solvent system; wherein the recovery rate is higher than a reference recovery rate when step a) is absent.

[0018] In some embodiments, the present disclosure provides a method for decreasing decomposition of a phosphoramidite when the phosphoramidite contacts a purification medium, comprising:

a) removing water from the purification medium.

[0019] In some embodiments, the present disclosure provides a method comprising steps of:

a) deactivating a purification medium; and

b) using the purification medium to purify a phosphoramidite;

so that the deactivated purification medium provides a higher recovery rate than the purification medium without deactivation.

[0020] In some embodiments, the present disclosure provides a method comprising 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 the phosphoramidite.

[0021] In some embodiments, the present disclosure provides a method comprising steps of: a) removing water from a purification medium;

b) contacting the purification medium with a phosphoramidite; and

c) using the purification medium to purify the phosphoramidite.

[0022] In some embodiments, the present disclosure provides a method comprising steps of: a) removing water from a purification medium; and

b) using the purification medium to purify a phosphoramidite.

[0023] In some embodiments, the present disclosure provides a method comprising steps of: a) mechanically 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.

[0024] In some embodiments, the present disclosure provides a method comprising steps of: a) mechanically removing water from a purification medium;

b) contacting the purification medium with a phosphoramidite; and

c) using the purification medium to purify a phosphoramidite.

[0025] In some embodiments, the present disclosure provides a method comprising steps of: a) mechanically removing water from a purification medium; and

b) using the purification medium to purify a phosphoramidite.

[0026] In some embodiments, the present disclosure provides a method comprising steps of: a) treating a purification medium with a hygroscopic solvent; and

b) using the purification medium to purify a phosphoramidite.

[0027] In some embodiments, the present disclosure provides a method comprising steps of: a) treating a purification medium with an alcohol; and

b) using the purification medium to purify a phosphoramidite.

[0028] In some embodiments, the present disclosure provides a method comprising steps of: a) treating a purification medium with one or more alcohols;

b) re-equilibrating the purification medium with a solvent system less polar than the one or more alcohols;

c) using the silica gel to purify a phosphoramidite, wherein the elution solvent system is less polar than the one or more alcohols.

[0029] In some embodiments, the present disclosure provides a method comprising steps of: a) treating a purification medium with methanol;

b) re-equilibrating the purification medium with a solvent system comprising ethyl acetate and hexanes;

c) using the purification medium to purify a phosphoramidite, wherein the elution solvent system comprises ethyl acetate, hexanes, and optionally a modifier.

[0030] In some embodiments, the present disclosure provides a method comprising steps of: a) treating a purification medium with methanol;

b) re-equilibrating the purification medium with a solvent system comprising ethyl acetate and hexanes;

c) using the purification medium to purify a phosphoramidite, wherein the elution solvent system is a mixture of ethyl acetate and hexanes.

[0031] In some embodiments, the present disclosure provides a method comprising steps of: a) treating a purification medium with methanol;

b) re-equilibrating the purification medium with a solvent system comprising ethyl acetate and hexanes;

c) using the purification medium to purify a phosphoramidite, wherein the elution solvent system is a mixture of ethyl acetate, hexanes and triethylamine.

[0032] In some embodiments, the present disclosure provides a method, comprising:

contacting a compound with a medium;

wherein the medium is de-activated.

[0033] In some embodiments, the present disclosure provides a method, comprising:

purifying a compound with a medium;

wherein the medium is de-activated.

[0034] In some embodiments, a compound is a phosphoramidite. In some embodiments, a compound is a nucleoside. In some embodiments, a compound is a nucleotide. In some embodiments, a compound is an oligonucleotide. In some embodiments, a medium is a purification medium. In some embodiments, a medium comprises silica gel. In some embodiments, a medium is silica gel. In some embodiments, a medium is de-activated mechanically as described in the present disclosure. In some embodiments, a medium is deactivated using a solvent system or solvent as described in the present disclosure. In some embodiments, a medium is de-activated by contact with a solvent system is or comprises a hygroscopic solvent as described in the present disclosure.

[0035] In some embodiments, a provided method is a chromatography method comprising using a medium as described in the present disclosure. In some embodiments, a medium is or comprises silica gel. In some embodiments, a medium is de-activated through, e.g., mechanical means, contact with a hygroscopic solvent (system), etc.

[0036] In some embodiments, the present disclosure provides compositions from preparing a phosphoramidite. In some embodiments, the present disclosure provides a composition comprising:

a) a de-activated purification medium;

b) a phosphoramidite.

In some embodiments, a phosphoramidite is in contact with a de-activated purification medium. In some embodiments, a phosphoramidite is adsorbed on a de-activated purification medium.

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

[0038] In some embodiments, a phosphoramidite is a nucleoside phosphoramidite. In some embodiments, a phosphoramidite is one 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/2011/0294124, WO/2014/010250, US/2015/0197540, WO/2011/108682, US/2013/0184450, WO/2012/039448, US/2013/0178612, WO/2012/073857, or US/2013/0253178, the phosphoramidites of each of which are hereby incorporated by reference.

[0039] In some embodiments, a phosphoramidite is stereochemically enriched. In some embodiments, a phosphoramidite is stereochemically pure. In some embodiments, a phosphoramidite is used for preparing chirally controlled oligonucleotide compositions, such as those described in 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/2011/0294124, WO/2014/010250, US/2015/0197540, WO/2011/108682, US/2013/0184450, WO/2012/039448, US/2013/0178612, WO/2012/073857, or US/2013/0253178, the chirally controlled oligonucleotide compositions of each of which are hereby incorporated by reference.

[0040] In some embodiments, a phosphoramidite has the structure of formula I:

I

wherein each variable is independently as defined and described infra.

BRIEF DESCRIPTION OF THE DRAWING

[0041] Figure 1. Chromatogram: Non-pre-treated silica.

[0042] Figure 2. Chromatogram: Pre-treated silica.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

1. Definitions

[0043] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0044] Aliphatic: As used herein, "aliphatic" means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is completely saturated or that contains one or more units of unsaturation, or combinations thereof. Unless otherwise specified, aliphatic groups contain 1-100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups

contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof.

[0045] Alkyl: As used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C20 for straight chain, C2-C20 for branched chain), and alternatively, about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyls).

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

[0047] Alkynyl: As used herein, the term "alkynyl" 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 as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term "aryl," as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. In some embodiments, an aryl group has a radical or point of attachment on an aromatic ring.

[0049] Cycloaliphatic: The term "cycloaliphatic," as used herein, refers to saturated or partially unsaturated aliphatic monocyclic, bicyclic, or poly cyclic ring systems having, e.g., from 3 to 30, members, wherein the aliphatic ring system is optionally substituted. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons. The terms "cycloaliphatic" may also include aliphatic rings that are fused to one or more aromatic or nonaromatic 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, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon, or a C8-Ci0 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule, or a C9-C16 tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.

[0050] Halogen: The term "halogen" means F, CI, Br, or I.

[0051] Heteroaliphatic: The term "heteroaliphatic" is given its ordinary meaning in the art and refers to aliphatic groups as described herein in which one or more carbon atoms is replaced with a heteroatom (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).

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

[0053] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl," or "heteroaralkoxy," refer to monocyclic, bicyclic or polycyclic ring systems having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a

heteroaryl group is a group 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, a heteroaryl group has 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroar omatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroar omatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. A heteroaryl group 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 by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0054] Heteroatom: The term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl); etc.).

[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 membered) 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, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical," are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic or poly cyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

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

[0057] Protecting Group: The phrase "protecting group," as used herein, refers to temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. A "Si protecting group" is a protecting group comprising a Si atom, such as Si-trialkyl (e.g., trimethyl silyl, tributylsilyl, t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g., t-butyldiphenylsilyl), or Si-aryl-dialkyl (e.g., Si-phenyldialkyl). Generally, a Si protecting group is attached to an oxygen atom. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991). Such protecting groups (and associated protected moieties) are described in detail below.

[0058] Protected hydroxyl groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitably protected hydroxyl groups further include, but are not limited to, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of suitable esters include formates, acetates, proprionates, pentanoates, crotonates, and benzoates. Specific examples of suitable esters include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate, p-benznylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitable carbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate. Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropyl silyl ether, and other trialkylsilyl ethers. Examples of suitable alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether, or derivatives thereof. Alkoxyalkyl ethers include acetals such as methoxy methyl, 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, aralkylamines, carbamates, allyl amines, amides, and the like. Examples of suitable mono-protected amino moieties include t-butyloxycarbonylamino (- HBOC), ethyloxycarbonylamino, methyloxycarbonylamino, trichloroethyloxycarbonylamino, allyloxycarbonylamino (-NHAlloc), benzyloxocarbonylamino (- HCBZ), allylamino, benzylamino (- HBn), fluorenylmethylcarbonyl (- HFmoc), formamido, acetamido, chloroacetamido, dichloroacetamido, tri chloroacetamido, phenylacetamido, trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like. Suitable di-protected amines include amines that are substituted with two substituents independently selected from those described above as mono-protected amines, and further include cyclic imides, such as phthalimide, maleimide, succinimide, and the

like. Suitable di-protected amines also include pyrroles and the like, 2,2,5,5-tetramethyl-[l,2,5]azadisilolidine and the like, and azide.

[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-dioxanes, 1,3-dioxolanes, 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, but are not limited to, optionally substituted Ci_6 aliphatic esters, optionally substituted aryl esters, silyl esters, activated esters, amides, hydrazides, and the like. Examples of such ester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, and phenyl ester, wherein each group is optionally substituted. Additional suitable protected carboxylic acids include oxazolines and ortho esters.

[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, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, and trichloroethoxycarbonyl thioester, to name but a few.

[0063] Substitution: As described herein, compounds of the disclosure may contain 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 designated 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 substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0064] Suitable monovalent substituents include halogen; -(CH2)o-4R°; -(CH2)0^OR°; -0(CH2)o-4R0, -0-(CH2)o-4C(0)OR°; -(CH2)0-4CH(OR°)2; -(CH2)0^Ph, which may be substituted with R°; -(CH2)0-4O(CH2)0-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o-40(CH2)0_i-pyridyl which may be substituted with R°; -N02; -CN; -N3; -(CH2)0_4N(R°)2; -(CH2)0_4N(Ro)C(O)R°; -N(R°)C(S)R°; -(CH2)0_ 4N(R°)C(0) R°2; -N(R°)C(S) R°2; -(CH2)0_4N(R°)C(O)OR°; -N(R°)N(R°)C(0)R°; -N(R°)N(R°)C(0) R°2; -N(R°)N(R°)C(0)OR°; -(CH2)0_4C(O)Ro; -C(S)R°; -(CH2)0_ 4C(0)OR°; -(CH2)o_4C(0)SR°; -(CH2)0^C(O)OSiR°3; -(CH2)0^OC(O)R°; -OC(O)(CH2)0_4SR, -SC(S)SR°; -(CH2)o^SC(0)R°; -(CH2)0_4C(O) Ro2; -C(S) R°2; -C(S)SR°; -SC(S)SR°, -(CH2)o_4OC(0) R°2; -C(0)N(OR°)R°; -C(0)C(0)R°; -C(0)CH2C(0)R°; -C(NOR°)R°; -(CH2)o_4SSR°; -(CH2)0_4S(O)2Ro; -(CH2)0^S(O)2OR°; -(CH2)0^OS(O)2R°; -S(0)2 R°2; -(CH2)o^S(0)R°; -N(R°)S(0)2 R°2; -N(R°)S(0)2R°; -N(OR°)R°; -C( H) R°2; -P(0)2R°; -P(0)R°2; -OP(0)R°2; -OP(0)(OR°)2; -SiR°3; -OSiR°3; -(Ci_4 straight or branched alkylene)0-N(R°)2; or -(Ci_4 straight or branched alkylene)C(0)0-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci_20 aliphatic, Ci_20 heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, -CH2-(C6.14 aryl), -O(CH2)0_1(C6-14 aryl), -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 definition above, two independent occurrences of R°, taken together with their intervening atom(s), 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] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)0_2R*, -(haloR*), -(CH2)0_2OH, -(CH2)0_2OR*, -(CH2)0_2CH(OR*)2; -O(haloR'), -CN, -N3, -(CH2)0_2C(O)R*, -(CH2)0_2C(O)OH, -(CH2)0_2C(O)OR*, -(CH2)0_2SR*, -(CH2)o_2SH, -(CH2)o_2NH2, -(CH2)0_2NHR*, -(CH2)0_2NR*2, -N02, -SiR\ -

OSiR*3, -C(0)SR* -(Ci-4 straight or branched alkylene)C(0)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from
aliphatic, -CH2Ph, -O(CH2)0_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0066] Suitable divalent substituents include the following: =0, =S, =NNR*2, =N HC(0)R*, =N HC(0)OR*, =N HS(0)2R*, = R*, =NOR*, -0(C(R*2))2_30- or -S(C(R*2))2_3S- wherein each independent occurrence of R* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group include: -0(CR*2)2_30- wherein each independent occurrence of R* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0067] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, - H2, - HR*, - R*2, or -N02, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4 aliphatic, -CH2Ph, -O(CH2)0_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0068] In some embodiments, suitable substituents on a substitutable nitrogen include -R†, - R†2, -C(0)R†, -C(0)OR†, -C(0)C(0)R†, -C(0)CH2C(0)R†, -S(0)2R†, -S(0)2 R†2, -C(S) R†2, -C( H) R†2, or -N(R†)S(0)2R†; wherein each R† is independently hydrogen, Ci-e aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0069] Suitable substituents on the aliphatic group of RT are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR'), -CN, -C(0)OH, -C(0)OR*, - H2, - HR*, - R*2, or

-N02, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4 aliphatic, -CH2Ph, -O(CH2)0_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

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

2. Detailed Description of Certain Embodiments

[0071] Phosphoramidites have a variety of applications, for example, nucleoside phosphoramidites are widely used as monomers for oligonucleotide synthesis. Many technologies for preparing phosphoramidites suffer from low yield and/or purity. Among other things, such low yield and/or purity can lead to high cost and/or low quality of phosphoramidites, and any products downstream therefrom, for example, oligonucleotides prepared therefrom.

[0072] Among other things, the present disclosure encompasses the recognition that in some instances, a low yield and/or purity can be due to the purification process when a phosphoramidite is separated from other substances through, e.g., chromatography. In some embodiments, the present disclosure appreciates that one source of a problem with prior technologies that utilize chromatography purification {e.g., purification using silica gel, alumina, etc.) in preparation of phosphoramidites may be that a significant amount of phosphoramidite may be lost during chromatography. Without wishing to be bound by any particular theory, the present disclosure proposes that, in some instances, phosphoramidites may be lost through irreversible absorption/adsorption to a purification medium and/or decomposition when in contact with a purification medium during chromatography. In some embodiments, a phosphoramidite may be lost due to irreversible absorption/adsorption to a purification medium in that it cannot be readily eluted. In some embodiments, a phosphoramidite may decompose during chromatography purification. For example, as described in the exemplification section, in some embodiments, a phosphoramidite may decompose during purification using silica gel as a purification medium.

[0073] The low yield and/or purity of phosphoramidite, among other things, can significantly

increase the cost of compositions made from them, particularly at large, process scale. In some embodiments, when they are used for oligonucleotide synthesis, the high cost of phosphoramidites can make oligonucleotides, compositions thereof and medicaments therefrom unusually expensive.

[0074] The present disclosure encompasses certain surprising findings, including that certain processes for pre-treating a purification medium can surprisingly improve utility and/or effectiveness of that medium, particularly for use in preparation of phosphoramidites. In some embodiments, such processes may be or comprise a deactivation process. In some embodiments, the pre-treatment is a deactivation process. In some embodiments, the pre-treatment comprises deactivation of a purification medium. In some embodiments, when a purification medium is deactivated, there is less irreversible absorption/adsorption and/or decomposition of phosphoramidites when such a purification medium is used for purification of phosphoramidites compared to the same, non-deactivated 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, a pre-treated medium provides higher recovery yield and /or purity of phosphoramidites than medium not pre-treated. In some embodiments, a pre-treated medium provides higher recovery yield. In some embodiments, a pre-treated medium provides higher purity. In some embodiments, a pre-treated medium provides higher recovery yield and higher purity.

[0075] In some embodiments, the pre-treatment may be or comprise removal or exclusion of water from a purification medium. In some embodiments, the pre-treatment comprises removal or exclusion of water from a purification medium. In some embodiments, the pre-treatment removes water from a purification medium. In some embodiments, the pre-treatment excludes water from a purification medium.

[0076] In some embodiments, the pre-treatment is performed mechanically. In some embodiments, a purification medium is pre-treated with heat. In some embodiments, a purification medium is pre-treated with vacuum. In some embodiments, a purification medium is pre-treated with heat and vacuum. In some embodiments, such mechanical pre-treatment processes deactivate the purification medium. As a person of ordinary skill in the art would appreciate, heat and/or vacuum can, among other things, remove water or other solvents from a purification medium in accordance with the present disclosure. In some embodiments, 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, a vacuum is no more than 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, a treatment is for a prolonged time. In some embodiment, a treatment lasts 5 min, 10 min, 30 min, 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, a purification medium is pre-treated with a first solvent system. In some embodiments, a first solvent system is or comprises a hygroscopic solvent. In some embodiments, a first solvent system is a hygroscopic solvent system. In some embodiments, a hygroscopic solvent system is a first solvent system as described in the present disclosure. In some embodiments, a first solvent system comprises a hygroscopic solvent. In some embodiments, a first solvent is a hygroscopic solvent. In some embodiments, a purification medium is deactivated by contacting the medium with a first solvent system. In some embodiments, a first solvent system comprises a water-miscible solvent. In some embodiments, a first solvent system comprises a water-miscible organic solvent. In some embodiments, a first solvent system is a water-miscible solvent. In some embodiments, a first solvent system is a water-miscible organic solvent. In some embodiments, a first solvent system comprises two or more solvents. In some embodiments, a first solvent system comprises two or more water-miscible solvents. In some embodiments, a first solvent system comprises two or more water-miscible organic solvents. Exemplary water-miscible organic solvent includes acetaldehyde, acetic acid, acetone, acetonitrile, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2-butoxyethanol, butyric acid, diethanolamine, diethylenetriamine, dimethylformamide, dimethoxy ethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol, methyl diethanolamine, methyl isocyanide, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propanoic acid, propylene glycol, pyridine, tetrahydrofuran, triethylene glycol, etc.

[0078] In some embodiments, a first solvent system comprises a hygroscopic solvent. In some embodiments, a first solvent system is a hygroscopic solvent. In some embodiments, a first solvent system comprises two or more hygroscopic solvents, e.g., a hygroscopic solvent system

which is a mixture of two or more hygroscopic solvents, a hygroscopic solvent system comprises two or more hygroscopic solvents and one or more dry, non-hygroscopic solvents. In some embodiments, a first hygroscopic solvent is a water-miscible solvent. In some embodiments, a hygroscopic solvent is a water-miscible organic solvent. In some embodiments, as a person having ordinary skill in the art appreciates, a hygroscopic solvent system can absorb water from its environment, for example, when contacted with a purification medium, a hygroscopic solvent system can absorb water, if any, from the purification medium. When run through a column packed with a purification medium as a stationary phase, a hygroscopic solvent may remove water from the purification medium. In some embodiments, a hygroscopic solvent used for deactivation may contain no more than 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%, 0.007%, 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%) of water (v/v). In some embodiments, a hygroscopic solvent is used as an anhydrous solvent directly purchased from commercial vendors (e.g., Sigma- Aldrich, Acros Organics, etc.). Alternatively or additionally, a hygroscopic solvent can be further purified and/or dried according to known protocols in the art.

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

solvent is a solvent more hygroscopic than ethanol. In some embodiments, a hygroscopic solvent is a solvent more hygroscopic than isopropanol. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than chloroform. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than methylene chloride. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than ethyl acetate. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than acetone. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than acetonitrile. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than dimethyl carbonate. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than methanol. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than ethanol. In some embodiments, a hygroscopic solvent is a solvent no less hygroscopic than isopropanol.

[0080] In some embodiments, a first solvent system comprises an alcohol. In some embodiments, a first solvent system is an alcohol. In some embodiments, a first solvent system comprises an alcohol and another organic solvent. In some embodiments, a first solvent system comprises at least 10% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 20%) (v/v) alcohol. In some embodiments, a first solvent system comprises at least 30%> (v/v) alcohol. In some embodiments, a first solvent system comprises at least 40% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 50% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 60% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 70% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 80% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 90% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 95% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 96% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 97% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 98% (v/v) alcohol. In some embodiments, a first solvent system comprises at least 99% (v/v) alcohol. In some embodiments, an alcohol is a Ci-6 alcohol. In some embodiments, an alcohol is methanol. In some embodiments, an alcohol is an ethanol. In some embodiments, an alcohol is isopropanol. In some embodiments, an alcohol is 1-propanol. In some embodiments, an alcohol is a butanol.

[0081] In some embodiments, a first solvent system comprises methanol. In some

embodiments, a first solvent system is methanol. In some embodiments, a first solvent system comprises at least 10% (v/v) methanol. In some embodiments, a first solvent system comprises at least 20% (v/v) methanol. In some embodiments, a first solvent system comprises at least 30%) (v/v) methanol. In some embodiments, a first solvent system comprises at least 40% (v/v) methanol. In some embodiments, a first solvent system comprises at least 50% (v/v) methanol. In some embodiments, a first solvent system comprises at least 60% (v/v) methanol. In some embodiments, a first solvent system comprises at least 70% (v/v) methanol. In some embodiments, a first solvent system comprises at least 80% (v/v) methanol. In some embodiments, a first solvent system comprises at least 90% (v/v) methanol. In some embodiments, a first solvent system comprises at least 95% (v/v) methanol. In some embodiments, a first solvent system comprises at least 96% (v/v) methanol. In some embodiments, a first solvent system comprises at least 97% (v/v) methanol. In some embodiments, a first solvent system comprises at least 98% (v/v) methanol. In some embodiments, a first solvent system comprises at least 99% (v/v) methanol. In some embodiments, a first solvent system is methanol.

[0082] In some embodiments, a first solvent system comprises ethanol. In some embodiments, a first solvent system is ethanol. In some embodiments, a first solvent system comprises at least 10% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 20%) (v/v) ethanol. In some embodiments, a first solvent system comprises at least 30% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 40% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 50% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 60% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 70% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 80% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 90% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 95% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 96% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 97% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 98% (v/v) ethanol. In some embodiments, a first solvent system comprises at least 99% (v/v) ethanol. In some embodiments, a first solvent system is ethanol.

[0083] In some embodiments, a first solvent system comprises 1-propanol. In some embodiments, a first solvent system is 1-propanol. In some embodiments, a first solvent system comprises at least 10% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 20% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 30%) (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 40% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 50% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 60% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 70% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 80% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 90% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 95% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 96% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 97% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 98% (v/v) 1-propanol. In some embodiments, a first solvent system comprises at least 99% (v/v) 1-propanol. In some embodiments, a first solvent system is 1-propanol.

[0084] In some embodiments, a first solvent system comprises isopropanol. In some embodiments, a first solvent system is isopropanol In some embodiments, a first solvent system comprises at least 10% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 20% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 30% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 40% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 50% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 60% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 70% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 80% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 90% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 95% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 96% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 97% (v/v) isopropanol. In some embodiments, a first solvent system comprises at least 98% (v/v) isopropanol. In some embodiments, a first solvent system

comprises at least 99% (v/v) isopropanol. In some embodiments, a first solvent system is isopropanol.

[0085] In some embodiments, a first solvent system comprises acetone. In some embodiments, a first solvent system is acetone. In some embodiments, a first solvent system comprises at least 10%> (v/v) acetone. In some embodiments, a first solvent system comprises at least 20%) (v/v) acetone. In some embodiments, a first solvent system comprises at least 30%> (v/v) acetone. In some embodiments, a first solvent system comprises at least 40% (v/v) acetone. In some embodiments, a first solvent system comprises at least 50% (v/v) acetone. In some embodiments, a first solvent system comprises at least 60%> (v/v) acetone. In some embodiments, a first solvent system comprises at least 70% (v/v) acetone. In some embodiments, a first solvent system comprises at least 80%> (v/v) acetone. In some embodiments, a first solvent system comprises at least 90% (v/v) acetone. In some embodiments, a first solvent system comprises at least 95% (v/v) acetone. In some embodiments, a first solvent system comprises at least 96% (v/v) acetone. In some embodiments, a first solvent system comprises at least 97% (v/v) acetone. In some embodiments, a first solvent system comprises at least 98% (v/v) acetone. In some embodiments, a first solvent system comprises at least 99% (v/v) acetone. In some embodiments, a first solvent system is acetone.

[0086] In some embodiments, a first solvent system comprises acetonitrile. In some embodiments, a first solvent system is acetonitrile. In some embodiments, a first solvent system comprises at least 10% (v/v) acetonitrile. In some embodiments, a first solvent system comprises at least 20% (v/v) acetonitrile. In some embodiments, a first solvent system comprises at least 30%) (v/v) acetonitrile. In some embodiments, a first solvent system comprises at least 40% (v/v) acetonitrile. In some embodiments, a first solvent system comprises at least 50% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 60% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 70% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 80% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 90% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 95% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 96% (v/v) acetonitrile In some embodiments, a first solvent system comprises at least 97% (v/v)

acetonitrile. In some embodiments, a first solvent system comprises at least 98% (v/v) acetonitrile. In some embodiments, a first solvent system comprises at least 99% (v/v) acetonitrile. In some embodiments, a first solvent system is acetonitrile.

[0087] In some embodiments, a first solvent system comprises dimethyl carbonate. In some embodiments, a first solvent system is dimethyl carbonate. In some embodiments, a first solvent system comprises at least 10°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 20°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 30°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 40°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 50°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 60°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 70°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 80°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 90°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 95°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 96°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 97°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 98°/o (v/v) dimethyl carbonate, n some embodiments, a first solvent system comprises at least 99°/o (v/v) dimethyl carbonate. n some embodiments, a first solvent system is dimethyl carbonate.

[0088] In some embodiments, a first solvent system comprises ethyl acetate. In some embodiments, a first solvent system is ethyl acetate. In some embodiments, a first solvent system comprises at least 10% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 20% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 30% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 40% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 50% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 60% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 70% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 80% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 90% (v/v) ethyl acetate. In some embodiments, a first solvent system

comprises at least 95% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 96% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 97% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 98% (v/v) ethyl acetate. In some embodiments, a first solvent system comprises at least 99% (v/v) ethyl acetate. In some embodiments, a first solvent system is ethyl acetate.

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

[0090] In some embodiments, a first solvent system optionally comprises a modifier. In some embodiments, a first solvent system comprises a modifier. In some embodiments, a first solvent system comprises no modifier.

[0091] Those skilled in the art appreciate that many mobile phase modifiers can be utilized in accordance with the present disclosure to, e.g., modify certain properties of a solvent system as mobile phase. In some embodiments, a modifier is a base. In some embodiments, a modifier is ammonia. In some embodiments, a modifier is triethylamine. In some embodiments, a modifier is an acid. In some embodiments, a modifier is formic acid. A modifier can be added in various amount, 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 (v/v).

[0092] In some embodiments, a first solvent system removes water from a purification medium. In some embodiments, a first solvent may react with reactive sites of a purification medium and deactivates a purification medium, so that compounds such as phosphoramidites decompose less when contacted with the purification medium. In some embodiments, a first solvent system may provide pre-treatment by heat. For example, when methanol is contacted with silica gel, heat may be generated.

[0093] A person of ordinary skill in the art will appreciate that various procedures can be used to pre-treat a purification medium in accordance to provided methods. For example, a first solvent system can be run through a column packed with a purification medium as stationary

phase to pre-treat the stationary phase. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more column volumes of a first solvent system is run through a column to pre-treat the purification medium.

[0094] In some embodiments, the present disclosure demonstrate that provided pre-treatment process, including those that were previously understood or expected to degrade or otherwise damage or wash out a purification medium during chromatography, can in fact improve utility and/or effectiveness of such a medium, particularly in the purification and/or preparation of phosphoramidites. For example, at the time of the present disclosure it is generally taught that high percentage of certain alcohols, such as methanol, should not be run through columns packed with certain purification media, for example, silica gel. As exemplified by the present disclosure, however, pure methanol can be run through silica columns, and silica pre-treated with methanol can provide unexpectedly higher yield and/or purity of phosphoramidites compared to non-pre-treated silica.

[0095] In some embodiments, a provided method comprises an equilibration process after a pre-treatment process. In some embodiments, an equilibration process comprises contacting a pre-treated purification medium with a second solvent system. In some embodiments, a second solvent system is less polar than the first solvent system used to pre-treat the purification medium.

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

[0097] In some embodiments, a second solvent system comprises an isocratic system during the equilibration process. In some embodiments, a second solvent system is isocratic during the equilibration process. In some embodiments, a second solvent system comprises a gradient system. In some embodiments, a 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 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 that the first one, e.g., containing more ethyl acetate in for an ethyl acetate/hexanes system. In some embodiments, the second isocratic process uses a less polar solvent system that the first one, e.g., containing less ethyl acetate for an ethyl acetate/hexanes system. In some embodiments, the first isocratic process for equilibration uses the same solvent system as the first solvent system.

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

[0099] A person of ordinary skill in the art appreciates that a variety of methods can be used to equilibrate a purification medium. In some embodiments, for a column packed with a purification medium as 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 volume of a second solvent system (including isocratic and/or gradient) is run through the column for equilibration a pre-treated purification medium.

[00100] In some embodiments, after an equilibration process, a phosphoramidite is loaded to the pre-treated and equilibrated purification medium, and a third solvent system is utilized to elute, separate and/or purify the phosphoramidite. A person of ordinary skill in the art appreciates that many suitable solvent systems can be employed as a third solvent system in accordance with the present disclosure. In some embodiments, a third solvent system is less polar than the first solvent system. In some embodiments, a third solvent system is the same as the second solvent system for equilibration. In some embodiments, a third solvent system is the same as the second solvent system at the end of the equilibration process.

[00101] In some embodiments, a third solvent system consists of one solvent and optionally a modifier. In some embodiments, a third solvent system comprises two or more solvents and optionally a modifier. In some embodiments, a modifier is a base. In some embodiments, a modifier is triethylamine.

[00102] In some embodiments, a third solvent system comprises an isocratic system during the elution process. In some embodiments, a third solvent system is isocratic during the elution process. In some embodiments, a third solvent system comprises a gradient system. In some embodiments, a 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 that the first one, e.g., containing more ethyl acetate for an ethyl acetate/hexanes 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.

[00103] In some embodiments, a third solvent system comprises ethyl acetate. In some embodiments, a third solvent system comprises ethyl acetate and a base as a modifier. In some embodiments, a third solvent system comprises ethyl acetate and an amine as a modifier. In some embodiments, a third solvent system comprises ethyl acetate and triethylamine as a modifier. In some embodiments, a third solvent system comprises hexanes. In some embodiments, a third solvent system comprises hexanes and triethylamine as a modifier. In some embodiments, a third solvent system comprises hexanes and ethyl acetate. In some embodiments, a third solvent system is a mixture of hexanes and ethyl acetate. In some embodiments, a third solvent system comprises hexanes, ethyl acetate, and triethylamine. In some embodiments, a third solvent system is a mixture of hexanes, ethyl acetate, and triethylamine. Exemplary solvent systems for elution are illustrated in the examples.

[00104] A person of ordinary skill in the art appreciates that a variety of methods can be used to elute, separate and/or purity a phosphoramidite. In some embodiments, for a column packed with a purification medium as 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 volume of a third solvent system (including isocratic and/or gradient) is run through the column.

[00105] Those skilled in the art appreciate that various purification media can be used for purification of phosphoramidites, and can be pre-treated, equilibrated and/or eluted using

provided methods to improve yields and/or purity of phosphoramidites in accordance with the present disclosure. In some embodiments, a purification medium is a silica medium. In some embodiments, a purification medium is silica gel. In some embodiments, a purification medium is alumina. In some embodiments, a purification medium is basis alumina. In some embodiments, a purification medium can be used for other purposes other than purification.

[00106] Purification media of various particle sizes can be pre-treated and utilized according to the present disclosure. In some embodiments, a particle size is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, or more micrometer in diameter. In some embodiments, a 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 micrometer in diameter. In some embodiments, a 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 micrometer in diameter. In some embodiments, a purification medium is silica of about 10 micrometer in diameter. In some embodiments, a purification medium is silica of about 15 micrometer in diameter. In some embodiments, a purification medium is silica of about 20 micrometer in diameter. In some embodiments, a purification medium is silica of about 25 micrometer in diameter. In some embodiments, a purification medium is silica of about 30 micrometer in diameter. In some embodiments, a purification medium is silica of about 35 micrometer in diameter. In some embodiments, a purification medium is silica of about 40 micrometer in diameter. In some embodiments, a purification medium is silica of about 45 micrometer in diameter. In some embodiments, a purification medium is silica of about 50 micrometer in diameter. In some embodiments, a purification medium is silica of about 60 micrometer in diameter. In some embodiments, a purification medium is silica of about 70 micrometer in diameter. In some embodiments, a purification medium is silica of about 80 micrometer in diameter. In some embodiments, a purification medium is silica of about 90 micrometer in diameter. In some embodiments, a purification medium : is silica of about 100 micrometer in diameter. In some embodiments, a purification medium is silica of greater than about 100 micrometer in diameter.

[00107] In some embodiments, a purification medium is packed into a column. In some embodiments, a 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, several column volumes of mixtures of ethyl acetate and hexanes with

triethylamine as modifier are used to equilibrate the treated silica gel before crude phosphoramidite is loaded. In some embodiments, phosphoramidite is eluted with mixtures of ethyl acetate and hexanes with triethylamine as modifier.

[00108] In some embodiments, the present disclosure provides a composition comprising a pre-treated purification medium as described herein, and a phosphoramidite. In some embodiments, the present disclosure provides a composition comprising a pre-treated and equilibrated purification medium as described herein, and a phosphoramidite. In some embodiments, a purification medium is silica gel. In some embodiments, the silica gel is pre-treated with an alcohol. In some embodiments, the silica gel is pre-treated with methanol. In some embodiments, the silica gel is equilibrated with a solvent system comprising hexanes. 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 comprises ethyl acetate and hexanes. In some embodiments, the silica gel is equilibrated with a solvent system consisting of ethyl acetate and hexanes. In some embodiments, the silica gel is equilibrated with a solvent system comprising ethyl acetate, hexanes and triethylamine. In some embodiments, the silica gel is equilibrated with a solvent system consisting of ethyl acetate, hexanes and triethylamine. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with hexanes. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with ethyl acetate. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with triethylamine. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with hexanes and ethyl acetate. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with hexanes and triethylamine. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with ethyl acetate and triethylamine. In some embodiments, the silica gel is equilibrated, wherein the equilibration comprises contact with hexane, ethyl acetate and triethylamine. In some embodiments, the silica gel is equilibrated, wherein the equilibration consists of contact with hexane, ethyl acetate and triethylamine.

[00109] In some embodiments, a phosphoramidite is a nucleoside phosphoramidite.

[00110] In some embodiments, a phosphoramidite has the structure of formula I:

R2

— R3

/

BA— SU—

O

/

R

I

wherein:

BA is R, or an optionally substituted group selected from a 3-30 membered cycloaliphatic 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, -L-O- or
, wherein SU is

connected to the phosphorus atom in formula I through the oxygen atom;

L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-30 aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci-6 alkenylene,— C≡C— -C(R')2- -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')-, -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0- -OC(0)N(R')-, -S(O)- "S(0)2-, -S(0)2N(R')-, -N(R')S(0)2- -SC(O)- -C(0)S- -OC(O)-, or -C(0)0-;

R5s is R' or -OR';

R2s is -F, -CN, -N3, -NO, -N02, -R' -OR', -SR', -N(R')2, -L-R\ -O-L-OR', -O-L-SR', or -0-L-N(R')2, or R2s is L connecting C2 with CI, C2, C3, C4 or C5;

-Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered

heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

1 2 3 1 2 3

each of R , R , and R is independently R', or two or three of R , R , and R are taken together with their intervening atoms to form:

— @-(Rs)t .

Ring A is an optionally substituted multivalent, 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 Rs is independently R' or -L-R';

t is 0-5;

each R' is independently -R, -C(0)R, -C02R, or -S02R, or:

two or more R' are taken together with their intervening atoms to 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; and

each R is independently hydrogen, or an optionally substituted group selected from Ci-3o

aliphatic, Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 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.

[00111] In some embodiments, a phosphoramidite has the structure of formula I:

I

wherein:

BA is an optionally substituted group selected from C1-30 cycloaliphatic, C6-3o aryl, C3-3o

heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety and a modified nucleobase moiety;

SU is -L-O- or
, wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;

L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from Ci-3o aliphatic and Ci-3o heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted Ci-6 alkylene, Ci-6 alkenylene,— C≡C— -C(R')2- -Cy- -0-, -S- -S-S- -N(R')- -C(O)-, -C(S)-, -C(NR')- -C(0)N(R')- -N(R')C(0)N(R')-, -N(R')C(0)- -N(R')C(0)0-, -OC(0)N(R')-, -S(O)- "S(0)2-, -S(0)2N(R')- -N(R')S(0)2- -SC(O)- -C(0)S- -OC(O)-, or -C(0)0-;

R5s is R' or -OR';

R2s is -F, -CN, -N3, -NO, -N02, -R' -OR', -SEC, -N(R')2, -O-L-OR', -O-L-SR', or

-0-L-N(R')2, or R2s is L connecting C2 with CI, C2, C3, C4 or C5;

-Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6- 30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

1 2 3 1 2 3

each of R , R , and R is independently R', or two or three of R , R , and R are taken together with their intervening atoms to form:

— ®-(Rs)t .

Ring A is an optionally substituted multivalent, 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 Rs is independently R' or -L-R';

t is 0-5;

each R' is independently -R, -C(0)R, -C02R, or -S02R, or:

two or more R' are taken together with their intervening atoms to 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; and

each R is independently hydrogen, or an optionally substituted group selected from Ci-3o

aliphatic, Ci-3o heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-3o aryl, 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.

[00112] In some embodiments, BA is an optionally substituted group selected from a 3-30 membered cycloaliphatic 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. In some embodiments, BA is optionally substituted Ci-30 cycloaliphatic. In some embodiments, BA is optionally substituted C6-3o aryl. In some embodiments, BA is optionally substituted 3-30 membered heterocyclyl. In some embodiments, BA is optionally substituted C3- 3o heterocyclyl. In some embodiments, BA is optionally substituted 5-30 membered heteroaryl. In some embodiments, BA is optionally substituted C5-30 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 an optionally substituted group selected from Ci-3o cycloaliphatic, C6-3o aryl, C3-30 heterocyclyl, and C5-3o heteroaryl. In some embodiments, BA is an optionally substituted group selected from Ci-30 cycloaliphatic, C6-3o aryl, C3-30 heterocyclyl, C5-30 heteroaryl, and a natural nucleobase moiety. In some embodiments, BA is optionally substituted A, T, C, or G. In some embodiments, BA is A, T, C, G, or 5-mC.

CLAIMS
1. A method for purifying a phosphoramidite, comprising 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.

2. A method for improving recovery rate of a phosphoramidite, comprising steps of:

a) removing water from a purification medium;

b) adding the phosphoramidite to the purification medium; and

c) eluting the phosphoramidite from the purification medium with a solvent system;

wherein the recovery rate is higher than a reference recovery rate when step a) is absent.

3. The method of claim 2, wherein the difference between the improved recovery rate and the reference recovery rate is at least about 10%, 20%, 30%, 40% or 50%.

4. A method for decreasing decomposition of a phosphoramidite when the phosphoramidite contacts a purification medium, comprising:

a) removing water from the purification medium.

5. The method of any one of the preceding claims, wherein the step of removing water from the purification medium comprises heating the purification medium.

6. The method of any one of the claims 1-4, wherein the step of removing water from the purification medium comprises contacting the purification medium with a hygroscopic solvent system.

7. The method of claim 6, wherein the hygroscopic solvent system consists of one hygroscopic solvent.

8. The method of claim 7, 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, dimethyl carbonate,

dimethylformamide, dimethoxy ethane, dimethyl sulfoxide, 1,4-dioxane, ethanol, ethylamine, ethylene glycol, formic acid, furfuryl alcohol, glycerol, methanol, methyl diethanolamine, methyl isocyanide, 1-propanol, 1,3-propanediol, 1,5-pentanediol, 2-propanol, propanoic acid, propylene glycol, pyridine, tetrahydrofuran, and tri ethylene glycol.

9. The method of claim 8, wherein the hygroscopic solvent is selected from methanol,

acetonitrile, and ethyl acetate, optionally with triethylamine up to 50%.

10. The method of claim 8, wherein the hygroscopic solvent is methanol.

11. A method comprising steps of:

a) pre-treating a purification medium;

b) contacting the purification medium with a phosphoramidite; and

b) optionally using the purification medium to purify a phosphoramidite.

12. The method of claim 11, wherein the pre-treatment comprises heating the purification medium.

13. The method of claim 11, wherein the pre-treatment comprises contacting the purification medium with a first solvent system comprises a hygroscopic solvent.

14. The method of any one of claims 11-13, wherein the purification medium is silica gel.

15. The method of any one of the preceding claims, comprising chromatography purification of the phosphoramidite using the purification medium at a temperature higher and/or lower than room temperature.

16. A composition comprising:

a) a purification medium pre-treated by step a) of any one of the preceding claims;

b) a phosphoramidite.

17. A composition comprising:

a) silica gel pre-treated with a hygroscopic solvent;

b) a phosphoramidite.

18. The method or composition of any one of the preceding claims, wherein the phosphoramidite has the structure of formula II.

19. A method or a composition of any one of embodiments 1-144.