TECHNOLOGIES FOR OLIGONUCLEOTIDE PREPARATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No.

62/560,169, filed September 18, 2017, the entirety of which is incorporated herein by reference.

BACKGROUND

[0002] Oligonucleotides may contain a variety of modifications. Certain modifications, such as phosphorothioate internucleotidic linkages, may introduce new chiral centers into oligonucleotides.

SUMMARY

[0003] Oligonucleotides are useful for many purposes. However, natural oligonucleotides have been found to suffer disadvantages, such as low stability, low activity, etc., that can reduce or negate their usefulness as therapeutics.

[0004] Certain technologies have been developed that can improve oligonucleotide properties and usefulness. For example, certain modifications, e.g., to nucleobases, sugars, and/or internucleotidic linkages, etc., have been described that can improve oligonucleotide properties and usefulness. Moreover, technologies that permit control of stereochemistry, and/or preparation of stereocontrolled oligonucleotides have been demonstrated to provide particularly useful and effective oligonucleotide compositions. Certain exemplary useful technologies are described, for example, in one or more of: US20150211006, US20170037399, WO2017/015555, WO2017/015575, WO2017/062862, etc., each of which is incorporated herein by reference.

[0005] Particularly given the demonstrated desirability and usefulness of stereocontrolled oligonucleotide compositions, the present Applicant appreciated that developments of technologies that could improve or facilitate production of oligonucleotide compositions, particularly stereocontrolled oligonucleotide compositions, could provide significant benefits. The present disclosure describes certain such developments, and provides technologies relating to oligonucleotide compositions, particularly to stereocontrolled oligonucleotide compositions. Provided technologies may be particularly useful, for example, with respect to therapeutic oligonucleotides.

[0006] Among other things, the present disclosure encompasses the recognition that certain conditions and/or sequences of steps that have been utilized in the preparation of oligonucleotides, particularly stereocontrolled (e.g., stereopure) oligonucleotide compositions, can be associated with generation of certain impurities. In some embodiments, the present disclosure thus identifies the source of a problem with strategies that have utilized such conditions and/or steps; the present disclosure provides technologies including methods that utilize certain conditions and/or sequences of steps that are described and demonstrated to dramatically improve crude product purity and yield, in some embodiments significantly improving efficiency and/or reducing production cost. For example, as demonstrated in the Examples, in some embodiments, provided technologies can deliver chirally controlled oligonucleotide compositions with crude purity of over 70% (full-length product purity), much higher than reference technologies (crude full-length product purity of can be around 30% or lower).

[0007] Oligonucleotide synthesis typically utilizes highly efficient chemical transformations in its steps. However, despite the high efficiency, products of one or more steps often contain one or more reactive functional groups that can introduce significant impurities if uncapped, e.g., for coupling products, unreacted 5'-OH groups, and/or newly formed reactive groups (e.g., primary and/or secondary amino groups) when chiral auxiliaries are utilized for chirally controlled oligonucleotide synthesis. In many cases, such reactive functional groups are capped during oligonucleotide synthesis in order to reduce impurities from them. In some embodiments, the present disclosure encompasses the recognition of a source of problem that capping steps as those typically used in traditional phosphoramidite-based oligonucleotide synthesis can lead to generation of a significant amount of byproducts, particularly for many chirally controlled (stereocontrolled, stereoselective) oligonucleotide synthesis processes. Among other things, the present disclosure provides technologies that address the problems.

[0008] In some embodiments, the present disclosure provides methods comprising a post-modification capping step, e.g., after a modification step but before the next de-blocking and/or coupling step. In some embodiments, a post-modification capping step is after a modification step that provides a chirally controlled internucleotidic linkage but before the next de-blocking and/or coupling step.

[0009] In some embodiments, the present disclosure provides methods comprising capping

steps of different chemistry strategies compared to a reference capping step in traditional oligonucleotide synthesis. For example, in some embodiments, the present disclosure provides methods comprising one or more capping steps, each of which selectively caps amino groups over hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis). In some embodiments, provided capping steps are selective for amidation over esterification. In some embodiments, capping reagent systems for capping steps contain no or reduced levels (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis) of strong nucleophiles and/or esterification catalysts (or reagents that can provide them when contacted with a composition to be capped), e.g., no or reduced levels of DMAP, NMI, etc.. In some embodiments, the present disclosure provides methods comprising capping steps that can cap both amino groups and hydroxyl groups efficiently, e.g., capping steps that are comparable or identical to a reference capping step in traditional oligonucleotide synthesis.

[0010] In some embodiments, the present disclosure provides methods that comprise capping steps of the same or different chemistry strategies to achieve oligonucleotide synthesis and can provide various advantages, e.g., improved crude purity, improved yield, etc., particularly for chirally controlled (stereocontrolled, stereoselective) oligonucleotide synthesis. In some embodiments, the present disclosure provides methods of a pre-modification capping step (after a coupling step but before the next modification step) and a post-modification capping step (after a modification step but before the next de-blocking and/or coupling step). In some embodiments, a pre-modification capping step and post-modification capping step are different. In some embodiments, a pre-modification capping step and post-modification capping step have different chemistry strategies. In some embodiments, a pre-modification capping step caps amino groups selectively over hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis). In some embodiments, a post-modification capping step can cap both amino and hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis). In some embodiments, a pre-modification capping step caps amino groups selectively over hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis). In some embodiments, a pre-modification capping step can cap both amino and hydroxyl groups (e.g., compared to a reference capping reagent system in traditional oligonucleotide synthesis).

[0011] In some embodiments, provided methods comprise two or more capping steps in an oligonucleotide synthesis cycle. In some embodiments, provided methods comprise two capping steps in an oligonucleotide synthesis cycle, wherein the two steps are separated by a modification step, e.g., oxidation, sulfurization, etc. In some embodiments, provided methods comprise a step in which a chiral modified internucleotidic linkage comprising a chiral linkage phosphorus is formed with a stereoselectivity of at least 80:20, 85:15, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1, favoring either the Rp or Sp configuration.

[0012] In some embodiments, the present disclosure provides a method comprising:

providing a chiral nucleoside phosphoramidite which comprises a chiral atom that is not the phosphorus atom or a sugar carbon atom; and

a capping step immediately following a sulfurization or oxidation step.

[0013] In some embodiments, the present disclosure provides a method comprising:

providing a chiral nucleoside phosphoramidite which comprises a chiral atom that is not the phosphorus atom and is not an atom of the nucleoside unit; and

a capping step immediately following a sulfurization or oxidation step.

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

providing an oligonucleotide intermediate comprising a chiral linkage phosphorus atom, which is bonded to a chiral unit which does not comprise a nucleoside unit or a part thereof; and a capping step immediately following a sulfurization or oxidation step.

[0015] In some embodiments, the present disclosure provides a method comprising:

providing an oligonucleotide intermediate comprising a chiral linkage phosphorus atom, which is bonded to a chiral unit which does not comprise an atom of a nucleoside unit; and a capping step immediately following a sulfurization or oxidation step.

[0016] In some embodiments, the present disclosure provides a method for preparing a composition comprising a plurality of oligonucleotides comprising:

a) a coupling step comprising:

contacting a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is de- blocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and

coupling a partner compound with the free hydroxyl groups of a plurality of de-blocked oligonucleotides or nucleosides;

wherein the coupling step provides a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

b) optionally a pre-modification capping step comprising:

contacting a coupling product composition with a pre-modification capping reagent system; and

capping one or more functional groups of the coupling product composition; wherein the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides;

c) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more internucleotidic linkages of one or more coupling product oligonucleotides; or

contacting a pre-modification capping product composition with a modification reagent system and modifying one or more linkages of one or more pre-modification capping product oligonucleotides;

wherein the modification step provides a modification product composition comprising a plurality of modification product oligonucleotides;

d) optionally a post-modification capping step comprising:

contacting a modification product composition with a post-modification capping reagent system; and

capping one or more functional groups of a plurality of oligonucleotides of the modification product composition;

wherein the post-modification capping step provides a post-modification capping product composition comprising a plurality of post-modification capping product oligonucleotides;

e) optionally a de-blocking step comprising:

contacting a modification product composition, or a post-modification capping product composition, with a de-blocking reagent system;

wherein the deblocking step provides a de-blocking product composition comprising a plurality of de-blocking product oligonucleotides, each of which independently comprises a free hydroxyl group; and

f) optionally repeating steps b) through e) a number of times.

[0017] In some embodiments, provided methods comprise one or more pre-modification capping steps. In some embodiments, provided methods comprise one or more post-modification capping steps. In some embodiments, provided methods comprise one or more pre-and post-modification capping steps. In some embodiments, provided methods comprise one or more de-blocking steps.

[0018] In some embodiments, the present disclosure provides a method for preparing a composition comprising a plurality of oligonucleotides comprising one or more cycles, each cycle independently comprises:

a) a coupling step comprising:

contacting a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is de- blocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and coupling a partner compound with the free hydroxyl groups of a plurality of de- blocked oligonucleotides or nucleosides;

wherein the coupling step provides a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

b) optionally a pre-modification capping step comprising:

contacting a coupling product composition with a pre-modification capping reagent system; and

capping one or more functional groups of the coupling product composition;

wherein the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides;

c) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more internucleotidic linkages of one or more coupling product oligonucleotides; or

contacting a pre-modification capping product composition with a modification reagent system and modifying one or more linkages of one or more pre-modification capping product oligonucleotides;

wherein the modification step provides a modification product composition comprising a plurality of modification product oligonucleotides;

d) optionally a post-modification capping step comprising:

contacting a modification product composition with a post-modification capping reagent system; and

capping one or more functional groups of a plurality of oligonucleotides of the modification product composition;

wherein the post-modification capping step provides a post-modification capping product composition comprising a plurality of post-modification capping product oligonucleotides;

e) optionally a de-blocking step comprising:

contacting a modification product composition, or a post-modification capping product composition, with a de-blocking reagent system;

wherein the deblocking step provides a de-blocking product composition comprising a plurality of de-blocking product oligonucleotides, each of which independently comprises a free hydroxyl group.

[0019] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises the following steps:

(1) a coupling step;

(2) optionally a pre-modification capping step;

(3) a modification step;

(4) optionally a post-modification capping step; and

(5) a de-blocking step.

[0020] In some embodiments, a cycle comprises one or more pre-modification capping steps. In some embodiments, a cycle comprises one or more post-modification capping steps. In some embodiments, a cycle comprises one or more pre- and post-modification capping steps. In some embodiments, a cycle comprises one or more de-blocking steps.

[0021] In some embodiments, the present disclosure encompasses the recognition that traditional capping conditions when used as in traditional oligonucleotide synthesis may be a significant source of various problems under certain circumstances, and may contribute to formation of one or more by-products (impurities) and significantly lower oligonucleotide crude purity and yield, particularly for stereoselective preparation of oligonucleotides comprising one or more chiral internucleotidic linkages. Among other things, the present disclosure provides technologies comprising capping strategies that can deliver unexpectedly high crude impurity and yield compared to an appropriate reference technology, for example, through designed capping strategies in combination with other steps in oligonucleotide synthesis.

[0022] In some embodiments, a reference technology uses a traditional capping condition as in traditional phosphoramidite-based oligonucleotide synthesis, which typically is or comprises an esterification condition that acrylates hydroxyl groups, e.g., by using a mixture comprising an acylating agent (e.g., acetic anhydride), a base (e.g., 2,6-lutidine), and a catalyst (e.g., N-methylimidazole, DMAP, etc.) to contact oligonucleotides to cap hydroxyl groups (e.g., unreacted 5'-OH groups). Traditional capping conditions typically use a substantial amount of acylating agent, base and catalyst for capping, generally each independently about 5%-15% volume, and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 equivalents relative to the first nucleoside incorporated into an oligonucleotide (before any cycle forming internucleotidic linkage) or oligonucleotide loading capacity of a support (e.g., loading capacity of the support used for preparing an oligonucleotide, can be calculated by multiplying unit loading capacity of a support (e.g., umol/g) by amount of support (g)). In some embodiments, as used in traditional oligonucleotide synthesis, each synthetic cycle of a reference technology contains a single capping step. In some embodiments, a reference technology comprises no more than one capping step in each of its synthetic cycle, wherein capping is

performed using an esterification condition, e.g., comprising an acylating agent (e.g., acetic anhydride), a base (e.g., 2,6-lutidine), and a catalyst (e.g., N-methylimidazole (NMI), DMAP, etc.), each independently no less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by volume of the capping reagent solution, and/or the catalyst is no less than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.5, or 2 equivalents relative to the acylating agent and/or the base.

[0023] In some embodiments, the present disclosure provides technologies comprising one or more capping steps, e.g., pre-modification capping steps, post-modification capping steps, etc., each of which is independently comparable or identical to a reference capping step, e.g., of traditional oligonucleotide synthesis based on phosphoramidite chemistry. In some embodiments, the present disclosure reduces by-products that may be formed in such capping steps by strategically positioning their positions (or timing) in oligonucleotide synthesis methods and/or cycles. In some embodiments, such capping steps are positioned after amino groups (typically primary and secondary) are, in many instances selectively, capped (over free hydroxyl groups, particularly, 5'-OH), either as individual separate capping steps or in combination with other capping steps (e.g., capping steps capping amino groups, in many instances selectively 5'-OH).

[0024] In some embodiments, the present disclosure provides technologies comprising one or more capping steps that each independently comprise a condition that is selective or specific for amidation over esterification. In some embodiments, the present disclosure provides technologies comprising one or more capping steps that use an amidation condition which is not an efficient and/or typical esterification condition. As readily appreciated by those skilled in the art, esterification and amidation have been extensively studied, and various conditions selective or specific for amidation over esterification, and various methods for assessing selectivity and/or specificity for amidation over esterification, are widely known in the art and can be utilized in accordance with the present disclosure. For example, a typical condition selective or specific for amidation over esterification is an anhydride and a base without a catalyst (e.g., Ac2O and 2,6-lutidine), as a corresponding efficient esterification condition typically requires an anhydride, a base, and a catalyst (e.g., Ac2O, 2,6-lutidine, and NMI) as traditional capping conditions. In some embodiments, the present disclosure provides technologies that comprise one or more synthetic cycles each independently comprising a coupling step, a modification step (e.g.,

oxidation, sulfurization, etc.), and one or more capping steps, wherein each capping step after a coupling step and before a modification step comprising an amidation condition and no estenfication condition. In some embodiments, an amidation condition comprises no more than 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% by volume of a catalyst for esterification under an appropriate corresponding condition (having the same acylating agent and base), and/or no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents of a catalyst for esterification under an appropriate corresponding condition (having the same acylating agent and base), relative to an acylating agent and/or relative to oligonucleotide loading capacity of a support. In some embodiments, an acylating agent is an anhydride. In some embodiments, an acylating agent is Ac2O. In some embodiments, a catalyst is NMI. In some embodiments, a catalyst is DMAP. In some embodiments, a catalyst is a nucleophilic nitrogen base.

[0025] Without the intention to be limited by any theory, in some embodiments, the present disclosure encompasses the recognition of a source of a problem in oligonucleotide synthesis, that an nucleophilic agent, particularly when used in a capping step that is after a coupling step and before a modification step in stereoselective oligonucleotide preparation, may contribute to generation of byproducts and lower overall preparation efficiency and/or crude purity through, e.g., degradation of oligonucleotides, lowering performance of another step, etc. Thus, in some embodiments, the present disclosure provides capping technologies comprising greatly reduced levels of or no strong nucleophiles, e.g., catalysts used in typical capping conditions such as DMAP, NMI, etc., in contrast to traditional capping conditions which can comprise a large amount of a nucleophilic catalyst (e.g., in some cases, 5%-15% NMI by volume of capping solutions). In some embodiments, each of one or more capping steps after a coupling step and before a modification step within an oligonucleotide preparation cycle independently comprises greatly reduced levels of or no strong nucleophiles. In some embodiments, a reduced level is no more than 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5% by volume of a capping reagent solution. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents relative to an acylating agent. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, or 1.2 equivalents relative to oligonucleotide loading capacity of a support.

[0026] In some embodiments, a strong nucleophile is a nucleophilic base. In some

embodiments, a nucleophilic base is a nitrogen base. In some embodiments, a nucleophilic base is a nitrogen base wherein the basic nitrogen atom (e.g., =N– or –N(–)–) has no alpha substituents. In some embodiments, a nucleophilic base is a nitrogen base wherein the basic nitrogen atom (e.g., =N– or –N(–)–) has no alpha substituent that is not part of a ring. In some embodiments, a nucleophilic base is optionally substituted 5-10 membered heteroaryl compound comprising a basic nitrogen atom =N–, wherein the nitrogen atom has less than two, or no, alpha-sub stituents. In some embodiments, a nucleophilic base is a nucleophilic nitrogen base. In some embodiments, a nucleophilic nitrogen base is a compound of the structure of formula B-I:

N(RN)3,

B-I

wherein each RN is independently R and the three R groups are taken together with the nitrogen atom to form an optionally substituted bicyclic or polycyclic ring as described in the present disclosure for R groups (and groups can be R), wherein the nitrogen of N(RN)3 (underlined) is a tertiary nitrogen, and there are no substitutions at any of the positions alpha to the nitrogen atom. In some embodiments, a formed ring is saturated. In some embodiments, a nucleophilic base is DABCO (1,4-diazabicyclo[2.2.2]octane). In some embodiments, a formed ring contains one or more unsaturation.

[0027] In some embodiments, a nucleophilic nitrogen base is a base comprising =N–, wherein there are no substitutions at any of the positions alpha to the nitrogen atom. In some embodiments, a nucleophilic nitrogen base is a base comprising an aromatic moiety comprising =N–, wherein there are no substitutions at any of the positions alpha to the nitrogen atom. In some embodiments, a nucleophilic nitrogen base is a compound of the structure of formula B-II:

RN–CH=N–CH=CH–RN,

B-II

wherein each RN is independently R and the two R groups are taken together with their intervening atoms to form an optionally substituted ring as described in the present disclosure, wherein the compound comprises –CH=N–CH=. In some embodiments, a formed ring is an optionally substituted C5-30 heteroaryl ring comprising 0-10 hetereoatoms in addition to the nitrogen atom. In some embodiments, a formed ring is an optionally substituted 5-membered heteroaryl ring. In some embodiments, a formed ring is a substituted 5-membered heteroaryl ring. In some embodiments, a formed ring is a substituted imidazolyl ring. In some

embodiments, a nucleophilic base is substituted imidazole. In some embodiments, a nucleophilic nitrogen base is NMI. In some embodiments, a formed ring is an optionally substituted 6-membered heteroaryl ring. In some embodiments, a formed ring is a substituted 6-membered heteroaryl ring. In some embodiments, a formed ring is a substituted pyridinyl ring. In some embodiments, a nucleophilic base is substituted pyridine. In some embodiments, a nucleophilic nitrogen base is DMAP.

[0028] As appreciated by those skilled in the art, nucleophilicity, e.g., of basic nitrogen atoms in bases, is related to several factors, e.g., steric hindrance, electron density, etc.

Technologies for assessing nucleophilicity are widely known in the art and can be utilized in accordance with the present disclosure. Additionally or alternatively, bases of various levels of nucleophilicity are well-known and can be assessed and/or utilized in accordance with the present disclosure. In some embodiments, a base that can efficiently catalyze esterification reactions, e.g., a base that can be used for efficient capping of unreacted 5'-OH together with anhydride and 2,6-lutidine in traditional oligonucleotide synthesis (e.g., DMAP, NMI, etc.) is a strong nucleophilic base and should be avoided or used at reduced levels for capping steps that comprise greatly reduced levels of or no strong nucleophiles, e.g., any capping step after a coupling step and before a modification step. In some embodiments, a strong nucleophilic base is a base that can effectively replace DMAP or NMI in esterification. In some embodiments, a strong nucleophilic base is a base that can effectively replace DMAP or NMI in a capping step of traditional oligonucleotide synthesis (which typically uses phosphoramidite chemistry and does not use chiral auxiliaries and is considered non-stereoselective/non-stereocontrolled).

[0029] In some embodiments, provided methods comprise a capping step, which capping step comprises no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents of a strong nucleophilic base relative to oligonucleotides or loading capacity of a support, or no strong nucleophilic bases. In some embodiments, such a capping step is immediately followed by a non-capping step. In some embodiments, such a capping step is immediately after a non-capping step. In some embodiments, such a capping step is immediately followed by a non-capping step, and is immediately after a non-capping step. In some embodiments, a non-capping step is a coupling step. In some embodiments, a non-coupling step is a modification step. In some embodiments, a non-capping step immediately before such a capping step is a coupling modification step.

[0030] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises no strong nucleophile, or if it comprises one or more strong nucleophiles, level of each of the one or more strong nucleophiles is independently reduced compared to an appropriate reference capping condition.

[0031] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises no strong nucleophile, or if it comprises one or more strong nucleophiles, level of each of the one or more strong nucleophiles is independently no more than no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.

[0032] In some embodiments, the first incorporated nucleoside of the oligonucleotide is the first nucleoside loaded to a support before the first cycle that forms an internucleotidic linkage. In some embodiments, equivalent of the first incorporated nucleoside of an oligonucleotide to oligonucleotide loading capacity of a support used for preparing the oligonucleotide is 1.

[0033] In some embodiments, a strong nucleophile is a strong nucleophile base as described in the present disclosure. In some embodiments, a strong nucleophilic base is a compound of formula B-I. In some embodiments, a strong nucleophilic base is a compound of formula B-I and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis. In some embodiments, a strong nucleophilic base is a compound of formula B-II. In some embodiments, a strong nucleophilic base is a compound of formula B-II and can be used

for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis. In some embodiments, a strong nucleophilic base is DMAP. In some embodiments, a strong nucleophilic base in NMI.

[0034] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises no catalyst that promotes capping of 5'-OH as in an appropriate reference capping condition, or if it comprises one or more such catalysts, level of each of the one or more such catalysts is independently reduced compared to an appropriate reference capping condition.

[0035] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises no catalyst that promotes capping of 5'-OH as in an appropriate reference capping condition, or if it comprises one or more such catalysts, level of each of the one or more such catalysts is independently no more than 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.

[0036] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises no catalyst for esterification, or if it comprises one or more catalysts for esterification, level of each of the one or more such catalysts is independently reduced compared to an appropriate reference capping condition.

[0037] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises no catalyst for esterification, or if it comprises one or more catalysts for esterification, level of each of the one or more such catalysts is independently no more than 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.

[0038] In some embodiments, a catalyst, e.g., that promotes capping of 5'-OH as in oligonucleotide synthesis, for esterification, etc., is a compound of formula B-I. In some embodiments, a catalyst is a compound of formula B-I and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis. In some embodiments, a catalyst is a compound of formula B-II. In some embodiments, a catalyst is a compound of formula B-II and can be used for efficient capping in traditional, phosphoramidite-based oligonucleotide synthesis. In some embodiments, a catalyst is DMAP. In some embodiments, a strong nucleophilic base in NMI.

[0039] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises a selective condition for amidation over esterification. [0040] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an internucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an internucleotidic linkage, and which modification step modifies the internucleotidic linkage formed in the coupling step;

wherein each capping step between the coupling step and the modification step (pre-modification capping step) comprises a selective condition for amidation over esterification, and no condition identical to or comparable to an appropriate reference condition.

[0041] In some embodiments, selective conditions for amidation over esterification comprise reduced levels of or no catalysts for esterification, e.g., no DMAP, NMI, etc. In some embodiments, a condition identical to or comparable to an appropriate reference condition can be used to replace capping conditions in traditional phosphoramidite-based oligonucleotide synthesis without significantly reducing (or with no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of reduction of) efficiency, crude purity, and/or yield.

[0042] In some embodiments, provided methods comprise a second capping step after a modification step in one or more cycles. In some embodiments, provided methods comprise a second capping step after a modification step and before a deblocking step (which de-blocks blocked hydroxyl groups) in one or more cycles. In some embodiments, a second capping step comprises a strong nucleophile. In some embodiments, a second capping step comprises a strong nucleophile at a level comparable to a reference capping condition. In some embodiments, a second capping step comprises an esterification catalyst. In some embodiments, a second capping step comprises an esterification catalyst at a level comparable to a reference capping condition. In some embodiments, a second capping step comprises an esterification condition. In some embodiments, a second capping step comprises an esterification condition that is identical or comparable with a reference capping condition, e.g., in terms of capping unreacted 5'-OH in oligonucleotide synthesis. In some embodiments, a strong nucleophile is DMAP or NMI. In some embodiments, a strong nucleophile is DMAP. In some embodiments, a strong nucleophile is NMI. In some embodiments, an esterification catalyst is DMAP or NMI. In some embodiments, an esterification catalyst is DMAP. In some embodiments, an esterification catalyst is NMI.

[0043] In some embodiments, the present disclosure provides a method for preparing an oligonucleotide, comprising a cycle comprising steps of:

(1) a coupling step;

(2) a first capping step;

(3) a modification step;

(4) a second capping step;

(5) a de-blocking step;

wherein the cycle comprises steps in the order of (2)-(3)-(4);

wherein the cycle is repeated until the length of the oligonucleotide is achieved.
CLAIMS

1. A method for preparing a composition comprising a plurality of oligonucleotides comprising:

a) a coupling step comprising:

contacting a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is de- blocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and coupling a partner compound with the free hydroxyl groups of a plurality of de- blocked oligonucleotides or nucleosides;

wherein the coupling step provides a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

b) a pre-modification capping step comprising:

contacting a coupling product composition with a pre-modification capping reagent system; and

capping one or more functional groups of the coupling product composition; wherein the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides;

c) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more internucleotidic linkages of one or more coupling product oligonucleotides; or

contacting a pre-modification capping product composition with a modification reagent system and modifying one or more linkages of one or more pre-modification capping product oligonucleotides;

wherein the modification step provides a modification product composition comprising a plurality of modification product oligonucleotides;

d) a post-modification capping step comprising:

contacting a modification product composition with a post-modification capping reagent system; and

capping one or more functional groups of a plurality of oligonucleotides of the modification product composition;

wherein the post-modification capping step provides a post-modification capping product composition comprising a plurality of post-modification capping product oligonucleotides;

e) optionally a de-blocking step comprising:

contacting a modification product composition, or a post-modification capping product composition, with a de-blocking reagent system;

wherein the deblocking step provides a de-blocking product composition comprising a plurality of de-blocking product oligonucleotides, each of which independently comprises a free hydroxyl group; and

f) optionally repeating steps b) through e) a number of times.

2. A method for preparing a composition comprising a plurality of oligonucleotides comprising one or more cycles, each cycle independently comprises:

a) a coupling step comprising:

contacting a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is de- blocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and coupling a partner compound with the free hydroxyl groups of a plurality of de- blocked oligonucleotides or nucleosides;

wherein the coupling step provides a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

b) a pre-modification capping step comprising:

contacting a coupling product composition with a pre-modification capping reagent system; and

capping one or more functional groups of the coupling product composition;

wherein the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides;

c) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more internucleotidic linkages of one or more coupling product oligonucleotides; or

contacting a pre-modification capping product composition with a modification reagent system and modifying one or more linkages of one or more pre-modification capping product oligonucleotides;

wherein the modification step provides a modification product composition comprising a plurality of modification product oligonucleotides;

d) a post-modification capping step comprising:

contacting a modification product composition with a post-modification capping reagent system; and

capping one or more functional groups of a plurality of oligonucleotides of the modification product composition;

wherein the post-modification capping step provides a post-modification capping product composition comprising a plurality of post-modification capping product oligonucleotides;

e) optionally a de-blocking step comprising:

contacting a modification product composition, or a post-modification capping product composition, with a de-blocking reagent system;

wherein the deblocking step provides a de-blocking product composition comprising a plurality of de-blocking product oligonucleotides, each of which independently comprises a free hydroxyl group.

3. The method claim 2, wherein there are no steps other than modification steps between the pre-modification capping and post-modification capping steps, wherein a post-modification capping step comprises contacting a modification product composition comprising a plurality of modification product oligonucleotides, each of which independently comprises a linkage phosphorus bonded to an atom that is not oxygen.

4. The method of claim 3, wherein the pre-modification capping reagent system caps a plurality of non-hydroxyl groups of a plurality of coupling product oligonucleotides, and a modification step that comprises sulfurization, which sulfurization provides a modification product composition comprising a plurality of modification product oligonucleotides, each of which independently comprises a P=S moiety.

5. The method of claim 4, wherein the method comprises a post-modification capping step, comprising contacting a modification product composition comprising a plurality of modification product oligonucleotide, each of which independently comprises a linkage that comprises at least one chirally controlled chiral center in that at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% oligomeric compounds within the modification product composition comprising the chiral center and having the same constitution share the same stereochemical configuration at the chiral center.

6. The method of claim 5, wherein the method comprises a post-modification capping step, and a coupling reagent system comprising a chiral partner compound that comprises a monomelic unit of the oligomeric compound, wherein the chiral partner compound comprises a chiral atom that is not within the monomeric unit.

7. The method of claim 3, wherein the method comprises a coupling step, a modification step, and one or more pre-modification capping steps between the coupling step and the modification step, wherein for each pre-modification capping step between the coupling step and the modification step, the pre-modification capping step caps amino groups selectively over hydroxyl groups.

8. A method for preparing an oligonucleotide, comprising:

providing a chiral nucleoside phosphoramidite which comprises a chiral atom that is not the phosphorus atom or a sugar carbon atom; and

a post-modification capping step after a modification step but before the next de-blocking step or the next coupling step.

9. A method for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises:

a pre-modification capping step immediately before a modification step, which comprises sulfurization or oxidation, and a post-modification capping step immediately after a modification step.

10. A method for preparing an oligonucleotide, comprising:

providing an oligonucleotide intermediate comprising a chiral linkage phosphorus atom, which is bonded to a chiral center which is not within a nucleoside unit; and

a post-modification capping step after a modification step but before the next de-blocking step or the next coupling step.

11. The claim of any one of claims 8-10, comprising a coupling step.

12. The claim of claim 11, comprising a pre-modification capping step.

13. The claim of claim 12, comprising a modification step.

14. The claim of claim 13, comprising a de-blocking step.

15. A method for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises the following steps:

(1) coupling;

(2) a pre-modification capping;

(3) a modification step;

(4) a post-modification capping; and

(5) de-blocking.

16. A method for oligonucleotide synthesis, comprising:

one or more pre-modification capping steps after a coupling step and before the next modification step,

wherein the capping condition of each pre-modification capping step after a coupling step and before the next modification step is independently selective or specific for amidation over esterification.

17. A method for oligonucleotide synthesis, comprising:

one or more pre-modification capping steps after a coupling step and before the next modification step,

wherein each pre-modification capping step after a coupling step and before the next modification step independently comprises no strong nucleophile, or if it comprises one or more strong nucleophiles, the level of each of the one or more strong nucleophiles is independently no more than no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.

18. A method for oligonucleotide synthesis, comprising:

one or more pre-modification capping steps after a coupling step and before the next modification step,

wherein each pre-modification capping step after a coupling step and before the next modification step independently comprises no esterification catalyst, or if it comprises one or more esterification catalysts, the level of each of the one or more esterification catalysts is independently reduced compared to an appropriate reference capping condition.

19. A method for oligonucleotide synthesis, comprising:

one or more pre-modification capping steps after a coupling step and before the next modification step,

wherein each pre-modification capping step after a coupling step and before the next modification step independently comprises no esterification catalyst, or if it comprises one or more esterification catalysts, the level of each of the one or more esterification catalysts is independently no more than no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalents relative to the first incorporated nucleoside of the oligonucleotide.

20. The claim of any one of claims 16-19, comprising a post-modification capping step.

21. The claim of claim 20, comprising a de-blocking step.

22. The method of claim 21, comprising a coupling step wherein the plurality of the de-blocked oligonucleotides or nucleosides is loaded on a support.

23. The method of claim 22, wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof.

24. The method of claim 23, wherein for each coupling step, each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII.

25. The method of claim 24, wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each LP is independently of formula VII or a salt form thereof, wherein each PL is not P, or wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each LP is independently of formula VII or a salt form thereof, wherein each PL is independently P(=W), wherein W is O or S, or wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each LP is independently of formula VII or a salt form thereof, wherein each –X–Ls–R5 independently contains no free amino group, or

wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each LP is independently of formula VII or a salt form thereof, wherein each –X–Ls–R5 is independently –L7–R1.

26. The method of claim 25, wherein at least one ofR5 or R6 is –C(O)CH3.

27. The method of claim 24, wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each LP is independently of formula VII or a salt form thereof, wherein each X is independently –O– or –S–; and/or wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each LP is independently of formula VII or a salt form thereof, wherein each of Y and Z is –O–; and/or wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each –X–Ls–R5 independently contains no free amino group; and/or wherein each oligonucleotide of the plurality of the de-blocked oligonucleotides independently has the structure of formula VIII or a salt thereof, wherein each R5s is –OH.

28. The method of claim 24, wherein a de-blocked oligonucleotide composition comprising a plurality of de-blocked oligonucleotides is a chirally controlled oligonucleotide composition, wherein:

oligonucleotides of the plurality share the same constitution; and

oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage; and

wherein no less than ((DS)Nc*100)% of all oligonucleotides sharing the same base sequence in the de-blocked product composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.

29. The method of claim 28, wherein a partner compound is of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, VI-a, VI-b, VI-c-1, VI-c-2, VI-d, or VI-e, or a salt thereof; or wherein a partner compound is a phosphoramidite of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, VI-a, VI-b, VI-c-1, VI-c-2, VI-d, or VI-e, or a salt thereof, wherein PL is P; or wherein for each coupling step, a partner compound is a phosphoramidite of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, VI-a, VI-b, VI-c-1, VI-c-2, VI-d, or VI-e, or a salt thereof, wherein PL is P.

30. The method of claim 29, wherein a coupling reagent system comprises a partner compound and an activator, wherein an activator is an optionally substituted tetrazole, or wherein an activator is selected from cyanomethyl imidazole triflate, cyanomethyl pyrrolidine triflate, ETT, phenyl(2H-tetrazol-5-yl)methanone, 2-(dimethylamino)acetonitrile/trifluorosulfonic acid(2/1), 2-(1H-imidazol-1-yl)acetonitrile/trifluorosulfonic acid(2/1), and 2-(pyrrolidin-1-yl)acetonitrile /trifluorosulfonic acid(2/1) or wherein an activator is CMTMT, or wherein an activator is CMPT, or wherein an activator is ETT.

31. The method of claim 29, wherein each coupling step independently forms an internucleotidic linkage of formula VII-b, or a salt form thereof.

32. The method of claim 31, wherein a coupling product composition comprising a plurality of coupling product oligonucleotides, and the coupling product composition comprising the plurality of oligonucleotides is a chirally controlled oligonucleotide composition wherein:

oligonucleotides of the plurality share the same constitution; and

oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;

wherein at least ((DS)Nc*100)% of all oligonucleotides sharing the same base sequence in the coupling product composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.

33. The method of claim 31, comprising a pre-modification capping reagent system which comprises an acylating agent and a base.

34. The method of claim 33, wherein a pre-modification capping reagent system comprises an acylating agent, wherein the acylating agent is of formula R'–C(O)–Ls–Rs.

35. The method of claim 34, wherein a base is of formula N(R3), wherein the nitrogen atom has no alpha-substitution.

36. The method of claim 35, wherein a pre-modification capping step is selective for amidation over esterification.

37. The method of claim 36, wherein no more than 1%, 5%, 10%, 20%, 30%, 40%, or 50% of free hydroxyl groups is converted into –O–C(O)R.

38. The method of claim 35, wherein a pre-modification capping step caps at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of free primary and secondary amino groups of a coupling product composition.

39. The method of claim 38, wherein a pre-modification capping reagent system is a solution of 2,6-Lutidine/Ac2O.

40. The method of claim 33, comprising a modification step which comprises modifying an internucleotidic linkage formed in the immediate preceding coupling step.

41. The method of claim 40, wherein a modification step comprises modifying an internucleotidic linkage of formula VII or a salt thereof, wherein PL is P to form an internucleotidic linkage of formula VII or a salt form thereof, wherein PL is not P.

42. The method of claim 40, wherein a modification step comprises sulfurization, which sulfurization comprises converting a –P(–)– linkage phosphorus atom into a –P(=S)(–)– linkage phosphorus atom, or wherein a modification step comprises sulfurization, which sulfurization comprises converting an internucleotidic linkage of formula VII-b or a salt form thereof, wherein each of X, Y, and Z is –O–, and –X–Ls–R5 comprises a –Si(R)3 group, into an internucleotidic linkage of formula VII or a salt thereof, wherein PL is P(=O), and X is –S–, Y is –O–, and Z is –O–.

43. The method of claim 42, wherein a modification reagent system is a sulfurization reagent system comprising one or more sulfurization reagent, wherein wherein a sulfurization reagent system comprises a sulfurization reagent selected from POS (3-phenyl-1,2,4-dithiazolin-5-one), DDTT (((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione), DTD (dimethylthiarum disulfide), xanthane hydride (XH), S-(2-cyanoethyl) methanesulfonothioate (MTS-CNE), or phenylacetyl disulfide.

44. The method of claim 42, comprising a post-modification capping step which caps a plurality of hydroxyl groups.

45. The method of claim 44, wherein a post-modification capping reagent system is a solution of 2,6-Lutidine/NMI/Ac2O.

46. The method of claim 44, comprising a de-blocking step, wherein a de-blocking reagent system comprises a de-blocking reagent, wherein the de-blocking reagent is an acid.

47. The method of claim 46, comprising a cleavage/deprotection step that comprises:

contacting a plurality of oligonucleotides with one or more cleavage/deprotection reagent systems;

wherein the cleavage/deprotection step provides a final product composition comprising a plurality of final product oligonucleotides.

48. The method of claim 47, wherein a final product composition comprising a plurality of final product oligonucleotides, each of which is independently an oligonucleotide of formula VIII or a salt thereof.

49. The method of of claim 48, wherein a final product composition comprising a plurality of final product oligonucleotides is a chirally controlled oligonucleotide composition wherein: oligonucleotides of the plurality share the same constitution; and

oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;

wherein at least ((DS)Nc*100)% of all oligonucleotides sharing the same base sequence in the final product composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage.

50. The method of any one of the preceding claims, wherein the method provides a final product composition with at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% crude purity, wherein the crude purity is % full-length product.

51. A crude chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein:

oligonucleotides of the plurality share the same base sequence;

oligonucleotides of the plurality share the same pattern of backbone linkages; and

oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;

wherein at least ((DS)Nc*100)% of all oligonucleotides sharing the same base sequence in the crude composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage; or

a crude chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein:

oligonucleotides of the plurality share the same constitution; and

oligonucleotides of the plurality comprise at least one chirally controlled internucleotidic linkage, which internucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the internucleotidic linkage;

wherein at least ((DS)Nc*100)% of all oligonucleotides sharing the same base sequence in the crude composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled internucleotidic linkage;

wherein the crude chirally controlled oligonucleotide composition has a crude purity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%; or

a composition, comprising a plurality of oligonucleotides and a reagent of a reagent system, wherein:

the plurality of oligonucleotides is a plurality of oligonucleotides of a modification product composition of any one of the preceding claims;

the reagent system is a pre-modification or post-modification capping reagent system of any one of the preceding claims; and

the post-modification capping reagent system is in contact with the plurality of oligonucleotides.

52. The method or composition of any one of the preceding claims, wherein oligonucleotides of a plurality have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases, or wherein the final product composition is a chirally controlled oligonucleotide composition of WV-887, WV-892, WV-896, WV-1714, WV-2444, WV-2445, WV-2526, WV-2527, WV-2528, WV-2530, WV-2531, WV-2578, WV-2580, WV-2587, WV-3047, WV-3152, WV-3472, WV-3473, WV-3507, WV-3508, WV-3509, WV-3510, WV-3511, WV-3512, WV-3513, WV-3514, WV-3515, WV-3545, WV-3546, WV-2603, WV-2595, WV-1510, WV- 2378, WV-2380, WV-1092, WV-1497, WV-1085, WV-1086, or WV-2623.

53. A method or composition of any one of Example Embodiments 1-46