0001] This application claims priority to United States Provisional Application No. 62/821,423, filed March 20, 2019, the entirety of which is incorporated herein by reference.

BACKGROUND

[0002] Oligonucleotides may contain a variety of modifications. Certain modifications, such as phosphorothioate intemucleotidic 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, e.g., 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 intemucleotidic linkages, etc., have been described that can improve oligonucleotide properties and usefulness.

Moreover, technologies that permit control of stereochemistry, and/or preparation of chirally controlled oligonucleotide compositions have been demonstrated to provide particularly useful and effective oligonucleotide compositions. Certain exemplary useful technologies are described, for example, in one or more of: US 20150211006, US 20170037399, US 20180216107, US 20180216108, US 20190008986, US 20180216107, US 20180216108, US 20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2018/067973, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, WO 2019/032607, etc., each of which is incorporated herein by reference.

[0005] Particularly given the demonstrated desirability and usefulness of chirally controlled oligonucleotide compositions, the present Applicant appreciated that developments of technologies that could improve or facilitate production of oligonucleotide compositions, particularly chirally controlled oligonucleotide compositions, could provide significant benefits. The present disclosure describes certain such developments, and provides technologies relating to oligonucleotide compositions, particularly to chirally controlled 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 technologies, including conditions and/or sequences of steps, etc., that have been utilized in the preparation of oligonucleotides, particularly chirally controlled (e.g., stereopure) oligonucleotide compositions, can be associated with generation of certain impurities and/or use of certain reagents and/or conditions, the associated production cost of which could be further lowered, and the associated operation of which can be further improved. In some embodiments, the present disclosure thus identifies the source of a problem and/or challenge with strategies that have utilized such technologies. In some embodiments, the present disclosure provides technologies (e.g., reagents, conditions, reactions, sequences of steps, cycles, methods, etc.) that are described and demonstrated to dramatically improve crude product purity and yield, significantly increase operation efficiency and/or reduce production cost.

[0007] For example, in some embodiments, certain provided technologies utilize chiral auxiliaries that can be readily removed using bases without the utilization of HF, and/or basic conditions and/or elevated temperatures that may cause significant break of oligonucleotide chain and/or undesired transformation of certain intemucleotidic linkages (e.g., formation of natural phosphate linkage from phosphorothioate intemucleotidic linkages (or precursors thereof) and/or neutral intemucleotidic linkage (or precursors thereof)).. Such technologies can provide new chemical compatibility, and can provide high stereoselectivity, cmde purity and yield as demonstrated herein. In some embodiments, a useful compound is a compound of formula I, I-a, I-a-1, I-a-2, I-b, I-c, I-d, I-e, II, II-a, II-b, III, III-a, or III-b, or a salt thereof. Among other things, such compounds may be utilized as chiral auxiliaries, e.g., for preparation of chirally controlled oligonucleotide compositions as demonstrated here.

[0008] 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.

[0009] 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 intemucleotidic linkage but before the next de-blocking and/or coupling step.

[0010] 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.

[0011] 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).

[0012] 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 intemucleotidic 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.

[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 or a sugar carbon atom; and

a capping step immediately following a sulfurization or oxidation step.

[0014] 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.

[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 a nucleoside unit or a part thereof; and

a capping step immediately following a sulfurization or oxidation step.

[0016] 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.

[0017] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising:

(1) a coupling step;

(2) optionally a pre-modification capping step;

(3) a modification step;

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

(5) optionally a de-blocking step.

[0018] Example coupling steps, pre -modification capping steps, modification steps, post-modification capping steps, and de-blocking steps are described herein. In some embodiments, a cycle comprises all optional steps.

[0019] In some embodiments, the present disclosure provides a method, e.g., for preparing a composition comprising a plurality of oligonucleotides comprising:

(1) 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 intemucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

(2) 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;

(3) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more intemucleotidic 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;

(4) 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;

(5) 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

(6) optionally repeating steps (1) through (5) a number of times (e.g., 1-100, 1-90, 1-80, 1-70, 1- 60, 1-50, 1-40, 1-30, 1-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, or 25, etc.; or such that a desired length of an oligonucleotide has been achieved).

[0020] 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.

[0021] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises:

(1) a coupling step;

(2) optionally a pre-modification capping step;

(3) a modification step;

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

(5) optionally a de-blocking step.

[0022] In some embodiments, the present disclosure provides a method, e.g., for preparing a composition comprising a plurality of oligonucleotides comprising one or more cycles, each cycle independently comprising:

(1) 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 intemucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

(2) 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;

(3) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more intemucleotidic 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;

(4) 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;

(5) 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.

[0023] 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. In some embodiments, a cycle comprises a coupling step, a pre -modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, a cycle comprises a coupling step, a pre -modification capping step, a modification step, and a de-blocking step. In some embodiments, a cycle comprises a coupling step, a modification step, a post-modification capping step and a de-blocking step. In some embodiments, comprise a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, one or more cycles comprise a coupling step, a pre-modification capping step, a modification step, and a de-blocking step. In some embodiments, one or more cycles comprise a coupling step, a modification step, a post-modification capping step and a de -blocking step.

[0024] In some embodiments, a cycle comprises one or more but not all optional steps. In some embodiments, a cycle comprises a pre -modification step. In some embodiments, a cycle does not contain a pre-modification step. In some embodiments, a cycle comprises a post-modification step. In some embodiments, a cycle does not contain a post-modification step. In some embodiments, a cycle does not contain a de-blocking step, e.g., the last cycle when a desired length of an oligonucleotide is achieved.

[0025] In some embodiments, each step in a cycle is independently selected from a coupling step, a pre -modification capping step, a modification step, a post-modification capping, and a de-blocking step.

In some embodiments, a cycle may comprise two or more of the same steps (e.g., two coupling steps), each of which may utilize the same or different reagents, conditions, etc. As appreciated by those skilled in the art, in some embodiments, e.g., when a reaction of a step does not go to completion, it may be beneficial to repeat such a step, either immediately after the first such step or after one or more next steps. In many embodiments, repeat is performed before a next de-blocking step is performed. For example, in some embodiments, a coupling step is repeated immediately after another coupling step one or more times. In some embodiments, a sequence of steps (e.g., (1)-(2), (1)-(3), (1)-(2)-(3), (1)-(3)-(4), (1)-(2)-(3)-(4)) is repeated one or more times.

[0026] As used in the present disclosure, in some embodiments, “one or more” is one. In some embodiments, “one or more” is two or two or more. In some embodiments, “one or more” is 1-200, 1- 150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 5-200, 5-150, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 15-200, 15-150, 15-100, 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20,

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32, 33, 34, 35, 36, 37, 38, 39, 40, or more.

[0027] As used in the present disclosure, in some embodiments, “at least one” is one. In some embodiments, “at least one” is two or two or more. In some embodiments, “at least one” is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 5-200, 5-150, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 15-200, 15-150, 15-100, 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, or more.

[0028] In some embodiments, one or more cycles comprise a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step, and one or more cycles comprise a coupling step, a pre-modification capping step, a modification step, and a de-

blocking step. In some embodiments, one or more cycles comprise a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step, and one or more cycles comprise a coupling step, a modification step, a post-modification capping step and a de-blocking step.

[0029] In some embodiments, a cycle consists of a coupling step, a pre -modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, a cycle consists of a coupling step, a pre-modification capping step, a modification step, and a de-blocking step. In some embodiments, a cycle consists of a coupling step, a modification step, a post-modification capping step and a de -blocking step.

[0030] In some embodiments, a coupling step, e.g., in a cycle, is immediately followed by a pre-modification capping step. In some embodiments, a coupling step, e.g., in a cycle, is immediately followed by a modification step. In some embodiments, a coupling step, e.g., in a cycle, is immediately followed by a modification step which comprises an oxidation reaction converting a P(III) linkage into a P(VI) phosphate linkage (e.g., comprising installation of =O to a P(III) linkage phosphorus). In some embodiments, a pre-modification step, e.g., in a cycle, is immediately followed by a modification step. In some embodiments, a modification step comprises formation of a bond (e.g., single bond, double bond, etc.) between a linkage phosphorus and a sulfur or a nitrogen atom. In some embodiments, a modification step is immediately followed by a post-modification capping step. In some embodiments, a post-modification capping step is immediately followed by a de-blocking step.

[0031] As appreciated by those skilled in the art, one or more washes (e.g., of oligonucleotides on support) using various suitable solvents (in some embodiments, can be mixtures of chemicals) may be included in steps described herein and may be performed before and/or after reactions of such steps, e.g., those steps in various methods and/or cycles described herein. For example, as demonstrated herein in the Examples, after performing a reaction of a step, oligonucleotides on solid support are typically extensive washed (e.g., to remove excess reagents, to remove undesired products, to switch solvents, to condition for a next reaction, etc.) before performance of another reaction of the same or an immediate following step. In some embodiments, a step described herein, e.g., a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, or a de-blocking step, optionally comprises one or more washes. In some embodiments, as demonstrated in the Examples, a reagent, solvent, and/or reagent system for a reaction is removed after the reaction is performed. In some embodiments, removal is performed by filtration and/or washes when product oligonucleotides are on solid support, e.g., when using solid support for oligonucleotide synthesis.

[0032] 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 intemucleotidic 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.

[0033] 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), abase (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 intemucleotidic 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.

[0034] 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).

[0035] 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 esterification 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.

[0036] 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.

[0037] 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-substituents. 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.

[0038] 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.

[0039] 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., abase 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).

[0040] 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.

[0041] In some embodiments, the present disclosure provides a method, e.g., for preparing an

oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0042] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0043] 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 intemucleotidic 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.

[0044] 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.

[0045] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0046] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0047] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0048] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0049] 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.

[0050] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0051] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, wherein:

each cycle independently forms an intemucleotidic linkage;

each cycle independently comprising a coupling step, one or more capping steps, and a modification step, which coupling step forms an intemucleotidic linkage, and which modification step modifies the intemucleotidic 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.

[0052] 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, cmde purity, and/or yield.

[0053] 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 orNMI. 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.

[0054] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles each independently comprising:

(1) a coupling step;

(2) optionally a first capping step;

(3) a modification step;

(4) optionally a second capping step; and

(5) optionally a de-blocking step.

[0055] In some embodiments, a cycle comprises a first capping step. In some embodiments, a cycle comprises a second capping step. In some embodiments, a cycle comprises a first and a second capping step. In some embodiments, a cycle comprises a de-blocking step. In some embodiments, a cycle comprises a first capping step and a de-blocking step. In some embodiments, a cycle comprises a second capping and a de-blocking step.

[0056] In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles each independently comprising:

(1) a coupling step;

(2) a first capping step;

(3) a modification step;

(4) a second capping step; and

(5) a de-blocking step.

[0057] In some embodiments, the present disclosure provides a method, e.g., for preparing an

oligonucleotide, comprising one or more cycles each independently comprising:

(1) a coupling step;

(2) a pre-modification capping step;

(3) a modification step;

(4) a post-modification capping; and

(5) a de-blocking step.

[0058] In some embodiments, provided methods comprise repeating a number of steps or cycles, e.g., until a desired length (e.g., a desired length of an oligonucleotide or an intermediate) is achieved.

[0059] Some or all steps in a provided method or a cycle may be performed in certain orders. In some embodiments, an order is, or comprises (1)-(2), ( l)-(3), (2)-(3), (3)-(4), (3)-(5), (4)-(5), (5)-(1), (1)-(2)-(3), (1)-(3)-(4), (1)-(2)-(3)-(4) or any combination thereof (as appreciated by those skilled in the art, each of (1)-(5) independently represents a corresponding step of a method or a cycle, e.g., (1) - a coupling step; (2)- a pre -modification capping step or a first capping step; (3) - a modification step; (4) - a post-modification capping or a second capping step; (5)- a de-blocking step). In some embodiments, an order is or comprises (1)-(2). In some embodiments, an order is or comprises (1)-(3). In some embodiments, an order is or comprises (2)-(3). In some embodiments, an order is or comprises (3)-(4). In some embodiments, an order is or comprises (3)-(5). In some embodiments, an order is or comprises (4)-(5).

In some embodiments, an order is or comprises (5)-(1). In some embodiments, an order is or comprises (1)-(2)-(3). In some embodiments, an order is or comprises (1)-(3)-(4). In some embodiments, an order is or comprises (1)-(2)-(3)-(4). In some embodiments, an order is or comprises (1)-(2)-(3)-(5). In some embodiments, an order is or comprises (1)-(3)-(4)-(5). In some embodiments, an order is or comprises (1)-(2)-(3)-(4)-(5). In some embodiments, an order is or comprises (5)-(1)-(2)-(3). In some embodiments, an order is or comprises (5)-(1)-(3)-(4). In some embodiments, an order is or comprises (5)-(1)-(2)-(3)-(4).

[0060] In some embodiments, a cycle or each cycle independently consists of steps (1)-(5). In some embodiments, a cycle or each cycle independently consists of steps (1)-(5) in the order of (1)-(2)-(3)-(4)-(5). In some embodiments, a cycle or each cycle independently consists of steps (1)-(5) in the order of (5)-(1)-(2)-(3)-(4). In some embodiments, a cycle or each cycle independently consists of steps (1)-(5) in the order of (1)-(2)-(4)-(3)-(5). In some embodiments, a cycle or each cycle independently consists of steps (1)-(5) in the order of (5)-(1)-(2)-(4)-(3).

[0061] In some embodiments, a provided method optionally or additionally comprises one or more cycles each independently comprising steps (1), (2), (3), and (5), optionally in that order. In some embodiments, a provided method optionally or additionally comprises one or more cycles each independently consisting of steps (1), (2), (3), and (5), optionally in that order. In some embodiments,

such a cycle provides a natural phosphate linkage, e.g., optionally after cleavage, deprotection, etc. of an oligonucleotide. In some embodiments, such a cycle provides a non-chirally controlled phosphorothioate intemucleotidic linkage, e.g., optionally after cleavage, deprotection, etc. of an oligonucleotide. In some embodiments, each such cycle independently provides a natural phosphate linkage, e.g., optionally after cleavage, deprotection, etc. of an oligonucleotide. In some embodiments, each natural phosphate linkage in an oligonucleotide is independently formed by such a cycle optionally after cleavage, deprotection, etc. of an oligonucleotide.

[0062] In some embodiments, a provided method optionally or additionally comprises one or more cycles each independently comprising steps (1), (3), (4), and (5), optionally in that order. In some embodiments, a provided method optionally or additionally comprises one or more cycles each independently consisting of steps (1), (3), (4), and (5), optionally in that order. In some embodiments, such a cycle provides a natural phosphate linkage, e.g., optionally after cleavage, deprotection, etc. of an oligonucleotide. In some embodiments, such a cycle provides a non-chirally controlled phosphorothioate intemucleotidic linkage, e.g., optionally after cleavage, deprotection, etc. of an oligonucleotide. In some embodiments, each such cycle independently provides a natural phosphate linkage, e.g., optionally after cleavage, deprotection, etc. of an oligonucleotide. In some embodiments, each natural phosphate linkage in an oligonucleotide is independently formed by such a cycle optionally after cleavage, deprotection, etc. of an oligonucleotide.

[0063] In some embodiments, a first capping comprising no strong nucleophiles, or if any, of reduced levels, as described in the present disclosure. In some embodiments, a first capping step comprises no esterification catalysts, of if any, of reduced levels. In some embodiments, a first capping step comprises a selective condition for amidation over esterification. In some embodiments, a first capping step comprises no condition identical to or comparable to an appropriate reference condition.

[0064] In some embodiments, a first capping step is a pre -modification capping step as described in the present disclosure. In some embodiments, a first capping step utilizes a capping reagent system that is a pre-modification capping reagent system. In some embodiments, a second capping step is a post-modification capping step as described in the present disclosure. In some embodiments, a second capping step utilizes a capping reagent system that is a post-modification capping reagent system.

[0065] In some embodiments, an appropriate reference capping condition is a capping condition of traditional oligonucleotide synthesis based on phosphoramidite chemistry. Example cycles for traditional phosphoramidite-based oligonucleotide synthesis are described below, wherein the illustrated modification step is an oxidation step installing P=O:

[0066] In some embodiments, a cycle of is a DPSE cycle depicted below (DPSE chiral auxiliary

compounds: for different configurations of linkage

phosphorus):

[0067] In some embodiments, a cycle of is a PSM cycle depicted below (PSM auxiliary compounds:

for different configurations of linkage phosphorus):

[0068] In some embodiments, Capping- 1 is a pre-modification or first capping step. In some embodiments, Capping-2 is a post-modification or second capping step. In some embodiments, cycle exit is after de-blocking before the next coupling. In some embodiments, cycle exit is before de-blocking (e.g., to keep a 5’-blocking group such as DMTr on). In some embodiments,
is nucleoside, nucleotide, or oligonucleotide on support (optionally linked to support via a linker). Example steps, reagents, modifications, intermediates and products, etc., are illustrated. Those skilled in the art will appreciate that other steps, reagents, modifications, intermediates and products, etc., may also be utilized in accordance with the present disclosure.

[0069] In some embodiments, a first or pre-modification capping step comprises reduced levels of a strong nucleophilic base or no strong nucleophilic base. In some embodiments, a first or pre -modification capping step comprises a reduced level of NMI. In some embodiments, a first or pre-modification

capping step comprises no NMI. In some embodiments, a first or pre -modification capping step comprises a reduced level of DMAP. In some embodiments, a first or pre-modification capping step comprises no DMAP. In some embodiments, a second or post-modification capping step comprises a strong nucleophilic base. In some embodiments, a second or post-modification capping step comprises NMI. In some embodiments, a second or post-modification capping step comprises DMAP.

[0070] In some embodiments, a reduced level of the present disclosure is no more than a percentage, e.g., 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, etc., by volume of a capping reagent solution. In some embodiments, a percentage is 0.01%. In some embodiments, a percentage is 0.02%. In some embodiments, a percentage is 0.05%. In some embodiments, a percentage is 0.1%. In some embodiments, a percentage is 0.2%. In some embodiments, a percentage is 0.5%. In some embodiments, a percentage is 1%. In some embodiments, a percentage is 2%. In some embodiments, a percentage is 3%. In some embodiments, a percentage is 4%. In some embodiments, a percentage is 5%.

[0071] 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 a reference agent. In some embodiments, a reference agent is an acylating agent. In some embodiments, a reference agent is a support (by oligonucleotide loading capacity). 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, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 equivalents relative to oligonucleotide. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 equivalents relative to the first nucleoside incorporated to an oligonucleotide. In many instances, equivalent of the first nucleoside incorporated into an oligonucleotide to oligonucleotide loading capacity of a support used to prepare the oligonucleotide is 1. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or

100 equivalents relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, or 10 equivalents relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, or 5 equivalents relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalent relative to oligonucleotide loading capacity of a support. In some embodiments, a reduced level is no more than about 0.01 equivalent. In some embodiments, a reduced level is no more than about 0.02 equivalent. In some embodiments, a reduced level is no more than about 0.05 equivalent. In some embodiments, a reduced level is no more than about 0.1 equivalent. In some embodiments, a reduced level is no more than about 0.2 equivalent. In some

embodiments, a reduced level is no more than about 0.5 equivalent. In some embodiments, a reduced level is no more than about 1 equivalent. In some embodiments, a reduced level is no more than about 1.1 equivalents. In some embodiments, a reduced level is no more than about 1.2 equivalents.

[0072] Among other things, provided technologies are particularly useful for preparing chirally controlled oligonucleotide compositions. In some embodiments, provided technologies comprise formation of one or more chiral intemucleotidic linkages each independently comprising a chiral linkage phosphorus, wherein each of the chiral linkage phosphorus chiral center is independently formed with a stereoselectivity as described in the present disclosure, e.g., of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0073] In some embodiments, provided technologies comprise use of one or more chiral auxiliary to stereoselectively form one or more chirally controlled intemucleotidic linkages. In some embodiments, provided technologies comprise providing monomeric phosphoramidites of diastereomeric purity as described in the present disclosure, e.g., of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In many embodiments, phosphoramidites of provided diastereomeric purity comprise a chiral auxiliary moiety. In some embodiments, phosphoramidites of traditional oligonucleotide synthesis are utilized for non-chirally controlled intemucleotidic linkages, and/or non-chiral intemucleotidic linkages. Suitable chiral auxiliaries and phosphoramidites for chirally controlled oligonucleotide synthesis that can be utilized in accordance with the present disclosure include those described in US 9598458, US 9744183, US 9605019, US 9394333, US 8859755, US 20130178612, US 8470987, US 8822671, US 20150211006, US 20170037399, US 20180216107, US 20180216108, US 20190008986, US 20180216107, US 20180216108, US 20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/022473, WO 2018/067973, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, or WO 2019/217784, chiral auxiliaries and phosphoramidites of each of which are independently incorporated herein by reference. In some embodiments, a chiral auxiliary is of formula I, I-a, I-a-1, I-a-2, I-b, I-c, I-d, I-e, II, II-a, II-b, III, III-a, or III-b, or a salt thereof, as described in the present disclosure. In some embodiments, a phosphoramidite has the structure of formula IV, IV-a, IV-b, IV-c-1, IV-c-2, IV-d, IV-e, IVa, IVa-a, IVa-b, IVa-c-1, IVa-c-2, IVa-d, IVa-e, V, V-a, V-b, V-c-1, V-c-2, V-d, V-e, VI, VI-a, VI-b, VI-c-1, VI-c-2, VI-d, or VI-e, or a salt thereof, as described in the present disclosure.

[0074] In some embodiments, provided technologies comprising formation of an intemucleotidic linkage having the stmcture of formula VII, VII- a- 1, VII-a-2, VII-b, VII-c, VII-d, VII-e, NL-n-1, NL-n-2, NL-n-3, NL-n-4, NL, NL-a-1, NL-a-2, NL-b-1, NL-b-2, NL-c-1, NL-c-2, NL-d-1, NL-d-2, or a

salt form thereof, as described in the present disclosure.

[0075] In some embodiments, provided technologies provides oligonucleotides as intermediates and/or products. In some embodiments, a provided oligonucleotide is of formula O-I or a salt thereof as described in the present disclosure. In some embodiments, an oligonucleotide, e.g., a final product, a product of a reaction, a product of a step, e.g., is one described in US 9598458, US 9744183, US 9605019, US 9394333, US 8859755, US 20130178612, US 8470987, US 8822671, US 20150211006, US 20170037399, US 20180216107, US 20180216108, US 20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/022473, WO 2018/067973, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, or WO 2019/217784, oligonucleotides of each of which are independently incorporated herein by reference, or shares the same or comprises the base sequence, a sugar modification or patterns thereof, an intemucleotidic linkage or patterns thereof, a base modification or a pattern thereof, and/or a pattern of backbone chiral centers (linkage phosphorus) of such an oligonucleotide. In some embodiments, provided intermediates and/or products are chirally controlled oligonucleotide compositions. In some embodiments, provided intermediates and/or products are chirally controlled oligonucleotide compositions of a plurality of oligonucleotides of formula O-I or salts thereof. In some embodiments, provided intermediates and/or products are chirally controlled oligonucleotide compositions of US 9598458, US 9744183, US 9605019, US 9394333, US 8859755, US 20130178612, US 8470987, US 8822671, US 20150211006, US 20170037399, US 20180216107, US 20180216108, US 20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/022473, WO 2018/067973, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, or WO 2019/217784, oligonucleotide compositions of each of which are independently incorporated herein by reference.

[0076] In some embodiments, the present disclosure provides a composition comprising:

a plurality of oligonucleotides of a modification product composition; and

a post-modification capping reagent system;

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

[0077] In some embodiments, the present disclosure provides a composition comprising:

a capping reagent system comprising a first compound having the structure of formula B-I or B- II,

a plurality of oligonucleotides each comprising at least one intemucleotidic linkage comprising a -C(O)-N(-)- moiety or a -P-S- moiety;

wherein the first compound is at a level of at least 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 equivalents relative to the plurality of oligonucleotides.

[0078] In some embodiments, the present disclosure provides a composition comprising:

a capping reagent system comprising a first compound having the structure of formula B-I or B- II,

a plurality of oligonucleotides, wherein each intemucleotidic linkage of oligonucleotides of the plurality is independently an intemucleotidic linkage comprising a -C(O)-N(-)- moiety and a linkage phosphoms that is tetravalent;

wherein the first compound is at a level of at least 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, or 100 equivalents relative to the plurality of oligonucleotides.

[0079] In some embodiments, the plurality of oligonucleotides is a plurality of oligonucleotides of a modification product composition.

[0080] In some embodiments, in a oligonucleotide composition comprising a plurality of oligonucleotides:

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 intemucleotidic linkage; wherein at least ((DS)Nc* 100)% of all oligonucleotides sharing the same base sequence in the composition are oligonucleotides of the plurality, wherein DS is at least 80%, 85%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98% or 99%, and Nc is the number of chirally controlled intemucleotidic linkage.

[0081] At a chirally controlled intemucleotidic linkage, oligonucleotides of the plurality share the same linkage phosphoms configuration (Rp or rip). In some embodiments, a chirally controlled intemucleotidic linkage is referred to as a “stereodefined” intemucleotidic linkage.

[0082] In some embodiments, a first compound is of formula B-I. In some embodiments, a first compound is of formula B-II. In some embodiments, a first compound is a strong nucleophile as described in the present disclosure. In some embodiments, a first compound is an esterification catalyst as described in the present disclosure. In some embodiments, a first compound is of formula B-I. In some embodiments, a first compound is of formula B-II. In some embodiments, a first compound is a base comprising =N-, wherein there are no substitutions at any alpha-positions relative to the nitrogen of =N-. In some embodiments, a first compound is a base comprising a heteroaryl moiety, which heteroaryl moiety comprises =N-, wherein there are no substitutions at any alpha-positions relative to the nitrogen of =N-.

[0083] In some embodiments, a first compound is NMI. In some embodiments, a first compound is DMAP.

[0084] In some embodiments, oligonucleotides of a plurality are attached to a support, e.g., a solid support used to prepare the oligonucleotides. In some embodiments, molar amount of the oligonucleotides of a plurality equals loading capacity of the solid support they are attached to. In some embodiments, oligonucleotides of a plurality are attached to a support via linkers. Various linkers are known in the art and may be utilized in accordance with the present disclosure; certain examples are described herein.

[0085] In some embodiments, a plurality of oligonucleotides share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the plurality of oligonucleotides share the same linkage phosphorus stereochemistry at one or more chiral intemucleotidic linkages (chirally controlled or stereodefined intemucleotidic linkages). In some embodiments, about 1%-100%, (e.g., 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all intemucleotidic linkages in oligonucleotides of a plurality are chirally controlled intemucleotidic linkages. In some embodiments, in addition to one or more chirally controlled intemucleotidic linkages, one or more intemucleotidic linkages are natural phosphate linkages. In some embodiments, all chiral intemucleotidic linkages which comprise chiral linkage phosphoms are chirally controlled intemucleotidic linkages. In some embodiments, one or more chiral intemucleotidic linkages which comprise chiral linkage phosphoms are stereorandom chiral intemucleotidic linkages (not chirally controlled intemucleotidic linkages, typically prepared by non-chirally controlled methods, e.g., traditional oligonucleotide synthesis without utilization of chiral auxiliaries or chiral modification (e.g., sulfurization) reagents). In some embodiments, oligonucleotides of a plurality share the same constitution. In some embodiments, oligonucleotides of a plurality share the same stmcture (structurally identical). In some embodiments, a plurality of oligonucleotides share the same stereochemistry at least one intemucleotidic linkage comprising a -C(O)-N(-)- moiety or a -P-S- moiety. In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide

composition that share the common base sequence, or that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications, or share the same constitution of oligonucleotides of the plurality, are oligonucleotides of the plurality.

[0086] In some embodiments, oligonucleotides of a plurality are each of the structure of formula O-I or a salt thereof. In some embodiments, oligonucleotides of a plurality are oligonucleotides of US 9598458, US 9744183, US 9605019, US 9394333, US 8859755, US 20130178612, US 8470987, US 8822671, US 20150211006, US 20170037399, US 20180216107, US 20180216108, US 20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/022473, WO 2018/067973, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, WO 2019/032607, WO 2019/055951, WO 2019/075357, WO 2019/200185, or WO 2019/217784, oligonucleotides of each of which are independently incorporated herein by reference. In some embodiments, oligonucleotides of a plurality share the same constitution. In some embodiments, oligonucleotides of a plurality are identical.

[0087] In some embodiments, a level of the present disclosure is at least 0.1 equivalent. In some embodiments, a level is at least 0.2 equivalent. In some embodiments, a level is at least 0.5 equivalent.

In some embodiments, a level is at least 1 equivalent. In some embodiments, a level is at least 2 equivalents. In some embodiments, a level is at least 3 equivalents. In some embodiments, a level is at least 4 equivalents. In some embodiments, a level is at least 5 equivalents. In some embodiments, a level is at least 6 equivalents. In some embodiments, a level is at least 7 equivalents. In some embodiments, a level is at least 8 equivalents. In some embodiments, a level is at least 9 equivalents. In some embodiments, a level is at least 10 equivalents. In some embodiments, a level is at least 20 equivalents.

In some embodiments, a level is at least 50 equivalents. In some embodiments, a level is at least 100 equivalents.

[0088] In some embodiments, a -C(O)-N(-)- is part of a capped amino group in a chiral auxiliary moiety bonded to a linkage phosphorus, wherein the corresponding chiral auxiliary (replacing bonding to -C(O)- of-C(O)-N(-)- with -H, and replacing bonding to the linkage phosphorus with -H) is a compound of formula I, I-a, I-a-1, 1-a-2, 1-b, I-c, I-d, I-e, II, II-a, II-b, III, III-a, III-b, or a salt thereof.

[0089] Among other things, the present disclosure provides oligonucleotide compositions of high crude purity. In some embodiments, the present disclosure provides a crude chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides, wherein:

oligonucleotides of the plurality share the same base sequence;

oligonucleotides of the plurality share the same pattern of backbone linkages; and oligonucleotides of the plurality comprise at least one chirally controlled intemucleotidic linkage,

which intemucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the intemucleotidic 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 intemucleotidic linkage.

[0090] In some embodiments, the present disclosure provides a cmde 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 intemucleotidic linkage, which intemucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphoms of the intemucleotidic linkage;

wherein at least ((DS)Nc* 100)% of all oligonucleotides sharing the same base sequence in the cmde 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 intemucleotidic linkage.

[0091] In some embodiments, a provided cmde chirally controlled oligonucleotide composition has a cmde purity of 30%-80%, 30%-90%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, or more. In some embodiments, a cmde chirally controlled oligonucleotide composition is cleaved from a support, and before any further purification. In some embodiments, cmde chirally controlled oligonucleotide composition is cleaved from a support, after de-salting, and before any further purification. In some embodiments, cmde chirally controlled oligonucleotide composition is before any chromatograph or gel purification. In some embodiments, a cmde purity is % full-length product. In some embodiments, a cmde purity is % full-length product as assessed by LC-UV monitored at UV 260 nm.

[0092] In some embodiments, DS is about 80%-100%, 85%-100%, 87%-100%, 89%-100%, 90-100%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more. In some embodiments, DS is about 85% or more. In some embodiments, DS is about 86% or more. In some embodiments, DS is about 87% or more. In some embodiments, DS is about 88% or more. In some embodiments, DS is about 89% or more. In some embodiments, DS is about 90% or more. In some embodiments, DS is about 91% or more. In some embodiments, DS is about 92% or more. In some embodiments, DS is about 93% or more. In some embodiments, DS is about 94% or more. In some embodiments, DS is about 95% or more. In some embodiments, DS is about 96% or more. In some embodiments, DS is about 97% or more. In some embodiments, DS is about 98% or more. In some embodiments, DS is about 99% or more.

[0093] In some embodiments, diastereoselectivity at, and/or diastereopurity of, chiral linkage phosphorus of a chiral intemucleotidic linkage in an oligonucleotide may be measured or represented through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same intemucleotidic linkage as the chiral intemucleotidic linkage, the 5’-nucleoside of the dimer is the same as the nucleoside to the 5’-end of the chiral intemucleotidic linkage, and the 3 ’-nucleoside of the dimer is the same as the nucleoside to the 3’-end of the chiral intemucleotidic linkage. For example, diastereopurity of the underlined linkage in NNNNNNNG*SGNNNNNNN can be assessed from coupling two G moieties under the same or comparable conditions, e.g., monomers, chiral auxiliaries, solvents, activators, temperatures, etc. In some embodiments, diastereopurity (and/or diastereoselectivity) of the linkage of a dimer (G*SG) is used as diastereopurity (and/or diastereoselectivity) of a corresponding linkage in an oligonucleotide (NNNNNNNG*SGNNNNNNN).

In some embodiments, diastereopurity of a compound comprising multiple chiral elements is product of diastereomeric purity of all its chiral elements. In some embodiments, diastereopurity (i.e.. diastereomeric purity) of a provided oligonucleotide is product of diastereomeric purity of all its chiral linkage phosphoms in its chiral intemucleotidic linkages.

WE CLAIMS

1. 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) optionally a de-blocking step, and

wherein in at least one cycle, the coupling step independently comprises reacting a free hydroxyl group of an oligonucleotide or a nucleoside with a coupling partner compound comprising a chiral

auxiliary group having the structure of wherein:

R2 comprises an electronic-withdrawing group and is -Ls-R’ ;

each Ls is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene, -CºC-, a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R’)2-,

-Cy-, -O-, -S-, -S-S-, -N(R’)-, -C(O)-, -C(S)-, -C(NR’)-, -C(O)N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R’)-, -C(O)S- -C(O)O-, -P(O)(OR’)-, -P(O)(SR’)--P(O)(R’)-, -P(O)(NR’)-, -P(S)(OR’)-, -P(S)(SR’)-, -P(S)(R’)- -P(S)(NR’)-, -P(R’)- -P(OR’)--P(SR’)-, -P(NR’)-, -P(OR’)[B(R’)3]-, -OP(O)(OR’)O-, -OP(O)(SR’)O-, -OP(O)(R’)O--OP(O)(NR’)O-, -OP(OR’)O-, -OP(SR’)O-, -OP(NR’)O-, -OP(R’)O-, or -OP(OR’)[B(R’)3]O-, and one or more carbon atoms are optionally and independently replaced with CyL;

each -Cy- is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each CyL is independently an optionally substituted tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms

independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R’ is independently -R, -C(O)R, -CO2R, or -SO2R;

R6 is -L-R’;

L is a covalent bond, or optionally substituted C1-6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L’-; or L is Ls;

each L’ is independently a covalent bond, optionally substituted bivalent C1-3 alkylene, -C(R3)(R4)-, -C(R3)(R4)-C(R3)(R4)-, -Cy- or -C(R3)[C(R4)3]-; and

each R is independently -H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

2. The method of claim 1, comprising one or more cycles, each cycle independently comprises:

(1) 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 intemucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide with a nucleoside unit of a partner compound;

(2) 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;

(3) a modification step comprising:

contacting a coupling product composition with a modification reagent system comprising a modification reagent, and modifying one or more intemucleotidic 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;

(4) 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;

(5) 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

wherein in at least one cycle, the coupling partner compound independently comprises a chiral auxiliary group having the structure of

wherein:

R2 comprises an electronic-withdrawing group and is -Ls-R’;

each Ls is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene, -CºC-, a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R’)2-,

-Cy-, -O-, -S-, -S-S-, -N(R’)-, -C(O)-, -C(S)- -C(NR’)-, -C(O)N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R’)-, -C(O)S-, -C(O)O-, -P(O)(OR’)-, -P(O)(SR’)-, -P(O)(R’)-, -P(O)(NR’)-, -P(S)(OR’)-, -P(S)(SR’)-, -P(S)(R’)-, -P(S)(NR’)-, -P(R’)-, -P(OR’)-, -P(SR’)-, -P(NR’)-, -P(OR’)[B(R’)3]-, -OP(O)(OR’)O-, -OP(O)(SR’)O-, -OP(O)(R’)O-, -OP(O)(NR’)O-, -OP(OR’)O-, -OP(SR’)O-, -OP(NR’)O-, -OP(R’)O-, or -OP(OR’)[B(R’)3]O-, and one or more carbon atoms are optionally and independently replaced with CyL;

each -Cy- is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each CyL is independently an optionally substituted tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R’ is independently -R, -C(O)R, -CO2R, or -SO2R;

R6 is -L-R’;

L is a covalent bond, or optionally substituted C1-6 alkylene, wherein one or more methylene units are optionally and independently replaced with -L’-; or L is Ls;

each L’ is independently a covalent bond, optionally substituted bivalent C1-3 alkylene,

-C(R3)(R4)-, -C(R3)(R4)-C(R3)(R4)-, -Cy-, or -C(R3)[C(R4)3]-; and

each R is independently -H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

3. The method of claim 1, wherein a cycle comprises a de-blocking step.

4. The method of any one of claims 1-3, wherein a cycle comprises a pre-modification capping step or a post-modification capping step.

5. The method of any one of claims 1-4, wherein a cycle comprises a pre- and a post-modification capping step.

6. The method of claim 1, wherein a cycle comprises a modification step that forms an intemucleotidic linkage of *PS or *PR, wherein *PS is independently of formula

or a salt form thereof, *PR is independently of formula

or a salt form thereof, wherein -X-Ls-R5 is

7. The method of claim 6, wherein a cycle comprises a modification step that forms an intemucleotidic linkage of *NS or *NR, wherein *NS is independently of formula

or a salt form thereof, *NR is independently of formula

or a salt form thereof, wherein -X-Ls-R5 is

8. The method claim 6, wherein R4 and R5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.

9. The method claim 7, wherein R4 and R5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.

10. The method of claim 1, wherein each partner compound comprising a chiral auxiliary group

independently has the structure of

or a salt thereof, wherein:

BA is an optionally substituted group selected from C3-30 cycloaliphatic, C6-30 aryl, C5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety;

s is 0-20;

Ring As is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each of R2s, R4s and R5s is independently Rs;

each Rs is independently -H, halogen, -CN, -N3, -NO, -NO2, -Ls-R’, -Ls-Si(R)3, -Ls-OR’, -Ls-SR’, -Ls-N(R’)2, -O-Ls-R’, -O-Ls-Si(R)3, -O-Ls-OR’, -O-Ls-SR’, or -O-Ls-N(R’)2;

R2 comprises an electronic-withdrawing group and is -Ls-R’ ;

each Ls is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene, -C=C- . a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, -C(R’)2-, -Cy-, -O-, -S-, -S-S-, -N(R’)-, -C(O)-, -C(S)-, -C(NR’)-, -C(O)N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R’)-, -C(O)S-, -C(O)O-, -P(O)(OR’)-, -P(O)(SR’)--P(O)(R’)-, -P(O)(NR’)-, -P(S)(OR’)-, -P(S)(SR’)-, -P(S)(R’)-, -P(S)(NR’)-, -P(R’)-, -P(OR’)--P(SR’)-, -P(NR’)-, -P(OR’)[B(R’)3]-, -OP(O)(OR’)O-, -OP(O)(SR’)O-, -OP(O)(R’)O--OP(O)(NR’)O-, -OP(OR’)O-, -OP(SR’)O-, -OP(NR’)O-, -OP(R’)O-, or -OP(OR’)[B(R’)3]O-, and one or more carbon atoms are optionally and independently replaced with CyL;

each -Cy- is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each CyL is independently an optionally substituted tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R’ is independently -R, -C(O)R, -C(O)OR, or -S(O)2R;

each R is independently -H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

11. The method of claim 10, wherein R4 and R5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.

12. The method of claim 10, wherein each partner compound comprising a chiral auxiliary group

independently has the structure of

13. The method of claim 12, wherein in a partner compound, R4s is -H.

14. The method of claim 13, wherein in a partner compound, R2s is -H.

15. The method of claim 13, wherein in a partner compound, R2s is -OR, wherein R is optionally substituted C1-6 aliphatic.

16. The method of claim 13, wherein in a partner compound, R2s is -O-Si(R)3, wherein each R is independently not -H.

17. The method of claim 13, wherein in a partner compound, R2s is -OTBS, wherein each R is independently not -H.

18. The method of any one of claims 6-17, wherein R2 is -CH2SO2R’, wherein R’ is an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1- 10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms.

19. The method of claim 18, wherein R’ is optionally substituted phenyl.

20. The method of claim 18, wherein R’ is phenyl.

21. The method of claim 18, wherein R’ is tert-butyl.

22. The method of claim 6 or 10, comprising removal of a chiral auxiliary group by contact the oligonucleotides comprising a chiral auxiliary group with a base under an anhydrous condition.

23. The method of claim 22, wherein the base is DEA.

24. The method of claim 6 or 10, wherein a product oligonucleotide comprises a phosphorothioate intemucleotidic linkage.

25. The method of claim 6 or 10, wherein a product oligonucleotide comprises a sugar comprising 2’-OH.

26. The method of claim 24 or 25, wherein a product oligonucleotide further comprises a non-negatively charged intemucleotidic linkage.

27. The method of claim 26, wherein the non-negatively charged intemucleotidic linkage is n001.

28. The method of claim 24 or 25, wherein a product oligonucleotide further comprises a natural phosphate linkage.

29. The method of any one of the preceding claims, wherein in at least one cycle, a partner compound does not contain a chiral auxiliary group (e.g., a partner compound is a 2-cyanoethyl N,N-diisopropylchlorophosphoramidite).

30. The method of any one of the preceding claims, 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 intemucleotidic linkage, which intemucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphoms of the intemucleotidic 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 intemucleotidic linkage.

31. An oligonucleotide, wherein the oligonucleotide comprises an intemucleotidic linkage of *PS or

*PR, wherein *PS is independently of formula or a salt form thereof, *PR is

independently of formula
or a salt form thereof, wherein -X-Ls-R5 is

32. The oligonucleotide of claim 31, wherein the oligonucleotide further comprise an intemucleotidic

linkage of *NS or *NR, wherein *NS is independently of formula or a salt form

thereof, *NR is independently of formula
or a salt form thereof, wherein

-X-Ls-R5 is

33. The oligonucleotide of claim 31 or 32, wherein R4 and R5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 1-5 heteroatoms.

34. The oligonucleotide of claim 33, wherein R2 is -CH2SO2R’, wherein R’ is an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1- 10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms.

35. The oligonucleotide of claim 34, wherein R’ is optionally substituted phenyl.

36. The oligonucleotide of claim 34, wherein R’ is phenyl.

37. The oligonucleotide of claim 34, wherein R’ is tert-butyl.

38. A 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 intemucleotidic linkage, which intemucleotidic linkage is chirally controlled in that oligonucleotides of the plurality share the same stereochemical configuration at the chiral linkage phosphorus of the intemucleotidic 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 intemucleotidic linkage; and

wherein each oligonucleotide is independently an oligonucleotide of any one of claims 31-37.

39. A phosphoramidite having the stmcture of

or a salt thereof in any

one of claims 10-21.

40. A compound, composition, method or oligonucleotide described in the specification, or of any one of embodiments 1-665.