Technologies for oligonucleotide preparation
Inventors
Bowman, Keith Andrew • Vargeese, Chandra • Butler, David Charles Donnell • Kandasamy, Pachamuthu • Alam, Mohammed Rowshon • Shimizu, Mamoru • Standley, Stephany Michelle • Aduda, Vincent • Bommineni, Gopal Reddy • Tripathi, Snehlata • Korboukh, Ilia
Assignees
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Publication Number
US-11608355-B2
Publication Date
2023-03-21
Expiration Date
Abstract
Among other things, the present disclosure provides technologies for oligonucleotide preparation, particularly chirally controlled oligonucleotide preparation, which technologies provide greatly improved crude purity and yield, and significantly reduce manufacturing costs.
Core Innovation
The invention relates to a method for preparing an oligonucleotide using one or more cycles, each cycle independently comprising coupling, a pre-modification capping step, a modification step, a post-modification capping step, and de-blocking. The capping condition of each pre-modification capping step after a coupling step and before the next modification step is independently selective or specific for amidation over esterification.
The method further includes a modification step in which sulfurization converts a linkage phosphorus atom in an internucleotidic linkage of formula VII-b, or a salt form, into a P(S) linkage. The disclosed subject matter also describes chirally controlled oligonucleotide compositions with shared constitution and shared stereochemical configuration at a chiral linkage phosphorus, together with DS/Nc-based membership thresholds for base-sequence sharing.
The description additionally refers to sulfurization reagent systems comprising named sulfurization reagents, including POS, DDTT, DTD, xanthane hydride (XH), MTS-CNE, and phenylacetyl disulfide. It also states that the resulting oligonucleotides can be chirally controlled and that the synthesis framework supports improved crude purity/yield, scalability, and related structural control.
Claims Coverage
The independent claim is a cyclic oligonucleotide synthesis method with one or more cycles comprising coupling, pre-modification capping, modification, post-modification capping, and de-blocking. The core inventive feature across the independent claim is that each pre-modification capping step is independently selective or specific for amidation over esterification, with dependent claims adding quantitative capping constraints, sulfurization-based linkage modification, named sulfurization reagents, and chirally controlled final product composition features.
Cyclic oligonucleotide synthesis with amidation-selective pre-modification capping
A method for preparing an oligonucleotide comprising one or more cycles, each cycle independently comprising coupling, a pre-modification capping, a modification step, a post-modification capping, and de-blocking, wherein the capping condition of each pre-modification capping step after a coupling step and before the next modification step is independently selective or specific for amidation over esterification.
Limited strong nucleophile during pre-modification capping
Each pre-modification capping step after a coupling step and before the next modification step independently contains no strong nucleophile, or any strong nucleophile level is no more than about 0.1 equivalent relative to the first incorporated nucleoside.
Limited esterification catalyst during pre-modification capping
Each pre-modification capping step after a coupling step and before the next modification step independently has no esterification catalyst, or any esterification catalyst level is independently no more than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, or 1 equivalent relative to the first incorporated nucleoside.
Sulfurization converts linkage phosphorus to a P(S) linkage
A modification step includes sulfurization that converts a linkage phosphorus atom in an internucleotidic linkage of formula VII-b, or a salt form, into a P(S) linkage.
Sulfurization reagent system
The modification reagent system is a sulfurization reagent system comprising one or more sulfurization reagents selected from POS, DDTT, DTD, xanthane hydride (XH), MTS-CNE, or phenylacetyl disulfide.
Chirally controlled oligonucleotide composition with DS/Nc threshold
A final product composition is a chirally controlled oligonucleotide composition where oligonucleotides share the same constitution and share the same stereochemical configuration at the chiral linkage phosphorus, with at least ((DS)/Nc*100)% of oligonucleotides sharing the same base sequence belonging to the plurality, with DS at least 80-99%.
The claim coverage centers on cyclic oligonucleotide synthesis in which pre-modification capping is independently selective or specific for amidation over esterification. Dependent coverage further constrains pre-modification capping by limiting strong nucleophile and esterification catalyst content, defines sulfurization of linkage phosphorus to a P(S) linkage using selected sulfurization reagents, and requires a chirally controlled final composition with shared stereochemical configuration at a chiral linkage phosphorus and DS/Nc-based membership thresholds.
Stated Advantages
Improved crude purity/yield.
Scalability.
Improves reagent compatibility and crude purity/yield for modified and chirally controlled oligonucleotides.
Enables pre-determination of chirally controlled crude oligonucleotide compositions by shared base sequence, backbone linkage pattern, and backbone stereochemistry/phosphorus modifications using stereocontrol metrics (DS/Nc) and crude purity/full-length product thresholds.
Supports scalable/parallel synthesis formats including plates and microarrays.
Documented Applications
Scalable or parallel synthesis formats including plates and microarrays.
Therapeutic utility is noted in the context of example final product compositions (WV-series) within chirally controlled oligonucleotide compositions.
Isotopic labeling/deuteration effects are noted for the example final product compositions (WV-series).
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