Production method for bifurcated lipid-linked oligonucleotide, and intermediate
Inventors
TANINO, Tetsuya • Sekiguchi, Mitsuaki • OCHI, Shunsuke • FUKUI, Nobuaki
Assignees
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Publication Number
US-12351804-B2
Publication Date
2025-07-08
Expiration Date
Abstract
Provided are production methods capable of controlling quality of a bifurcated lipid-linked oligonucleotide, and intermediates which is useful for the production method, has good stability, and is easy to manage and analyze. Specifically, it is a method for producing a bifurcated lipid-linked oligonucleotide including a step of reacting a compound of formula (II):wherein Pro1 to Pro4 are each independently a protecting group;Pro1 and Pro2 or Pro3 and Pro4 may be taken together to form a protecting group;m, n and p are each independently an integer of 0 to 5; andY is a group of a formula: —P(OC2H4CN)(N(i-Pr)2), or the like, with an oligonucleotide.
Core Innovation
The disclosure describes solid-phase production of bifurcated (diacyl) lipid-linked oligonucleotides using specialized phosphoramidite-type intermediates, including a compound of formula (II) having a Y group selected from groups of formula (Y-1), (Y-2), or (Y-3). The approach reacts a protecting-group-bearing compound of formula (II) with an oligonucleotide on a solid phase carrier to form a lipid-linked oligonucleotide with controlled structural connectivity at the 3′ and 5′ ends.
The disclosure addresses poor condensation efficiency and oligonucleotide dimer formation in prior methods, and states that protecting both hydrogens on the nitrogen before lipid introduction suppresses dimer formation and improves condensation. It also links the intermediate design to crystallinity, powder X-ray diffraction quality, UV-detectable impurity behavior, and improved stability of the intermediate.
The disclosure further addresses poor crystallinity or amorphous PXRD, lack of a UV-detectable band, instability at −20 °C with decomposition over time, and relatively poor synthesis efficiency. The claimed intermediate provides good crystallinity enabling crystallization purification, UV-detectable impurity control, stability up to 40 °C for 50 days, and improved synthesis efficiency.
Claims Coverage
The partial content includes three independent claims: one directed to a compound of formula (I), and two directed to methods for producing specific formula compounds (VII and X). Across these independent claims, the inventive coverage centers on a defined phosphorous-containing Y group selection (Y-1/Y-2/Y-3) within a general structural framework, and a solid-phase production sequence involving reaction of a compound of formula (II), deprotection and cutting from a solid phase carrier, and subsequent reactions using compound formula definitions and linkage constraints.
Defined compound structure with phosphorous-containing Y group
A compound of formula (I), or its salt, where A and B are each independently O or S, X1–X4 are each independently O or S, q is each independently an integer of 0 to 4, r is an integer of 0 to 3, s is each independently an integer of 0 to 3, substituent variables R1–R3 are defined options, and Y is a group of formula (Y-1), (Y-2), or (Y-3).
Solid-phase method producing compound of formula (VII)
A method for producing a compound of formula (VII) comprising reacting a compound of formula (II) with an oligonucleotide of formula (III) on a solid phase, deprotecting a protecting group in a compound of formula (IV), cutting out from a solid phase carrier in the presence of a cleavage reagent and a deprotecting agent to obtain a compound of formula (V), and reacting a compound of formula (VI) with the compound of formula (V).
Solid-phase method producing compound of formula (X) with further reactions
A method for producing a compound of formula (X) comprising reacting a compound of formula (II) with an oligonucleotide of formula (III) on a solid phase, obtaining a compound of formula (IV), deprotecting and cutting out from a solid phase carrier in the presence of a cleavage reagent and a deprotecting agent to obtain a compound of formula (V), reacting a compound of formula (VI) to obtain a compound of formula (VIII), and reacting a compound of formula (IX) with the compound of formula (VIII).
Overall, the independent claims provide coverage for a defined phosphorous-containing Y-group compound (formula (I) with Y selected from (Y-1), (Y-2), or (Y-3), including salts) and for solid-phase production workflows that couple an oligonucleotide with a phosphoramidite-type intermediate of formula (II), followed by deprotection/cutting from a solid phase carrier and further reactions to reach formula (VII) or formula (X).
Stated Advantages
Reduced byproduct formation.
Ability to obtain target high-quality pharmaceutical-grade product.
Crystallizable intermediate(s) enable QC.
Suppresses dimer formation by protecting both hydrogens on the nitrogen before lipid introduction.
Improves condensation efficiency.
Provides good crystallinity enabling crystallization purification.
Enables UV-detectable impurity control.
Shows improved stability up to 40 °C for 50 days.
Improves synthesis efficiency.
Documented Applications
Double-stranded oligonucleotide formation by annealing a complementary sequence to obtain a double strand.
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