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BR-112021025026-B1 - PROCESS FOR THE PRODUCTION OF AN OLIGONUCLEOTIDE

BR112021025026B1BR 112021025026 B1BR112021025026 B1BR 112021025026B1BR-112021025026-B1

Abstract

PROCESS FOR THE PRODUCTION OF AN OLIGONUCLEOTIDE. The invention relates to a process for the production of a mixed P=O/P=S oligonucleotide comprising a selective oxidation of an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II, according to the scheme with an oxidation solution obtained by mixing iodine, an organic solvent and water, characterized in that the oxidation solution has been aged for a period of time sufficient to selectively oxidize the phosphite triester compound of formula I to the phosphodiester compound of formula II without oxidizing the phosphorothioate internucleotide bonds.

Inventors

  • Hongrim CHOI
  • Alec Fettes
  • Achim Geiser
  • Leonhard JAITZ
  • KYEONG EUN JUNG
  • SUNG WON KIM

Assignees

  • F. HOFFMANN-LA ROCHE AG

Dates

Publication Date
20260317
Application Date
20200609
Priority Date
20190611

Claims (7)

  1. 1. PROCESS FOR THE PRODUCTION OF A MIXED P=O/P=S OLIGONUCLEOTIDE, characterized by comprising the oxidation of an intermediate phosphite triester compound of formula I into a phosphodiester compound of formula II according to the scheme: with an oxidation solution obtained by mixing iodine, an organic solvent and water, wherein the oxidation solution has been aged for a period of time of at least 3 days, 5 days, 10 days, 15 days or at least 20 days at a temperature of 30°C to 60°C, so that the oxidation solution can selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II minimising the conversion of phosphorothioate internucleotide bonds, wherein the organic solvent is pyridine or a C1-6 alkyl substituted pyridine.
  2. 2. PROCESS, according to claim 1, characterized in that the volume ratio of pyridine or C1-6 alkyl-substituted pyridine to water is from 1:1 to 20:1.
  3. 3. PROCESS, according to any one of claims 1 to 2, characterized in that the iodine concentration in the oxidation solution is from 10 mM to 100 mM.
  4. 4. PROCESS, according to any one of claims 1 to 3, characterized in that the process comprises monitoring pH and conductivity to determine the time period sufficient to selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II while minimizing the conversion of phosphorothioate internucleotide bonds.
  5. 5. PROCESS, according to any one of claims 1 to 4, characterized in that the amount of oxidant used in the oxidation reaction is selected from between 1.1 equivalents and 15 equivalents.
  6. 6. PROCESS, according to any one of claims 1 to 5, characterized in that the reaction temperature for the oxidation reaction is selected between 15 °C and 27 °C.
  7. 7. PROCESS, according to any one of claims 1 to 6, characterized in that the oligonucleotide consists of optionally modified DNA or RNA nucleoside monomers or combinations thereof and has a length of 10 to 40 nucleotides.

Description

[0001] The invention relates to a new process for the production of a mixed P=O/P=S oligonucleotide comprising the oxidation of an intermediate phosphite triester compound of formula I to a phosphodiester compound of formula II according to the scheme: in which the oxidation follows a particular oxidation protocol. [0002] Oligonucleotide synthesis, in principle, is a gradual addition of nucleotide residues to the 5' end of the growing chain until the desired sequence is assembled. [0003] As a rule, each addition is referred to as a synthetic cycle and, in principle, consists of the following chemical reactions: a1) unlocking the protected hydroxyl group on the solid support; a2) coupling the first nucleoside as an activated phosphoramidite with the free hydroxyl group on the solid support; a3) oxidizing or sulfurizing the respective nucleoside linked to P (triester phosphite) to form the respective phosphodiester (P=O) or the respective phosphorothioate (P=S); a4) optionally, capping any unreacted hydroxyl groups on the solid support; a5) unlocking the 5' hydroxyl group of the first nucleoside linked to the solid support; a6) coupling the second nucleoside as an activated phosphoramidite to form the respective P-linked dimer; a7) oxidizing or sulfurizing the respective P-linked dinucleotide (triester phosphite) to form the respective phosphodiester. (P=O) or the respective phosphorothioate (P=S);a8) optionally, cap any unreacted 5' hydroxyl groups; anda9) repeat the previous steps from a5 to a8 until the desired sequence is assembled. [0004] The principles of oligonucleotide synthesis are well known in the art (see, for example, Oligonucleotide synthesis; Wikipedia, the free encyclopedia; https://en.wikipedia.org/wiki/Oligonucleotide synthesis, accessed March 15, 2016). [0005] The oxidation step is typically carried out with an oxidation solution comprising iodine, an organic solvent, which is usually pyridine, and water. [0006] However, it was observed that when a freshly prepared oxidation solution was applied, not only did the desired oxidation of the intermediate phosphite triester compound of formula I into a phosphodiester compound of formula II occur, but also, as a secondary reaction, phosphorothioate internucleotide bonds present in the molecule could be affected by a conversion of P=S to P=O in the internucleotide bonds, resulting in a higher than expected content of phosphodiester bonds within the compound of formula II. [0007] The objective of the invention was, therefore, to find an oxidation protocol that allows selective oxidation of the phosphite triester compound of formula I to the phosphodiester compound of formula II without affecting the internucleotide bond of phosphorothioate. [0008] It has been found that the object of the invention can be achieved with the process for the production of a mixed P=O/P=S oligonucleotide structure comprising the oxidation of an intermediate phosphite triester compound of formula I into a phosphodiester compound of formula II according to the scheme: with an oxidation solution obtained by mixing iodine, an organic solvent and water, and characterized in that the oxidation solution has been aged for a period of time sufficient to selectively oxidize the phosphite triester compound of formula I into the phosphodiester compound of formula II without oxidizing the internucleotide bonds of phosphorothioate. [0009] The following definitions are presented to illustrate and define the meaning and scope of the various terms used to describe the invention in this document. [0010] The term "C1-6-alkyl" denotes a saturated linear or branched monovalent hydrocarbon group of 1 to 6 carbon atoms, and in a more particular embodiment, 1 to 4 carbon atoms. Typical examples include methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, sec-butyl or t-butyl, preferably methyl or ethyl. [0011] The term oligonucleotide, as used in this document, is defined as is generally understood by those skilled in the art as a molecule comprising two or more covalently linked nucleotides. For use as a therapeutically valuable oligonucleotide, oligonucleotides are typically synthesized as 10 to 40 nucleotides, preferably 10 to 25 nucleotides in length. [0012] Oligonucleotides may consist of optionally modified DNA or RNA nucleoside monomers or combinations thereof. [0013] Optionally modified, as used in this document, refers to nucleosides that are modified compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or nucleobase moiety. [0014] Typical modifications may be the (2'-MOE) substitution of the 2'-O-(2-Methoxyethyl) substitution in the sugar moiety or the blocked nucleic acid (LNA), which is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. [0015] The term modified nucleoside may also be used interchangeably in this document with the term "nuc