Acid-free thermal deprotection of trityl-protected oligonucleotides

Abstract

This disclosure relates to a method for deprotecting an oligonucleotide, such as an oligonucleotide modified with a 5'-amine group, from a trityl protecting group from an amine group or a hydroxyl group. The method generally involves heating the oligonucleotide in an acid-free aqueous solution at a temperature of at least 60°C, and optionally removing an insoluble trityl-hydroxyl byproduct to further advance the deprotection reaction. The method allows for easy deprotection of the trityl protecting group with little to no depurination of the oligonucleotide product.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 534,076, filed on 22 August 2023, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to a method for easily removing a trityl protecting group from an oligonucleotide, including a 5'-amino modified oligonucleotide, comprising heating the protected oligonucleotide in an acid-free aqueous solution at a temperature of at least 60°C, and optionally removing an insoluble trityl-hydroxyl byproduct to further advance the deprotection reaction.

[0003] Sequence listing reference A formal copy of the sequence listing will be submitted simultaneously with this Specified [Background technology]

[0004] Nucleic acid conjugates have revolutionized entirely different fields, from therapeutic interventions to early diagnostic detection and DNA-based data storage. The impact of nucleic acid conjugates is accelerated by the continuous evolution of synthetic methods for incorporating chemically reactive handles for downstream derivatization. One obvious example is the field of nucleic acid therapeutics, where ligand incorporation has significantly improved both the drug-like properties and cell-specific delivery of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) (see, e.g., References 1–4). The placement of reactive chemical handles during synthesis, enabling efficient post-synthesis conjugation, is crucial to the production of these conjugates. While several handles exist with robust conjugation reactions that are widely used (see, e.g., References 5–6), amide bond formation reactions between amines and activated esters are widely applied due to their cost-effectiveness and the relatively simple preparation of starting materials (see, e.g., References 7–9).

[0005] There are two main classes of phosphoramidite reagents capable of labeling oligonucleotides with 5'-amines, differing in their amino protecting groups and therefore in their deprotection methods. The first group includes both trifluoroacetyl (TFA) (compound 1) and phthalic acid diamide (PDA) (compound 2), whose structures are shown below, and which contain base-unstable protection.

[0006] [ka]

[0007] In the former case, the TFA group is readily deprotected during standard cleavage and deprotection in concentrated ammonia water, while in the latter case, the PDA group requires a 1:1 mixture of concentrated ammonia water and methylamine (AMA). This method requires that conjugation occur in the crude synthesis mixture, and that the subsequent conjugation be chemically distinct enough to allow isolation from the truncated oligonucleotide sequence generated during synthesis. This requires the use of HPLC purification, which is expensive, time-consuming, and not available in all laboratories.

[0008] The second group contains reagents having trityl protecting groups. MMTr (compound 3) and 5'-DMS(O)MT (compound 4), whose structures are shown below, are two of the most widely used reagents for labeling oligonucleotides with 5'-amines, due to the convenient hydrophobic handle provided by the trityl protecting group, which enables routine cartridge purification.

[0009] [ka]

[0010] The compatibility of these reagents with cartridge purification significantly accelerates the ability to produce many high-quality conjugates without the need for laborious purification. However, a significant challenge when using these reagents with trityl protecting groups (as stated on the supplier's website) is that the trityl group tends to remain bound during deprotection or to detach unexpectedly during routine handling (see, e.g., Reference 10). In addition to these challenges, the acidic treatment required to deprotect these reagents can cause undesirable depurination of oligonucleotides, resulting in debasication sites and cleavage products (see, e.g., References 11-13). Previous studies have explored alternative methods to minimize depurination using less acidic deprotection cocktails for DMTr and MMTr protected oligonucleotides, for example (see, e.g., References 14-15). While many alternative protecting groups to aminoamidites have attracted attention, none have significantly improved upon the MMTr protected amine linker phosphoramidite reagent of compound (3), which remains the most practical and widely used option to date (see, e.g., Reference 16). [Overview of the project]

[0011] A practical method for synthesizing 5'-amine labeled oligonucleotides using phosphoramidite reagents with trityl protecting groups, such as compound (3), which solve the problems of incomplete deprotection and / or depurination of oligonucleotides, would greatly facilitate the routine synthesis of oligonucleotide conjugates and open the way for oligonucleotide modifications that are unsuitable for conventional deprotection strategies. Therefore, an improved method for deprotecting trityl protecting groups commonly used in the synthesis of oligonucleotides, such as 5'-amino modified oligonucleotides, is still needed.

[0012] This disclosure relates to a method for deprotecting an amine or hydroxyl group from a trityl protecting group during the synthesis of oligonucleotides, such as oligonucleotides modified with a 5'-amino group. The method generally involves heating a protected oligonucleotide containing a trityl protecting group on an amine or hydroxyl group in an acid-free aqueous solution at a temperature of at least 60°C to obtain an oligonucleotide having a deprotected amine or hydroxyl group. The method optionally includes simultaneous removal of the deprotection trityl-hydroxyl byproduct to further accelerate the completion of the deprotection reaction. The method allows for easy deprotection of the trityl protecting group with little to no depurination of the oligonucleotide product. This summary is intended to introduce the subject matter of this disclosure but does not exhaust all embodiments, combinations, or variations contemplated and described within this disclosure. Further embodiments are contemplated and described in the detailed description, drawings, and claims disclosure.

[0013] In at least one embodiment, the present disclosure provides a method for deprotecting an oligonucleotide containing an amine or hydroxyl group protected by a trityl protecting group, comprising heating the oligonucleotide in an acid-free aqueous solution at a temperature of at least 60°C to cleave the trityl protecting group and thereby obtain a deprotected oligonucleotide containing an amine or hydroxyl group.

[0014] In at least one embodiment of this method, the trityl protecting group is selected from trityl, monomethoxytrityl (MMTr), dimethoxytritryl (DMTr), and dimethoxymethylsulfonyltrityl (DMS(O)MTr). In at least one embodiment, the trityl protecting group is

[0015] [ka] (In the formula, [ka] represents a covalent bond to the nitrogen atom of an amine group or the oxygen atom of a hydroxyl group) having a structure selected from.

[0016] In at least one embodiment of the method, deprotection of the amine group or hydroxyl group produces an insoluble trityl by-product, and the method further comprises separating the insoluble trityl by-product from the deprotected oligonucleotide. In at least one embodiment, the insoluble trityl by-product is separated by extraction, desalting, or precipitation and filtration. In at least one embodiment, the insoluble trityl by-product is selected from MMTr-OH, DMTr-OH, Tr-OH, and DMS(O)Tr-OH. In at least one embodiment, the insoluble trityl by-product is

Chemical Formula

[0017] In at least one embodiment of the method, the amine group or hydroxyl group is bonded to the 5' phosphate group of the oligonucleotide.

[0018] In at least one embodiment of the method, the amine group or hydroxyl group is bonded to the 5' phosphate group of the oligonucleotide via a linker group. In at least one embodiment, the linker group comprises an unbranched alkylene chain of 5 to 12 carbons. In at least one embodiment, the linker group comprises an unbranched alkylene chain of 4 to 12 carbons having at least one ether bond.

[0019] In at least one embodiment, the linker is selected from succinate linkers, PEG3 linkers, PEG4 linkers, sarcosine-glutarate linkers, hydrazone linkers, disulfide linkers, valine-citrulline linkers, valine-alanine linkers, tris-hexylamino linkers, hydroquinone-O,O'-diacetate ("Q") linkers, hexylamine linkers, hexyloxy linkers, pentaethylene glycol linkers, and their derivatives. In one embodiment, the linker is selected from PEG3 linkers (-(CH2CH2O)3-) and PEG4 linkers (-(CH2CH2O)4-).

[0020] In at least one embodiment of this method, the amine group of the oligonucleotide protected by the trityl protecting group is a primary amine group.

[0021] In at least one embodiment of the present method, the method further comprises conjugating a ligand to an oligonucleotide by reacting a deprotected primary amine group of an oligonucleotide with a ligand molecule containing an amine-reactive group, thereby forming a ligand-oligonucleotide conjugate. In at least one embodiment, the amine-reactive group is selected from esters, NHS esters, and carboxylic acids. In at least one embodiment, the ligand is selected from N-acetylgalactosamine (GalNAc), triantennary clusters of N-acetylgalactosamine moieties, lipid molecules, small peptides (e.g., RGD, cell-permeable peptides, integrins), proteins, and antibodies.

[0022] In at least one embodiment of this method, the amine group of the oligonucleotide protected by the trityl protecting group is a secondary amine linked to a phosphorodiamidate morpholino oligomer (PMO).

[0023] In at least one embodiment of this method, the conversion rate from oligonucleotides having a trityl protecting group to oligonucleotides having a deprotected amine group or hydroxyl group is at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 100%.

[0024] In at least one embodiment, the present disclosure provides a method for deprotecting a 5'-amine-modified oligonucleotide compound of formula (I), comprising heating the 5'-amino-modified oligonucleotide compound of formula (I) in an acid-free aqueous solution at a temperature of at least 60°C to form an oligonucleotide compound of formula (II) and a by-product compound of formula (III): [ka] or its salt or a pharmaceutically acceptable salt thereof, (In the formula, R 1 , R 2 , and R 3 Each of them is independently hydrogen, -OCH3, or -S(O)CH3. L is any linker, X is either O or S, Y is either OH or SH, [ka] This represents a covalent bond to the 5' end of an oligonucleotide. [ka] or its salt, or a pharmaceutically acceptable salt thereof, (In the formula, X is either O or S, Y is -OH or -SH, L is any linker, [ka] This represents a covalent bond to the 5' end of an oligonucleotide.

Chemical Structure

[0025] In at least one embodiment of the method for deprotecting an oligonucleotide compound of formula (I), the method further comprises separating the oligonucleotide compound of formula (II) from the by-product compound of formula (III) by extraction, desalting, or precipitation and filtration.

[0026] In at least one embodiment of the method for deprotecting an oligonucleotide compound of formula (I), the oligonucleotide compound of formula (I) is prepared by reacting a phosphoramidite compound of formula (A) with an oligonucleotide compound of formula (B):

Chemical Structure

Chemical Structure

Chemical Structure

[0027] In at least one embodiment of a method for deprotecting an oligonucleotide compound of formula (I), the method reacts an oligonucleotide compound of formula (II) with a ligand molecule containing an amine-reactive group to form a ligand-oligonucleotide conjugate compound of formula (IV): [ka] or its salt, or a pharmaceutically acceptable salt thereof, (In the formula, L is a divalent linker, X is either S or O, Y is either -SH or -OH, R 4 is a ligand, [ka] (This represents a covalent bond to the 5' end of an oligonucleotide.) This further includes forming

[0028] In at least one embodiment of a method for deprotecting an oligonucleotide compound of formula (I), the amine-reactive group is selected from esters, NHS esters, and carboxylic acids.

[0029] In at least one embodiment of a method for deprotecting an oligonucleotide of formula (I), ligand R 4 These are selected from N-acetylgalactosamine (GalNAc), a three-branched cluster of the N-acetylgalactosamine moiety, lipid molecules, small molecule peptides (e.g., RGD, cell-permeable peptides, integrins), proteins, and antibodies.

[0030] In at least one embodiment of a method for deprotecting an oligonucleotide compound of formula (I), the linker portion L is a divalent linker of formula (Li): [ka] (In the formula, [ka] This represents the covalent bond of the primary amine group to the nitrogen atom. [ka] (This represents the covalent bond between the phosphate group and the oxygen atom.) That is the case.

[0031] In at least one embodiment of the method for deprotecting an oligonucleotide compound of formula (I), linker L comprises (i) a 5- to 20-carbon branched or unbranched alkylene chain; or (ii) a 4- to 20-carbon branched or unbranched alkylene chain having at least one ether bond (or at least one carbon atom in the chain being replaced by an oxygen atom). In another embodiment of the method for deprotecting an oligonucleotide compound of formula (I), linker L comprises an unbranched alkylene chain of 4- to 12 carbon, for example, an unbranched alkylene chain of 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbons. In another embodiment of the method for deprotecting an oligonucleotide compound of formula (I), the linker L comprises a 4- to 12-carbon unbranched alkylene chain having at least one ether linkage (or at least one carbon atom in the chain being replaced by an oxygen atom), such as a 4-carbon, 5-carbon, 6-carbon, 7-carbon, 8-carbon, 9-carbon, 10-carbon, 11-carbon, or 12-carbon unbranched alkylene chain.

[0032] In at least one embodiment of a method for deprotecting an oligonucleotide compound of formula (I), linker L includes succinate linker, PEG3 linker, PEG4 linker, sarcosine-glutarate linker, hydrazone linker, disulfide linker, valine-citrulline linker, valine-alanine linker, tris-hexylamino linker, hydroquinone-O,O'-diacetate ("Q") linker, hexylamine linker, hexyloxy linker, pentaethylene glycol, or derivatives thereof. In one embodiment, linker L includes PEG3 linker (-(CH2CH2O)3-). In another embodiment, linker L includes PEG4 linker (-(CH2CH2O)4-).

[0033] In at least one embodiment of the method for deprotecting oligonucleotides of the present disclosure, an acid-free aqueous solution is heated to a temperature of at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C. In at least one embodiment, an acid-free aqueous solution is heated to a temperature of about 60°C to about 100°C, about 60°C to about 80°C, about 60°C to about 75°C, or about 60°C to about 70°C.

[0034] In at least one embodiment of the deprotection method of the present disclosure, an acid-free aqueous solution is heated for at least about 1 hour, or at least about 2 hours. In at least one embodiment, an acid-free aqueous solution is heated for about 1 to about 2 hours.

[0035] In at least one embodiment of the method for deprotecting oligonucleotides of the present disclosure, the acid-free aqueous solution has a pH of about 6.5 to about 8.0, about 6.8 to about 7.8, about 6.8 to about 7.6, about 7.0, about 7.2, or about 7.4.

[0036] In at least one embodiment of a method for deprotecting an oligonucleotide compound of formula (I), the proportion of the oligonucleotide compound of formula (I) converted to an oligonucleotide compound of formula (II) is at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 100%.

[0037] In at least one embodiment of the method for deprotecting oligonucleotides of the present disclosure, the ratio of depurinated oligonucleotides to total oligonucleotides detected by MS analysis after deprotection is less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%.

[0038] In at least one embodiment of the method for deprotecting an oligonucleotide of the present disclosure, the length of the oligonucleotide is 2mer to 100mer, and optionally the length of the oligonucleotide is 3mer to 75mer, 5mer to 50mer, 15mer to 50mer, 15mer to 40mer, 15mer to 30mer, or 2mer to 10mer.

[0039] A more comprehensive understanding of the novel features and advantages of this disclosure can be gained by referring to the following detailed description, which describes explanatory embodiments in which the principles of this disclosure are utilized, and to the accompanying drawings (hereinafter also referred to as "Figure" and "FIG."). [Brief explanation of the drawing]

[0040] [Figure 1A] Figure 1A is a time-course liquid chromatography (LC) plot showing the incomplete deprotection of oligonucleotide MMTr-ON #5 under a standard acid-catalyzed deprotection protocol as described in Example 2. [Figure 1B-1C]Figures 1B and 1C plot the MS peaks detected for acid-catalyzed deprotection of oligonucleotide MMTr-ON #5 (Figure 1B) versus the MS peaks detected for non-acid thermal deprotection (Figure 1C) as described in Example 2. The MS peaks in Figure 1B indicate the detection of >5% depurinated species due to the acid-catalyzed deprotection protocol. [Figure 1D] Figure 1D is an LC plot showing how the varying conditions of acetic acid percentage, temperature, and incubation time described in Example 2 affect the acid-catalyzed deprotection of oligonucleotide MMTr-ON #5. [Figure 1E] Figure 1E is a plot of MS peaks observed for the acid-catalyzed deprotection reaction of oligonucleotide MMTr-ON #5 carried out at 45°C, as described in Example 2. The MS peaks indicate increased depurination of the deprotected oligonucleotide MMTr-ON #5 product. [Figure 1F] Figure 1F is a time-course LC plot showing that the use of the acid-free thermal deprotection conditions described in Example 2 resulted in complete deprotection of oligonucleotide MMTr-ON #5 in 60 minutes. [Figure 2A] Figure 2A shows the NMR spectrum confirming that the white precipitate formed during the acid-free thermal deprotection protocol is the reaction byproduct, MMTr-OH. The upper spectrum in Figure 2A is from a commercially available sample of MMTr-OH, and the lower spectrum in Figure 2A is from the isolated white precipitate, which is the reaction byproduct. [Figure 2B]Figure 2B is a schematic comparison of acid-catalyzed deprotection and acid-free thermal deprotection of MMTr-protected oligonucleotides (indicated by "A") to form deprotected 5'-amine linker-modified oligonucleotide products (indicated by "B"). These reactions illustrate how the insolubility of the MMTr-OH byproduct in water helps to facilitate the completion of the acid-free thermal deprotection reaction, whereas the solubility of the cation byproduct, MMTr+, formed during the acid-catalyzed reaction, can allow for the re-formation of the MMTr-protected oligonucleotide starting material, where the reverse reaction at equilibrium is undesirable. [Figure 3A-3F] Figures 3A, 3B, 3C, 3D, 3E, and 3F are illustrative LC plots comparing acid-catalyzed deprotection and acid-free thermal deprotection of six MMTr-protected oligonucleotides with different sequences, as described in Example 4. As shown in Figure 3A, "A" indicates a peak attributable to the protected MMTr-ON starting material, and "B" indicates a peak attributable to the deprotected 5'-amine linker-modified oligonucleotide product. Figure 3A: MMTr-ON #6; Figure 3B: MMTr-ON #7; Figure 3C: MMTr-ON #9; Figure 3D: MMTr-ON #11; Figure 3E: MMTr-ON #12; and Figure 3F: MMTr-ON #13. [Figure 4A] Figure 4A is a schematic diagram of the comparative reactions and conditions used for the deprotection of the 5'-DMTr-O protected oligonucleotide (indicated by "A") to obtain a deprotected oligonucleotide having a 5'-OH group, as described in Example 5. [Figure 4B] Figure 4B is an LC plot showing that complete deprotection of the 5'-DMTr-ON starting material (peak indicated by "A") for forming a deprotected oligonucleotide having a 5'-OH group (peak indicated by "B") was achieved using the acid-free thermal deprotection conditions described in Example 5, at 95°C for 1.5 hours. [Modes for carrying out the invention]

[0041] In this description and the appended claims, the singular “a” and “an” refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to “an oligonucleotide” includes two or more oligonucleotides, and a reference to “a compound” refers to two or more compounds. It should be further noted that the claims may be constructed to exclude any optional element. This statement is therefore intended to be used as prior art for the use of exclusive terms such as “exclusively,” “only,” or “negative” limitations relating to the enumeration of elements of the claims. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” is interchangeable and not intended to be restrictive. Where descriptions of various embodiments use the term “comprising,” it should be understood that in some specific cases the embodiments may be described alternatively using the phrases “essentially consisting of” or “consisting of.”

[0042] Where a range of values ​​is presented, unless the context clearly indicates otherwise, each integer between the upper and lower limits of that range, and each tenth of each integer between the upper and lower limits, as well as any other stated or intervening values ​​within that described range, are understood to be included within the invention. The upper and lower limits of these smaller ranges may independently be included within smaller ranges, which are also included within the invention, subject to any explicitly excluded limits within the described range. If the described range includes one or both of these limits, the range excluding (i) either or (ii) both of the limits they include is also included within the invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0043] A range may be expressed herein as "approximately" from one particular value and / or "approximately" to another particular value. Where such a range is expressed, another aspect includes the range from one particular value and / or other particular values ​​within that range. It is further understood that each endpoint of those ranges is important, both in relation to the other endpoints and independently of the other endpoints. Similarly, where a value is expressed as an approximation by the preceding use of "approximately," it is understood that a particular value forms another aspect.

[0044] In general, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly used by those skilled in the art. Singleton et al., *Dictionary of Microbiology and Molecular Biology*, 2nd edition, John Wiley and Sons, New York (1994), and Hale & Marham, *The Harper Collins Dictionary of Biology*, Harper Perennial, NY (1991) provide many common dictionaries of the terms used in this invention. Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, but preferred techniques and methods are described, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Volumes 1–3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter referred to as "Sambrook"); and Current Protocols in Molecular Biology, Volumes 00–130 (1987–2020), edited by FMAusubel et al. (hereinafter referred to as "Ausubel"), which was first published in book form in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., regularly supplemented until 2011, and is now available in online journal format as Current Protocols in Molecular Biology, Volumes 00–130 (1987–2020), published by Wiley & Sons, Inc. in the Wiley Online Library. Since the specific methods, protocols, and reagents described may vary, it should be understood that the present invention is not limited thereto.

[0045] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. It should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting.

[0046] As used herein, “oligonucleotides” are defined as molecules comprising two or more covalently linked nucleotides, as commonly understood by those skilled in the art. Such covalently linked nucleotides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly prepared in the laboratory by solid-phase chemical synthesis followed by purification, but they may also be produced enzymatically using purifying enzymes, such as ligases and / or polymerases. When referring to the sequence of an oligonucleotide, the sequence or order, or modification thereof, of the nucleic acid base portions of the covalently linked nucleotides or nucleosides is referred to. Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxy direction, respectively. This disclosure generally deals with oligonucleotides that are chemically synthesized (i.e., artificially produced) and then typically purified or isolated. However, oligonucleotides used in the methods of this disclosure may also be produced enzymatically and then, if desired, purified and / or isolated. The oligonucleotides used herein are intended to include modified oligonucleotides, such as those having modified sugars, modified internucleotide bonds, and / or bonded conjugate groups, such as 5'-joint amino groups protected by trityl protecting groups. The oligonucleotides used herein may also be therapeutic oligonucleotides, such as antisense oligonucleotides and small interfering RNAs (siRNAs).

[0047] As used herein, “nucleotide” refers to a glycoside comprising a sugar moiety, a nucleic acid base moiety, and a phosphate or modified phosphate group (e.g., a phosphorothioate), encompassing both naturally occurring nucleotides found in DNA (dA, dC, dG, and dT) and RNA (A, C, G, U), as well as non-naturally occurring nucleotides with modified sugar and / or base moieties that are well known in the art of therapeutic antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), such as locked nucleic acid (LNA) nucleotides and phosphorodiamidate morpholino oligomer (PMO) nucleotides.

[0048] As used herein, “phosphorothioate linkage” or “PS linkage” refers to an internucleotide phosphate linkage in an oligonucleotide in which one of the non-crosslinking oxygen atoms is substituted with sulfur. The substitution of one of the non-crosslinking oxygen atoms with sulfur introduces a chiral center, and therefore, within a single phosphorothioate oligonucleotide, the phosphorothioate internucleotide linkage can be either an S(Sp) or R(Rp) stereoisoform. Such an internucleotide linkage is referred to as a “chiral internucleotide linkage.” In contrast, a phosphodiester internucleotide linkage is non-chiral because it has two non-terminal oxygen atoms. As those skilled in the art will understand, a given phosphorothioate oligonucleotide may have a mixture of different S(Sp) or R(Rp) links.

[0049] As used herein, “nucleic acid base” includes nucleosides that form hydrogen bonds in nucleic acid hybridization and purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleotides. In the context of this disclosure, the term nucleic acid base also includes modified (or non-naturally occurring) nucleic acid bases that are functional in nucleic acid hybridization and / or suitable for incorporation into therapeutic or diagnostic oligonucleotides, although these may differ from naturally occurring nucleic acid bases.

[0050] The nucleic acid base moiety may be represented by the corresponding letter code of each nucleic acid base, for example, A, T, G, C, or U, where each letter may optionally contain equally functional modified nucleic acid bases. For example, in the illustrated oligonucleotide, the nucleic acid base moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, 5-methylcytosine LNA nucleoside may be used for LNA gapmers.

[0051] A "modified nucleotide" refers to a nucleotide that has a modified sugar moiety, a modified bond, or a modified base.

[0052] A "modified oligonucleotide" refers to an oligonucleotide containing one or more nucleic acid base modifications, one or more sugar-modified nucleotides, one or more modified internucleotide bonds, and / or one or more 5' or 3' terminal modifications, such as a 5'-aminolinker moiety that can act as a reactive handle for attaching a ligand or conjugate moiety to the oligonucleotide.

[0053] As used herein, "trityl protecting group" refers to a protecting group containing a triphenylmethyl group.

[0054] A "ligand" or "conjugate moiety" refers to a group of molecules or parts that can bind to an oligonucleotide, either directly or indirectly via a linker.

[0055] As used herein, “linker” refers to any divalent molecular part that covalently links two or more molecules, groups of molecules, and / or molecular parts, such as an oligonucleotide and a ligand. Useful linkers in the methods of this disclosure include, but are not limited to, saturated divalent aliphatic chains of carbon atoms (also referred herein as “alkylene” chains). For example, exemplary linkers may include branched or unbranched alkylene chains of 5 to 12 carbon atoms, as described elsewhere herein, and may optionally further include one or more ether bonds (e.g., PEG).

[0056] "Locked nucleic acid" or "LNA" refers to a bicyclic nucleoside analog (2'-4' bicyclic nucleotide analog) that contains a bridge between the 2' and 4' positions in the ribose ring. For example, LNA contains one or more nucleotides having an extra methylene bridge that fixes the ribose portion to either the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation. As those skilled in the art will understand, LNA is also referred to as BNA (crosslinked nucleic acid or bicyclic nucleic acid), and the two terms may be used interchangeably. The term LNA may also refer to an LNA monomer, or, when used in the context of "LNA oligonucleotide," LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogs. In some embodiments, the bicyclic nucleoside analog is an LNA nucleotide, and therefore these terms may be used interchangeably, in such embodiments both being characterized by the presence of a linker (such as a cross-link) between the C2' and C4' of the ribose sugar ring. LNAs provide, for example, stability against enzymatic degradation and improved specificity and affinity in base pairing as monomers or components of oligonucleotides.

[0057] As used herein, “phosphodiamidate morpholino oligomer” or “PMO” refers to an oligomer molecule containing nucleic acid bases attached to a skeleton of a six-membered methylenemorpholine ring linked via phosphorodiamidate bonds, as illustrated by the skeletal structure shown below.

[0058] [ka]

[0059] "Antisense oligonucleotides," abbreviated as "ASO," refer to single-stranded oligomers of nucleoside polymers having a nucleic acid base sequence that enables hybridization to a corresponding region (target region) or segment of a target nucleic acid. As those skilled in the art will understand, antisense oligonucleotides can regulate the expression of target genes by hybridizing to a target nucleic acid, particularly a continuous sequence (subsequence) on the target nucleic acid.

[0060] Acid-free thermal deprotection of trityl protecting groups This disclosure provides a useful method for deprotecting trityl protecting groups from oligonucleotides containing trityl-protecting amine groups or hydroxyl groups, such as oligonucleotides modified with a 5'-amino group. The method of this disclosure has the advantages of achieving easy and complete deprotection of the trityl protecting group and little to no depurination of the oligonucleotide product.

[0061] The deprotection method involves heating an oligonucleotide containing a trityl protecting group that protects an amine or hydroxyl group in an acid-free aqueous solution (e.g., about pH 6.5 to about pH 8.0) at a temperature of at least 60°C for a certain period of time, typically at least about 1 hour. As described in detail below and in the examples, this disclosure intends that the method can be carried out using various trityl protecting groups under various temperature and pH conditions.

[0062] Furthermore, as described in the examples, the deprotection method can act on any protected oligonucleotide, regardless of its sequence or length. Therefore, the deprotection method can be carried out when the oligonucleotide length is 2-100 mer, and optionally when the oligonucleotide length is 3-75 mer, 5-50 mer, 15-50 mer, 15-40 mer, 15-30 mer, or 2-10 mer. Also, as described in the examples, the deprotection method can act on oligonucleotides having modifying groups commonly used in therapeutic oligonucleotides (such as ASOs or siRNAs), including but not limited to phosphorothioate bonds, phosphorodiamidate bonds, 2'-glycosylation, and / or LNA nucleotides.

[0063] The combination of heating in an acid-free aqueous solution used in the methods of this disclosure results in the complete or near-complete deprotection of the trityl protecting group protecting an amine or hydroxyl group, such as 5'-amino or 5'-hydroxyl. An acid-free thermal reaction resulting in the deprotection of a trityl protecting group on a 5'-amine modified oligonucleotide according to one embodiment of the methods described herein is illustrated in Scheme 1 below. [ka]

[0064] As illustrated by the reaction in Scheme 1, the acid-free conditions used in the deprotection methods of this disclosure result in the formation of a free primary amine and trityl hydroxide byproduct on the C6 linker bonded to the 5' end of the oligonucleotide. Under acid-free reaction conditions, this byproduct is insoluble and precipitates. Without attempting to confine to any particular mechanism, the formation of this insoluble trityl hydroxide byproduct helps to expedite the completion of the reaction. Simultaneous removal or sequestration of the insoluble byproduct from the reaction can further accelerate the deprotection reaction and allow it to complete rapidly. Therefore, in at least one embodiment, an acid-free thermal deprotection method is employed in which deprotection of an amine group or a hydroxyl group produces an insoluble trityl byproduct, and the method further comprises separating the insoluble trityl byproduct from the deprotected oligonucleotide. The separation may be carried out using standard methods and techniques for separating insoluble byproducts from reactions in aqueous solution. In at least one embodiment, the method may further comprise separating the insoluble trityl byproduct by extraction, desalting, or precipitation and filtration.

[0065] The formation of this insoluble byproduct during trityl deprotection is in contrast to byproducts formed under standard acidic conditions used in the art for such deprotection (e.g., 20% acetic acid, 25°C, 1 hour). Standard acidic deprotection conditions result in the formation of a soluble, positively charged triphenyl ion byproduct, which undergoes a reverse "protection" reaction in equilibrium with the deprotection reaction. Therefore, the presence of a soluble, positively charged byproduct can slow the progress of the deprotection reaction and reduce the overall yield of the desired deprotection product.

[0066] Trityl protecting groups share a common triphenylmethyl (or trityl) moiety structure that has been found to be particularly useful as protecting groups. A broad range of trityl groups with different groups substituted on the phenyl ring are known in the art of protecting groups. Any of these known trityl protecting groups is intended for use in the methods of the present disclosure. Exemplary trityl protecting groups useful in the compositions and methods of the present disclosure include, but are not limited to, the chemical moieties shown in Table 1 below.

[0067] [Table 1]

[0068] Table 1 (above) shows the structures of several exemplary trityl protecting groups that are well known and can be used with acid-free thermal deprotection methods: trityl-(Tr), dimethoxytrityl-(DMTr), monomethoxytrityl-(MMTr), and dimethoxymethylsulfonyltrityl-(DMS(O)MTr).

[0069] Trityl protecting groups, such as DMTr, are widely used as 5'-hydroxyl protecting groups in standard automated solid-phase oligonucleotide synthesis (SPOS). During the SPOS cycle, an acidic solution at room temperature is added to remove DMTr and obtain a free 5'-OH group that can be coupled to the next phosphoramidite reagent. Oligonucleotides prepared in SPOS also typically contain a protected 5' "handle" in the final step, which can be deprotected to obtain a reactive group for subsequent 5'-conjugation to the oligonucleotide. This reactive 5' handle is usually a primary amine group or a hydroxyl group. Any of these groups can be readily conjugated to a ligand using a variety of well-known chemical reactions. Standard SPOS conditions for deprotecting trityl protecting groups are acidic. However, as described in the background art and elsewhere in this specification, deprotection of trityl protecting groups under acidic conditions can be incomplete or result in adverse effects such as depurination of the oligonucleotide, particularly in the case of 5'-protected amine or hydroxyl groups. As described elsewhere in this specification and shown in the examples, the acid-free thermal deprotection methods of this disclosure result in little to no depurination of the deprotected oligonucleotides. Accordingly, in at least one embodiment of the methods disclosed herein, the ratio of depurinated oligonucleotides to total oligonucleotides detected by mass spectral (MS) analysis after deprotection is less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%.

[0070] As described above, oligonucleotides modified with reactive amines or trityl protecting groups that protect hydroxyl groups can be prepared using standard automated SPOS systems and commercially available phosphoramidite reagents from well-known SPOS reagent vendors such as Glen Research Inc. (Sterling, VA, USA) and Hongene Biotech Corporation (Shanghai, China). For example, the phosphoramidite reagent, "MMTr-amine linker phosphoramidite," can be used during the final step of SPOS to add an MMTr-protected amine group bonded to a C6 alkyl linker to the 5' end of an oligonucleotide. This MMTr-protected oligonucleotide may be cleaved from a solid support, purified, and then stored. The MMTr-protected oligonucleotide may then, if desired, be subjected to an acid-free thermal deprotection method in aqueous solution disclosed herein to produce a deprotected oligonucleotide having a reactive 5'-amine group that can be used for further reactions, e.g., conjugation to a ligand, as described below.

[0071] The deprotection method of this disclosure is carried out in an acid-free aqueous solution. As used in this context, the term “acid-free” means any aqueous solution having a pH between approximately pH 6.5 and approximately pH 8.0. Buffer salts may be included in the aqueous solution. Exemplary acid-free aqueous solutions useful in the method of this disclosure include a neutral, unbuffered aqueous solution (e.g., water at pH 7.0) or a buffer solution (e.g., PBS at pH 7.4 or TES buffer at pH 8.0). Generally, the acid-free aqueous solution is preferably not strongly basic (e.g., 28% NH4OH). Strongly basic solutions (e.g., >pH 9) can adversely affect the deprotection reaction, as described in the examples.

[0072] The presence of an organic solvent (e.g., CH3CN) in an acid-free aqueous solution can lead to a decrease in the efficiency of the deprotection reaction, as shown in the examples. This decrease in efficiency is likely due to the presence of the organic solvent, which solubilizes the trityl hydroxide by-products formed during the deprotection reaction. Therefore, in at least one embodiment, the acid-free aqueous solution contains less than 25%, less than 10%, less than 5%, or less than 1% of an organic solvent, preferably CH3CN.

[0073] The acid-free thermal deprotection method of this disclosure generally uses a temperature of at least 60°C, with higher temperatures intended depending on the specific trityl protecting group and the specific amine or hydroxyl group it protects. As described in the examples disclosed herein, 1 hour at 60°C or 80°C resulted in 100% deprotection of the MMTr-protected 5'-amine group of the oligonucleotide. Lower temperatures of 35°C or 45°C resulted in only 45% or 94% deprotection, respectively. Acid-free thermal deprotection of the DMTr 5'-hydroxyl group of the oligonucleotide can also be carried out, but as shown in the examples, a higher temperature of 95°C (1.5 hours) was used to achieve complete deprotection to obtain a 5'-hydroxyl oligonucleotide. Therefore, deprotection of hydroxyl groups protected by trityl protecting groups is preferably carried out at temperatures higher than 60°C, for example, 95°C.

[0074] Acid-free thermal deprotection methods are further intended to be used in other deprotection reactions, such as the deprotection of trityl groups that protect secondary amine groups in phosphorodiamidate morpholino oligomer (PMO) bonds generated in PMO oligonucleotide synthesis. In such deprotection reactions, temperatures up to 100°C are intended to be used.

[0075] Therefore, in at least one embodiment, an acid-free thermal deprotection method is employed in which the temperature of the aqueous solution is at least 60°C, at least 70°C, at least 80°C, at least 90°C, or at least 95°C. In some embodiments, the temperature of the aqueous solution is about 60°C to about 100°C.

[0076] As described elsewhere in this specification, a remarkable advantage of the deprotection methods of this disclosure is that acid-free conditions can be used to achieve complete deprotection of the trityl protecting group and generate a free amine or hydroxyl group on the modified oligonucleotide. For example, in at least one embodiment, the acid-free thermal deprotection method of this disclosure produces a conversion rate of at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 100% from oligonucleotides having a trityl protecting group to oligonucleotides having a deprotected amine or hydroxyl group. These fully deprotected reactive groups can then be used as handles for further modification of the oligonucleotide, for example, by conjugation at the 5' end to obtain a ligand-oligonucleotide conjugate. Thus, in at least one embodiment of the deprotection method of this disclosure, the reactive amine or hydroxyl group resulting from the deprotection of the trityl group is bonded to the 5' phosphate group of the oligonucleotide. The amine or hydroxyl group can be bonded directly or indirectly via a linker group to the 5' phosphate group of the oligonucleotide. Suitable linker groups include, but are not limited to, those known and used for attaching 5'-reactive groups to oligonucleotides in SPOS.

[0077] Examples of linkers useful in the compositions and methods of the present disclosure include (i) a 5- to 20-carbon branched or unbranched alkylene chain; (ii) a branched or unbranched alkylene chain having at least 4- to 20 carbon atoms and at least one ether linkage; (iii) a polymer chain of 2-100 polyethylene glycol (PEG) moieties, which may further comprise alkyl, alkene, alkyne, ester, ether, amide, imide, and / or phosphodiester groups; or (iv) succinate linkers, PEG3 linkers, PEG4 linkers, sarcosine-glutarate linkers, hydrazone linkers, disulfide linkers, valine-citrulline linkers, valine-alanine linkers, tris-hexylamino linkers, hydroquinone-O,O'-diacetate ("Q") linkers, hexylamine linkers, hexyloxy linkers, pentaethylene glycol, or derivatives thereof. Other exemplary linkers include those shown in Table 2 below.

[0078] [Table 2-1] [Table 2-2] [Table 2-3]

[0079] Other exemplary linkers can be found, for example, in Doronina et al., Bioconjug Chem. 2006 Jan-Feb; 17(1):114-24; Sheyi et al., Pharmaceutics 2022, 14(2), 396; Acchione et al., MAbs. 2012 May 1; 4(3):362-372; Lu et al., Int J Mol Sci. 2016 April; 17(4):561; and Giese et al., Bioconjugate Chem. 2021, 32, 10, 2257-2267, each of which is incorporated herein in whole.

[0080] As described elsewhere in this specification, the deprotection reaction of the present disclosure yields a deprotected reactive group, such as a 5'-amine or 5'-hydroxyl (hydroxl), which can be used as a handle for further conjugation to an oligonucleotide. Thus, the method of the present disclosure may further include conjugating the deprotected reactive group of an oligonucleotide to a ligand, thereby forming a ligand-oligonucleotide conjugate. For example, in at least one embodiment, the deprotected reactive group is a primary amine, and the further conjugation reaction involves contacting the deprotected oligonucleotide with an amine reactive group, such as an ester, an N-hydroxysuccinimide (NHS) ester, or a carboxylic acid.

[0081] In such embodiments of methods involving further conjugation to a deprotected reactive group, the disclosure intends that a wide range of ligands may be used, including but not limited to carbohydrate complexes (e.g., N-acetylgalactosamine (GalNAc)); peptide molecules (e.g., RGD, cell-permeable peptides, integrins); lipophilic molecules (aromatic and non-aromatic), including steroid molecules; protein molecules (e.g., antibodies, enzymes, serum proteins); vitamins (water-soluble or lipid-soluble); polymers (water-soluble or lipid-soluble); small molecules, e.g., drugs, toxins, reporter molecules, or receptor ligands; nucleic acid cleavage complexes; metal chelators (e.g., porphyrins, texaphyllins, crown ethers, etc.); intercalators, including hybrid photonucleases / intercalators; crosslinkers (e.g., photoactive, redox-active); or combinations or derivatives thereof.

[0082] In one exemplary embodiment, the method comprises conjugating a deprotected oligonucleotide to a ligand that is a polyhydric carbohydrate. Such a polyhydric carbohydrate may comprise two, three, or four identical or non-identical carbohydrate moieties and be conjugated directly or indirectly via a linker to an oligonucleotide (such as an ASO compound or siRNA compound). For example, the ligand may be N-acetylgalactosamine (GalNAc) in the form of a monovalent, divalent, trivalent, or tetravalent conjugated moiety, as described in WO2014 / 076196, WO2014 / 207232, and WO2014 / 179620, each of which is incorporated herein by reference. GalNAc has a strong binding affinity to the asialoclyoglycoprotein (ASGP) receptor, and trivalent GalNAc conjugated to an oligonucleotide (such as an ASO or siRNA) may be used to target the compound to the liver. In another exemplary embodiment, the ligand or conjugate moiety can be galactose or a galactose derivative, i.e., any galactose-based molecule having an affinity for the ASGP receptor equal to or greater than that of galactose itself for the ASGP receptor.

[0083] As described elsewhere in this specification, the deprotection method of this disclosure may be carried out in which a trityl protecting group protects a reactive amine group bonded to the 5' end of the oligonucleotide, optionally via a linker. Thus, in at least one embodiment, the method involves a 5'-amine modified oligonucleotide of formula (I) (or a salt thereof or a pharmaceutically acceptable salt thereof) [ka] (In the formula, R 1 , R 2 , and R 3 Each of these is independently hydrogen, -OCH3, or -S(O)CH3, L is any linker, X is O or S, and Y is OH or SH. The oligonucleotide bonded at the 5' end of the phosphate oxygen is, [ka] (As shown by) It may be used to deprotect it.

[0084] The deprotection method involves heating the 5'-amine-modified oligonucleotide of formula (I) in an acid-free aqueous solution at a temperature of at least 60°C to obtain the compound of formula (II) (or its salt, or a pharmaceutically acceptable salt thereof). [ka] (In the formula, X is O or S, Y is -OH or -SH, L is any linker, and the oligonucleotide bonded to the 5' end of its phosphate oxygen is shown above for the compound of formula (I).) This includes forming.

[0085] Deprotection methods for compounds of formula (I) are also available for insoluble trityl-hydroxyl by-product compounds of formula (III): [ka] (In the formula, R 1 , R 2 , and R 3 Each of these is independently hydrogen, -OCH3, or -S(O)CH3. Generates.

[0086] A method for deprotecting the compound of formula (I) may further include separating the insoluble by-product compound of formula (II) from the compound of formula (III) by extraction, desalting, or precipitation and filtration.

[0087] As with the general deprotection methods of this disclosure, a wide range of linker structures may be used when the linker, L, is present in the compound of formula (I). In at least one embodiment, the linker L in the compound of formula (I) is the divalent linker of formula (Li): [ka] (In the formula, [ka] This represents the covalent bond of the primary amine group to the nitrogen atom. [ka] (This represents the covalent bond between the phosphate group and the oxygen atom.) That is the case.

[0088] The compound of formula (II), as described above, represents a 5'-amine-modified oligonucleotide, which is the desired product of the acid-free thermal deprotection reaction of the compound of formula (I). As described elsewhere in this specification, the compound of formula (II) is further reacted with a ligand molecule containing an amine-reactive group to form the ligand-oligonucleotide conjugate compound of formula (IV): [ka] or its salt, or a pharmaceutically acceptable salt thereof, (In the formula, L is a divalent linker, X is either S or O, Y is either -SH or -OH, R 4 is a ligand,

[0089] [ka] (This represents a covalent bond to the 5' end of an oligonucleotide.) You may obtain it.

[0090] Ligand R 4These ligands may include, but are not limited to, N-acetylgalactosamine (GalNAc), a tribranched cluster of the N-acetylgalactosamine moiety, lipid molecules, low molecular weight peptides (e.g., RGD, cell-permeable peptides, integrins), proteins, and antibodies, as described elsewhere in this specification. Ligand molecules, such as these, may, if necessary, be modified with amine-reactive groups, such as esters, NHS esters, or carboxylic acids, using standard techniques, or in some cases, are commercially available with amine-reactive groups for conjugation.

[0091] As described elsewhere in this specification, oligonucleotides having a trityl protecting group that protects a reactive primary amine at the 5' end of the oligonucleotide can be prepared, for example, in the final step of an automated SPOS system using a suitable protecting phosphoramidite reagent. Therefore, a compound of formula (I) useful in the method of this disclosure is obtained by reacting a compound of formula (A) with a compound of formula (B): [ka] (In the formula, R 1 , R 2 , and R 3 Each of these is independently hydrogen, -OCH3, or -S(O)CH3, and L is any linker. [ka] or its salt, or a pharmaceutically acceptable salt thereof, (In the formula, X is O or S, and Y is -OH or -SH, [ka] (This represents a covalent bond to the 5' end of an oligonucleotide.) It can be prepared by [method].

[0092] As described elsewhere in this specification and shown in the examples, the acid-free thermal deprotection method results in complete or near-complete conversion from the protected oligonucleotide compound of formula (I) to the desired deprotected oligonucleotide compound of formula (II) with little or no depurination detectable by MS analysis. Thus, in at least one embodiment of the method, the proportion of the oligonucleotide compound of formula (I) converted to the oligonucleotide compound of formula (II) is at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 100%. In addition, in at least one embodiment of the method, the proportion of depurinated oligonucleotide species detectable by MS analysis to the total deprotected oligonucleotide compound of formula (II) after deprotection treatment is less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%. [Examples]

[0093] Various features and embodiments of this disclosure are described in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily understand that the specific examples merely illustrate the invention as fully described by the subsequent claims. All embodiments and features described herein should be understood to be interchangeable and combinable with all embodiments contained herein.

[0094] [Example 1] Synthesis of 5'-amino modified oligonucleotides having MMTr protective amines This example illustrates an exemplary synthesis of an oligonucleotide having an MMTr-protected 5' amino group used in the examples of the present disclosure.

[0095] material and method Synthesis of 5'-MMTr-protected oligonucleotides The 5'-MMTr-protected oligonucleotides ("MMTr-ON") used in the examples were synthesized on a 1 μmol scale using a standard phosphoramidite method in an LGC MerMade-192x synthesizer, with phosphoramidites from Glen Research Inc. (Sterling, VA, USA) and Hongene Biotech Corporation (Shanghai, China). Exemplary phosphoramidite reagents used in oligonucleotide synthesis are shown in Table 3 below. [Table 3]

[0096] Glen UnySupport™ CPG 1000 (G110408) was purchased from Glen Research. (I) Coupling: Phosphoamidites in dry acetonitrile (0.05 M) were activated with 0.25 M 5-(ethylthio)-1H-tetrazole (ETT) in dry acetonitrile, and then coupled to a solid support for the coupling time (120 sec for DNA, 180 sec for LNA); (II) Capping: (A) Ac2O / 2,6-lutidine / THF, and (B) 16% 1-methylimidazole in THF; (III) Oxidation (for phosphodiester bonds): 30 sec with 0.02 M I2 in THF / Py / H2O; or sulfidation (for phosphorothioate bonds) for 180 sec with 0.1 M DDTT in pyridine; (IV) Detritylation: 3% dichloroacetic acid in dichloromethane.

[0097] After synthesis, the synthesized MMTr-ON was removed from the solid support and deprotected with concentrated ammonia water containing 10% ammonium acetate at 65°C for 12 hours. Crude MMTr-ON was then separated from the CPG by washing with 100 mg / mL sodium chloride solution and subsequently purified using a Glen-Pak® DNA purification cartridge (Glen Research, cat#60-5100). Failure sequences were removed by washing with 15% MeCN in 100 mg / mL aqueous NaCl solution, followed by elution of MMTr-ON from the cartridge with 50% MeCN / H2O. Organic matter was removed by evaporation using a vacuum concentrator. To monitor the reaction, samples were analyzed using a Waters Acquity H-class UPLC equipped with a QDA detector.

[0098] LC-MS analysis Synthesized oligonucleotides were isolated and characterized using LC-MS analysis on a Waters Acquity H-Class UPLC. The following LC-MS conditions were used: Analytical LC / MS: Reverse-phase chromatography; Mobile phase A: 14.3 mM triethylamine, 114 mM hexafluoro-2-propanol, 2.5% methanol in water; Mobile phase B: 14.3 mM triethylamine, 114 mM hexafluoro-2-propanol, 60% methanol in water; Mobile phase gradient elution: 100% A to 100% B in 3.5 mins; Column: Aquity UPLC® Oligonucleotide BEH C18, 130 Å, 1.7 μm, 2.1 mm × 50 mm (P#186003949); Column temperature: 65 °C; Sample injection volume: approx. 1 ug; Flow rate: 1.0 mL / min; UV detector: 260 nm.

[0099] result An exemplary 16-mer, MMTr-ON #5, having a 5'-MMTr-protected 5'-amino group, was synthesized according to the method described above. The sequence (SEQ ID NO: 1) and mass of MMTr-ON #5 are shown in Table 4. As indicated by the sequence of SEQ ID NO: 1 depicted in Table 4, the MMTr-protected 5'-amino modified oligonucleotide of MMTr-ON #5 contained three locked nucleic acids at each of the 5' and 3' ends adjacent to the 10-mer DNA, and all internucleotide bonds were phosphorothioates. [Table 4]

[0100] [Example 2] Acid-catalyzed deprotection compared to acid-free thermal deprotection This example illustrates a comparative study of standard acid-catalyzed deprotection reaction conditions recommended for the removal of trityl protecting groups from 5'-amino groups, compared to the acid-free thermal deprotection method of the present disclosure.

[0101] material and method Acid-catalyzed deprotection An industry-standard protocol for the deprotection of 5'-trityl-protected 5'-amine-modified oligonucleotides was performed. This protocol requires incubation in 20% acetic acid in water for 60 minutes at room temperature (RT). Exemplary MMTr-ON #5 synthesized as described in Example 1 (see Table 4) was treated according to a standard acid-catalyzed deprotection protocol. Additional acid-catalyzed deprotection of MMTr-ON #5 under varying conditions of time, acetic acid %, and temperature is as follows: (i) 20% acetic acid, room temperature, 90 min; (ii) 30% acetic acid, room temperature, 60 min; (iii) 20% acetic acid, 45°C, 60 min; (iv) 80% acetic acid, room temperature, 60 min; and (v) 80% acetic acid, room temperature, 30 min.

[0102] Acid-free thermal deprotection MMTr-ON #5 (see Table 4) was synthesized on a 1 micromolar scale according to Example 1 and purified. After purification, the eluted oligonucleotide was concentrated under vacuum to remove acetonitrile, and then diluted to 1.0 mL with water. The solution was then heated at 60°C for 60 minutes in a dry heat block to completely remove the 5'-MMTr protecting group. A precipitate of MMTr-OH, a byproduct of the deprotection reaction, was observed and subsequently removed by extraction with ethyl acetate (3 × 1 mL). (Alternatively, the insoluble MMTr-OH byproduct may be removed by using an optimal desalting method.) Pure 5'-amine-modified ON #5 was then confirmed by LC-MS and quantified by nanodrop (A260) to obtain 400 nmol of ON #5.

[0103] result As shown by the LC plot over time in Figure 1A, the standard acid-catalyzed deprotection protocol resulted in incomplete deprotection of MMTr-ON #5. In addition, the MS peak for acid-catalyzed deprotection (Figure 1B) indicated the detection of >5% of the depurinated species, whereas no such peak was detected after thermal deprotection without acid (Figure 1C). As shown by the LC plot in Figure 1D, the use of increased acetic acid ratios (30% or 80%) and / or increased incubation time at room temperature (90 minutes) did not result in any increase in conversion to the deprotected product. Furthermore, as explained by the MS results for deprotection in 20% acetic acid at 45°C for 60 minutes shown in Figure 1E, the use of a higher temperature of 45°C (rather than room temperature) resulted in an increase in overall conversion compared to the reaction carried out in 20% acetic acid at room temperature, but with the trade-off of a substantially increased level of the depurinated oligonucleotide product (compare the MS peak in Figure 1D with the MS peak in Figure 1B). In contrast to the results shown in Figure 1A, the use of acid-free thermal deprotection conditions (i.e., heating in unbuffered water at 60°C for 60 minutes) in MMTr-ON #5 resulted in near-complete deprotection (Figure 1F). These results demonstrate that, after elution, the 5'-MMTr-protected oligonucleotide can be nearly completely deprotected by simply heating in the elution buffer without the use of depurinating acidic conditions, once acetonitrile has been removed.

[0104] [Example 3] Evaluation of reaction variables for thermal deprotection without the use of acid This example illustrates the study of the effects of different reaction variables (temperature, buffer system, solvent) on the acid-free thermal deprotection protocol of Example 2.

[0105] material and method Acid-free thermal deprotection of 5'-MMTr protection ON #5, as described in Example 2, was performed under various conditions of temperature, buffer / solvent, and pH, and the conversion percentage to MMTr deprotected species and depurination of oligonucleotides were observed. The conversion percentage and the presence of depurinated species were determined using LC-MS as described in Example 1.

[0106] result The results are summarized in Table 5 below (LC-MS traces are not shown). [Table 5]

[0107] During temperature evaluation, it was found that complete deprotection was not achieved below 60°C (Table 5, items 1-3). Therefore, all other experiments were performed at 60°C. Various solutions commonly used in the purification and handling of oligonucleotides were evaluated.

[0108] Complete deprotection was observed in phosphate-buffered saline (PBS, pH 7.4) and TE buffer (pH 8.0), indicating that an acidic pH is not necessary to promote the completion of the reaction (Table 5, items 7-8). However, concentrated ammonium hydroxide (NH4OH) resulted in the formation of only trace amounts of product (Table 5, item 6).

[0109] A white precipitate was observed after incubation in pH 7.0 and buffered samples (pH 7.4 and pH 8). As described in Example 3, this white precipitate was confirmed by NMR to be the by-product, MMTr-OH, as shown by comparing the spectrum of the commercially available sample (top, Figure 2A) with the spectrum of the isolated white precipitate (bottom, Figure 2A). The insolubility of the MMTr-OH by-product was hypothesized to facilitate the acid-free thermal deprotection reaction by minimizing the known reversibility of MMTr deprotection (see, e.g., Reference 17) through the sequestration of the by-product as an insoluble species. Figure 2B shows MMTr in 20% acetic acid.+ This diagram illustrates the mechanism by which the insolubility of MMTr-OH in water can drive the deprotection reaction. Supporting evidence is found in Table 5, item 5, which shows that incubation of ON #5 in a 50:50 CH3CN / H2O mixture results in incomplete (55%) deprotection. This is likely due to increased solubility of MMTr-OH in the 50% CH3CN solvent, which leads to an unfavorable equilibrium shift for MMTr deprotection.

[0110] [Example 4] Effect of oligonucleotide sequence on acid-free deprotection This example describes a study on whether the oligonucleotide sequence affects the deprotection of oligonucleotides using the acid-free thermal deprotection method of this disclosure. More specifically, it is to rule out the possibility that the acid-free thermal deprotection may be biased by a particular oligonucleotide sequence.

[0111] material and method Eight different MMTr-ONs, each with a variant sequence relative to MMTr-ON #5 (see Table 4), were synthesized according to the method of Example 1. The variant sequences of the eight different MMTr-ONs contained all possible bases at the first position, all adjacent bases at the second position, and several variants at the third position. In addition, oligonucleotides with a 5'-DMTr protected 5'-hydroxyl group were synthesized. The sequences (SEQ ID NOs. 2-10) and masses of these 5'-protected oligonucleotides are summarized in Table 6 below. [Table 6]

[0112] After synthesis and purification, each of the 5'-MMTr protected ONs #5 through #14 (Table 6) was concentrated under vacuum to remove acetonitrile, and then diluted to 1.0 mL with water. This solution was then heated at 60°C for 60 minutes in a dry heat block to completely remove the 5'-MMTr protecting group. In addition, all control samples of 5'-MMTr-ONs in Table 6 were subjected to standard acid-catalyzed deprotection conditions.

[0113] result Using an acid-free thermal deprotection protocol, complete 5'-MMTr deprotection was achieved for all 5'-MMTr-ON #6–13. In contrast, acid-catalyzed deprotection of 5'-MMTr-ON #6–13 resulted in incomplete deprotection. Figures 3A–3F depict exemplary LC plots comparing the results of acid-catalyzed deprotection and acid-free thermal deprotection for 5'-MMTr-ON #6, #7, #9, #11, #12, and #13. In each case, acid-free thermal deprotection resulted in complete or near-complete deprotection, demonstrating the sequence independence of this method.

[0114] [Example 5] Thermal deprotection of DMTr-O protected oligonucleotide This example illustrates a comparative study to determine whether the acid-free thermal deprotection protocol of the present disclosure can be used to deprotect 5'-DMTr-protected oligonucleotides, e.g., DMTr-ON #14 (Table 6), as well as or better than the acid-catalyzed deprotection protocol. In this study, 5'-DMTr protects the 5'-hydroxyl moiety rather than the amino group. Figure 4A illustrates the deprotection reaction conditions compared in this example.

[0115] material and method Deprotection of 5'-DMTr-ON #14 (see Table 6) was carried out using either acid-free thermal deprotection at 95°C for 1.5 hours, or standard 5% trifluoroacetic acid treatment at room temperature for 15 minutes.

[0116] result As shown by the LC plot in Figure 4B, complete deprotection of 5'-DMTr-ON to obtain 5'-OH ON was achieved under slightly more rapid, acid-free thermal deprotection conditions of 1.5 hours at 95°C.

[0117] conclusion The simplicity of the acid-free thermal deprotection method of this disclosure and its compatibility with standard oligonucleotide purification techniques are demonstrated by the examples described above. After elution of the MMTr-ON product from the oligonucleotide purification column, the resulting protected oligonucleotide can be deprotected simply by heating in an elution buffer after removal of acetonitrile. Insoluble MMTr-OH byproducts may be removed, for example, by extraction with ethyl acetate or by any optimal desalting method. The streamlined purification and deprotection method reduces overall complexity and the time required for the purification of amino-modified oligonucleotides, while eliminating the use of corrosive acids and the corresponding waste flow. Thus, the method described herein makes oligonucleotide conjugate materials more readily available and expands the use of these species for human health and other applications.

[0118] While the foregoing disclosure of the present invention is described in some detail with examples and illustrations for the purposes of clarity and understanding, the present disclosure, including the examples, descriptions and embodiments set forth herein, is for illustrative purposes only and is intended to be illustrative and should not be construed as limiting the present disclosure. Additional embodiments of the present invention are described in the appended claims.

[0119] Various modifications or changes to the examples, descriptions, and embodiments described herein may be made and will be apparent to those skilled in the art as being within the spirit and scope of this disclosure and the appended claims. Furthermore, those skilled in the art will recognize a number of methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of this disclosure and are covered by the appended claims.

[0120] All publications, patents, patent applications, and other documents referenced herein are incorporated herein by reference in whole for all purposes to the same extent as each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes. In case of any inconsistency, including the terms specified herein, this specification shall prevail.

[0121] References JPEG2026530457000042.jpg251170JPEG2026530457000043.jpg131170

Claims

1. A method for deprotecting an oligonucleotide comprising an amine group or a hydroxyl group protected by a trityl protecting group, comprising heating the oligonucleotide in an acid-free aqueous solution at a temperature of at least 60°C to cleave the trityl protecting group and thereby obtain the oligonucleotide comprising a deprotected amine group or hydroxyl group.

2. The method according to claim 1, wherein the trityl protecting group is selected from trityl, monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), and dimethoxymethylsulfonyltrityl (DMS(O)MTr).

3. The aforementioned trityl protecting group 【Chemistry 1】 (In the formula, 【Chemistry 2】 (However, this indicates a covalent bond to the nitrogen atom of the amine group or the oxygen atom of the hydroxyl group.) The method according to any one of claims 1 to 2, having a structure selected from the following.

4. The method according to any one of claims 1 to 3, wherein the deprotection of the amine group or hydroxyl group generates an insoluble trityl byproduct, and the method further comprises separating the insoluble trityl byproduct from the deprotected oligonucleotide.

5. The method according to claim 4, wherein the insoluble trityl by-product is separated by extraction, desalting, or precipitation and filtration.

6. The method according to claim 4 or 5, wherein the insoluble trityl by-product is MMTr-OH, DMTr-OH, Tr-OH, or DMS(O)Tr-OH.

7. The insoluble trityl by-product is 【Transformation 3】 The method according to any one of claims 4 to 6, having a structure selected from the following.

8. A method comprising the amine group or hydroxyl group being bonded to the 5' phosphate group of the oligonucleotide, according to any one of claims 1 to 7.

9. A method wherein the amine group or hydroxyl group is bonded to the 5' phosphate group of the oligonucleotide via a linker group, the method according to any one of claims 1 to 8.

10. The method according to claim 9, wherein the linker group comprises a non-branched alkylene chain of 5 to 12 carbon atoms, and optionally the alkylene chain further comprises at least one ether bond.

11. The method according to claim 9, wherein the linker group is selected from succinate linker, PEG3 linker, PEG4 linker, sarcosine-glutarate linker, hydrazone linker, disulfide linker, valine-citrulline linker, valine-alanine linker, tris-hexylamino linker, hydroquinone-O,O'-diacetate ("Q") linker, hexylamine linker, hexyloxy linker, pentethylene glycol linker, and derivatives thereof.

12. The method according to any one of claims 1 to 11, wherein the amine group of the oligonucleotide protected with a trityl protecting group is a primary amine group.

13. The method according to claim 12, further comprising reacting the deprotected primary amine group of the oligonucleotide with a ligand molecule containing an amine-reactive group to conjugate the ligand to the oligonucleotide, thereby forming a ligand-oligonucleotide conjugate.

14. The method according to claim 13, wherein the amine-reactive group is selected from esters, NHS esters, and carboxylic acids.

15. The method according to claim 13, wherein the ligand molecule is selected from N-acetylgalactosamine (GalNAc), a tribranched cluster of the N-acetylgalactosamine moiety, a lipid molecule, a low molecular weight peptide, a protein, and an antibody.

16. The method according to any one of claims 1 to 15, wherein the conversion rate from the oligonucleotide having a trityl protecting group to the oligonucleotide having a deprotected amine group or hydroxyl group is at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 100%.

17. A method for deprotecting a 5'-amine modified oligonucleotide compound of formula (I), comprising heating the 5'-amino modified oligonucleotide compound of formula (I) in an acid-free aqueous solution at a temperature of at least 60°C to form an oligonucleotide of formula (II) and a by-product compound of formula (III): 【Chemistry 4】 (In the formula, R １ , R ２ , and R ３ Each of them independently contains hydrogen, -OCH ３ , or -S(O)CH ３ And, L is any linker, X is either O or S, Y is either OH or SH, and 【Transformation 5】 (This represents a covalent bond to the 5' end of the oligonucleotide.) or its salt or a pharmaceutically acceptable salt thereof; 【Transformation 6】 (In the formula, X is either O or S, Y is -OH or -SH, L is any linker, and 【Transformation 7】 (This represents a covalent bond to the 5' end of the oligonucleotide.) or its salt, or a pharmaceutically acceptable salt thereof; 【Transformation 8】 (In the formula, R １ , R ２ , and R ３ are each independently hydrogen, -OCH ３ , or -S(O)CH ３ ) A method that includes the following:

18. The method according to claim 17, further comprising extracting, desalting, or separating the oligonucleotide compound of formula (II) from the by-product compound of formula (III) by precipitation and filtration.

19. The compound of formula (I) reacts with the phosphoramidite compound of formula (A) with the oligonucleotide compound of formula (B): 【Chemistry 9】 (In the formula, R １ , R ２ , and R ３ Each of them independently contains hydrogen, -OCH ３ , or -S(O)CH ３ and (L is any linker); 【Chemistry 10】 or its salt, or a pharmaceutically acceptable salt thereof, (In the formula, X is either O or S, Y is -OH or -SH, and 【Chemistry 11】 (This represents a covalent bond to the 5' end of the oligonucleotide.) The method according to any one of claims 17 to 18, prepared by...

20. L is a divalent linker represented by the formula (L - i): 【Chemistry 12】 (In the formula, 【Chemistry 13】 This represents the covalent bond of the primary amine group to the nitrogen atom. 【Chemistry 14】 (This represents the covalent bond between the phosphate group and the oxygen atom.) The method according to any one of claims 17 to 19.

21. The method according to any one of claims 17 to 19, wherein the linker L is (i) a 5-carbon to 20-carbon branched or unbranched alkylene chain; or (ii) a 4-carbon to 20-carbon branched or unbranched alkylene chain comprising at least one ether bond.

22. The method according to any one of claims 17 to 19, wherein the linker L comprises a succinate linker, a PEG3 linker, a PEG4 linker, a sarcosine-glutarate linker, a hydrazone linker, a disulfide linker, a valine-citrulline linker, a valine-alanine linker, a tris-hexylamino linker, a hydroquinone-O,O'-diacetate ("Q") linker, a hexylamine linker, a hexyloxy linker, a pentaethylene glycol linker, or a derivative thereof.

23. The oligonucleotide compound of formula (II) is reacted with a ligand molecule containing an amine-reactive group to obtain the ligand-oligonucleotide conjugate compound of formula (IV): 【Chemistry 15】 (In the formula, L is a divalent linker, X is either S or O, Y is -SH or -OH, R ４ , is a ligand and 【Chemistry 16】 (This represents a covalent bond to the 5' end of the oligonucleotide.) or its salt, or its pharmaceutically acceptable salt The method according to any one of claims 18 to 23, further comprising forming a

24. The method according to claim 23, wherein the amine-reactive group is selected from esters, NHS esters, and carboxylic acids.

25. The ligand R ４ The method according to any one of claims 23 to 24, wherein is selected from N-acetylgalactosamine (GalNAc), a tribranched cluster of N-acetylgalactosamine residues, a lipid molecule, a low molecular weight peptide, a protein, and an antibody.

26. The method according to any one of claims 1 to 25, wherein the acid-free aqueous solution is heated to a temperature of at least about 65°C, at least about 70°C, at least about 75°C, at least about 80°C, at least about 85°C, at least about 90°C, or at least about 95°C.

27. The method according to any one of claims 1 to 26, wherein the acid-free aqueous solution is heated to a temperature of about 60°C to about 100°C, about 60°C to about 80°C, about 60°C to about 75°C, or about 60°C to about 70°C.

28. The method according to any one of claims 1 to 27, wherein the acid-free aqueous solution is heated for at least about 1 hour or at least about 2 hours.

29. The method according to any one of claims 1 to 28, wherein the acid-free aqueous solution is heated for about 1 to 2 hours.

30. The method according to any one of claims 1 to 29, wherein the acid-free aqueous solution has a pH of approximately 6.5 to approximately 8.0, approximately 6.8 to approximately 7.8, approximately 6.8 to approximately 7.6, approximately 7.0, approximately 7.2, or approximately 7.

4.

31. The method according to any one of claims 17 to 30, wherein the proportion of the oligonucleotide compound of formula (I) converted to the oligonucleotide compound of formula (II) is at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 100%.

32. The method according to any one of claims 1 to 31, wherein the ratio of depurinated oligonucleotides to total deprotected oligonucleotides detected by MS analysis after deprotection is less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.01%.

33. The method according to any one of claims 1 to 32, wherein the length of the oligonucleotide is 2 mer to 100 mer, and optionally the length of the oligonucleotide is 3 mer to 75 mer, 5 mer to 50 mer, 15 mer to 50 mer, 15 mer to 40 mer, 15 mer to 30 mer, or 2 mer to 10 mer.

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