Compositions comprising dichloroacetic acid, methods of making and uses thereof

Abstract

A composition comprising dichloroacetic acid and glyoxylic acid, wherein the content of glyoxylic acid is below 1000 ppm, and a method for preparing and using the composition. A method for preparing the composition, which comprises mixing a dichloroacetic acid raw material having a glyoxylic acid content of more than 1000 ppm with a glyoxylic acid capturing reagent, and a method for preparing oligonucleotides, which comprises using the composition. It has been found that glyoxylic acid is an impurity in commercially available DCA products, which can cause failure in oligonucleotide synthesis. Ion chromatography can be used to detect glyoxylic acid in dichloroacetic acid and accurately determine its concentration.

Description

[0001] Reference to Related Applications

[0002] This application claims priority to International Application PCT / CN2020 / 091643, filed May 21, 2020, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to a composition comprising dichloroacetic acid, a method for preparing the same and uses thereof. BACKGROUND

[0004] One method of inhibiting the expression of a particular gene is to use oligonucleotides, particularly those that are complementary to a particular target messenger RNA (mRNA) sequence. Several oligonucleotides are in clinical trials for such use. Oligonucleotides can also act as competitive inhibitors of transcription factors, interacting with double-stranded DNA during the process of transcription, thereby modulating its action.

[0005] The use of oligonucleotides and their analogs also exists in diagnostic tests, research reagents, and other experimental procedures. The widespread use of such oligonucleotides increases the need for rapid, inexpensive, and efficient procedures for their modification and synthesis.

[0006] DCA (dichloroacetic acid) is a commonly used reagent for deblocking nucleotides in oligonucleotide synthesis. Since the addition of new nucleotides involves the repeated use of DCA to deprotect 5'-hydroxyl groups, it is essential that the reagent be as free of contaminants as possible, as these contaminants can spread impurities and produce incorrect target oligonucleotide sequences. It has been found that impurities in DCA, such as chloral hydrate and 2,2-dichloroacetic anhydride (DCAA), can cause oligonucleotide synthesis to fail.

[0007] SUMMARY OF THE DISCLOSURE

[0008] Since DCA is repeatedly used to remove protecting groups from oligonucleotides, it is critical that DCA be free of contaminants that can spread impurities and produce incorrect target oligonucleotide sequences. It has been found that glyoxylic acid present in DCA can have a detrimental effect when such DCA is used to deprotect acid-labile protecting groups in oligonucleotide synthesis. It has further been found that glyoxylic acid in DCA can be detected and its concentration accurately measured by ion chromatography (IC method).

[0009] It is an object of the present disclosure to provide a composition comprising DCA and glyoxylic acid, wherein the content of glyoxylic acid is below 1000 ppm.

[0010] It is another object of the present disclosure to provide a method for preparing the composition.

[0011] Another object of the present disclosure is to provide a method for preparing an oligonucleotide by using the composition.

[0012] Another object of the present disclosure is to provide a method for detecting glyoxylic acid in a DCA sample.

[0013] The above and other important objects are realized by the detailed description.

[0014] Detailed description of the present disclosure

[0015] In a first aspect, the present disclosure provides a composition comprising DCA and glyoxylic acid, wherein the content of glyoxylic acid is 1000 ppm or less.

[0016] In some embodiments, the content of glyoxylic acid in the composition is 950 ppm, 900 ppm, 800 ppm, 700 ppm, 600 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.1 ppm, 1.05 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm or less.

[0017] In some embodiments, the composition has a content of glyoxylic acid of 500 ppm, 450 ppm, 400 ppm, 350 ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm, 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, 15 ppm, 12 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm or less.

[0018] In some embodiments, the composition has a glycolate content of 50 ppm, 45 ppm, 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 12 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm or less.

[0019] In some embodiments, the glyoxylic acid content in the composition is below 15 ppm, 14.5 ppm, 14 ppm, 13.5 ppm, 13 ppm, 12.5 ppm, 12 ppm, 11.5 ppm, 11 ppm, 10.5 ppm, 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm.

[0020] In some embodiments, the DCA content in the composition is higher than 50%, higher than 55%, higher than 60%, higher than 65%, higher than 70%, higher than 75%, higher than 80%, higher than 85%, higher than 90%, higher than 95%, higher than 98%, higher than 99%, higher than 99.5%, higher than 99.8%, for example 99.8153% (by weight, GC / IC area, or any combination thereof).

[0021] In some embodiments, the glyoxylic acid content in the composition is > 0, meaning that the glyoxylic acid content is not 0 (in some embodiments of the disclosure, the glyoxylic acid content is preferably > 0.05 ppm, or > 1 ppm).

[0022] In some embodiments, the DCA and glyoxylic acid content in the composition is determined, for example by ion chromatography. The ion chromatography is preferably as defined below. Preferably, the DCA content is quantified by area normalization. Preferably, the glyoxylic acid is quantified by external standard.

[0023] In the external standard method, the method for preparing the standard curve comprises:

[0024] i) preparing glyoxylic acid standard working solutions with concentrations of 2.5 ppm, 5 ppm, 15 ppm, 20 ppm and 25 ppm, respectively; and

[0025] ii) performing ion chromatography analysis; linearly regressing the concentrations of the glyoxylic acid series standard working solutions and the corresponding peak areas of the response ion chromatogram to obtain a standard curve.

[0026] In the external standard method, the conditions of ion chromatography analysis are as described in the present disclosure. The concentrations of the glyoxylic acid series standard working solutions and the corresponding peak areas of the response ion chromatogram are shown in Table 1.

[0027] Table 1

[0028]

[0029]

[0030] The regression equation of the standard curve is: y = 0.00003x - 0.00004 (R 2 = 0.99861).

[0031] In some embodiments, the composition defined in the present disclosure is used for deblocking nucleotides in oligonucleotide synthesis.

[0032] In a second aspect, the present disclosure provides a DCA reagent, wherein the content of glyoxylic acid is below 1000 ppm.

[0033] In some embodiments, the DCA reagent has a content of glyoxylic acid of 950 ppm, 900 ppm, 800 ppm, 700 ppm, 600 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.1 ppm, 1.05 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm or less.

[0034] In some embodiments, the DCA reagent has a content of glyoxylate of 500 ppm, 450 ppm, 400 ppm, 350 ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm, 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, 15 ppm, 12 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm or less.

[0035] In some embodiments, the DCA reagent has a content of glyoxylic acid of 50 ppm, 45 ppm, 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 12 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm or less.

[0036] In some embodiments, the DCA reagent contains less than 15 ppm, 14.5 ppm, 14 ppm, 13.5 ppm, 13 ppm, 12.5 ppm, 12 ppm, 11.5 ppm, 11 ppm, 10.5 ppm, 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm of glyoxylic acid.

[0037] In some embodiments, the DCA reagent contains substantially no glyoxylic acid, in particular, no glyoxylic acid or no detectable glyoxylic acid.

[0038] In some embodiments, the DCA reagent contains more than 90% (e.g., more than 95%, more than 98%, more than 99%, or more than 99.5%, more than 99.8%, e.g., 99.8153%) of DCA by weight, GC area, ion chromatography, or any combination thereof.

[0039] In some embodiments, the DCA reagent contains more than 0 of glyoxylic acid, which means that the content of glyoxylic acid is not 0 (in some embodiments of the present disclosure, the content of glyoxylic acid is preferably more than 0.05 ppm, or more than 1 ppm).

[0040] In some embodiments, the content of DCA and glyoxylic acid in the DCA reagent is determined, for example, by ion chromatography. Ion chromatography is preferably as defined below. Preferably, the content of DCA is quantified by area normalization. Preferably, the glyoxylic acid is quantified by external standard method as described in the present disclosure.

[0041] In some embodiments, the DCA reagent as defined in the present disclosure is used for deblocking nucleotides in oligonucleotide synthesis.

[0042] In a third aspect, the present disclosure provides a method for preparing the composition as described, comprising mixing a DCA raw material with a content of glyoxylic acid higher than 1000 ppm with a glyoxylic acid capturing reagent.

[0043] In some embodiments, the capturing reagent can physically bind to glyoxylic acid, or can chemically react with glyoxylic acid, and the product has no or little effect on the synthesis of oligonucleotides.

[0044] In some embodiments, the capturing reagent is selected from the group consisting of amino acids, chemicals with bifunctional or multifunctional groups, hydroxylamine compounds, reducing agents, and mixtures thereof.

[0045] In some embodiments, the amino acid is a commonly used amino acid, including but not limited to cysteine, lysine, phenylalanine, and mixtures thereof.

[0046] In some embodiments, the chemical with bifunctional or multifunctional groups is a commonly used chemical with bifunctional or multifunctional groups, including but not limited to diols, for example, (2R)-propane-1,2-diol.

[0047] In some embodiments, the hydroxylamine compound is a commonly used hydroxylamine compound, including but not limited to hydroxylamine salts, such as hydroxylamine hydrochloride.

[0048] In some embodiments, the reducing agent is a commonly used reducing agent, including but not limited to silanes, for example, triethylsilane.

[0049] In some embodiments, the amount of the capturing reagent is selected according to the content of glyoxylic acid in the composition. For example, the molar ratio of the capturing reagent to glyoxylic acid is ≥1, such as ≥2 or ≥5. In embodiments, in order to save costs, the molar ratio of the capturing reagent to glyoxylic acid is 1-10, such as 5.

[0050] The purpose of mixing is to mix the DCA raw material and the capturing reagent sufficiently. Therefore, in some embodiments, the mixing time of the DCA raw material and the capturing reagent is selected according to the content of glyoxylic acid in the system. For example, the mixing time is 12-36 hours (hrs). The temperature of the mixing is, for example, room temperature.

[0051] In some embodiments, the content of glyoxylic acid in the DCA feedstock is, for example, >1100 ppm, >1200 ppm, >1300 ppm, >1400 ppm, >1500 ppm, >1600 ppm, >1700 ppm, >1800 ppm, >1900 ppm, >2000 ppm, >2100 ppm, >2500 ppm, >2600 ppm (e.g., 2142.7 ppm). In some embodiments, the content of glyoxylic acid in the DCA feedstock will adversely affect the synthesis of oligonucleotides.

[0052] In some embodiments, the process of preparing the composition further comprises distillation (e.g., vacuum distillation) after the DCA feedstock and the trapping reagent are mixed thoroughly (e.g., by standing or stirring) to obtain the target product.

[0053] In some embodiments, the product has any one of the following properties.

[0054] (i) it is a solid (e.g., crystalline); thus, it does not distill out under DCA vacuum distillation conditions;

[0055] (ii) it is a liquid, provided that it does not distill out under DCA vacuum distillation conditions; or

[0056] (iii) it is a liquid, provided that although it distills out under DCA vacuum distillation conditions, it is readily separable from DCA by fractional distillation or other methods.

[0057] In a fourth aspect, the disclosure provides a method of preparing a DCA reagent, which is the same as the method of preparing the composition defined above.

[0058] In a fifth aspect, the disclosure provides a method of preparing an oligonucleotide, which comprises using the composition or DCA reagent defined above as a deprotecting reagent.

[0059] In some embodiments, the method of preparing an oligonucleotide comprises contacting an oligonucleotide with the composition or DCA reagent defined above.

[0060] In some embodiments, the oligonucleotide bears a protecting group.

[0061] Oligonucleotide

[0062] The basic subunit of an oligonucleotide, such as RNA or DNA, is described as follows.

[0063]

[0064] In an oligonucleotide, Bx is the binding member, the phosphate moiety [P(=G')(G"H)OH] is the linking member, and the residue known as the sugar backbone is the backbone member. The phosphate member forms a covalent bond by condensation with the 5'-OH of an adjacent subunit, thereby forming a phosphodiester linkage. When each of G' and G" is O, it is known as a phosphodiester linkage; when one of G' or G" is S and the other is O, it is known as a phosphorothioate linkage, and when both G' and G" are S, it is known as a phosphorodithioate linkage.

[0065] Those skilled in the art will recognize that, in naturally occurring nucleotides, R2' is H for DNA (deoxyribonucleic acid) and OH for RNA (ribonucleic acid), each of G' and G" is O, and Bx is one of the following structures:

[0066]

[0067] where G, C, A, U, and T are guanine, cytosine, adenine, uracil, and thymine, respectively.

[0068] In the above formula, G' and G" can be O or S, and R2' can be H, OH, or other values.

[0069] In naturally occurring RNA, the binding member is a nucleosyl group selected from G, C, A, and U, the backbone includes a sugar residue (ribosyl, i.e., R2' is OH) and a phosphate (G'=G"=O). The ribosyl sugar residue is the backbone member, and the phosphate links adjacent monomers through the 5'- and 3'-oxygen atoms on the ribosyl ring. The sugar is covalently bound to the nucleoside base at the 1 '-position, with the -beta-D configuration predominating.

[0070] Naturally occurring DNA is similar to RNA, except that the sugar is 2'-deoxyribosyl (R2' is H).

[0071] Oligonucleotides of the present disclosure generally include both naturally occurring and non-naturally occurring oligonucleotides. Generally, oligonucleotides of the present disclosure include compounds of Formula (I):

[0072]

[0073] where each Bx is a nucleobase as defined in the present disclosure, each q is 0 or 1, each R2' is H or OH, reversibly protected OH, or a substituent or forms a bridge with R4'; R3' is H or a substituent; R4' is H, a substituent or forms a bridge with R2' or R5'; R5' is H, a substituent or forms a bridge with R4', and each curved bond indicates that the bond can be in the up or down configuration.

[0074] Each Bx in a naturally occurring oligonucleotide is selected from G, C, A, U (for RNA) and T (DNA), each G' and G" is O, each R3', each R4', each R5' is H, each q is 1, n is an integer, and the sugar oxygens are in the ribo configuration. Conversely, a non-naturally occurring oligonucleotide includes at least one of the following: at least one Bx is a nucleobase other than G, C, A, U (for RNA) and T (DNA), and at least one sugar oxygen is not in the ribo configuration. As used in the present disclosure, the terms "oligonucleotide" and non-naturally occurring oligonucleotide, or mixtures thereof. In specific embodiments of the present disclosure, the term oligonucleotide has both naturally occurring and non-naturally occurring nucleotide subunits. In specific embodiments of the present disclosure, one or more of the nucleobases, sugar backbone, and / or phosphate linking members are non-naturally occurring. These features are described in more detail below.

[0075] Sugar backbone

[0076] In general, the sugar backbone has the following structure:

[0077]

[0078] where each Bx is a nucleobase as defined in the present disclosure, q is 0 or 1, each R2' is H, OH, reversibly protected OH, or a substituent or together with R4' forms a bridge; R3' is H or a substituent; R4' is H, a substituent or together with R2' or R5' forms a bridge; R5' is H, a substituent or together with R4' forms a bridge. The dotted line (— ) indicates the position of the sugar group binding to the phosphate linker to form a nucleotide linkage.

[0079] Those skilled in the art will recognize that when R2' is in the down configuration and q is 1, the ring is a ribo configuration, and when R2' is in the up configuration and q is 1, the ring is an arabino configuration. Likewise, when q is 0 and R2' is in the down configuration, the ring is a mer configuration. As described in more detail in the present disclosure, when R2' and R4' are linked to form a bridge, the ring is referred to as a locked nucleic acid (LNA). In some embodiments, the bridge formed by R2' and R4' is R2'-O-(CH2) r -R4' (where r is 1 or 2) or R2'-CH2-O-CH2-R4' (using R2' and R4' in the subformula to indicate the point of attachment). LNAs can exist in either an alpha-L- or beta-D- conformation. Each of these analogs possesses some useful properties, including resistance to exonuclease activity, induction of endonuclease activity, and modulation of hybridization.

[0080] When R4' and R5' form a bridge, it can form a tricycle with the sugar ring to which it is attached. The structure of a tricyclic nucleoside is as follows.

[0081]

[0082] Those skilled in the art will recognize that similar thiophosphates, and 2'-substituted tricyclic deoxynucleosides, can be prepared by using a thiooxidizing agent instead of an oxidizing agent. In the case of ribonucleic acid, 2'-substituted tricyclic deoxynucleosides can be prepared from a similar 2'-OH protecting group.

[0083] Suitable 2'-substituents corresponding to R2' include: F, O-alkyl (e.g., O-methyl), S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-ynyl, S-ynyl, N-ynyl, O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups can be substituted or unsubstituted C1-C1 groups, respectively. 10 Alkyl or C2-C 10 Alkenyl or ynyl. O[(CH2)] is particularly preferred. g O] h CH3, O(CH2) g OCH3, O(CH2) g NH2, O(CH2) g CH3, O(CH2) g ONH2 and O(CH2) g ON[(CH2) g [CH3]2, wherein g and h are 1 to about 10. Other preferred oligonucleotides include C1 to C2 at the 2' position. 10 The substituents include lower alkyl groups, substituted lower alkyl groups, alkenyl groups, alkynyl groups, aryl groups, O-alkaneyl groups or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SO2CH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkaneyl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups that improve the pharmacokinetic properties of oligonucleotides, or groups that improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. A preferred 2'-modification is 2'-deoxy-2'-methoxyethoxy (2'-OCH2CH2OCH3, also known as 2'-O-(2-methoxyethoxy) or 2'-MOE ribosyl). Other preferred modifications include 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0084] Other preferred modifications include 2'-methoxy (2'-0-CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-0-CH2-CH=CH2), and 2'-fluoro (2'-F). The 2'-modification can be in the arabino (up) position or the ribo (down) position. The preferred 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions including the 3' position of the oligonucleotide, particularly the 3' terminal nucleotide, or in a 2'-5' linked oligonucleotide.

[0085] Further representative substituents include groups of the formula (I) a or formula (II) a :

[0086]

[0087] wherein:

[0088] R b is O, S, or NH;

[0089] R d is a single bond, O, or C(=0);

[0090] R e is C1-C 10 alkyl, N(R k )(R m ), N=C(R p )(R q ), N=C(R p )(R r ), or formula (Ilia);

[0091]

[0092] each R c , R q , R r , R s , R t , R u , and R v is independently hydrogen, C(O)R w , substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C2-C 10 alkenyl, substituted or unsubstituted C2-C 10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group, or a conjugating group, wherein the substituents are selected from the group consisting of hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl;

[0093] or optionally, R u and R v , together with the nitrogen atom to which they are attached, form a phthalimide moiety.

[0094] each R w is independently substituted or unsubstituted C1-C 10 alkyl, trifluoromethyl, cyanoethoxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, isobutyryl, phenyl, or aryl;

[0095] R k is hydrogen, a nitrogen protecting group, or -R x ; y ;

[0096] R p is hydrogen, a nitrogen protecting group, or -R x ; y ;

[0097] R x is a bond or a linking group;

[0098] R y is a chemical functional group, a conjugating group, or a solid support medium;

[0099] each R m and R n is independently H, a nitrogen protecting group, substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C2-C 10 alkenyl, substituted or unsubstituted C2-C 10 alkynyl, wherein the substituents are selected from the group consisting of hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH 3+ , N(R u )(R y ), guanidinyl, and acyl, wherein the acyl is an amide or an ester;

[0100] or R m and R n , together are a nitrogen protecting group, are linked into a cyclic structure, optionally including an additional heteroatom selected from N and O, or are a chemical functional group;

[0101] R i is OR z , SR z , or N(R z )2; each R zindependently H, C1-C8alkyl, C1-C8haloalkyl, C(=NH)N(H)R u , C(=O)N(H)R u or OC(=O)N(H)R u ;

[0102] R f , R g and R h include ring systems having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen and sulfur, the ring system being aliphatic, unsaturated aliphatic, aromatic or saturated or unsaturated heterocyclic;

[0103] R j is alkyl or haloalkyl having from 1 to about 10 carbon atoms, alkenyl having from 2 to about 10 carbon atoms, alkynyl having from 2 to about 10 carbon atoms, aryl having from 6 to about 14 carbon atoms, N(R k )(R m )OR k , halogen, SR k or CN;

[0104] ma is 1 to about 10;

[0105] mb is independently 0 or 1;

[0106] mc is 0 or an integer from 1 to 10;

[0107] md is an integer from 1 to 10;

[0108] me is 0, 1 or 2; and

[0109] with the proviso that when mc is 0, md is greater than 1.

[0110] Particularly preferred sugar substituents include O[(CH2) g O] h CH3, O(CH2) g OCH3, O(CH2) g NH2, O(CH2) g CH3, O(CH2) g ONH2and O(CH2) g ON[(CH2) g CH3)]2, wherein g and h are 1 to about 10.

[0111] Some preferred oligomeric compounds of the present disclosure comprise at least one nucleoside having one of the following substituents: C1-C 10lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycle alkyl, heterocycle alkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligomers, or groups for improving the pharmacodynamic properties of oligomers, and other substituents having similar properties. A preferred modification is 2'-methoxyethoxy [2'-0-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE, a group that is an alkoxyl ether. A further preferred modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE.

[0112] Nucleobase

[0113] A nucleobase Bx(also referred to in the art as a nucleic acid base or simply a base) can be a naturally occurring G, C, A, U, or T, or can be selected from a variety of non-naturally occurring bases described herein. The two most common classes of nucleobases are purines and pyrimidines. The naturally occurring purine bases are guanine (G) and adenine (A), which are attached to the sugar through a 9-N nitrogen on the 13-endo position of the sugar ring. The naturally occurring pyrimidine bases are uracil (U), thymine (T), and cytosine (C), which are attached to the sugar through a 1-N nitrogen. In double-stranded DNA (dsDNA), Watson-Crick base pairing occurs between G and C, and between A and T. In double-stranded RNA (dsRNA), Watson-Crick base pairing occurs between G and C, and between A and U, and Watson-Crick base pairing for DNA and RNA is shown below.

[0114]

[0115] In RNA-DNA hybridization, and in hybridization between naturally occurring RNA or DNA and synthetic oligonucleotides composed of non-naturally occurring monomer subunits, similar base pairing can be observed.

[0116] In the synthetic oligonucleotides of the present disclosure, one or more of the naturally occurring nucleobases can be replaced by similar binding members (nucleobase analogs). Thus, the term "nucleobase" includes both naturally occurring nucleobases and non-naturally occurring nucleobases. The term "nucleobase analog" (also referred to as a nucleobase mimic or nucleic acid base mimic in the present disclosure) refers to a non-naturally occurring nucleobase and refers to a residue that has a nucleobase-like action by providing sequence-specific binding to a heterocyclic residue on a complementary oligomer. In some embodiments of the present disclosure, a nucleobase analog is a residue that is capable of establishing one or more non-covalent bonds with a nucleobase on a separate oligonucleotide strand. The non-covalent bonds are hydrogen bonds, ionic bonds, and polar interactions (other interactions with non-complementary nucleobases are possible, such as base stacking interactions). In some embodiments of the present disclosure, the non-covalent bonds are formed by hydrogen bonds between the nucleobase ring components and / or exocyclic substituents, which can be analogous to Watson-Crick pairing, Hoogsteen pairing, some combination thereof, or some other method described in the present disclosure or known in the art.

[0117] As used herein, "unmodified" or "natural" nucleobases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases (nucleobase analogs) include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, and other 5- substituted uracils and cytosines, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2- aminoadenine, 8-azaguanine and 8-azadenine, 7-aza guanine and 7-azadeanine, and 3- deazaguanine and 3-deazadeanine, 7-propynyl-7-deaza-8-adenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytosine (1H-pyrimido[5,4b][1,4]benzoxazin-2(3H)-one), phenothiazine cytosine (1H-pyrimido[5,4b][1,4]benzothiazin-2(3H)-one), G-clamp such as a substituted phenoxazine cytosine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytosine (2H-pyrimido[4,5-b]indole-2-one), pyridoindole cytosine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidine-2-one). Modified nucleobases can also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, for example 7-deazadeanine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone.

[0118] Certain of the nucleobases described are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. The nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6, and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

[0119] In general, the term "base" includes the nucleobases described above. The term "base" refers to a binding member as described above. While nucleobases are generally heterocyclic groups, the term "base" as used herein refers to any group or residue capable of participating in specific binding with a naturally occurring nucleobase.

[0120] In some embodiments of the disclosure, oligomeric compounds are prepared by replacing one or more heterocyclic groups with polycyclic heterocyclic compounds. Certain tricyclic heterocyclic compounds have been previously reported. Such compounds are typically used in antisense applications to improve the binding properties of the modified strand to the target strand.

[0121] In the present disclosure, "hybridization" refers to hydrogen bonding between complementary nucleoside or nucleotide bases, which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by hydrogen bond formation. "Complementary" as used in the present disclosure refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bond formation with the nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered complementary to each other at that position. When a sufficient number of corresponding positions in each molecule are occupied by such nucleotides that can form hydrogen bonds with each other, the molecules are considered substantially complementary. Thus, the terms "specifically hybridizable" and "complementary" are used to refer to a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that an antisense compound need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.

[0122] Phospholink

[0123] Oligonucleotides are generally those oligomers that are composed of monomeric subunits comprising linking members that are phosphodiester. Phosphodiester linkages include phosphodiester, phosphorothioate, and phosphorodithioate linkages.

[0124] Naturally occurring nucleosides are linked to one another by phosphodiester linkages. Antisense compounds can be prepared with phosphodiester linkages, which are generally suitable for diagnostic and other non-nuclease applications. However, antisense therapeutic compounds preferably include at least one phosphorothioate linkage, which confers superior nuclease stability. Both phosphodiester and phosphorothioate diester linkages are generally referred to as phosphodiester linkages. When a plurality of nucleotides are linked by consecutive phosphodiester linkages, the resulting oligomer is referred to as an oligonucleotide.

[0125] Oligonucleotide synthesis

[0126] As noted above, the term "oligonucleotide" includes naturally occurring RNA and DNA, as well as phospholinked oligonucleotides having various sugar backbones and nucleobases. Oligonucleotides are made by phosphotriester, H-phosphonate, and phosphoramidite methods, as noted above. Of these three methods, the phosphoramidite method has become the standard of fact for oligonucleotide synthesis, particularly where one or more modifications to the sugar backbone or nucleobase are made, or where special purity, yield, or scale is critical. The phosphoramidite method (amidite method) is described below.

[0127] Amidite method

[0128] While the present disclosure is directed primarily to oligonucleotides, certain oligonucleotide analogs can also be prepared by the methods of the present disclosure, with appropriate changes to the starting materials. Oligonucleotide analogs include compounds in which the oligonucleotide sugar has been replaced by a heterocyclic or carbocyclic structure. Such compounds and isomers, salts, and solvates thereof are described below in Formula (I-1),

[0129]

[0130] wherein G', G", Bx, n, R2', R3', R4', and R5' each have the meanings defined above. Groups T' and T" are each H, or a conjugate group such as a protecting group and a substituent. Each Q" is independently O, S, NR"', C(R'")2, or -CR'"=CR"'-, wherein each R'" is H, alkyl, or when two R'" groups are on the same or adjacent carbon atoms, they can form a carbocyclic or heterocyclic ring, wherein the ring contains one or two of N, O, or S. Preferred values of R'" are H and C l ~C4alkyl.

[0131] The above-described oligonucleotides and oligonucleotide analogs can be synthesized by solid phase synthesis, for example, by the amidite method. In addition, other or alternative synthetic methods known in the art for such synthesis can also be employed.

[0132] Support-bound oligonucleotide synthesis relies on the sequential addition of nucleotides at the end of a growing chain. Typically, the first nucleotide (with a protecting group on any present exocyclic amine functionality) is attached to an appropriate glass bead support, and then activated phosphite compounds (typically nucleotide phosphoramidites, with appropriate protecting groups) are added stepwise to extend the growing oligonucleotide.

[0133] The amidite method of oligonucleotide synthesis can generally be carried out by reacting a suitable nucleotide or modified nucleotide (Formula (4)) with a phosphoryl diamide (Formula (5)) to form a phosphoramidite (Formula (6)):

[0134]

[0135] wherein each variable Q', Bx, R2', R3', R4', R5', G" and q' are as defined above. L is an amine leaving group; pg is a phosphate protecting group; T'" is a hydroxyl protecting group, each as more specifically defined herein. In some embodiments of the disclosure, T'" is DMT in at least one cycle of the synthesis method.

[0136] The support-bound nucleotide of formula (7) is first deprotected at the 5'-position (to generate a 5'-OH radical). In some embodiments of the disclosure, at least one 5-protecting group (T'") is DMT and the deprotecting reagent is the composition or DCA reagent defined above. In more specific embodiments of the disclosure, multiple 5'-deprotection steps are carried out in the presence of the composition or DCA reagent defined above. In some embodiments of the disclosure, each 5'-deprotection step is carried out in the presence of the composition or DCA reagent defined above, which step can optionally be carried out in a suitable solvent such as acetonitrile or toluene.

[0137] Following 5'-deprotection, the first amide of (7) is coupled to the support-bound nucleotide to form a support-bound dimer of formula (8), which is then oxidized and end-capped to form a support-bound dimer of formula (9).

[0138]

[0139]

[0140] The 5'-deprotection, coupling, oxidation and end-capping steps are then repeated n-2 times to form a support-bound oligomer of formula (10).

[0141]

[0142] The compound (10) is then cleaved from the solid support, deprotected at the 5' terminus if necessary, and purified to yield an oligomer of Formula (I). The oligonucleotides can be further derivatized, purified, precipitated, or otherwise treated as described in greater detail herein. In certain embodiments of the application, the final protecting group is left on the oligonucleotide (10, SS is replaced by H) and the oligonucleotide is first subjected to high performance liquid chromatography (HPLC) and then the final 5'-protecting group is removed. In certain embodiments of the application, the final 5'-protecting group is removed by contacting the purified oligonucleotide with acetic acid. In other embodiments, the 5'-protecting group can be removed while the oligonucleotide is left on the solid support (SS). The deprotected oligonucleotide (10, where T" is replaced by H) can then be removed from the column and subjected to a purification step as described above. In certain embodiments of the application, the deprotected oligonucleotide can be subjected to ion exchange chromatography, such as soft anion exchange (SAX) chromatography. The anion exchange chromatography can be performed directly after the deprotected oligonucleotide is removed from the solid phase synthesis support, or after the 5'-protected oligonucleotide is purified by liquid chromatography and then deprotected.

[0143] In each of the above formulae, SS represents a solid support to which the 3'-terminal nucleoside is attached via a cleavable linker, each pg is a phosphate protecting group as defined herein, n is an integer, G' and G" are independently O or S, and each Bx, R2', R3', R4', R5', Q', and q' are independently as defined above.

[0144] Amide

[0145] Phosphoramidites (amidites) for use in oligonucleotide synthesis are available from a variety of commercial sources. Commercially available phosphoramidites are mostly prepared for automated synthesis of DNA, and are therefore prepared for immediate use in the synthesis of oligonucleotides of the desired sequence.

[0146] Carrier medium

[0147] As noted above, oligonucleotides are typically prepared on a support medium (a support), such as a solid support medium. Generally, first monomers (e.g., monomers such as nucleosides) are attached to the support medium, and then the oligonucleotide is synthesized by sequentially coupling the monomers to the support-bound polymer. This iterative extension ultimately results in the final oligomeric compound or other polymer, such as a polypeptide. Suitable support media can be soluble, insoluble, or have different solubilities in different solvents to enable the growing support-bound polymer to enter or exit solution as desired. Traditional support media, such as solid supports, are generally insoluble and are conventionally placed in a reaction vessel while reagents and solvents are reacted with the growing chain and / or washed until the oligomer reaches the target length, and then cleaved from the support, and further processed if necessary to yield the final polymeric compound. More recent methods have introduced soluble supports, including soluble polymeric supports, to enable the iterative synthesis of the growing product to be precipitated and solubilized at desired stages in the synthesis.

[0148] The term support medium (support) is intended to include supports known to those of skill in the art for use in the synthesis of oligomeric compounds and related compounds such as peptides. Some representative support media that can be used in the methods of the present disclosure include, but are not limited to, controlled pore glass (CPG), oxalyl controlled pore glass, silica-containing particles (e.g., porous glass beads and silica gel, such as that formed by the reaction of trichloro-[3-(4-chloromethyl)phenyl]propylsilane with porous glass beads), monoesters of l,4-dihydroxymethylbenzene and silica, TENTAGEL, cross-linked styrene / divinylbenzene copolymer bead matrices or POROS, copolymers of polystyrene / divinylbenzene, soluble support media, polyethylene glycol (PEG).

[0149] In some embodiments, the support medium is, for example, a polystyrene primer dT350 support.

[0150] Synthesis apparatus

[0151] Commercially available equipment is generally used for the synthesis of oligomeric compounds and related compounds based on support media. In some embodiments, the equipment used for support media is, for example, an automated AKTA OP100 synthesizer with 6.3 milliliter reaction columns. Any other means known in the art for such synthesis can additionally or alternatively be employed.

[0152] Phosphate protecting group

[0153] Generally, a phosphorus protecting group (pg) is an alkyl group or a beta-elimination group having the formula -CH2CH2-G w where G ware electron withdrawing groups. Generally, alkyl or cyanoethyl electron withdrawing groups are preferred because commercially available phosphoramidites generally contain methyl or cyanoethyl phosphoric acid protecting groups.

[0154] Methods for removing phosphoric acid protecting groups (pg’s) depend on the specific pg that needs to be removed. Beta-elimination groups are generally removed in weak base solution, whereby the acidic beta-hydrogen is abstracted and the -CH2CH2-G w group is eliminated by rearrangement to form the corresponding acrylic compound CH2=CH-G w In contrast, alkyl groups are generally removed by nucleophilic attack on the alpha-carbon of the alkyl group.

[0155] Coupling

[0156] Prior to coupling, the amide needs to be activated. In some embodiments, the amide is activated with tetrazole, 5-(ethylthio)-lH-tetrazole (ETT) or 5-(benzylthio)-lH-tetrazole (BTT). Various solvents, acetonitrile, and the like can be used for coupling. In some embodiments, the described coupling cycle includes co-transporting 2.0 equivalents of a 0.2 M solution of the amide in acetonitrile and a 0.6 M solution of ETT in acetonitrile at a flow ratio of 2:3 for 0.5 minutes, followed by a 4 minute column cycle.

[0157] Oxidation (including sulfurization)

[0158] One skilled in the art will recognize that P(III) oxidation to P(V) can be performed by a variety of reagents. Furthermore, one skilled in the art will recognize that P(V) species can exist in the form of phosphotriesters, phosphorothioate diesters, or phosphorodithioate diesters. As described in the present disclosure, each type of P(V) linkage has its uses and advantages. Thus, the term “oxidizing agent” should be broadly understood as any reagent capable of converting a P(III) species, such as a phosphite, to a P(V) species. Thus, the term “oxidizing agent” includes “sulfurizing agent”, and oxidation will be understood to include the introduction of oxygen and the introduction of sulfur, or sulfurization. When it is desired to note that the oxidizing agent introduces oxygen to the P(III) species to make the P(V) species, the oxidizing agent here will be referred to as an “oxygen-introducing oxidizing agent”.

[0159] In the phosphoramidite approach, the oxidizing agent used to prepare the phosphodiester linkages is known in the art, such as iodine. Examples of sulfurizing reagents that have been used to synthesize oligonucleotides containing phosphorothioate linkages include elemental sulfur, dibenzoyl tetrasulfide, 3-H-l,2-benzodithiol-3-one-l,l-dioxide (also known as the Beaucage reagent), tetraethyl thiuram disulfide (TETD), and bis-(0,0-diisopropoxyphosphoryl sulfur) disulfide (known as the Stec reagent). Oxidizing agents used to prepare phosphorothioate diester linkages include phenylacetyl disulfide (PADS). In some embodiments of the present disclosure, the phosphorothioate diester linkages and the phosphodiester linkages can be alternated between sugar subunits. In other embodiments of the present disclosure, phosphorothioate linkages alone can be employed.

[0160] Various solvents can be used in the oxidation reaction, acetonitrile, toluene, xylene, dichloromethane, pyridine, water, etc. In some embodiments, the solvent in the oxidation reaction is a mixture of pyridine and water (e.g., pyridine:water = 9: 1, v / v).

[0161] Cleavage and processing

[0162] Reagents for cleaving oligonucleotides from a support are common reagents in the art.

[0163] Oligonucleotides can be processed by standard procedures known in the art, for example, by size exclusion chromatography, high performance liquid chromatography (e.g., reverse phase HPLC), differential precipitation, etc. In some embodiments of the present disclosure, the oligonucleotide is cleaved from the solid support when the 5'-OH protecting group is still on the final nucleoside. This so-called DMT-on (or trityl-on) oligonucleotide is then chromatographed, followed by removal of the DMT group by treatment in an organic acid, followed by desalting and further purification of the oligonucleotide to form the final product.

[0164] After cleavage of the oligonucleotide from the support, removal of the 5- protecting group is typically carried out using acetic acid.

[0165] As used herein, "oligonucleotide synthesis" is intended to have the art-recognized meaning of preparing the oligonucleotide using synthetic methods well known to those of ordinary skill in the art. See, e.g., US7169916, US6069243, and US6399765, the entire contents of each of which are incorporated herein by reference.

[0166] In a sixth aspect, the present disclosure provides a method of detecting glyoxylic acid in a DCA sample.

[0167] The DCA sample of the present disclosure can be selected from the compositions or DCA reagents defined above or commercially available DCA products.

[0168] In some embodiments, the method for detecting glyoxylic acid is ion chromatography.

[0169] In the described ion chromatography, the standard substance is glyoxylic acid, for example glyoxylic acid monohydrate, which is commercially available (e.g. Aladin; Lot # F1714025).

[0170] In the described ion chromatography, preferably, the qualitative analysis is based on the retention time.

[0171] In the described ion chromatography, preferably, the content of glyoxylic acid in the DCA sample is quantified by using an external standard method.

[0172] In the described external standard method, the content of glyoxylic acid in the DCA sample is calculated according to the content of the STD solution using conventional methods in the art.

[0173] The mobile phase in the described ion chromatography is, for example, 20 mM KOH in water.

[0174] The chromatographic column in the described ion chromatography is, for example, Dionex IonPac AS18.

[0175] The column specifications in the described ion chromatography are, for example, 4 mm x 250 mm.

[0176] The flow rate of the chromatographic column in the described ion chromatography is, for example, 1.0 mL / min.

[0177] The injection volume of the chromatographic column in the described ion chromatography is, for example, 25 μL.

[0178] The analysis time of the chromatographic column in the described ion chromatography is, for example, 20 minutes.

[0179] The conditions of the described ion chromatography are as follows:

[0180] Instrument: ICS-6000;

[0181] Chromatographic column: Dionex IonPac AS18, 4 mm x 250 mm;

[0182] Mobile phase: KOH (RFC): 20 mM in water (RFC: Reagent-Free Controller).

[0183] Flow rate: 1.0 mL / min;

[0184] Aers-4 mm suppressor: 50 mA;

[0185] Injection volume: 25 μL; and

[0186] Analysis time: 20 minutes.

[0187] In a seventh aspect, the present disclosure provides a method for measuring the concentration of glyoxylic acid in the above-defined composition or DCA reagent or commercially available DCA product, in particular detecting glyoxylic acid and measuring its concentration in the above-defined composition or DCA reagent or commercially available DCA product. In some embodiments, the method is the above-defined ion chromatography.

[0188] In some embodiments, the present disclosure provides an analysis method comprising determining whether the ion chromatogram extracted from the DCA sample comprises a chromatographic peak associated with glyoxylic acid.

[0189] Existing DCA products (such as commercially available DCA products) are prepared by different processes, which makes it almost impossible for DCA to be a pure chemical substance (i.e. 100% dichloroacetic acid). Therefore, the dichloroacetic acid product contains more or less impurities. Therefore, in some embodiments, each of the above-defined composition and DCA reagent of the present disclosure further comprises one or some impurities (not including glyoxylic acid) that can have no effect on the synthesis of oligonucleotides.

[0190] In some embodiments, the total content of other impurities in the above-defined composition or DCA reagent is, for example, below 0.2%, such as below 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% and 0.01%. In some embodiments, the content of a single impurity (other impurities) in the above-defined composition or DCA reagent is determined, for example, by ion chromatography. The ion chromatography is as defined in the present disclosure. Preferably, the content of a single impurity (other impurities) is quantified by area normalization.

[0191] In some embodiments, the compositions of the disclosure comprise, consist essentially of, or consist of DCA and one or more impurities that have no adverse effect on oligonucleotide synthesis. In some embodiments, the compositions of the disclosure comprise, consist essentially of, or consist of DCA, glyoxylic acid, and other impurities. In some embodiments, an impurity can be considered to have no adverse effect on oligonucleotide synthesis (e.g., in the model synthesis of oligonucleotide T10) when removal or reduction of the impurity does not increase the yield and / or purity of the target oligonucleotide. In some embodiments, an impurity can be considered to have no adverse effect on oligonucleotide synthesis when the impurity is present in the DCA composition at a level that is sufficiently low such that there is no decrease in the yield and / or purity of the target oligonucleotide (e.g., in the model synthesis of oligonucleotide T10) compared to the use of a DCA composition that does not contain the impurity in the synthesis of the oligonucleotide. The levels of DCA and glyoxylic acid in the compositions described include those compositions defined above.

[0192] Each of the compositions and DCA reagents of the disclosure do not include the known impurities in the art (e.g., chloral hydrate) that have been reported in the prior art DCA products to have an effect on oligonucleotide synthesis. If present, such impurities are present in an amount that does not have an effect on oligonucleotide synthesis.

[0193] In an eighth aspect, the disclosure provides a method of synthesizing an oligonucleotide, comprising:

[0194] a) selecting or identifying substantially pure dichloroacetic acid having a glyoxylic acid content of less than 1000 ppm (as described herein, e.g., less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, e.g., not detected); and

[0195] b) combining the substantially pure dichloroacetic acid with a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby producing a deprotected oligonucleotide. As used herein, “not detected” is understood to be below the limit of detection or limit of quantitation of a suitable analytical method as described herein, e.g., ion chromatography.

[0196] In some embodiments of the disclosure, in the above method, the selecting or identifying comprises the step of determining or having determined that the tested substantially pure dichloroacetic acid has a glyoxylic acid content of less than 1000 ppm (as described herein, e.g., less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, e.g., not detected).

[0197] In some embodiments of the disclosure, in the above method, the selecting or identifying comprises a step of determining or having determined whether the tested substantially pure dichloroacetic acid comprises less than 1000 ppm (as described herein, for example less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, such as not detected) of glyoxylic acid by ion chromatography (as described herein).

[0198] In some embodiments of the disclosure, in the above method, the protected oligonucleotide comprises a 5'-hydroxyl group protected with an acid-labile protecting group. As will be understood by those skilled in the art, the methods herein are not particularly limited to the preparation of any particular oligonucleotide, and those known in the art that use an acid-labile protecting group (such as trityl) in synthesis can be prepared by the methods herein. The methods herein are also not particularly limited to any particular protected oligonucleotide. Any oligonucleotide known in the art having an acid-labile protecting group that can be deprotected by DCA can be prepared by the methods herein.

[0199] In some embodiments of the disclosure, in the above method, the acid-labile protecting group is trityl, preferably 4,4'-dimethoxytrityl. Other suitable acid-labile protecting groups include those known in the art.

[0200] In some embodiments of the disclosure, in the above method, the protected oligonucleotide is bound to a solid support (such as any suitable support known in the art).

[0201] In some embodiments of the disclosure, in the above method, the mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent, such as in toluene. In particular, in such embodiments, the substantially pure dichloroacetic acid is typically first mixed with a solvent, such as the solvent as a solution comprising 5%, 10%, 20%, 30%, 40%, 50%, 80%, 90%, or any range between the aforementioned values by weight, which can then be mixed with the protected oligonucleotide. Further, more than one solvent can be used. For example, in some embodiments, the protected oligonucleotide can be contacted with a second solvent (which can be the same as or different from the solvent), and then the substantially pure dichloroacetic acid solution is added to mix the protected oligonucleotide with the second solvent together with the substantially pure dichloroacetic acid. Similar understanding should be made for other expressions of mixing in a solvent as described herein.

[0202] In some embodiments of the disclosure, the above method further comprises converting the deprotected oligonucleotide to an oligonucleotide having a desired sequence.

[0203] In a ninth aspect, the disclosure provides a method of synthesizing an oligonucleotide, comprising:

[0204] a) preparing or having prepared substantially pure dichloroacetic acid having a glyoxylic acid content of less than 1000 ppm (as described herein, for example less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, for example not detected); and

[0205] b) mixing said substantially pure dichloroacetic acid with a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby producing a deprotected oligonucleotide. Conditions suitable for removal of acid-labile protecting groups with dichloroacetic acid are well known in the art and are exemplified in the present disclosure.

[0206] In some embodiments of the present disclosure, in the above method, prior to said mixing, the glyoxylic acid content of said substantially pure dichloroacetic acid is determined to be less than 1000 ppm (as described herein, for example less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, for example not detected).

[0207] In some embodiments of the present disclosure, in the above method, prior to said mixing, the glyoxylic acid content of said substantially pure dichloroacetic acid is determined by ion chromatography to be less than 1000 ppm (as described herein, for example less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, for example not detected).

[0208] In some embodiments of the present disclosure, in the above method, said substantially pure dichloroacetic acid is prepared by reducing the glyoxylic acid content of a starting dichloroacetic acid composition to less than 1000 ppm (as described herein, for example less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, for example not detected).

[0209] In some embodiments of the present disclosure, in the above method, said substantially pure dichloroacetic acid is prepared by reacting a starting dichloroacetic acid composition with a glyoxylic acid trapping reagent to reduce the glyoxylic acid content to less than 1000 ppm (as described herein, for example less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, for example not detected).

[0210] In some embodiments of the present disclosure, in the above method, said protected oligonucleotide comprises a 5'-hydroxyl group protected by an acid-labile protecting group.

[0211] In some embodiments of the present disclosure, in the above method, said acid-labile protecting group is a trityl group, preferably a 4,4'-dimethoxytrityl group.

[0212] In some embodiments of the disclosure, in the above method, the protected oligonucleotide is bound to a solid support.

[0213] In some embodiments of the disclosure, in the above method, the mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent, such as toluene.

[0214] In some embodiments of the disclosure, the above method further comprises converting the deprotected oligonucleotide to an oligonucleotide having a desired sequence.

[0215] In a tenth aspect, the disclosure provides a method of synthesizing an oligonucleotide, the method comprising:

[0216] a) determining or having determined that the substantially pure dichloroacetic acid has a glyoxylic acid content of less than 1000 ppm (as described herein, such as less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, such as not detected); and

[0217] b) mixing the substantially pure dichloroacetic acid with a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby producing a deprotected oligonucleotide.

[0218] In some embodiments of the disclosure, the above method comprises determining or having determined that the substantially pure dichloroacetic acid has a glyoxylic acid content of less than 1000 ppm (as described herein, such as less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, such as not detected) by ion chromatography.

[0219] In some embodiments of the disclosure, in the above method, the protected oligonucleotide comprises a 5'-hydroxyl group protected by an acid-labile protecting group.

[0220] In some embodiments of the disclosure, in the above method, the acid-labile protecting group is a trityl group, preferably a 4,4'-dimethoxytrityl group.

[0221] In some embodiments of the disclosure, in the above method, the protected oligonucleotide is bound to a solid support.

[0222] In some embodiments of the disclosure, in the above method, the mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent, such as toluene.

[0223] In some embodiments of the disclosure, the above method further comprises converting the deprotected oligonucleotide to an oligonucleotide having a desired sequence.

[0224] In an eleventh aspect, the present disclosure provides a method of synthesizing an oligonucleotide, the method comprising:

[0225] a) mixing substantially pure dichloroacetic acid with a protected oligonucleotide of a first sequence having an acid-labile protecting group under conditions suitable for removal of the acid-labile protecting group, thereby providing an oligonucleotide of the first sequence;

[0226] b) reacting the oligonucleotide of the first sequence with a desired nucleotide protected by a second protecting group under conditions suitable for elongation of the nucleotide chain, thereby forming a protected oligonucleotide of a second sequence having the second protecting group;

[0227] c) deprotecting the protected oligonucleotide of the second sequence to remove the second protecting group; and optionally

[0228] d) repeating steps b) and c) until a desired sequence is achieved,

[0229] wherein the substantially pure dichloroacetic acid is determined to have a glyoxylic acid content of less than 1000 ppm (as described herein, e.g., less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, e.g., not detected).

[0230] In a twelfth aspect, the present disclosure provides a method of removing an acid-labile protecting group from a protected oligonucleotide, the method comprising:

[0231] a) determining or having determined that substantially pure dichloroacetic acid has a glyoxylic acid content of less than 1000 ppm (as described herein, e.g., less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, e.g., not detected); and

[0232] b) mixing the substantially pure dichloroacetic acid with the protected oligonucleotide under conditions suitable for removal of the acid-labile protecting group.

[0233] In some embodiments of the present disclosure, the above method comprises determining or having determined that the substantially pure dichloroacetic acid has a glyoxylic acid content of less than 1000 ppm (as described herein, e.g., less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, e.g., not detected) by ion chromatography.

[0234] In some embodiments of the present disclosure, in the above method, the protected oligonucleotide comprises a 5'-hydroxyl group protected by an acid-labile protecting group.

[0235] In some embodiments of the present disclosure, in the above method, the acid-labile protecting group is a trityl group, preferably a 4,4'-dimethoxytrityl group.

[0236] In some embodiments of the disclosure, in the above method, the protected oligonucleotide is bound to a solid support.

[0237] In some embodiments of the disclosure, in the above method, the mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent, such as toluene.

[0238] In a thirteenth aspect, the disclosure provides a method of selecting or identifying a substantially pure dichloroacetic acid for use in oligonucleotide synthesis, the method comprising: 1) determining or having determined the glyoxylic acid content of a test substantially pure dichloroacetic acid, such as by ion chromatography; and optionally, 2) selecting or identifying a substantially pure dichloroacetic acid having a glyoxylic acid content of less than 1000 ppm (as described herein, such as less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, such as not detected) for use in oligonucleotide synthesis.

[0239] In a fourteenth aspect, the disclosure provides a method of analyzing a dichloroacetic acid composition, comprising determining the glyoxylic acid content of the dichloroacetic acid composition, such as by ion chromatography.

[0240] In a fifteenth aspect, the disclosure provides a method of preparing a dichloroacetic acid composition, comprising: 1) analyzing the glyoxylic acid content of a starting dichloroacetic acid composition, such as by ion chromatography; and optionally, 2) reducing the glyoxylic acid content of the starting dichloroacetic acid composition to less than 1000 ppm (as described herein, such as less than 50 ppm, preferably less than 15 ppm, more preferably less than 2.5 ppm, such as not detected), such as by mixing the starting dichloroacetic acid composition with a glyoxylic acid trapping reagent (as described herein), thereby preparing the dichloroacetic acid composition.

[0241] The term "dichloroacetic acid (DCA)" refers to a compound having the structure Cl2CHC(=0)(OH).

[0242] The term "substantially pure dichloroacetic acid" refers to dichloroacetic acid having a purity of greater than 90% (such as greater than 95%, greater than 98%, greater than 99%, or greater than 99.5%) by weight, GC area, ion chromatography, or any combination thereof.

[0243] In any one of the embodiments described herein, the substantially pure dichloroacetic acid reagent described has a content of glyoxylic acid of less than 950 ppm, 900 ppm, 800 ppm, 700 ppm, 600 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 100 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.05 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm (by weight, GC area, ion chromatography, or any combination thereof).

[0244] In any one of the embodiments described herein, the substantially pure dichloroacetic acid reagent described has a content of glyoxylic acid of less than 500 ppm, 450 ppm, 400 ppm, 350 ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm, 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, 30 ppm, 20 ppm, 15 ppm, 12 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm by weight, GC area, ion chromatography, or any combination thereof, unless otherwise specified or made clear from the context.

[0245] In any one of the embodiments described herein, the substantially pure dichloroacetic acid reagent described has a content of glyoxylic acid of less than 50 ppm, 45 ppm, 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 12 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm (by weight, GC area, ion chromatography, or any combination thereof), unless otherwise specified or made clear from the context.

[0246] In any one of the embodiments described herein, the substantially pure dichloroacetic acid reagent described has an amount of glyoxylic acid of less than 15 ppm, 14.5 ppm, 14 ppm, 13.5 ppm, 13 ppm, 12.5 ppm, 12 ppm, 11.5 ppm, 11 ppm, 10.5 ppm, 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm, 4 ppm, 3.5 ppm, 3.09 ppm, 3 ppm, 2.5 ppm, 2.45 ppm, 2.4 ppm, 2.35 ppm, 2.3 ppm, 2.25 ppm, 2.2 ppm, 2.15 ppm, 2.1 ppm, 2.05 ppm, 2 ppm, 1.95 ppm, 1.9 ppm, 1.85 ppm, 1.8 ppm, 1.75 ppm, 1.7 ppm, 1.65 ppm, 1.6 ppm, 1.55 ppm, 1.5 ppm, 1.45 ppm, 1.4 ppm, 1.35 ppm, 1.3 ppm, 1.25 ppm, 1.2 ppm, 1.15 ppm, 1.04 ppm, 1.1 ppm, 1.05 ppm, 1.01 ppm, 1 ppm, 0.95 ppm, 0.9 ppm, 0.85 ppm, 0.8 ppm, 0.75 ppm, 0.7 ppm, 0.65 ppm, 0.6 ppm, 0.55 ppm, 0.5 ppm, 0.45 ppm, 0.4 ppm, 0.35 ppm, 0.3 ppm, 0.25 ppm, 0.2 ppm, 0.15 ppm, 0.1 ppm, or 0.05 ppm (by weight, GC area, ion chromatography, or any combination thereof).

[0247] In some embodiments of the disclosure, the substantially pure dichloroacetic acid described is substantially free of glyoxylic acid, and in particular, for example, is free of glyoxylic acid or has no detectable amount of glyoxylic acid (by weight, GC area, ion chromatography, or any combination thereof).

[0248] The term "glyoxylic acid" refers to a compound having the structure CHOCOOH.

[0249] The term "DCA reagent" refers to pure dichloroacetic acid, or has the same definition as the composition described in the disclosure.

[0250] The term "DCA sample" refers to any DCA product, such as the composition described in the disclosure or the DCA reagent, or a DCA product prepared by any method, or any commercially available DCA product.

[0251] In some embodiments, each of the terms "essentially pure dichloroacetic acid tested", "starting dichloroacetic acid composition" can have the same definition as the term "DCA sample".

[0252] Abbreviations:

[0253] DCA stands for dichloroacetic acid;

[0254] DMT stands for 4,4'-dimethoxytrityl;

[0255] ETT stands for 5-(ethylthio)-lH-tetrazole;

[0256] CV stands for column volume;

[0257] GA-H2O stands for glyoxylic acid monohydrate;

[0258] GA stands for glyoxylic acid;

[0259] RFC stands for reagent free controller;

[0260] min stands for minute;

[0261] hr(s) stands for hour(s);

[0262] STD stands for standard. BRIEF DESCRIPTION OF DRAWINGS

[0263] Figure 1 H-NMR spectrum of the white solid of Example 2. 1 H-NMR spectrum.

[0264] Figure 2 C-NMR spectrum of the white solid of Example 2. 13 C-NMR spectrum.

[0265] Figure 3 LC-MS spectrum of the white solid of Example 2.

[0266] Figure 4 HPLC profile of the GA standard.

[0267] Figure 5 HPLC profile of the DCA sample - 01. EXAMPLE

[0268] The present application is further illustrated by the following examples, but without being restricted thereto.

[0269] Apparatus

[0270] 1 H-NMR: Bruker NMR.

[0271] 13C-NMR: Bruker NMR.

[0272] LC-MS: Waters Q-TOF, and Agilent LC-MS model: Agilent 1290 + MSD.

[0273] HPLC: Agilent HPLC model: Agilent 1260.

[0274] Oligonucleotide synthesizer: Automated AKTA OP100 synthesizer (6.3 mL reaction column).

[0275] DCA sample-01 was purchased from Changzhou Wujin Changxinke Chemical Co., Ltd.

[0276] DCA sample-02 was purchased from Cabbe, Acros.

[0277] DCA reagent was prepared according to the method of the present disclosure.

[0278] Example 1: Oligonucleotide synthesis

[0279] 1.1. Oligonucleotide T10 synthesis

[0280] Oligonucleotide T10 was synthesized on a polystyrene primer dT350 support in 0.2 mmol scale using standard phosphoramidite chemistry with an automated AKTA OP100 synthesizer with a 6.3 mL reaction column.

[0281] For each amide compound, four chemical reactions were performed, including de-tritylation (5'-deprotection), coupling, oxidation, and capping.

[0282] De-tritylation was performed using 10% DCA sample-02 in toluene (v / v) and monitored with 350 nm UV control. The coupling cycle included co-delivery of 2.0 equivalents of 0.2 M amide solution in acetonitrile and 0.6 M ETT in acetonitrile at a flow rate ratio of 2:3 for 0.5 min, followed by a 4 min column cycle, oxidation with 0.05 M iodine in 9:1 pyridine:water (v / v) for 0.5 min, capping with 0.5 CV (column volume) of acetic anhydride acetonitrile (1:4, v / v) and N-methylimidazole-pyridine-acetonitrile (2:3:5, v / v / v) capping mixture (1:1, v / v) for 0.5 min, and washing with acetonitrile after each block. After 10 rounds of solid-phase assembly, a 10mer oligonucleotide modified solid support was obtained.

[0283] 1.2. Oligonucleotide T11 synthesis

[0284] Then, when the 11th dT amide was assembled onto the solid support, the de-tritylation time using 10% DCA sample-01 in toluene was increased. After increasing the de-tritylation time to 90 minutes, the coupling did not run anymore, which indicated that some groups were trapping the 5'-OH on the solid support.

[0285] The oligonucleotide was cleaved from the support and detected by LC-MS, the main product was the 10mer dT product, which indicated that the oligonucleotide T11 synthesis failed.

[0286]

[0287] Example 2: DCA sample-01 analysis

[0288] When 0.1% cysteine was added to 10% DCA sample-01 in toluene, the inventors found some white solid precipitated out. The white solid was filtered and dried. 1 H-NMR, 13 C-NMR and LC-MS indicated that the main impurities were the condensates of glyoxylic acid with cysteine (compounds A and B). The results of the structure identification information are shown in Figure 1 、 Figure 2 and Figure 3 .

[0289]

[0290] 0.058% glyoxylic acid in DCA sample-01 was observed by HPLC method. In the HPLC method, the STD was glyoxylic acid monohydrate (source: Aladdin; Lot# F1714025). The HPLC profiles of GA and DCA sample-01 are shown in Figure 4 and Figure 5 .

[0291] Example 3: Preparation and detection of DCA reagents

[0292] 3.1. Preparation of DCA reagents

[0293] 5 grams of trapping reagents (1% w / w or 1% v / v) including cysteine, lysine, phenylalanine (amino acids), (2R)-propane-1,2-diol (difunctional chemical), hydroxylamine hydrochloride, triethylsilane (reducing agent), etc. were added to 500 grams of DCA sample-01. The mixture was incubated (e.g., left to stand or stirred) for 12-36 hours, and then vacuum distilled to obtain the DCA reagents.

[0294] 3.2. Detection of DCA reagents

[0295] 3.2.1. Sample preparation (4 mg / mL of DCA reagents)

[0296] Accurately weigh about 200 mg of sample into different 50 mL volumetric flasks, make up to volume with diluent (i.e. water) and mix well.

[0297] Note: Sample refers to DCA reagent.

[0298] 3.2.2. STD preparation (0.01 pg / mL of GA)

[0299] 3.2.2.1. Accurately weigh about 31 mg of GA-H20 (Aladin; Lot# F1714025) into a 25 mL volumetric flask, make up to volume with diluent (i.e. water) and mix well, label as GA-1.

[0300] 3.2.2.2. Accurately pipette 200 pL of GA-1 into a 100 mL volumetric flask, make up to volume with diluent (i.e. water) and mix well, label as GA-2.

[0301] 3.2.2.3. Accurately pipette 0.5 mL of GA-2 into a 100 mL volumetric flask, make up to volume with diluent (i.e. water) and mix well, label as GA-3 (0.01 pg / mL of GA).

[0302] 3.2.3. Ion Chromatography detection

[0303] Instrument: ICS-6000;

[0304] Column: Dionex IonPac AS18, 4 x 250 mm;

[0305] Mobile phase: KOH (RFC): 20 mM in water (RFC: Reagent Free Controller);

[0306] Flow rate: 1.0 mL / min;

[0307] Aers-4 mm Suppressor: 50 mA;

[0308] Injection volume: 25 pL;

[0309] Analysis time: 20 minutes.

[0310] 3.3. Results: The results are summarized in Table 2.

[0311] Table 2

[0312] DCA sample Capture reagent Content Content of glyoxylic acid in DCA reagent DCA reagent 1 Cysteine 1% (w / w) 1.65 ppm DCA reagent 2 Lysine 1% (w / w) 1.04 ppm DCA reagent 3 Phenylalanine 1% (w / w) 1.5 ppm DCA reagent 4 (2R)-propane-1,2-diol 1% (v / v) 304 ppm DCA reagent 5 Hydroxylamine hydrochloride 1% (v / v) 50.75 ppm DCA reagent 6 Triethylsilane 1% (v / v) 1.01 ppm DCA sample-01 - - 2142.7 ppm

[0313] Conclusion:

[0314] When cysteine, lysine, triethylsilane or phenylalanine is selected as the capture reagent, the content of glyoxylic acid in the DCA reagent is less than 2.5 ppm. And when other capture reagents are selected, the glyoxylic acid in the DCA sample-01 is also effectively reduced.

[0315] The detection method of the present disclosure can be used to detect the content of glyoxylic acid in the DCA sample.

[0316] Example 4: Evaluation of glyoxylic acid in DCA reagent

[0317] 4.1. Sample preparation (DCA reagent at 4 mg / mL)

[0318] The preparation method is the same as that in section 3.2.1.

[0319] 4.2. STD preparation (GA at 0.01 μg / mL)

[0320] The preparation method is the same as that in section 3.2.2.

[0321] 4.3. Specification of residual GA: X (X is the content of glyoxylic acid in the DCA sample that has no effect on oligonucleotide synthesis according to actual needs, in this case, X is 2.5 ppm)

[0322] 4.4. Report results (limit method):

[0323] Compare the peak areas of the GA peaks in the STD and sample chromatograms with the blank chromatogram, and integrate the GA peaks in the STD and sample solutions.

[0324] If the GA peak area in the sample injection solution is greater than the GA peak area in the standard solution, the result is reported as "> X ppm".

[0325] If the GA peak area in the sample injection solution is equal to the GA peak area in the standard solution, the result is reported as "= X ppm".

[0326] If the GA peak area in the sample injection solution is less than the GA peak area in the standard solution, the result is reported as "< X ppm".

[0327] Table 3

[0328] Number Peak area (μs*min) Reported result STD 0.000296 / DCA reagent 1 0.000286 <2.5 ppm DCA reagent 2 0.000214 <2.5 ppm DCA reagent 3 0.000189 <2.5 ppm DCA reagent 4 0.034351 > 2.5 ppm DCA reagent 5 0.008913 > 2.5 ppm DCA reagent 6 > 2.5 ppm 0.000271 <2.5 ppm

[0329] The specification of residual GA can be set according to actual needs.

[0330] Conclusion:

[0331] The evaluation method of the present disclosure can be used to detect whether the content of glyoxylic acid in the DCA sample meets specific requirements.

[0332] Example 5: Oligonucleotide synthesis

[0333] 5.1. Oligonucleotide T10 (TTTTT TTTTT) synthesis

[0334] Oligonucleotide T10 was synthesized on a polystyrene primer dT350 support at a 0.1 mmol scale using standard phosphoramidite chemistry with an automated AKTA OP100 synthesizer with a 6.3 mL reaction column.

[0335] For each amide compound, four chemical reactions were performed, including detritylation, coupling, oxidation and capping.

[0336] Detritylation was performed using 10% DCA reagent (see Table 4) in toluene (v / v) and monitored using 350 nm UV light. Coupling cycles included co-delivery of 2.0 equivalents of 0.2 M amide solution in acetonitrile and 0.6 M ETT in acetonitrile at a flow rate ratio of 2:3 for 0.5 minutes, followed by a 4 minute column cycle, oxidation with 0.05 M iodine in 9:1 pyridine:water (v / v) for 0.5 minutes, capping with 0.5 CV of acetic anhydride acetonitrile (1:4, v / v) and N-methylimidazole-pyridine-acetonitrile (2:3:5, v / v / v) capping mixture (1:1, v / v) for 0.5 minutes and washing with acetonitrile after each block.

[0337] A 40 wt% aqueous methylamine solution and aqueous ammonium hydroxide solution (10 mL per gram of synthesized oligonucleotide) were added to the solid support in a 1:1 mixture to cleave T10 from the solid support and remove the nucleoside protecting groups, and the resulting mixture was incubated at 30-40 °C on a shaker for 2-3 hours.

[0338] The mixture was filtered through a glass fibre filter, the support was washed with pure water and the filtrate was combined. After adjusting the pH to 7.0-9.0 with 20% acetic acid, samples were taken for MS and HPLC analysis.

[0339] 5.2. Oligonucleotide 17mer (CCCGGGTTTCGTCGTAA) synthesis

[0340] Oligonucleotide 17mer DMT-CCCGGGTTTCGTAA was synthesized on a PS Primer Unylinker 350 support at a 0.2 mmol scale using standard phosphoramidite chemistry with an automated AKTA OP100 synthesizer with a 6.3 mL reaction column.

[0341] For each amide compound, four chemical reactions were performed, including detritylation, coupling, oxidation and capping.

[0342] Deprotecting was performed using 10% DCA reagent (see Table 4) in toluene (v / v) and monitored using 350 nm UV light. The coupling cycle consisted of co-transporting 2.0 equivalents of 0.2 M amide solution in acetonitrile and 0.6 M ETT in acetonitrile at a flow rate ratio of 2:3 for 0.5 min, followed by a 4 min column cycle, oxidation with 0.05 M iodine in 9:1 pyridine:water (v / v) for 0.5 min, capping with 0.5 CV of capping mixture (1:1, v / v) of acetic anhydride in acetonitrile (1:4, v / v) and N-methylimidazole-pyridine-acetonitrile (2:3:5, v / v / v) for 0.5 min, and washing with acetonitrile after each block.

[0343] Ammonium hydroxide aqueous solution (10 mL per gram of synthesized oligonucleotide) was added to the solid support to cleave the 17mer from the solid support and remove the nucleoside protecting groups, and the resulting mixture was incubated at 50-60 °C on a shaker for 15-17 hours.

[0344] The mixture was filtered through a glass fiber filter, the support was washed with pure water, and the filtrates were combined. Samples were taken for MS and HPLC analysis.

[0345] Table 4

[0346]

[0347]

[0348] 5.3. Preparation of DCA reagents

[0349] DCA reagent 7:

[0350] To 500 g of DCA sample-01 was added an excess of cysteine. The mixture was left for 16 hours, then vacuum distilled to give DCA reagent 7, which was tested using the method described in section 3.2 of Example 3 and no GA was detected (N / A).

[0351] DCA reagent 8:

[0352] To 500 g of DCA sample-01 was added 2.5 g of cysteine. The mixture was left for 16 hours, then vacuum distilled to give DCA reagent 8, which was tested using the method described in section 3.2 of Example 3 and had a GA content of 2.5 ppm.

[0353] DCA reagent 9 and DCA reagent 10:

[0354] To 50 g of DCA reagent 7, 0.5 g of glyoxylic acid was added, and the mixture was incubated at 25-50 °C for 0.5 h. Then the mixture was filtered to obtain a DCA sample-03 containing 7200 ppm of glyoxylic acid. The DCA sample-03 containing 7200 ppm of glyoxylic acid was diluted 1200-fold and 600-fold with DCA to obtain DCA reagent 9 and DCA reagent 10.

[0355] CONCLUSION:

[0356] After treatment with the capture reagent of the present disclosure and vacuum distillation, the DCA sample-01 can also be used for oligonucleotide synthesis. Therefore, glyoxylic acid is an impurity in commercially available DCA products, which can cause failure of oligonucleotide synthesis.

[0357] It should be understood that the above description of the preferred embodiments is intended to be purely illustrative of the principles of the present disclosure, and not exhaustive, and that changes and variations will be apparent to those skilled in the art, and that the present disclosure is not intended to be limited, except as explicitly defined in the following claims. SEQUENCE LISTING <110> Changzhou Hequan Pharmaceutical Co., Ltd. Shanghai Hequan Pharmaceutical Research and Development Co., Ltd. <120> Compositions comprising dichloroacetic acid, methods of making the same, and uses thereof <130> P22117404CP <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> DNA Sequence 1 <400> 1 tttttttttt 10 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> DNA Sequence 2 <400> 2 cccgggtttc gtcgtaa 17

Claims

1. Use of a composition comprising dichloroacetic acid and glyoxylic acid for deblocking nucleotides in oligonucleotide synthesis; characterized in that, The glyoxylic acid content of the composition is below 15 ppm.

2. Use according to claim 1, characterized in that, The glyoxylic acid content of the composition is below 12 ppm.

3. Use according to claim 2, characterized in that, The glyoxylic acid content of the composition is below 6 ppm.

4. Use according to claim 3, characterized in that, The glyoxylic acid content of the composition is below 2.5 ppm.

5. A process for the preparation of a dichloroacetic acid composition having a glyoxylic acid content of 15 ppm or less, characterized in that, It comprises: mixing a dichloroacetic acid feedstock having a glyoxylic acid content above 1000 ppm with a glyoxylic acid trapping reagent; The trapping reagent is selected from the group consisting of an amino acid, a reducing agent, and mixtures thereof; The amino acid is selected from the group consisting of cysteine, lysine, and phenylalanine, or mixtures thereof; The reducing agent is selected from the group consisting of triethylsilane.

6. The production method according to claim 5, wherein It further comprises distilling the dichloroacetic acid after mixing the dichloroacetic acid feedstock and the trapping reagent.

7. The production method according to claim 6, wherein It further comprises vacuum distilling the dichloroacetic acid after mixing the dichloroacetic acid feedstock and the trapping reagent.

8. A method of preparing an oligonucleotide, characterized by, It comprises using a dichloroacetic acid composition having a glyoxylic acid content below 15 ppm as a deprotection reagent.

9. The production method according to claim 8, wherein The glyoxylic acid content of the composition is below 12 ppm.

10. The production method according to claim 9, wherein The glyoxylic acid content of the composition is below 10 ppm.

11. The production method according to claim 10, wherein The glyoxylic acid content of the composition is below 9 ppm.

12. The production method according to claim 11, wherein The glyoxylic acid content of the composition is below 8 ppm.

13. The production method according to claim 12, wherein The glyoxylic acid content of the composition is below 7 ppm.

14. The production method according to claim 13, wherein The glyoxylic acid content of the composition is below 6 ppm.

15. The production method according to claim 14, wherein The glyoxylic acid content of the composition is below 5 ppm.

16. The production method according to claim 15, wherein The glyoxylic acid content of the composition is below 4 ppm.

17. The production method according to claim 16, wherein The glyoxylic acid content of the composition is below 3.5 ppm.

18. The production method according to claim 17, wherein The glyoxylic acid content of the composition is below 3 ppm.

19. The production method according to claim 18, wherein The glyoxylic acid content of the composition is below 2.5 ppm.

20. A method of synthesizing an oligonucleotide, comprising: It comprises: a) selecting or identifying a substantially pure dichloroacetic acid having a glyoxylic acid content below 15 ppm; and b) mixing the substantially pure dichloroacetic acid with a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby producing a deprotected oligonucleotide.

21. The method of synthesis of claim 20, wherein, It comprises: selecting or identifying a substantially pure dichloroacetic acid having a glyoxylic acid content below 2.5 ppm.

22. The method of synthesis of claim 21, wherein, It comprises: selecting or identifying a substantially pure dichloroacetic acid in which no glyoxylic acid is detected.

23. The method of synthesis of claim 20, wherein, The selecting or identifying comprises the step of determining or having determined whether the glyoxylic acid content of the tested substantially pure dichloroacetic acid is comprised below 15 ppm.

24. The method of synthesis of any one of claims 20-23, wherein, The protected oligonucleotide comprises a 5'-hydroxyl group protected by an acid-labile protecting group.

25. The method of synthesis of any one of claims 20-23, wherein, The acid-labile protecting group is trityl.

26. The method of synthesis as claimed in claim 24, wherein, The acid-labile protecting group is 4,4'-dimethoxytrityl.

27. The method of synthesis of any one of claims 20-23, wherein, The protected oligonucleotide is bound to a solid support.

28. The method of synthesis of any one of claims 20-23, wherein, The mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent.

29. The method of synthesis of any one of claims 20-23, wherein, It further comprises converting the deprotected oligonucleotide into an oligonucleotide having a desired sequence.

30. A method of synthesizing an oligonucleotide, comprising: It comprises: a) producing or having produced a substantially pure dichloroacetic acid having a glyoxylic acid content below 15 ppm; and b) mixing the substantially pure dichloroacetic acid with a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby producing a deprotected oligonucleotide.

31. The method of synthesis of claim 30, wherein, It comprises: preparing or having prepared substantially pure dichloroacetic acid having a glyoxylic acid content of 2.5 ppm or less.

32. The method of synthesis of claim 31, wherein, It comprises: preparing or having prepared substantially pure dichloroacetic acid having a glyoxylic acid content of 2.5 ppm or less.

33. The method of synthesis of claim 30, wherein, The substantially pure dichloroacetic acid has a glyoxylic acid content of 15 ppm or less prior to mixing.

34. The method of synthesis of claim 30, wherein, The substantially pure dichloroacetic acid has a glyoxylic acid content of 15 ppm or less prior to mixing as determined by ion chromatography.

35. The method of synthesis of any one of claims 30-34, wherein, The substantially pure dichloroacetic acid is prepared from a starting dichloroacetic acid composition having glyoxylic acid by reducing the glyoxylic acid content to 15 ppm or less.

36. The method of synthesis of claim 35, wherein, The substantially pure dichloroacetic acid is prepared from a starting dichloroacetic acid composition by reacting the starting dichloroacetic acid composition with a glyoxylic acid trapping reagent to reduce the glyoxylic acid content to 15 ppm or less.

37. The method of synthesis of any one of claims 30-34, wherein, The protected oligonucleotide comprises a 5'-hydroxyl group protected with an acid-labile protecting group.

38. The method of synthesis of any one of claims 30-34, wherein, The acid-labile protecting group is trityl.

39. The method of synthesis of claim 37, wherein, The acid-labile protecting group is 4,4'-dimethoxytrityl.

40. The method of synthesis of any one of claims 30-34, wherein, The protected oligonucleotide is bound to a solid support.

41. The method of synthesis of any one of claims 30-34, wherein, The mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent.

42. The method of synthesis of claim 41, wherein, The solvent is toluene.

43. The method of synthesis of any one of claims 30-34, wherein, It further comprises converting the deprotected oligonucleotide to an oligonucleotide having a desired sequence.

44. A method of synthesizing an oligonucleotide, comprising: The synthetic method comprises: a) determining or having determined a substantially pure dichloroacetic acid having a glyoxylic acid content of 15 ppm or less; and b) mixing the substantially pure dichloroacetic acid with a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby preparing a deprotected oligonucleotide.

45. The method of synthesis of claim 44, wherein, It comprises: determining or having determined a substantially pure dichloroacetic acid having a glyoxylic acid content of 2.5 ppm or less.

46. The method of synthesis of claim 45, wherein, It comprises: determining or having determined a substantially pure dichloroacetic acid having a glyoxylic acid content of 2.5 ppm or less.

47. The method of synthesis of claim 44, wherein, It comprises determining or having determined a substantially pure dichloroacetic acid having a glyoxylic acid content of 15 ppm or less by ion chromatography.

48. The method of synthesis of any one of claims 44-47, wherein, The protected oligonucleotide comprises a 5'-hydroxyl group protected with an acid-labile protecting group.

49. The method of synthesis of any one of claims 44-47, wherein, The acid-labile protecting group is trityl.

50. The method of synthesis of claim 48, wherein, The acid-labile protecting group is 4,4'-dimethoxytrityl.

51. The method of synthesis of any one of claims 44-47, wherein, The protected oligonucleotide is bound to a solid support.

52. The method of synthesis of any one of claims 44-47, wherein, The mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent.

53. The method of synthesis of claim 52, wherein, The mixing comprises mixing the substantially pure dichloroacetic acid with the protected oligonucleotide in toluene.

54. The method of synthesis of any one of claims 44-47, wherein, It further comprises converting the deprotected oligonucleotide to an oligonucleotide having a desired sequence.

55. A method of synthesizing an oligonucleotide, comprising: The synthetic method comprises: a) mixing a substantially pure dichloroacetic acid with a first sequence of a protected oligonucleotide having an acid-labile protecting group under conditions suitable to remove the acid-labile protecting group, thereby providing a first sequence of an oligonucleotide; wherein the substantially pure dichloroacetic acid has a glyoxylic acid content of 15 ppm or less as determined; and b) reacting the oligonucleotide of the first sequence with a desired nucleotide protected by a second protecting group under conditions to extend the nucleotide chain to form a protected oligonucleotide of the second sequence having the second protecting group; c) deprotecting the protected oligonucleotide of the second sequence to remove the second protecting group; and optionally d) repeating steps b) and c) until the desired sequence is achieved.

56. The method of synthesis of claim 55, wherein, The method of synthesis comprises: The glyoxalic acid content of the substantially pure dichloroacetic acid is determined to be less than 2.5 ppm.

57. The method of synthesis of claim 56, wherein, The method of synthesis comprises: The glyoxalic acid content of the substantially pure dichloroacetic acid is determined to be not detectable.

58. A method of removing an acid-labile protecting group from a protected oligonucleotide, characterized in that, The method comprises: a) determining or having determined the glyoxalic acid content of the substantially pure dichloroacetic acid to be less than 15 ppm; and b) combining the substantially pure dichloroacetic acid with the protected oligonucleotide under conditions suitable to remove the acid-labile protecting group.

59. The method of claim 58, wherein, The method comprises determining or having determined the glyoxalic acid content of the substantially pure dichloroacetic acid to be less than 2.5 ppm.

60. The method of claim 59, wherein, The method comprises determining or having determined the glyoxalic acid content of the substantially pure dichloroacetic acid to be not detectable.

61. The method of claim 58, wherein, It comprises determining or having determined the glyoxalic acid content of the substantially pure dichloroacetic acid to be less than 15 ppm by ion chromatography.

62. The method of any one of claims 58-61, wherein, The protected oligonucleotide comprises a 5'-hydroxyl group protected by an acid-labile protecting group.

63. The method of any one of claims 58-61, wherein, The acid-labile protecting group is trityl.

64. The method of claim 62, wherein, The acid-labile protecting group is 4,4'-dimethoxytrityl.

65. The method of any one of claims 58-61, wherein, The protected oligonucleotide is bound to a solid support.

66. The method of any one of claims 58-61, wherein, The combining comprises combining the substantially pure dichloroacetic acid with the protected oligonucleotide in a solvent.

67. The method of claim 66, wherein, The combining comprises combining the substantially pure dichloroacetic acid with the protected oligonucleotide in toluene.

68. A method of making a dichloroacetic acid composition, comprising: It comprises: 1) analyzing the glyoxalic acid content of a starting dichloroacetic acid composition; and, 2) reducing the glyoxalic acid content of the starting dichloroacetic acid composition to less than 15 ppm by combining the starting dichloroacetic acid composition with a glyoxalic acid trapping reagent, thereby producing a dichloroacetic acid composition; The trapping reagent is selected from the group consisting of an amino acid, a reducing agent, and mixtures thereof; The amino acid is selected from the group consisting of cysteine, lysine, and phenylalanine, or mixtures thereof; The reducing agent is selected from the group consisting of triethylsilane.

69. The production method according to claim 68, wherein The glyoxalic acid content of the starting dichloroacetic acid composition is reduced to less than 2.5 ppm.

70. The method of claim 69, wherein the step of preparing is performed by a method comprising: The glyoxalic acid content of the starting dichloroacetic acid composition is not detectable.

71. The method of making of any of any of claims 68-70, wherein, It further comprises distilling the dichloroacetic acid after combining the dichloroacetic acid feedstock and the trapping reagent.

72. The method of claim 71, wherein the step of preparing is performed by a method comprising: It further comprises vacuum distilling the dichloroacetic acid after combining the dichloroacetic acid feedstock and the trapping reagent.

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