COMPOSITIONS COMPRISING DICHLOROACETIC ACID, PROCESSES FOR PREPARING SAME AND USES THEREOF

Abstract

Disclosed is a composition comprising dichloroacetic acid, a process for preparing the same and a use thereof. It has been discovered that the novel impurity is glyoxylic acid, and glyoxylic acid in dichloroacetic acid can be detected and its concentration accurately measured, by ion chromatography method.

Claims

1-14. (canceled)

15. A process for preparing a dichloroacetic acid composition having less than 1000 ppm glyoxylic acid, comprising: mixing a dichloroacetic acid material having greater than 1000 ppm glyoxylic acid with a glyoxylic acid capture reagent.

16. The process as defined in claim 15, wherein the capture reagent is selected from amino acids, chemicals having bifunctional or multifunctional groups, hydroxylamine compounds, reductants, and mixtures thereof.

17. The process as defined in claim 16, wherein 1) the amino acids are selected from cysteine, lysine and phenylalanine, or mixtures thereof; 2) the chemicals having bifunctional or multifunctional groups are selected from dihydric alcohols; 3) the hydroxylamine compounds are selected from hydroxylamine hydrochloride, and/or, 4) the reductants are selected from silanes.

18. The process as defined in claim 15, further comprising distillation of the dichloroacetic acid after mixing the dichloroacetic acid material and the capture reagent.

19-28. (canceled)

29. A method of synthesizing an oligonucleotide, comprising: a) preparing or having prepared a substantially pure dichloroacetic acid having less than 50 ppm glyoxylic acid; 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.

30. The method of claim 29, wherein prior to the mixing, the substantially pure dichloroacetic acid is determined to comprise less than 50 ppm glyoxylic acid.

31. The method of claim 29, wherein prior to the mixing, the substantially pure dichloroacetic acid is determined to comprise less than 50 ppm glyoxylic acid by ion chromatography.

32. The method of claim 29, wherein the substantially pure dichloroacetic acid is prepared from a starting dichloroacetic acid composition having glyoxylic acid by reducing the level of glyoxylic acid to less than 50 ppm.

33. The method of claim 32, wherein the substantially pure dichloroacetic acid is prepared from reacting the starting dichloroacetic acid composition with a glyoxylic acid capture reagent to reduce the level of glyoxylic acid to less than 50 ppm.

34. The method of claim 29, wherein the protected oligonucleotide comprises a 5′-hydroxyl protected with the acid labile protecting group.

35. The method of claim 29, wherein the acid labile protecting group is a trityl group.

36. The method of claim 29, wherein the protected oligonucleotide is bound to a solid support.

37. (canceled)

38. (canceled)

39. A method of synthesizing an oligonucleotide, the method comprising: a) determining or having determined a substantially pure dichloroacetic acid as having less than 50 ppm glyoxylic acid; 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.

40. The method of claim 39, comprising determining or having determined the substantially pure dichloroacetic acid as having less than 50 ppm glyoxylic acid by ion chromatography.

41. The method of claim 39, wherein the protected oligonucleotide comprises a 5′-hydroxyl protected with the acid labile protecting group.

42. The method of claim 39, wherein the acid labile protecting group is a trityl group.

43. The method of claim 39, wherein the protected oligonucleotide is bound to a solid support.

44-54. (canceled)

55. A method of producing a dichloroacetic acid composition, comprising 1) analyzing the level of glyoxylic acid in a starting dichloroacetic acid composition; and 2) reducing the level of glyoxylic acid in the starting dichloroacetic acid composition to be less than 50 ppm by mixing the starting dichloroacetic acid composition with a glyoxylic acid capture agent, thereby producing the dichloroacetic acid composition.

56. The process as defined in claim 55, wherein the capture reagent is selected from amino acids, chemicals having bifunctional or multifunctional groups, hydroxylamine compounds, reductants, and mixtures thereof.

57. The process as defined in claim 56, wherein 1) the amino acids are selected from cysteine, lysine and phenylalanine, or mixtures thereof; 2) the chemicals having bifunctional or multifunctional groups are selected from dihydric alcohols; 3) the hydroxylamine compounds are selected from hydroxylamine hydrochloride, and/or, 4) the reductants are selected from silanes.

58. The process as defined in claim 55, further comprising distillation of the dichloroacetic acid after mixing the starting dichloroacetic acid composition and the glyoxylic acid capture reagent.

59. The dichloroacetic acid composition produced by claim 55.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0223] FIG. 1 is the .sup.1H-NMR spectrum of the white solid in example 2.

[0224] FIG. 2 is the .sup.13C-NMR spectrum of the white solid in example 2.

[0225] FIG. 3 is the LC-MS spectrum of the white solid in example 2.

[0226] FIG. 4 is the HPLC spectrum of GA standard substance.

[0227] FIG. 5 is the HPLC spectrum of the DCA sample-01.

EXAMPLES

[0228] The following examples further illustrate the present disclosure, but the present disclosure is not limited thereto.

Equipment

[0229] .sup.1-NMR: Bruker NMR.

[0230] .sup.13-C NMR: Bruker NMR.

[0231] LC-MS: Waters Q-TOF, and Agilent LC-MS model: Agilent 1290+MSD.

[0232] HPLC: Agilent HPLC model: Agilent 1260.

[0233] Oligonucleotide Synthesizer: Automated AKTA OP100 Synthesizer (6.3 mL reaction column).

[0234] DCA sample-01 is purchased Changzhou Wujin Changxin Teaching Chemical Co., Ltd.

[0235] DCA sample-02 is purchased from Cabbe, Acros.

[0236] DCA reagents are prepared according to the methods of the present disclosure.

Example 1: Oligonucleotide Synthesis

[0237] 1.1. Synthesis of Oligodeoxyribonucleotide T10

[0238] Oligodeoxyribonucleotide T10 was synthesized using standard phosphoramidite chemistry at 0.2 mmol scale on polystyrene primer dT 350 support using an automated AKTA OP100 Synthesizer with 6.3 mL reaction column.

[0239] For each amidite assembly, four chemical reactions were conducted including detritylation (5′-deprotection), coupling, oxidization and capping.

[0240] The detritlyation was conducted using 10% DCA sample-02 in toluene (v/v) with UV 350 nm monitoring control. The coupling recycle consists of co-delivery of 2.0 equivalents of 0.2M amidite solution in acetonitrile and 0.6 M ETT in acetonitrile in a 2:3 flow ratio over the course of 0.5 min, recirculation through the column for 4 min, oxidation with 0.05 M iodine in 9:1 pyridine:water (v/v), capping with 0.5 CV (column volume) of a capping mixture (1:1, v/v) of acetic anhydride acetonitrile (1:4, v/v) and N-methylimidazole-pyridine-acetonitrile (2:3:5, v/v/v) in 0.5 min), and washing with acetonitrile at each block. After 10 rounds of solid phase assembly, 10mer of oligonucleotide modified solid support was obtained.

[0241] 1.2. Synthesis of Oligodeoxyribonucleotide T11

[0242] Then the detritylation time using 10% DCA sample-01 in toluene was increased when assembling the 11th dT amidite on to the solid support. After increasing detritylation time to 90 min, the coupling wasn't run again, which indicated some moieties captured the 5′-OH on the solid support.

[0243] After cleaving the oligonucleotide from support and checking by LC-MS, the main product was 10mer dT product, which indicated that the synthesis of oligodeoxyribonucleotide T11 failed.

##STR00013##

Example 2: Analysis of DCA Sample-01

[0244] When charging 0.1% cysteine into 10% DCA sample-01 in toluene, the inventors found that some white solid precipitated out. The white solid was filtered and dried. .sup.1-NMR, .sup.13C-NMR and LC-MS showed that the main impurity was the condensate of glyoxylic acid with cysteine (Compounds A and B). Results of structure identification information were shown in FIG. 1, FIG. 2 and FIG. 3.

##STR00014##

[0245] 0.058% of glyoxylic acid in the DCA sample-01 was observed by HPLC method. In the HPLC method, STD is glyoxylic acid monohydrate (Source: Aladdin; Lot #F1714025). The HPLC spectra of GA and the DCA sample-01 are as shown in FIG. 4 and FIG. 5.

Example 3: Preparation and Detection of DCA Reagents

[0246] 3.1. Preparation of DCA reagents

[0247] To 500 g DCA sample-01 was charged 5 g of capture reagents (1% w/w or 1% v/v) including cysteine, lysine, phenylalanine (amino acids), (2R)-propane-1,2-diol (difunctional groups chemicals), hydroxylamine hydrochloride, triethylsilane (reductants) etc. The mixtures were incubated (e.g., stood or stirred) for 12-36 hrs, then vacuum distillation was conducted to obtain DCA reagents.

[0248] 3.2. Detection of DCA reagents

[0249] 3.2.1. Samples preparation (4 mg/mL DCA reagents)

[0250] About 200 mg of samples were accurately weighed and added into different 50 mL volumetric flasks, then diluted to volume with diluent (i.e. water), and mixed well.

[0251] Note: Samples referred to DCA reagents.

[0252] 3.2.2. STD preparation (0.01 μg/mL of GA)

[0253] 3.2.2.1. About 31 mg of GA.H.sub.2O (Aladin; Lot #F1714025) was accurately weighed and added into a 25 mL volumetric flask, then diluted to volume with diluent (i.e. water), and mixed well, labeled as GA-1.

[0254] 3.2.2.2. 200 μL of GA-1 was accurately removed into a 100 mL volumetric flask, then diluted to volume with diluent (i.e. water), and mixed well, labeled as GA-2.

[0255] 3.2.2.3. 0.5 mL of GA-2 was accurately removed into a 100 mL volumetric flask, then diluted to volume with diluent (i.e. water), and mixed well, labeled as GA-3 (0.01 μg/mL of GA).

[0256] 3.2.3. IC detection

[0257] Instrument: ICS-6000;

[0258] Column Dionex IonPac AS18, 4×250 mm;

[0259] Mobile phase: KOH (RFC):20 mM aqueous solution (RFC: Reagent free controller);

[0260] Flow rate: 1.0 mL/min;

[0261] Aers-4 mm Suppressor: 50 mA;

[0262] Injection volume: 25 μL;

[0263] Analysis time: 20 min.

[0264] 3.3. Results: The results were summarized in Table 2.

TABLE-US-00002 TABLE 2 Glyoxylic acid in the DCA samples Capture reagents Content DCA reagents 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 1% (v/v)  50.75 ppm hydrochloride DCA reagent 6 Triethylsilane 1% (v/v)  1.01 ppm DCA sample-01 — — 2142.7 ppm

[0265] Conclusions:

[0266] The content of glyoxylic acid in the DCA reagent was less than 2.5 ppm when choosing cysteine, lysine, triethylsilane or phenylalanine as capture reagents. And when choosing other capture reagents, glyoxylic acid in the DCA sample-01 was also effectively decreased.

[0267] The detection method of the present disclosure can be used to detect the content of glyoxylic acid in DCA samples.

Example 4: Evaluation of Glyoxylic Acid in DCA Reagents

[0268] 4.1. Samples preparation (4 mg/mL DCA reagents)

[0269] The preparation method was the same as section 3.2.1.

[0270] 4.2. STD preparation (0.01 μg/mL of GA)

[0271] The preparation method was the same as section 3.2.2.

[0272] 4.3. Specification of residual GA: X (X is the content of glyoxylic acid in DCA sample which does not have effect on oligonucleotide synthesis according to actual needs, in the example, X is 2.5 ppm)

[0273] 4.4. Report results (limit method):

[0274] Compare STD and sample chromatogram with blank chromatogram, and integrate GA peak in STD solution and sample solution, compare their peak areas.

[0275] If the peak area of GA in sample injection is more than that in standard solution, report the result as “>X ppm”.

[0276] If the peak area of GA in sample injection is equal to that in standard solution, report the result as “=X ppm”.

[0277] If the peak area of GA in sample injection is less than that in standard solution, report the result as “ <X ppm”.

TABLE-US-00003 TABLE 3 Area No. (μs*min) Report results 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 0.000271 <2.5 ppm

[0278] Specification of residual GA may be set according to actual needs.

[0279] Conclusions:

[0280] The evaluation method of the present disclosure can be used to detect whether the content of glyoxylic acid in the DCA samples meets specific requirements.

Example 5: Oligonucleotide Synthesis

[0281] 5.1. Synthesis of oligodeoxyribonucleotide T10 (TTTTT TTTTT)

[0282] Oligodeoxyribonucleotide T10 was synthesized using standard phosphoramidite chemistry at 0.1 mmol scale on polystyrene primer dT 350 support using an automated AKTA OP100 Synthesizer with 6.3 mL reaction column.

[0283] For each amidite assembly, four chemical reactions were conducted including detrilyation, coupling, oxidization and capping.

[0284] The detritlyation was conducted using 10% DCA reagents (see table 4) in toluene (v/v) with UV 350 nm monitoring control. The coupling recycle consists of co-delivery of 2.0 equivalents of 0.2M amidites solution in acetonitrile and 0.6 M ETT in acetonitrile in a 2:3 flow ratio over the course of 0.5 minutes, recirculation through the column for 4 min, oxidation with 0.05 M iodine in 9:1 pyridine:water (v/v), capping with 0.5 CV of a capping mixture (1:1, v/v) of acetic anhydride acetonitrile (1:4, v/v) and N-methylimidazole-pyridine-acetonitrile (2:3:5, v/v/v) in 0.5 min) in 0.5 min, and washing with acetonitrile at each block.

[0285] T10 was cleaved from the solid support with concomitant removal of nucleoside protecting groups by addition of a 1:1 mixture of 40 wt % methylamine aqueous solution and ammonium hydroxide aqueous solution (10 mL per gram of synthesized oligonucleotide) to the support and the resulting mixture was incubated at 30-40° C. for 2-3 hrs in a shaker.

[0286] The mixture was filtered through glass fiber filter, wash the support with purified water, and combine the filtrate. Take sample for analysis by MS and HPLC after adjusting the pH to 7.0-9.0 by 20% acetic acid.

[0287] 5.2. Synthesis of Oligodeoxyribonucleotide 17mer (CCCGGGTTTCGTCGTAA)

[0288] Oligodeoxyribonucleotide 17mer DMT-CCCGGGTTTCGTCGTAA was synthesized using standard phosphoramidite chemistry at 0.2 mmol scale on PS Primer Unylinker350 support using an automated AKTA OP100 Synthesizer with 6.3 mL reaction column.

[0289] For each amidites assembly, four chemical reactions were conducted including detrilyation, coupling, oxidization and capping.

[0290] The detritlyation was conducted using 10% DCA reagents (see table 4) in toluene (v/v) with UV 350 nm monitoring control. The coupling recycle consists of co-delivery of 2.0 equivalents of 0.2M amidites solution in acetonitrile and 0.6M ETT in acetonitrile in a 2:3 flow ratio over the course of 0.5 minute, recirculation through the column for 4 minutes, oxidation with 0.05M iodine in 9:1 pyridine:water (v/v), capping with 0.5 CV of a capping mixture (1:1, v/v) of acetic anhydride acetonitrile (1:4, v/v) and N-methylimidazole-pyridine-acetonitrile (2:3:5, v/v/v) in 0.5 min) in 0.5 minute, and washing with acetonitrile at each block.

[0291] 17 mer was cleaved from the solid support with concomitant removal of nucleoside protecting groups by addition ammonium hydroxide aqueous solution (10 mL per gram of synthesized oligonucleotide) to the support and the resulting mixture was incubated at 50-60° C. for 15-17 hrs in a shaker.

[0292] The mixture was filtered through glass fiber filter, wash the support with purified water, and combine the filtrate. Take sample for analysis by MS and HPLC.

TABLE-US-00004 TABLE 4 Glyoxylic acid DCA Samples DNA sequence content (ppm) Purity (HPLC) DCA reagent TTTTT TTTTT (SEQ ID N/A 95.52% 7 NO: 1) DCA reagent TTTTT TTTTT (SEQ ID  2.5 ppm 94.58% 8 NO: 1) DCA reagent TTTTT TTTTT (SEQ ID  6.0 ppm 93.24% 9 NO: 1) DCA reagent TTTTT TTTTT (SEQ ID 12.0 ppm 92.68% 10 NO: 1) DCA reagent CCCGGGTTTCGTCGTAA  2.5 ppm 86.42% 8 (SEQ ID NO: 2) DCA reagent CCCGGGTTTCGTCGTAA  6.0 ppm 79.71% 9 (SEQ ID NO: 2)

[0293] 5.3. Preparation of DCA reagents

[0294] DCA reagent 7:

[0295] To 500 g DCA sample-01 was charged much excessive amount of cysteine. The mixtures were stood for 16 hrs, then vacuum distillation was conducted to obtain DCA reagent 7, which was detected by using the method as described in section 3.2 in example 3, and no GA was detected (N/A).

[0296] DCA reagent 8

[0297] To 500 g DCA sample-01 was charged 2.5 g of cysteine. The mixtures were stood for 16 hrs, then vacuum distillation was conducted to obtain a DCA reagent 8, which was detected by using the method as described in section 3.2 in example 3, and the content of GA was 2.5 ppm.

[0298] DCA reagent 9 & DCA reagent 10:

[0299] To 50 g of DCA reagent 7 was charged 0.5 g glyoxylic acid and the mixture was incubated under 25-50° C. for 0.5 hrs. Then the mixture was filtered to get a DCA sample-03 containing 7200 ppm glyoxylic acid. The DCA sample-03 with 7200 ppm glyoxylic acid was diluted with DCA for 1200 times and 600 times to get DCA reagent 9 and DCA reagent 10.

[0300] Conclusions:

[0301] After treating with capture reagents of the present disclosure and vacuum distillation, the DCA sample-01 could also be used for oligonucleotide synthesis. Therefore, glyoxylic acid is an impurity in commercially available DCA products, which may cause the oligonucleotide synthesis failure.

[0302] It is to be understood that the foregoing description of preferred examples is intended to be purely illustrative of the principles of the disclosure, rather than exhaustive thereof, 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 other than expressly set forth in the following claims.