PURIFICATION OF TRIPHOSPHORYLATED OLIGONUCLEOTIDES USING CAPTURE TAGS
20170145410 ยท 2017-05-25
Assignee
Inventors
Cpc classification
C07H21/00
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a method of preparing triphosphate-modified oligonucleotides using a capture tag. The method allows the synthesis and purification of triphosphate-modified oligonucleotides in high yield and purity suitable for pharmaceutical applications.
Claims
1-15. (canceled)
16. A method of preparing an oligonucleotide of formula (I), ##STR00007## wherein V.sub.1, V.sub.3 and V.sub.5 are independently selected from the group consisting of O, S and Se; V.sub.2, V.sub.4 and V.sub.6 are independently selected from the group consisting of OH, OR.sup.1, SH, SR.sup.1, F, NH.sub.2, NHR.sup.1, N(R.sup.1).sub.2 and BH.sub.3.sup.M.sup.+, W.sub.1 is O or S, W.sub.2 is O, S, NH or NR.sup.2, W.sub.3 is O, S, NH, NR.sup.2, CH.sub.2, CHHal or C(Hal).sub.2, R.sup.1, R.sup.2 and R.sup.3 are selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.2-6 acyl and a cyclic group, each substituted or unsubstituted, or wherein two R.sup.1 may form a ring together with an N-atom bound thereto, M.sup.+ is a cation, X is NH, NR.sup.3, O or S, Z represents a capture tag which is a long-chain alkyl residue, a perfluoroalkyl entity, an azide, an alkynyl group, a chemical entity containing a second amino group in an NH.sub.2YXH type reagent, a chloroformate group, a lipidic residue, or a first partner of a non-covalent binding pair which has a binding constant of 10.sup.6 l/mol or less to its complementary binding partner, Y represents a bond or a linker connecting the capture tag to X, and ON represents an oligonucleotide comprising at least 4 nucleotides or nucleotide analogue building blocks, comprising the steps: (a) reacting a compound of formula (IIa) ##STR00008## with an oxidizing agent to obtain a compound of formula (IIb) ##STR00009## (b) reacting a compound of formula (IIb) with a capture tag agent of formula (III),
ZYXH(III) to obtain a reaction product comprising the oligonucleotide of formula (I), and (c) contacting the reaction product of step (b) with a capture reagent capable of interacting with the capture tag, wherein the contacting takes place under conditions which allow separation of the oligonucleotide (I) from other species contained in said reaction product.
17. The method of claim 16, wherein Y is an alkylene linker, or an aralkylene linker selected from the group consisting of (i) an alkylene linker, or an aralkylene linker comprising heteroatoms or heteroatom containing groups, (ii) an alkylene linker, or an aralkylene linker comprising CC or CC bonds, and (iii) an alkylene linker, or an aralkylene linker comprising both heteroatoms or heteroatom containing groups, and CC or CC bonds.
18. The method of claim 17, wherein the heteroatom containing groups are selected from the group consisting of O, S, NH, CO, and CS.
19. The method of claim 16, wherein Y is a polyalkylene oxide linker selected from (i) a poly-C.sub.2-C.sub.6-alkylene oxide, (ii) a poly-C.sub.2-C.sub.3-alkylene oxide, (iii) a linker with a number average molecular weight in the range of 30-800 g/mol, and (iv) a linker which is [CH.sub.2CHR.sup.4O-].sub.n, wherein n is 1-10, and R.sup.4 represents H or C.sub.1-6-alkyl.
20. The method of claim 16, wherein Y is CH.sub.2CH.sub.2[(OCH.sub.2CH.sub.2)].sub.3.
21. The method of claim 16, wherein Z is a lipidic residue selected from cholesteryl and tocopheryl.
22. The method of claim 16, wherein Z is a perfluoralkyl entity selected from a 4-(1H,1H,2H,2H-perfluorodecyl)benzyl and 3-(perfluorooctyl)propyl residue.
23. The method of claim 16, wherein Z is a first partner of a non-covalent binding pair selected from biotin, desthiobiotin, a hapten, and an antigen, and the complementary binding partner is a streptavidin, an avidin, or an antibody.
24. The method of claim 16, wherein ZYX is propargylamino.
25. The method of claim 16, wherein the capture tag and the capture reagent capable of interacting therewith are selected from: (i) a hydrophobic or fluorinated group and a chromatographic material with affinity for hydrophobic or fluorinated groups; (ii) a first partner of a non-covalent binding pair and a second partner of a non-covalent binding pair, and (iii) a first partner of a covalent binding pair and a second partner of a covalent binding pair, where the first and second partner form covalent bonds.
26. The method of claim 16, wherein the triphosphate/triphosphate analogue group is attached to the 5-terminus of the oligonucleotide.
27. The method of claim 26, wherein the triphosphate/triphosphate analogue group is attached to the 5-OH-group of the 5-terminal sugar thereof.
28. The method of claim 16, further comprising the step: (d) removing the capture tag to obtain an oligonucleotide of formula (IV): ##STR00010## wherein V.sub.1, V.sub.3, V.sub.5, V.sub.2, V.sub.4, V.sub.6, W.sub.1, W.sub.2, W.sub.3 and ON are as defined in claim 16.
29. The method of claim 16, wherein the oligonucleotide is selected from desoxyribonucleotides, ribonucleotides and oligonucleotide analogues.
30. The method of claim 16, wherein the oligonucleotide is single-stranded or double stranded.
31. The method of claim 30, wherein the oligonucleotide is double-stranded and the duplex is closed by a loop at the distal end thereof, wherein the loop comprises nucleotide and/or non-nucleotide building blocks.
32. The method of claim 30, wherein the oligonucleotide is double-stranded and the duplex is blunt-ended at the proximal end thereof.
33. Oligonucleotide of Formula (I), obtainable by a method comprising (a) reacting a compound of formula (IIa) ##STR00011## with an oxidizing agent to obtain a compound of formula (IIb) ##STR00012## (b) reacting a compound of formula (IIb) with a capture tag agent of formula (III),
ZYXH(III) to obtain a reaction product comprising the oligonucleotide of formula (I), and (c) contacting the reaction product of step (b) with a capture reagent capable of interacting with the capture tag, wherein the contacting takes place under conditions which allow separation of the oligonucleotide (I) from other species contained in said reaction product, wherein V.sub.1, V.sub.3, V.sub.5, V.sub.2, V.sub.4, V.sub.6, W.sub.1, W.sub.2, W.sub.3, X, Y, Z and ON are defined as in claim 16, and wherein X, Z and Y are defined as in claim 16.
34. A kit for preparing an oligonucleotide of formula (I) ##STR00013## wherein V.sub.1, V.sub.3, V.sub.5, V.sub.2, V.sub.4, V.sub.6, W.sub.1, W.sub.2, W.sub.3, X, Y, Z and ON are defined in claim 16, wherein the kit comprises: (a) a capture tag agent of formula (III)
ZYXH(III) wherein X, Z and Y are defined as in claim 16, and (b) a capture reagent capable of interacting with the capture tag.
Description
[0072] Further, the present invention shall be explained in more detail by the following Figures and Examples.
[0073]
[0074]
(A) crude reaction mixture containing 65% n-decyl-NH-pppRNA (peak at 14 min);
(B) isolated n-decyl-NH-pppRNA;
(C) pppRNA; the pH=3.8 60 min hydrolysis product from B
[0075] In
[0076] The broad peak at 10 min retention time in A contains the nonphosphory-lated 24-mer, shorter synthesis failure sequences, the minor pppRNA hydrolysis product and the 5-H-phosphonate derivative of the 24-mer. The insert shows the position of pppRNA and 5-OH RNA in this system.
[0077] Column: Hamilton PRP-1 4.1250 mm, 10 m
[0078] Gradient: 1-100% B in 18 min, A=0.05 M TEAB; B=80% Methanol 0.05 M TEAB
[0079]
[0080] (A) spectrum recorded from the crude reaction mixture after desalting showing the presence of n-decyl-NH-ppp RNA (24d), pppRNA (24c), 5-H-phosphonate RNA(24b) and 5-OH -RNA(24a) and shorter synthesis failure sequences indicated as peaks 12-23;
[0081] (B) spectrum recorded from HPLC isolated n-decyl-NHpppRNA (B),
[0082] (C) spectrum of pure pppRNA as obtained from the direct EtOH precipitation of the pH=3.8 hydrolysis product of n-decyl-NH-pppRNA
[0083]
[0084]
[0085]
[0086]
[0087] Column: Hamilton PRP-1 7250 mm, 10 m Flow rate 3 mL/min.
[0088] Gradient: 1-80% B in 50 min, A=0.1M TEAB; B=80% Methanol 0.1 M TEAB
[0089]
[0090] In all figures the x-axis is volume [ml] and the y-axis is absorbance at 260 nm [mAu].
[0091]
[0092]
[0093]
[0094]
EXAMPLE 1
Preparation of a 5-Triphosphate Modified Oligonucleotide Using a Decyl Amine Capture Tag Purification Step
[0100] An overview of the reaction scheme described in Example 1 is shown in
[0101] Step 1:
[0102] Dissolve 203 mg (1 mmol) of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in 1 mL of dry dioxane in a 10 mL septum vial under argon.
[0103] Step 2:
[0104] Dry the synthesis column containing the fully protected RNA that has been detitrylated and thoroughly washed with acetonitrile, in vacuum for 12 h. Wash the column contents thoroughly by repeatedly drawing in and expelling 2 mL of anhydrous dioxane/pyridine solution, 3:1 (v/v) in an argon atmosphere.
[0105] Step 3:
[0106] Add into a vial first 2 mL of pyridine/dioxane, 3:1 v/v followed by 100 L of 1 M 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one solution in dry dioxane to give a 50 mM solution of the phosphitylating reagent, e.g. 2-chloro-4H-1,3,2-benzodioxaphosphorin-2-one, in dioxane/pyridine, 3:1 (v/v). Homogenize the solution by gently shaking. Start the reaction by drawing the 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one solution through the synthesis column from the vial.
[0107] During the reaction, repeatedly draw in and expel the 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one containing solution from the synthesis column, in order to allow thorough contact and good mixing with the solid phase supported RNA. A 30 min reaction time usually gives near quantitative reaction of the free 5-OH group of the support bound oligomer in the 20-40 nt range.
[0108] Step 4:
[0109] After a 30 min reaction time expel the dioxane/pyridine solution containing the excess phosphitylating agent into a waste container, fill a new syringe with a vortexed mixture of 1 mL of 0.5 M (Bu.sub.3NH).sub.2 pyrophosphate in dry DMF and 238 L (1 mmol) of dry Bu.sub.3N to give a 0.5 M (Bu.sub.3N).sub.4 pyrophosphate solution. Push this solution through the column thereby replacing the dioxane/pyridine solution. The large excess of the pyrophosphate ensures a quantitative conversion of the intermediate to the P(III)-P(V) cyclic anhydride IIa.
[0110] Step 5:
[0111] Wash the column with 3 mL of CH.sub.3CN to remove the DMF and excess PP.sub.i, and to fill the column reactor with dry CH.sub.3CN.
[0112] Step 6:
[0113] Dissolve 300 L of t-BuOOH (5.5 M solution in decane, Sigma-Aldrich) in 2 mL of anhydrous CH.sub.3CN to give an approximately 0.7 M homogeneous solution. Contact the synthesis support with this solution for 15 min in order to obtain the oxidized P(V) cyclic anhydride IIb.
[0114] Step 7:
[0115] Wash the column with 3 mL of dry CH.sub.3CN to remove the excess peroxide and fill it with dry CH.sub.3CN.
[0116] Step 8:
[0117] Dissolve 300 L of dry decylamine in 1 mL of dry CH.sub.3CN under argon and bring the solution in contact with the support in the column. Move the decylamine solution through the support. The contact time of the CPG with the amine solution should be 3 min.
[0118] Step 9: Wash the column thoroughly with 9 mL acetonitrile, then dry the column contents by flushing argon through it.
[0119] Step 10First Stage of the Deprotection:
[0120] Pass 1 mL of deprotection solution (40% aq. methylamine/conc. aq. ammonia 1:1 v/v. AMA reagent) through the support for 2-3 times. After a contact of 30 min transfer the solution into a new vial. Wash the support with same volume of AMA deprotection solution and combine the washings. Heat the combined solution and washings for 10 min at 65 C. After cooling on ice, concentrate the solution to a volume of 300-500 L, then evaporate to dryness.
[0121] Step 11Removal of the 2-O-TBDMS Protecting Groups:
[0122] Dry the residue by addition and coevaporation of 300 L of dry EtOH, add 1 mL of dry 1 M TBAF (tetra-n-butylammonium fluoride) in THF, seal tightly and put on a shaker for 16 h. Quench the reaction with 1 mL of sterile aqueous 1 M TEAB (triethylammonium bicarbonate), and desalt it on a NAP-25 (Nucleic Acid Purification) column using sterile water as eluent. Filtration through a sterile 2 m filter may be necessary at this step. Combine and evaporate the UV-absorbing fractions to a volume of 150 L, add 100 mL of 1 M TEAB pH8 and store the solution frozen at 20 C. until the HPLC purification can be performed. The decyl-NHpppRNA product is stable at 20 C. for weeks at pH 7-8.
[0123] Step 12HPLC Purification:
[0124] The reaction product from an 1 mol scale reaction mixture from step 11 was loaded into a 725 mm PRP-1 column (Hamilton). Purification was performed using a linear gradient buffer B from 0 to 80% in 50 min at a flow rate of 3 mL/min. Buffer A is 100 mM TEAB and buffer B is 100 mM TEAB in methanol/water 8:2 v/v. A typical example of a 27-mer purification is shown in
[0125] Fractions 5 and 6 are collected, evaporated on a rotary evaporator and desalted by several coevaporations with dry methanol, The residue (approx. 200-250 nmol of decyl-NHpppRNA) was dissolved in water and transferred into a screw cap Eppendorf vial.
[0126] Step 13Removal of the Decylamine Tag:
[0127] 100 nmol of decyl-NHpppRNA was dissolved in 400 L of pH 3.8 deprotection buffer in a 2 mL Eppendorf tube, and the sealed tube was heated at 60 C. for 70 min. These conditions result in quantitative cleavage of the phosphoramidate bond with no degradation of the triphosphate moiety. Then the reaction mixture was cooled on ice and 25 I.sub. of sterile 5 M NaCl solution and 1.2 mL of absolute EtOH were added. After thorough mixing the solution was kept at 20 C. overnight to precipitate the pppRNA. The precipitate was collected by centrifugation, washed with cold ethanol, dried on a SpeedVac, then dissolved in 500 mL of sterile water and stored frozen at 20 C.
TABLE-US-00001 TABLE 1 Summary of the reaction conditions for introduction of the 5-terminal decyl-NHppp-residue. Step Reagent Time 1 3 mL dioxane/pyridine, 3:1 v/v wash .fwdarw. 2 50 mM 2-chloro-4H-1,3,2-benzodioxaphosphorin- 30 min .fwdarw. 4-one in 2 mL of dioxane/pyridine, 3:1 v/v 3 1 mL of 0.5M (Bu.sub.3NH).sub.2PP.sub.i in DMF plus 10 min .fwdarw. 238 L of Bu.sub.3N 4 3 mL of dry acetonitrile wash .fwdarw. 5 300 L of t-BuOOH (5.5M in decane) in 2 mL of 15 min .fwdarw. CH.sub.3CN 6 3 mL of dry acetonitrile Wash .fwdarw. 7 300 L of n-decylamine in 1 mL of dry 3 min .fwdarw. acetonitrile (1.1M decylamine) 8 10 mL of acetonitrile wash .fwdarw. 1 mol scale synthesis column containing support bound detitrylated RNA bidirectional movements of reagents, .fwdarw. unidirectional washing step
[0128] In analogous manner, a 5-triphosphate modified oligonucleotide was also synthesized and purified using an octadecyl or a cholesteryl capture tag.
EXAMPLE 2
Preparation of Triphosphate Oligonucleotides Using Non-Lipophilic Capture Tags
(F-TAG-pppRNA and N.SUB.3.-TAG-pppRNA)
[0129] In order to demonstrate the utility of non-lipophilic interaction based purification strategies the pppRNA derivatives F-TAG-RNA and N3-TAG-RNA were prepared (see
F-TAG-RNA and N3-TAG-RNA Analytical Data (See FIG. 10):
[0130]
TABLE-US-00002 (theRNAsequenceintheseexamplesis 5-GACGCUGACCCUGAAGUUCAUCUU) Time required for complete P-N Calcu- Mass cleavage HPLC lated measured atpH retention Mass, by 3.8at time* Da MALDI,Da 60 C. F-TAG-pppRNA 15.1min 8287.74 8290.30 70min N3-TAG-ppRNA 11min 8033.20 8033.92 70min * PRP-1 column 0-100% B in 20 min (A = 100 mM Triethylammoniumbicarbonate (TEAB), B = 100 mM TEAB 80% MeOH)
[0131] pppRNA oligonucleotides containing fluorous tags (F-TAG-pppRNA) can be purified using commercial fluorous cartridges, or fluorous HPLC columns which enable the exploitation of the strong nonconvalent interaction between perfluorinated alkyl chains. The gamma azide modified pppRNA derivatives (N3-TAG-pppRNA) can be covalently bound to commercially available propyne modified solid phases by RNA compatible versions of the copper(I)-catalysed-alkyne-azide cycloaddition reaction (click chemistry). This procedure enables the purification of highly structured pppRNA sequences because in the resin bound form denaturing conditions can be applied to remove non-triphosphorylated by-products.
[0132] Upon acid hydrolysis both F-TAG-RNA and N3-TAG-RNA release the pppRNA end product with comparable kinetics to the simple PN alkyl amide as described in
EXAMPLE 3
Variation of the RP-HPLC Elution Position of Tag-pppRNA by n-Alkyl Capture Tags of Increasing Chain Length
[0133] Besides the n-decyl-tag described in Example 1, aliphatic n-alkyl residues with longer chain lengths (C.sub.12, C.sub.14, C.sub.18) can be used to increase the retention time of the Tag-pppRNA product during RP-HPLC purification enabling an efficient separation from impurities that do not contain the tag. N-dodecyl-NH-pppRNA, n-tetradecyl-NH-pppRNA and n-octadecyl-NH-pppRNA can be prepared following the procedure described in example 1 by variation of step 8: A 0.1 M solution of n-alkylamine (n-dodecylamine, n-tetradecylamine or n-octadecylamine) in dry CH.sub.2Cl.sub.2 is prepared and 2 mL of the solution is brought in contact with the support in the column. The alkylamine solution is pushed to and fro through the support. After a contact time of 3 h an additional washing step with 2 mL of CH.sub.2Cl.sub.2 is required prior to continuing with the next workup steps.
Analytical Data:
[0134]
TABLE-US-00003 RP-HPLC* Mass Time for retention Calculated measured by complete P-N time Mass MALDI cleavage at (min) (Da) (Da) pH 3.8 at 60 C. C.sub.12NH- 15.5 7995.7 7999.2 70 min pppRNA C.sub.14NH- 17.3 8023.7 8028.1 70 min pppRNA C.sub.18NH- 19.7 8079.8 8082.2 70 min gives > pppRNA 80% product *PRP-1 column 0-100% B in 20 min (A = 100 mM Triethylammoniumbicarbonate, B = 100 mM TEAB 80% MeOH)
[0135]