Purification of triphosphorylated oligonucleotides using capture tags

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. 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.2-6alkenyl, 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 C.sub.8-24 alkyl residue, a perfluoroalkyl entity, an azide, an alkynyl group, a chemical entity containing an 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, wherein said first partner is selected from the group consisting of biotin, desthiobiotin, a hapten, and an antigen, and the complementary binding partner is a streptavidin, an avidin, or an antibody, 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 (lla) ##STR00008## with an oxidizing agent to obtain a compound of formula (llb) ##STR00009## (b) reacting a compound of formula (llb) with a capture tag agent of formula (lll),
ZYXH (lll) 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.

2. The method of claim 1, 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.

3. The method of claim 2, wherein the heteroatom containing groups are selected from the group consisting of O, S, NH, CO, and CS.

4. The method of claim 1, wherein Y is a polyalkylene oxide linker selected from (i) a polyC.sub.2C.sub.6alkylene oxide, (ii) a polyC.sub.2C.sub.3alkylene 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.1-6alkyl.

5. The method of claim 1, wherein Y is CH.sub.2CH.sub.2[(OCH.sub.2CH.sub.2)].sub.3.

6. The method of claim 1, wherein Z is a lipidic residue selected from cholesteryl and tocopheryl.

7. The method of claim 1, wherein Z is a perfluoralkyl entity selected from a 4-(1H, 1H, 2H, 2H-perfluorodecyl)benzyl and 3-(perfluorooctyl)propyl residue.

8. The method of claim 1, wherein ZYX is propargylamino.

9. The method of claim 1, 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.

10. The method of claim 1, wherein the triphosphate/triphosphate analogue group is attached to the 5-terminus of the oligonucleotide.

11. The method of claim 10, wherein the triphosphate/triphosphate analogue group is attached to the 5-OH-group of the 5-terminal sugar thereof.

12. The method of claim 1, 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 1.

13. The method of claim 1, wherein the oligonucleotide is selected from desoxyribonucleotides, ribonucleotides and oligonucleotide analogues.

14. The method of claim 1, wherein the oligonucleotide is single-stranded or double stranded.

15. The method of claim 14, 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.

16. The method of claim 14, wherein the oligonucleotide is double-stranded and the duplex is blunt-ended at the proximal end thereof.

17. Oligonucleotide of Formula (I), obtainable by a method comprising (a) reacting a compound of formula (lla) ##STR00011## with an oxidizing agent to obtain a compound of formula (llb) ##STR00012## (b) reacting a compound of formula (llb) with a capture tag agent of formula (lll),
ZYXH (lll) 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 1, and wherein X, Z and Y are defined as in claim 1.

18. 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 1, wherein the kit comprises: (a) a capture tag agent of formula (lll)
ZYXH (III) wherein X, Z and Y are defined as in claim 1, and (b) a capture reagent capable of interacting with the capture tag.

Description

(1) Further, the present invention shall be explained in more detail by the following Figures and Examples.

(2) FIG. 1 shows a schematic overview of the method of the invention using a decyl residue as capture tag Z

(3) FIGS. 2A-C shows RP-HPLC purification of pppRNA via n-decyl-NH-pppRNA intermediate

(4) (A) crude reaction mixture containing 65% n-decyl-NH-pppRNA (peak at 14 min);

(5) (B) isolated n-decyl-NH-pppRNA;

(6) (C) pppRNA; the pH=3.8 60 min hydrolysis product from B

(7) In FIG. 2 the x-axis means time [min] and the y-axis means absorbance at 260 nm [mAu].

(8) The broad peak at 10 min retention time in A contains the nonphosphorylated 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.

(9) Column: Hamilton PRP-1 4.1250 mm, 10 m

(10) Gradient: 1-100% B in 18 min, A=0.05 M TEAB; C=80% Methanol 0.05 M TEAB

(11) FIGS. 3A-C shows MALDI-TOF spectra (x-axis: mass [Da]) corresponding to HPLC traces A, B and C in FIG. 2 respectively.

(12) (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;

(13) (B) spectrum recorded from HPLC isolated n-decyl-NHpppRNA (B),

(14) (C) spectrum of pure pppRNA as obtained from the direct EtOH precipitation of the pH=3.8 hydrolysis product of n-decyl-NH-pppRNA

(15) FIGS. 4A-B shows a reaction scheme explaining the generation of side products 24a-c

(16) FIG. 5 shows the time course for the conversion of n-decyl-NH-pppRNA to pppRNA via acidic hydrolysis of the phosphoramidate bond.

(17) FIGS. 6A-C shows typical MALDI spectra (x-axis: mass [Da]) of 21-mer, 24-mer, 27-mer pppRNA products as obtained after capture tag removal and EtOH precipitation as Na+salt. The correct mass peak is observed at m/z 6911.6 (A), m/z 7828 (B), m/z 8808.1 (C) and the peaks at m/z 3462 (A), m/z 3918 (B), 4408 (C) are due to the doubly charged pppRNA, respectively. Similar quality spectra have been obtained in more than 50 examples with a variety of sequences containing nucleoside analogs and 3 modifications in the 15-42-mer range.

(18) FIG. 7A shows a semipreparative scale reversed phase HPLC purification of a 1 mol scale reaction of decyl-NHpppRNA 21 mer on a 7 mm Hamilton PRP-1 column

(19) Column: Hamilton PRP-1 7250 mm, 10 m Flow rate 3 mL/min.

(20) Gradient: 1-80% B in 50 min, A=0.1 M TEAB; B=80% Methanol 0.1 M TEAB

(21) FIG. 7B and FIG. 7C show semipreparative scale reversed phase HPLC purifications, in particular showing how the inventive method is able to deal with sub-optimal synthesis and/or 5-phosphorylation conditions.

(22) In all figures the x-axis is volume [ml] and the y-axis is absorbance at 260 nm [mAu].

(23) FIG. 8 shows especially preferred modified oligonucleotides of formula (I).

(24) FIGS. 9A-B shows the synthesis of compounds F-TAG-pppRNA and N3-TAG-pppRNA (A) and the strategy for reversible covalent immobilisation using N3-TAG RNA (B)

(25) FIGS. 10A-B shows MALDI spectra of F-TAG-pppRNA (A) N3-TAG-pppRNA (B)

(26) FIG. 11 shows the RP-HPLC analysis of pppRNA and n-alkyl-NH-pppRNAs with alkyl residues of increasing chain length: A. pppRNA, RT=9.3 min B. n-decyl-NH-pppRNA, RT=13.8 min, C. n-dodecyl-NH-pppRNA, RT=15.5 min D. n-tetradecyl-NH-pppRNA, RT=17.3 min E. n-octadecyl-NH-pppRNA, RT=19.7 min

EXAMPLE 1

(27) Preparation of a 5-Triphosphate Modified Oligonucleotide Using a Decyl Amine Capture Tag Purification Step.

(28) An overview of the reaction scheme described in Example 1 is shown in FIG. 1.

(29) Step 1: 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.

(30) Step 2: 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.

(31) Step 3: 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.

(32) 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.

(33) Step 4: 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. Step 5: 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.

(34) Step 6: 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.

(35) Step 7: Wash the column with 3 mL of dry CH.sub.3CN to remove the excess peroxide and fill it with dry CH.sub.3CN.

(36) Step 8: 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.

(37) Step 9: Wash the column thoroughly with 9 mL acetonitrile, then dry the column contents by flushing argon through it.

(38) Step 10First stage of the deprotection: 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.

(39) Step 11Removal of the 2-O-TBDMS protecting groups: 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.

(40) Step 12HPLC purification: 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 FIG. 7A.

(41) 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.

(42) Step 13Removal of the decylamine tag: 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 L 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.

(43) TABLE-US-00001 TABLE 1 Summary of the reaction conditions for introduction of the 5-terminal decyl-NHppp-residue. 1 mol scale synthesis column containing support bound detitrylated RNA 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 4-one in 2 mL of dioxane/pyridine, 3:1 v/v 3 1 mL of 0.5M (Bu.sub.3NH).sub.2PP, in DMF plus 238 L 10 min 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 CH.sub.3CN 6 3 mL of dry acetonitrile Wash .fwdarw. 7 300 L of n-decylamine in 1 mL of dry acetonitrile 3 min (1.1M decylamine) 8 10 mL of acetonitrile wash .fwdarw. bidirectional movements of reagents, .fwdarw. unidirectional washing step

(44) In analogous manner, a 5-triphosphate modified oligonucleotide was also synthesized and purified using an octadecyl or a cholesteryl capture tag.

EXAMPLE 2

(45) Preparation of Triphosphate Oligonucleotides Using Non-Lipophilic Capture Tags

(46) (F-TAG-pppRNA and N.sub.3-TAG-pppRNA)

(47) 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 FIG. 9). All steps of the synthesis are identical with the procedure described in Example 1 except that in step 8 of FIG. 1, 2 mL of a 0.1 M solution of 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-Heptadecafluoroundecylamine in anhydrous acetonitrile was used for the ring opening of the solid phase bound cyclotriphosphate with an increased 3 h reaction time to give F-TAG-RNA; and 2 mL of a 0.1 M solution of 11-azido-3,6,9-trioxaundecan-1-amine in dry acetonitrile for 3 h was used to give N.sub.3-TAG-pppRNA. The following deprotection steps are identical with those given in the detailed description for DecNHpppRNA in Example 1.

(48) F-TAG-RNA and N3-TAG-RNA analytical data (see FIG. 10):

(49) (the RNA sequence in these examples is 5-GACGCUGACCCUGAAGUUCAUCUU)

(50) TABLE-US-00002 Mass Time required measured for complete HPLC by P-N cleavage retention Calculated MALDI, at pH 3.8 time* Mass, Da Da at 60 C. F-TAG-pppRNA 15.1 min 8287.74 8290.30 70 min N3-TAG-ppRNA 11 min 8033.20 8033.92 70 min *PRP-1 column 0-100% B in 20 min (A = 100 mM Triethylammoniumbicarbonate (TEAB), B = 100 mM TEAB 80% MeOH)

(51) 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.

(52) 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 FIG. 5.

EXAMPLE 3

(53) Variation of the RP-HPLC Elution Position of Tag-pppRNA by n-alkyl Capture Tags of Increasing Chain Length

(54) 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.

(55) Analytical Data:

(56) TABLE-US-00003 Mass Time for RP-HPLC* measured complete P-N retention Calculated by MALDI cleavage at pH time (min) Mass (Da) (Da) 3.8 at 60 C. C12-NH-pppRNA 15.5 7995.7 7999.2 70 min C14-NH-pppRNA 17.3 8023.7 8028.1 70 min C18-NH-pppRNA 19.7 8079.8 8082.2 70 min gives >80% product *PRP-1 column 0-100% B in 20 min (A = 100 mM Triethylammoniumbicarbonate, B = 100 mM TEAB 80% MeOH)

(57) FIG. 11 shows the RP-HPLC analysis of pppRNA and n-alkyl-NH-pppRNAs with alkyl residues of increasing chain length.