GB2125798A - Solid phase synthesis of oligonucleotides - Google Patents

Abstract

A method for synthesizing deoxyribooligonucleotides and ribooligonucleotides on a solid support is disclosed.

Description

SPECIFICATION Solid phase synthesis of oligonucleotides Technical field This invention relates to a method of synthesizing deoxyribooligonucleotides and ribooiigonucleotides on a solid support.

Background art In recent years scientists have developed methods for synthesizing oligonucleotides of difined sequences which can be used to manipulate DNA and RNA. For example, synthetic pieces of DNA of 10 to 14 units in size can be joined together by specific enzymatic techniques to form whole genes or can serve as probes to aid in identifying a desired DNA sequence. Chains of synthetic oligonucleotides can serve as linkers to aid in splicing together pieces of DNA or in constructing recombinant DNA molecules.

Oligonucleotides were first mde synthetically by Khorana et al. by means of a diester synthesis, J. Molec.

Biol. 72:251 (1972). Narang et al., J. Biological Chemistry 250:4592 (1975), expanded this procedure with the development of a triester synthesis. These early methods were difficult and highly time consuming. Much time was necessary to successfully join nucleotides together, to prepare reagents for condensation and to purify the products following each condensation step, and to isolate and purify the final nucleotide sequence.

Letsinger, et al., Am. Chem. Society 97:3278-79 (1975), improved the methods for synthesizing oligonucleotides with the disclosure of the "phosphite triester" procedure. This phosphite triester approach later was extended to solid-phase synthesis of oligonucleotides. Solid phase, or "polymer support," methods of synthesis are advantageous in that they facilitate separation and purification steps. Silica gel, a rigid, non-swellable colloid frequently is used as the support. In the solid phase procedure a protected nucleoside is reacted with a phosphitylating agent such as methoxydichlorophosphine. The resulting nucleoside phosphomonochloridite is then reacted with a second protected nucleoside attached to a support. Subsequent mild oxidation using iodine in tetrahydrofuran, lutidine and water converts the phosphite to a phosphate.Although this process is much faster than those that proceded it, a considerable amount of the 3'-3' isomer is formed, even when the reaction is carried out at -780C and the ratio of nuceloside to phosphitylating agent is carefully controlled. Another drawback was the instability of the reactive intermediates to hydrolysis and oxidation by the air.

Caruthers et al., J. Am. Chem. Soc. 103: 3185-3191 (1981), modified this procedure by introducing a "tetrazolide" derivative of nucleoside phosphite. This intermediate, however, is not very stable and preparation of the derivative introduces an additional step to be carried out at -780C.

Caruthers et al., Tetrahedron Letters 22, 1859-62(1981), also modified the basic process such that methoxydichlorophosphine, the phosphitylating agent, is converted to an N,N'-dialkylamino derivative which produces a fairly stable "nucleoside phosphite" when treated with a nucleoside. The product then is treated with silica gel carrying a nucleoside in the presence of tetrazole. Although this procedure provides an intermediate with greater stability than before, this advantage is offset by the time consuming method of preparation of the starting material.

Thus, there is a continuing need for a procedure for synthesizing an oligonucleotide on a solid support that is relatively simple and easy to carry out and which avoids the problems described above. It herefore is an object of the invention to develop a method for the solid-phase synthesis of oligonucleotides which can be carried out readily and economically and yields a stable product.

Summary of the invention It has been discovered that oligonucleotides can be synthesized on a solid support easily by a method which involves treating the solid support with a phosphitylating agent followed by treatment with the nucleoside. This is in contrast to the prevailing methods which involve treating the nucleoside with the phosphitylating agent followed by treatment with the support.

This invention relates to a novel method for synthesizing oligonucleotides on a solid-phase support, principally silica gel. A nucleoside is coupled to the solid-phase support at its 3'-hydroxyl group. The 5'-hydroxyl group of the nucleoside is protected by a protecting group. The protecting group is removed, and the free 5'-hydroxyl reacts with a bifunctional phosphitylating agent. The support then is treated with a protected nucleoside. The phosphite groups are oxidized to phosphates, and the 5'-hydroxyl protecting group is removed from the second nucleoside. Chain extension then is carried out by the same cycle of reactions: treatment with phosphitylating agent, treatment with another nucleoside, and oxidation. At the end of the synthesis the oligonucleotide is cleaved from the support, deblocked, and purified.This basic procedure can be followed in the synthesis of both deoxyribooligonucleotides and ribooligonucleotides; however, in the synthesis of ribooligonucleotides both the 5'-and the 2'-hydroxyl groups are protected by a protecting group.

With this procedure, oligonucleotides can be synthesized at moderate temperatures. The synthesis can take place in a single vessel and is easily adaptable for automation. In addition, a large excess of reacting materials can be used and recovered easily after the reaction.

Detailed description of the invention In accordance with the present invention, oligonucleotides can be synthesized on a solid-phase support.

As used herein, the term oligonucleotide is intended to include both ribooligonucleotides and deoxyribooligonucleotides.

In the first steps of the process of the present invention, a nucleoside is coupled to a solid support. As a general rule, a solid support which allows molecules to freely diffuse into it, and does not irreversibly absorb reagents can be used as a support in the process of this invention. A preferred support is silica gel. The coupling advantageously occurs between the 3'-hydroxyl group of the nucleoside and the support. To ensure that the coupling occurs at this site on the nucleoside, the other hydroxy group(s) of the nucleoside (the 5'-hydroxyl group of deoxyribonucleoside and the 2'-and 5'-hydroxyl groups of a ribonucleoside) is protected by a protecting group. Suggested protecting groups for the 5-hydroxyl group are monomethoxytrityl or dimethoxytrityl; the dimethoxytrityl being preferred.Suggested protecting groups for the 2'-hydroxyl group of a ribonucleoside are tetrahydroxlpyran, o-nitrobenzyl, and tert-butyidimethyisilyl (Ohtsuka, E. etal.mJ.Am. Chem. Soc., 100:821 (1978); Ogilvie, K., et al., Can J. Chem. 57:2230 (1979)).

The first nucleoside can be coupled to the support in accordance with conventional procedures. See, for example, Matteuci and Caruthers,J.Am. Chem. Soc, 103:3185(1981); and Chow, F.,etal. TheAcidsRes.

9:2807 (1981). After the nucleoside has been coupled to the solid support the group protecting the 5'-hydroxyl group is removed. If the nucleoside is a ribonucleoside the group protecting the 2'-hydroxyl group advantageously is removed only after the synthesis of the oligonucleotide is completed, prior to cleaving the oligonucleotide form the support. The nucleoside is treated with a bifunctional phosphitylating agent to form a nucleoside phosphomonochloridite attached to the support. Any alkyl or aryl phosphodichloridite can be used as the phosphitylating agent, methoxydichlorophosphine being preferred.

This bifunctional phosphitylating agent can be added to the nucleoside directly, or it first can be modified by converting it to a tetrazole derivative. Modifying the phosphitylating agent in this way can prevent the possible formation of 5'-5' crosslinking between two adjacent oligonucleotide chains. Such cross-linking is not believed to be a serious problem when the incorporation of nucleosides on the solid support is moderately small (approximately 30 u mole/g.), but may become significant when the incorporaton of nucleosides becomes larger. The conversion of the phosphitylating agent can be accomplished either by reacting it directly with the tetrazole before it is added to the nucleoside or by adding the tetrazole to the nucleoside prior to treating the nucleoside with the phosphitylating agent.

The molar ratio of nucleoside to phosphitylating agent generally is about 1:10 to about 1 :20, preferably about 1:15. The reaction mixture may be quickly shaken and centrifuged and the excess phosphodichloridite removed. The total reaction time generally ranges from about 2 to about 10 minutes.

When the phosphitylating agent is modified with tetrazole, the molar ratio of phosphitylating agent to tetrazole generally is from about 1:2 to about 1 :10, preferably about 1:3. Reaction conditions for this embodiment of the present invention generally include a total reaction time of about 15 minutes at a temperature of about 0 C.

Following treatment with the phosphitylating agent the support is reacted with an excess of a second nucleoside. The reaction is generally allowed to proceed for about 10 to 15 minutes at about 27on. Following the reaction with the second nucleoside, the internucleotide phosphite bonds are oxidized to phosphates in accordance with conventional techniques, as, for example, with iodine in an aqueous solution of tetrahydrofuran and lutidine. A preferred ratio of tetrahydrofuran to lutidine to water is about 2:1:1.

The protecting group of the second nucleoside is then removed. The dimer may be cleaved from the support, deblocked and purified if desired. The yield of this reaction typically is about 95%.

Chain extension may be carried out following the same cycle of reactions outlined above; treatment of nucleoside with phosphitylating agent, treatment with another nucleoside, and oxidation of the phosphite groups. At the end of the synthesis the oligonucleotide advantageously is cleaved from the support, deblocked and purified.

The intermediate centrifugation step to remove excess phosphitylating agent may introduce moisture into the reaction mixture. It has been discovered that centrifuging the reaction mixture containing the silica and the phosphitylating agent is not necessary to assure high yeilds of the desired final oligonucleotide. The procedure may be carried out as outlined above, omitting the centrifugation step. Then, after the oligonucleotide has been oxidized by a conventional oxidizing technique, the support can be washed thoroughly several times with organic solvents. In a preferred embodiment, the support is washed with 50% aqueoustetrahydrofuran followed by tetrahydrofuran and finally with ether. As before, the yield of the desired dimer is about 90 to 100%.These high yields indicate that even if an excess of the phosphitylating agent is present the desired 5'-3' coupling can be achieved by using an excess of nucleoside. Furthermore, thin layer chromatography (TLC) analysis of the supernatant from the reaction mixtures suggests that at least about 80% of the nucleoside added remains intact and thus can be recovered and reused if desired.

In an alternative embodiment, rather than synthesizing an oligonucleotide by adding one nucleoside at a time to a growing chain, the chain extension also can be done using suitably protected nucleotide blocks.

As indicated above, there are a number of advantages to this procedure in view of those taught by the prior art. The procedure does not require any preformed starting materials, can be carried out at temperatures much more moderate than previously has been possible and can take place in a single vessel.

It is easily adaptable for automation. Both ribooligonucleotides and deoxyribooligonucleotides can be synthesized by this method. Finally, excess nucleoside can be recovered once the reaction has been run making this method very economical.

The following examples illustrate, but are not intended to limit, the process of this invention.

EXAMPLE I Coupling of first nucleoside to support A. Synthesis of the support Silica gel (29, Vydac TP-20) was treated with triethoxy silyl propylamine (3ml) and toluene (1 Oml) and the mixture was refluxed for 7 hours. After cooling the reaction mixture, silica was filtered and washed with pyridine (3x20ml). Trimethylsilycylchloride (2.2ml) was then added and the reaction proceeded for 4 hours at room temperature. The silica was filtered, washed with pyridine (3 x 10ml) and ethyl ether (3 x 10ml) and dried in a desiccator. The silica was then ready for reaction with 5'-0-dimethoxytrityi-2'-deoxy nuceloside-3'0-succinic acids which were prepared as follows: B. Preparation of 5'-0-Dimethoxytrityl-2'-Deoxy Nucleoside-3'-0-Succinic Acids.

The 5'-0-dimethoxytrityl-2'-deoxynucleoside (2 mmole) and N,N-dimethylamino pyridine (2 mmole) were dissolved in pyridine (50ml) and succinic anhydride (2.1 mmole) was added slowly at room temperature.

After 48 hours reaction, water (3 ml) was added and the pyridine was removed by carefully coevaporating with toluene. The result was dissolved in dichloromethane (200 ml) and extracted with an aqueous solution (100 ml) of citric acid (4 mmole). The solution was dried over sodium sulfate, concentrated and the residue was dissolved in dichloromethane (50 ml) and precipitated from hexane (300 ml).

C. Reaction of 5'-O-Dimethoxytrityl-2'-Deoxy Nucleoside-3'-0-Succinic Acid with Amino-Propyl Silica Aminopropylsilica (1 g) in pyridine (2 ml) was treated with triethylamine (0.17 g) for a few hours and the excess triethylamine was carefully removed by evaporation with pyridine. To the silica was added deoxynucleoside-3'-0-succinic acid (1 mmole) in pyridine (20 ml) and dicyclohexylcarbodiimide (7 mmole, 1.5 g). The mixture was shaken at room temperature for tw days. The silica was then filtered, washed with pyridine (50 ml) and reacted with benzoylcyanide (17 mmole, 2g) in 8 ml pyridine for 3 hours. The silica was again filtered, washed with pyridine (30 ml) and ethylether (40 ml) and dried.The incorporation of nucleoside was determined by breaking a known quantity of silica with 2 y benzene sulfonic acid in acetonitrile and measuring the absorption at 498 nm.

This procedure was used as the first step in each of the following examples to attach a first nucleoside to a silica support.

EXAMPLE II Preparation of dimer by direct treatment with phophitylating agent Silica (Vydak TP, 100 mg) carrying deoxythymidine (4 umole) was treated with methoxydichlorophosphine (60 umole) in anhydrous pyridine (0.2 ml) at room temperature. The reaction mixture was quickly shaken and centrifuged (the total reaction time including centrifugation was 2 minutes at room temperature). The supernatant was removed and a 10 fold excess of 5'-dimethoxytritylcytidine was added in pyridine (0.2 ml) to the silica. After 10' at room temperature, the reaction mixture was oxidized with iodine in tetrahydrofuran/lutidine/water (2:1:1) mixture. The yield of the reaction as estimated by removal of trityl group and the HPLC after deblocking was found to be 95%.

EXAMPLE ill Preparation of dimer by direct treatment with phosphitylating agent Silica (Vydak TP, 100 mg) carrying deoxythymidine (4 umole) was treated with methoxydichlorophosphine (60 umole) in pyridine (0.2 ml) for 10' at 0 C or 1-2 minutes at room temperature. 5'-dimethoxytritylcytidine (200 umole) in pyridine (0.2 to 0.25 ml) was then added to the reaction mixture without centrifugation. After 10' at room temperature, oxidation with iodine in tetrahydrofuran/lutidine/water (2:1:1) was carried out. The silica was then washed thoroughly with 50% aqueous tetrahydrofuran, tetrahydrofuran and finally with ether. The yield was estimated to be 95% by the absorption of trityl cation at 498 nm.The yield was also confirmed by reverse phase HPLC analysis after deblocking. (12-16 hours at 50"C with concentrated ammonium hydroxide followed by 80% acetic acid treatment.) The dimer peak was collected during HPLC analysis and the UV spectrum was found to agree very well with the computer generated spectrum.

EXAMPLE IV Other dimers were prepared by the method of Example II. The isolated yield of each dimer is provided in the following table: TABLE 1 Isolated Yield Compound (o) d-CpC 96 d-GpT 87 d-CpT 95 d-GpG 98 d-CpG 84 d-GpA 81 EXAMPLE V Synthesis of C-T dimer using modified bifunctional phosphitylating agent Silica (Vydac TP, 100 mg), carrying N-benzoyldeoxycytidine (4.2 umole) and tetrazole (630 umole) were azeotroped with pyridine. The silica and the tetrazole were treated with methoxydichlorophosphine (63 umole) in pyridine (0.3 moi) for 10 minutes at 0 C. Then 5'-dimethoxytrityl thymidine (210 umole) was added to the reaction mixture.After 15 minutes at room temperature, the reaction mixture was oxidized with iodine in tetrahydrofuran/lutidine/water (2:1:1). The silica was thoroughly washed with 50% aqueous tetrahydrofuran, tetrahydrofuran, and, finally, ether. The yield was estimated to be 98% by the absorption of the trityl cations at 498 nm.

EXAMPLE VI Synthesis ofpentamer using modifiedphosphitylating agent Silica (Vydac 100 mg) carrying N-benzoyldeoxycytidine (4.2 umole) and tetrazole (630 umole) were azeotroped with pyridine. The silica and the tetrazole then were treated with 15 vold methoxydichlorophosphine (63 umole) in pyridine (0.3 ml) for 10 minutes at 0 C. Fifty-fold 5'-dimethoxytritylthymidine in pyridine (0.3 ml) was added to the reaction mixture. After 15 minutes at room temperature, oxidation with iodine in tetrahydrofuran/lutidine/water (2:1:1) was carried out. The silica was then washed as in Example IV. The trityl group was removed by treatment with 5% trichloroacetic acid in chloroform. The silica was thoroughly washed with chloroform.

This cycle was repeated to extend the chain to the pentamer sequence T-T-T-T-C. The ratio for reagents was left constant during the entire synthesis; the volume of pyridine was adjusted as necessary.

The overall yield for the synthesis of the T-T-T-T-C pentamer was 53%.

EXAMPLE VII The octamer C-A-A-G-C-T-A-G was synthesized by the method of Example V with an overall yield of 24%.

EXAMPLE VIII Silica (Vydac TP, 100 mg) carrying N-isobutyldeoxyguanosine (2.9 umole) and 45 fold tetrazole (130 umole) were azeotroped with pyridine. Anhydrous pyridine (0.1 mL) was added to the silica-tetrazole mixture befofe the silica was treated with 15 fold methoxydichlorophosphine (43 umole) for 10' at 0 C.

5'-dimethoxytrityl -N-benzoyldeoxyadenosine in 0.2 mL pyridine was added. After 15' at room temperature oxidation with iodine in tetrahydrofuranilutidine/water (2:1:1) was carried out. The silica then was washed with 50% aqueous tetrahydrofuran, tetahydrofuran, and ether. The yield was estimated to be about 100% based on the trityl absorption of the trityl cation at 498 nm.

EXAMPLE IX Silica (Vydac TP, 100 mg) carrying N-isobutyryldeoxyguanosine (2.9 umole) was treated with 0.2 ml of pyridine containing methoxydichlorophosphine (43.5 umole) and tetrazole (130 micromole) for 15' at 0 C, 5'-Dimethoxytrityl-N-benzoyladenosine in 0.2 ml was added to the reaction mixture. After 15' at room temperature, the reaction mixture was oxidized and washed as per the previous examples. The yield was estimated to be about 100%.

EXAMPLE X Synthesis ofA-T-G-CA-C-A-C-A-A-G-A-G Silica (Vydak TP, 500 mg) carrying N-isobutyryldeoxyguanosine (14.5 umole) was dried by washing with anhydrous pyridine (2x10 ml) and anhydrous diethyl ether (10 ml). The silica was further dried in a vacuum dessicator until used.

A 45-fold excess of tetrazole (652 umole) was dissolved in 1 ml pyridine at 0 C. A 15-fold excess of methoxydichlorophosphine (217 umole) was added to the tetrazole-pyridine solution, then the solution was added to the silica. After 15 minutes at 0 C, a 50 fold excess of 5'-0- (dimethoxytrityl)-N benzoyldeozyadenosine in 1.0 ml of pyridine was added. The reaction continued for 15 minutes at room temperature, then the reaction mixture was oxidized and washed per the method of the previous examples.

The silica was treated with 5% trichloroacetic acid in chloroform to remove the dimethoxytrityl group. After washing with chloroform, an hydrous pyridine and anhydrous ether, the solution was ready for the addition of the next nucleoside.

The remaining 11 nucleosides were added exactly as indicated above. The amount and concentration of the reagents remained the same during the entire synthesis.

A qualitative estimate of the yield was obtained after each addition based upon the orange color resulting from the 5% trichloroacetic acid treatment. In all cases the yield appeared to be excellent.

Quantitative estimates of the yield were obtained by treating an an accurately weighed amount of silica with 2% benzenesulfonic acid in acetonitrile and observing the absorptions of the trityl cations at 498 nm.

The overall yield at several stages are given below: Length of oligonucleotide Yield (%) 2 100 5 90 9 78-83 13 60-70

Claims (23)

1. A method for synthesizing a deoxyribooligonucleotide on a solid support comprising the steps of: (a) attaching a first nucleoside, the 5'-hydroxyl group of which is protected by a protecting group, to a solid phase support through the 3'-hydroxyl group of said first nucleoside, (b) removing said protecting group on the 5'-hydroxyl of said first nucleoside, (c) reacting said 5'-hydroxyl group with a bifunctional phosphitylating group, (d) attaching a subsequent nucleoside, the 5'-hydroxyl of which is protected by a protecting group, to said support by coupling the 3'-hydroxyl of said subsequent nucleoside go the 5'-hydroxyl group of said first nucleosode, (e) oxidizing the phosphite bond formed between said nucleosides, and (f) repeating steps b) through e) above until the desired number of nucleosides have been added to said support.

2. A method for synthesizing a ribooligonucleotide on a solid support comprising the steps of: (a) attaching a first ribonucleoside, the 2'-hydroxyl group and the 5'-hydroxyl group of which are each protected by a protecting group, to a solid phase support through the 3'-hydroxyl group of said first nucleoside, (b) removing said protecting group on the 5'-hydroxyl of said first nucleoside, (c) reacting said 5'-hydroxyl group with a bifunctional phosphitylating group, (d) attaching a subsequent ribonucleoside, the 2'-hydroxyl group and the 5'-hydroxyl of which are each protected by a protecting group, to said spport by coupling the 3'-hydroxyl of said subsequent nucleoside to the 5'-hydroxyl group of said first nucleoside, (e) oxidizing the phosphite bond formed between said nucleosides, (f) repeating steps b) through e) above until the desired number of nucleosides have been added to said support and (g) removing the protecting groups from the 2'-hydroxyl groups of the ribooligonucleotide,

3. A method according to claim 1 or 2 comprising the additional steps of (a) cleaving said oligonucleotide from said support, and (b) deblocking and purifying said oligonucleotide.

4. A method according to claims 1 or 2 wherein said bifunctional phosphitylating agent of step (c) is converted to a derivative of tetrazole before reactinvg with said 5'-hydroxyl of said nucleoside.

5. A method according to claims 1 or 2 wherein said nucleoside is mixed with tetrazole before being treated with said bifunctional phosphitylating agent.

6. A method according to claim 1 or 2 wherein said solid support is silica gel.

7. A method according to claim 4 wherein said solid support is silica gel.

8. A method according to claim 1 or 2 wherein the protecting group for said 5'-hydroxyl group is monomethyltrityl or dimethoxytrityl.

9. A method according to claim 2 wherein the protecting group for said 2'-hydroxyl is selected from the group consisting of tetrahydrylpyran, o-nitrobenzyl, and tert-butyldimethylsilyl.

10. A method according to claims 1 or 2 wherein said bifunctional phosphitylating agent is an alkyl or aryl phosphodichloridite.

11. A method according to claim 1 or 2 wherein said bifunctional phosphitylating agent is methoxydichlorophosphine.

12. A method according to claim 1 or 2 wherein the molar ratio of nucleoside to phosphitylating agent ranges from about 1:10 to about 1:20.

13. A method according to claim 1 or 2 wherein the molar ratio of nucleoside to phosphitylating agent is about 1:15.

14. A method according to claim 4 wherein the molar ratio of phosphitylating agent to tetrazole ranges from about 1:2 to about 1:10.

15. A method according to claim 5 wherein the molar ratio of phosphitylating agent to tetrazole ranges from about 1:2 to about 1:10.

16. A method according to claim 4 wherein the molar ratio of phosphitylating agent to tetrazole is about 1:3.

17. A method according to claim 5 wherein the molar ratio of phosphitylating agent to tetrazole is about 1:3.

18. A method according to claim 1 to 2 wherein the reactions are carried out at about 0 C. to about 27"C.

19. A method according to claim 1 or 2 wherein the reactions are carried out at OOC.

20. A method according to claim 1 or 2 wherein excess nucleoside can be recovered by extraction procedures and reused.

21. A modification of the method as claimed in any of the preceding claims in which a suitably protected nucleotide is used in place of said first nucleoside and/or said subsequent nucleoside.

22. A method according to any of the previous claims substantially as described herein with reference to the Examples.

23. An oligonucleotide whenever produced by a method according to any of the preceding claims.