NL8302921A - METHOD FOR THE SYNTHESIS OF AN OLIGONUCLEOTIDE ON A FIXED CARRIER. - Google Patents

Description

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Method for the synthesis of an oligonucleotide on a solid support.

The invention relates to a method for the synthesis of deoxyribodligonucleotides and riboligonucleotides on a solid support.

In recent years, scientists have developed methods for the synthesis of oligonucleotides with defined sequences, which can be used to manipulate DNA and RNA. For example, synthetic stretches of DNA of 10 to 14 units in size can be joined by specific enzymatic techniques to form whole genes or serve as probes to aid in identifying a desired DNA sequence. Synthetic oligonucleotide chains can act as linkers (link bridges) to aid in the joining of pieces of DNA or in the construction of recombinant DNA molecules.

Oligonucleotides were first synthesized by Khorana et al. By diester synthesis, J. Molec. Biol. 72: 251 (1972). Narang et al., J. Biological Chemistry 250: 4592 (1975), extend this procedure with the development of a triester synthesis. These older methods were difficult and time consuming. Much time was required to successfully pool nucleotides, to prepare condensation reagents and to purify the products after each condensation step, as well as to isolate and purify the final nucleotide sequence.

Letsinger, et al., J. am. Chem. Society 97: 3278-79 (1975) improved the synthesis methods for oligonucleotides by the description of the phosphite triester procedure. This phosphite triester approach was. later expanded to a solid-phase synthesis of oligonucleotides. Solid phase or polymeric carrier synthesis methods are preferred because they facilitate the separation and purification steps. Silica gel, a rigid, non-swellable colloid, is often 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 an Ö Ö J -α i i 2 support. Subsequent mild oxidation using iodine in tetrahydrofuran, lutidine and water converts the phosphite into a phosphate. Although this method is much faster than the older methods, a significant amount of the 3'-31 isomer is formed even when the reaction is conducted at -78 ° C and the ratio of nucleoside to phosphitylating agent is precisely controlled. Another drawback was the instability of the reactive intermediates to hydrolysis and air oxidation.

Caruthers et al., J. Am. Chem. Soc. 103: 3185 - 3191 10 (19811, modified this procedure by introducing a tetrazolid derivative of nucleoside phosphite. However, this intermediate is not very stable and preparation of the derivative introduces an additional step, which should be at -78 ° C are carried out.

Caruthers et al., Tetrahedron Letters 22, 1859 - 62 15 (1981), also modified the basic procedure to convert methoxydichlorophosphine, the phosphitylating agent, into a Ν, Ν'-dialkylamino derivative which, when treated with a nucleoside, gives quite stable nucleoside phosphite. The product is then treated with silica gel bearing a nucleoside in the presence of tetrazole. Although this procedure gives an intermediate with greater stability than before, this drawback is mitigated by the time-consuming preparation method of the starting material.

There is therefore a continuing need for a procedure for the synthesis of an oligonucleotide on a solid support, which procedure can be performed relatively simply and easily and which avoids the above described problems. The object of the present invention is therefore to develop a method for the solid-phase synthesis of oligonucleotides, which can be carried out easily and economically and gives a stable product.

It has been found that oligonucleotides can be easily synthesized on a solid support by a method in which the solid support is treated with a phosphitylating agent followed by treatment with the nucleoside. This is in contrast to the existing methods, in which the nucleoside is treated with the phosphine. Tylating agent, followed by treatment with the carrier.

The invention relates to a new method '8302921

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3 for the synthesis of oligonucleotides on a solid phase support, primarily silica gel. A nucleoside is coupled to the solid phase support with its 31-hydroxyl group. The 5'-hydroxyl group of the nucleoside is protected by a protecting group. The protection group.

5 is removed, and the free 5'-hydroxyl group reacts with a bifunctional phosphitilating agent. The support is then treated with a protected nucleoside. The phosphite groups are oxidized to phosphates, and the 5'-hydroxyl protecting group is removed from the second nucleoside. Then the chain is extended according to the same cycle of 10 reactions: treatment with phosphitilating agent, treatment with another nucleoside, and oxidation. At the end of the synthesis, the oligonucleotide is cleaved from the support, unblocked, and purified.

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

According to the present invention, oligonucleotides can be synthesized on a solid phase support. The term "oligonucleotide" as used herein includes both riboligonucleotides and deoxyribo-oligonucleotides.

In the first steps of the method of the present invention, a nucleoside is coupled to a solid support.

As a general rule, as a support in the present process, a solid support can be used, which allows molecules to diffuse freely therein, and which does not irreversibly absorb reagents. A preferred carrier is silica gel. The coupling takes place between the 2'-hydroxyl group of the nucleoside and the support; the 5'-hydroxyl group is protected by a protecting group such as a monomethoxytrityl-30 or dimethoxytrityl group, the dimethoxytrityl group being preferred. This protecting group is removed and the nucleoside is treated with a bifunctional phosphitilating agent to form a support bound nucleoside phosphomonochloridite. As the phosphitilating agent, alkyl or aryl phosphodichloridite can also be used, with methoxydichlorophosphine being preferred.

This bifunctional phosphitilating agent can be used directly on A. ·> “Ί - \ ^.!« C - ·; The nucleoside may be added, or may first be modified by converting it to a tetrazole derivative. Modification of the phosphilizing agent in this manner can prevent the possible formation of 5'-5 'cross-linking between two adjacent oligonucleotide chains. Such cross-linking is not considered to be a serious problem when the uptake of nucleosides on the solid support is moderately small (about 30 µmol / g), but can become significant as the uptake of nucleosides increases. The conversion of the phosphitilating agent can be carried out by reacting it directly with the tetrazole before adding it to the nucleoside or by adding the tetrazole to the nucleoside before treating the nucleoside with the phosphilizing agent.

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

When the phosphitilating agent is modified with tetrazole, the molar ratio of phosphitilating agent to tetrazole is generally about 1: 2 to about 1:10, preferably about 1: 3. The 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.

After treatment with the phosphitilating agent, the support is reacted with an excess of a second nucleoside.

The reaction is generally allowed to proceed at about 27 ° C for 10 to 15 minutes. After the reaction with the second nucleoside, the intemucleotide phosphite bands are oxidized to phosphates according to conventional techniques, such 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 protection group of the second nucleoside is then removed Λ. If desired, the dimer can be cleaved from the support, unblocked and purified. The yield of this reaction is typically about 95%.

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The chain can be extended according to the same cycle of reactions as described above: treatment of nucleoside with phosphitilating agent, treatment with another nucleoside, and oxidation of the phosphite groups. Preferably, at the end of the synthesis, the oligo-5 nucleotide is cleaved from the support, unblocked and purified.

The intermediate centrifugation step to remove excess phosphitizing agent can introduce moisture into the reaction mixture. It has been found that centrifugation of the reaction mixture containing the silicon oxide and the phosphilizing agent is not necessary to ensure high yields of the desired final oligonucleotide.

The procedure can be performed as described above, with the centrifugation step omitted. Then, after the oligonucleotide has been oxidized by a conventional oxidation technique, the support can be thoroughly washed several times with organic solvents. In a preferred embodiment, the support is washed with 50% aqueous tetrahydrofuran, followed by tetrahydrofuran and finally ether. As before, the yield of the desired dimer is about 90 to 100%. These high yields show that even when an excess of the phosphitilating 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 of the reaction mixtures shows that at least about 80% of the added nucleoside remains intact and therefore can be recovered and reused if desired.

As indicated above, this procedure has a number of advantages over the known procedures. The present procedure does not require preformed starting materials, can be performed at much more moderate temperatures than previously possible, and can be performed in a single vessel. It can be easily automated. Both riboligonucleotides and deoxyribodligonucleotides can be synthesized by this method. Finally, excess nucleoside can be recovered once the reaction has been carried out, making this method very economical.

The present method is further illustrated in the following examples, but not limited.

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Example I

Preparation of dimer by direct treatment with phosphitilating agent. 100 mg of silica (Vydak TP) bearing 4 µmole deoxythymidine was treated with 60 µmole methoxydichlorophosphine in 0.2 ml anhydrous pyridine at room temperature. The reaction mixture was shaken quickly and centrifuged (the total reaction time including centrifugation was 2 minutes at room temperature). The supernatant was removed and a tenfold excess of 51-dimethoxytritylcytidine in 0.2 ml of pyridine was added to the silica.

After 10 'at room temperature, the reaction mixture was oxidized with iodine in a tetrahydrofuran / lutidine / water (2: 1: 1) mixture. The reaction yield, estimated by removal of the trityl group and HPLC after deblocking, was found to be 95%.

Example II

Preparation of dimer by direct treatment with phosphitilating agent

100 mg of silica (Vydak.TP) bearing 4 µmole deoxythymidine was treated with 60 µmole methoxy dichlorophosphine in 0.2 ml pyridine for 10 'at 0 ° C or 1-2 minutes at room temperature. 200 µmol of 5'-dimethoxytritylcytidine in 0.2 to 0.25 ml of pyridine was then added to the reaction mixture without centrifugation. After 10 'at room temperature, an oxidation with iodine in tetrahydrofuran / lutidine / water (2: 1: 1) was performed. The silica was then thoroughly washed with 50% aqueous tetrahydrofuran, tetrahydrofuran and finally ether. The yield was estimated to be 95% due to the absorption of trityl cations 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 treatment with 80% acetic acid). The dimer peak was collected during the HPLC analysis and the UV spectrum appeared to correspond very well to the computer-made spectrum.

Example III

Other dimers were prepared by the method of Example II. The isolated yield of each dimer is shown in the following table: 35 fi ”Γ. - 9 1 V 'm' / ·. o '3 7

TABLE A

Compound Isolated yield,% d-CpC 96 d-GpT 87 d-CpT 95 5 d-GpG 98 d-CpG 84 d-GpA 81

Example IV

Synthesis of C-T dimer, using modified bifunctional 10-phosphitilating agent 100 mg Silicon oxide (Vydac TP), which bore 4.2 µmol N-ben-2oyldeoxycytidine and 630 µmol tetrazole, was azeotroped with pyridine. The silica and tetrazole were treated with 63 µmol methoxydichlorophosphine in 0.3 ml pyridine for 10 minutes at 0 ° C. 210 µmol of 51-dimethoxy-trityl-thymidine was then 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 washed thoroughly with 50% aqueous tetrahydrofuran, tetrahydrofuran, and finally ether. The yield was estimated to be 98% due to the absorption of the trityl cations at 498 nm.

Example V

Synthesis of pentamer, using modified phosphylating agent 100 mg of silicon oxide (Vydac TP), which carried 4.2 µmol N-benzoyl-deoxycytidine and 630 µmol tetrazole, was subjected to an azeotropic treatment with pyridine. The silica and the tetrazole were then treated with 15-fold (63 µmol) methoxydichlorophosphine in 0.3 ml pyridine for 10 minutes at 0 ° C. A 50-fold of 5'-dimethoxytritylthymidine in 0.3 ml of pyridine was added to the reaction mixture. After 15 minutes at room temperature, an oxidation with iodine in tetrahydrofuran / lutidine / water (2: 1: 1) was performed.

The silica was then washed as in Example IV. The trityl group was removed by treatment with 5% trichloroacetic acid 35 in chloroform. The silica was washed thoroughly with chloroform.

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This cycle was repeated to extend the chain to the pentamer sequence T-T-T-T-C. The ratio for the reagents was kept constant throughout the synthesis; the pyridine volume was adjusted as required.

5 The total yield for the synthesis of the T-T-T-T-C

pentamer was 53%.

Example VI

The octamer C-A-A-G-C-T-A-G was synthesized by the method of Example V with a total yield of 24%.

Example VII

100 mg of silicon oxide (Vydac TP), which carried 2.9 µmol N-iso-butyldeoxyguanosine and a 45-fold (130 µmol) tetrazole, were azeotroped with pyridine. 0.1 ml of anhydrous pyridine was added to the silica-tetrazole mixture 15 before the silica was treated with a 15-fold (43 µMol) methoxydichlorophosphine for 10 'at 0 ° C. 5'-Dimethoxytrityl-N-benzoyldeoxyadenosine in 0.2 ml pyridine was added. After 15 'at room temperature, an oxidation with iodine in tetrahydrofuran / lutidin-ne / water (2: 1: 1) was performed. The silica was then washed with 50% aqueous tetrahydrofuran, tetrahydrofuran, and ether. The yield was estimated to be about 100% based on the trityla absorption of the trityl cations at 498 nm.

Example VIII

100 mg of silicon oxide (Vydac TP), which bore 2.9 µmoles of N-isobutyryldeoxyguanosine, were treated for 15 minutes at 0 ° C with 0.2 ml of pyridine, containing 43.5 µmoles of methoxydichlorophosphine and 130 µmoles of tetrazole . 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 in the previous examples. The yield was estimated to be about 100%.

Example IX

Synthesis of A-T-G-C-A-C-A-C-A-A-G-A-G

500 mg of silica (Vydak TP), carrying 14.5 µmol N-isobutyryldeoxyguanosine, were dried by washing with 2 times 10 ml of anhydrous pyridine and 10 ml of anhydrous diethyl ether. The silica was further dried in a vacuum desiccator until used.

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A 45-fold excess of tetrazole (652 µmol) was dissolved in 1 ml of pyridine at 0 ° C. A 15-fold excess (217 µM) of methoxydichlorophosphine 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'-O- (dimethoxytriethyl) r-N-benzoyldeoxyadenosine in 1.0 ml pyridine was added. The reaction was continued at room temperature for 15 minutes, after which the reaction mixture was oxidized and washed according to 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, anhydrous pyridine and anhydrous ether, the solution was ready for the addition of the next nucleoside.

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

A qualitative yield estimate was obtained after each addition, based on the orange color resulting from the treatment with 5% trichloroacetic acid. In all cases, the yield turned out to be excellent.

Quantitative yield estimates were obtained by treating a carefully weighed amount of silica with 2% benzenesulfonic acid in acetonitrile and observing the absorptions of the trityl cations at 498 nm. The total yield at various times is given below: 25

Oligonucleotide Length Yield (%).

2 100 5 90 9 78-73 13 60 - 70 0 7 ^ ί

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Claims (15)

A method for the synthesis of an ologonucleotide on a solid support, wherein a) a first nucleoside, the 5 'hydroxyl group of which is protected by a protecting group, by the 3' hydroxyl group of the first nucleoside on a solid phase support is bound; b) removing the protecting group on the 5 * hydroxyl group of the first nucleoside; c) the 5'-hydroxyl group is reacted with a bifunctional phosphitylation group; D) a subsequent nucleoside, the 5'-hydroxyl group of which is protected by a protecting group, is bound to the support by coupling the 3'-hydroxyl group of this next nucleoside with the 51-hydroxyl group of the first nucleoside; d) the phosphite bond formed between the nucleosides is oxidized; f) steps b to e are repeated until the desired number of nucleosides have been added to the support; g) the oligonucleotide is cleaved from the support; and 20 h) the oligonucleotide is unblocked and purified. The method of claim 1, wherein the bifunctional phosphitylating agent of step c is converted to a tetrazole derivative before the reaction with the 51-hydroxyl group of the nucleoside is performed. The method of claim 1, wherein the nucleoside is mixed with tetrazole before it is treated with the bi-functional phosphitylating agent. The method of any one of claims 1 to 3, wherein the solid support is silicon oxide. A method according to any one of claims 1-3, wherein the protecting group is the monomethoxy-trityl group. The method of any one of claims 1-3, wherein the protecting group is dimethoxytrityl. 35 o "Z r O Γ · P 1 Si V ixf iilfl V * e π A method according to any one of claims 1 to 3, wherein the biotuctive phosphitylating agent is an alkyl or aryl phosphodichloride diet. 8. A method according to any one of claims 1-3, wherein the bifunctional phosphitylating agent is methoxydichlorophosphine. The method of any one of claims 1 to 3, wherein the nucleoside to phosphitylating agent molar ratio ranges from about 1r10 to about 1:20. 10. The method of any one of claims 1-3, wherein the molar ratio of nucleoside to phosphitylating agent is about 1:15. The method of claim 2 or 3, wherein the molar ratio of phosphitylating agent to tetrazole ranges from about 1: 2 to about 1:10. The method of claim 2 or 3, wherein the mole ratio of phosphitylating agent to tetrazole is about 1: 3. The method of any one of claims 1-3, wherein the reactions are conducted at about 0 to about 27 ° C. 14. A method according to any one of claims 1-3, wherein the reactions are performed at 0 ° C. A method according to any one of claims 1 to 3, wherein excess nucleoside can be recovered and reused by extraction procedures. -. i