Process for preparing polynucleotides

• US 5,132,418 A
• Filed: 06/18/1984
• Issued: 07/21/1992
• Est. Priority Date: 02/29/1980
• Status: Expired due to Fees

• Current Assignee: University Patents, Inc.
• Original Assignee: University Patents, Inc.
• Inventors: Matteucci, Mark D., Caruthers, Marvin H.
• Primary Examiner(s): Michl, Paul R.
• Assistant Examiner(s): McDonald, Thomas
• Application Number:
  US06/601,813
• Time in Patent Office: 2,955 Days
• Field of Search: 536/22, 536/23, 536/24, 536/25, 536/26, 536/27, 536/28, 536/29
• US Class Current: 536/25.3
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/0068 Means for controlling the apparatus of the process
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

This invention is a process for the production of oligonucleotides which comprises:

(a) converting an inorganic polymer into a coupling agent-polymer;

(b) combining the coupling agent-polymer with a blocked nucleoside to obtain a blocked nucleoside-modified support; and

(c) removing the blocking group from the blocked nucleoside-modified support to provide for the coupling of nucleotides to the nucleoside-modified support.

Claims

1. A process for the production of oligonucleotides which comprises:

(a) converting an inorganic polymer into a coupling agent-polymer;

(b) combining the coupling agent-polymer with a blocked nucleoside to obtain a blocked nucleoside-modified support; and

(c) removing the blocking group from the blocked nucleoside-modified support to provide for the coupling of nucleotides to the nucleoside-modified support.

2. The process according to claim 1, wherein the blocking group is dimethoxytrityl.

3. The process according to claim 1 wherein additional nucleotides are added to the nucleoside-modified support in the presence of a coupling agent, and any unreacted hydroxyl groups on the nucleotide on the modified support is blocked by a blocking group.

4. A process for the production of oligonucleotides which comprises:

(a) treating an inorganic polymer with a coupling agent which agent has a reactive group capable of reacting with a reactive group carried on a nucleoside; and

(b) combining the treated polymer with a nucleoside or a blocked nucleoside to obtain a nucleoside-modified support or a blocked nucleoside-modified support.

5. A process for the production of oligonucleotides which comprises:

(a) providing an inorganic polymer;

(b) converting the polymer into an amino-polymer;

(c) providing a nucleoside

(d) combining the amino-polymer and the nucleoside to obtain a nucleoside-modified support; and

(e) attaching nucleotides to the nucleoside-modified support.

6. The process of claim 5 wherein the nucleotides are added to the resulting support in the presence of a coupling agent.

7. The process of claim 5 wherein the step (e) is repeated to couple additional nucleotides to the already coupled nucleotides.

8. A process for the production of oligonucleotides which comprises:

(a) converting an inorganic silica into a coupling agent-silica,

(b) combining the coupling agent-silica with a blocked nucleoside to obtain a blocked nucleoside-modified support; and

(c) removing the blocking group from the blocked nucleoside-modified support to provide for the coupling of nucleotides to the nucleoside-modified support.

9. A process for the production of oligonucleotides which comprises:

(a) treating an inorganic silica with a coupling agent which agent has a reactive group capable of reacting with a reactive group carried on a nucleoside; and

(b) combining the treated silica with a nucleoside or a blocked nucleoside to obtain a nucleoside-modified support or a blocked nucleoside-modified support.

10. A process for the production of oligonucleotides which comprises:

(a) providing an inorganic silica;

(b) converting the silica gel into a coupling agent amino-silica;

(c) providing a nucleoside;

(d) combining the amino-silica and the nucleoside to obtain a nucleoside-modified support; and

(e) attaching nucleotides to the nucleoside-modified support.

11. A process for the production of oligonucleotides which comprises:

(a) converting a silica gel into a coupling agent-silica gel;

(b) combining the coupling agent-silica gel with a blocked nucleoside to obtain a blocked nucleoside-modified support; and

(c) removing the blocking group from the blocked nucleoside-modified support to provide for the coupling of nucleotides to the nucleoside-modified support.

12. A process for the production of oligonucleotides which comprises:

(a) treating a silica gel with a coupling agent which agent has a reactive group capable of reacting with a reactive group carried on a nucleoside;

(b) combining the treated silica gel with a nucleoside or a blocked nucleoside to obtain a nucleoside-modified support or a blocked nucleoside-modified support, and

(c) attaching nucleotides to the nucleoside-modified support.

13. A process for the production of oligonucleotides which comprises:

(a) providing a silica gel;

(b) converting the silica gel into a coupling agent amino-silica gel;

(c) providing a nucleoside;

(d) combining the amino-silica gel and the nucleoside to obtain a nucleoside-modified support; and

(e) attaching nucleotides to the nucleoside-modified support.

14. A process for the production of oligonucleotides which comprises:

(a) providing a silica gel support material;

(b) converting said silica gel to a derivatize silica gel containing aminoalkyl or hydroxyalkyl groups;

(c) providing a nucleoside modified to contain the half ester of a dicarboxylic acid; and

(d) combining the nucleoside ester with the derivatized silica gel, to obtain a nucleoside modified silica gel support.

15. A process according to claim 14 in which the derivative silica gel contains aminoalkyl groups.

16. A process according to claim 15 in which the dicarboxylic acid is succinic acid.

17. A process according to claim 15 in which the succinic anhydride is use in step (c).

18. A process for the production of oligonucleotides which comprises:

(a) providing a silica gel support material;

(b) converting the silica gel to a derivatized silica gel containing aminoalkyl groups or hydroxyalkyl groups;

(c) reacting the derivatized silica gel with a dicarboxylic acid to form an amine or ester linkage having carboxy functionality; and

(d) condensing the free carboxy groups on the silica with a free hydroxy group of a nucleoside, to obtain a nucleoside modified silica gel support.

19. A process according to claim 18 in which the derivatized silica contains aminoalkyl groups.

20. A process according to claim 18 in which the dicarboxylic acid is succinic acid.

21. A process for production of oligonucleotides which comprises the step of condensing the 3'"'"'-OH or 5'"'"'-OH group of a nucleoside or oligonucleotide covalently linked to an inorganic polymer by a coupling agent through the 5'"'"'-O-- or 3'"'"'-O--, respectively, of said nucleoside or oligonucleotide with a phosphite compound of the formula:

##STR5## wherein R is a blocking group;

B is a nucleoside or deoxynucleoside base;

A is H or OR;

R₁ is lower alkyl; and

X is formed by the removal of the hydrogen atom from a ring nitrogen of a nitrogen heterocyclic compound which contains unsaturated bonds in the ring structure.

22. The process according to claim 21 wherein the phosphite compound is of the formula:

##STR6## wherein substituents R, R₁, X, A and B are previously defined and said condensation occurs through the 5'"'"'-OH of said nucleoside or oligonucleotide.

23. The process according to claim 21 in which the heterocyclic compound is triazole.

24. The process according to claim 21 in which the heterocyclic compound is tetrazole.

25. A method of creating polynucleotides including the following steps:

providing a resin support;

providing an activated ester of a nucleoside; and

combining the activated ester of the nucleoside and the resin support to obtain a resin-supported nucleoside.

26. In a method of producing a polynucleotide, the steps of:

providing a resin-supported nucleoside;

combining a monotrizolide and the resin-supported nucleoside in the presence of a nucleophilic catalyst to provide a dimethoxytrityl resin; and

substituting the dimethoxytritly group in the dimethoxytrityl resin with a hydroxyl group to form the polynucleotide.