Linkers for the synthesis of multiple oligonucleotides in seriatim from a single support attachment

• US 5,393,877 A
• Filed: 04/05/1993
• Issued: 02/28/1995
• Est. Priority Date: 04/03/1992
• Status: Expired due to Fees

• Current Assignee: Nitto Denko Avecia, Inc. (Nitto Denko Corporation)
• Original Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Inventors: Holland, David, Sheppard, Robert C., Garman, Andrew J., McLean, Michael J.
• Primary Examiner(s): Rollins, John W.
• Assistant Examiner(s): Crane, L. Eric
• Application Number:
  US08/041,599
• Time in Patent Office: 694 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.33, 536/25.34, 558/199
• US Class Current: 536/25.3
• CPC Class Codes: C07F 9/65515 the oxygen atom being part of a five-membered ring
• 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

Abstract

A method for the synthesis of a plurality of oligonucleotides comprising the steps of

(i) forming a first oligonucleotide on a first cleavable link attached to a solid support;

(ii) attaching to the first oligonucleotide a cleavable linker moiety;

(iii) forming a second oligonucleotide on the cleavable linker moiety; and

(iv) cleaving the first cleavable link and the cleavable linker moiety to give a plurality of oligonucleotides; wherein the cleavable linker moiety is of the Formula (1): ##STR1## in which A¹, A² and E are as defined herein, and novel compounds which may be used in the operation of the method.

Claims

1. A method for the synthesis of a plurality of oligonucleotides comprising the steps of

(i) forming a first oligonucleotide on a first cleavable link attached to a solid support;

(ii) attaching to the first oligonucleotide a cleavable linker moiety;

(iii) forming a second oligonucleotide on the cleavable linker moiety; and

(iv) cleaving the first cleavable link and the cleavable linker moiety to give a plurality of oligonucleotides;

wherein the cleavable linker moiety is of the Formula (1);

##STR35## wherein one or both of A¹ and A² is a divalent group of the formula (a);

##STR36## R¹ and R² are each independently H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryloxy, halogen, cyano, nitro, optionally protected hydroxy, optionally protected oxycarbonyl, optionally protected NH₂ or an electron withdrawing group;

Y is CH₂, CH₂ CH₂, NH, S or O;

E is an organic spacer group; and

any remaining group represented by A¹ or A² is of the formula (b);

##STR37## wherein n has a value of from 1 to 5;

the carbon atom marked with an asterisk is attached to an oxygen atom shown in Formula (1); and

each R³ independently represents H or optionally substituted alkyl.

2. A method according to claim 1 wherein cleavage of the cleavable linker moiety results in a plurality of oligonucleotides each having at the 3'"'"' and 5'"'"' position a group selected from hydroxy and phosphate.

3. A method according to claim 1 or 2 wherein cleavage of the cleavable linker moiety results in a plurality of oligonucleotides each having a hydroxy at the 3'"'"' position.

4. A method according to claim 1 wherein steps (i) to (iii) are carried out using an automated oligonucleotide synthesizer and step (iv) is carried out under basic conditions at a temperature from 20°

to 100°

C. for up to 48 hours.

5. A method according to claim 1 wherein the cleavable linker moiety of formula (1) is derived from a compound of formula (3):

##STR38## wherein one or both of A¹ and A² is a divalent group of the formula (a);

##STR39## in which R¹ and R² are each independently H, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryloxy, or an electron withdrawing group;

Y is CH₂ ;

CH₂ CH₂, NH, S or O;

E is an organic spacer group; and

any remaining group represented by A¹ or A² is of the formula (b);

##STR40## wherein;

n has a value of from 1 to 5;

the carbon atom marked with an asterisk is attached to an oxygen atom shown in Formula (3); and

each R³ independently represents H or optionally substituted alkyl;

Z¹ is an acid labile protecting group; and

--O--PA is a phosphoramidite group, a phosphate ester group or a H-phosphonate group.

6. A method according to claim 5 wherein A¹ and A² are both of formula (a).

7. A method according to claim 6 wherein A¹ is the group of formula (a) and A² is the group of formula (b).

8. A method according to claim 7 wherein Y is O or CH₂.

9. A method according to claim 8 wherein E is an alkyl, aryl or aralkyl spacer group, optionally interrupted by an ether, thioether, amino or amido group.

10. A method according to claim 9 wherein E is optionally substituted phenylene or C_2-6 -alkylene.

11. A method according to claim 10 wherein R¹ and R² are each independently H, alkyl or alkoxy.

12. A method according to claim 11 wherein R³ is independently C_1-4 -alkyl or H.

13. A method according to claim 12 wherein Z¹ is dimethoxytrityl.

14. A method according to claim 13 wherein O--PA is a phosphoramidite of formula ##STR41## wherein R₅ and R₆ are each independently optionally subsituted alkyl, optionally substituted aralkyl, cycloalkyl and cycloalkylalkyl containing up to 10 carbon atoms, or taken togther with the nitrogen atom to which they are attached form an optionally substituted pyrrolidine or piperidine ring or R₅ and R₆ when taken together with the nitrogen atom to which they are attached form a saturated nitrogen heterocycle which optionally includes one or more additional hetero atom(s) from the group consisting of nitrogen, oxygen and sulphur;

and R₇ represents a hydrogen or a protecting group.

15. A method according to claim 14 wherein the group of formula (a) is where Y is O and R¹ and R² are both either hydrogen or methoxy;

and the group of formula (b) is --*CH₂ CH₂ --SO₂ --CH₂ --CH₂.

16. A method according to claim 15 wherein A¹ and and A² are both of formula (a), Y is O, R¹ and R² are both hydrogen or methoxy, E is --CH₂ CH₂ --, Z¹ is dimethoxytrityl, and O--PA is a phosphoramidite of formula ##STR42## wherein R₅ and R₆ are each iso-propyl and R₇ is 2-cyanoethyl.

17. A method according to claim 16 wherein A¹ is of formula (a) in which Y is O and R¹ and R² are both hydrogen or methoxy and A² is --*CH₂ CH₂ --SO₂ --CH₂ --CH₂ --, Z¹ is dimethoxytrityl, and O--PA is a phosphoramidite of formula ##STR43## wherein R⁵ and R⁶ are both iso-propyl and R is 2-cyanoethyl.