Orthoester protecting groups in RNA synthesis

• US 5,889,136 A
• Filed: 06/09/1995
• Issued: 03/30/1999
• Est. Priority Date: 06/09/1995
• Status: Expired due to Term

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Scaringe, Stephen, Caruthers, Marvin H.
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/488,878
• Time in Patent Office: 1,390 Days
• Field of Search: 536/25.34, 536/26.1, 536/25.3
• US Class Current: 536/25.34
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Phosphoramidite oligonucleotide synthesis is facilitated by the use of fluoride-labile 5'"'"' silyl protecting groups. RNA synthesis is improved by the use of 2'"'"' orthoester protecting groups. Reactions are conducted on a solid phase support, and acidic deprotection conditions are avoided, as is the necessity of oxidizing the phosphite linkage between each coupling reaction.

Claims

1. A protected monomer for use in stepwise oligonucleotide synthesis having a molecular formula comprising:

##STR1## wherein R'"'"' is an orthoester protecting group, H, ethers, alkyl ethers, esters, halogens, protected amines, and protected hydroxyl moieties;

R" comprises a phosphorous moiety;

R₁, R₂, and R₃ include at least one alkoxy or siloxy substituent; and

BASE is a nucleic acid base.

2. The monomer as set forth in claim 1, wherein said phosphorous moiety is a phosphoramidite moiety.

3. The monomer of claim 2, wherein said phosphoramidite moiety has a formula ##STR2## wherein R₄ is any organic ligand.

4. The monomer as of claim 1, wherein said R₁, R₂, and R₃ are any combination of the following formulas:

##STR3##

5. The monomer of claim 1, wherein said orthoester protecting group is a noncyclic orthoester.

6. The monomer of claim 5, wherein said orthoester protecting group has a formula ##STR4##

7. The monomer of claim 6, wherein said R and R₁, or wherein R equals R₁ are any combination of the following structures:

##STR5## wherein X₁, X₂, X₃, X₄, and X₅, is a compatible ligand, or hydrogen, or halogen, alkyl, or cyano substituent; and

R₅ is a compatible ligand.

8. The monomer of claim 1, wherein said BASE comprises adenine, guanine, cytosine, thymine, uracil, N-protected adenine, guanine, cytosine, and functional homologues thereof.

9. A method for site-specific stepwise olionucleotide synthesis comprising the steps of:

preparing a first monomer chain including at least one substituted monomer having a first silyl protecting group and a first phosphorous moiety;

deprotecting said one substituted monomer by adding a fluoride ion source to remove said first silyl protecting group thereby yielding a deprotected chain;

providing a second substituted monomer having a second silyl protecting group and a second phosphorous moiety at specific sites on said second substituted monomer; and

reacting said second substituted monomer with said deprotected chain to yield an elongated monomer chain.

10. The method of claim 9, wherein said deprotecting step takes place in neutral to basic conditions.

11. The method of claim 9, wherein said reacting step includes converting said first phosphorous moiety into a phosphitetriester linkage by reaction of said deprotected monomer with said phosphorous moiety for elongation of said first chain.

12. The method of claim 11, wherein said one substituted monomer and said second substituted monomer are respectively coupled to corresponding bases independently selected from a group comprising adenine, guanine, cytosine, thymine, uracil, and functional homologues thereof.

13. The method of claim 11, wherein said second substituted monomer includes a second phosphorous moiety, thereby becoming said one substituted monomer in said first monomer chain after elongation of said chain, and said method includes repeating said providing, deprotecting, reacting, and converting steps.

14. A method of synthesizing an oligonucleotide sequence, comprising the steps of:

coupling a first nucleotide to an insoluble non-glass support, wherein a neutral to base labile silyl group is positioned at the 5'"'"' position of said first nucleotide;

exposing said coupled nucleotide to a fluoride ion source thereby yielding a deprotected chain;

providing a second nucleotide protected with a neutral to base labile silyl group; and

reacting said deprotected chain with said second protected phosphoramidite nucleotide to form a chain two nucleotides in length having one phosphorous linkage.

15. The method of claim 14, wherein in said providing step said protected nucleotide has a formula ##STR6## wherein R'"'"' is an orthoester protecting group, H, ethers, alkyl ethers, halogens, protected amines, and protected hydroxyl moieties;

R" is a phosphormidite moiety;

R₁, R₂, and R₃ include at least one alkoxy or siloxy substituent; and

BASE is a nucleic acid base.

16. The method of claim 15, wherein R₁, R₂, and R₃, or wherein R₁ equals R₂, and R₃, or wherein R₁ equals R₃, and R₂, or wherein R₁ equals R₂ equals R₃, have the following structure:

##STR7##

17. The method as set forth in claim 15, wherein said R'"'"' is an orthoester.

18. The method as of claim 17, wherein said orthoester has a formula ##STR8## wherein R and R₁ are substituents having up to about twenty carbon atoms.

19. The method of claim 18, wherein said R and R₁, or wherein R equals R₁ are any combination of the following formulas:

##STR9## wherein X₁, X₂, X₃, X₄, and X₅, is a compatible ligand, hydrogen, halogen, alkyl, or cyano substituent; and

R₅ is a compatible ligand.

20. The method of claim 14 further comprising repeating said exposing, providing, and reacting steps through a plurality of cycles without oxidizing to stabilize said phosphorous linkages in said chain.

21. The method of claim 20, wherein said plurality of cycles exceeds five cycles.

22. The method of claim 14, wherein said insoluble non-glass support is a polystyrene bead.

23. A protected nucleoside monomer for use in a stepwise oligonucleotide polymerization reaction, wherein said protected nucleoside monomer has a plurality of protecting groups to facilitate said reaction and wherein at least one of said protecting groups is either a silyl group as shown below ##STR10## wherein at least one of substituents R1, R2 and R3 is alkoxy or siloxy;

or an orthoester.