Substituted propargylethoxyamido nucleosides, oligonucleotides and methods for using same

• US 5,770,716 A
• Filed: 04/10/1997
• Issued: 06/23/1998
• Est. Priority Date: 04/10/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/833,855
• Time in Patent Office: 439 Days
• Field of Search: 514/45, 514/46, 514/47, 514/49, 514/50, 514/51, 536/22.1, 536/23.1, 536/24.33, 536/25.3, 536/25.32, 536/25.33, 536/25.34, 536/26.26, 536/26.6, 536/26.8, 536/27.14, 536/27.21, 536/27.6 , 536/27.8, 536/27.81, 536/28.1, 536/28.2, 536/28.5, 536/28.53, 536/28.54, 435/87, 435/88, 435/89, 435/91.1
• US Class Current: 536/23.1
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Substituted propargylethoxyamido nucleosides are disclosed having the structure ##STR1## wherein X is selected from the group consisting of amino alkanoic acid, alkylamino benzoic acid, α-amino acid, and 4-amino-2-butynoic acid. R₁ and R₂ taken separately are selected from the group consisting of --H, lower alkyl, protecting group, and label; R₃ is selected from the group consisting of --H and lower alkyl. B is a 7-deazapurine, purine, or pyrimidine nucleoside base. When B is purine or 7-deazapurine, the sugar moiety is attached at the N⁹ -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N¹ -position of the pyrimidine. When B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine. W₁ is selected from the group consisting of --H and --OH. W₂ is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'"'"'-position. W₃ is selected from the group consisting of --PO₄, --P₂ O₇, --P₃ O₁₀, phosphate analog, and --OH. Additionaly, a primer extension method is provided employing the above X-substituted propargylethoxyamido nucleosides, and polynucleotides including the above X-substituted propargylethoxyamido nucleosides is provided.

Claims

1. A polynucleotide comprising a nucleotide compound having the structure:

##STR20## wherein;

X is selected from the group consisting of ##STR21## where n ranges from 1 to 5, ##STR22## where n ranges from 1 to 5, ##STR23## R₁ and R₂ taken separately are selected from the group consisting of --H, lower alkyl, protecting group, and label;

R₃ is selected from the group consisting of --H and lower alkyl;

B is a 7-deazapurine, purine, or pyrimidine nucleoside base;

wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N⁹ -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N¹ -position of the pyrimidine; and

wherein when B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;

W₁ is selected from the group consisting of --H and --OH;

W₂ is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'"'"'-position; and

W₃ is selected from the group consisting of --PO₄, phosphate analog, and --OH.

2. A nucleoside compound having the structure:

##STR24## wherein;

X is selected from the group consisting of ##STR25## where n ranges from 1 to 5, ##STR26## where n ranges from 1 to 5, ##STR27## R₁ and R₂ taken separately are selected from the group consisting of --H, lower alkyl, protecting group, and label;

R₃ is selected from the group consisting of --H and lower alkyl;

B is a 7-deazapurine, purine, or pyrimidine nucleoside base;

wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N⁹ -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N¹ -position of the pyrimidine; and

wherein when B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;

W₁ is selected from the group consisting of --H and --OH;

W₂ is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'"'"'-position; and

W₃ is selected from the group consisting of --PO₄, --P₂ O₇, --P₃ O₁₀, phosphate analog, and --OH.

3. The nucleoside compound of claim 2 wherein one of R₁ and R₂ is label.

4. The nucleoside compound of claim 3 wherein the label is a fluorescein-type dye.

5. The nucleoside compound of claim 3 wherein the label is selected from a group consisting of a rhodamine-type dye and a FLAN-type dye.

6. The nucleoside compound of claim 2 wherein:

W₁ is --H;

W₂ is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'"'"'-position; and

W₃ is --P₃ P₁₀.

7. The nucleoside compound of claim 2 wherein W₂ selected from the group consisting of --OH, --H, azido, amino, halo, and methoxy.

8. The nucleoside compound of claim 2 wherein W₂ selected from the group consisting of --H and fluoro.

9. The nucleoside of claim 2 wherein B is selected from the group consisting of uracil, cytosine, 7-deazaadenine, and 7-deazaguanosine.

10. The nucleoside compound of claim 2 wherein X is alkylamino benzoic acid having a para configuration and n=1.

11. A method for performing a primer extension reaction comprising the steps of:

providing a template nucleic acid;

annealing an oligonucleotide primer to a portion of the template nucleic acid; and

adding primer-extension reagents to the primer-template hybrid for extending the primer, the primer extension reagents including a nucleoside compound having the structure;

##STR28## wherein X is selected from the group consisting of ##STR29## where n ranges from 1 to 5, ##STR30## where n ranges from 1 to 5, ##STR31## R₁ and R₂ taken separately are selected from the group consisting of --H, lower alkyl, protecting group, and label;

R₃ is selected from the group consisting of --H and lower alkyl;

B is a 7-deazapurine, purine, or pyrimidine nucleoside base;

wherein when B is purine or 7-deazapurine, the sugar moiety is attached at the N⁹ -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N¹ -position of the pyrimidine; and

wherein when B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine;

W₁ is selected from the group consisting of --H and --OH;

W₂ is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'"'"'-position; and

W₃ is selected from the group consisting of --PO₄, --P₂ O₇, --P_3O10, phosphate analog, and --OH.

12. The method of claim 11 wherein one of R₁ and R₂ is label and the other is --H.