Optically active DNA probe having phosphonic diester linkage

• US 6,211,354 B1
• Filed: 05/05/1999
• Issued: 04/03/2001
• Est. Priority Date: 05/06/1998
• Status: Active Grant

• Current Assignee: Tosoh Corporation
• Original Assignee: Tosoh Corporation
• Inventors: Horie, Ryuichi, Ishiguro, Takahiko
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/305,223
• Time in Patent Office: 699 Days
• Field of Search: 536/24.3, 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34, 536/25.4, 536/25.6
• US Class Current: 536/24.3
• 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 DNA probe of the following structural formula (1) (wherein P* is an optically active phosphorus atom, each of R₁ and R₂ is a DNA oligomer having an arbitrary sequence, and R₃ is a fluorescent intercalative dye attached via an appropriate linker) which has an optically active configuration about P*.

Claims

1. An optically active DNA probe of formula (1),

wherein P* is an optically active phosphorus atom, each of R₁ and R₂ is a DNA oligomer having a desired nucleotide sequence, and R₃ is a fluorescent intercalative dye attached via a linker, wherein said probe has an optically active configuration about P*.

2. The DNA probe according to claim 1, wherein said probe has a R- or S-configuration about P*.

3. The DNA probe according to claim 1, wherein a fluorescence characteristic of the fluorescent intercalative dye chances when the dye is intercalated into a complementary binding portion between a target nucleotide molecule and the DNA probe.

9. The optically active DNA probe of claim 1, wherein the linker comprises an aminoalkylphosphate.

4. An optically active DNA oligomer of formula (2),

wherein P* is an optically active phosphorus atom, each of R₄ and R₅ is a DNA oligomer having a desired nucleotide sequence, and R₆ is a hydrogen atom or an amino-protecting group, and wherein said probe has an optically active configuration about P*.

5. The DNA oligomer according to claim 4, wherein said probe has a R- or S-configuration about P*.

6. A method of preparing an optically active DNA comprising attaching a fluorescent intercalative dye to the nitrogen atom in the aminoethylphosphonate of the optically active DNA oligomer of formula (2) via a linker,

wherein P* is an optically active phosphorus atom, each of R₄ and R₅ is a DNA oligomer having a desired nucleotide sequence, and R₆ is a hydrogen atom or an amino-protecting group, having an optically active configuration about P*.

10. The method of claim 6, wherein the linker comprises an aminoalkylphosphate.

7. A method of preparing an optically active DNA oligomer comprising the steps of:

(A) reacting an optically active dinucleotide of formula (4),

 

wherein P* is an optically active phosphorus atom, B₁ and B₂ are desired nucleotide bases, R₁₀ is an amino-protecting group, R₁₁ is a hydroxyl-protecting group, and R₁₂ is a hydroxyl-protecting group, a hydrogen atom, a phosphate group or a phosphoroamidite group, with the 5′

-hydroxyl group of a first DNA oligomer having a desired nucleotide sequence; and

then

(B) reacting a second DNA oligomer having a desired nucleotide sequence to the 5′

-terminal.

11. The method of claim 7, wherein the amino protecting group is a trifluoroacetyl (TFA) group and a protecting group at the 3′

position at R₁₂ is a phosphoroamidite group.

8. A method of preparing an optically active dinucleotide of formula (8),

wherein P* is an optically active phosphorus atom, B₁ and B₂ are desired nucleotide bases, R₁₆ is an amino-protecting group, R₁₇ is a hydroxyl-protecting group, and R₁₈ is a t-butyldimethylsilyl group, which comprises the steps of:

(A) reacting a phosphonic nucleotide of formula (5),

 

wherein B₁ is a desired nucleotide base, R₁₃ is an amino-protecting group, and R₁₄ is a hydroxyl-protecting group, with a nucleoside of formula (6),

 

wherein B₂ is a desired nucleotide base, and R₁₅ is a t-butyldimethylsilyl group, to give a dinucleotide of formula (7),

 

wherein each of B₁ and B₂ is a desired nucleotide base, R₁₆ is an amino-protecting group, R₁₇ is a hydroxyl-protecting group, and R₁₈ is a t-butyldimethylsilyl group; and

(B) optically resolving the resulting dinucleotide.

12. The method of claim 8, wherein the amino protecting group is a trifluoroacetyl (TFA) group.