COMPOSITIONS, PROBES AND CONJUGATES AND USES THEREOF

• US 20100152431A1
• Filed: 03/01/2010
• Published: 06/17/2010
• Est. Priority Date: 05/22/2006
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Skrzypczynski, Zbigniew, Wayland, Sarah R.
• Granted Patent: US 8,003,771 B2
• Time in Patent Office: Days
• US Class Current: 536/23.100
• CPC Class Codes: A61K 49/0039 Coumarin dyes
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• 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

The present invention relates to compositions useful as probes and in other applications and methods of their use. In some embodiments, nucleotides are prepared and functionalized with dyes. In some embodiments a first molecule is functionalized with an alkynyl group, a second molecule is functionalized with an azide group, and said first and second molecules are mixed under conditions to form a conjugate with a 1,2,3-triazol group. In further embodiments, a nucleotide is functionalized with an alkynyl group, a dye is functionalized with an azide group, and mixing the nucleotide and the dye forms a conjugate capable of emitting light.

Claims

1. A composition comprising a conjugate, said conjugate comprising a nucleotide and an ethynyl group functioning to emit a detectable signal when reacted with a compound comprising an azide group.

2. The composition of claim 1, wherein said conjugate comprises at least one phosphorus atom between said ethynyl group and the base portion of said nucleotide.

3. The composition of claim 1, comprising a compound selected from the group consisting of:

5-(4-ethynylphenyl)amino-5-oxo-pentanoic acid;

N′

-(6-hydroxyhexyl)-N-(4-ethynylphenyl)-pentandiamide;

N′

-(6-aminohexyl 2-cyanoethyl diisopropylamidophosphite)-N-(4-ethynylphenyl)-pentandiamide.

4. A composition comprising a substituted or unsubstituted unreacted compound functioning to react with an azide to form a reacted compound with a 1,2,3-triazol group, said unreacted compound having the following formula:

5. A composition comprising a compound with a 1,2,3-triazol group, wherein said compound has the following formula:

6. The composition of claim 5, wherein said compound is attached to a solid surface.

7. A composition comprising an unreacted compound functioning to react with an azide to form a reacted compound comprising a 1,2,3-triazol group, said unreacted compound having the following formula:

8. The composition of claim 7, wherein said unreacted compound comprises the structure:

9. A method for making a nucleic acid molecule comprising a label, comprising:

reacting a first unreacted compound, wherein said first unreacted compound is linked to a nucleic acid molecule and comprises an ethynyl group, with a second unreacted compound, wherein said second unreacted compound comprises an azide group, to create a nucleic acid molecule comprising a label.

10. The method of claim 9, wherein in said first unreacted compound, at least one phosphorus atom is between said ethynyl group and said nucleic acid molecule.

11. The method of claim 9, wherein said second unreacted compound comprises a label.

12. The method of claim 11, wherein said label comprises a fluorescent label.

13. A method of making 5-(4-ethynylphenyl)amino-5-oxo-pentanoic acid comprising:

mixing 4-ethynylaniline with glutaric anhydride under conditions such that 5-(4-ethynylphenyl)amino-5-oxo-pentanoic acid is formed.

14. A method of making N′

-(6-aminohexyl 2-cyanoethyl diisopropylamidophosphite)-N-(4-ethynylphenyl)-pentandiamide comprising mixing N′

-(6-hydroxyhexyl)-N-(4-ethynylphenyl)-pentandiamide with 2-Cyanoethyl-N,N,N′

,N′

-tetraisopropylphosphoramidite under conditions such that N′

-(6-aminohexyl 2-cyanoethyl diisopropylamidophosphite)-N-(4-ethynylphenyl)-pentandiamide is formed.

15. A method of making N′

-(6-hydroxyhexyl)-N-(4-ethynylphenyl)-pentandiamide comprising;

i) mixing 5-(4-ethynylphenyl)amino-5-oxo-pentanoic acid with N-hydroxysuccinimide under conditions such that (2,5-dioxopyrrolidin-1-yl) 5-(4-ethynylphenyl)amino-5-oxo-pentanate is formed and ii) mixing (2,5-dioxopyrrolidin-1-yl) 5-(4-ethynylphenyl)amino-5-oxo-pentanate with 1-amino-6-hexanol under conditions such that N′

-(6-hydroxyhexyl)-N-(4-ethynylphenyl)-pentandiamide is formed.

16. A method of making N′

-(6-hydroxyhexyl)-N-(4-ethynylphenyl)-pentandiamide comprises i) mixing 5-(4-ethynylphenyl)amino-5-oxo-pentanoic acid with a leaving group reagent under conditions such that a compound comprising a leaving group is formed and ii) mixing said compound comprising said leaving group with 1-amino-6-hexanol under conditions such that N′

-(6-hydroxyhexyl)-N-(4-ethynylphenyl)-pentandiamide is formed.