Pyrimidines linked to a quencher

• US 6,117,986 A
• Filed: 06/10/1998
• Issued: 09/12/2000
• Est. Priority Date: 06/10/1998
• Status: Expired due to Term

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.), Oncor, Inc
• Original Assignee: Intergen Company, L.P.
• Inventors: Nardone, Glenn, Boal, Jila, Nazarenko, Irena
• Primary Examiner(s): POWERS, FIONA
• Application Number:
  US09/095,014
• Time in Patent Office: 825 Days
• Field of Search: 534/727, 536/25.32
• US Class Current: 534/727
• 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

Abstract

Compounds are described comprising pyrimidines substituted at the C-5 position with a linker and quencher. These compounds when incorporated into hairpin oligonucleotides quench the fluorescence of fluorophores linked to the 5'"'"'-terminus of the oligonucleotide. These nucleotide-quencher compounds are also easy to incorporate into oligonucleotides using conventional, automated, oligonucleotide synthetic techniques.

Claims

1. A derivatized pyrimidine compound having the structure ##STR10## wherein Pyr represents a pyrimidine substituted at the 5 position with a linker L and said linker comprising at least three carbon atoms and is linking the pyrimidine with the quencher;

wherein Q is a quencher and

whenever x is an activated phosphorus group capable of intermolecular bond formation with a 3'"'"' hydroxyl of another nucleotide, then y is a protected hydroxyl group and z is --H, --F, --OCH₃, --O-allyl or a protected hydroxyl or amine group and

whenever y is an activated phosphorus group capable of intermolecular bond formation with a 5'"'"' hydroxyl of another nucleotide then x is a protected hydroxyl group and z is --H, --F, --OCH₃, --O-allyl or a protected hydroxyl or amine group.

2. The compound of claim 1 wherein the linker L is selected from the following structures:

--CH═

CH--C(O)--NH--(CH₂)_n --NH--;

--CH═

CH--C(O)--NH--CH₂ --CH₂ --(O--CH₂ --CH₂)_m --NH--;

--C.tbd.C--C(O)--(CH₂)_n --NH--; and

--CH═

CH--C(O)--NH--(CH₂)_n --CO₂ -- and wherein n is 2-12 and m is 1 or 2.

3. The compound of claim 1 wherein x, y and z are selected from --H, HO--, HO--P(═

O)(--OH)--O--, HOP(═

O)(--OH)--O--P(═

O)(--OH)--O--, HOP(═

O)(--OH)--O--P(═

O)(--OH)--O--P(═

O)(--OH)--O--, --OCH₃, --F, --NH₂, --O-allyl, benzyl, dimethoxytrityl, trityl, acetyl, phosphothioates, (HO)₂ P(═

S)--O--, (HO)₂ P(═

O)--O--(HO)P(═

O)--O--(HO)P(═

S)--O--, methyl phosphoamidite, methyl phosphonoamidite, H-phosphonate , phosphotriesters, --O-propargyl, silyl, support bound, --O--C(O)--(CH₂)₂ --C(O)-- alkylamino-support, and phosphoamidites, ((CH₃)₂ CH)₂ --N--P(OCH₂ CH₂ CN)--O--, ((CH₃)₂ CH)₂ --N--P(OCH₃)--O--.

4. The compound of claim 1 wherein the quencher is a non-fluorescent diazo dye.

5. The compound of claim 4 wherein the quencher is selected from compounds having the structure ##STR11## wherein T is independently selected from --NH--, --SO₂ --, --O--, --S--, --C(O)--S--, --C(O)--, --C(O)--NH--, --C(O)--O--, --C(S)--NH-- and --C(S)--O-- and a, a'"'"', b, b'"'"', c, c'"'"', d, d'"'"' and e'"'"' are independently selected from the group consisting of H, Cl, Br, F, --OCH₃, --OH, --CH₃, --SO₃^-, diazophenyl and diazo-1-naphthyl, --CO₂ R and N(R)₂ and wherein R is independently selected from H, --CH₃, --CH₂ --CH₃ and O.

6. The compound of claim 5 wherein T is --SO₂ -- a, a'"'"', b, b'"'"', c, c'"'"', d and d'"'"' are H and e'"'"' is selected from N--(CH₃)₂ and N(CH₂ CH₃)₂.

7. The compound of claim 4 wherein the nonfluorescent azo dye is selected from the group consisting of acid alizarin violet N, acid black 24, acid blue 29, acid blue 92, acid blue 113, acid blue 120, and blue 161, acid orange 8, acid orange 51, acid orange 74, acid red 1, acid red 4, acid red 8, acid red 37, acid red 40, acid red 88, acid red 97, acid red 106, acid red 151, acid red 183, acid violet 7, acid yellow 17, acid yellow 25, acid yellow 29, acid yellow 34, acid yellow 38, acid yellow 40, acid yellow 42, acid yellow 65, acid yellow 76, acid yellow 99, alizarin yellow 66, crocein orange G, alizarin blue black B, palatine chrome black 6BN, mordant black 3, basic red 29, basic blue 66, brilliant yellow chrysophine, chrysoldin, crocein orange G, crystal scarlet, fast black K salt, fast corinth V salt, fast garnet GBC, fat brown B, fat brown RR, mordant blue 9, mordant brown 1, mordant brown 4, mordant brown 6, mordant brown 24, mordant brown 33, mordant brown 48, mordant orange 1, mordant orange 6, mordant orange 10, oil red E6N, oil red O, orange 11, orange G, palatine chrome black 6BN, palatine fast yellow BLN and tropaeolin O.

8. The compounds of claim 7, wherein the compounds are selected from the group acid alizarin violet N, acid black 24, acid blue 92, acid blue 113, acid blue 120, acid orange 8, acid orange 51, acid orange 74, acid red 1, acid red 4, acid red 8, acid red 88, acid red 151, acid red 183, acid yellow 34, acid yellow 40, acid yellow 76, crocein orange 6, crystal scarlet, mordant blue 9, mordant brown 1, mordant brown 33, mordant orange 1, mordant orange 6, mordant orange 10, orange 11, orange G, palatine chrome black 6BN, palatine fast yellow BLN and tropaeolin O.

9. The compound of claim 1 wherein the quencher is a non-fluorescent triphenyl methyl dye.

10. The compound of claim 9 wherein the triphenyl methyl dye is selected from the group consisting of alkali blue 6B, aniline blue, aurintricarboxcylic acid, basic violet 14, basic red 9, brilliant green, bromochlorophenol blue, bromocresol purple, chlorophenol red, crystal violet, ethyl violet, fast green FCF, guinea green B, malachite green, methyl green, new fuchsia, pyrocatechol violet, thymol blue, thymolphthalin, victoria blue B, victoria blue R, and victoria pure blue BO.

11. The compound of claim 10 wherein the compound is derivatized with an isocyanate, isothiocyanate or N-succinimidyl ester group.

12. The compound of claim 10 wherein the triphenyl methyl dye selected is malachite green.

13. The compound of claim 12 wherein the malachite green is derivatized at the 4 position with an amine.

14. The compound of claim 3 wherein z is H.

15. The compound of claim 3 wherein the linker is selected from the group consisting of:

--CH═

CH--C(O)--NH--(CH₂)_n --NH--;

--CH═

CH--C(O)--NH--CH₂ --CH₂ --(O--CH₂ --CH₂)_m --NH--;

--C.tbd.C--C(O)--(CH₂)_n --NH--; and

--CH═

CH--C(O)--NH--(CH₂)_n --CO₂ -- and wherein n is 2-12 and m is 1 or 2.

16. The compound of claim 15 wherein z is H and x and y are selected from dimethoxytrityl, trityl and ((CH₃)₂ CH)₂ --N--P(--OCH₂ CH₂ CN)--O--.

17. The compound of claim 2, wherein the quencher is selected from the group consisting of uniblue B alizarin blue black B, alizarin red S, alizarin violet 3R, fast blue BB, and fast blue RR.

18. The compound of claim 2 wherein the linker is selected from --CH═

CH--C(O)--NH--(CH₂)_n --NH-- and --CH═

CH--C(O)--NH--CH₂ --CH₂ --(O--CH₂ --CH₂)_m --NH-- and wherein n is 2-12 and m is 1, 2 or 3.

19. The compound of claim 1 wherein z is H, x is selected from dimethoxytrityl and trityl and y is ((CH₃)₂ CH)₂ --N--P(--OCH₂ CH₂ CN)--O--.

20. The compound of claim 19 wherein the linker is selected from --CH═

CH--C(O)--NH--(CH₂)_n --NH-- and --CH═

CH--C(O)--NH--CH₂ --CH₂ --(O--CH₂ --CH₂)_m --NH-- and wherein n is 2-12 and m is 1 or 2.

21. T he compound of claim 20 wherein Q is dabsyl.

22. The compound of claim 20 wherein Q is malachite green.

23. A method of forming a hairpin oligonucleotide with a fluorophore and a quencher, said fluorphore and quencher being in close proximity so as to substantially quench the fluorescence of the fluorophore when the hairpin oilgonucleotide is in the hairpin conformation and said quencher being far enough away so as to have no quenching effect when the oligonucleotide is in a double helix or unfolded conformation said method comprising the steps of:

attaching a starting nucleotide to a solid support;

selecting the appropriate nucleotides and reacting the selected nucleotides with the bound nucleotides so as to form a hairpin oligonucleotide;

reacting the derivatized pyrimidine compound of claim 1 with the oligonucleotide at a preselected point in the oligonucleotide synthesis;

reacting a fluorophore with the hairpin oligonucleotide at the 5'"'"' position of the nucleotide after the hairpin oligonucleotide has been synthesized.