Purines and pyrimidines linked to a quencher

• US 6,531,581 B1
• Filed: 10/26/2000
• Issued: 03/11/2003
• Est. Priority Date: 06/10/1998
• Status: Expired due to Fees

• Current Assignee: Intergen Company, EMD Millipore Corporation (Merck & Co., Inc.), Oncor, Inc
• Original Assignee: Serologicals Corporation (Merck & Co., Inc.)
• Inventors: Boal, Jila, Nazarenko, Irena, Nardone, Glenn
• Primary Examiner(s): Shah, Mukund J.
• Assistant Examiner(s): MCKENZIE, THOMAS C
• Application Number:
  US09/646,669
• Time in Patent Office: 866 Days
• Field of Search: 544/243, 544/244, 534/560, 536/25.32, 536/26.6, 536/24.3
• US Class Current: 534/560
• 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 purines substituted at the C-8 position and pyrimidines substituted at the C-4 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 easily incorporated into oligonucleotides using conventional or automated oligonucleotide synthetic techniques.

Claims

1. A derivatized pyrimidine or purine compound having the following structure:

wherein, L is a linker group 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 C—

C(O)—

(CH₂)_n—

NH—

, —

CH═

CH—

C(O)—

NH—

(CH₂)_n—

CO₂—

, —

CH═

CH—

C(O)—

NH—

(CH₂)_n—

NH—

, —

(CH₂)_n—

NH—

, and —

NH—

(CH₂)_n—

NH—

, wherein n is an integer from 2 to 12, and m is 1 or 2, and Q is a quencher group selected from the group consisting of radicals of azo dyes and triphenyl methane dyes;

P represents a pyrimidine selected from the group consisting of uridine, thymidine and cytosine or the purine adenine;

substituted with a linker L at the 4 position, when P is a pyrimidine and at the 8 position when P is a purine;

wherein said pyrimidine is linked to the sugar at the 1-position nitrogen and said guanine is linked to the sugar at the 9-position nitrogen and either;

x is an activated phosphorus group capable of bonding to a 3′

-hydroxyl group of a nucleotide or polynucleotide selected from the group consisting of HO—

P(═

O)(—

OH)—

O—

, HO—

P(═

O)(—

OH)—

O—

P(═

O)(—

OH)—

O—

, HO—

P—

(═

O)(—

OH)—

O—

P(═

O)(—

OH)—

O—

P(═

O)(—

OH)—

O—

, (HO)₂P(═

S)—

O—

, (HO)₂P(═

O)—

O—

(HO)P(═

O)—

O—

(HO)P(═

S)—

O—

, —

O—

P(OCH₃)(N(iso-propyl)₂), —

O—

P(OCH₂CH₂CN)(N(isopropyl)₂) and —

O—

P(CH₃)(N(iso-propyl)₂);

or x is NH₂, H, OH or a protected hydroxyl;

y is a protected hydroxyl group, OH, H or NH₂, and z is —

H, —

OH, —

F, —

OCH₃, —

O-allyl, a protected hydroxyl group, —

NH₂ or a protected amine group;

or

y is an activated phosphorus group capable of bonding to a 5′

hydroxyl group of a nucleotide or polynucleotide selected from the group consisting of HO—

P(═

O)(—

OH)—

O—

, HO—

P(═

O)(—

OH)—

O—

P(═

O)(—

OH)—

O—

, HO—

P—

(═

O)(—

OH)—

O—

P(═

O)(—

OH)—

O—

P(═

O)(—

OH)—

O—

, (HO)₂P(═

S)—

O—

, (HO)₂P(═

O)—

O—

(HO)P(═

O)—

O—

(HO)P(═

S)—

O—

, —

O—

P(OCH₃)(N(iso-propyl)₂), —

O—

P(OCH₂CH₂CN)(N(isopropyl)₂) and —

O—

P(CH₃)(N(iso-propyl)₂);

or y is NH₂, H, OH or a protected hydroxyl;

x is a protected hydroxyl group, OH, H or NH₂, and z is —

H, —

OH, —

F, —

OCH₃, —

O-allyl, a protected hydroxyl group, NH₂ or a protected amine group.

2. The compound of claim 1, wherein P is uridine, cytosine, or adenine.

3. The compound of claim 1, wherein L is —

CH═

CH—

C(O)—

NH—

(CH₂)₆—

NH—

, —

NH—

(CH₂)₃—

NH—

, or —

NH—

(CH₂)₃—

NH—

.

4. The compound of claim 1, wherein said protected hydroxyl group is selected from the group consisting of —

O-allyl, —

O-benzyl, —

O-dimethoxytrityl, —

O-trityl, —

O-propargyl, —

O-silyl (wherein the silyl group is selected from the group consisting of trimethylsilyl, triisopropylsilyl and t-butyldimethylsilyl) and —

O-acetyl.

5. The compound of claim 1, wherein z is H.

6. The compound of claim 4, wherein y is (HO)₂P(═

S)—

O—

or x is (HO)₂P(═

O)—

O—

(HO)P(═

O)—

O—

(HO)P(═

S)—

O—

.

7. The compound of claim 1, wherein Q is a radical of an azo dye.

8. The compound of claim 1, wherein the radical of the azo dye or triphenyl methane dye is acid alizarin violet N, acid black 24, acid blue 29, acid blue 92, acid blue 113, acid blue 120, 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, 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 33, mordant brown 48, mordant orange 1, mordant orange 6, mordant orange 10, oil red E6N, orange 11, orange G, palatine chrome black 6BN, palatine fast yellow BLN, tropaeolin O, acid yellow 36, 4-dimethylamino-2-methylazobenzene, disperse orange 1, disperse orange 3, disperse orange 13, disperse orange 25, disperse red 1, disperse red 13, disperse red 19, disperse yellow 3, disperse yellow 5, disperse yellow 7, ethyl red, methyl red, naphthyl red, 4-phenylazoaniline, 4-phenylazomalcinanil, 4-phenylazophenol, 4(4-nitrophenylazo) resorcinol, nitro red, orange II, para red, Sudan I, Sudan II, Sudan III, Sudan IV, Sudan Orange G, or Sudan Red 7B.

9. The compound of claim 1, wherein the radical of the azo dye is 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 G, 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, tropaeolin O, 4-dimethylamino-2-methylazobenzene, ethyl red, methyl red, naphthyl red, 4-phenylazoaniline, 4-phenylazophenol, disperse orange 3, disperse orange 13, disperse orange 25, disperse yellow 7, orange 2, para red, Sudan I, Sudan II, Sudan III, or Sudan Orange G.

10. The compound of claim 1, wherein Q is a radical of a triphenyl methane dye.

11. The compound of claim 1, wherein the radical of the triphenyl methane dye is alkali blue 6B, aurintricarboxylic acid, basic violet 14, basic red 9, brilliant green, bromochlorophenyl blue, bromcresol purple, chlorophenol red, crystal violet, ethyl violet, fast green FCF, guinea green B, malachite green, methyl green, new fuchsia, pyrocatechol violet, thymol blue, thymophtalin, victoria blue B, victoria blue R, or victoria pure blue BO.

12. The compound of claim 11, wherein the triphenyl methane dye is linked to the linker through reaction with a dye containing an isocyanate group, an isothiocyanate group, or an N-succinimidyl ester group.

13. The compound of claim 11, wherein the radical of the triphenyl methane dye is malachite green.

14. The compound of claim 1, wherein Q is uniblue B, alizarin blue black B, alizarin red S, alizarin violet 3R, fast blue BB, or fast blue RR.

15. The compound of claim 1, wherein Q is

T is — NH—

, —

SO₂—

, —

O—

, —

S—

, —

C(O)—

S—

, —

C(O)—

, —

C(O)—

NH—

, —

C(O)—

O—

, —

C(S)—

NH—

, or —

C(S)—

O—

,

a, a′

, b, b′

, c, c′

, d, d′

, and e′

are each —

H, —

Cl, —

Br, —

F, —

OCH₃, —

OH, —

CH₃, SO₃, diazophenyl, diazo-1-naphthyl, NO₂, —

CO₂R, or —

N(R)₂, and

R is —

H, —

CH₃, or —

CH₂—

CH₃.

16. The compound of claim 15, wherein T is —

SO₂—

;

a, a′

, b, b′

, c, c′

, d, and d′

each are H; and

e′

is —

N—

(CH₃)₂ or —

N—

(CH₂—

CH₃)₂.

17. The compound of claim 16, wherein e′

is —

N—

(CH₃)₂.

18. The compound of claim 16, wherein e′

is —

N—

(CH₂—

CH₃)₂.

19. A method for producing a compound containing at least two nucleotides, wherein the method comprises linking the compound of claim 1 to another compound containing at least one nucleotide.

20. The method of claim 19, wherein the compound containing at least two nucleotides is an oligonucleotide.