US2005164225A1PendingUtilityA1
Non-fluorescent quencher compounds and biomolecular assays
Priority: Aug 27, 2001Filed: Jul 23, 2004Published: Jul 28, 2005
Est. expiryAug 27, 2021(expired)· nominal 20-yr term from priority
C07D 207/46C07C 245/08G01N 33/533C07C 255/67C12Q 1/68C07C 317/32C07D 241/16C07C 245/10C07F 9/1411C07F 9/2404G01N 21/6486C07F 7/1804
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Claims
Abstract
Bis-diazo,triaryl and aryldiazo-N-arylphenazonium quencher moieties, substituted with electron-withdrawing and electron-donating substituents which induce polarity in the delocalized aryl/diazo ring systems, are useful as labels when attached to biomolecules such as polynucleotides, nucleosides, nucleotides, and polypeptides. The quencher moieties are non-fluorescent and accept energy from fluorescent reporter labels by any energy-transfer mechanism, such as FRET. Fluorescence quencher compositions are useful in preparing quencher labelled biomolecules for various molecular biology assays based on fluorescence detection.
Claims
exact text as granted — not AI-modified1 . (canceled)
2 . A compound comprising:
(i) a quencher moiety comprising structure Q: at least one aryl carbon of Q is substituted with an electron-withdrawing group; and at least one aryl carbon of Q is substituted with an electron-donating group; and (ii) an optionally protected reactive linking group linked to an aryl carbon of the quencher moiety, either directly or by way of an optional linker moiety.
3 . The compound of claim 2 which has the structure (I):
L 1 and L 3 are each, independently of one another, selected from a bond, C 1 -C 12 alkyldiyl, C 1 -C 12 alkoxyldiyl, C 1 -C 12 alkylaminodiyl, C 1 -C 12 alkylamidediyl, C 5 -C 14 aryldiyl and 1-20 ethyleneoxy units;
L is an C 1 -C 12 alkoxyldiyl;
X is an acid-labile protecting group;
Y is selected from N and CR, where R is selected from H, C 1 -C 6 alkyl and C 5 -C 14 aryl; and
Z is a phosphoramidite of the formula —O—P(NR 21 R 22 )(OR 23 ), where R 21 and R 22 are each, independently of one another, selected from C 1 -C 12 alkyl, C 5 -C 14 aryl and a saturated ring comprising up to 10 ring atoms or, alternatively, R 21 and R 22 are taken together with the nitrogen atom to which they are bonded form a saturated ring comprising from 5 to 12 ring atoms and R 23 is a phosphite ester protecting group.
4 . The compound of claim 2 in which X is selected from DMT, MMT, trityl, substituted trityl, pixyl and trialkylsilyl.
5 . The compound of claim 3 in which R 23 is selected from C 1 -C 6 alkyl, cyanoethyl, C 5 -C 14 aryl, phenyl and 2-(4-nitrophenyl)ethyl.
6 . The compound of claim 3 in which R 21 and R 22 are isopropyl and R 23 is cyanoethyl.
7 . The compound of claim 2 in which the quencher moiety Q has the structure Q1:
R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R 4 is taken together with R 5 and/or R 6 is taken together with R 7 to form a benzo group;
with the provisos that: (i) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-withdrawing group; and (ii) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-donating group.
8 . The compound of claim 7 in which the electron-withdrawing groups are each, independently of one another, selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R′, C(O)NH 2 , N(O)NHR′, C(O)NR′R′, CHO, C(O)R′, SO 2 R′, SO 2 CF 3 , SO 2 OR′, SO 3 H, NO and C 5 -C 14 aryl, where R′ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
9 . The compound of claim 7 in which the electron-donating groups are each, independently of one another, selected from O − , S − , NR″R″, NHR″, NH 2 , NHC(O)R″, OR″, OH, OC(O)R″, SR″, SH, Br, I, Cl, F, R″ and C 5 -C 14 aryl, where R″ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
10 . The compound of claim 7 in which R 10 is NO 2 .
11 . The compound of claim 7 in which at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 12 , R 13 and R 14 is OCH 3 .
12 . The compound of claim 7 in which Q1 is selected from:
13 . The compound of claim 2 which is selected from compounds 75 and 88.
14 . The compound of claim 2 in which the reactive linking group is selected from a nucleophile and an electrophile.
15 . The compound of claim 14 in which the reactive linking group is a carboxylic acid or an ester.
16 . The compound of claim 15 in which the ester is an NHS ester.
17 . The compound of claim 14 in which the quencher moiety Q has the structure Q1:
R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R 4 is taken together with R 5 and/or R 6 is taken together with R 7 to form a benzo group;
with the provisos that: (i) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-withdrawing group; and (ii) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-donating group.
18 . The compound of claim 17 in which the electron-withdrawing groups are each, independently of one another, selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R′, C(O)NH 2 , N(O)NHR′, C(O)NR′R′, CHO, C(O)R′, SO 2 R′, SO 2 CF 3 , SO 2 OR′, SO 3 H, NO and C 5 -C 14 aryl, where R′ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
19 . The compound of claim 17 in which the electron-donating groups are each, independently of one another, selected from O − , S − , NR″R″, NHR″, NH 2 , NHC(O)R″, OR″, OH, OC(O)R″, SR″, SH, Br, I, Cl, F, R″ and C 5 -C 14 aryl, where R″ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
20 . The compound of claim 17 in which R 10 is NO 2 .
21 . The compound of claim 17 in which at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 12 , R 13 and R 14 is OCH 3 .
22 . The compound of claim 17 in which Q1 is selected from:
23 . The compound of claim 17 which is selected from compounds 40, 63, 64, 31, 30, 36, 34, 41, 32, 70, 60, 71, 59, 58 and 35.
24 . A composition useful for synthesizing a quencher-labeled polynucleotide, comprising:
(i) a quencher moiety comprising structure Q: at least one aryl carbon of Q is substituted with an electron-withdrawing group; and at least one aryl carbon of Q is substituted with an electron-donating group; (ii) a hydroxyl protected with an acid-labile protecting group; and (iii) a solid support, wherein the protected hydroxyl group and the quencher moiety are linked to the solid support via an optionally cleavable linker.
25 . The composition of claim 24 which has the structure (II):
L 1 , L 3 and L 4 are each, independently of one another, selected from a bond, C 1 -C 12 alkyldiyl, C 1 -C 12 alkoxyldiyl, C 1 -C 12 alkylaminodiyl, C 1 -C 12 alkylamidediyl, C 5 -C 14 aryldiyl and 1-20 ethyleneoxy units;
L 2 is an C 1 -C 12 alkoxyldiyl;
A is a cleavable linker;
X is an acid-labile protecting group;
Y is selected from N and CR, where R is selected from H, C 1 -C 6 alkyl and C 5 -C 14 aryl; and
represents the solid support.
26 . The composition of claim 25 in which A is selected from:
where R a is selected from H, C 1 -C 12 alkyl and C 1 -C 12 alkoxy.
27 . The composition of claim 25 in which X is selected from DMT, MMT, trityl, substituted trityl, pixyl and trialkylsilyl.
28 . The composition of claim 25 in which the solid support is selected from polystyrene, controlled-pore glass, silica gel, silica, polycarylamide, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of such polymers.
29 . The composition of claim 25 in which the quencher moiety Q has the structure Q1:
R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R 4 is taken together with R 5 and/or R 6 is taken together with R 7 to form a benzo group;
with the provisos that: (i) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-withdrawing group; and (ii) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-donating group.
30 . The compound of claim 29 in which the electron-withdrawing groups are each, independently of one another, selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R′, C(O)NH 2 , N(O)NHR′, C(O)NR′R′, CHO, C(O)R′, SO 2 R′, SO 2 CF 3 , SO 2 OR′, SO 3 H, NO and C 5 -C 14 aryl, where R′ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
31 . The compound of claim 29 in which the electron-donating groups are each, independently of one another, selected from O − , S − , NR″R″, NHR″, NH 2 , NHC(O)R″, OR″, OH, OC(O)R″, SR″, SH, Br, I, Cl, F, R″ and C 5 -C 14 aryl, where R″ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
32 . The compound of claim 29 in which R 10 is NO 2 .
33 . The compound of claim 29 in which at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 12 , R 13 and R 14 is OCH 3 .
34 . The compound of claim 29 in which Q1 is selected from:
35 . The compound of claim 24 which is selected from compounds 65 and 69.
36 . A quencher-labeled conjugate, comprising a quencher moiety and a conjugated substance, wherein the quencher moiety comprises structure Q:
at least one aryl carbon is substituted with an electron-withdrawing group; and
at least one aryl carbon is substituted with an electron-donating group,
and further wherein the quencher moiety is covalently linked to the conjugated substance at an aryl carbon, optionally by way of a linker.
37 . The quencher-labeled conjugate of claim 36 in which the conjugated substance is selected from an amino acid, a polypeptide, a nucleoside, a nucleotide, a polynucleotide and protected forms thereof.
38 . The quencher-labeled conjugate of claim 36 in which the conjugated substance is a polynucleotide.
39 . The quencher-labeled conjugate of claim 38 which further comprises a fluorescent moiety covalently attached to the polynucleotide, the fluorescence of which is capable of being quenched by the quencher moiety when the fluorescent and quencher moieties are in close proximity to one another.
40 . The quencher-labeled conjugate of claim 39 in which the 3′-terminus of the polynucleotide cannot be extended by a polymerase.
41 . The quencher-labeled conjugate of claim 40 in which the quencher moiety is attached to the 3′-terminal nucleotide of the polynucleotide and the fluorescent moiety is attached to the 5′-terminal nucleotide of the polynucleotide.
42 . The quencher-labeled conjugate of claim 40 in which the quencher moiety is attached to the 3′-terminus of the polynucleotide.
43 . The quencher-labeled conjugate of claim 40 in which the fluorescent moiety is attached to the 5′-terminus of the polynucleotide.
44 . The quencher-labeled conjugate of claim 40 in which the fluorescent moiety is attached to the 3′-terminal nucleotide of the polynucleotide and the quencher moiety is attached to the 5′-terminal nucleotide of the polynucleotide.
45 . The quencher-labeled conjugate of claim 40 in which the fluorescent moiety is attached to the 3′-terminus of the polynucleotide.
46 . The quencher-labeled conjugate of claim 40 in which the quencher moiety is attached to the 5′-terminus of the polynucleotide.
47 . The quencher-labeled conjugate of claim 37 in which the conjugated substance is a polypeptide.
48 . The quencher-labeled conjugate of claim 36 in which the quencher moiety Q comprises structure Q1:
R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each, independently of one another, selected from hydrogen, an electron-withdrawing group and an electron-donating group, or, alternatively R 4 may be taken together with R 5 or R 6 may be taken together with R 7 to form a benzo group, with the provisos that: (i) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-withdrawing group; and (ii) at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 or R 14 is an electron-donating group.
49 . The quencher-labeled conjugate of claim 48 in which the electron-withdrawing groups are each, independently of one another, selected from NO 2 , CN, CF 3 , CO 2 H, CO 2 R′, C(O)NH 2 , N(O)NHR′, C(O)NR′R′, CHO, C(O)R′, SO 2 R′, SO 2 CF 3 , SO 2 OR′, SO 3 H, NO and C 5 -C 14 aryl, where R′ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
50 . The quencher-labeled conjugate of claim 48 in which the electron-donating groups are each, independently of one another, selected from O − , S − , NR″R″, NHR″, NH 2 , NHC(O)R″, OR″, OH, OC(O)R″, SR″, SH, Br, I, Cl, F, R″ and C 5 -C 14 aryl, where R″ is H, C 1 -C 12 alkyl or C 5 -C 14 aryl.
51 . The quencher-labeled conjugate of claim 48 in which R 10 is NO 2 .
52 . The quencher-labeled conjugate of claim 48 in which at least one of R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 11 , R 12 , R 13 and R 14 is OCH 3 .
53 . The quencher-labeled conjugate of claim 48 in which Q1 is selected from:
54 . A method of labeling a polynucleotide with a quencher moiety, comprising contacting the polynucleotide with a compound according to claim 2 under conditions in which the reactive linking group forms a covalent attachment to a group on the polynucleotide.
55 . A method of labeling a polypeptide with a quencher moiety, comprising contacting the polypeptide with a compound according to claim 2 under conditions in which the reactive linking group forms a covalent attachment to a group on the polypeptide.Join the waitlist — get patent alerts
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