U.S. patent application number 09/942342 was filed with the patent office on 2003-05-01 for non-fluorescent quencher compounds and biomolecular assays.
Invention is credited to Ewing, Gregory J., Graham, Ronald J., Mullah, Khairuzzaman Bashar.
Application Number | 20030082547 09/942342 |
Document ID | / |
Family ID | 25477956 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082547 |
Kind Code |
A1 |
Ewing, Gregory J. ; et
al. |
May 1, 2003 |
Non-fluorescent quencher compounds and biomolecular assays
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. 1
Inventors: |
Ewing, Gregory J.;
(Sunnyvale, CA) ; Mullah, Khairuzzaman Bashar;
(Union City, CA) ; Graham, Ronald J.; (San Ramon,
CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.
APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Family ID: |
25477956 |
Appl. No.: |
09/942342 |
Filed: |
August 27, 2001 |
Current U.S.
Class: |
435/6.12 ;
530/409; 534/727; 534/753; 534/824 |
Current CPC
Class: |
C07F 9/2404 20130101;
C07D 241/16 20130101; C07F 9/1411 20130101; C07C 245/10 20130101;
C12Q 1/68 20130101; C07C 317/32 20130101; C07C 245/08 20130101;
C07C 255/67 20130101; G01N 33/533 20130101; G01N 21/6486 20130101;
C07D 207/46 20130101; C12Q 1/68 20130101; C07F 7/1804 20130101;
C12Q 2563/107 20130101 |
Class at
Publication: |
435/6 ; 534/727;
534/753; 534/824; 530/409 |
International
Class: |
C12Q 001/68; C07K
014/00; C07H 021/04; C09B 035/08 |
Claims
We claim:
1. A fluorescence quencher composition having the structure:
85wherein Y is selected from N and CR, where R is H,
C.sub.1-C.sub.6 alkyl or C.sub.5-C.sub.14 aryl; L.sub.1, L.sub.2,
and L.sub.3 are independently selected from a bond,
C.sub.1-C.sub.12 alkyldiyl, C.sub.1-C.sub.12 alkoxyldiyl,
C.sub.1-C.sub.12 alkylaminodiyl, C.sub.1-C.sub.12 alkylamidediyl,
C.sub.5-C.sub.14 aryldiyl, and 1-20 ethyleneoxy units; X is an
amino acid, a polypeptide, a nucleoside, a nucleotide, a
polynucleotide, or a protected form thereof; or X is an acid-labile
protecting group; Z is selected from H, CO.sub.2H, OH, NH.sub.2,
NHR, NR.sub.2, SH, an ester, a cleavable linker, a solid support, a
reactive linking group, and a label selected from a fluorescent
dye, a hybridization-stabilizing moiety, a chemiluminescent dye,
and an affinity ligand; and Q is selected from the diazo
structures: 86wherein Ar is C.sub.5-C.sub.14 aryl; one of the aryl
carbons of the diazo structures is the site of attachment to
L.sub.1; at least one aryl carbon of each diazo structure is
substituted with an electron-withdrawing group and at least one
aryl carbon of each diazo structure is substituted with an
electron-donating group.
2. The fluorescence quencher composition of claim 1 wherein the
electron-withdrawing groups are selected from NO.sub.2, CN,
CF.sub.3, CO.sub.2H, CO.sub.2R, C(O)NH.sub.2, C(O)NHR,
C(O)NR.sub.2, CHO, C(O)R, SO.sub.2R, SO.sub.2CF.sub.3, SO.sub.2OR,
SO.sub.3H, NO, and C.sub.5-C.sub.14 aryl, where R is H,
C.sub.1-C.sub.12 alkyl or C.sub.5-C.sub.14 aryl.
3. The fluorescence quencher composition of claim 2 wherein a
NO.sub.2 is para to a diazo group.
4. The fluorescence quencher composition of claim 1 wherein the
electron-donating groups are selected from O.sup.-, S.sup.-,
NR.sub.2, NHR, NH.sub.2, NHC(O)R, OR, OH, OC(O)R, SR, SH, Br, I,
Cl, F, R, and C.sub.5-C.sub.14 aryl, where R is H, C.sub.1-C.sub.12
alkyl or C.sub.5-C.sub.14 aryl.
5. The fluorescence quencher composition of claim 4 wherein a
OCH.sub.3 is ortho or meta to a diazo group.
6. The fluorescence quencher composition of claim 1 where Z is
OH.
7. The fluorescence quencher composition of claim 1 where Z is an
ester selected from the structures: 87
8. The fluorescence quencher composition of claim 1 selected from
the structures: 88where n is 1 to 12.
9. The fluorescence quencher composition of claim 1 wherein X is
selected from DMT, MMT, trityl, substituted trityl, pixyl, and
trialkylsilyl.
10. The fluorescence quencher composition of claim 1 having the
structure: 89wherein A is a cleavable linker selected from the
structures: 90where R' is H, C.sub.1-C.sub.12 alkyl or
C.sub.1-C.sub.12 alkoxy; L.sub.4 is selected from a bond,
C.sub.1-C.sub.12 alkyldiyl, C.sub.1-C.sub.12 alkoxyldiyl,
C.sub.1-C.sub.12 alkylaminodiyl, C.sub.1-C.sub.12 alkylamidediyl,
C.sub.5-C.sub.14 aryldiyl, and 1-20 ethyleneoxy units; and 91is a
solid support.
11. The fluorescence quencher composition of claim 10 wherein X is
a nucleotide.
12. The fluorescence quencher composition of claim 10 wherein the
solid support is selected from polystyrene, controlled-pore-glass,
silica gel, silica, polyacrylamide, polyacrylate,
hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy,
and copolymers and grafts of such.
13. The fluorescence quencher composition of claim 10 wherein the
form of the solid support is selected from a particle, a bead, a
membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a
micromachined chip, an alkanethiol-gold layer, a magnetic bead, a
non-porous surface, an addressable array, and
polynucleotide-immobilizing medium.
14. The fluorescence quencher composition of claim 1 having the
structure: 92wherein A is a cleavable linker selected from the
structures: 93where R is H, C.sub.1-C.sub.12 alkyl or
C.sub.1-C.sub.12 alkoxy; L.sub.4 and L.sub.5 are independently
selected from a bond, C.sub.1-C.sub.12 alkyldiyl, C.sub.1-C.sub.12
alkoxyldiyl, C.sub.1-C.sub.12 alkylaminodiyl, C.sub.1-C.sub.12
alkylamidediyl, C.sub.5-C.sub.14 aryldiyl, and 1-20 ethyleneoxy
units; G is a hybridization-stabilizing moiety; and 94is a solid
support.
15. The fluorescence quencher composition of claim 14 in which G
comprises: 95where L are the sites of attachment to L.sub.3 and
L.sub.5.
16. The fluorescence quencher composition of claim 14 wherein the
solid support is selected from polystyrene, controlled-pore-glass,
silica gel, silica, polyacrylamide, magnetic beads, polyacrylate,
hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy,
and copolymers and grafts of such.
17. The fluorescence quencher composition of claim 14 wherein the
form of the solid support is selected from a particle, a bead, a
membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a
micromachined chip, an alkanethiol-gold layer, a magnetic bead, a
non-porous surface, an addressable array, and
polynucleotide-immobilizing medium.
18. The fluorescence quencher composition of claim 1 having the
structure: 96wherein X is an acid-labile protecting group; R.sub.1
and R.sub.2 are individually selected from isopropyl, morpholino,
methyl, ethyl and C.sub.5-C.sub.14 aryl; R.sub.1 and R.sub.2 taken
together are C.sub.4-C.sub.11 cycloalkyl or morpholino; and R.sub.3
is C.sub.1-C.sub.6 alkyl or C.sub.5-C.sub.14 aryl.
19. The fluorescence quencher composition of claim 18 wherein
R.sub.1 and R.sub.2 are each isopropyl and R.sub.3 is
cyanoethyl.
20. The fluorescence quencher composition of claim 18 wherein X is
selected from DMT, MMT, trityl, substituted trityl, pixyl, and
trialkylsilyl.
21. The fluorescence quencher composition of claim 11 having the
structure: 97wherein X is an acid-labile protecting group; B is a
nucleobase; and R.sub.3 is selected from H, C.sub.1-C.sub.6 alkyl,
and C.sub.5-C.sub.14 aryl.
22. The fluorescence quencher composition of claim 11 having the
structure: 98wherein X is an acid-labile protecting group; B is a
nucleobase; and R.sub.3 is selected from H, C.sub.1-C.sub.6 alkyl,
and C.sub.5-C.sub.14 aryl.
23. The fluorescence quencher composition of claim 1 where X is a
polynucleotide.
24. The fluorescence quencher composition of claim 23 wherein the
polynucleotide comprises one or more N-[2-(aminoethyl)]glycine
units having a nucleobase attached to nitrogen through a methylene
carbonyl linkage.
25. The fluorescence quencher composition of claim 23 wherein the
polynucleotide comprises one or more 2'-4' or 3'-4' bicyclic sugar
modifications.
26. A labelled nucleoside or nucleotide having the formula:
99wherein Q is a quencher moiety selected from the diazo
structures: 100wherein Ar is C.sub.5-C.sub.14 aryl; one of the aryl
carbons of the diazo structures is the site of attachment to L; at
least one aryl carbon of each diazo structure is substituted with
an electron-withdrawing group and at least one aryl carbon of each
diazo structure is substituted with an electron-donating group; B
is a nucleobase; R.sup.19 is H, monophosphate, diphosphate,
triphosphate, thiophosphate, phosphate analog, or acid-labile
protecting group; R.sup.20 and R.sup.21, when taken alone, are each
independently H, HO, F, or a moiety which terminates
polymerase-mediated target-directed polymerization; or when taken
together form 2'-3'-didehydroribose; and L is a linker comprising
an alkynyl, propargyl, propargylethoxyamido, vinyl, or allyl
group.
27. The labelled nucleoside or nucleotide of claim 26 in which L
comprises: 101wherein n is 0, 1, or 2.
28. The labelled nucleoside or nucleotide of claim 26 which is
enzymatically incorporatable.
29. The labelled nucleoside or nucleotide of claim 26 which is
enzymatically extendable.
30. The labelled nucleoside or nucleotide of claim 26 which is a
terminator.
31. The labelled nucleoside or nucleotide of claim 26 wherein Q
further comprises a fluorescent dye, wherein the fluorescent dye
and quencher moiety are covalently attached by a linker; and the
fluorescent dye is selected from a fluorescein dye, a rhodamine
dye, a benzophenoxazine, and a cyanine dye.
32. A nucleobase-labelled polynucleotide having the formula:
102comprising 2 to 100 nucleotides, wherein Q is a quencher moiety
selected from the diazo structures: 103wherein Ar is
C.sub.5-C.sub.14 aryl; one of the aryl carbons of the diazo
structures is the site of attachment to L; at least one aryl carbon
of each diazo structure is substituted with an electron-withdrawing
group and at least one aryl carbon of each diazo structure is
substituted with an electron-donating group; B is a nucleobase;
R.sup.21 is H, OH, halide, azide, amine, C.sub.1-C.sub.6
aminoalkyl, C.sub.1-C.sub.6 alkyl, allyl, C.sub.1-C.sub.6 alkoxy,
--OCH.sub.3, or --OCH.sub.2CH.dbd.CH.sub.2; R.sup.22 is H.
phosphate, internucleotide phosphodiester, or internucleotide
analog; R.sup.23 is H, phosphate, internucleotide phosphodiester,
or internucleotide analog; and L is a linker comprising an alkynyl,
propargyl, propargylethoxyamido, vinyl, allyl, or C.sub.1-C.sub.12
alkyldiyl group.
33. The nucleobase-labelled polynucleotide of claim 32 in which L
comprises: 104wherein n is 0, 1, or 2.
34. The nucleobase-labelled polynucleotide of claim 32 which
further comprises one or more N-[2-(aminoethyl)]glycine units
having a nucleobase attached to nitrogen through a methylene
carbonyl linkage.
35. The nucleobase-labelled polynucleotide of claim 32 which
further comprises one or more 2'-4' or 3'-4' bicyclic sugar
modifications.
36. The nucleobase-labelled polynucleotide of claim 32 wherein Q
further comprises a fluorescent dye, wherein the fluorescent dye
and quencher moiety are covalently attached by a linker; and the
fluorescent dye is selected from a fluorescein dye, a rhodamine
dye, a benzophenoxazine, and a cyanine dye.
37. A method of labelling a polypeptide comprising the step of
reacting a linking moiety of a fluorescence quencher with a
polypeptide to form a labelled quencher-polypeptide conjugate,
wherein the linking moiety is selected from the group consisting of
an azido, a monosubstituted primary amine, a disubstituted
secondary amine, a thiol, an hydroxyl, a halide, an epoxide, an
N-hydroxysuccinimidyl ester, a carboxyl, and an activated ester;
whereby the quencher moiety is attached to a location of the
polypeptide selected from the amino terminus, the carboxyl
terminus, and an amino acid side-chain.
38. A method of polynucleotide labelling comprising: a) providing
the fluorescence quencher composition of claim 1 wherein Z is a
solid support, L.sub.2 is C.sub.1-C.sub.12 alkoxydiyl, and X is an
acid-labile protecting group; b) reacting the labelled
solid-support with acid to remove X; c) adding a
3'-phosphoramidite, 5' protected nucleoside and an activator,
thereby forming a bond between L.sub.2 and the 3' terminus of the
nucleoside; d) adding an oxidizing reagent; and e) repeating steps
b) to d) until a labelled polynucleotide is synthesized.
39. The method of polynucleotide labelling of claim 38 further
comprising capping any unreacted sites on the solid-support after
step c).
40. The method of polynucleotide labelling of claim 38 wherein the
5' terminus is attached to a fluorescent dye by a linkage, wherein
the fluorescent is selected from a fluorescein, a rhodamine, a
benzophenoxazine, and a cyanine.
41. The method of polynucleotide labelling of claim 38 wherein a
nucleobase of the polynucleotide is labelled with a fluorescent
dye, selected from a fluorescein, a rhodamine, a benzophenoxazine,
and a cyanine, by a linkage at a position on the polynucleotide
selected from the 8-position of a purine nucleobase, the 7- or
8-position of a 7-deazapurine nucleobase, and the 5-position of a
pyrimidine nucleobase.
42. The method of polynucleotide labelling of claim 38 further
comprising deprotecting the labelled polynucleotide.
43. The method of polynucleotide labelling of claim 38 wherein the
solid-support is selected from polystyrene, controlled-pore-glass,
silica gel, silica, polyacrylamide, magnetic beads, polyacrylate,
hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy,
and copolymers and grafts of such.
44. The method of polynucleotide labelling of claim 38 wherein the
form of the solid support is selected from a particle, a bead, a
membrane, a frit, a fiber, a tube, a capillary, a slide, a plate, a
micromachined chip, an alkanethiol-gold layer, a magnetic bead, a
non-porous surface, an addressable array, and
polynucleotide-immobilizing medium.
45. The method of polynucleotide labelling of claim 38 wherein a
plurality of polynucleotides covalently attached to a solid support
in an addressable array are synthesized.
46. A method of polynucleotide labelling comprising coupling a
polynucleotide with the fluorescence quencher composition of claim
18, whereby a 5' quencher labelled polynucleotide is formed.
47. The method of polynucleotide labelling of claim 46 wherein the
3' terminus of the polynucleotide is covalently attached to a solid
support.
48. A 5' quencher labelled polynucleotide having the formula:
105comprising 2 to 100 nucleotides, wherein Q is a quencher moiety
selected from the diazo structures: 106wherein Ar is
C.sub.5-C.sub.14 aryl; one of the aryl carbons of the diazo
structures is the site of attachment to L; at least one aryl carbon
of each diazo structure is substituted with an electron-withdrawing
group and at least one aryl carbon of each diazo structure is
substituted with an electron-donating group; B is a nucleobase; X
is O, NH, or S; R.sup.21 is H, OH, halide, azide, amine,
C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6 alkyl, allyl,
C.sub.1-C.sub.6 alkoxy, --OCH.sub.3, or --OCH.sub.2CH.dbd.CH.sub.2;
R.sup.22 is internucleotide phosphodiester or internucleotide
analog; and L is C.sub.1-C.sub.12 alkyldiyl, aryldiyl, or
polyethyleneoxy.
49. A 3' quencher labelled polynucleotide having the formula:
107comprising 2 to 100 nucleotides, wherein Q is a quencher moiety
selected from the diazo structures: 108wherein Ar is
C.sub.5-C.sub.14 aryl; one of the aryl carbons of the diazo
structures is the site of attachment to L; at least one aryl carbon
of each diazo structure is substituted with an electron-withdrawing
group and at least one aryl carbon of each diazo structure is
substituted with an electron-donating group; B is a nucleobase; X
is O, NH, or S; R.sup.21 is H, OH, halide, azide, amine,
C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6 alkyl, allyl,
C.sub.1-C.sub.6 alkoxy, --OCH.sub.3, or --OCH.sub.2CH.dbd.CH.sub.2;
R.sup.23 is internucleotide phosphodiester or internucleotide
analog; and L is C.sub.1-C.sub.12 alkyldiyl, aryldiyl, or
polyethyleneoxy.
50. A method of primer extension comprising: annealing a
polynucleotide primer to a target polynucleotide; and extending the
primer by polymerase-mediated incorporation of a 2'-deoxynucleotide
5'-triphosphate; wherein the primer or the nucleotide
5'-triphosphate is covalently attached by a linkage to an aryl
carbon of a quencher moiety selected from the diazo structures:
109wherein Ar is C.sub.5-C.sub.14 aryl; at least one aryl carbon of
each diazo structure is substituted with an electron-withdrawing
group and at least one aryl carbon of each diazo structure is
substituted with an electron-donating group; whereby a labeled
polynucleotide is formed.
51. The method of claim 50 further comprising amplifying the target
polynucleotide with nucleotide 5'-triphosphates, a polymerase, and
two or more primers; wherein the primers are complementary to the
target polynucleotide sequence and at least one primer is
covalently attached by a linkage to an aryl carbon of a quencher
moiety.
52. The method of claim 50 further comprising amplifying the target
polynucleotide with nucleotide 5'-triphosphates, a polymerase, and
two or more primers; wherein the primers are complementary to the
target polynucleotide sequence and at least one nucleotide
5'-triphosphate is covalently attached by a linkage to an aryl
carbon of a quencher moiety.
53. The method of claim 50 further comprising amplifying the target
polynucleotide with nucleotide 5'-triphosphates, a polymerase, two
or more primers; wherein the primers are complementary to the
target polynucleotide sequence, and a detectable probe; wherein the
detectable probe is complementary to the target polynucleotide and
is covalently attached to a fluorescent dye and a quencher
moiety.
54. The method of claim 53 further comprising detecting a signal
from the fluorescent dye of said detectable probe.
55. The method of claim 54 wherein the signal is detected at each
thermal cycle during amplification.
56. The method of claim 53 wherein said polymerase cleaves the
detectable probe during amplification; whereby the fluorescent dye
and the quencher moiety are separated.
57. The method of claim 56 further comprising detecting a signal
from the fluorescent dye of said cleaved, detectable probe.
58. The method of claim 57 wherein the signal is detected at each
thermal cycle during amplification.
59. The method of claim 53 wherein said fluorescent dye is attached
to the 5' terminus or 3' terminus of the detectable probe.
60. The method of claim 53 wherein said quencher moiety is attached
to the 5' terminus or 3' terminus of the detectable probe.
61. The method of claim 53 wherein the detectable probe is further
labelled with a hybridization-stabilizing moiety.
62. The method of claim 61 wherein the hybridization-stabilizing
moiety comprises the structure: 110where L is an attachment site to
the detectable probe.
63. The method of claim 50 further comprising: forming one or more
labeled polynucleotide fragments by polymerase-directed primer
extension of a primer; resolving the one or more labeled
polynucleotide fragments; and detecting the resolved labeled
polynucleotide fragments.
64. The method of claim 63 wherein the resolving step is an
electrophoretic size-dependent separation process and the one or
more labeled polynucleotide fragments are detected by
fluorescence.
65. The method of claim 64 wherein the primer is covalently
attached by a linkage to an aryl carbon of a quencher moiety.
66. The method of claim 64 wherein a nucleotide 5'-triphosphate is
covalently attached by a linkage to an aryl carbon of a quencher
moiety.
67. A method of oligonucleotide ligation comprising annealing two
probes to a target sequence and forming a phosphodiester bond with
a ligase enzyme between the 5' terminus of one probe and the 3'
terminus of the other probe; wherein one probe is covalently
attached to a fluorescent dye and the other probe is covalently
attached by a linkage to an aryl carbon of a quencher moiety
selected from the diazo structures: 111wherein Ar is
C.sub.5-C.sub.14 aryl; at least one aryl carbon of each diazo
structure is substituted with an electron-withdrawing group and at
least one aryl carbon of each diazo structure is substituted with
an electron-donating group; whereby an oligonucleotide ligation
product is formed.
68. A method of hybridization detection comprising annealing a
probe to a target polynucleotide sequence, wherein the probe is
covalently attached to a fluorescent dye and a quencher moiety
selected from the diazo structures: 112wherein Ar is
C.sub.5-C.sub.14 aryl; at least one aryl carbon of each diazo
structure is substituted with an electron-withdrawing group and at
least one aryl carbon of each diazo structure is substituted with
an electron-donating group; and detecting a signal from the
fluorescent dye.
69. The method of hybridization detection of claim 68 wherein the
probe comprises one or more N-[2-(aminoethyl)]glycine units having
a nucleobase attached to nitrogen through a methylene carbonyl
linkage.
70. The method of hybridization detection of claim 68 wherein the
probe comprises one or more 2'-4' or 3'-4' bicyclic sugar
modifications.
71. The method of hybridization detection of claim 68 wherein the
probe sequence comprises a self-complementary hairpin sequence,
whereby an increase in fluorescence signal is detectable when the
probe is annealed to the target sequence.
72. A kit for primer extension comprising one or more nucleotide
5'-triphosphates and one or more primers wherein at least one
primer is covalently attached by a linkage to an aryl carbon of a
quencher moiety selected from the diazo structures: 113wherein Ar
is C.sub.5-C.sub.14 aryl; at least one aryl carbon of each diazo
structure is substituted with an electron-withdrawing group and at
least one aryl carbon of each diazo structure is substituted with
an electron-donating group.
73. The kit of claim 72 further comprising a polymerase.
74. The kit of claim 72 further comprising a chain-terminating
nucleotide analog.
75. A kit for nucleic acid amplification comprising two or more
primers, and a detectable probe covalently attached to a
fluorescent dye and a quencher moiety; wherein the detectable probe
is covalently attached by a linkage to an aryl carbon of a quencher
moiety selected from the diazo structures: 114wherein Ar is
C.sub.5-C.sub.14 aryl; at least one aryl carbon of each diazo
structure is substituted with an electron-withdrawing group and at
least one aryl carbon of each diazo structure is substituted with
an electron-donating group.
Description
I. FIELD OF THE INVENTION
[0001] This invention relates generally to the field of molecular
biology and more particularly, to methods and reagents of
biomolecular detection and fluorescence-based assays.
II. INTRODUCTION
[0002] Researchers use fluorescence quenching biomolecular assays
to detect the interaction, assembly, cleavage, dissociation and
conformations of proteins, nucleic acids, and other biomolecules.
Fluorescence resonance energy transfer (FRET) is an important
technique for investigating a variety of biological phenomena that
produce changes in molecular proximity, conformation, and bond
formation and cleavage. FRET has been applied in detection of
labelled biomolecules to important areas of genomics:
identification of single-nucleotide polymorphisms and other allelic
variants, protein-protein interactions, and genome-wide analysis of
regulatory sequences. Genetic information is being rapidly gleaned
by automated sequencing of genomes from all kinds of organisms. As
this information becomes available, functions are sought for
individual gene products, factors that influence the expression
level of these gene products will be identified; and allelic
variants that act alone or in combination to give rise to complex
traits will be characterized. Because of the size and number of
genomes of interest, methods and reagents that can streamline or
automate these processes are highly desirable. Fluorescence is an
attractive readout for such high-throughput tasks because of the
availability of instruments, reagents, methods, and software which
are designed to detect light-emitting compounds with great speed,
accuracy and at high throughput (De Angelis, D. (1999) Physiol.
Genomics 1:93-99).
[0003] Fluorescent quenching acceptors, e.g. TAMRA
(tetramethylrhodamine), have been employed in FRET assays, but have
at least two significant limitations: background fluorescence and
the preclusion of detection of reporter fluorescence at the
acceptor fluorescence emission. For example, the emission maxima at
about 582 mn of TAMRA-labelled oligonucleotide probes hinders the
use of reporter dyes with comparable emission maxima. There is a
need for non-fluorescent quencher compositions for FRET assays with
reporter dyes that fluoresce above about 550 nm. Reporter dyes with
absorption maximum at wavelengths at about 600 nm allow for the use
of cheaper He-Ne lasers.
III. SUMMARY OF THE INVENTION
[0004] The present invention provides bis-diazo,triaryl I and
aryldiazo-N-arylphenazonium II compounds which are useful as
quencher labels when attached to biomolecules such as
polynucleotides, nucleosides, nucleotides, carbohydrates, and
polypeptides. 2
[0005] In one aspect of the invention, the fluorescence quencher
compositions have structure III: 3
[0006] Y is N or CR, where R is H, C.sub.1-C.sub.6 alkyl or
C.sub.5-C.sub.14 aryl. L.sub.1, L.sub.2, and L.sub.3 are each,
independently, a bond, or a linker such as C.sub.1-C.sub.12
alkylidyl, C.sub.1-C.sub.12 alkoxydiyl, C.sub.1-C.sub.12
alkylaminodiyl, C.sub.1-C.sub.12 alkylamidediyl, C.sub.5-C.sub.14
aryldiyl, and 1-20 ethyleneoxy units.
[0007] X is a biomolecule such as an amino acid, a polypeptide, a
nucleoside, a nucleotide, a polynucleotide, or protected form
thereof; or X may be an acid-labile protecting group which can be
removed to form a reactive linking group which can form an
attachment to a biomolecule.
[0008] Z is a reactive linking group to form attachments to
biomolecules and labels, a solid support, or a label.
[0009] Q is a bis-diazo,triaryl quencher moiety I or
aryldiazo-N-arylphenazonium quencher moiety II. One of the aryl
carbons of a quencher moiety, Q, is attached to linker L.sub.1. At
least one aryl carbon of the quencher moiety is substituted with an
electron-withdrawing group, and at least one aryl carbon of a
quencher moiety is substituted with an electron-donating group. The
electron-withdrawing group may be on the same or different aryl
ring as the electron-donating group.
[0010] Other aspects of the invention include compositions
comprising nucleosides, nucleotides, polynucleotides, or
polypeptides labelled with the bis-diazo,triaryl and
aryldiazo-N-arylphenazonium quencher moieties. The quencher
labelled biomolecules may further contain fluorescent reporter
moieties which form energy transfer pairs.
[0011] Other aspects of the invention include methods of labelling
polynucleotides and polypeptides with the bis-diazo,triaryl and
aryldiazo-N-arylphenazonium quencher moieties of the invention. For
example, polynucleotides can be labelled at the 3' terminus with
fluorescence quencher solid support compositions. Polynucleotides
can be labelled at the 5' terminus with fluorescence quencher
phosphoramidite compositions.
[0012] Another aspect of the invention are methods of primer
extension where a polynucleotide primer is annealed to a target
polynucleotide and extended by polymerase-mediated incorporation of
a nucleotide 5'-triphosphate. The primer or a nucleotide
5'-triphosphate may be labelled with a quencher moiety of the
invention. The 3' terminus of the quencher labelled nucleotide may
be modified to terminate primer extension. The resulting extension
fragments may be separated and analyzed.
[0013] Embodiments of primer extension methods include nucleic acid
amplification. A target polynucleotide may be amplified by the
polymerase chain reaction, or other nucleic acid amplification
method, with nucleotide 5'-triphosphates, a polymerase, and two or
more primers. In one embodiment, a primer is labelled with a
quencher moiety of the invention. In another embodiment, a
nucleotide 5'-triphosphate is labelled with a quencher moiety of
the invention. A detectable probe may be labelled with a
fluorescent dye and a quencher moiety. Hybridization to the target
polynucleotide may be monitored by FRET and used to detect the
target sequence. The probe may be cleaved by nuclease activity of
an enzyme during nucleic acid amplification. Cleavage may generate
a detectable signal and used to monitor and detect nucleic acid
hybridization and amplification. The primers and probes may be
further labelled with hybridization-stabilizing moieties, such as
minor groove binders.
[0014] Another embodiment of primer extension is a method of
fragment analysis where polynucleotide fragments are formed by
polymerase-directed primer extension of a primer. The primer can be
labelled with a quencher moiety of the invention. The fragments are
resolved, i.e. separated, and detected.
[0015] Another aspect of the invention is a method of
oligonucleotide ligation. A probe labelled with a quencher moiety
is hybridized to a complementary sequence, and adjacent to another
probe labelled with a fluorescent reporter. Ligation may be
performed with a ligase, generating a polynucleotide bearing the
quencher moiety and the fluorescent reporter which interact to give
a detectable fluorescent change.
[0016] Another aspect of the invention includes a method for
hybridization detection. In one embodiment, a probe labelled with a
fluorescent dye and a quencher moiety of the invention is annealed
to a target polynucleotide sequence and a signal is detected from
the fluorescent dye.
[0017] The invention further includes kits of reagents for
performing the methods and uses detailed herein. The kits may
contain the fluorescence quencher compositions, biomolecules
labelled with the quencher moieties, and/or other reagents.
IV. DETAILED DESCRIPTION
[0018] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying Examples. While the invention will be described in
conjunction with the exemplary embodiments, it will be understood
that they are not intended to limit the invention to those
embodiments. On the contrary, the invention is intended to cover
all alternatives, modifications, and equivalents, which may be
included within the scope of the claimed invention.
[0019] IV.1 Definitions
[0020] "Biomolecule" means an amino acid, a polypeptide, a
nucleoside, a nucleotide, a polynucleotide, a carbohydrate, a
vitamin, a hormone, and any other compound produced by an
organism.
[0021] "Nucleobase" means any nitrogen-containing heterocyclic
moiety capable of forming Watson-Crick hydrogen bonds in pairing
with a complementary nucleobase or nucleobase analog, e.g. a
purine, a 7-deazapurine, or a pyrimidine. Typical nucleobases are
the naturally occurring nucleobases adenine, guanine, cytosine,
uracil, thymine, and analogs of the naturally occurring
nucleobases, e.g. 7-deazaadenine, 7-deazaguanine,
7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine, nebularine,
nitropyrrole, nitroindole, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine,
pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine,
7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,
4-thiouracil, O.sup.6-methylguanine, N.sup.6-methyladenine,
O.sup.4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,
4-methylindole, pyrazolo[3,4-D]pyrimidines (U.S. Pat. Nos.
6,143,877 and 6,127,121; WO 01/38584), and ethenoadenine (Fasman
(1989) in Practical Handbook of Biochemistry and Molecular Biology,
pp. 385-394, CRC Press, Boca Raton, Fla.).
[0022] "Nucleoside" refers to a compound consisting of a nucleobase
linked to the C-1' carbon of a sugar, such as ribose, arabinose,
xylose, and pyranose. The sugar may be substituted or
unsubstituted. Substituted ribose sugars include, but are not
limited to, those riboses in which one or more of the carbon atoms,
for example the 2'-carbon atom, is substituted with one or more of
the same or different Cl, F, --R, --OR, --NR.sub.2 or halogen
groups, where each R is independently H, C.sub.1-C.sub.6 alkyl or
C.sub.5-C.sub.14 aryl. Ribose examples include ribose,
2'-deoxyribose, 2',3'-dideoxyribose, 2'-haloribose,
2'-fluororibose, 2'-chlororibose, and 2'-alkylribose, e.g.
2'-O-methyl, 4'-.alpha.-anomeric nucleotides, 1'-.alpha.-anomeric
nucleotides, 2'-4'- and 3'-4'-linked and other "locked" or "LNA",
bicyclic sugar modifications (WO 98/22489; WO 98/39352; WO
99/14226). Exemplary LNA sugar analogs within a polynucleotide
include the structures: 4
[0023] where B is any nucleobase.
[0024] Modifications at the 2'- or 3'-position of ribose include
hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy,
isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino,
fluoro, chloro and bromo. Nucleosides and nucleotides include the
natural D optical isomer, as well as the L optical isomer forms
(Garbesi (1993) Nucl. Acids Res. 21:4159-65; Fujimori (1990) J.
Amer. Chem. Soc. 112:7435; Urata, (1993) Nucleic Acids Symposium
Ser. No. 29:69-70). When the nucleobase is purine, e.g. A or G, the
ribose sugar is attached to the N.sup.9-position of the nucleobase.
When the nucleobase is pyrimidine, e.g. C, T or U, the pentose
sugar is attached to the N.sup.1-position of the nucleobase
(Komberg and Baker, (1992) DNA Replication, 2.sup.nd Ed., Freeman,
San Francisco, Calif.).
[0025] "Nucleotide" refers to a phosphate ester of a nucleoside, as
a monomer unit or within a nucleic acid. "Nucleotide
5'-triphosphate" refers to a nucleotide with a triphosphate ester
group at the 5' position, and are sometimes denoted as "NTP", or
"dNTP" and "ddNTP" to particularly point out the structural
features of the ribose sugar. The triphosphate ester group may
include sulfur substitutions for the various oxygens, e.g.
.alpha.-thio-nucleotide 5'-triphosphates. For a review of nucleic
acid chemistry, see: Shabarova, Z. and Bogdanov, A. Advanced
Organic Chemistry of Nucleic Acids, VCH, New York, 1994.
[0026] As used herein, the terms "polynucleotide" and
"oligonucleotide" are used interchangeably and mean single-stranded
and double-stranded polymers of nucleotide monomers, including
2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotide phosphodiester bond linkages, or internucleotide
analogs, and associated counter ions, e.g., H.sup.+,
NH.sub.4.sup.+, trialkylammonium, Mg.sup.2+, Na.sup.+ and the like.
A polynucleotide may be composed entirely of deoxyribonucleotides,
entirely of ribonucleotides, or chimeric mixtures thereof.
Polynucleotides may be comprised of nucleobase and sugar analogs.
Polynucleotides typically range in size from a few monomeric units,
e.g. 5-40 when they are more commonly frequently referred to in the
art as oligonucleotides, to several thousands of monomeric
nucleotide units. Unless denoted otherwise, whenever a
polynucleotide sequence is represented, it will be understood that
the nucleotides are in 5' to 3' order from left to right and that
"A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise
noted.
[0027] "Internucleotide analog" means a phosphate ester analog or a
non-phosphate analog of a polynucleotide. Phosphate ester analogs
include: (i) C.sub.1-C.sub.4 alkylphosphonate, e.g.
methylphosphonate; (ii) phosphoramidate; (iii) C.sub.1-C.sub.6
alkyl-phosphotriester; (iv) phosphorothioate; and (v)
phosphorodithioate. Non-phosphate analogs include compounds wherein
the sugar/phosphate moieties are replaced by an amide linkage, such
as a 2-aminoethylglycine unit, commonly referred to as PNA
(Buchardt, WO 92/20702; Nielsen (1991) Science 254:1497-1500).
[0028] "Polypeptide" refers to a polymer including proteins,
synthetic peptides, antibodies, peptide analogs, and
peptidomimetics in which the monomers are amino acids and are
joined together through amide bonds. When the amino acids are
.alpha.-amino acids, either the L-optical isomer or the D-optical
isomer can be used. Additionally, unnatural amino acids, for
example, valanine, phenylglycine and homoarginine are also
included. Commonly encountered amino acids that are not
gene-encoded may also be used in the present invention. All of the
amino acids used in the present invention may be either the D- or
L- optical isomer. In addition, other peptidomimetics are also
useful in the present invention. For a general review, see Spatola,
A. F., in Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267
(1983). The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner choices can be found in many art
recognized references.
[0029] "Attachment site" refers to a site on a moiety or a
molecule, e.g. a quencher, a fluorescent dye, a polynucleotide, or
a PNA, to which is covalently attached, or capable of being
covalently attached, a linker or another moiety.
[0030] "Linker" refers to a chemical moiety in a molecule
comprising a covalent bond or a chain of atoms that covalently
attaches one moiety or molecule to another, e.g. a quencher to a
polynucleotide. A "cleavable linker" is a linker which has one or
more covalent bonds which may be broken by the result of a reaction
or condition. For example, an ester in a molecule is a linker that
may be cleaved by a reagent, e.g. sodium hydroxide, resulting in a
carboxylate-containing fragment and a hydroxyl-containing
product
[0031] "Reactive linking group" refers to a chemically reactive
substituent or moiety, e.g. a nucleophile or electrophile, on a
molecule which is capable of reacting with another molecule to form
a covalent bond. Reactive linking groups include active esters,
which are commonly used for coupling with amine groups. For
example, N-hydroxysuccinimide (NHS) esters have selectivity toward
aliphatic amines to form aliphatic amide products which are very
stable. Their reaction rate with aromatic amines, alcohols, phenols
(tyrosine), and histidine is relatively low. Reaction of NHS esters
with amines under nonaqueous conditions is facile, so they are
useful for derivatization of small peptides and other low molecular
weight biomolecules. Virtually any molecule that contains a
carboxylic acid or that can be chemically modified to contain a
carboxylic acid can be converted into its NHS ester. NHS esters are
available with sulfonate groups that have improved water
solubility.
[0032] "Substituted" as used herein refers to a molecule wherein
one or more hydrogen atoms are replaced with one or more
non-hydrogen atoms, functional groups or moieties. For example, an
unsubstituted nitrogen is --NH.sub.2, while a substituted nitrogen
is --NHCH.sub.3. Exemplary substituents include but are not limited
to halo, e.g., fluorine and chlorine, C.sub.1-C.sub.8 alkyl,
sulfate, sulfonate, sulfone, amino, ammonium, amido, nitrile,
nitro, alkoxy (--OR where R is C.sub.1-C.sub.12 alkyl), phenoxy,
aromatic, phenyl, polycyclic aromatic, heterocycle,
water-solubilizing group, and linking moiety.
[0033] "Alkyl" means a saturated or unsaturated, branched,
straight-chain, branched, cyclic, or substituted hydrocarbon
radical derived by the removal of one hydrogen atom from a single
carbon atom of a parent alkane, alkene, or alkyne. Typical alkyl
groups consist of 1-12 saturated and/or unsaturated carbons,
including, but not limited to, methyl, ethyl, cyanoethyl,
isopropyl, butyl, and the like.
[0034] "Alkyldiyl" means a saturated or unsaturated, branched,
straight chain, cyclic, or substituted hydrocarbon radical of 1-12
carbon atoms, and having two monovalent radical centers derived by
the removal of two hydrogen atoms from the same or two different
carbon atoms of a parent alkane, alkene or alkyne. Typical
alkyldiyl radicals include, but are not limited to, 1,2-ethyldiyl
(--CH.sub.2CH.sub.2--), 1,3-propyldiyl
(--CH.sub.2CH.sub.2CH.sub.2--), 1,4-butyldiyl
(--CH.sub.2CH.sub.2CH.sub.2- CH.sub.2--), and the like.
"Alkoxydiyl" means an alkoxyl group having two monovalent radical
centers derived by the removal of a hydrogen atom from the oxygen
and a second radical derived by the removal of a hydrogen atom from
a carbon atom. Typical alkoxydiyl radicals include, but are not
limited to, methoxydiyl (--OCH.sub.2--) and 1,2-ethoxydiyl or
ethyleneoxy (--OCH.sub.2CH.sub.2--). "Alkylaminodiyl" means an
alkylamino group having two monovalent radical centers derived by
the removal of a hydrogen atom from the nitrogen and a second
radical derived by the removal of a hydrogen atom from a carbon
atom. Typical alkylaminodiyl radicals include, but are not limited
to --NHCH.sub.2--, --NHCH.sub.2CH.sub.2--, and
--NHCH.sub.2CH.sub.2CH.sub.2--. "Alkylamidediyl" means an
alkylamide group having two monovalent radical centers derived by
the removal of a hydrogen atom from the nitrogen and a second.
radical derived by the removal of a hydrogen atom from a carbon
atom. Typical alkylamidediyl radicals include, but are not limited
to --NHC(O)CH.sub.2--, --NHC(O)CH.sub.2CH.sub.2--, and
--NHC(O)CH.sub.2CH.sub.2CH.sub.2--.
[0035] "Aryl" means a monovalent aromatic hydrocarbon radical of
5-14 carbon atoms derived by the removal of one hydrogen atom from
a single carbon atom of a parent aromatic ring system. Typical aryl
groups include, but are not limited to, radicals derived from
benzene, substituted benzene, naphthalene, anthracene, biphenyl,
and the like, including substituted aryl groups.
[0036] "Aryl carbon" means any carbon atom part of an aromatic ring
system.
[0037] "Aryldiyl" means an unsaturated cyclic or polycyclic
hydrocarbon radical of 5-14 carbon atoms having a conjugated
resonance electron system and at least two monovalent radical
centers derived by the removal of two hydrogen atoms from two
different carbon atoms of a parent aryl compound, including
substituted aryldiyl groups.
[0038] "Substituted alkyl", "substituted alkyldiyl", "substituted
aryl" and "substituted aryldiyl" mean alkyl, alkyldiyl, aryl and
aryldiyl respectively, in which one or more hydrogen atoms are each
independently replaced with another substituent. Typical
substituents include, but are not limited to, F, Cl, Br, I, R, OH,
--OR, --SR, SH, NH.sub.2, NMR, NR.sub.2, --.sup.+NR.sub.3,
--N.dbd.NR.sub.2, --CX.sub.3, --CN, --OCN, --SCN, --NCO, --NCS,
--NO, --NO.sub.2, --N.sub.2.sup.+, --N.sub.3, --NHC(O)R, --C(O)R,
--C(O)NR.sub.2 --S(O).sub.2O.sup.-, --S(O).sub.2R, --OS(O).sub.2OR,
--S(O).sub.2NR, --S(O)R, --OP(O)(OR).sub.2, --P(O)(OR).sub.2,
--P(O)(O.sup.-).sub.2, --P(O)(OH).sub.2, --C(O)R, --C(O)X, --C(S)R,
--C(O)OR, --CO.sub.2.sup.-, --C(S)OR, --C(O)SR, --C(S)SR,
--C(O)NR.sub.2, --C(S)NR.sub.2, --C(NR)NR.sub.2, where each R is
independently --H, C.sub.1-C.sub.6 alkyl, C.sub.5-C.sub.14 aryl,
heterocycle, or linking group. Substituents also include divalent,
bridging functionality, such as diazo (--N.dbd.N--), ester, ether,
ketone, phosphate, alkyldiyl, and aryldiyl groups.
[0039] "Electron-donating group" means a functional group that
donates electron density into a bond or delocalized resonance
system, and includes: O.sup.-, S.sup.-, NR.sub.2, NHR, NH.sub.2,
--NHC(O)R, --OR, OH, --OC(O)R, --SR, SH, Br, I, Cl, F, R, and
C.sub.5-C.sub.14 aryl, where R is C.sub.1-C.sub.12 alkyl.
[0040] "Electron-withdrawing group" means a functional group that
removes electron density into a bond or delocalized resonance
system, and includes: NO.sub.2, CN, CO.sub.2H, CO.sub.2R,
C(O)NH.sub.2, C(O)NHR, C(O)NR.sub.2, CHO, C(O)R, SO.sub.2R,
SO.sub.2OR, NO, and C.sub.5-C.sub.14 aryl, where R is
C.sub.1-C.sub.12 alkyl (J. March (1985) Advanced Organic Chemistry,
Third Ed., John Wiley & Sons, New York, p. 238).
[0041] "Heterocycle" refers to a molecule with a ring system in
which one or more ring atoms is a heteroatom, e.g. nitrogen,
oxygen, and sulfur (as opposed to carbon).
[0042] "Enzymatically extendable" refers to a nucleotide which is:
(i) capable of being enzymatically incorporated onto the terminus
of a polynucleotide chain through the action of a polymerase
enzyme, and (ii) capable of supporting further primer extension.
Enzymatically extendable nucleotides include nucleotide
5'-triphosphates, i.e. dNTP and NTP.
[0043] "Enzymatically incorporatable" refers to a nucleotide which
is capable of being enzymatically incorporated onto the terminus of
a polynucleotide chain through the action of a polymerase enzyme.
Enzymatically incorporatable nucleotides include dNTP, NTP, and
2',3'-dideoxy, nucleotide 5'-triphosphates, i.e. ddNTP.
[0044] "Target sequence" means a polynucleotide sequence that is
the subject of hybridization with a complementary polynucleotide,
e.g. a primer or probe. The target sequence can be composed of DNA,
RNA, an analog thereof, and including combinations thereof.
[0045] The term "probe" means a polynucleotide that is capable of
forming a duplex structure by complementary base pairing with a
sequence of a target nucleic acid. For example, probes may be
labelled, e.g. with a quencher moiety, or an energy transfer pair
comprised of a fluorescent reporter and quencher.
[0046] The term "label" refers to any moiety which can be attached
to a molecule and: (i) provides a detectable signal; (ii) interacts
with a second label to modify the detectable signal provided by the
second label, e.g. FRET; (iii) stabilizes hybridization, i.e.
duplex formation; or (iv) provides a capture moiety, i.e. affinity,
antibody/antigen, ionic complexation. Labelling can be accomplished
using any one of a large number of known techniques employing known
labels, linkages, linking groups, reagents, reaction conditions,
and analysis and purification methods. Labels include
light-emitting or light-absorbing compounds which generate or
quench a detectable fluorescent, chemiluminescent, or
bioluminescent signal (Kricka, L. in Nonisotopic DNA Probe
Techniques (1992), Academic Press, San Diego, pp. 3-28).
Fluorescent reporter dyes useful for labelling biomolecules include
fluoresceins (U.S. Pat. Nos. 5,188,934; 6,008,379; 6,020,481),
rhodamines (U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087;
6,051,719; 6,191,278), benzophenoxazines (U.S. Pat. No. 6,140,500),
energy-transfer dye pairs of donors and acceptors (U.S. Pat. Nos.
5,863,727; 5,800,996; 5,945,526), and cyanines (Kubista, WO
97/45539), as well as any other fluorescent label capable of
generating a detectable signal. Examples of fluorescein dyes
include 6-carboxyfluorescein; 2',4',1,4,-tetrachlorofluorescein;
and 2',4',5',7',1,4-hexachlorofluorescein. See Example 50 and
Menchen, U.S. Pat. No. 5,118,934.
[0047] Another class of labels are hybridization-stabilizing
moieties which serve to enhance, stabilize, or influence
hybridization of duplexes, e.g. intercalators, minor-groove
binders, and cross-linking functional groups (Blackburn, G. and
Gait, M. Eds. "DNA and RNA structure" in Nucleic Acids in Chemistry
and Biology, 2.sup.nd Edition, (1996) Oxford University Press, pp.
15-81). Yet another class of labels effect the separation or
immobilization of a molecule by specific or non-specific capture,
for example biotin, digoxigenin, and other haptens (Andrus, A.
"Chemical methods for 5' non-isotopic labelling of PCR probes and
primers" (1995) in PCR 2: A Practical Approach, Oxford University
Press, Oxford, pp. 39-54). Non-radioactive labelling methods,
techniques, and reagents are reviewed in: Non-Radioactive
Labelling, A Practical Introduction, Garman, A. J. (1997) Academic
Press, San Diego.
[0048] As used herein, "energy transfer" refers to the process by
which the excited state energy of an excited group, e.g.
fluorescent reporter dye, is conveyed through space or through
bonds to another group, e.g. a quencher moiety, which may attenuate
(quench) or otherwise dissipate or transfer the energy. Energy
transfer can occur through fluorescence resonance energy transfer,
direct energy transfer, and other mechanisms. The exact energy
transfer mechanisms is not limiting to the present invention. It is
to be understood that any reference to energy transfer in the
instant application encompasses all of these
mechanistically-distinct phenomena.
[0049] "Energy transfer pair" refers to any two moieties that
participate in energy transfer. Typically, one of the moieties acts
as a fluorescent reporter, i.e. donor, and the other acts as a
fluorescence quencher, i.e. acceptor ("Fluorescence resonance
energy transfer." Selvin P. (1995) Methods Enzymol 246:300-334; dos
Remedios C. G. (1995) J. Struct. Biol. 115:175-185; "Resonance
energy transfer: methods and applications." Wu P. and Brand L.
(1994) Anal Biochem 218:1-13). Fluorescence resonance energy
transfer (FRET) is a distance-dependent interaction between two
moieties in which excitation energy, i.e. light, is transferred
from a donor ("reporter") to an acceptor without emission of a
photon. The acceptor may be fluorescent and emit the transferred
energy at a longer wavelength, or it may be non-fluorescent and
serve to diminish the detectable fluorescence of the reporter
(quenching). FRET may be either an intermolecular or intramolecular
event, and is dependent on the inverse sixth power of the
separation of the donor and acceptor, making it useful over
distances comparable with the dimensions of biological
macromolecules. Thus, the spectral properties of the energy
transfer pair as a whole change in some measurable way if the
distance between the moieties is altered by some critical amount.
Self-quenching probes incorporating fluorescent
donor-nonfluorescent acceptor combinations have been developed
primarily for detection of proteolysis (Matayoshi, (1990) Science
247:954-958) and nucleic acid hybridization ("Detection of Energy
Transfer and Fluorescence Quenching" Morrison, L., in Nonisotopic
DNA Probe Techniques, L. Kricka, Ed., Academic Press, San Diego,
(1992) pp. 311-352; Tyagi S. (1998) Nat. Biotechnol. 16:49-53;
Tyagi S. (1996) Nat. Biotechnol 14:303-308). In most applications,
the donor and acceptor dyes are different, in which case FRET can
be detected by the appearance of sensitized fluorescence of the
acceptor or by quenching of donor fluorescence.
[0050] The term "quenching" refers to a decrease in fluorescence of
a fluorescent reporter ran moiety caused by a quencher moiety by
energy transfer, regardless of the mechanism. Hence, illumination
of the fluorescent reporter in the presence of the quencher leads
to an emission signal that is less intense than expected, or even
completely absent.
[0051] "Chimera" as used herein refers to a polynucleotide
including one or more modification or analog to a sugar, a
nucleobase, or an internucleotide linkage.
[0052] The terms "annealing" and "hybridization" are used
interchangeably and mean the base-pairing interaction of one
nucleic acid with another nucleic acid that results in formation of
a duplex or other higher-ordered structure. The primary interaction
is base specific, i.e. A/T and G/C, by Watson/Crick and
Hoogsteen-type hydrogen bonding.
[0053] The term "solid support" refers to any solid phase material
upon which a nucleic acid or polypeptide is synthesized, attached
or immobilized. Solid support encompasses terms such as "resin",
"solid phase", and "support". A solid support may be composed of
organic polymers such as polystyrene, polyethylene, polypropylene,
polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as
co-polymers and grafts thereof. A solid support may also be
inorganic, such as glass, silica, controlled-pore-glass (CPG), or
reverse-phase silica. The configuration of a solid support may be
in the form of beads, spheres, particles, granules, or a surface.
Surfaces may be planar, substantially planar, or non-planar. Solid
supports may be porous or non-porous, and may have swelling or
non-swelling characteristics. A solid support may be configured in
the form of a well, depression or other vessel. A plurality of
solid supports may be configured in an array, addressable for
robotic delivery of reagents, or by detection means including
scanning by laser illumination and confocal or deflective light
gathering.
[0054] "Array" means a predetermined spatial arrangement of
polynucleotides present on a solid support or in an arrangement of
vessels.
[0055] The term "end-point analysis" refers to a method where data
collection occurs only when a reaction is substantially
complete.
[0056] The term "real-time analysis" refers to periodic monitoring
during PCR. Certain systems such as the ABI 7700 and 7900HT
Sequence Detection Systems (Applied Biosystems, Foster City,
Calif.) conduct monitoring during each thermal cycle at a
pre-determined or user-defined point. Real-time analysis of PCR
with FRET probes measures fluorescent dye signal changes from
cycle-to-cycle, preferably minus any internal control signals.
[0057] IV.2 Fluorescence Quencher Compositions
[0058] Novel compositions including quencher moieties are disclosed
which are used to conduct, or are used to prepare other novel
compositions which conduct, fluorescence-based biomolecular assays
and methods. When attached to biomolecules such as polynucleotides,
nucleotides, nucleosides, and polypeptides, quencher moieties of
the invention undergo efficient energy transfer with fluorescent
dyes. The fluorescent dyes may be: (1) attached to the same
biomolecule (intramolecular), or (2) attached to another
biomolecule or reagent (intermolecular), which is free in solution
or bound to a solid support. A quencher moiety can be matched with
one or more fluorescent reporters to form an energy transfer pair,
based on spectral overlap and other spectral properties. Typically,
the fluorescent reporter in an energy transfer pair will have a
longer wavelength absorbance or excitation maximum than the
absorbance maximum of the quencher moiety. The quencher moieties of
the invention include bis-diazo, triaryl structure I: 5
[0059] and aryldiazo-N-arylphenazonium structure II: 6
[0060] Various counterions may be associated with charged
structures I and II. Ar in structure II is a C.sub.5-C.sub.14 aryl
group, such as phenyl or substituted phenyl. One of the aryl
carbons in each of I and II is the site of a covalent attachment,
e.g. to the linker, L.sub.1 of structure III.
[0061] Structures I and II have extended aromaticity for efficient
spectral overlap with longer wavelength (redder) fluorescent dye
reporters. Quencher moieties of the invention have broad absorbance
above 500 nm and enable energy transfer of fluorescence emitted
from fluorescent dyes in the range of about 500-700 nm. Quenchers
of the invention also have the surprising and beneficial properties
of non-fluorescence and accept energy from fluorescent reporter
labels by an energy-transfer mechanism. Quencher moieties of the
invention are effective at quenching reporter dyes across a broad
spectra of fluorescence detection, especially at longer
wavelengths, and eliminate problems of background fluorescence
resulting from direct (i.e. nonsensitized) acceptor excitation
(Lee, U.S. Pat. No. 6,080,868; Reed, WO 01/42505; Cook, WO
00/5378). Quencher moieties of the invention also have the
surprising and unexpected properties of: ease and efficiency of
preparation and purification, good chemical stability, good
water-solubility, and efficient labelling of biomolecules.
[0062] The quencher moieties of the present invention are
substituted with electron-withdrawing and electron-donating
substituents which induce polarity in the delocalized aryl/diazo
ring systems. In an unexpected discovery, it was found that when
the aryl carbons of structures I and II were substituted with
combinations of electron-withdrawing and electron-donating groups,
efficient quenching of fluorescent dyes was attained. At least one
aryl carbon of structures I and II is substituted with an
electron-withdrawing group, and at least one aryl carbon of
structures I and II is substituted with an electron-donating group.
The electron-withdrawing group may be on the same or different aryl
ring as the electron-donating group.
[0063] Electron-withdrawing groups are selected from NO.sub.2, CN,
CF.sub.3, CO.sub.2H, CO.sub.2R, CONH.sub.2, CONHR, CONR.sub.2, CHO,
C(O)R, SO.sub.2R, SO.sub.2OR, SO.sub.2CF.sub.3, SO.sub.3H, NO, and
C.sub.5-C.sub.14 aryl, where R is H, C.sub.1-C.sub.12 alkyl or
C.sub.5-C.sub.14 aryl. Electron-donating groups are selected from
O.sup.-, S.sup.-, NR.sub.2, NHR, NH.sub.2, NHCOR, OR, OH, OCOR, SR,
SH, Br, I, Cl, F, C.sub.1-C.sub.12 alkyl, and C.sub.5-C.sub.14
aryl, where R is H, C.sub.1-C.sub.12 alkyl or C.sub.5-C.sub.14
aryl.
[0064] The electron-withdrawing and electron-donating groups on the
aryl carbons of the aromatic rings of I and II maybe in any
configuration, i.e. ortho, meta, or para to the diazo group.
[0065] Procedures for the preparation of monocyclic (Examples 1-7)
and bicyclic (Examples 8-27) precursors are detailed therein. The
synthesis of bis-diazo, triaryl structures I from these precursors
and from commercially available reagents are detailed in Examples
28-40. An exemplary synthesis of aryldiazo-N-arylphenazonium
structures II is detailed in Example 41. Diazonium coupling
reactions to form the aryl diazo compounds are typical
electrophilic aromatic substitutions (J. McMurry in Organic
Chemistry, Fifth Edition, Brooks/Cole, Pacific Grove, Calif.,
pp1006-07). Compounds of the present invention may be prepared by
reacting aryl compounds substituted with electron-donating groups
and aryl diazonium compounds. The diazonium compounds may be
purchased from commercial sources or prepared by diazotization of
aryl amines with diazotizing reagents such as NaNO.sub.2/HCl,
nitrosylsulfuric acid, and nitrosonium tetrafluoroborate. The
diazonium compounds can be isolated or used in situ by the
subsequent addition of the aryl compound substituted with one or
more electron-donating group such as an alkylamine or alkoxy group,
e.g. methoxy. The compounds described in the Examples (1-88) may be
prepared by other known reactions, using routes known in the art,
or from other commercial sources. For a general review of azo dye
chemistry, see: Zollinger, H. Color Chemistry; VCH, New York,
1987.
[0066] A fluorescence quencher composition of the invention may
have structure III: 7
[0067] Y may be N or CR, where R is H, C.sub.1-C.sub.6 alkyl or
C.sub.5-C.sub.14 aryl. The linkers, L.sub.1, L.sub.2, and L.sub.3,
are independently selected from a bond, C.sub.1-C.sub.12 alkyldiyl,
C.sub.1-C.sub.12 alkoxyldiyl, C.sub.1-C.sub.12 alkylaminodiyl,
C.sub.1-C.sub.12 alkylamidediyl, C.sub.5-C.sub.14 aryldiyl, and
1-20 ethyleneoxy units. X is an amino acid, a polypeptide, a
nucleoside, a nucleotide, a polynucleotide, and protected forms
thereof, or an acid-labile protecting group, e.g.
4,4'-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), trityl,
substituted trityl, 9-phenylxanthen-9-yl (pixyl), and trialkylsilyl
(Beaucage (1992) Tetrahedron 48:2223-2311 at 2233-2242). Z is
selected from H, CO.sub.2H, OH, NH.sub.2, NUR, NR.sub.2, SH, an
ester, a cleavable linker, a solid support, a reactive linking
group, and a label selected from a fluorescent dye, a
hybridization-stabilizing moiety, a chemiluminescent dye, and an
affinity ligand. Q is selected from the diazo structures I and
II.
[0068] Z may be any ester, such as an oxalate, phenoxymethyl,
quinone, diglycolate, succinate, or allyloxycarbonyl. Z may be
selected from the structures: 8
[0069] Exemplary embodiments of fluorescence quencher compositions
include the structures: 9
[0070] where L.sub.1 is alkylamidediyl, L.sub.2 is alkoxydiyl,
L.sub.3 is alkyldiyl, and n is 1 to 12.
[0071] Fluorescence quencher compositions of the invention may
include a quencher moiety linked to a solid support through a
cleavable linker, represented by the general structures IV and V:
10
[0072] where Q, Y, L.sub.1, L.sub.2, and L.sub.3 are selected from
structures I, II, and III. A may be any cleavable linker, including
the structures: 11
[0073] R' is H, C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12 alkoxy.
Additionally, the cleavable linker may be an allyl or propargyl
linker, cleavable by a metal reagent, such as a palladium
complex.
[0074] L.sub.4 and L.sub.5 may be a bond, C.sub.1-C.sub.12
alkyldiyl, C.sub.1-C.sub.12 alkoxyldiyl, C.sub.1-C.sub.12
alkylaminodiyl, C.sub.1-C.sub.12 alkylamidediyl, C.sub.5-C.sub.14
aryldiyl, or 1-20 ethyleneoxy units.
[0075] The ester linkers A may be cleaved by basic reagents such as
aqueous or gaseous ammonium hydroxide, anhydrous amines, aqueous
hydroxide reagents, and aqueous amines. The ester linkers may be
selected on the basis of their cleavage rate and desired stability
of the linkage between the quencher moiety and the solid support.
For example, an oxalate linkage is relatively labile, being
virtually completely cleaved within a few minutes in concentrated
ammonium hydroxide at room temperature. The succinate linkage may
require one hour or more under the same conditions. The quinone and
diglycolate linkages have intermediate stability to basic cleavage.
Alkoxysilyl linkers may be cleaved by strong base or fluoride
reagents. Disulfide linkers may be cleaved by reducing agents such
as dithiothreitol (DTT).
[0076] The solid support, 12
[0077] may be polystyrene, controlled-pore-glass, silica gel,
silica, polyacrylamide, polyacrylate, hydroxyethylmethacrylate,
polyamide, polyethylene, polyethyleneoxy, or copolymers and grafts
of such. The solid support may be in any form, e.g. a particle, a
bead, a membrane, a frit, a fiber, a tube, a capillary, a slide, a
plate, a micromachined chip, an alkanethiol-gold layer, a magnetic
bead, a non-porous surface, an addressable array, or any
polynucleotide-immobilizing medium.
[0078] G is a hybridization-stabilizing moiety, such as a minor
groove binder, intercalator, polycation, such as polylysine and
spermine, and cross-linking functional group.
Hybridization-stabilizers may increase the stability of
base-pairing, i.e. affinity, or the rate of hybridization (Corey
(1995) J. Amer. Chem. Soc. 117:9373-74) of the primer and target,
or probe and target. Hybridization-stabilizers serve to increase
the specificity of base-pairing, exemplified by large differences
in Tm between perfectly complementary oligonucleotide and target
sequences and where the resulting duplex contains one or more
mismatches of Watson/Crick base-pairing (Blackburn, G. and Gait, M.
Eds. "DNA and RNA structure" in Nucleic Acids in Chemistry and
Biology, 2.sup.nd Edition, (1996) Oxford University Press, pp.
15-81 and 337-46). Minor groove binders include Hoechst 33258
(Rajur (1997) J. Org. Chem. 62:523-29), distamycin, netropsin,
(Gong (1997) Biochem. and Biophys. Res. Comm. 240:557-60), and
CDPI.sub.1-3 (U.S. Pat. No. 5,801,155; WO 96/32496). An example of
a minor groove binder is CDPI.sub.3, represented by the structure:
13
[0079] where L are the sites of attachment to L.sub.3 and L.sub.5
(Dempcy, WO 01/31063).
[0080] Fluorescence quencher compositions of the invention include
quencher-phosphoramidites according to the structure VI: 14
[0081] where Q, Y, L.sub.1, L.sub.2, and L.sub.3 are selected from
structures I, II, and III. Quencher-phosphoramidite reagents VI are
particularly useful for the automated synthesis of labelled
polynucleotides. The phosphoramidite reagents can be nucleosidic
(X=nucleoside) or non-nucleosidic, according to structure VI, which
can effect labelling of a polynucleotide or polypeptide with one or
more protected or unprotected quencher moieties, Q. When taken
separately, R.sub.1 and R.sub.2 are C.sub.1-C.sub.12 alkyl such as
methyl, ethyl, or isopropyl; C.sub.5-C.sub.14 aryl; or cycloalkyl
containing up to 10 carbon atoms such as, morpholino. When taken
together with the phosphoramidite nitrogen atom, R.sub.1 and
R.sub.2 may be C.sub.4-C.sub.11 cycloalkyl, e.g. morpholino.
R.sub.3 is a phosphite ester protecting group which prevents
unwanted extension of the polynucleotide. Generally, R.sub.3 is
stable to polynucleotide or polypeptide synthesis conditions yet is
able to be removed from a synthetic polynucleotide product with a
reagent that does not adversely affect the integrity of the
polynucleotide or the dye. R.sub.3 may be C.sub.1-C.sub.6 alkyl,
such as methyl, tert-butyl, or cyanoethyl; C.sub.5-C.sub.14 aryl,
such as phenyl or 2-(4-nitrophenyl)ethyl.
[0082] Exemplary quencher-phosphoramidite 75 (Example 42) was
prepared by coupling linker compound 76 with quencher NHS ester 36
by amide bond formation (Example 34) to form 77 which was
phosphitylated to give 75.
[0083] Fluorescence quencher compositions of the invention include
quencher-supports where a nucleotide is attached at its 3' or 5'
position, according to the structures VII and VIII: 15
[0084] where Q, Y, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are
selected from structures I, II, III, and IV. X is H or an
acid-labile protecting group. B may be any protected or unprotected
nucleobase. The exocyclic amino groups of nucleobase may be
protected with acyl groups, e.g. isobutyryl or benzoyl, and as
formamidines and acetamidines. R.sub.3 may be unprotected, e.g.
phosphodiester; or protected, e.g. cyanoethyl, C.sub.1-C.sub.6
alkyl, or C.sub.5-C.sub.14 aryl.
[0085] Compositions VII and VIII may be intermediates formed during
automated oligonucleotide synthesis on a solid support. Composition
VII may be formed by: (1) deprotecting quenchers-support IV, i.e.
removing the acid-labile protecting group X with acid, (2) coupling
a 3'-phosphoramidite nucleoside, and (3) oxidizing the
internucleotide phosphite linkage to phosphate. For example, where
L.sub.2 is alkoxydiyl and X is DMT, IV is treated with
trichloroacetic acid or dichloroacetic acid. Addition of subsequent
nucleosides by iteration of the above steps (1), (2), and (3) in a
cycle results in the 3' to 5' direction synthesis of an
oligonucleotide. Optionally, a capping step may be conducted to
prevent any unreacted sites from subsequent propagation, e.g.
acetylation of 5' OH with acetic anhydride. Cleavage of linker A
results in a 3'-quencher labelled oligonucleotide. Composition VIII
may be formed by the same steps (1), (2), and (3), with iteration
to give an oligonucleotide synthesized in the 5' to 3' direction,
when 5'-phosphoramidites are employed in step (2). Cleavage of
linker A then results in a 5'-quencher labelled
oligonucleotide.
[0086] IV.3 Quencher Labelled Nucleosides and Nucleotides
[0087] The sugar or nucleobase moieties of nucleotide
5'-triphosphates may be labelled with the quencher moieties I and
II for use in certain embodiments of the invention. Nucleobase
labelling sites include the 8-C of a purine nucleobase, the 7-C or
8-C of a 7-deazapurine nucleobase, and the 5-position of a
pyrimidine nucleobase (Nardone, U.S. Pat. No. 6,117,986). The
labelled nucleotide may be enzymatically incorporatable and
enzymatically extendable by polymerases or reverse transcriptase.
Quencher labelled nucleosides and nucleotides may have the
following formula: 16
[0088] where Q is a protected or unprotected quencher.
Alternatively, quencher labelled nucleosides and nucleotides may
have the following formula: 17
[0089] where E is an energy transfer pair comprising a reporter dye
and a quencher moiety attached by a linker. B is a nucleobase, e.g.
uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and
8-deazaguanosine. R.sup.19 is triphosphate, thiophosphate, or
phosphate ester analog. Alternatively, the quencher labelled
nucleosides may be intermediates useful for conversion to
nucleotide 5'-triphosphates or other compounds, where R.sup.19 is H
or an acid-labile protecting group. The acid-labile protecting
group may be a trityl group, such as DMT. R.sup.20 and R.sup.21,
when taken alone, are each independently H, HO, and F. Linker L may
include alkynyl, propargyl, propargylethoxyamido, vinyl, and allyl
groups. For example, L may be: 18
[0090] wherein n is 0, 1, or 2 (Khan, U.S. Pat. Nos. 5,770,716 and
5,821,356; Hobbs, U.S. Pat. No. 5,151,507).
[0091] IV.4 Quencher Labelled Polynucleotides
[0092] Polynucleotides are commonly synthesized on solid supports
by the phosphoramidite method (Caruthers, U.S. Pat. No. 4,973,679;
Beaucage (1992) Tetrahedron 48:2223-2311), using commercially
available phosphoramidite nucleosides (Caruthers, U.S. Pat. No.
4,415,732), supports, e.g. silica, controlled-pore-glass
(Caruthers, U.S. Pat. No. 4,458,066) and polystyrene (Andrus, U.S.
Pat. Nos. 5,047,524 and 5,262,530) and automated synthesizers such
as Models 392, 394, 3948, 3900 DNA/RNA Synthesizers (Applied
Biosystems, Foster City, Calif.).
[0093] Labelled polynucleotides may be formed by coupling a
reactive linking group on a label, e.g. a quencher moiety, with a
polynucleotide in a suitable solvent in which both are soluble or
appreciably soluble, using methods well-known in the art. For
labelling methodology, see Hermanson, Bioconjugate Techniques,
(1996) Academic Press, San Diego, Calif. pp. 40-55, 643-71; Garman,
1997, Non-Radioactive Labelling: A Practical Approach, Academic
Press, London. Crude, labelled polynucleotides may be purified from
any starting materials or unwanted by-products, and stored dry or
in solution for later use, preferably at low temperature.
[0094] The label may bear a reactive linking group at one of the
substituent positions, e.g. an aryl-carboxyl group of a quencher
moiety I or II, or the 5- or 6-carboxyl of fluorescein or
rhodamine, for covalent attachment through a linkage. Generally,
the linkage linking a label and the polynucleotide or nucleotide
should not (i) interfere with hybridization affinity or
specificity, (ii) diminish quenching, (iii) interfere with primer
extension, (iv) inhibit polymerase activity, or (v) adversely
affect the fluorescence or quenching properties of the label.
Electrophilic reactive linking groups form a covalent bond with
nucleophilic groups such as amines and thiols on a polynucleotide
or polypeptide. Examples of electrophilic reactive linking groups
include active esters, isothiocyanate, sulfonyl chloride, sulfonate
ester, silyl halide, 2,6-dichlorotriazinyl, phosphoramidite,
maleimide, haloacetyl, epoxide, alkylhalide, allyl halide,
aldehyde, ketone, acylazide, anhydride, and iodoacetamide. Active
esters include succinimidyl (NHS), hydroxybenzotriazolyl (HOBt) and
pentafluorophenyl esters.
[0095] An NHS ester of a quencher moiety may be preformed,
isolated, purified, and/or characterized, or it may be formed in
situ and reacted with a nucleophilic group of a polynucleotide or
polypeptide. Typically, a quencher moiety carboxyl group is
activated by reacting with some combination of: (1) a carbodiimide
reagent, e.g. dicyclohexylcarbodiimide- , diisopropylcarbodiimide,
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiim- ide); or a uronium
reagent, e.g. TSTU (O-(N-Succinimidyl)-N,N,N',N'-tetram-
ethyluronium tetrafluoroborate, HBTU
(O-benzotriazol-1-yl)-N,N,N',N'-tetra- methyluronium
hexafluorophosphate), or HATU (O-(7-azabenzotriazol-1-yl)-N,-
N,N',N'-tetramethyluronium hexafluorophosphate); (2) an activator,
such as HOBt (1-hydroxybenzotriazole) or HOAt
(1-hydroxy-7-azabenzotriazole; and (3) N-hydroxysuccinimide to give
the NHS ester.
[0096] The general quencher-phosphoramidite VI reacts with a
hydroxyl group, e.g. 5' terminal OH of a polynucleotide bound to a
solid support, under mild acid activation, to form an
internucleotide phosphite group which is then oxidized to an
internucleotide phosphate group. Alternatively, the
quencher-phosphoramidite reagent reacts with a hydroxyl group on
the side chain of an amino acid of a polypeptide such as serine,
threonine, or tyrosine. In some instances, the quencher moiety
contains functional groups that require protection either during
the synthesis of the phosphoramidite reagent or during its
subsequent use to label polynucleotides or polypeptides. The
protecting group(s) used will depend upon the nature of the
functional groups, and will be apparent to those having skill in
the art (Greene, T. and Wuts, P. Protective Groups in Organic
Synthesis, 2nd Ed., John Wiley & Sons, New York, 1991). The
quencher will be attached at the 5' terminus of the polynucleotide,
as a consequence of the common 3' to 5' direction of synthesis
method. Alternatively, the 3' terminus of a polynucleotide may be
labelled with a quencher-phosphoramidite when synthesis is
conducted in the less-common 5' to 3' direction with 3'-protected,
5' phosphoramidite nucleosides (Wagner, (1997) Nucleosides &
Nucleotides 16:1657-60). Other quencher-phosphoramidite reagents,
nucleosidic and non-nucleosidic, allow for labelling at other sites
of a polynucleotide, e.g. 3' terminus, nucleobase, internucleotide
linkage, sugar. Labelling at the nucleobase, internucleotide
linkage, and sugar sites allows for internal and multiple
labelling.
[0097] A nucleobase-labelled polynucleotide may have the following
formula: 19
[0098] where the primer or probe comprises 2 to 100 nucleotides and
Q is a protected or unprotected quencher. Alternatively, a
nucleobase-labelled polynucleotide may have the following formula:
20
[0099] where E is an energy transfer pair comprising a reporter dye
and a quencher moiety attached by a linker. B is a nucleobase, e.g.
uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and
8-deazaguanosine. L is a linker, e.g. propargyl,
propargylethoxyamido, allyl, vinyl, or C.sub.1-C.sub.12 alkyldiyl.
R.sup.21 is H, OH, halide, azide, amine, C.sub.1-C.sub.6
aminoalkyl, C.sub.1-C.sub.6 alkyl, allyl, C.sub.1-C.sub.6 alkoxy,
--OCH.sub.3, or --OCH.sub.2CH.dbd.CH.sub.2. R.sup.22 is H,
phosphate, internucleotide phosphodiester, or internucleotide
analog. R.sup.23 is H, phosphate, internucleotide phosphodiester,
or internucleotide analog. In this embodiment, the
nucleobase-labelled polynucleotide may bear multiple fluorescent
labels, e.g. dyes, attached through the nucleobases.
Nucleobase-labelled polynucleotides may be formed by: (i) enzymatic
incorporation of enzymatically incorporatable nucleotide reagents
where R.sup.19 is triphosphate, by a DNA polymerase or ligase, and
(ii) coupling of a nucleoside phosphoramidite reagent by automated
synthesis (Theisen (1992) "Fluorescent dye phosphoramidite
labelling of polynucleotides", in Nucleic Acid Symposium Series No.
27, Oxford University Press, Oxford, pp. 99-100). 5'
quencher-labelled polynucleotides of the invention include the
following structure: 21
[0100] where Q is structure I or II. X is O, NH, or S; R.sup.21 is
H, OH, halide, azide, amine, C.sub.1-C.sub.6 aminoalkyl,
C.sub.1-C.sub.6 alkyl, allyl, C.sub.1-C.sub.6 alkoxy, --OCH.sub.3,
or --OCH.sub.2CH.dbd.CH.sub.2- ; and R.sup.22 is internucleotide
phosphodiester, or internucleotide analog. L is a linker, including
C.sub.1-C.sub.12 alkyldiyl, e.g. n-hexyldiyl, aryldiyl, or
polyethyleneoxy (U.S. Pat. No. 4,757,141; Andrus, "Chemical methods
for 5' non-isotopic labelling of PCR probes and primers" (1995) in
PCR 2: A Practical Approach, Oxford University Press, Oxford, pp.
39-54; Hermanson, in Bioconjugate Techniques, (1996) Academic
Press, San Diego, Calif. pp. 40-55, 643-71; Mullah (1998) Nucl.
Acids Res. 26:1026-1031). The 5' quencher-polynucleotide may have
additional covalently attached labels, such as a fluorescent dye or
a minor groove binder.
[0101] A solid support bearing a quencher of the present invention,
or bearing functionality which can be labelled by a quencher in a
post-synthesis reaction, can be utilized as a solid support for
polynucleotide synthesis (U.S. Pat. Nos. 5,141,813; 5,231,191;
5,401,837; 5,736,626). By this approach, the quencher or the
functionality is present during synthesis of the polynucleotide.
During cleavage and deprotection, the quencher or the functionality
remains covalently attached to the polynucleotide. Polynucleotides
labelled at the 3' terminus may have the following structure:
22
[0102] where the probe comprises 2 to 100 nucleotides. Q is
structure I or II. B is any nucleobase, e.g. uracil, thymine,
cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine. L
is a linker, including C.sub.1-C.sub.12 alkyldiyl, e.g.
n-hexyldiyl, aryldiyl, or polyethyleneoxy. R.sup.21 is H, OH,
halide, azide, amine, C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6
alkyl, allyl, C.sub.1-C.sub.6 alkoxy, --OCH.sub.3, or
--OCH.sub.2CH.dbd.CH.sub.2. R.sup.23 is internucleotide
phosphodiester or internucleotide analog. The 3'
quencher-polynucleotide may have additional covalently attached
labels, such as a fluorescent dye or a minor groove binder.
[0103] In another embodiment of the invention, 3'
quencher-polynucleotides bound to a solid support may be derived
from VII and have the general structure: 23
[0104] where the polynucleotide comprises 2 to 100 nucleotides.
Alternatively, 5' quencher labelled polynucleotides bound to a
solid support may be derived from VIII and have the general
structure: 24
[0105] where the polynucleotide comprises 2 to 100 nucleotides.
[0106] Polynucleotides can be immobilized on solid supports by a
covalent bond attachment, ionic interaction, hybridization to an
immobilized probe, hydrophobic interaction, or ligand/receptor
interaction, using a variety of known procedures (Lane, U.S. Pat.
No. 5,902,724; Mirkin, WO 98/04740). Covalent bonds between a
polynucleotide and solid support include Schiff-base type linkages
(Lukhtanov, WO 01/09385) and phosphoramidate linkages (Rampal, U.S.
Pat. No. 6,013,789).
[0107] Nucleic acid analogs are structural analogs of DNA and RNA
and which are designed to hybridize to complementary nucleic acid
sequences. Through modification of the internucleotide linkage, the
sugar, and/or the nucleobase, nucleic acid analogs of the invention
may attain any or all of the following desired properties: 1)
optimized hybridization specificity or affinity, 2) nuclease
resistance, 3) chemical stability, 4) solubility, 5)
membrane-permeability, and 6) ease or low costs of synthesis and
purification. The fluorescence quencher compositions of the
invention include nucleic acid analogs labelled with quencher
moieties, e.g. structures III, IV, and V, where X=a nucleic acid
analog.
[0108] One useful and accessible class of nucleic acid analogs is
the family of peptide nucleic acids (PNA) in which the
sugar/phosphate backbone of DNA or RNA has been replaced with
acyclic, achiral, and neutral polyamide linkages. The
N-[2-(aminoethyl)]glycine polyamide linkage with nucleobases
attached to nitrogen through a methylene carbonyl linkage by an
amide bond has been well-studied as an embodiment of PNA and shown
to possess exceptional hybridization specificity and affinity
(Buchardt, WO 92/20702; Nielsen (1991) Science 254:1497-1500;
Egholm (1993) Nature, 365:566-68; Nielsen, P. and Egholm, M. (1999)
in Peptide Nucleic Acids; Protocols and Applications, Horizon
Scientific Press, Norfolk, England).
[0109] PNA may be synthesized at any scale, e.g. 2-100 .mu.mole
scale or more, using Fmoc/Bhoc, tBoc/Z, or MMT protecting group
monomers on an Expedite Synthesizer (Applied Biosystems) on XAL or
PAL support; or on the Model 433A Synthesizer (Applied Biosystems)
with MBHA support; or on other automated synthesizers. PNA may be
synthesized on many of the solid supports commonly used for peptide
synthesis (M. W. Pennington and B. M. Dunn (Eds.) "Methods in
molecular biology, Vol. 35: Peptide synthesis protocols", Humana
Press, Totowa, N.J. (1994), pp. 91; G. Grant (Ed.), "Synthetic
peptides", W. H. Freeman & Co., New York, N.Y., 1992; G. B.
Fields, Int. J. Peptide Protein Res. (1990) 35:161; G. B. Fields
(Eds.), "Methods in enzymology: Vol. 289", Academic Press, New
York, N.Y., 1997; W. C. Chan and P. D. White, "Fmoc solid phase
peptide synthesis: a practical approach, Oxford University Press,
Oxford, UK, 2000; P. Lloyd-Williams and F. Albericio (Eds.),
"Chemical approaches to the synthesis of peptides and proteins",
CRC Press, New York, N.Y. 1997.
[0110] PNA-DNA chimera are oligomer molecules with discrete PNA and
nucleotide moieties. They can be synthesized by covalently linking
PNA monomers and nucleotides in virtually any combination or
sequence. Efficient and automated methods have been developed for
synthesizing PNA-DNA chimera (Vinayak (1997) Nucleosides &
Nucleotides 16:1653-56; Uhlmann (1996) Angew. Chem., Intl. Ed. Eng.
35:2632-35; Uhlmann, EP 829542; Van der Laan (1997) Tetrahedron
Lett. 38:2249-52; Van der Laan (1998) Bioorg. Med. Chem. Lett.
8:663-68. PNA-DNA chimera are designed to have desirable properties
found in PNA and DNA, e.g. superior hybridization properties of PNA
and biological functions like DNA, including primer extension
through the 3' OH terminus of the DNA moiety (Uhlmann (1998) Biol.
Chem. 379:1045-52). PNA and PNA-DNA chimera can be labelled with a
quencher moiety I or II, and a fluorescent reporter, to form PNA
FRET probes (Hyldig-Nielsen, U.S. Pat. No. 5,985,563; Coull, WO
98/24933; Coull, WO 99/22018; Gildea, WO 99/21881; Coull, WO
99/49293).
[0111] The linker between a PNA monomer unit and a label is: (i) a
covalent bond; (ii) an C.sub.1-C.sub.12 alkyldiyl; (iii)
ethyleneoxy --(CH.sub.2CH.sub.2O).sub.n--, where n is 1 to 12, (iv)
C.sub.5-C.sub.14 aryldiyl; (v) 2-(2-aminoethoxy)acetic acid; or
(vi) one or more amino acids (Gildea (1998) Tetrahedron Letters
39:7255-58). Lysine, aspartic acid, and glutamic acid side chains
in the linker may be quencher-linkage sites in quencher-labelled
PNA. The .epsilon.-amino group of the sidechain of lysine may be
the reactive linking group for attachment of a label, e.g.
fluorescent reporter or quencher moiety. Linkers are typically
attached to the amino and/or carboxyl terminus of the PNA by the
corresponding monomer units with compatible protecting groups and
reactive functionality for condensation with PNA monomer units and
the solid support. For example, the "O linker", units of
2-(2-aminoethoxy)acetic acid, can be attached to the amino terminus
of any PNA backbone amino group, or on amino functionality of a
solid support.
[0112] IV.5 Quencher Labelled Polypeptides
[0113] Polypeptides that are labeled with a quencher of the
invention can be used in both in vivo and in vitro enzymatic
assays. For example, the present invention provides generally for
methods to determine if a sample contains an enzyme. A method may
comprise: (a) contacting the sample with a polypeptide labelled
with a fluorescent reporter and a quencher moiety; (b) exciting the
fluorophore; and (c) determining a fluorescence property of the
sample, wherein the presence of the enzyme in the sample results in
a change in the fluorescence property. The change in fluorescence
may be due to cleavage or assembly of a recognition site for the
enzyme.
[0114] When the labelled polypeptide is used to detect an enzyme,
such as a degradative enzyme (e.g. protease), the degree of
donor-acceptor energy transfer in the sample can be determined, for
example, as a function of the amount of fluorescence from the donor
moiety, the amount of fluorescence from the acceptor moiety, the
ratio of the amount of fluorescence from the donor moiety to the
amount of fluorescence from the acceptor moiety or the excitation
state lifetime of the donor moiety. The assay methods can also be
used to determine whether a compound alters the activity of an
enzyme, i.e. screening assays, and determining the amount of
activity of an enzyme in a sample from an organism. Screening
compounds produced by combinatorial chemistry requires convenient
enzymatic assays. In one embodiment, the amount of enzyme activity
in the sample is determined as a function of the degree of
donor-acceptor energy transfer in the sample and the amount of
activity in the sample is compared with a standard activity for the
same or different concentration of enzyme.
[0115] Representative enzymes with which the present invention can
be practiced include, for example, trypsin, enterokinase, HTV-1
protease, prohormone convertase, kininogenase, and proteases in
general. The most convenient assays for proteases are based on
donor-acceptor energy transfer from a donor fluorophore to a
quencher placed at opposite ends of a short peptide chain
containing the potential cleavage site (Knight C. G., (1995)
Methods in Enzymol. 248:1 8-34). Proteolysis separates the
fluorophore and quencher, resulting in increased intensity in the
emission of the donor fluorophore.
[0116] In one embodiment, the polypeptide sequence includes a
cleavage recognition site, specific for an enzyme or other cleavage
agent of interest. A cleavage recognition sequence for a protease
is a specific amino acid sequence recognized by the protease during
proteolytic cleavage. Many protease cleavage sites are known in the
art, and these and other cleavage sites can be included in the
linker moiety (Matayoshi, (1990) Science 247:954; Dunn in Meth. in
Enzymol. 241:254(1994); Seidahin Meth. in Enzymol. 244: 175 (1994);
Thomberryin Meth. in Enzymol. 244:615 10 (1994); Weber in Meth. in
Enzymol. 244:595 (1994); Smith in Meth. in Enzymol. 244:412 (1994);
Bouvier in Meth. in Enzymol. 248:614 (1995), Hardy in Amyloid
Protein Precursor In Development, Aging, And Alzheimer's Disease,
ed. Masters, pp. 190-198 (1994)). Cleavage of the sequence results
in separation of the fluorescent dye and the quencher moiety of a
FRET peptide. The cleavage may be measurable as a change in
donor-acceptor energy transfer, with a resulting increase in
detectable fluorescence. Conversely, peptide assembly can be
detected by an increase in donor-acceptor energy transfer
(quenching) between a peptide fragment bearing quencher moiety and
a peptide fragment bearing a fluorescent reporter.
[0117] Labelling of polypeptides with reporter moieties and other
labels for research in immunology, histochemistry, and cell biology
has been reviewed. See: Lundblad, R. L. and Noyes, C. M. (1984)
Chemical Reagents for Protein Modification, Vols. I and II, CRC
Press, New York; Pfleiderer, G. (1985) Chemical Modification of
Proteins, In Modern Methods in Protein Chemistry, H. Tschesche,
Ed., Walter DeGryter, Berlin and New York; Wong (1991) Chemistry of
protein Conjugation and Cross-linking, CRC Press, Boca Raton,
Fla.
[0118] An electrophilic reactive linking group of a quencher moiety
of the invention can react with a nucleophilic side chain of an
amino acid in a polypeptide (Brinkley, 1992, Bioconjugate Chem.
3:2). The aliphatic .epsilon.-amine of the amino acid lysine is
often a good nucleophile above pH 8.0 (pK.sub.a=9.18) (Fasman, G.
D. Ed. (1989) Practical Handbook of Biochemistry and Molecular
Biology, p13, CRC Press, Boca Raton, Fla.) and therefore reacts
easily and cleanly with a variety of electrophilic reagents to form
stable bonds. Other reactive amines that are found in proteins are
the a-amino groups of the N-terminal amino acids. The a-amino
groups are less basic than lysines and are reactive at around pH
7.0. Sometimes they can be selectively modified in the presence of
lysines. There is usually at least one .alpha.-amino acid in a
protein, and in the case of proteins that have multiple peptide
chains or several subunits, there can be more (one for each peptide
chain or subunit). Since either N-terminal amines or lysines are
almost always present in any given protein or peptide, and since
they are easily reacted, the most commonly used method of protein
modification is through these aliphatic amine groups. The quencher
moiety may be conjugated to the N-terminus of a resin-bound
polypeptide before removal of other protecting groups and release
of the labeled peptide from the resin. About five equivalents of an
amine-reactive quencher reagent may be used per amine of the
immobilized peptide (Haugland, 1996, Molecular Probes Handbook of
Fluorescent Probes and Research Chemicals, Molecular Probes,
Inc.)
[0119] Another reactive linking group in many polypeptides is a
thiol residue from the sulfur-containing amino acid cystine and its
reduction product cysteine (or half-cystine), which are counted
together as one of the 20 amino acids. Cysteine contains a free
thiol group, which is more nucleophilic than amines and is
generally the most reactive functional group in a protein. It
reacts with some of the same modification reagents as do amines and
in addition can react with reagents that are not very reactive
toward amines. Unlike most amines, thiols are reactive at neutral
pH, and therefore they can be coupled to other molecules
selectively in the presence of amines. Since free sulfhydryl groups
are relatively reactive, proteins with these groups often exist in
their oxidized form as disulfide-linked oligomers or have
internally bridged disulfide groups. Reduction of the disulfide
bonds with a reagent such as dithiothreitol (DTT) is required to
generate the reactive free thiol. In addition to cystine and
cysteine, some proteins also have the amino acid methionine, which
contains sulfur in a thioether linkage.
[0120] Acylation reactions may be performed at polypeptide amino
groups with quencher reagents. Acylation reactions may be described
by the following general scheme:
Q--CO--X+Pep--NH.sub.2.fwdarw.Pep--NHCO--Q+HX
[0121] where Pep is the polypeptide, X is a leaving group and Q is
the quencher moiety. The reactive reagent Q--CO--X may be produced
in situ by the action of an activating agent, such as a
carbodiimide, on the free carboxylic acid, but more preferable is
the use of stable active esters that may be stored as solid
reagents, e.g. N-hydroxysuccinimide (NHS) esters, and also more
water-soluble sulfonated forms. Other amine-reactive reagents which
may serve as reactive linking groups for the quencher moiety to
prepare quencher-labelled polypeptide include other succinimidyl
esters, isothiocyanates (--NCS), sulphonyl halides (--SO.sub.2Cl)
and dichlorotriazine derivatives. Thiol-reactive reagents include
iodoacetyl (--C(O)CH.sub.2I) and maleimido derivatives.
[0122] IV.6 Nucleic Acid Assay Methods
[0123] Oligonucleotides labelled with a fluorescent dye and a
quencher of the invention are useful in a wide variety of important
nucleic acid hybridization and amplification applications,
including allelic discrimination and real-time PCR quantitation.
The specificity of such oligonucleotides as primers and probes is
effective for distinguishing single base-pair differences and
mismatches.
[0124] The fluorescent/quencher probes of the invention are useful
as detection agents in a variety of DNA
amplification/quantification strategies including, for example,
5'-nuclease assay, Strand Displacement Amplification (SDA), Nucleic
Acid Sequence-Based Amplification (NASBA), Rolling Circle
Amplification (RCA), Oligonucleotide Ligation Assay (OLA), Ligase
Chain Reaction (LCR), Transcription-Mediated Amplification (TMA)
and Q-beta replicase. Fluorescent/quencher probes are also useful
for direct detection of targets in other solution phase or solid
phase (e.g., array) assays. Furthermore, the fluorescent/quencher
probes can be used in any format, including, for example, molecular
beacons, Scorpion probes.TM., Sunrise probes.TM., light up probes,
Invader.TM. Detection probes, and TaqMan.TM. probes. See, for
example, Cardullo, R. (1988) Proc. Natl. Acad. Sci. USA,
85:8790-8794; Stryer, L., (1978) Ann. Rev. Biochem., 47:819-846;
Rehman, F. N., (1999) Nucleic Acids Research, 27:649-655; Gibson,
E. M., (1996) Genome Methods, 6:995-1001; Livak, U.S. Pat. No.
5,538,848; Wittwer, C. T., (1997) BioTechniques, 22:176-181;
Wittwer, C. T., (1997) BioTechniques, 22:130-38; Tyagi, WO
95/13399, U.S. Pat. Nos. 6,037,130, 6,150,097, and 6,103,476;
Uehara, H. (1999) BioTechniques, 26:552-558; Whitcombe, (1999)
Nature Biotechnology, 17:804-807; Lyamichev, V. (1999) Nature
Biotechnology, 17:292; Daubendiek, (1991) Nature Biotechnology,
15:273-350; Nardone, WO 99/64432; Nadeau, U.S. Pat. Nos. 5,846,726
and 5,928,869; and Nazarenko, U.S. Pat. No. 5,866,336.
[0125] A method of the invention includes nucleic acid
amplification, i.e. amplifying a target polynucleotide, i.e.
template DNA, with nucleotide 5'-triphosphates, a polymerase, and
two or more primers. The primers are complementary to the target
polynucleotide sequence. One or more of the primers can be
covalently attached by a linkage to an aryl carbon of a quencher
moiety I or II. A detectable probe, complementary to the target
polynucleotide sequence, may also be included in the amplification
reaction.
[0126] The ability to detect and quantitate gene targets rapidly,
in real time or by end-point PCR detection, can be carried out with
the 5'-nuclease assay, often referred to as "TaqMan.RTM." (Gelfand,
U.S. Pat. No. 5,210,015; Holland, Proc. Natl. Acad. Sci. USA, 88:
7276-7280 (1991); Lee, Nucleic Acids Res., 21: 3761-3766 (1993);
Livak, U.S. Pat. No. 5,723,591; Lyamichev, (1993) Science,
260:778-783). The process uses the 5' to 3' exonuclease activity of
TAQ polymerase, and other thermo-stable polymerases, to digest the
double-labelled, self-quenched, detectable probe during the PCR
amplification process. The probe is cleaved by the 5'-exonuclease
activity of DNA polymerase if, and only if, it hybridizes within
the target sequence being amplified, i.e. between the
primer-binding sites. Cleavage of the probe generates an increase
in the fluorescence intensity of the fluorescent reporter. The
fluorescent donor and quencher are preferably each located at or
near the 3'- and 5'-ends of the probe. For example, a probe may be
designed with a 5' fluorescent reporter dye, e.g.
carboxyfluorescein, and a 3' quencher moiety of the present
invention. The Tm of the probe (about 65.degree. C.) is selected
primarily on the basis of length and G+C content and known
algorithms to insure binding to the target polynucleotide under
extension conditions.
[0127] In one embodiment, the detectable probe is synthesized using
a 3'-quencher support, e.g. 65 (Example 46) or 69 (Example 47). The
dual-labelled, detectable probe is synthesized on the support
including the step of covalent attachment of the fluorescent
reporter. The fluorescent reporter may be added as a
phosphoramidite reagent (e.g. VI, X=DMT) within the
oligonucleotide, or as the last base (3' to 5' direction) at the 5'
terminus of the oligonucleotide. Alternatively, the fluorescent
reporter can be attached via a reactive linking group, e.g. NHS
ester, at the 5' terminus of a 5' nucleophile (amino or thiol)
oligonucleotide which has been cleaved from the support and
deprotected. The fluorescent reporter may then be deprotected, if
necessary, and the dual labelled oligonucleotide can be analyzed
and purified. To function as a nuclease assay probe, i.e. "TaqMan"
probe, the 3'-terminal nucleotide of the probe is blocked or
rendered incapable of extension by a nucleic acid polymerase. Such
blocking is achieved conveniently by the quencher moiety or the
fluorescent reporter at the terminal 3'-hydroxyl. Alternatively,
the 3' hydroxyl may be phosphorylated or blocked by other
functionality.
[0128] One method of the invention includes nucleic acid
amplification, i.e. amplifying a target polynucleotide with
nucleotide 5'-triphosphates, a polymerase, two or more primers, and
a detectable probe. The detectable probe may be labelled with a
fluorescent reporter dye and a quencher moiety. The primers are
complementary to the target polynucleotide sequence. The probe is
complementary to the target polynucleotide at a binding site
between the primer binding sites. The probe is covalently attached
by a linkage to an aryl carbon of a quencher moiety I or II. A
signal can be detected from the detectable probe, either upon
hybridization of the probe to the target, or upon cleavage of the
probe by the polymerase, during amplification.
[0129] Cleavage of the dual labelled, self-quenching probe by TAQ
polymerase with 5'-nuclease activity generates free fluorescent
reporter each cycle of amplification, leading to exponential
accumulation of fluorescent signal, correlated to the exponential
production of PCR product ("amplicon"). By plotting the fluorescent
signal vs. PCR cycle number and comparing with a standard reference
curve, the number of starting gene copies can be determined. A
threshold cycle, CT, is designated, as the fractional cycle number,
at which the increase in fluorescence crosses some fixed threshold
above baseline. Thus, the 5'-nuclease assay determines both the
presence of a gene as well as the amount of gene present. By using
different probes with unique reporter fluorophores, several gene
determinations can be performed simultaneously in the same reaction
tube. As probes can be designed to discern a single base mismatch,
specific alleles may be determined.
[0130] The power and utility of the 5'-nuclease assay has been
expanded by the introduction of instrument systems (e.g. ABI
PRISM.RTM. 7700 and 7900HT Sequence Detector Systems, Applied
Biosystems) which automatically conduct the 5'-nuclease PCR process
in 96- and 384-tube or well microtiter formats. Since the vessels
are closed throughout the process, by-products derived by
contamination are prevented. The instrument monitors the
fluorescence (at several wavelengths to allow multiplexing with
multiple reporter dyes) in each well. Fluorophore multiplexing
further expands the number of assays possible by allowing more than
one target sequence to be interrogated per vessel, with multiple
probes labelled with different dyes.
[0131] By configuring standard primer pairs and probes as reagent
kits and robotic dispensing into the vessels (i.e. tubes, wells,
array loci, or spots), high-throughput assays for the rapid
profiling of single-nucleotide polymorphisms (SNP), allelic
discrimination, or disease related genes are enabled.
[0132] Sensitivity and accuracy of 5'-nuclease PCR and
hybridization assays are limited where the self-quenching probes
have inherent noise, i.e. background fluorescence. The
non-fluorescent quencher moieties of the invention have the
advantages of not adding fluorescence to detection, and obviating:
(1) labor-intensive or costly steps, such as post-PCR purification;
(2) elaborate controls; and (3) complicated data processing.
[0133] A series of 5'-fluorescent reporter, 3'-quencher probes were
synthesized with a 3' quencher support 69 (Example 48) and
fluorescent dye phosphoramidites for the 5' labelling (Example 50).
These probes were used in the 5' nuclease assay, i.e. "TaqMan", on
the ABI Prism 7700 instrument (Example 51). By way of comparison,
the same probe sequence was labelled with the same set of 5'
fluorescent dyes. The variable of study was the quencher moiety.
The second set of probes was labelled with TAMRA
(tetramethylrhodamine), a fluorescent quencher. Efficiency of
amplicon production and detection was measured by the change in
fluorescence, relative to baseline (.DELTA.Rn) and the threshold
cycle number (C.sub.T). The results are summarized in the table
below. No significant differences in the average .DELTA.R.sub.n and
C.sub.T values were observed for quencher 34 and TAMRA. .DELTA.Rn
is the increase in reporter fluorescence at any cycle minus
baseline fluorescence. The .DELTA.Rn values reported are after 40
cycles of PCR. Threshold cycle C.sub.T is the fractional PCR cycles
number at which ARn crosses some fixed threshold above baseline.
Four replicates were run to determine the average and standard
deviation for each value. By all comparisons, the non-fluorescent
quencher probes (Example 51) gave equal or superior performance
relative to the TAMRA probes.
1 .DELTA.Rn C.sub.T Reporter dye 34 TAMRA 34 TAMRA F.sub.1 Average
1.74 1.66 19.58 19.71 Standard deviation 0.04 0.01 0.21 0.10
F.sub.2 Average 1.47 1.82 20.08 19.92 Standard deviation 0.02 0.02
0.02 0.09 F.sub.3 Average 0.70 0.63 20.96 21.41 Standard deviation
0.05 0.04 0.06 0.19 F.sub.4 Average 0.84 0.87 20.71 20.75 Standard
deviation 0.02 0.02 0.08 0.11
[0134] Probes containing a fluorescent dye-quencher pair have been
developed for hybridization assays where the probe forms a hairpin
structure due to a self-complementary sequence. A hairpin probe
hybridizes to itself to form a stem/loop structure. In this
conformation and in the absence of a complementary nucleic acid
sequence, the quencher molecule may brought into proximity with the
fluorescent reporter dye to effectively quench the fluorescence of
the fluorescent dye (WO 90/03446; EP 0601889 A2). When a
complementary target sequence is present, hybridization of the
hairpin FRET probe to the complementary target sequence disrupts
the hairpin structure and causes the probe to adopt a conformation
where the quencher molecule is no longer close enough to the
reporter molecule to quench the reporter molecule. As a result, the
probes provide an increased fluorescent signal when hybridized to a
target sequence than when they are unhybridized (Tyagi (1996)
(Nature Biotech., 14:303-309; U.S. Pat. Nos. 5,119,801 and
5,312,728). In one embodiment, the hybridization probe may be
labelled at the 5' or 3' terminus with the fluorescent dye and at
the other terminus with a quencher moiety of the invention. When
the FRET probe is hybridized to a target polynucleotide, i.e. open
conformation, the fluorescence of the fluorescent dye is
detectable, whereas when the FRET probe is in hairpin (closed)
conformation, the fluorescence of the donor fluorophore is
quenched. The probes for hybridization detection may comprise a
nucleic acid analog, e.g. one or more PNA
(N-[2-(aminoethyl)]glycine) units having a nucleobase attached to
nitrogen through a methylene carbonyl linkage, or LNA (2'-4' or
3'-4' bicyclic sugar modifications). When employed in PCR, the FRET
probe hybridized to one of the strands of the PCR product is "open"
and is detected, while those that remain closed in the hairpin
conformation, i.e. unbound to target, do not fluoresce because the
fluorescent dye is quenched. As a result, the amount of
fluorescence will increase as the amount of PCR product increases,
and thus can be used as a measure of the progress of the PCR. In
other words, fluorescent intensity is correlated with PCR.
[0135] Polynucleotide fragments labelled with quencher moieties I
and II may be generated through template-directed enzymatic
synthesis using quencher-labelled primers or quencher-labelled
nucleotides, e.g. by ligation or polymerase-directed primer
extension. The resulting polynucleotide fragments may be further
labelled with fluorescent reporters to form energy-transfer pairs.
Quencher labelled polynucleotide fragments may serve to
intermolecularly quench the fluorescence of other
fluorescent-labelled compounds. Polynucleotide fragments may be
subjected to a size-dependent separation process, e.g.,
electrophoresis or chromatography, and the separated fragments are
detected subsequent to the separation, e.g., by laser-induced
fluorescence (Hunkapiller, U.S. Pat. No. 4,811,218). Multiple
classes of polynucleotides may be separated simultaneously and the
different classes are distinguished by spectrally resolvable
fluorescent reporters. In electrophoresis, the classes separate on
the basis of electrophoretic migration rate.
[0136] The chain termination methods of DNA sequencing, i.e.
dideoxy DNA sequencing, or Sanger-type sequencing, and fragment
analysis maybe employed (Sanger (1977) "DNA sequencing with
chain-terminating inhibitors", Proc. Natl. Acad. Sci. USA
74:5463-5467) with the fluorescence quenching compositions of the
invention Exemplary chain-terminating nucleotide analogs
"terminators" include the 2',3'-dideoxynucleoside 5'-triphosphates
(ddNTP) which lack the 3'-OH group necessary for 3' to 5' DNA chain
elongation. A primer or ddNTP may be labelled with a quencher
moiety and a fluorescent reporter to form an energy transfer pair
within the primer extension product. The change in fluorescence
from each fragment may be detected after separation of the
fragments by high-resolution electrophoresis.
[0137] In another embodiment of the invention, a ligation event may
be detected and monitored with oligonucleotide probes labelled with
quencher moieties I and II. Covalent joining of nucleic acid probes
by ligase enzymes is one of the most useful tools available to
molecular biologists. When two probes are annealed to a template
nucleic acid where the two probes are adjacent and without
intervening gaps, a phosphodiester bond can be formed between a 5'
terminus of one probe and the 3' terminus of the other probe by a
ligase enzyme, (Whiteley, U.S. Pat. No. 4,883,750; Landegren,
(1988) "A ligase mediated gene detection technique", Science
241:1077-80; Nickerson, "Automated DNA diagnostics using an
ELISA-based oligonucleotide assay" (1990) Proc. Natl. Acad. Sci USA
87:8923-27). Oligonucleotide ligation assays detect the presence of
specific sequences in target DNA sample. Where one probe is
labelled with a fluorescent reporter and the other probe is
labelled with a quencher moiety of the invention, the ligation
product may be detected by fluorescence. Ligation products may be
separated by electrophoresis, chromatography, or other size- or
charge-based separation method.
[0138] In one embodiment, a quencher-oligonucleotide of the present
invention may be immobilized on a solid support and used as a
capture probe. The probe may be covalently attached directly to a
solid support, for example by attachment of the 3'- or 5'-terminal
nucleotide of the probe to the solid support. The probe may be
attached to the solid support by a linker which serves to distance
the probe from the solid support. The linker may be about 5 to
about 30 atoms in length, or more.
[0139] Examples of preferred types of solid supports for
immobilization of the oligonucleotide probe include controlled pore
glass, glass plates, polystyrene, avidin-coated beads, cellulose,
nylon, polystyrene grafted with polyethylene glycol, polyacrylamide
gel and activated dextran. These solid supports are preferred for
hybridization and diagnostic studies because of their chemical
stability, ease of functionalization and well defined surface area.
Solid supports such as controlled pore glass (CPG) and
non-swelling, high cross-linked polystyrene are advantageous due to
their compatibility with oligonucleotide synthesis, precluding the
necessity to cleave and reattach the oligonucleotide.
[0140] The probe may be attached to the solid support through a
variety of structures and synthetic routes. For example, the probe
may be attached to the solid support by attachment of the 3' or 5'
hydroxyl of the terminal nucleotide of the probe to the solid
support. In other embodiments, the probe is attached to the solid
support through a nucleobase, a sugar, or an internucleotide
linkage. In one embodiment, the solid support is used as the
synthesis support in preparing the fluorescent/quencher
oligonucleotide probe. For oligonucleotide synthesis, the linker
arm can be attached to the 3'-OH of the 3'-terminus by an ester
linkage which can be cleaved with basic reagents to free the
oligonucleotide from the solid support.
[0141] A wide variety of linkers are known in the art which may be
used to attach the oligonucleotide probe to the solid support. The
linker should not significantly interfere with the hybridization of
the target sequence to the probe attached to the solid support. The
linker may be formed of a non-target complementary oligonucleotide
which can be readily added on to the linker by automated synthesis.
Alternatively, polymers such as functionalized polyethylene glycol
can be used as the linker. Polyethylene glycol is commercially
available, soluble in both organic and aqueous media, easy to
functionalized, and stable under oligonucleotide synthesis and
post-synthesis conditions. The linker between the solid support and
the probe is preferably not cleaved during removal of base
protecting groups under basic conditions at high temperature. These
linkers can, however, be selected from groups that are cleavable
under a variety of conditions. Exemplary linkers include carbamate
and amide functionality.
[0142] IV.7 Kits
[0143] The invention includes kits comprising polynucleotides
labelled with quencher moieties of structures I or II, and other
reagents such as nucleotide 5'-triphosphates, polymerase, primers,
a detectable probe, and buffer. Such kits are useful in primer
extension, hybridization assay, and 5' nuclease "TaqMan.RTM." PCR
detection assay. The kits may farther comprise one or more target
polynucleotides, or standard/control polynucleotides.
[0144] In another embodiment, the kits are useful for coupling
fluorescence quencher compositions of the invention to
polynucleotides and polypeptides. Such kits generally comprise a
fluorescence quencher composition with a reactive linking group,
e.g. a phosphoramidite or active ester, and any activator, coupling
agent, buffer, solvent, or other reagent necessary to effect
coupling to a polynucleotide or polypeptide. One such kit may
contain a fluorescence quencher on solid-support and other reagents
for the synthesis of a 3'-quencher labelled polynucleotide.
IV.8 EXAMPLES
[0145] The invention having been described, the following Examples
are offered by way of illustration, and not limitation.
Example 1
Synthesis of Ethyl 4-[(2,5-dimethoxyphenyl)ethylamino]butanoate
4
[0146] 25
[0147] 2,5-Dimethoxyaniline 1 (7.7 gm, 0.050 moles) was dissolved
in 40 ml tetrahydrofuran (THF) and chilled to 0.degree. C. under
argon. Acetic anhydride (7.72 ml, 0.082 moles) was added. After
stirring for 2 hours, thin-layer chromatography (TLC) indicated the
reaction was complete. The mixture was evaporated under reduced
pressure and kept under vacuum overnight.
[0148] Crude 2,5-dimethoxyacetanilide 2 was dissolved in 80 ml THF
and stirred under argon. Borane-methylsulfide complex (62.5 ml,
2.0M in THF, 0.125 moles) was added by syringe over 10 minutes.
After 10 minutes at ambient temperature, the mixture was heated to
reflux for 3 hours. Analysis by TLC showed only a trace of 2. After
cooling to room temperature, 42 ml water was cautiously added with
stirring, followed by 4.5 ml conc. HCl. The reaction was heated for
1 hour. Next added sequentially with vigorous stirring were 125 ml
water, 250 ml dichloromethane (DCM), and 16.6 ml 10N NaOH. The
mixture was extracted with three 100 ml portions of DCM which were
combined, dried over Na.sub.2SO.sub.4, and filtered. After
concentration by rotary evaporation under reduced pressure, the
crude product was purified by flash chromatography on silica gel,
eluting with a gradient mixture of ethyl acetate and hexane.
(2,5-Dimethoxyphenyl)ethylamine 3 (8.33 gm, 91% yield) was isolated
after removing the solvent as a colorless liquid.
[0149] (2,5-Dimethoxyphenyl)ethylamine 3 (8.33 gm, 0.046 moles),
ethyl 4-bromobutyrate (11.64 gm, 0.060 moles) and
diisopropylethylamine (7.76 gm, 0.060 moles) were stirred at
110-120.degree. C. for 18 hours, cooled, and partitioned between 50
ml DCM and 50 ml water. The aqueous layer was extracted thrice with
50 ml portions of DCM. The combined organic extracts were washed
with saturated NaCl, dried over Na.sub.2SO.sub.4, filtered, and
concentrated by rotary evaporation under reduced pressure. The
crude product was purified by flash chromatography on silica gel,
eluting with a gradient mixture of ethyl acetate and hexane. Ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (12.73 gm, 94%
yield) was isolated after removing the solvent. .sup.1H NMR
(CDCl.sub.3) .delta. 6.44-6.78, 3H, m; 4.11, 2H, q; 3.79, 3H, s;
3.76, 3H, s; 3.08-3.19, 4H, m; 2.30, 2H, t; 1.79, 2H, m; 1.24, 3H,
t; 1.03, 3H, t.
Example 2
Synthesis of Ethyl 4-(ethylphenylamino)butanoate 6
[0150] 26
[0151] N-ethylaniline 5 (2.0 gm, 0.016 moles), triethylamine (5.0
ml, 0.036 moles), and ethyl 4-bromobutyrate (3.45 gm, 0.018 moles)
were heated at 110.degree. C. for 14 hours under argon. The mixture
was cooled to room temperature, diluted with 50 ml ethyl acetate,
washed with 50 ml water and 50 ml saturated NaCl, dried over
MgSO.sub.4, filtered, and evaporated under reduced pressure to give
crude ethyl 4-(ethylphenylamino)butanoate 6 (3.0 gm, 77% yield).
.sup.1H NMR (CDCl.sub.3) .delta. 7.2, 2H, m; 6.6, 3H, m; 4.15, 2H,
q; 3.3, 4H, m; 2.38, 2H, t; 1.9, 2H, m; 1.25, 3H, t; 1.10, 3H,
t.
Example 3
Synthesis of Methyl 2-(ethylphenylamino)acetate 7
[0152] 27
[0153] N-Ethylaniline 5 (2 g, 16.52 mmol) was added to a stirred
suspension of sodium hydride (436 mg, 18.17 mmol) in DMF (20 ml) at
ambient temperature under argon. After 45 minutes, bromomethyl
acetate (3.02 g, 18.17 mmol) was added and the reaction mixture was
stirred for 22 hr. DMF was removed under reduced pressure and the
residue was treated with ethyl acetate (60 ml) and water (50 ml).
The organic extract was washed with saturated brine (50 ml), dried
(Na.sub.2SO.sub.4), filtered and evaporated to give crude Methyl
2-(ethylphenylamino) acetate 7 as liquid (3.06 g, 96%).
Example 4
Synthesis of Ethyl 4-(ethylnaphthylamino)butanoate 9
[0154] 28
[0155] N-Ethyl-1-naphthylamine 8 (11.4 gm, 0.066 moles),
diisopropylethylamine (11.2 gm, 0.087 moles), and ethyl
4-bromobutyrate (19 gm, 0.097 moles) were heated at 110-120.degree.
C. for 16 hours under argon at which time, HPLC indicated the
reaction was complete. The mixture solidified upon cooling to room
temperature. The solid was partitioned between DCM and water. The
aqueous layer was extracted with two portions of DCM. The combined
organic extracts were washed with saturated NaCl, dried over
MgSO.sub.4, filtered, concentrated under reduced pressure by rotary
evaporation to give 22 gm of a crude solid. The solid was purified
by flash chromatography, eluting with ethyl acetate and hexane, to
give ethyl 4-(ethylnaphthylamino)butanoate 9 (6.04 gm, 32% yield)
as an orange oil. .sup.1H NMR (CDCl.sub.3) .delta. 8.3, 1H, m; 7.8,
1H, m; 7.35-7.55, 4H, m; 7.15, 1H, m; 4.05, 2H, q; 3.18, 4H, m;
2.32, 2H, t; 1.8, 2H, m; 1.20, 3H, t; 1.05, 3H, t.
Example 5
Synthesis of Ethyl 4-[ethyl(3-methoxyphenyl)amino]butanoate 15
[0156] 29
[0157] 3-Methoxyaniline 12 (5 gm, 0.041 moles) was dissolved in 30
ml tetrahydrofuran (THF) at room temperature under argon. Acetic
anhydride (5.8 ml, 0.061 moles) was added. After stirring for 2
hours, the mixture was evaporated under reduced pressure, dissolved
in several mls of toluene and purified by flash chromatography on
silica gel, eluting with a mixture of ethyl acetate and hexane.
3-Methoxyacetanilide 13 (6.63 gm, 99% yield) was isolated after
removing the solvent. .sup.1H NMR (CDCl.sub.3) .delta. 7.6, 1H, br;
7.2, 2H, m; 6.95, 1H, d; 6.66, 1H, d; 3.78, 3H, s; 2.16, 3H, s.
[0158] 3-Methoxyacetanilide 13 (6.63 gm, 0.040 moles) was dissolved
in 65 ml THF and stirred under argon. Borane-methylsulfide complex
(52 ml, 2.0 M, 0.10 moles) was added by syringe over 10 minutes.
After 10 minutes at ambient temperature, the mixture was heated to
reflux for 3 hours. After cooling to room temperature, 40 ml water
was cautiously added with stirring over 10 minutes, followed by 3.6
ml conc. HCl. After heating for 30 minutes, 100 ml DCM and 67 ml 2N
NaOH were added sequentially with vigorous stirring. An emulsion
resulted which was eventually separated. The aqueous portion was
extracted with three 50 ml portions of DCM which were combined,
dried over MgSO.sub.4, and filtered. After concentration by rotary
evaporation under reduced pressure, the crude product was purified
by flash chromatography on silica gel, eluting with a gradient
mixture of ethyl acetate and hexane. (3-Methoxyphenyl)ethylamine 14
(5.73 gm, 93% yield) was isolated after removing the solvent as a
colorless oil. .sup.1H NMR (CDCl.sub.3) .delta. 7.07, 1H, t;
6.15-6.28, 3H, m; 3.76, 3H, s; 3.13, 2H, q; 1.23, 3H, t.
[0159] (3-Methoxyphenyl)ethylamine 14 (5.73 gm, 0.038 moles), ethyl
4-bromobutyrate (9.60 gm, 0.049 moles) and diisopropylethylamine
(6.40 gm, 0.050 moles) were heated at 110-120.degree. C. overnight,
cooled, and partitioned between 100 ml DCM and 100 ml water. The
aqueous layer was extracted twice with 50 ml portions of DCM. The
combined organic extracts were washed with saturated NaCl, dried
over Na.sub.2SO.sub.4, filtered, and concentrated by rotary
evaporation under reduced pressure. The crude product was purified
by flash chromatography on silica gel, eluting with a gradient
mixture of ethyl acetate and hexane. Ethyl
4-[ethyl(3-methoxyphenyl)amino]butanoate 15 (9.6 gm, 95% yield) was
isolated as a slightly yellow oil after removing the solvent.
.sup.1H NMR (CDCl.sub.3) .delta. 7.12, 1H, t; 6.31, 1H, d; 6.22,
2H, m; 4.12, 2H, q; 3.79, 3H, s; 3.3, 4H, m; 2.35, 2H, t; 1.9, 2H,
m; 1.26, 3H, t; 1.14, 3H, t.
Example 6
Synthesis of Ethyl 4-[(2,5-dimethylphenyl)ethylamino]butanoate
19
[0160] 30
[0161] 2,5-Dimethylaniline 16 (5.0 gm, 0.041 moles) was dissolved
in 30 ml tetrahydrofuran (THF) at room temperature under argon.
Acetic anhydride (5.9 ml, 0.062 moles) was added. Within 15
minutes, solids formed and the mixture became a solid white mass
after 1 hour. Methanol was added which dissolved the mass. The
mixture was evaporated under reduced pressure, dissolved in hot DCM
and purified by flash chromatography on silica gel, eluting with a
mixture of methanol and DCM. 2,5-Dimethylacetanilide 17 (6.67 gm,
99% yield) was isolated after removing the solvent. .sup.1H NMR
(CDCl.sub.3) .delta. 7.53, 1H, br s; 6.89-7.06, 3H, m; 2.30, 3H, s;
2.19, 3H, s; 2.17, 3H, s.
[0162] 2,5-Dimethylacetanilide 17 (6.67 gm, 0.041 moles) was
dissolved with heating in 65 ml THF and stirred under argon. The
solution was cooled to room temperature and borane-methylsulfide
complex (52 ml, 2.0M, 0.10 moles) was added by syringe over 10
minutes. After 10 minutes at ambient temperature, the mixture was
heated to reflux for 3 hours. After cooling to room temperature, 40
ml water was cautiously added with stirring over 10 minutes,
followed by 3.6 ml conc. HCl. After heating for 30 minutes, 100 ml
DCM and 67 ml 2N NaOH were added sequentially with vigorous
stirring. An emulsion resulted which was eventually separated. The
aqueous portion was extracted with three 50 ml portions of DCM
which were combined, dried over MgSO.sub.4, and filtered. After
concentration by rotary evaporation under reduced pressure, the
crude product was purified by flash chromatography on silica gel,
eluting with a gradient mixture of ethyl acetate and hexane.
(2,5-Dimethylphenyl)ethylamine 18 (5.36 gm, 88% yield) was isolated
after removing the solvent as a colorless oil. .sup.1H NMR
(CDCl.sub.3) .delta. 6.92, 1H, d; 6.63, 2H, m; 3.18, 2H, q; 2.29,
3H, s; 2.08, 3H, s; 1.29, 3H, t.
[0163] (2,5-Dimethylphenyl)ethylamine 18 (5.36 gm, 0.036 moles),
ethyl 4-bromobutyrate (9.11 gm, 0.046 moles) and
diisopropylethylamine (6.07 gm, 0.047 moles) were heated at
110-120.degree. C. overnight, cooled, and partitioned between 100
ml DCM and 100 ml water. The aqueous layer was extracted twice with
50 ml portions of DCM. The combined organic extracts were washed
with saturated NaCl, dried over Na.sub.2SO.sub.4, filtered, and
concentrated by rotary evaporation under reduced pressure. The
crude product was purified by flash chromatography on silica gel,
eluting with a gradient mixture of methanol and DCM. Ethyl
4-[(2,5-dimethylphenyl)ethy- lamino]butanoate 19 (4.66 gm, 49%
yield) was isolated as a slightly yellow oil after removing the
solvent. .sup.1H NMR (CDCl.sub.3) .delta. 7.07, 1H, d; 6.88, 1H, br
s; 6.79, 1H, d; 4.10, 2H, q; 2.9, 4H, m; 2.35, 2H, t; 2.29, 3H, s;
2.24, 3H, s; 1.74, 2H, m; 1.24, 3H, t; 0.98, 3H, t.
Example 7
Synthesis of [3-(Ethylamino)-4-methoxyphenyl]dimethylamine 27
[0164] 31
[0165] 2-Methoxy-5-nitroaniline 23 (13.17 gm, 0.078 moles) was
dissolved in 60 ml tetrahydrofuran (THE) at room temperature under
argon. Acetic anhydride (11.1 ml, 0.118 moles) was added. An orange
solution was formed. The mixture was evaporated under reduced
pressure, and kept under vacuum overnight to give
N-(2-Methoxy-5-nitrophenyl)acetamide 24 (.sup.1H NMR (CDCl.sub.3)
.delta. 9.20, 1H, br s; 7.99, 1H, d; 6.95, 1H, d; 4.02, 3H, s;
3.12, 1H, s; 2.26, 3H, s), which was suspended in 115 ml ethanol.
5% Palladium on carbon (8.05 gm) was added, followed by the slow
addition of 8.05 ml hydrazine hydrate (1 ml/5 minutes). After 90
minutes of heating, TLC indicated the reduction was complete. The
mixture was filtered and the cake was washed with six 100 ml
portions of methanol. The combined filtrate was concentrated under
reduced pressure, taken up in 100 ml methanol and filtered again
through Celite. The filtrate was concentrated to an oil and
triturated with DCM and methanol to give crystals of
N-(3-amino-6-methoxyphenyl)acetamide 25 (12.8 gm, 91% from 23) in
two crops. .sup.1H NMR (CDCl.sub.3) .delta. 7.85, 1H, m; 7.80, 1H,
br s; 6.67, 1H, d; 6.37, 1H, dd; 3.79, 3H, s; 3.5, 2H, br; 2.17,
3H, s.
[0166] N-(3-amino-6-methoxyphenyl)acetamide 25 (1.0 gm, 0.0055
moles) was dissolved in 30 ml DCM at room temperature under argon
with stirring. Diisopropylethylamine (4.83 ml, 0.028 moles) and
dimethylsulfate (2.36 ml, 0.025 moles) were added. After stirring
at room temperature for 8 hours, 30 ml DCM and 40 ml 0.3 N NaOH
were added. The mixture was partitioned and the aqueous layer was
washed with three 30 ml portions of DCM. The combined organic
extract was washed with saturated NaCl, dried over MgSO.sub.4,
filtered, and concentrated to give
N-(3-dimethylamino-6-methoxyphenyl)acetamide 26 (0.57 gm, 50%).
.sup.1H NMR (CDCl.sub.3) .delta. 8.01, 1H, m; 7.75, 1H, br s; 6.79,
1H, d; 6.43, 1H, dd; 3.82, 3H, s; 2.89, 6H, s, 2.19, 3H, s.
[0167] N-(3-dimethylamino-6-methoxyphenyl)acetamide 26 (0.10 gm,
0.48 mmoles) was dissolved in 2 ml THF and stirred under argon at
room temperature. Borane-methylsulfide complex (0.6 ml, 2.0M, 1.2
mmoles) was added by syringe. After 10 minutes at ambient
temperature, the mixture was heated to reflux for 3 hours. After
cooling to room temperature, 0.6 ml water was cautiously added with
stirring 0.1 ml conc. HCl. After heating for 1 hour, 5 ml DCM and
0.3 ml 2N NaOH were added sequentially with vigorous stirring. The
aqueous portion was extracted with three 30 ml portions of DCM
which were combined, dried over MgSO.sub.4, filtered, and
concentrated by rotary evaporation under reduced pressure to give
95 mg of crude [3-(ethylamino)-4-methoxyphenyl]dimethylamine
27.
Example 8
Synthesis of Diazo-diaryl compound 39
[0168] 32
[0169] In a first flask, crushed 2-chloro-4-nitrophenylamine 10
(6.25 gm, 0.036 moles) was suspended in 110 ml water at 0.degree.
C. and 10.8 ml conc. HCl was added. After 10 minutes, NaNO.sub.2
(2.57 gm, 0.037 moles) in 20 ml water was added over 10 minutes.
After stirring at 0.degree. C. for one hour, 420 mg sulfuric acid
(0.004 mole) in 5 ml water was added to the reaction solution. The
reaction solution was directly filtered through a fritted funnel,
collecting and retaining the filtrate.
[0170] To a one-liter three neck flask with overhead stirring was
added 2,5-dimethoxyaniline 1 (5.73 gm, 0.037 moles), 500 ml water,
and 3.6 ml conc. HCl. The mixture was heated to 80.degree. C. to
achieve a yellow solution. The solution was cooled to 0.degree. C.
and the filtrate from the first flask containing the diazonium
compound was added. The thick orange-brown suspension was stirred
at 0.degree. C. for 2 hours. After adding 20 ml 10 N NaOH, the
reaction was stirred for 5 minutes during which it became a
homogeneous purple. The mixture was extracted exhaustively with
DCM. The combined organic extracts were dried over MgSO.sub.4,
filtered, concentrated under reduced pressure and triturated with
methanol and DCM to give crystals of diazo-diaryl compound 39
(11.85 gm, 97%).
Example 9
Synthesis of Diazo-diaryl compound 61
[0171] 33
[0172] Following the procedure of Example 8,
2-chloro-4-nitrophenylamine 10 (1.21 gm, 7.0 mmoles) was diazotized
and reacted with aniline (0.67 gm, 7.2 mmoles) to give diazo-diaryl
compound 61 (1.48 gm, 76%). .sup.1H NMR (CDCl.sub.3) .delta. 9.97,
1H, br s; 8.31, 1H, d; 8.17, 1H, d; 7.83, 1H, d; 7.63, 1H, d; 7.4,
3H, m.
Example 10
Synthesis of Diazo-diaryl compound 72
[0173] 34
[0174] Following the procedure of Example 8,
2-chloro-4-nitrophenylamine 10 (1.21 gm, 7.0 mmoles) was diazotized
and reacted with 1-aminonaphthalene (1.03 gm, 7.2 mmoles) to give
diazo-diaryl compound 72 (1.48 gm, 76%).
Example 11
Synthesis of Diazo-diaryl compound 62
[0175] 35
[0176] Following the procedure of Example 13,
2,6-dichloro-4-nitrophenylam- ine 28 (0.57 gm, 2.73 mmoles) was
diazotized and reacted with ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (0.765 gm, 2.6
mmoles) to give diazo-diaryl compound 62 (0.55 gm, 41%). .sup.1H
NMR (CDCl.sub.3) .delta. 8.24, 2H, s; 7.38, 1H, s; 6.50, 1H, s;
4.13, 2H, q; 4.01, 3H, s; 3.85, 3H, s; 3.44, 4H, m; 2.36, 2H, m;
1.98, 2H, m; 1.27, 3H, t; 1.24, 3H, t. Absorbance max. 474 nm
(methanol.
Example 12
Synthesis of Diazo-diaryl compound 57
[0177] 36
[0178] Following the procedure of Example 8,
2-methoxy-4-nitrophenylamine 23 (0.40 gm, 2.4 mmoles) was
diazotized and reacted with ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (0.65 gm, 2.80
mmoles) to give diazo-diaryl compound 57 (0.71 gm, 68%). .sup.1H
NMR (CDCl.sub.3) .delta. 7.9, 2H, m; 7.69, 1H, d, 7.36, 1H, s,
6.52, 1H, s; 4.13, 2H, q; 4.08, 3H, s; 4.03, 3H, s; 3.87, 3H, s;
3.41, 4H, m; 2.35, 2H, t; 1.94, 2H, m; 1.26, 3H, t; 1.20, 3H, t.
Absorbance max. 525 nm (methanol.
Example 13
Synthesis of Diazo-diaryl ester compound 42
[0179] 37
[0180] 2,4-Dinitroaniline (0.64 gm, 3.5 mmoles) was dried under
vacuum with P.sub.2O.sub.5 and suspended in 3 ml acetic acid at
room temperature under argon with stirring. Nitrosylsulfuric acid
(40%, 0.75 ml, 3.8 mmoles) was added and stirred as a yellowish
solution for 2 hours. The solution was cooled to 0.degree. C. and
ethyl 4-(ethylphenylamino)butanoa- te 6 (0.77 gm, 3.27 mmoles) in 2
ml acetic acid was added which caused an immediate dark red
solution. After stirring at room temperature for 2.5 hours, 10 ml
water was cautiously added, followed by 100 ml DCM, 10 ml 2 N NaOH,
and approximately 14.5 gm of solid sodium carbonate. The mixture
was transferred to a separatory funnel, shaken, and the organic
phase was removed. The aqueous phase was extracted twice with 50 ml
DCM. The combined organic extracts was washed with saturated sodium
chloride, dried over Na.sub.2SO.sub.4, filtered, and concentrated
under reduced pressure. The crude product was purified by silica
gel chromatography, eluting with ethyl acetate and hexane, to give
diazo-diaryl ester 42 (0.92 gm, 66%) after removal of solvent. Abs.
max. 519 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta. 8.68, 1H,
s; 8.41, 1H, d; 7.9, 2H, m; 6.88, 1H, d; 4.15, 2H, q; 3.5, 4H, m;
2.40, 2H, t; 1.98, 2H, m; 1.29, 3H, t; 1.26, 3H, t.
Example 14
Synthesis of Diazo-diaryl ester 47
[0181] 38
[0182] Following the procedure of Example 13, 2,4-dinitroaniline
(0.51 gm, 2.8 mmoles) and ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (0.765 gm, 2.59
mmoles) were converted to diazo-diaryl ester 47 (0.48 gm, 38%).
Abs. max. 563 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta. 8.62,
1 H, s; 8.38, 1H, d; 7.93, 1H, d; 7.34, 1H, s; 6.42, 1H, s; 4.15,
2H, q; 4.04, 3H, s; 3.82, 3H, s; 3.5, 4H, m; 2.4, 2H, t; 2.0, 2H,
m; 1.27, 3H, t; 1.26, 3H, t.
Example 15
Synthesis of Diazo,diaryl acid 48
[0183] 39
[0184] Diazo-diaryl ester 47 (480 mg, 0.98 mmole) was stirred at
room temperature in 6 ml THF. Lithium hydroxide hydrate (0.164 gm,
3.9 mmoles) in 3 ml water was added. The mixture was stirred
overnight, then 10 ml 1 N acetic acid/sodium acetate buffer (pH
5.0) was added. The mixture was extracted with three portions of
DCM. The combined organic extract was washed with saturated NaCl,
dried over Na.sub.2SO.sub.4, filtered, and concentrated under
vacuum. The crude product was purified by silica gel chromatography
to give diazo,diaryl acid 48 (0.324 gm, 72%) after removal of
solvent. Absorbance max. 568 nm (methanol); 587 nm (0.1M TEAA).
.sup.1H NMR (CD.sub.3OD) .delta. 8.68, 1H, s; 8.39, 1H, d; 7.95,
1H, d; 7.33, 1H, s; 6.42, 1H, s; 4.03, 3H, s; 3.82, 3H, s; 3.5, 4H,
m; 2.4, 2H, t; 1.98, 2H, m; 1.26, 3H, t.
Example 16
Synthesis of Diazo-diaryl ester 46
[0185] 40
[0186] Following the procedure of Example 13, 2,4-dinitroaniline
(0.51 gm, 2.8 mmoles) and ethyl 4-(ethylnaphthylamino)butanoate 9
(0.739 gm, 2.59 mmoles) were converted to diazo-diaryl ester 46
(0.58 gm, 47%). Abs. max. 515 nm (methanol). .sup.1H NMR
(CDCl.sub.3) .delta. 8.90, 1H, d; 8.67, 1H, s; 8.41, 1H, d; 8.17,
1H, d; 7.96, 2H, m; 7.6, 2H, dt; 7.13, 1H, d; 4.10, 2H, q; 3.45,
4H, m; 2.38, 2H, t; 1.94, 2H, m; 1.22, 3H, t; 1.19, 3H, t.
Example 17
Synthesis of Diazo-diaryl ester 43
[0187] 41
[0188] Following the procedure of Example 13, 2,4-dinitroaniline
(0.51 gm, 2.8 mmoles) and ethyl
4-[ethyl(3-methoxyphenyl)amino]butanoate 15 (0.69 gm, 2.59 mmoles)
were converted to diazo-diaryl ester 43 (0.49 gm, 41%). Abs. max.
537 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta. 8.62, 1H, s;
8.35, 1H, d; 7.8, 2H, dd; 6.38, 1H, d; 6.30, 1H, s; 4.09, 2H, q;
4.05, 3H, s; 3.5, 4H, m; 2.4, 2H, m; 1.28, 3H, t; 1.27, 3H, t.
Example 18
Synthesis of Diazo,diaryl acid 44
[0189] 42
[0190] Following the procedure of Example 15, diazo-diaryl ester 43
(420 mg, 0.91 mmole) was stirred at room temperature in 6 ml THF.
Lithium hydroxide hydrate (0.153 gm, 3.6 mmoles) in 3 ml water was
added. The mixture was stirred overnight, then 10 ml 1 N acetic
acid/sodium acetate buffer (pH 5.0) was added. The mixture was
extracted with three portions of DCM. The combined organic extract
was washed with saturated NaCl, dried over Na.sub.2SO.sub.4,
filtered, and concentrated under vacuum. The crude product was
purified by silica gel chromatography to give diazo,diaryl acid 44
(0.346 gm, 88%) after removal of solvent. Absorbance max. 537 nm
(methanol); 556 nm (0.1M TEAA).
Example 19
Synthesis of Diazo-diaryl ester 45
[0191] 43
[0192] Following the procedure of Example 13, 2,4-dinitroaniline 11
(0.51 gm, 2.8 mmoles) and Ethyl
4-[(2,5-dimethylphenyl)ethylamino]butanoate 19 (0.68 gm, 2.59
mmoles) were converted to diazo-diaryl ester 45 (0.22 gm, 19%).
Abs. max. 479 nm (THF), 469 nm (methanol). .sup.1H NMR (CDCl.sub.3)
.delta. 8.66, 1H, s; 8.42, 1H, d; 7.80, 1H, d; 7.56, 1H, s; 6.89,
1H, s; 4.12, 2H, q; 3.20, 4H, m; 2.67, 3H, s; 2.32, 2H, m; 2.27,
3H, s; 1.86, 2H, m; 1.25, 3H, t; 1.11, 3H, t.
Example 20
Synthesis of Diazo-diaryl ester 49
[0193] 44
[0194] Following the procedure of Example 13,
2-amino-5-nitrobenzonitrile 20 (0.45 gm, 2.73 mmoles) and ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]bu- tanoate 4 (0.765 gm, 2.59
mmoles) were converted to diazo-diaryl ester 49 (0.27 gm, 22%).
Abs. max. 574 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta. 8.57,
1H, s; 8.38, 1H, d; 7.94, 1H, d; 7.56, 1H, s; 6.41, 1H, s; 4.15,
2H, q; 4.05, 3H, s; 3.86, 3H, s; 3.5, 4H, m; 2.39, 2H, t; 2.0, 2H,
m; 1.27, 6H, t.
Example 21
Synthesis of Diazo,diaryl acid 50
[0195] 45
[0196] Following the procedure of Example 15, diazo-diaryl ester 49
(145 mg, 0.31 mmole) was stirred at room temperature in 4 ml THF.
Lithium hydroxide hydrate (52 mg, 1.2 mmoles) in 1.5 ml water was
added. The mixture was stirred overnight, then 10 ml 1N acetic
acid/sodium acetate buffer (pH 5.0) was added. The mixture was
extracted with three portions of DCM. The combined organic extract
was washed with saturated NaCl, dried over Na.sub.2SO.sub.4,
filtered, and concentrated under vacuum. The crude product was
purified by silica gel chromatography to give diazo,diaryl acid 50
(112 mg, 82%) after removal of solvent. Abs. max. 577 nm
(methanol), 584 nm (0.1M TEAA).
Example 22
Synthesis of Bis-diazo,triaryl PFP ester 51
[0197] 46
[0198] Diazo,diaryl acid 50 (106 mg, 0.24 mmoles) was suspended in
2.5 ml DCM at room temperature under argon with stirring.
Triethylamine (67 .mu.l, 0.5 mmoles) and pentafluorophenyl
trifluoroacetate (53 .mu.l, 0.31 mmoles) and the mixture was
stirred overnight. The mixture was concentrated under vacuum and
purified by silica gel chromatography, eluting with ethyl acetate
and hexane to give the bis-diazo,triaryl PFP ester 51 (117 mg, 80%)
after removal of solvent. .sup.1H NMR (CDCl.sub.3) .delta. 8.55,
1H, s; 8.38, 1H, d; 7.95, 1H, d; 7.54, 1H, s; 6.39, 1H, s; 4.02,
3H, s; 3.88, 3H, s; 3.56, 4H, m; 2.78, 2H, t; 2.15, 2H, m; 1.27,
3H, t; 1.30, 3H, t.
Example 23
Synthesis of Diazo-diaryl ester 52
[0199] 47
[0200] Following the procedure of Example 13,
4-nitro-2-(trifluoromethyl)-- aniline 21 (0.56 gm, 2.73 mmoles) and
ethyl 4-[(2,5-dimethoxyphenyl)ethyla- mino]butanoate 4 (0.765 gm,
2.59 mmoles) were converted to diazo-diaryl ester 52 (0.47 gm,
35%). Abs. max. 549 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta.
8.62, 1H, s; 8.39, 1H, d, 7.95, 1H, d; 7.44, 1H, s, 6.42, 1H, s;
4.15, 2H, q; 4.05, 3H, s; 3.84, 3H, s; 3.49, 4H, m; 2.37, 2H, t;
1.98, 2H, m; 1.27, 3H, t; 1.24, 3H,
Example 24
Synthesis of Diazo,diaryl acid 53
[0201] 48
[0202] Following the procedure of Example 15, diazo-diaryl ester 52
(230 mg, 0.45 mmole) was stirred at room temperature in 4 ml THF.
Lithium hydroxide hydrate (75 mg, 1.8 mmoles) in 1.5 ml water was
added. The mixture was stirred overnight, then 10 ml 1N acetic
acid/sodium acetate buffer (pH 5.0) was added. The mixture was
extracted with three portions of DCM. The combined organic extract
was washed with saturated NaCl, dried over Na.sub.2SO.sub.4,
filtered, and concentrated under vacuum. The crude product was
purified by silica gel chromatography to give diazo,diaryl acid 53
(221 mg, 100%) after removal of solvent. Abs. max. 550 nm
(methanol), 556 nm (0.1M TEAA).
Example 25
Synthesis of Diazo-diaryl ester 54
[0203] 49
[0204] Following the procedure of Example 8,
2-chloro-4-nitrophenylamine 10 (0.21 gm, 1.2 mmoles) and ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]but- anoate 4 (0.325 gm, 1.1
mmoles) were converted to diazo-diaryl ester 54 (0.43 gm, 81%).
Abs. max. 538 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta. 8.38,
1H, s; 8.15, 1H, d; 7.78, 1H, d; 7.44, 1H, s; 6.46, 1H, s; 4.15,
2H, q; 4.04, 3H, s; 3.86, 3H, s; 3.46, 4H, m; 2.37, 2H, t; 1.98,
2H, m; 1.27, 3H, t; 1.23, 3H, t.
Example 26
Synthesis of Diazo,diaryl acid 55
[0205] 50
[0206] Following the procedure of Example 15, diazo-diaryl ester 54
(385 mg, 0.80 mmole) was stirred at room temperature in 6 ml THF.
Lithium hydroxide hydrate (134 mg, 3.2 mmoles) in 3 ml water was
added. The mixture was stirred overnight, then 10 ml 1 N acetic
acid/sodium acetate buffer (pH 5.0) was added. The mixture was
extracted with three portions of DCM. The combined organic extract
was washed with saturated NaCl, dried over Na.sub.2SO.sub.4,
filtered, and concentrated under vacuum. The crude product was
purified by silica gel chromatography to give diazo,diaryl acid 55
(197 mg, 55%) after removal of solvent. Abs. max. 543 nm
(methanol), 554 nm (0.1M TEAA).
Example 27
Synthesis of Diazo-diaryl ester 56
[0207] 51
[0208] Following the procedure of Example 8,
4-(trifluoromethylsulfonyl)an- iline 22 (0.27 gm, 1.2 mmoles) and
ethyl 4-[(2,5-dimethoxyphenyl)ethylamin- o]butanoate 4 (0.325 gm,
1.1 mmoles) were converted to diazo-diaryl ester 56 (0.43 gm, 73%).
Abs. max. 519 nm (methanol). .sup.1H NMR (CDCl.sub.3) .delta. 8.05,
4H, dd; 7.42, 1H, s; 6.51, 1H, s; 4.13, 2H, q; 4.04, 3H, s; 3.87,
3H, s; 3.45, 4H, m; 2.37, 2H, t; 1.99, 2H, m; 1.24, 3H, t; 1.22,
3H, t.
Example 28
Synthesis of Bis-diazo,triaryl compound 40
[0209] 52
[0210] A mixture of ethyl 4-(ethylnaphthylamino)butanoate 9 (611
mg, 2.14 mmoles) and diazo,diaryl compound 39 (793 mg, 2.35 mmoles)
in 42 ml DMF and 0.6 ml conc. HCl was stirred vigorously at room
temperature under argon. After 10 minutes, NaNO.sub.2 (403 mg, 5.8
mmoles) in 4.3 ml water was added as a 0.5 ml aliquot every 15
minutes. When addition was complete, the dark brown solution was
stirred another hour, then partitioned between 60 ml DCM and 30 ml
1 N acetic acid. The aqueous layer was extracted with two 20 ml
portions of DCM. The combined organic extracts were combined, dried
over MgSO.sub.4, filtered, and concentrated under reduced pressure.
The residue was purified by silica gel chromatography, eluting with
a gradient of ethyl acetate and hexane, then methanol and DCM to
give bis-diazo,triaryl 40 (690 mg, 51%) after removal of solvent as
a purple solid. Abs. max. 553 nm (methanol). .sup.1H NMR
(CDCl.sub.3) .delta. 9.0, 1H, d; 8.43, 1H, s; 8.2, 2H, m; 7.9, 2H,
dd; 7.6, 4H, m; 7.20, 1H, d; 4.15, 3H, s; 4.05, 3H, s; 3.6, 4H, m;
2.35, 2H, t; 1.9, 2H, m; 1.6, 2H, m; 1.22, 3H, t; 1.06, 3H, t.
Example 29
Synthesis of Diazo-diaryl compound 63
[0211] 53
[0212] Following the procedure of Example 28, diazo-diaryl 61 (650
mg, 2.35 mmoles) was diazotized and reacted with ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (632 mg, 2.14
mmoles) to give diazo-diaryl compound 63 (0.34 gm, 27%). .sup.1H
NMR (CDCl.sub.3) .delta. 8.35, 1H, s; 8.1, 2H, m, 7.78, 2H, d;
7.57, 1H, s; 7.46, 1H, s; 6.47, 2H, d; 4.15, 2H, q; 4.04, 3H, s;
3.96, 3H, s; 3.47, 4H, m; 2.35, 2H, m; 1.98, 2H, m; 1.27, 3H, t;
1.26, 3H, t. Absorbance max. 539 nm (methanol).
Example 30
Synthesis of Diazo-diaryl compound 64
[0213] 54
[0214] Following the procedure of Example 28, diazo-diaryl 72 (768
mg, 2.35 mmoles) was diazotized and reacted with ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (632 mg, 2.14
mmoles) to give diazo-diaryl compound 64 (0.17 gm, 12%). Absorbance
max. 582 nm (methanol).
Example 31
Synthesis of Bis-diazo,triaryl compound 31 from Fast Black K salt
29
[0215] 55
[0216] Fast Black K salt 29 (Aldrich, Abs. max. (methanol) 330+416
nm, 14.0 gm, 33.9 mmoles) was dissolved in 40 ml acetic acid and 10
ml water at 0.degree. C. under argon with stirring. Ethyl
4-(ethylphenylamino)buta- noate 6 (8.0.0 gm, 33.9 mmoles) dissolved
in 5 ml acetic acid was added. 56
[0217] After one hour, the mixture was allowed to rise to room
temperature and stirring was continued overnight. The solution was
adjusted to pH 4 with 1N NaOH, causing precipitation of the crude
product. The precipitate was dissolved in acetone filtered through
Celite, concentrated under reduced pressure and purified by silica
gel chromatography, eluting with ethyl acetate and hexane.
Bis-diazo,triaryl compound 31 (9.0 gm, 45%) was isolated after
removal of solvent.
Example 32
Synthesis of Bis-diazo,triaryl compound 31 from 38
[0218] Following the procedure of Example 28,
2,5-dimethoxy-4-[(4-nitrophe- nyl)diazenyl]phenylamine 38 (0.50 gm,
2.14 mmoles) was diazotized and reacted with ethyl
4-(ethylphenylamino)butanoate 6 (0.79 gm, 2.38 mmoles) to give
bis-diazo,triaryl 31 (950 mg, 76%). 57
Example 33
Synthesis of Bis-diazo,triaryl compound 30
[0219] 58
[0220] Tetramethoxy bis-diazo,triaryl compound 30 is prepared from
Fast Black K salt 29 and ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butanoate 4 (Example 1) by the
procedure of Example 31.
Example 34
Synthesis of Quencher-NHS compound 36
[0221] 59
[0222] Bis-diazo,triaryl compound 31 (2.40 gm, 4.74 mmoles) was
mixed with lithium hydroxide hydrate (2.00 gm, 47 mmoles), 80 ml
THF, and 20 ml water at room temperature for 26 hours. The mixture
was concentrated under vacuum and partitioned between 200 ml ethyl
acetate and 100 ml water and acidified with 0.5 N HCl. The organic
extract was concentrated under reduced pressure, dried over
Na.sub.2SO.sub.4, filtered, concentrated to give a dark blue solid
which was purified by silica gel chromatography, eluting with 1%
acetic acid in ethyl acetate to give bis-diazo,triaryl acid 34
after removal of solvent. 60
[0223] A mixture of bis-diazo,triaryl acid 34 (80 mg, 0.16 mmoles),
EDC (62 mg, 0.32 mmoles), N-hydroxysuccinimide (22 mg, 0.19 mmoles)
and 10 ml DMF was stirred for 18 hours at room temperature under
argon. The mixture was concentrated under reduced pressure,
dissolved in ethyl acetate and washed with two 40 ml portions of
sat. NaCl. The organic extract was dried over Na.sub.2SO.sub.4,
filtered, and evaporated to give quencher-NHS 36 as a dark blue
solid (60 mg).
Example 35
Synthesis of Bis-diazo, triaryl acid 41
[0224] 61
[0225] The ethyl ester of bis-diazo,triaryl 40 (460 mg, 0.73 mmole)
was hydrolyzed by the procedure in Example 15 to give bis-diazo,
triaryl acid 41.
Example 36
Synthesis of Bis-diazo, triaryl compound 32
[0226] 62
[0227] Fast Black K salt 29 (Aldrich, Abs. max. (methanol) 330+416
nm, 1.0 gm, 2.39 mmoles) was dissolved in 15 ml acetic acid and 5
ml water at 0.degree. C. under argon with stirring. Methyl
2-(ethylphenylamino)acetat- e 7 (0.462 gm, 2.39 mmoles) dissolved
in 2 ml acetic acid was added. After one hour, the mixture was
allowed to rise to room temperature and stirring was continued
overnight. The solution was neutralized with a slight excess of
conc. NH.sub.4OH, causing precipitation of the crude product. The
precipitate was dissolved in 70 ml ethyl acetate, dried over
MgSO.sub.4, filtered, concentrated under reduced pressure and
purified by silica gel chromatography, eluting with ethyl acetate
and hexane. Bis-diazo,triaryl compound 32 (0.504 gm, 40%) was
isolated after removal of solvent. Absorbance max. (methanol) 540
nm. Exact mass by mass spectroscopy: 506.19133, MW 506.51700
(C.sub.25H.sub.26N.sub.6O.sub.6).
Example 37
Synthesis of Bis-diazo,triaryl compound 70
[0228] 63
[0229] Following the procedure of Example 28, a mixture of ethyl
4-(ethylphenylamino)butanoate 6 (0.50 gm, 2.14 mmoles) and
diazo,diaryl compound 39 (793 mg, 2.35 mmoles) in 42 ml DMF and 0.6
ml conc. HCl was stirred vigorously at room temperature under
argon. After 10 minutes, NaNO.sub.2 (403 mg, 5.8 mmoles) in 4.3 ml
water was added as a 0.5 ml aliquot every 15 minutes. When addition
was complete, the dark brown solution was stirred another hour,
then partitioned between 60 ml DCM and 30 ml 1 N acetic acid. The
aqueous layer was extracted with two 20 ml portions of DCM. The
combined organic extracts were combined, dried over MgSO.sub.4,
filtered, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography, eluting with a gradient of
ethyl acetate and hexane, then methanol and DCM to give
bis-diazo,triaryl 70 (950 mg, 76%) after removal of solvent as a
blue-purple solid. Absorbance max. (methanol) 569 nm. .sup.1H NMR
(CDCl.sub.3) .delta. 8.40, 1H, s; 8.18, 1H, d; 7.9, 2H, d; 7.8, 1H,
d; 7.55, 1H, s; 7.42, 1H, s; 6.78, 1H, d; 4.18, 2H, q; 4.09, 3H, s;
4.04, 3H, s; 3.5, 4H, m; 2.4, 2H, t; 2.0, 2H, m; 1.29, 3H, t; 1.27,
3H, t.
Example 38
Synthesis of Bis-diazo,triaryl acid 60
[0230] 64
[0231] The ethyl ester of bis-diazo,triaryl 70 (950 mg, 1.63 mmole)
was hydrolyzed by the procedure in Example 15 to give bis-diazo,
triaryl acid 60 (527 mg, 58%) as a blue solid. Absorbance max. 571
nm (methanol); 487 nm (0.1M TEAA). .sup.1H NMR (CDCl.sub.3) .delta.
8.42, 1H, s; 8.20, 1H, d; 7.94, 2H, d; 7.82, 1H, d; 7.55, 1H, s;
7.43, 1H, s; 6.78, 2H, d; 4.10, 3H, s; 4.04, 3H, s; 3.5, 4H, m;
2.42, 2H, m; 2.0, 2H, m; 1.25, 3H, t.
Example 39
Synthesis of Bis-diazo,triaryl compound 71
[0232] 65
[0233] Following the procedure of Example 28, diazo,diaryl compound
39 (793 mg, 2.35 mmoles) and ethyl
4-[(2,5-dimethoxyphenyl)ethylamino]butano- ate 4 (632 mg, 2.14
mmoles) were reacted to give bis-diazo,triaryl compound 71 as a
blue solid (470 mg, 34%). Absorbance max. (methanol) 592 nm.
.sup.1H NMR (CDCl.sub.3) .delta. 8.62, 1H, s; 8.2, 1H, d; 7.82, 1H,
d; 7.54, 1H, s; 7.4, 2H, s; 6.52, 1H, s; 4.10, 3H, s; 4.06, 3H, s;
4.04, 3H, s; 3.89, 3H, s; 3.8, 2H, m; 3.40, 4H, m; 2.38, 2H, t;
1.9, 2H, m; 1.26, 3H, t; 1.21, 3H, t.
Example 40
Synthesis of Bis-diazo,triaryl PFP ester 59
[0234] 66
[0235] Following the procedure of Example 15, the ethyl ester of
bis-diazo,triaryl compound 71 (470 mg, 0.73 mmoles) was hydrolyzed
to give bis-diazo,triaryl acid 58 (275 mg, 61%) as a blue solid.
Absorbance max. 601 nm (methanol); 553 nm (0.1M TEAA). .sup.1H NMR
(CDCl.sub.3) .delta. 8.42, 1H, s; 8.20, 1H, d; 7.82, 1H, d; 7.57,
1H, s; 7.40, 2H, s; 6.55, 1H, s; 4.10, 3H, s; 4.04, 3H, s; 4.03,
3H, s; 3.88, 3H, s; 3.4, 4H, m; 2.75, 2H, t; 2.1, 2H, m; 1.23, 3H,
t. 67
[0236] Following the procedure of Example 22, bis-diazo,triaryl
acid 58 (261 mg, 0.42 mmoles) was suspended in 4 ml DCM at room
temperature under argon with stirring. Triethylamine (0.12 ml, 0.86
mmoles) and pentafluorophenyl trifluoroacetate (0.094 ml, 0.55
mmoles) and the mixture was stirred overnight. The mixture was
concentrated under vacuum and purified by silica gel
chromatography, eluting with ethyl acetate and hexane to give the
bis-diazo,triaryl PFP ester 59 (251 mg, 76%) after removal of
solvent.
Example 41
Synthesis of Hydroxy phenazonium compound 73
[0237] 68
[0238] A solution of 3-amino-7-(diethylamino)-5-phenyl-phenazinium
chloride 74 (Methylene violet 3RAX, Aldrich, 0.379 gm, 1.0 mmole)
in 12 ml sulfolane (tetramethylene sulfone) and 4 ml CH.sub.3CN was
added to nitrosonium tetrafluoroborate (NOBF.sub.4, 0.129 gm, 1.1
mmoles) dissolved in 4 ml sulfolane (tetramethylene sulfone) and 12
ml CH.sub.3CN at 0.degree. C. under argon with stirring. 69
[0239] After stirring for 15 minutes, 2-(N-ethylanilino)ethanol
(0.182 gm, 1.1 mmoles) was added. After stirring for 30 minutes,
the mixture was poured into 120 ml 1N HCl. The crude product was
purified by reverse phase HPLC to give hydroxy-phenazonium 73 (110
mg, 20%) as a blue solid. Absorbance max. (methanol) 662 nm. Exact
mass by mass spectroscopy: 519.29; MW 519.66;
C.sub.32H.sub.35N.sub.6O. .sup.1H NMR (CD.sub.3OD) .delta. 7.5-8.2,
11H, m; 7.1, 1H, d; 6.7, 2H, dd; 5.7, 1H, d; 3.5-3.9, 10H, m; 1.2,
9H, m. .sup.13C NMR (CD.sub.3OD) .delta. 157.22, 156.65, 154.34,
144.93, 144.43, 140.37, 138.90, 137.17, 135.77, 134.71, 133.79,
133.03, 132.97, 128.82, 128.22, 124.79, 121.44, 113.17, 111.73,
93.24, 60.38, 53.68, 47.97, 47.88, 47.23, 12.64.
Example 42
Synthesis of Quencher Phosphoramidite Compound 75
[0240] 70
[0241] Hydroxyl-quencher compound 77 (1.97 gm, 1.67 mmoles) and
diisopropylammonium 5-H-tetrazolide (0.337 gm, 1.69 mmoles) are
dissolved in 55 ml DCM. 2-Cyanoethyl-N,N-tetraisopropyl
phosphoramidite ((iPr.sub.2N).sub.2--P--OCH.sub.2CH.sub.2CN, 1.28
ml, 1.206 gm, 4.00 mmoles) was added. The solution was stirred at
room temperature under argon overnight, then diluted with DCM,
washed with saturated NaHCO.sub.3 and saturated NaCl, dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced
pressure. The crude product was purified by silica gel
chromatography, eluting with acetone, triethylamine, and DCM to
give quencher phosphoramidite 75 (2.30 gm, 85%) as a purple foam
after removal of solvent. 31P NMR showed 99.5% purity.
Example 43
Synthesis of Hydroxyl quencher linker 77
[0242] Fmoc linker compound 82 (3 g, 4.46 mmol) was treated with
tert-butyl dimethylsilyl chloride (807 mg, 5.35 mmol) and imidazole
(425 mg, 6.24 mmol) in DMF (15 ml) at room temperature for 18 h.
DMF was removed under reduced pressure. The residue was dissolved
in 70 ml ethyl acetate and washed with 50 ml water and 50 ml
saturated brine. The organic extract was dried over
Na.sub.2SO.sub.4 and evaporated to give Fmoc silyl 83 as a sticky
foam, which was treated with 20% piperidine in 70 ml DMF at ambient
temperature for 8 h. The DMF and piperidine were removed under
reduced pressure and the residual oil was applied to a silica gel
column. The column was eluted with 0-5% methanol in DCM gradient.
Appropriate fractions were combined and evaporated to give
silyl-amino compound 84 as an oil (1.78 g, 70% yield).
[0243] A mixture of silyl-amino 84 (1.77 g, 3.13 mmol), succinic
anhydride (408 mg, 4.08 mmol), triethylamine (474 mg, 4.69 mmol)
and dimethylaminopyridine (191 mg, 1.56 mmol) in dichloromethane
(20 ml) was stirred at ambient temperature for 18 h. The mixture
was diluted with DCM (20 ml) and washed with 30 ml 5% aqueous
citric acid solution and 30 ml saturated brine. The organic extract
was dried over Na.sub.2SO.sub.4 and concentrated. The crude product
was applied to a silica gel column and eluted with 5-10% methanol
in DCM gradient. Appropriate fractions were combined and evaporated
to give succinate amide ester 85 as a thick gum (1.78 g, 85%).
71
[0244] A solution of 79 (Example 48, 3.15 g, 4.59 mmol) in DCM (30
ml) was added to a stirred solution of
2,2'(ethylenedioxy)bis-ethylamine (6.78 g, 45.90 mmol) in 50 ml DCM
at ambient temperature under argon. After 1 hour the mixture was
washed with 40 ml water and 50 ml saturated brine. The organic
extract was dried (Na.sub.2SO.sub.4) and concentrated and loaded on
a silica gel column. The column was eluted with 5-15% methanol in
DCM gradient containing 5% triethylamine. Appropriate fractions
were combined and evaporated to give amino quencher 86 as purple
foam (2.79 g, 93%). 72
[0245] Diisopropylethyl amine (1.04 g, 8.04 mmol) and
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate (1.02 g, 2.68 mmol) were added to a solution of
succinate amide ester 85 (1.78 g, 2.68 mmol) and amino-quencher 86
(1.92 g, 2.94 mmol) in DMF (70 ml). The mixture was stirred at
ambient temperature for 24 h. DMF was removed under reduced
pressure. The residue was dissolved in DCM (100 ml) and washed with
water (1.times.50 ml) and sat. brine (1.times.50 ml). The organic
extract was dried (Na.sub.2SO.sub.4) and evaporated. The product
was purified by silica gel column eluting with a 0-5% methanol in
DCM gradient to give silyl 87 as a purple foam (3.2 g, 87%).
Tetrabutylammonium fluoride (1M, 5 ml, 5 mmol) was added to a
solution of silyl 87 (3.2 g, 2.46 mmol) in THF (50 ml) at ambient
temperature. After 48 h THF was removed and the residue was
dissolved in DCM (15 ml) and applied to a silica gel column. The
column was eluted with 0-10% methanol in DCM gradient containing 2%
triethylamine. Appropriate fractions were combined and evaporated
to give 77 as a purple foam (2.4 g, 82%). 73
Example 44
Synthesis of quencher phosphoramidite Compound 88
[0246] 74
[0247] Hydroxy-quencher compound 81 (Example 48, 2.2 g, 2.31 mmol)
and diisopropylammonium-5-H-tetrazole (230 mg, 1.15 mmol) dissolved
in DCM (30 ml). 2-Cyanoethyl-N,N-tetraisopropyl phosphoramidite
((iPr.sub.2N).sub.2P--OCH.sub.2CH.sub.2CN (1.04 g, 3.46 mmol) was
added. The solution was stirred at ambient temperature under argon
for 23 h. The solution was diluted with DCM (30 ml), washed with
10% NaHCO.sub.3 and saturated brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure. The crude product
was purified by silica gel column eluting with 10-30% ethyl acetate
in DCM containing 1% triethylamine. Appropriate fractions were
combined and evaporated to give quencher phosphoramidite 88 as a
purple foam (2.27 g, 84%). .sup.31P NMR showed 99.5% purity.
Example 45
Synthesis of quencher-oligonucleotides with quencher
phosphoramidite 88
[0248] 5' Q-T.sub.5 and 5' T.sub.2-Q-T.sub.5 3' oligonucleotides
were synthesized at the 0.2 .mu.mole scale using quencher
phosphoramidite 88, 3' phosphoramidite thymidine and 3' thymidine
on polystyrene support in a synthesis column. Automated trityl
monitoring showed 98.5% coupling efficiency for quencher
phosphoramidite 88
Example 46
Synthesis of Quencher-support Compound 65 from 32
[0249] 75
[0250] Bis-diazo,triaryl compound 32 (2.40 gm, 4.74 mmoles) was
mixed with lithium hydroxide hydrate (2.00 gm, 47 mmoles), 80 ml
THF, and 20 ml water at room temperature for 26 hours. The mixture
was concentrated under vacuum and partitioned between 200 ml ethyl
acetate and 100 ml water and acidified with 0.5 N HCl. The organic
extract was concentrated under reduced pressure, dried over
Na.sub.2SO.sub.4, filtered, concentrated to give a dark blue solid
which was purified by silica gel chromatography, eluting with 1%
acetic acid in ethyl acetate to give bis-diazo,triaryl acid 35
after removal of solvent. Abs. max: 548 nm, .epsilon. (extinction
coefficient): 15,023; 260 nm, .epsilon.: 4273 (methanol). Abs. max:
570 mn, .epsilon.: 8062; 260 nm, .epsilon.: 2160 (0.1M TEAA). Exact
mass by mass spectroscopy: 492.17568, MW 492.49020
(C.sub.24H.sub.24N.sub.6O.sub.6). 76
[0251] A mixture of
O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU, 20 mg, 52 .mu.moles),
1-hydroxybenzotriazole (HOBt, 7 mg, 52 .mu.moles),
diisopropylethylamine (23 .mu.l, 130 .mu.moles), bis-diazo,triaryl
acid 35 (13 mg, 26 .mu.moles), amino, DMT, CPG support 66 (200 mg,
26 .mu.moles/gm, 5.2 .mu.moles), 77
[0252] and 1.5 ml dimethylformamide (DMF) was shaken at room
temperature for 24 hours (Mullah, U.S. Pat. No. 5,736,626). The
support was washed with DMF, CH.sub.3CN, and capped with a mixture
of acetic anhydride, N-methylimidazole, pyridine, and THF for 45
minutes. The support was washed with CH.sub.3CN and DCM, and dried
under vacuum to give quencher support 65. From HPLC analysis, the
coupling yield of 35 to support 66 was measured at 78%.
Example 47
Synthesis of Quencher-support compound 69 from quencher-NHS 36
[0253] 78
[0254] A mixture of quencher-NHS 36 (21 mg, 44 .mu.moles), amino,
DMT, CPG support 66 (125 mg, 35 .mu.moles/gm, 4.4 .mu.moles),
diisopropylethylamine (23 .mu.l, 132 .mu.moles) and 1 ml DMF was
shaken at room temperature for 25 hours. The support was washed
with DMF, CH.sub.3CN, and capped with a mixture of acetic
anhydride, N-methyl imidazole, pyridine, and THF for 1 hour. The
support was washed with CH.sub.3CN and DCM, and dried under vacuum
to give quencher support 69. From HPLC analysis, the coupling yield
of 36 to support 66 was measured at 90%.
Example 48
Synthesis of Quencher-support compound 69 from diglycolate Ester
80
[0255] 79
[0256] Diglycolic anhydride (85 mg, 0.73 mmol) was added to a
mixture of compound 81 (500 mg, 0.52 mmol) and triethylamine (105
mg, 1.03 mmol) in DCM (15 ml) at 0.degree. C. (ice bath). After 30
min the reaction mixture was transferred to ambient temperature and
stirred for 2 h. The mixture was diluted with DCM (20 ml) and
washed with 20 ml 5% aqueous citric acid solution. Triethylamine
(0.5 ml) was added to the organic extract and the extract was dried
over Na.sub.2SO.sub.4. Evaporation of solvent gave diglycolate
ester 80 as a purple foam (triethylamine salt, 600 mg). 80
[0257] Hydroxyl-quencher 81 is prepared from a mixture of amino-DMT
compound 78 (2.5 g, 5.39 mmol) and PFP ester 79 (4.07 g, 5.90 mmol)
in DCM (60 ml), treated with triethylamine (1.09 g, 10.8 mmol) at
ambient temperature for 16 h (overnight). PFP ester 79 was prepared
from acid 34 by the procedure of Examples 22 and 40. The mixture
was concentrated and applied to a silica gel column. The column was
eluted with a 0-5% methanol in DCM containing 1% triethylamine .
Fractions containing products were combined and evaporated to give
hydroxyl-quencher 81 as a purple foam (4.65 g, 90%). 81
[0258] A mixture of 80 (428 mg, 450 .mu.mol), aminopropyl CPG (5 g,
225 .mu.mol amine per gram loading), diisopropylethylamine (87 mg,
675 .mu.mol), HBTU (85 mg, 225 .mu.mol) and DMF (12 ml) was shaken
at ambient temperature for 23 h. The support was washed with DMF,
THF, and capped with a mixture of acetic anhydride, N-methyl
imidazole, pyridine and THF for 1 h. The support was washed with
THF, CH.sub.3CN and DCM, and dried under vacuum. The dye loading
was calculated from trityl assay to be 33.6 .mu.mol/g.
Example 49
Synthesis of 3' Quencher-T.sub.5 Oligonucleotide 68
[0259] 82
[0260] Quencher support 65 (Example 46, 8 mg, 200 nmoles) was
loaded into a standard synthesis column and used to synthesize a
T.sub.5 oligonucleotide on an ABI 394 Synthesizer (Applied
Biosystems, Foster City, Calif.) by five successive couplings of
thymidine 3' cyanoethylphosphoramidite with the standard reagents,
conditions, and cycle recommended and provided by the manufacturer
(Applied Biosystems) to give about 8 odu (A260 nm) of approximately
58% pure, 3' quencher T.sub.5 on CPG support 67: 83
[0261] After cleavage of the diglycolate ester of 67 with conc.
NH.sub.4OH for 2 hours at room temperature, the mixture was
filtered and the eluate containing about 8 odu (A260 nm) of 68 was
dried under vacuum (Abs. max. 560 mn in 50 mM TEAA at pH 8.0) and
analyzed by reverse phase HPLC, eluting with CH.sub.3CN in aqueous
TEAA (triethylamine acetate) to be 58% pure.
[0262] The corresponding 3' quencher-T.sub.5 oligonucleotide
synthesized from support 69 was prepared and analyzed by the same
procedure above, in 78% purity (8 odu) using 69 prepared by the
method of Example 47, and in 92% purity (9 odu) using 69 prepared
by the method of Example 48.
Example 50
Synthesis of 3' Quencher, 5' Fluorescent Dye
Oligonucleotides--GAPDH Probes
[0263] Four 3'-quencher, 5'-fluorescent dye oligonucleotides
probes:
[0264] 5.degree. F-CGC CTG GTC ACC AGG GCT GC-Q 3' SEQ ID NO: 1
[0265] where F is a reporter dye and Q is a quencher moiety, were
synthesized on an ABI 394 Synthesizer (Applied Biosystems) with 3'
nucleoside cyanoethylphosphoramidites (A.sup.bz, G.sup.dmf,
C.sup.bz, T), ancillary reagents, cycle, and post-synthesis
procedures recommended and provided by the manufacturer (Applied
Biosystems) and in Mullah, U.S. Pat. No. 5,736,626. Syntheses were
conducted on quencher support 69 (Example 48) at the 200 nmole
scale. After addition of 3' nucleoside phosphoramidites to complete
the sequence from 3' to 5', the last coupling was with each of the
four fluorescent dye phosphoramidite reagents: 84
[0266] (Menchen, U.S. Pat. No. 5,188,934; Benson, U.S. Pat. Nos.
6,020,481 and 6,008,379) where X is the site of attachment to the
nitrogen of the linker to form an amide bond and the phenolic
oxygen atoms were protected as pivalate (tert-butyl) esters
(Theisen (1992) "Fluorescent dye phosphoramidite labelling of
polynucleotides", in Nucleic Acid Symposium Series No. 27, Oxford
University Press, Oxford, pp. 99-100). The average yield per cycle
was about 98%, as measured by trityl monitoring. The dual-labelled
probes were cleaved from the CPG support, and the nucleobase and
phosphate protecting groups were removed, with concentrated
NH.sub.4OH. Deprotection can also be accomplished with a mixture of
tert-butylamine/methanol/water: 1/1/2. The crude probes were
analyzed and purified by reverse phase HPLC.
Example 51
PCR Assay with 3'-quencher, 5'-fluorescent Dye GAPDH Probes
[0267] A 5' nuclease detection assay of PCR was conducted on the
ABI Prism 7700 Sequence Detection System (Applied Biosystems) with
the four dual-labelled probes of Example 50. PCR amplification
reactions (50 .mu.l) contained cDNA template nucleic acid from Raji
cell RNA, 2.times.TaqMan universal Master mix (25 .mu.l) including
PCR buffer, dNTPs (dATP, dGTP, dCTP, TTP), and MgCl.sub.2 (Applied
Biosystems), AmpliTaq Gold DNA polymerase, forward (SEQ ID NO: 2)
and reverse (SEQ ID NO: 3) primers:
2 Forward Primer: 5' GGG AAG GTG AAG GTC GGA GTC 3' SEQ ID NO:2
Reverse Primer: 5' CTG GAA GAT GGT GAT GGG ATT TC 3' SEQ ID
NO:3
[0268] and a probe from Example 50, each at 100 nM final
concentration. The target sequence was the GAPDH region. The
thermal cycling protocol began with 2 min at 50.degree. C. and 10
min at 95.degree. C., then proceeded with 40 cycles of 95.degree.
C. for 15 sec and 60.degree. C. for 1 min. Reaction conditions were
programmed on a Power Macintosh G3 (Apple Computer, Calif.) linked
directly to the ABI 7700 Sequence Detector. Analysis of data was
also performed on a Macintosh computer with collection and analysis
software (Applied Biosystems). Results of the real-time detection
assay using Example 50 probes with the bis-diazo, triaryl quencher
moiety (34) were compared with the same set of probes (SEQ ID NO:
1) labelled with the same fluorescent dye reporters,
F.sub.1-F.sub.4, but with TAMRA as the 3' quencher.
Example 52
Synthesis of Quencher-polypeptide with a quencher-isothiocyanate
Reagent
[0269] A procedure for labelling a polypeptide with a
quencher-isothiocyanate is illustrated by the steps:
[0270] 1. Prepare the polypeptide at 1-5 mg ml.sup.-1 in 0.1 M
NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, pH 8.5.
[0271] 2. Prepare a solution of the quencher-isothiocyanate at 50
mM in DMSO and add to the protein to give a final concentration of
2 mM. Mix and allow to react overnight at room temperature in the
dark.
[0272] 3. De-salt the product, e.g. on a size-exclusion gel media
cartridge, into the buffer of choice.
[0273] 4. Characterize as follows: record absorbance spectrum at
1:20 dilution, blanked against the column buffer. Determine protein
concentration by a standard method (e.g. Coomassie or BCA methods)
allowing for carrier BSA if present, or from the spectrum.
Example 53
Synthesis of Quencher-polypeptide with a quencher-NHS reagent
[0274] A procedure for labelling a polypeptide, where the
polypeptide is a relatively dilute protein (1 mg ml.sup.-1), with a
quencher-NHS ester is illustrated by the steps:
[0275] 1. Prepare protein in 50 mM sodium phosphate, pH 7.5 at ca 1
mg ml.sup.-1 (1.0 ml). Higher protein concentrations may be
used.
[0276] 2. Prepare a fresh 20 mM solution of quencher-NHS ester in
DMSO, and add 20 .mu.l of the reagent to the protein, stirring with
the pipette tip during addition. Final reagent concentration is 0.4
mM. Allow to react at room temperature for 1 h.
[0277] 3. Equilibrate a size exclusion gel media cartridge, e.g.
Sephadex.TM. G25 (Pharmacia PD-10) with PBS containing 1 mg
ml.sup.-1 BSA (heat-treated for 30 min at 56.degree. C.). BSA is
optional but helps improve protein recovery.
[0278] 4. Stop reaction by addition of 1/10th volume of 0.1 M
ethanolamine, pH 8.5 (0.1 ml) and allow to react for a further 10
min.
[0279] 5. Apply the reaction to the equilibrated cartridge,
followed by 1.8 ml buffer. Then add further 1.6 ml and collect the
colored product band.
[0280] 6. Characterize as follows: record absorbance spectrum at
1:20 dilution, blanked against the column buffer. Determine protein
concentration by a standard method (e.g. Coomassie, or BCA)
allowing for carrier BSA if present, or from the spectrum (less
accurate if carrier protein is present) or assume a column recovery
factor.
[0281] 7. Store at 4.degree. C. with 0.05% NaN.sub.3 (away from
light) or at -20.degree. C.
Example 54
Synthesis of Fluorescein/Quencher-polypeptide
[0282] A general procedure for labelling a polypeptide with a
quencher-NHS ester to prepare a fluorescein/quencher FRET protease
substrate is illustrated by the steps:
[0283] 1. Dissolve the polypeptide (having a free N-terminus, no
other amines and a cysteine in 0.1 M NaH2PO.sub.4/Na.sub.2HPO.sub.4
buffer, pH 7.5, to a concentration of 2 mM. Allow for non-peptidic
material in calculating the concentration. Peptides with free
thiols are prone to dimerization. Handle in degassed buffers and
react the thiol as soon as possible after dissolution. It is
advisable to check the free thiol content before reaction. If the
peptide does not dissolve, reduce the ionic strength of the buffer
and/or the peptide concentration. Water may be used provided the
resulting pH is in the range 68. Alternatively, co-solvents such as
DMSO may be added.
[0284] 2. Prepare a 10 mM solution of fluorescein-5-maleimide
(Molecular Probes) in DMSO. Add one molar equivalent to the peptide
and allow to react for 10 min. Because of uncertainties in the
peptide and fluorescein-5-maleimide concentration, it is advisable
to react, say, 0.75 equivalents of reagent, measure the thiol
content and then add smaller reagent aliquots until the thiol is
all reacted. The maleimido-thiol reaction is fast at these
concentrations, but will require longer incubations at lower
concentrations.
[0285] 3. Measure the thiol content using Ellman's method: take 10
.mu.l of the reaction, dilute to 1.0 ml in PBS (or above phosphate
buffer) in a cuvette and zero the spectrophotometer. Add 20 .mu.l
of 2 mM DTNB and record the OD at 412 nm. This should be zero
(check also unreacted peptide).
[0286] 4. Prepare a 50 mM solution of quencher NHS ester in DMSO.
Add to the fluoresceinylated peptide to a final concentration of 10
mM and react for 30 mm at room temperature.
[0287] 5. Prepare a size-exclusion gel media column, e.g.
Sephadex.TM. G10, of volume greater than 10 times the reaction
volume in a disposable column. Equilibrate in 50 mM Tris-HCI, pH
7.5, or running buffer of choice. If insolubility problems have
been encountered, there is a possibility of the product
precipitating on the column. Experiment to find a suitable buffer:
water may be a good choice.
[0288] 6. Apply the reaction mixture to the column. Add buffer to
elute and collect the first eluting colored band.
[0289] 7. Run spectrum to confirm that peaks corresponding to both
fluorescein (495 nm) and quencher are present. Analyze and purify
by reverse-phase HPLC and characterize by mass spectrometry.
[0290] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
[0291] The invention now having been fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the invention.
Sequence CWU 1
1
3 1 20 DNA Homo sapiens 1 cgcctggtca ccagggctgc 20 2 21 DNA Homo
sapiens 2 gggaaggtga aggtcggagt c 21 3 23 DNA Homo sapiens 3
ctggaagatg gtgatgggat ttc 23
* * * * *