U.S. patent application number 13/557065 was filed with the patent office on 2012-11-22 for aza-benzazolium containing cyanine dyes.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Richard P. Haugland, Stephen T. Yue.
Application Number | 20120295817 13/557065 |
Document ID | / |
Family ID | 26829787 |
Filed Date | 2012-11-22 |
United States Patent
Application |
20120295817 |
Kind Code |
A1 |
Haugland; Richard P. ; et
al. |
November 22, 2012 |
Aza-benzazolium Containing Cyanine Dyes
Abstract
Unsymmetrical cyanine dyes that incorporate an aza-benzazolium
ring moiety are described, including cyanine dyes substituted by a
cationic side chain, monomeric and dimeric cyanine dyes, chemically
reactive cyanine dyes, and conjugates of cyanine dyes. The subject
dyes are virtually non-fluorescent when diluted in aqueous
solution, but exhibit bright fluorescence when associated with
nucleic acid polymers such as DNA or RNA, or when associated with
detergent-complexed proteins. A variety of applications are
described for detection and quantitation of nucleic acids and
detergent-complexed proteins in a variety of samples, including
solutions, electrophoretic gels, cells, and microorganisms.
Inventors: |
Haugland; Richard P.;
(Eugene, OR) ; Yue; Stephen T.; (Eugene,
OR) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
26829787 |
Appl. No.: |
13/557065 |
Filed: |
July 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12984380 |
Jan 4, 2011 |
8252530 |
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13557065 |
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11756765 |
Jun 1, 2007 |
7871773 |
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12984380 |
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10683753 |
Oct 13, 2003 |
7226740 |
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11756765 |
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09557275 |
Apr 24, 2000 |
6664047 |
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10683753 |
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60131782 |
Apr 30, 1999 |
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60158859 |
Oct 12, 1999 |
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Current U.S.
Class: |
506/9 ; 435/5;
435/6.1; 436/86; 436/94 |
Current CPC
Class: |
Y10S 436/80 20130101;
G01N 33/5308 20130101; G01N 33/531 20130101; C09B 23/04 20130101;
G01N 2001/302 20130101; C12Q 1/6876 20130101; C12Q 1/6816 20130101;
G01N 1/30 20130101; G01N 33/68 20130101; G01N 33/52 20130101; G01N
33/582 20130101; C12Q 2563/107 20130101; C12Q 1/6816 20130101; C12Q
2563/173 20130101; C12Q 2563/107 20130101; G01N 27/44726 20130101;
C12Q 2563/173 20130101; Y10T 436/143333 20150115; C09B 23/02
20130101; C12Q 1/6816 20130101 |
Class at
Publication: |
506/9 ; 436/86;
436/94; 435/6.1; 435/5 |
International
Class: |
G01N 21/25 20060101
G01N021/25; C40B 30/04 20060101 C40B030/04 |
Claims
1. A method for determining the presence of an analyte of interest
in a sample, wherein said method comprises: a) combining said
sample with a staining solution, wherein said staining solution
comprises one or more dyes having formula ##STR00092## wherein A
represents the atoms necessary to form one to two fused aromatic
rings having 6 atoms in each ring, at least one of which is a
nitrogen atom, said ring or rings being optionally further
substituted one or more times by C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or
-L-Sc; wherein Rx is a reactive group; Sc is a conjugated
substance; and L and BRIDGE are independently a single covalent
bond, or a covalent linkage; X is O, S, Se, NR.sup.15, or
CR.sup.16R.sup.17, where R.sup.15 is H or an alkyl group having 1-6
carbons; and R.sup.16 and R.sup.17, which may be the same or
different, are independently alkyl groups having 1-6 carbons, or
R.sup.16 and R.sup.17 taken in combination complete a five or six
membered saturated ring; R.sup.2 is selected from the group
consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having
1-6 carbons that is optionally substituted by sulk), carboxy,
amino, substituted amino or substituted ammonium, wherein a is 0 or
1; and TAIL is a heteroatom-containing moiety; Y is
--CR.sup.3.dbd.CR.sup.4-- wherein p and m=0 or 1, such that p m=1;
R.sup.3, R.sup.4, R.sup.6, and R.sup.7 are independently selected
from the group consisting of hydrogen, a C.sub.1-C.sub.6 alkyl, a
halogen, a CYCLIC SUBSTITUENT, --OR.sup.8, --SR.sup.8,
--(NR.sup.8R.sup.9), TAIL; BRIDGE, -L-Rx and -L-Sc; where R.sup.8
and R.sup.9 are independently a C.sub.1-C.sub.6 alkyl group, 1-2
alicyclic or aromatic rings; or R.sup.8 and R.sup.9 taken in
combination are --(CH.sub.2).sub.2--V--(CH.sub.2).sub.2-- where V
is a single bond, --O--, --CH.sub.2--, or --NR.sup.10--, where
R.sup.10 is H or an alkyl having 1-6 carbons; wherein CYCLIC
SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or
C.sub.3-C.sub.10 cycloalkyl; or R.sup.6 and R.sup.7 form a fused
aromatic ring --R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14--
wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are
independently selected from the group consisting of hydrogen,
C.sub.1-C.sub.8 alkyl group, --OR.sup.8, --(NR.sup.8R.sup.9), a
CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; R.sup.5 is
independently selected from the group consisting of a
C.sub.1-C.sub.6 alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE,
-L-Rx and -L-Sc; or R.sup.5 is absent; R.sup.30, R.sup.31, and
R.sup.32 are independently selected from the group consisting of
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.10 cycloalkyl, aryl,
and heteroaryl, wherein n=0, 1 or 2; wherein, BRIDGE, when present,
is bound to a compound having formula I or another unsymmetrical
cyanine dye b) incubating said sample and said staining solution
for a sufficient amount of time to form a dye-analyte complex; c)
illuminating said complex with an appropriate wavelength whereby
the presence of said analyte is determined.
2. The method according to claim 1, wherein said analyte of
Interest is a poly(amino acid) or a nucleic acid.
3. The method according to claim 2, wherein said poly(amino acid)
is in solution or immobilized on a solid or semi solid matrix.
4. The method according to claim 3, wherein said matrix is a
polymeric gel, an array, a polymeric microparticle or a
membrane.
5. The method according to claim 4, wherein said gel is a
polyacrylamide gel.
6. The method according to claim 3, wherein said method further
comprises heating said sample prior to combining with said staining
solution, or healing combined sample and staining solution
mixture.
7. The method according to claim 3, wherein said method further
comprises removing, destroying, or dispersing below the critical
micelle concentration any biological membranes that are present in
said sample.
8. The method according to claim 3, wherein said method further
comprises adding an anionic detergent to the sample, staining
solution or combined sample and staining solution mixture.
9. The method according to claim 2, wherein said nucleic acid is in
solution, immobilized on a solid or semi-solid matrix or present in
a biological structure.
10. The method according to claim 9, wherein said matrix is a
polymeric gel, an array, a glass slide, a polymeric microparticle
or a membrane.
11. The method according to claim 10, wherein said gel is an
agarose gel.
12. The method according to claim 9, wherein said biological
structure is a biological cell, a virus particle or a tissue
section.
13. A method for the detection of immobilized poly(amino acids),
wherein method comprises: a) immobilizing said poly(amino acids) on
a solid or semi-solid matrix; b) combining said sample with a
staining solution prior, during or after immobilization of said
poly(amino acid), wherein said staining solution comprises a
compound having formula ##STR00093## wherein A represents the atoms
necessary to form one to two fused aromatic rings having 6 atoms in
each ring, at least one of which is a nitrogen atom, said ring or
rings being optionally further substituted one or more times by
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy trifluoromethyl,
halogen, BRIDGE, -L-Rx or -L-Sc; wherein Rx is a reactive group; Sc
is a conjugated substance; and L and BRIDGE are independently a
single covalent bond, or a covalent linkage; X is O, S, Se,
NR.sup.15, or CR.sup.16R.sup.17, where R.sup.15 is H or an alkyl
group having 1-6 carbons; and R.sup.16 and R.sup.17, which may be
the same or different, are independently alkyl groups having 1-6
carbons, or R.sup.16 and R.sup.17 taken in combination complete a
five or six membered saturated ring; R.sup.2 is selected from the
group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group
having 1-6 carbons that is optionally substituted by sulfo,
carboxy, amino, substituted amino or substituted ammonium, wherein
.alpha. is 0 or 1; and TAIL is a heteroatom-containing moiety; Y is
--CR.sup.3.dbd.CR.sup.4-- wherein p and m=0 or 1, such that p+m=1;
R.sup.3, R.sup.4, R.sup.6, and R.sup.7 are independently selected
from the group consisting of hydrogen, a C.sub.1-C.sub.6 alkyl, a
halogen, a CYCLIC SUBSTITUENT, --OR.sup.8, --SR.sup.8,
--(NR.sup.8R.sup.9), TAIL; BRIDGE, -L-Rx and -L-Sc; where R.sup.8
and R.sup.9 are independently a C.sub.1-C.sub.6 alkyl group, 1-2
alicyclic or aromatic rings; or R.sup.8 and R.sup.9 taken in
combination are --(CH.sub.2).sub.2--V--(CH.sub.2).sub.2-- where V
is a single bond, --O--, --CH.sub.2--, or --NR.sup.10--, where
R.sup.10 is H or an alkyl having 1-6 carbons; wherein CYCLIC
SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or
C.sub.3-C.sub.10 cycloalkyl; or R.sup.6 and R.sup.7 form a fused
aromatic ring --R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14--
wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are
independently selected from the group consisting of hydrogen,
C.sub.1-C.sub.6 alkyl group, --OR.sup.8, --SR.sup.8,
--(NR.sup.8R.sup.9), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and
-L-Sc; R.sup.5 is independently selected from the group consisting
of a C.sub.1-C.sub.6 alkyl group, a CYCLIC SUBSTITUENT, TAIL,
BRIDGE, -L-Rx and -L-Sc; or R.sup.5 is absent; R.sup.30, R.sup.31,
and R.sup.32 are independently selected from the group consisting
of hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.10 cycloalkyl,
aryl, and heteroaryl, wherein n=0, 1 or 2; wherein, BRIDGE, when
present, is bound to a compound having formula I or another
unsymmetrical cyanine dye c) incubating said sample and said
staining solution for a sufficient amount of time to form a
dye-poly(amino acid) complex; d) illuminating said complex with an
appropriate wavelength whereby the presence of said poly(amino
acid) is determined.
14. The method according to claim 13, wherein said compound is
overall neutral in charge.
15. The method according to claim 14, wherein said R.sup.2 is a
C.sub.2-C.sub.6 alkyl group substituted by a sulfo group or R.sup.5
is absent.
16. The method according to claim 13, wherein said X is O or S, n
is 0 or 1, R.sup.6 and R.sup.7 form a fused aromatic ring
--R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14-- wherein R.sup.11,
R.sup.12, R.sup.13, and R.sup.14 are independently selected from
the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl group,
--OR.sup.8; R.sup.4 and R.sup.5 are independently a C.sub.1-C.sub.6
alkyl group or a CYCLIC SUBSTITUENT.
17. The method according to claim 13, wherein said method further
comprises heating said sample prior to combining with said staining
solution, or heating combined sample and staining solution
mixture.
18. The method according to claim 13, wherein said method further
comprises removing, destroying, or dispersing below the critical
micelle concentration any biological membranes that are present in
said sample.
19. The method according to claim 13, wherein said method further
comprises adding an anionic detergent to the sample, staining
solution or combined sample and staining solution mixture.
20. The method according to claim 13, wherein said method further
comprising electrophoretically separating said sample before,
after, or while it is combined with said staining solution.
21. The method according to claim 20, wherein said matrix is a
polymeric gel.
22. The method according to claim 21, wherein said gel is a
polyacrylamide gel.
23. The method according to claim 22, wherein said method further
comprises adding an additional detection reagent to said sample,
said staining solution, or combined sample and staining solution
mixture.
24. The method according to claim 20, wherein said method further
comprises transferring said sample to a solid or semi-solid matrix
before or after combining with said staining solution.
25. A method for the detection of nucleic acids in a sample,
wherein said method comprises: a) combining said sample with a
staining solution, wherein said staining solution comprises one or
more dyes having formula ##STR00094## wherein A represents the
atoms necessary to form one to two fused aromatic rings having 6
atoms in each ring, at least one of which is a nitrogen atom, said
ring or rings being optionally further substituted one or more
times by C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy
trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc; wherein Rx is a
reactive group; Sc is a conjugated substance; and L and BRIDGE are
independently a single covalent bond, or a covalent linkage; X is
O, S, Se, NR.sup.15, or CR.sup.16R.sup.17, where R.sup.15 is Nor an
alkyl group having 1-6 carbons; and R.sup.16 and R.sup.17, which
may be the same or different, are independently alkyl groups having
1-6 carbons, or R.sup.16 and R.sup.17 taken in combination complete
a five or six membered saturated ring; R.sup.2 is selected from the
group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group
having 1-6 carbons that is optionally substituted by sulfo,
carboxy, amino, substituted amino or substituted ammonium, wherein
a is 0 or 1; and TAIL is a heteroatom-containing moiety; Y is
--CR.sup.3.dbd.CR.sup.4-- wherein p and m=0 or 1, such that p m=1;
R.sup.3, R.sup.4, R.sup.6, and R.sup.7 are independently selected
from the group consisting of hydrogen, a C.sub.1-C.sub.6 alkyl, a
halogen, a CYCLIC SUBSTITUENT, --OR.sup.8, --SR.sup.8,
--(NR.sup.8R.sup.9), TAIL; BRIDGE, -L-Rx and -L-Sc; where R.sup.8
and R.sup.9 are independently a C.sub.1-C.sub.6 alkyl group, 1-2
alicyclic or aromatic rings; or R.sup.8 and R.sup.9 taken in
combination are --(CH.sub.2).sub.2--V--(CH.sub.2).sub.2-- where V
is a single bond, --O--, --CH.sub.2--, or --NR.sup.10--, where
R.sup.10 is H or an alkyl having 1-6 carbons; wherein CYCLIC
SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or
C.sub.3-C.sub.10 cycloalkyl; or R.sup.6 and form a fused aromatic
ring --R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14-- wherein
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are independently
selected from the group consisting of hydrogen, C.sub.1-C.sub.8
alkyl group, --OR.sup.8, --SR.sup.8, --(NR.sup.8R.sup.9), a CYCLIC
SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; R.sup.5 is
independently selected from the group consisting of a
C.sub.1-C.sub.6 alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE,
-L-Rx and -L-Sc; or R.sup.5 is absent; R.sup.30, R.sup.31, and
R.sup.32 are independently selected from the group consisting of
hydrogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.10 cycloalkyl, aryl,
and heteroaryl, wherein n=0, 1 or 2; wherein, BRIDGE, when present,
is bound to a compound having formula I or another unsymmetrical
cyanine dye; b) incubating said sample and said staining solution
for a sufficient amount of time to form a dye-nucleic acid complex;
illuminating said complex with an appropriate wavelength whereby
the presence of said nucleic acid is determined.
26. The method according to claim 25, wherein said X is O, n is O,
R.sup.2 is a methyl group and R.sup.6 and R.sup.7 form a fused
aromatic ring --R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14--
wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are
independently selected from the group consisting of hydrogen,
C.sub.1-C.sub.8 alkyl group, --OR.sup.8.
27. The method according to claim 26, wherein said R.sup.5 is a
CYCLIC SUBSTITUENT that is an aryl group and R.sup.4 is an alkyl
group.
28. The method according to claim 26, wherein said R.sup.5 is an
alkyl group and R.sup.4 is a CYCLIC SUBSTITUENT that is an aryl
group.
29. The method according to claim 26, wherein said compound is
substituted by at least one TAIL substituent.
30. The method according to claim 29, wherein said TAIL is
according to formula LINK-SPACER-CAP; wherein LINK is single
covalent bond, an either linkage (--O--), a thioether linkage
(--S--) or an amine linkage (--NR.sup.20--); where R.sup.20 is
hydrogen, C.sub.1-C.sub.8 alkyl or SPACER-CAP; SPACER is a covalent
linkage; and, CAP is --O--R.sup.21, --S--R.sup.21,
--NR.sup.21R.sup.22 or --NR.sup.21R.sup.22R.sup.23; where R.sup.21,
R.sup.22, and R.sup.23 are independently selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl and a C.sub.1-C.sub.8
cycloalkyl wherein said alkyl or cycloalkyl are optionally
substituted by one or more substituents selected from the group
consisting of halogen, hydroxyl, C.sub.1-C.sub.8 alkoxy, amino,
carboxy, sulfo and phenyl where said phenyl is optionally
substituted by one or more substituents selected from the group
consisting halogen, hydroxyl, C.sub.1-C.sub.8 alkoxy, amino,
C.sub.1-C.sub.8 aminoalkyl, C.sub.1-C.sub.8 sulfoalkyl and
C.sub.1-C.sub.8 carboxyalkyl; or one or more R.sup.21, R.sup.22,
and R.sup.23, taken in combination with R.sup.20 and SPACER, or
with SPACER alone, forms a 5- or 6-membered ring.
31. The method according to claim 30, wherein said R.sup.4 or
R.sup.5 is independently a TAIL or a CYCLIC SUBSTITUENT substituted
by TAIL.
32. The method according to claim 31, wherein said R.sup.4 is a
TAIL and R.sup.5 is an alkyl group.
33. The method according to claim 32, wherein said CAP is
--NR.sup.21R.sup.22 where R.sup.21 and R.sup.22 are independently
C.sub.1-C.sub.6alkyl groups.
34. The method according to claim 32, wherein said CAP is
--NR.sup.21R.sup.22R.sup.23 where R.sup.21, R.sup.22 and R.sup.23
are independently C.sub.1-C.sub.6 alkyl groups.
35. The method according to claim 31, wherein said R.sup.5 is a
CYCLIC SUBSTITUENT that is an aryl or heteroaryl and said R.sup.4
is a TAIL where CAP is --O--R.sup.21 or --S--R.sup.21.
36. The method according to claim 31, wherein said R.sup.5 is a
TAIL.
37. The method according to claim 36, wherein said CAP is
--NR.sup.21R.sup.22R.sup.23 where R.sup.21, R.sup.22 and R.sup.23
are independently C.sub.1-C.sub.6 alkyl groups.
38. The method according to claim 26, wherein said nucleic acid is
in solution, immobilized on a solid or semi-solid matrix, or
present in a biological structure.
39. The method according to claim 38, wherein said solid or
semi-solid matrix is a polymeric gel, an array, a glass slide, a
polymeric microparticle or a membrane.
40. The method according to claim 39, wherein said gel is an
electrophoretic gel that is an agarose gel.
41. The method according to claim 38, wherein said biological
structure is a biological cell or portion thereof, virus particle
or tissue section.
42. The method according to claim 41, wherein said biological
structure is a cell or portion thereof.
43. The method according to claim 38, wherein said nucleic acid is
in solution that is free from cells or portions thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. Ser. No. 09/557,275,
filed Apr. 24, 2000, which claims the priority to provisional
application Ser. No. 60/131,782, filed Apr. 30, 1999, and
provisional application Ser. No. 60/158,859, filed Oct. 12, 1999,
which disclosures are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to cyanine dyes that possess
particular utility as fluorescent stains for nucleic acids and
poly(amino acids). In particular, the invention relates to
materials that are unsymmetrical cyanine dyes incorporating an
aza-benzazolium moiety. The subject dyes, which form fluorescent
complexes in combination with nucleic acids or lipid-complexed
poly(amino acids), can be used in analyzing a wide range of
materials, including biological and environmental samples.
BACKGROUND OF THE INVENTION
[0003] As researchers in the fields of molecular and cell biology
increasingly utilize fluorescent probes as research tools, the
ability to select the wavelength of fluorescence is becoming
critical. As increasing numbers of multiple-color applications are
developed, the ability to produce novel fluorescent probes with
detectably distinct fluorescent signals is becoming ever more
important.
[0004] Unsymmetrical cyanine dyes have previously been used as
fluorescent stains for nucleic acids, see for example U.S. Pat. No.
4,883,867 to Lee, at al. (1989) and U.S. Pat. No. 4,937,198 to Lee,
et al. (1990). The general spectral properties of the previously
described cyanine dyes could be selected somewhat by changing the
number of methine groups in the dye, and whether the dye
incorporated a pyridinium or quinolinium ring system (see, for
example, U.S. Pat. No. 5,321,130 to Yue et al. (1994); U.S. Pat.
No. 5,410,030 to Yue at al. (1995); U.S. Pat. No. 5,436,134 to
Haugland at al. (1995); U.S. Pat. No. 5,582,977 to Yue et al.
(1996); U.S. Pat. No. 5,658,751 to Yue et al. (1997); and U.S. Pat.
No. 5,863,753 to Haugland et al. (1999). In particular, monomethine
dyes that incorporate pyridinium ring systems typically exhibit
blue to blue-green fluorescence emission, while those that
incorporate quinolinium ring systems exhibit green to yellow-green
fluorescence emission. Trimethine dyes are substantially shifted
toward red wavelengths, and pentamethine dyes are typically shifted
even further, and may exhibit infrared fluorescence emission.
[0005] While the spectral properties of cyanine dyes can be finely
adjusted by selection of appropriate dye substituents, there were
nevertheless regions of the visible spectrum where suitable
fluorescent cyanine dyes that were useful as nucleic acid stains
either did not exist, or did not possess particularly favorable
fluorescence properties.
[0006] The dyes of the invention incorporate additional nitrogen
atoms in the aromatic benzazolium portion of the dye. The dyes of
the invention exhibit a bathochromic spectral shift (a shift to
longer wavelength) of approximately 30 to 50 nm relative to
otherwise structurally similar cyanine dyes known in the art. This
bathochromic spectral shift yields dyes that are particularly
useful for excitation in the wavelength ranges between 500 nm and
600 nm and at >630 nm. Of particular importance are the dyes of
the invention that exhibit absorbance maxima between 530 nm and 550
nm, as they match the principal emission lines of the mercury arc
lamp (546 nm), frequency-doubled Nd-Yag laser (532 nm), and HeNe
laser (543 nm).
[0007] Styryl dyes that complex with lipid-complexed poly(amino
acids) have been described previously (U.S. Pat. No. 5,616,502 to
Haugland et al., 1997). Cyanine dyes that incorporate a
3,4-diazaindene ring system have been described previously for use
as optical sensitizers in photographic materials (British patent
No. 870,753 to Ficken et al., (1961)), but their use in association
with either nucleic acids or lipid-complexed proteins as
fluorescent stains has not previously been described.
SUMMARY OF THE INVENTION
[0008] The present invention provides unsymmetrical aza-benzazolium
containing cyanine dyes (monomers and dimers), reactive versions of
the dyes, dye-conjugates and methods for detecting the presence of
an analyte of interest in a sample. The analyte of interest is
typically a nucleic acid or a poly(amino acid) wherein the present
dyes in the form of a staining solution are combined with the
sample for a sufficient amount of time to form a dye-analyte
complex and are illuminated with an appropriate wavelength whereby
the nucleic acid or a poly(amino acid) is detected.
[0009] The dyes of the invention form a staining solution by
typically being dissolved in an organic solvent such as DMSO to
form a stock solution and then diluted to an appropriate
concentration in a buffered solution. The staining solution is then
incubated with a sample to form the dye-analyte complex.
[0010] In one aspect of the invention the analyte is nucleic acids
that may be present in solution, immobilized on a solid or semi
solid matrix or present in a biological structure. Typically the
nucleic acid is immobilized on solid or semi solid matrix that is
selected from the group consisting of a polymeric gel, a membrane,
an array, a glass slide and a polymeric particle. Preferably the
nucleic acid is immobilized on a glass slide or in a gel such as an
agarose gel. Alternatively, the nucleic acid is present in a
biological structure that is a biological cell or portion thereof,
a virus particle or a tissue section.
[0011] In another aspect of the invention the analyte is a
poly(amino acid), typically the poly(amino acids) are complexed
with a lipid, such as an anionic detergent. Therefore, the
poly(amino acid) is typically present in solution or immobilized on
or in a solid- or semi-solid matrix wherein the matrix is a
polymeric gel, a membrane, an array or a polymeric particle.
Typically the poly(amino acid) is immobilized on a polyacrylamide
gel wherein the staining solution is combined before, during or
after immobilization on the gel. Preferably the poly(amino acid)
are electrophoretically separated on the polyacrylamide gel.
Alternatively, the poly(amino acids) may be spotted onto a
polyacrylamide gel surface such as a Hydrogel microarray slide.
[0012] Typically, the dyes of the invention are according to the
following formula;
##STR00001## [0013] wherein A represents the atoms necessary to
form one to two fused aromatic rings having 6 atoms in each ring,
at least one of which is a nitrogen atom, said ring or rings being
optionally further substituted one or more times by C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy trifluoromethyl, halogen, BRIDGE,
-L-Rx or -L-Sc; [0014] wherein. Rx is a reactive group; Sc is a
conjugated substance; and L and BRIDGE are independently a single
covalent bond, or a covalent linkage; [0015] X is O, S, Se,
NR.sup.15, or CR.sup.16R.sup.17, where R.sup.15 is H or an alkyl
group having 1-6 carbons; and R.sup.16 and R.sup.17, which may be
the same or different, are independently alkyl groups having 1-6
carbons, or R.sup.16 and R.sup.17 taken in combination complete a
five or six membered saturated ring; [0016] R.sup.2 is selected
from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an
alkyl group having 1-6 carbons that is optionally substituted by
sulfa, carboxy, amino, substituted amino or substituted ammonium,
wherein a is 0 or 1; and TAIL is a heteroatom-containing moiety;
[0017] Y is --CR.sup.3.dbd.CR.sup.4-- wherein p and m=0 or 1, such
that p m=1; [0018] R.sup.3, R.sup.4, R.sup.6, and R.sup.7 are
independently selected from the group consisting of hydrogen, a
C.sub.1-C.sub.6 alkyl, a halogen, a CYCLIC SUBSTITUENT, --SR.sup.8,
--(NR.sup.6R.sup.9), TAIL; BRIDGE, -L-Rx and -L-Sc; where R.sup.6
and R.sup.9 are independently a C.sub.1-C.sub.6 alkyl group, 1-2
acyclic or aromatic rings; or R.sup.8 and R.sup.9 taken in
combination are --(CH.sub.2).sub.2--V--(CH.sub.2).sub.2-- where V
is a single bond, --O--, --CH.sub.2--, or --NR.sup.19--, where
R.sup.19 is H or an alkyl having 1-6 carbons; [0019] wherein CYCLIC
SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or
C.sub.3-C.sub.10 cycloalkyl; [0020] or R.sup.6 and R.sup.7 form a
fused aromatic ring
--R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14-- wherein R.sup.11,
R.sup.12, R.sup.13, and R.sup.14 are independently selected from
the group consisting of hydrogen, C.sub.1-C.sub.6 alkyl group,
--OR.sup.8, --SR.sup.9, --(NR.sup.6R.sup.9), a CYCLIC SUBSTITUENT,
TAIL, BRIDGE, -L-Rx and -L-Sc; [0021] R.sup.5 is independently
selected from the group consisting of a C.sub.1-C.sub.6 alkyl
group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; or
R.sup.5 is absent; [0022] R.sup.30, R.sup.31, and R.sup.32 are
independently selected from the group consisting of hydrogen,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.10 cycloalkyl, aryl, and
heteroaryl, wherein n=0, 1 or 2; [0023] wherein, BRIDGE, when
present, is bound to a compound having formula I or another
unsymmetrical cyanine dye.
[0024] Dyes that are useful for detecting poly(amino acids) are
typically neutral in charge wherein R.sup.5 is absent or R.sup.2 is
a C.sub.2-C.sub.6 sulfoalkyl group. Alternatively, charged dyes
wherein X is O or S, n is 0 or 1, R.sup.8 and R.sup.7 form a fused
aromatic ring --R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14--
wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are
independently selected from the group consisting of hydrogen,
C.sub.1-C.sub.5 alkyl group, --OR.sup.8; and R.sup.4 and R.sup.5
are independently a C.sub.1-C.sub.6 alkyl group or a CYCLIC
SUBSTITUENT are also employed, and preferred, for the detection of
poly(amino acids).
[0025] Dyes that are useful for nucleic acid detection preferably
have the following properties wherein X is O, n is O, R.sup.2 is a
methyl group, R.sup.6 and R.sup.7 form a fused aromatic ring
--R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14-- wherein R.sup.11,
R.sup.12, R.sup.13, and R.sup.14 are independently selected from
the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl group,
--OR.sup.8, and R.sup.5 and R.sup.4 are independently a CYCLIC
SUBSTITUENT that is an aryl group or an alkyl group.
[0026] In one aspect of the invention, that is preferred for
detecting nucleic acids, the present dyes comprise at least one
TAIL substituent that has the formula LINK-SPACER-CAP. LINK is
single covalent bond, an either linkage (--O--), a thioether
linkage (--S--) or an amine linkage (--NR.sup.20--); where R.sup.20
is hydrogen, C.sub.1-C.sub.8 alkyl or SPACER-CAP; SPACER is a
covalent linkage; and, CAP is --O--R.sup.21, --S--R.sup.21,
--NR.sup.21R.sup.22 or --NR.sup.21R.sup.22R.sup.23; where R.sup.21,
R.sup.22, and R.sup.23 are independently selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl and a C.sub.1-C.sub.8
cycloalkyl wherein said alkyl or cycloalkyl are optionally
substituted by one or more substituents selected from the group
consisting of halogen, hydroxyl, C.sub.1-C.sub.6 alkoxy, amino,
carboxy, sulfo and phenyl where said phenyl is optionally
substituted by one or more substituents selected from the group
consisting halogen, hydroxyl, C.sub.1-C.sub.8 alkoxy, amino,
C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.8 sulfoalkyl and
C.sub.1-C.sub.8 carboxyalkyl; or one or more R.sup.21, R.sup.22,
and R.sup.23, taken in combination with R.sup.20 and SPACER, or
with SPACER alone, forms a 5- or 6-membered ring.
[0027] In this instance, R.sup.4 or R.sup.5 is independently a TAIL
or a CYCLIC SUBSTITUENT substituted by TAIL. Thus, in one aspect of
the invention, R.sup.4 is a TAIL or a CYCLIC SUBSTITUENT
substituted by TAIL and R.sup.5 is an alkyl group wherein CAP is
--NR.sup.21R.sup.22 where R.sup.21 and R.sup.22 are independently
C.sub.1-C.sub.6 alkyl groups. Alternatively, CAP is
--NR.sup.21R.sup.22R.sup.23 where R.sup.21, R.sup.22 and R.sup.23
are independently C.sub.1-C.sub.6 alkyl groups. In another aspect,
R.sup.5 is a CYCLIC SUBSTITUENT that is an aryl or heteroaryl and
R.sup.4 is a TAIL where CAP is --O--R.sup.21, --S--R.sup.21. In yet
another aspect, R.sup.5 is a TAIL, wherein CAP is
--NR.sup.21R.sup.22R.sup.23 where R.sup.21, R.sup.22 and R.sup.23
are independently C.sub.1-C.sub.6 alkyl groups.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1: Shows preferential fluorescent staining of
double-stranded DNA by Compound 22 in the presence of
single-stranded DNA and RNA, as described in Example 35.
[0029] FIG. 2: Shows preferential fluorescent staining of
double-stranded DNA by Compound 16 in the presence of
single-stranded DNA and RNA, as described in Example 35.
[0030] FIG. 3: Shows detection of bovine serum albumin using
Compound 51, as described in Example 38.
[0031] FIG. 4: Shows detection of bovine serum albumin using
Compound 44, as described in Example 38.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0032] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a fusion protein" includes a plurality of proteins
and reference to "a fluorescent compound" includes a plurality of
compounds and the like.
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. The
following terms are defined for purposes of the invention as
described herein.
[0034] The term "alkyl" as used herein refers to a straight,
branched or cyclic hydrocarbon chain fragment containing between
about one and about twenty five carbon atoms (e.g. methyl, ethyl
and the like). Straight, branched or cyclic hydrocarbon chains
having eight or fewer carbon atoms will also be referred to herein
as "lower alkyl". In addition, the term "alkyl" as used herein
further includes one or more substitutions at one or more carbon
atoms of the hydrocarbon chain fragment. Such substitutions
include, but are not limited to: aryl; heteroaryl; halogen; alkoxy;
amine (--NR'R''); carboxy and thio.
[0035] The term "amino" or "amine group" refers to the group
--NR'R'' (or N'RR'R'') where R, R' and R'' are independently
selected from the group consisting of hydrogen, alkyl, substituted
alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl,
heteroaryl, and substituted heteroaryl. A substituted amine being
an amine group wherein R' or R'' is other than hydrogen. In a
primary amino group, both R' and R'' are hydrogen, whereas in a
secondary amino group, either, but not both, R' or R'' is hydrogen.
In addition, the terms "amine" and "amino" can include protonated
and quaternized versions of nitrogen, comprising the group
--N.sup.+RR'R'' and its biologically compatible anionic
counterions.
[0036] The term "attachment site" as used herein refers to a site
on a moiety or a molecule, e.g. a quencher, a fluorescent dye, an
avidin, or an antibody, to which is covalently attached, or capable
of being covalently attached, to a linker or another moiety.
[0037] The term "aqueous solution" as used herein refers to a
solution that is predominantly water and retains the solution
characteristics of water. Where the aqueous solution contains
solvents in addition to water, water is typically the predominant
solvent.
[0038] The term "--Sc" or "conjugated substance" as used herein
refers to a biomolecule or non-biomolecule substance that contains
or is modified to contain a reactive group wherein the reactive
group will form a covalent bond with the present compounds that
contain an appropriate reactive group. A molecule capable of being
conjugated to the present compounds, conjugated substance,
includes, but not limited to, an amino acid, a peptide, a protein,
a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a
nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a
lipid assembly, a synthetic polymer, a polymeric microparticle, a
biological cell or a virus.
[0039] The term "detectable response" as used herein refers to a
change in or an occurrence of, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an optical response resulting
in a change in the wavelength distribution patterns or intensity of
absorbance or fluorescence or a change in light scatter,
fluorescence lifetime, fluorescence polarization, or a combination
of the above parameters.
[0040] The term "dye" as used herein refers to a compound that is
fluorescent upon binding or association with an analyte of
interest, e.g. nucleic acid or poly(amino acid). These dyes include
the present dyes and ones that can be employed as additional
detection reagents. The additional detection reagents include
fluorophores the are fluorogenic and ones that inherently
fluorescent. Numerous fluorophores are known to those skilled in
the art and include, but are not limited to, coumarin, acridine,
furan, indole, borapolyazaindacene and xanthenes including
fluoroscein, rhodamine, rhodol, cyanine, benzofuran, quinazolinone,
and benzazole as well as other fluorophores described in RICHARD P.
HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND
RESEARCH CHEMICALS (9.sup.th edition, CD-ROM, 2002).
[0041] The term "Linker", "L" or "BRIDGE" as used herein refers to
a covalent linkage that is a single covalent bond or a series of
stable covalent bonds incorporating 1-20 nonhydrogen atoms selected
from the group consisting of C, N, O, S and P that covalently
attach the present quenching compounds to another moiety such as a
chemically reactive group or a conjugated substance including
biological and non-biological substances. A "cleavable linker" is a
linker that has one or more covalent bonds that 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.
[0042] The term "poly(amino acids)" as used herein refers in a
generic sense to proteins and polypeptides that include polymers of
amino acid residues of any length. The term "peptide" is used
herein to refer to polypeptides having less than 250 amino acid
residues, typically less than 100 amino acid residues. The terms
apply to amino acid polymers in which one or more amino acid
residues are an artificial chemical analogue of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers.
[0043] The term "Rx" or "reactive group" as used herein refers to a
group that is capable of reacting with another chemical group to
form a covalent bond, i.e., is covalently reactive under suitable
reaction conditions, and generally represents a point of attachment
for another substance. The reactive group is a moiety, such as
malemide or succinimidyl ester, on the compounds of the present
invention that is capable of chemically reacting with a functional
group on a different compound to form a covalent linkage. Reactive
groups generally include nucleophiles, electrophiles and
photoactivatable groups.
[0044] The term "sample" as used herein refers to any material that
may contain an analyte of interest, which typically includes
poly(amino acids) and nucleic acids. Typically, the sample is a
live cell, a biological fluid that comprises endogenous host cell
proteins, nucleic acid polymers, nucleotides, oligonucleotides,
peptides and buffer solutions. The sample may be in an aqueous
solution, a viable cell culture or immobilized on a solid or semi
solid surface such as a polymeric gel, membrane blot or on a
microarray.
II. Compositions and Methods of Use
[0045] The Invention comprises unsymmetrical cyanine dyes that
incorporate an aza-benzazolium ring moiety. Included in the
invention are monomeric and dimeric cyanine dyes, including those
substituted by a cationic side chain, a chemically reactive
functional group, and a conjugated substance. The dyes of the
invention are virtually non-fluorescent when diluted in aqueous
solution but when associated with nucleic acid polymers such as DNA
or RNA, however, the resultant dye-nucleic acid complex is
extremely fluorescent upon illumination. Similarly, when associated
with lipid-complexed poly(amino acids), the resulting composition
is extremely fluorescent upon illumination. The present dyes of the
invention stain an analyte of interest, nucleic acids and
poly(amino acids), in a wide variety of samples, particularly in
aqueous solutions, electrophoretic gels, and a wide variety of
cells, including microorganisms.
A. Aza-Benzazolium Containing Cyanine Dyes
[0046] The dyes of the invention comprise a cyanine dye that
contains: 1) a first heterocyclic ring system that is a substituted
aza-benzazolium ring, 2) a bridging methine and 3) a second
heterocyclic ring system that is a pyridine, a quinoline, a
pyridinium or a quinolinium, in one embodiment of the invention,
the first or second ring system is substituted by a side chain,
TAIL, that contains at least one heteroatom. In another embodiment,
the first or second ring system is covalently bound to a second
cyanine dye, forming a homo- or heterodimer. The first and second
ring systems of the first or second cyanine dye is optionally
further substituted by a variety of substituents, including
chemically reactive groups as described below.
[0047] In one embodiment these dyes are pyridinium and
quinolinium-based dyes, wherein the dye has the following
formula:
##STR00002## [0048] wherein A represents the atoms necessary to
form one to two fused aromatic rings having 6 atoms in each ring,
at least one of which is a nitrogen atom, said ring or rings being
optionally further substituted one or more times by
C.sub.1-C.sub.22 alkyl, C.sub.1-C.sub.22 alkoxy trifluoromethyl,
halogen, BRIDGE, -L-Rx or -L-Sc; [0049] wherein Rx is a reactive
group; Sc is a conjugated substance; and L and BRIDGE are
independently a single covalent bond, or a covalent linkage; [0050]
X is O, S, Se, NR.sup.15, or CR.sup.16R.sup.17, where R.sup.15 is H
or an alkyl group having 1-22 carbons; and R.sup.16 and R.sup.17,
which may be the same or different, are independently alkyl groups
having 1-22 carbons, or R.sup.16 and R.sup.17 taken in combination
complete a five or six membered saturated ring; [0051] R.sup.2 is
selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE
and an alkyl group having 1-22 carbons that is optionally
substituted by sulfo, carboxy, amino, substituted amino or
substituted ammonium, wherein a is 0 or 1; and TAIL is a
heteroatom-containing moiety; [0052] Y is --CR.sup.3.dbd.CR.sup.4--
wherein p and m=0 or 1, such that p+m=1; [0053] R.sup.3, R.sup.4,
R.sup.6, and R.sup.7 are independently selected from the group
consisting of hydrogen, a C.sub.1-C.sub.22 alkyl, a halogen, a
CYCLIC SUBSTITUENT, --OR.sup.8, --SR.sup.9, --(NR.sup.9R.sup.9),
TAIL; BRIDGE, -L-Rx and -L-Sc; where R.sup.8 and R.sup.9 are
independently a C.sub.1-C.sub.8 alkyl group, 1-2 alicyclic or
aromatic rings; or R.sup.8 and R.sup.9 taken in combination are
--(CH.sub.2).sub.2--V--(CH.sub.2).sub.2-- where V is a single bond,
--O--, --CH.sub.2--, or --NR.sup.19--, where R.sup.10 is H or an
alkyl having 1-6 carbons; [0054] wherein CYCLIC SUBSTITUENT is a
substituted or unsubstituted aryl, heteroaryl or C.sub.3-C.sub.10
cycloalkyl; [0055] or R.sup.6 and R.sup.7 form a fused aromatic
ring --R.sup.11.dbd.R.sup.12--R.sup.13.dbd.R.sup.14 wherein
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are independently
selected from the group consisting of hydrogen, C.sub.1-C.sub.22
alkyl group, --OR.sup.8, --SR.sup.8, --(NR.sup.8R.sup.8), a CYCLIC
SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; [0056] R.sup.5 is
independently selected from the group consisting of a
C.sub.1-C.sub.22 alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE,
-L-Rx and -L-Sc; or R.sup.5 is absent; [0057] R.sup.30, R.sup.31,
and R.sup.32 are independently selected from the group consisting
of hydrogen. C.sub.1-C.sub.22 alkyl, C.sub.3-C.sub.10 cycloalkyl,
aryl, and heteroaryl, wherein n=0, 1 or 2; [0058] wherein, BRIDGE,
when present, is bound to a compound having formula I or another
unsymmetrical cyanine dye.
[0059] The A moiety represents the atoms necessary to form one or
more fused 6-membered aromatic rings, incorporating at least one
nitrogen atom .dbd.N(R.sup.2).sub..beta.--. The six membered rings
of the aza-benzazole ring system typically incorporate 1-3 nitrogen
atoms, more preferably 1-2 nitrogen atoms. The nitrogen atoms are
typically incorporated in the first 6-membered aromatic ring fused
to the azole ring. Preferred embodiments of the aza-benzazole
moiety include without limitation the following structures,
##STR00003##
[0060] At least one of the aza-benzazole nitrogen atoms is
quaternized by the substituent R.sup.2, resulting in a formal
positive charge. In one embodiment, .alpha.=1 and .beta.=0, such
that the azole nitrogen atom is quaternized, and the benzo nitrogen
atom is unsubstituted. In another embodiment, .alpha.=0 and
.beta.=1, such that the azole nitrogen atom is unsubstituted and at
one benzo nitrogen atom is quaternized by R.sup.2. Either .alpha.
or a .beta. is 1, and typically no more than one .beta. in a given
aza-benzazole is 1. The presence of an additional nitrogen atom
incorporated in the fused aromatic ring of the benzazole moiety
results in a shift of the emission spectra to longer wavelength.
The presence of additional nitrogen atoms (as in structure V,
above) shift the wavelength even further, as does the presence of
additional fused 6-membered rings.
[0061] The aza-benzazole moiety is optionally further substituted
at one or more aromatic carbons by an alkyl group having from 1-6
carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen,
BRIDGE, -L-Rx or -L-Sc. Although the carbon atoms of the
aza-benzazolium ring system are typically fully substituted by
hydrogen, incorporation of one or more non-hydrogen substituents
can be used to fine tune the absorption and emission spectrum of
the resulting dye. Typically, where present, there is only one
non-hydrogen substituent on the aza-benzazole, preferably an alkoxy
or halogen substituent.
[0062] R.sub.x is a reactive group that provides compounds of the
present invention that can be covalently attached to another
substance, a conjugated substance, such as a biomolecule or
non-biomolecule including proteins and non-protein molecules such
as dextran and a microparticle. The reactive group is typically a
nucleophile, electrophile or photoactivatable group that chemically
reacts with another appropriate reactive group on a biomolecule or
non-biomolecule to form a covalent bond between the compounds of
the present invention and another substance. Thus, a conjugated
substance (S.sub.c) can be any molecule with an appropriate
reactive group, or is modified to contain an appropriate reactive
group, that can be covalently bonded to the compounds of the
present invention.
[0063] L is a covalent linkage that attaches the reactive group or
conjugated substance to the present compounds wherein the covalent
linkage may be a single covalent bond or a series of stable
chemical bonds containing 1-20 nonhydrogen atoms. These atoms are
selected from the group consisting of C, N, P, O or S. As used
herein the term "BRIDGE" is also a covalent linkage having the same
properties as L, but that attaches any one of the present dyes to
another dye to form a homo- or hetero-dimer.
[0064] The substituent R.sup.2 is an alkyl group having 1-22
carbons, that is linear or branched, saturated or unsaturated, that
is optionally substituted one or more times by hydroxy, carboxy,
sulfo, amino, amino substituted by 1-2 C.sub.1-C.sub.6 alkyls, or
ammonium substituted by 1-3 C.sub.1-C.sub.6 alkyls. The R.sup.2
substituent is alternatively a TAIL moiety, BRIDGE, a reactive
group (-L-Rx) or a conjugated substance (-L-Sc). In one embodiment
particularly suited for staining nucleic acids, R.sup.2 is alkyl
having 1-6 carbons, preferably methyl or ethyl, more preferably
methyl. In another embodiment particularly suitable for staining
lipid-complexed poly(amino acids), R.sup.2 is a sulfoalkyl having
2-6 carbons.
[0065] As used herein the term "TAIL" refers to a
heteroatom-containing side chain that is described by the formula
MK-SPACER-CAP. LINK is the linking moiety by which TAIL is attached
to the dyes of the present invention. SPACER is a covalent linkage
that connects LINK to CAP. CAP is the portion of TAIL that
possesses a heteroatom component. Where the dye is substituted by a
TAIL moiety, typically one or more of R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13 and
R.sup.14 is a TAIL. In one embodiment of the invention, at least
one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11,
R.sup.13 and R.sup.14 is required to be a TAIL. Preferably, R.sup.4
or R.sup.5 is a TAIL or a TAIL-substituted CYCLIC SUBSTITUENT. When
R.sup.4 is a TAIL, LINK is preferably --NR.sup.20-- or --S--. When
TAIL is at any position other than R.sup.4 or R.sup.5, LINK is
preferably --O-- or a single bond.
[0066] As used herein the term "CYCLIC SUBSTITUENT" refers to a
substituent that is a substituted or unsubstituted aryl,
heteroaryl, or cycloalkyl having 3-10 carbons. As used herein, an
aryl is a phenyl or a naphthyl group, and a heteroaryl substituent
is a 5 or 6-membered heteroaromatic ring, wherein the heteroatom is
O, N or S. The CYCLIC SUBSTITUENT is optionally substituted by
halogen, amino, alkyl, perfluoroalkyl, alkylamino, dialkylamino,
alkoxy or carboxyalkyl, wherein each alkyl group has 1-6 carbons,
or by a TAIL moiety. The CYCLIC SUBSTITUENT is preferably a
substituted or unsubstituted naphthyl, phenyl, thienyl, or
cycloalkyl having 3-10 carbons, more preferably, the CYCLIC
SUBSTITUENT is phenyl.
[0067] Where the dye is substituted by a CYCLIC SUBSTITUENT,
typically one of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.11, R.sup.12, R.sup.13 and R.sup.14 is a CYCLIC SUBSTITUENT.
Preferably, one or more of R.sup.4, R.sup.5, R.sup.6, or R.sup.12
is TAIL or a CYCLIC SUBSTITUENT, including CYCLIC SUBSTITUENTS that
are substituted by TAIL moieties.
[0068] In one embodiment, X is one of O, S, Se or NR.sup.15, where
R.sup.15 is H or an alkyl group having 1-22 carbons, that is linear
or branched, saturated or unsaturated, that is optionally
substituted one or more times by hydroxy, carboxy, sulfo, amino,
amino substituted by 1-2 C.sub.1-C.sub.6 alkyls, or ammonium
substituted by 1-3 C.sub.1-C.sub.6 alkyls. The R.sup.15 substituent
is alternatively a TAIL moiety.
[0069] Alternatively, X is CR.sup.16R.sup.17, where R.sup.16 and
R.sup.17, which may be the same or different, are independently H
or alkyl groups having 1-22 carbons, that are linear or branched,
saturated or unsaturated, that are optionally substituted one or
more times by hydroxy, carboxy, sulfo, amino, amino substituted by
1-2 C.sub.1-C.sub.6 alkyls, or ammonium substituted by 1-3
C.sub.1-C.sub.8 alkyls. Alternatively, one of R.sup.16 and R.sup.17
is a TAIL moiety. Alternatively, the carbons of R.sup.16 and
R.sup.17 taken in combination complete a five or six membered
saturated ring. When X is CR.sup.16R.sup.17, R.sup.16 and R.sup.17
are typically alkyls having 1-6 carbons, preferably methyl or
ethyl.
[0070] Preferably, X is O or S, more preferably X is S.
[0071] The two heterocyclic ring systems of the cyanine dye are
linked by a bridging methine having the formula
--(CR.sup.32.dbd.CR.sup.31).sub.n--CR.sup.30.dbd.
(where n=0, 1, or 2) in such a way as to permit extensive
electronic delocalization. When n=0 the dyes are unsymmetrical
monomethine dyes; when n=1 the dyes are trimethine dyes; when n=2,
the dyes are pentamethine dyes. As with similar compounds, the
number of methine groups between the heteroaromatic rings
influences the spectral properties of the dye. The monomethine dyes
of the present invention typically have yellow to orange to red
fluorescence emission. The trimethine dye analogs are substantially
shifted toward red wavelengths beyond 600 nm, and the pentamethine
dyes are shifted even further, often exhibiting infrared
fluorescence emission. Preferably n=0 or 1, more preferably
n=0.
[0072] Methine substituents R.sup.30, R.sup.31, and R.sup.32 are
independently H, alkyl having 1-6 carbons, cycloalkyl having 3-10
carbons, aryl, or heteroaryl. Where n=1 or 2, each R.sup.31 and
R.sup.32 varies independently. In one embodiment, only the
substituent on the central carbon of the methine bridge is
nonhydrogen (R.sup.30 where n=0, R.sup.31 where n=1, the centrally
located R.sup.32 where n=2). In another embodiment, where a methine
substituent is nonhydrogen it is an alkyl. In another embodiment,
where a methine substituent is nonhydrogen it is an aryl.
Typically, each R.sup.30, R.sup.31, and R.sup.32 is hydrogen. Where
R.sup.30, R.sup.31, or R.sup.32 is nonhydrogen, preferably n=1.
[0073] In one aspect of the invention, selected cyanine dyes having
nonhydrogen methine substituents have particular utility as RNA
selective nucleic acid stains. In another aspect of the Invention,
cyanine dyes having nonhydrogen methine substituents increase the
signal-to-noise ratio when staining nucleic acids by decreasing
background fluorescence.
[0074] The incorporation of substituted methine bridges, as
described above, also provides useful derivatives of previously
described cyanine dyes (e.g., U.S. Pat. No. 5,321,130 to Yue et al.
(1994); U.S. Pat. No. 5,410,030 to Yue et al. (1995); U.S. Pat. No.
5,436,134 to Haugland et al. (1995); U.S. Pat. No. 5,582,977 to Yue
at al. (1996); U.S. Pat. No. 5,658,751 to Yue at al. (1997); and
U.S. Pat. No. 5,863,753 to Haugland at al, (1999)).
[0075] The second ring system contains a ring fragment Y that is
--CR.sup.3.dbd.CR.sup.4--, with subscripts p and m equal to 0 or 1,
such that p+m=1. The second ring system is a 6 membered heterocycle
according to one of these formulations
##STR00004##
[0076] In preferred embodiments of the invention, m=1 and p=0
("4-pyridiniums" and "4-quinoliniums").
[0077] The pyridinium and quinolinium-based cyanine dyes include an
N-substituent R.sup.5 that is an alkyl that is saturated or
unsaturated, linear or branched, having 1-22 carbons, that is
optionally substituted one or more times by hydroxy, carboxy,
sulfo, amino, amino substituted by 1-2 C.sub.1-C.sub.6 alkyls, or
ammonium substituted by 1-3 C.sub.1-C.sub.6 alkyls. Alternatively,
R.sup.5 is a CYCLIC SUBSTITUENT, a TAIL moiety, BRIDGE, a reactive
group, a conjugated substance, a pair of electrons, or sulfoalkyl.
Typically R.sup.5 is an alkyl having 1-6 carbons, preferably 1-2
carbons, or R.sup.6 is a CYCLIC SUBSTITUENT. In one embodiment,
R.sup.5 is a sulfoalkyl having 2-6 carbons. In another embodiment,
R.sup.5 is absent and replaced by a pair of electrons, resulting in
a neutral unsymmetrical cyanine dye, as described below.
[0078] The substituents of the second ring system, R.sup.3,
R.sup.4, R.sup.6, and R.sup.7, are independently H, halogen, an
alkyl that is saturated or unsaturated, linear or branched, having
1-6 carbons, a CYCLIC SUBSTITUENT, a TAIL moiety, BRIDGE a reactive
group (Rx) or a conjugated substance (Sc). R.sup.3, R.sup.4,
R.sup.6, and R.sup.7 are optionally --OR.sup.8, --SR.sup.8, or
--(NR.sup.8R.sup.9) where R.sup.6 and R.sup.9, which can be the
same or different, are independently alkyl groups having 1-6
carbons; or 1-2 alicyclic or aromatic rings; or R.sup.8 and R.sup.9
taken in combination form a cyclic structure having the formula
--(CH.sub.2).sub.2--W--(CH.sub.2).sub.2--, where W is a single
bond, --O--, --CH.sub.2--, or --NR.sup.10--, where R.sup.16 is H or
an alkyl having 1-6 carbons. In addition, R.sup.3 and R.sup.4 are
optionally and independently --OSO.sub.2R.sup.19 where R.sup.19 is
alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or
aryl.
[0079] Optionally, R.sup.6 and R.sup.7 taken in combination are
--(CH.sub.2).sub.2-- where v 3 or 4, forming a fused 5- or
6-membered ring, or R.sup.6 and R.sup.7, taken in combination form
a fused 6-membered aromatic ring.
[0080] Alternatively, any of R.sup.3, R.sup.4, R.sup.6 or R.sup.7
is a CYCLIC SUBSTITUENT. Preferred ring substituents are
independently H, alkyl, --OR.sup.8, or a CYCLIC SUBSTITUENT, or a
TAIL. In one aspect of the invention, R.sup.4 is not hydrogen. In
another aspect of the invention, R.sup.4 is a CYCLIC SUBSTITUENT, a
TAIL moiety, or a CYCLIC SUBSTITUENT that is further substituted by
a TAIL moiety.
[0081] Where R.sup.6 and R.sup.7 taken in combination form a fused
6-membered aromatic ring, embodiments of this invention are
quinolinium derivatives, and the second ring system has the
formula
##STR00005##
where ring substituents R.sup.11, R.sup.12, R.sup.13, and R.sup.14
are independently H, an alkyl that is saturated or unsaturated,
linear or branched, having 1-6 carbons; halogen, --OR.sup.8,
--SR.sup.8, --(NR.sup.6R.sup.9), where R.sup.8 and R.sup.9 are as
defined previously; or a CYCLIC SUBSTITUENT, a TAIL moiety, BRIDGE,
a reactive group or a conjugated substance. Typically no more than
one of R.sup.11, R.sup.13, and R.sup.14 is nonhydrogen, and is
preferably halogen or --OR.sup.8. Preferred embodiments of the
invention are quinoliniums wherein m=1 and p 0 ("4-quinoliniums").
The presence of the fused 6-membered ring at R.sup.6 and R.sup.7
typically increases the wavelength of the dye's fluorescence when
associated with nucleic acids or lipid-complexed poly(amino
acids).
[0082] In another embodiment of the invention, the dye of the
invention is a homodimer or heterodimer of unsymmetrical cyanine
dyes. That is, two cyanine dye subunits, at least one of which has
a structure as described above, are joined by a covalent linkage,
BRIDGE, to yield a dimeric compound having utility as a fluorescent
stain for nucleic acids or lipid-complexed poly(amino acids) (see
Compound 28, Example 12 and Compound 46, Example 13). While one
cyanine dye subunit is an aza-benzazolium based cyanine dye
subunit, the other portion of the dimeric dye may be a cyanine dye
as described previously by Haugland and Yue (U.S. Pat. No.
5,321,130; U.S. Pat. No. 5,436,134; U.S. Pat. No. 5,658,751; and
U.S. Pat. No. 5,869,689). Alternatively, the other portion of the
dimeric dye is dissimilar in structure, for example a
phenanthridium moiety.
[0083] The BRIDGE moiety is typically a combination of atoms having
stable chemical bonds. The BRIDGE moiety optionally includes
single, double, triple or aromatic carbon-carbon bonds, as well as
carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds
and carbon-sulfur bonds. The BRIDGE moiety typically includes
ether, thioether, carboxamide, sulfonamide, urea, urethane or
hydrazine moieties. Preferred BRIDGE moieties have 1-20 nonhydrogen
atoms selected from the group consisting of C, N, O and S; and are
composed of any combination of ether, thioether, amine, ester,
carboxamide, sulfonamide, hydrazide bonds and aromatic or
heteroaromatic bonds. Preferably BRIDGE is a combination of single
carbon-carbon bonds and carboxamide or thioether bonds. Examples of
BRIDGE include substituted or unsubstituted polymethylene, arylene,
alkylarylene or arylenealkyl.
[0084] In a preferred embodiment, the BRIDGE moiety is an aliphatic
chain containing a backbone of 4-19 carbon atoms. The carbon
backbone may be interspersed at one or more intervals with a
non-carbon heteroatom. The heteroatoms, which may be the same or
different, are N, O, or S, preferably the heteroatom is N. Where
the heteroatom is nitrogen, it is optionally substituted with one
or more alkyl substituents having 1-6 carbon atoms, which may be
the same or different. BRIDGE moieties that incorporate quaternary
nitrogens confer cationic charges on the dimers of the invention,
potentially increasing their affinity for nucleic acids, as well as
decreasing their ability to permeate cell membranes.
[0085] Where the dimeric dye incorporates two cyanine subunits, the
BRIDGE moiety is independently bound to each cyanine subunit at one
of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.13, or R.sup.14, as defined above. Preferably the
BRIDGE moiety is independently bound to each cyanine subunit at
R.sup.2, R.sup.4 or R.sup.5, more preferably at R.sup.4 or
R.sup.5.
[0086] By careful selection of the cyanine subunits that are
covalently joined to form the dimeric dye, a variety of dimers with
desirable spectral properties may be prepared. Selection of
subunits that function as an energy donor and energy acceptor,
respectively, via fluorescence resonance energy transfer typically
produces a dimeric stain with an enhanced Stokes shift. The
necessary difference in spectral properties for energy transfer to
occur may be created by covalently attaching an aza-benzothiazole
cyanine subunit to an aza-benzoxazole cyanine subunit.
Alternatively, a monomethine cyanine subunit may be attached to a
trimethine or pentamethine cyanine subunit, or a cationic cyanine
subunit may be attached to a neutral cyanine dye subunit. In
another aspect of the invention, an aza-benzoxazole cyanine subunit
is attached to a non aza-substituted cyanine subunit. In yet
another aspect of the invention, an aza-benzoxazole cyanine subunit
is attached to a non-cyanine subunit, typically a phenanthridium
subunit. Dimeric dyes incorporating non-aza-benzazolium containing
cyanine subunits have previously been described (U.S. Pat. No.
5,582,977 to Yue et al. (1996); U.S. Pat. No. 5,401,847 to Glazer,
(1995); U.S. Pat. No. 5,565,554 to Glazer (1996); and U.S. Pat. No.
5,760,201 to Glazer et. al. (1998)).
[0087] In a specific embodiment of the invention, the dyes of the
invention are 4-pyridiniums or 4-quinoliniums, wherein R.sup.5 is
an alkyl having 1-6 carbons, or R.sup.6 is a CYCLIC SUBSTITUENT,
and R.sup.4 is not hydrogen. Dyes having an R.sup.4 substituent
that is non-hydrogen typically possess enhanced quantum yields
relative to similar dyes wherein R.sup.4 is H. For this class of
dyes, R.sup.6 is preferably alkyl having 1-6 carbons. Preferably
R.sup.4 is a TAIL.
[0088] In an additional preferred embodiment of the Invention, the
second heterocyclic ring contains exactly two non-hydrogen
substituents, one of which is a TAIL.
[0089] Some of the dyes of the present invention that possess a
TAIL moiety at R.sup.4 or R.sup.5 exhibit particular utility for
staining nucleic acids in cells and microorganisms. The utility of
specific embodiments of the dyes of the present invention in
staining cells and microorganisms is generally dependent on the
chemical nature of the TAIL moiety, and the identity of the group
present at R.sup.6. For example, those compounds for which CAP is a
quaternary ammonium salt are generally impermeant to living cells,
with the exception of some yeast cells. However, the permeability
of those compounds for which CAP is a primary or secondary amine,
and LINK is a secondary or tertiary amine, is typically related to
the nature of R.sup.5 (the N-substituent).
[0090] Typically, dyes useful as impermeant cellular probes are
those dyes having 2 or more positive charges overall, more
preferably 3 or more positive charges. Preferred dyes for permeant
cellular probes are dyes wherein R.sup.5 is aryl or heteroaryl and
CAP is --O--R.sup.21 or --S--R.sup.21, or dyes wherein R.sup.4 is a
CYCLIC SUBSTITUENT substituted by a cationic TAIL (for example,
Compound 24, Example 10).
[0091] Dyes that are preferred for staining nucleic acids present
in electrophoretic gels typically have a CAP that is a dialkylamino
group, while dyes useful as stains for poly(amino acids) in
electrophoretic gels are overall neutral in charge, typically by
virtue of a sulfoalkyl group present at R.sup.2 or R.sup.5 that
balances the cationic charge on the aza-benzazole moiety.
[0092] Any net positive or negative charges possessed by the dyes
of the invention are balanced by a necessary counterion or
counterions. Typically, the dyes of the invention are positively
charged due to the presence of a quaternized aza-benzazole
nitrogen, however the overall charge on the dye is determined by
the charge present on each dye substituent (such as a negatively
charged sulfonic acid group, or a positively charged ammonium
group). Where necessary, the counterion is depicted as LP and the
polarity of the charge is indicated. Examples of useful counterions
for dyes having a net positive charge include, but are not limited
to, chloride, bromide, iodide, sulfate, alkanesulfonate,
arylsulfonate, phosphate, perchlorate, tetrafluoroborate,
tetraarylboride, nitrate and anions of aromatic or aliphatic
carboxylic acids. Preferred negative counterions are chloride,
iodide, perchlorate and various sulfonates. Examples of useful
counterions for dyes having a net negative charge include, but are
not limited to, alkali metal ions, alkaline earth metal ions,
transition metal ions, ammonium or substituted ammonium ions.
Preferably, any necessary counterion is biologically compatible, is
not toxic as used, and does not have a substantially deleterious
effect on biomolecules.
[0093] In one embodiment, the cyanine dyes of the invention are
substituted by at least one TAIL moiety having the formula
LINK-SPACER-CAP.
[0094] LINK is a single covalent bond, an ether linkage (--O--), a
thioether linkage (--S--), or an amine linkage (--NR.sup.20--). In
each embodiment, LINK forms the attachment between the cyanine dye
core structure and SPACER. When LINK is an amine, the amine
substituent (R.sup.20) is optionally H, such that LINK=--NH--.
Alternatively, R.sup.20 is a linear or branched alkyl having 1-8
carbons. In another embodiment of the invention, R.sup.20 is
--SPACER'-CAP', yielding a TAIL having the formula
##STR00006##
where SPACER' and CAP', respectively, may be the same as or
different from SPACER and CAP, and are selected from the same
alternatives defined for SPACER and CAP, respectively. For the sake
of simplifying the description, SPACER and CAP are defined with the
understanding that a description of SPACER includes SPACER', and a
description of CAP includes CAP'.
[0095] SPACER is a covalent linkage that joins LINK and CAP. SPACER
is a linear, branched, cyclic, heterocyclic, saturated or
unsaturated arrangement of 1-16 C, N, P, O or S atoms.
Alternatively, SPACER is a single covalent bond, such that both
LINK and SPACER are not simultaneously single covalent bonds.
Preferably, the SPACER linkage must begin and end with a carbon
atom. Typically, if SPACER consists of a single atom, it is
required to be a carbon atom, so that the first and last atom in
SPACER (in this specific instance, they are the same atom) is a
carbon. The 1-16 atoms making up SPACER are combined using any
appropriate combination of ether, thioether, amine, ester, or amide
bonds; or single, double, triple or aromatic carbon-carbon bonds;
or phosphorus-oxygen bonds; or phosphorus-sulfur bonds; or
nitrogen-nitrogen bonds; or nitrogen-oxygen bonds; or aromatic or
heteroaromatic bonds. SPACER is further substituted by hydrogen to
accommodate the valence state of each atom in SPACER.
[0096] Generally, the atoms of SPACER are arranged such that all
heteroatoms in the linear backbone of SPACER are separated by at
least one carbon atom, and preferably separated by at least two
carbon atoms. Typically, SPACER is 1-6 carbon atoms in a linear or
branched saturated chain. In one embodiment of the invention,
SPACER incorporates a 6-membered aromatic ring (phenylene linkage).
In another embodiment of the invention, SPACER incorporates a 5- or
6-membered heteroaromatic ring, wherein the heteroatoms are O, N,
or S. Alternatively, SPACER incorporates amide linkages, ester
linkages, simple ethers and thioethers, and amines in a linear
arrangement, such as
--CH.sub.2--CH.sub.2--(C.dbd.O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--.
Preferably, SPACER is an alkylene (--(CH.sub.2).sub.k--, where
k=1-8).
[0097] LINK and SPACER, in combination, serve to attach a
heteroatom-containing group, CAP, to the dye core structure. CAP
may contain oxygen, sulfur or nitrogen, according to the formulas
--O--R.sup.21, --S--R.sup.21, --NR.sup.21R.sup.22, or
--NR.sup.21R.sup.22R.sup.23. The substituents R.sup.21, R.sup.22,
and R.sup.23 are independently H, or a linear or branched alkyl or
cycloalkyl having 1-8 carbons, Where any of R.sup.21, R.sup.22 and
R.sup.23 are alkyl or cycloalkyl, they are optionally further
substituted by one or more halogen, hydroxy, alkoxy having 1-8
carbons, amino, carboxy, sulfo, or phenyl, where phenyl is
optionally further substituted by halogen, hydroxy, alkoxy having
1-8 carbons, amino, aminoalkyl having 1-8 carbons, or sulfoalkyl or
carboxyalkyl having 1-8 carbons. In another embodiment of the
invention, one or more of R.sup.21, R.sup.22 and R.sup.23, taken in
combination With R.sup.20 and SPACER, or with SPACER alone, forms a
5- or 6-membered ring that is aromatic, heteroaromatic, alicyclic
or heteroalicyclic ring. When the 5- or 6-membered ring is
heteroaromatic or heteroalicyclic, the ring contains 1-3
heteroatoms that are O, N or S. Preferably, R.sup.21 and R.sup.22
are independently hydrogen or alkyls having 1-8 carbons. R.sup.23
is typically H or alkyl having 1-8 carbons.
[0098] When CAP is --NR.sup.21R.sup.22R.sup.23, the CAP nitrogen
atom is formally positively charged, and adds to the cumulative
charge that is balanced by the presence of the counterion
.psi..
[0099] Additionally, there are several embodiments of the present
invention wherein CAP incorporates a cyclic structure. In these
embodiments, CAP typically incorporates a 5- or 6-membered
nitrogen-containing ring, optionally including an additional
heteroatom (typically oxygen), where the ring nitrogen is
optionally substituted by R.sup.23 to give an ammonium salt.
Specific versions of CAP include, but are not limited to, those
listed in Table 1.
TABLE-US-00001 TABLE 1 Examples of specific CAP moieties
##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011##
##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016##
[0100] CAP is preferably --NR.sup.21R.sup.22 or
--NR.sup.21R.sup.22R.sup.23, where R.sup.21, R.sup.22, and R.sup.23
are alkyls having 1-6 carbons. More preferably CAP is
--N(CH.sub.3).sub.2 or --N(CH.sub.3).sub.3.
[0101] Preferably TAIL contains 6-10 non-hydrogen atoms, including
LINK and CAP.
[0102] Selected examples of TAIL are listed in Table 2. For each
TAIL, the identities of LINK, SPACER and CAP are specified. Where
R.sup.21, R.sup.22, or R.sup.23 combined with either R.sup.20 or
SPACER, the combination is indicated in the table.
TABLE-US-00002 TABLE 2 Specific examples of TAIL moieties SPACER
CAP TAIL LINK SPACER' CAP' ##STR00017## ##STR00018##
--CH.sub.2--CH.sub.2--CH.sub.2-- --N(CH.sub.3).sub.2 ##STR00019##
##STR00020## --CH.sub.2--CH.sub.2--CH.sub.2--
--N.sup.+(CH.sub.3).sub.3 ##STR00021## ##STR00022##
--CH.sub.2--CH.sub.2--CH.sub.2-- --CH.sub.2--CH.sub.2--CH.sub.2--
--N(CH.sub.3).sub.2 --N(CH.sub.3).sub.2 ##STR00023## ##STR00024##
--CH.sub.2--CH.sub.2--CH.sub.2-- --N.sup.+(CH.sub.3).sub.3
##STR00025## --S-- --CH.sub.2--CH.sub.2-- --N(CH.sub.3).sub.2
##STR00026## --S-- --CH.sub.2--CH.sub.2-- --N.sup.+(CH.sub.3).sub.3
##STR00027## --N(--R.sup.22)-- --CH.sub.2--CH.sub.2--
--N(CH.sub.3)(CH.sub.2--CH.sub.2--R.sup.20) ##STR00028##
--N(--R.sup.23)-- --CH.sub.2--CH.sub.2--
--N.sup.+(CH.sub.3).sub.2--CH.sub.2--R.sup.20) ##STR00029## --S--
--C(--R.sup.22).dbd.N--CH.sub.2--CH.sub.2--CH.sub.2-- --NH(-SPACER)
##STR00030## bond (p-phenylene)-CH.sub.2--
--N.sup.+(CH.sub.3)(CH.sub.2CH.sub.3).sub.2 ##STR00031## --O--
--CH.sub.2--CH.sub.2--CH.sub.2-- --N(CH.sub.3).sub.2 ##STR00032##
bond (p-phenylene)-O--CH.sub.2--CH.sub.2--CH.sub.2--
--N(CH.sub.3).sub.2 ##STR00033## --S-- ##STR00034##
--OCH.sub.2CH.sub.3 --NH(CH.sub.3).sub.2 --NH-- bond
--N(CH.sub.3).sub.2 ##STR00035## --S--
--CH.sub.2--CH.sub.2--(C.dbd.O)--NH--CH.sub.2--CH.sub.2--CH.sub.2--
--N(CH.sub.3).sub.2
[0103] In another embodiment of the invention, the cyanine dyes of
the invention are described by the formula
##STR00036##
where A, X, R.sup.2, .alpha., n, Y.sub.m, Y.sub.p, R.sup.6,
R.sup.7, R.sup.30, R.sup.31, R.sup.32, and substitution patterns
and preferences are as defined previously. These `free base`
versions of the cyanine dyes of the invention are not derivatized
by an R.sup.5 hydrogen or non-hydrogen atom containing substituent,
instead a pair of electrons is present at the R.sup.5 position,
wherein the neural compound incorporates an aza-benzazole ring
system, a methine moiety, and a pyridine or quinoline ring system.
Related neutral cyanine dyes are described in U.S. Pat. No.
5,656,449 to Yue (1997),
B. Synthesis of the Present Dye Compounds
[0104] A useful synthetic route to the dyes of the present
invention can be described in three parts, following the natural
breakdown in the description of the compounds. In general, the
synthesis of these dyes requires three precursors: the appropriate
azabenzazolium, or polyazabenzazolium salt; the appropriate
pyridine, quinoline, pyridinium or quinolinium; and (where n=1 or
2) a source for the methine spacer. Typically each component is
selected so as to incorporate the appropriate chemical
substituents, or functional groups that can be converted to the
appropriate substituents. The chemistry that is required to prepare
and combine these precursors so as to yield any of the subject
derivatives is generally well understood by one skilled in the art
(see U.S. Pat. No. 5,436,134 to Haugland et al. (1995); U.S. Pat.
No. 5,656,449 to Yue (1997); U.S. Pat. No. 5,658,751 to Yue et al.
(1997), U.S. Pat. No. 5,863,753 to Haugland et al. (1999)).
Although there are many possible variations that may yield an
equivalent result, we provide herein some useful general methods
for their synthesis and incorporation of chemical
modifications.
1. The Azabenzazolium or Polyazabenzazollum Moiety
[0105] A wide variety of derivatives suitable for the preparation
of the dyes of the invention have been previously described (see,
for example, Brooker, et al., J. AM. CHEM. SOC., 64, 199 (1942);
Heravi, et al., INDIAN J. CHEM. 36B, 1025 (1997); Smith et al.
SULFUR LETTERS 17, 197 (1994); Chu-Moyer et al. J. ORG. CHEM. 60,
5721 (1995); Turner, J. ORG. CHEM. 48, 3401 (1983); Couture et al.
J. HETEROCYCLIC CHEM. 24, 1765 (1987); Petric et al. J.
HETEROCYCLIC CHEM. 14, 1045, (1977); Berlin et al. AUST. J. CHEM.,
37, 1729 (1984); Saikachi et al. CHEM. & PHARM. BULL. 9, 941
(1961); Berlin AUST. J. CHEM. 36, 983 (1983); Foye et al., J.
PHARM. SCl. 64, 1371 (1975); Khanna et al. J. ORG. CHEM. 60, 960
(1995)); British Patent No. 870,753 to Ficken at al. (1961); Ficken
at al., "Diazaindenes and Their Quaternary Salts--Part I" pp
3202-3212 (1959); Ficken at al., "Diazaindenes and Their Quaternary
Salts--Part II" pp 584-588 (1961); These synthetic precursors have
the common structure:
##STR00037##
where A, X, R.sup.2, and, c, have been defined previously. If X is
O, the precursor compound is an aza- or polyaza-benzoxazolium; if X
is S it Is an aza- or polyaza-benzothiazolium; if X is Se it is an
aza- or polyaza-benzoselenazolium; If X is N or an
alkyl-substituted N it is an aza- or polyaza-benzimidazolium; and
if X is CR.sup.16R.sup.17 then it is an aza- or polyaza-indolinium
derivative. Commonly R.sup.16 and R.sup.17 are both methyl.
However, methods for preparing compounds where R.sup.16 and
R.sup.17 are not methyl are known (Smith et al., SULFUR LETTERS,
18, 79 (1995); Smith et al., CHEM INDUSTRY, 9, 302 (1988); Couture
at al. HETEROCYCLES 22, 1383 (1984)).
[0106] The substituents on the aromatic carbons of the
aza-benzazolium moiety are typically Incorporated in the parent
aza- or polyaza-benzazole molecule prior to quaternization with an
alkylating agent. However, such substituents may also be
incorporated during the synthesis of the aza-benzazole moiety
(Examples 1-4). R.sup.2 is usually obtained by alkylation of the
parent heterocycle with an alkylating agent R.sup.2--Z, where
R.sup.2 is a substituent as described above, in particular where
R.sup.2 is an alkyl group having 1-6 carbons, that is optionally
substituted by sulfonate, carboxy, amino, or a chemically reactive
group, or a precursor necessary for formation of a TAIL moiety, or
attachment of a BRIDGE precursor, or second dimer subunit. Z of the
alkylating agent is an electronegative group that frequently
becomes the counterion on the resultant dye, W. The counterion may
be exchanged for another counterion by methods known in the art,
such as the use of ion exchange resins or by precipitation.
Selected examples of R.sup.2--Z include methyl iodide, diethyl
sulfate, hexyl p-toluenesulfonate, sulfopropyl iodide, and
sulfobutyliodide. Preferred R.sup.2--Z are compounds that yield
R.sup.2=methyl, such as methyl iodide, methyl methanesulfonate,
dimethyl sulfate, methyl trifluoromethanesulfonate or methyl
p-toluenesulfonate.
[0107] D is a substituent whose nature is determined by the
synthetic method utilized to couple the azabenzazolium or
polyazabenzazolium precursor with the desired precursor of the
second heterocyclic ring system. When n=0, D is typically
alkylthio, commonly methylthio, or D is chloro, bromo or iodo. When
n=1 or 2, D is methyl. Only in the case of D=methyl is any part of
D incorporated in the final compound.
[0108] The aza-benzoxazolium precursor compounds wherein X.dbd.O
have not been previously described.
[0109] The preparation of useful intermediates for the synthesis of
the dyes of the invention via acetylation of a
2-amino-3-hydroxypyridine has similarly not been previously
described (Examples 5 and 21).
2. The Second Heterocyclic Ring System that is a Pyridine, a
Quinoline, a Pyridinium or a Quinolinium
[0110] A variety of intermediate compounds useful as precursors for
the second heterocyclic ring system can be used to synthesize the
second heterocyclic ring system; many of which are available to one
skilled in the art. Compounds containing the quinolinium moiety in
this invention differ from those that contain a single pyridinium
ring only in the presence of an additional aromatic ring containing
four carbon atoms that is fused at the R.sup.6 and R.sup.7
positions of the parent structure. Except where reference is to a
specific pyridine or pyridinium salt, it is understood that mention
of pyridines or pyridinium salts encompasses benzopyridines and
benzopyridinium salts, which are formally called quinolines or
quinolinium salts. Mention of quinolines and quinolinium salts
refer only to structures containing at least two fused aromatic
rings. Similarly, pyridine and quinoline precursors are not
formally charged, and are not substituted by an R.sup.5
substituent.
[0111] In the synthesis of the dyes of the invention, the second
heterocyclic precursor is usually appropriately substituted.
Alternatively, substituents can be incorporated into the precursor
structure subsequent to attachment of the azabenzazolium portion of
the dye. One of the substituents that may be incorporated before or
after reaction with the azabenzazolium or polyazabenzazolium
precursor is TAIL.
[0112] There exist two major structural distinctions within the
family of dyes described in the invention that are related to the
point of attachment of the second heterocyclic ring system. In one
case (where m=0 and p=1) the position of attachment places the
methine moiety adjacent to the ring nitrogen ("2-substituted"). In
the more common case (where m=1 and p=0) the position of the
nitrogen atom is para to the point of attachment
("4-substituted").
[0113] Typically the required pyridinium salt precursor has the
structure
##STR00038##
and the quinolinium salt precursor has the general structure
##STR00039##
with the substituents as defined previously. At all times, the ring
is a 6-membered pyridinium-based heterocycle.
[0114] When n=0, B is methyl, or B is chloro, bromo or Iodo. When
n=1 or 2, B is methyl. Only when n=1 or n=2 is any part of B
incorporated in the final compound.
[0115] There are several general published methods for the
synthesis of derivatives of pyridinium and pyridine, including
those derivatives having substituents at any available position,
including substitutions that are TAIL or that can be converted to
TAIL before or after reaction with the azabenzazolium or
polyazabenzazolium portion to form the dye core structure (see U.S.
Pat. No. 5,436,134, U.S. Pat. No. 5,658,751, and U.S. Pat. No.
5,863,753). Typically, desired substituents are incorporated via
alkylation of the nitrogen atom of an appropriately substituted
quinoline, or R.sup.5 substituents that are aryl or heteroaryl are
Incorporated by an Ullmann reaction of aniline or a substituted
aniline or of a pyridone or quinolone derivative (pyridone and
quinolone precursors may also be prepared by an Ullmann reaction of
the appropriately substituted precursor if the nitrogen atom is
hydrogen-substituted).
[0116] Pyridone and quinolone intermediates containing a
non-hydrogen group at R.sup.5 are particularly important precursors
to a wide variety of other pyridinium and quinolinium salts that
are substituted at R.sup.4. In particular a salt is formed by
treatment of the appropriate pyridone or quinolone with a strong
chlorinating agent such as PCl.sub.5, POCl.sub.3 or SOCl.sub.2, for
instance in the reaction below. Similarly, a sulfonate can be
substituted at R.sup.4 by treating the pyridone or quinolone with
the appropriate sulfonic acid anhydride.
[0117] The reactivity of the 2-halogenated pyridinium or
quinolinium intermediate offers a variety of synthetic methods for
attachment of various substituents at the 2-position, including
TAIL moieties, TAIL precursors, -L-Rx moieties, and BRIDGE
precursors. However, the reactivity of the 2-halo derivatives is
preserved even after conjugation with the benzazolium precursor,
enabling conversion of the resulting dye in which R.sup.4 is
halogen into the appropriate alkoxy, amino and thiolate analogs, as
described for the pyridinium and quinolinium precursors. Of
particular utility for the dyes of the present invention is the
displacement of a 2-chloro substituent by amines.
[0118] Additionally, the 2-oxo group of pyridone or quinolone
precursors can be chemically reduced to derivatives in which
R.sup.4 is H using a variety of reagents including DIBAL-H
(diisobutylaluminum hydride).
3. The Methine Moiety
[0119] The specific synthetic reagents used in the synthesis of the
present dyes determine the number of methine groups that connect
the two heterocyclic ring systems. When n=0, the synthesis of
monomethine dyes commonly uses a combination of reagents where the
methine carbon atom results from either D on the azabenzazolium or
polyazabenzazolium salt or B on the pyridinium salt being methyl
and the other of D or B being a reactive "leaving group" that is
typically methylthio or chloro, but that can be any leaving group
that provides sufficient reactivity to complete the reaction, as
described by Brooker et al., supra. Whether D or B is methyl
depends primarily on the relative ease of synthesis of the
requisite precursor salts. Because 2-methyl and 4-methylpyridines
are usually easier to prepare than their corresponding methylthio
analogs, monomethine dyes are typically prepared from precursors in
which D=methylthio and B=methyl. The condensing reagent in the case
of monomethine dyes is typically a weak base such as triethylamine
or diisopropylethylamine.
[0120] To synthesize trimethine dyes (n=1) both D and B are methyl.
In this case the additional methine carbon is provided by a reagent
such as diphenylformamidine, N-methylformanilide or ethyl
orthoformate. Because under certain reaction conditions these same
reagents can yield symmetrical cyanine dyes that incorporate two
moles of a single quaternary salt, it is important to use the
proper synthetic conditions, and a suitable ratio of the
carbon-providing reactant to the first quaternary salt, so that the
proper Intermediate will be formed. This intermediate is treated
either before or after purification with the second quaternary salt
to form the asymmetric cyanine dye. If desired, the counterion can
be exchanged at this point. One can react either of the
heteroaromatic precursor salts with the carbon-providing reagent to
form the required intermediate, or form the intermediate from the
more readily available 2-methylazobenzazolium or
2-methylpolyazabenzazolium salts similar to the method described by
Brooker et at but modified by use of an azabenzazolium or
polyazabenzazolium salt.
[0121] Synthesis of the pentamethine dyes (n=2) raises the same
synthetic concerns about controlling the formation of an asymmetric
intermediate. The three-carbon fragment that is required for the
additional atoms in the methine moiety comes from a suitable
precursor to malonaldehyde such as malonaldehyde dianil,
1,1,3,3-tetramethoxypropane, 1,1,3-trimethoxypropene,
3-(N-methylanilino)propenal or other reagents. The condensing agent
for this reaction is usually 1-anilino-3-phenylimino-1-propene
(U.S. Pat. No. 2,269,234 to Sprague, 1942), which generates the
2-(2-anilinovinyl)-3-methylbenzazolium tosylate intermediate.
[0122] The introduction of desired methine substituents (R.sup.30,
R.sup.31, and/or R.sup.32) is typically accomplished by
modification of either the azabenzazolium moiety or the
quinolinium/pyridinium moiety prior to condensation of the desired
dye (Examples 28-31). Typically R.sup.32 is introduced by
incorporation into the azabenzazolium moiety (Example 29), while
R.sup.30 is introduced on the quinolinium/pyridinium moiety
(Example 28). R.sup.31 may be introduced on either heterocyclic
ring system, depending on the simplicity and accessibility of the
resulting synthesis.
[0123] Those dyes having a quinoline or pyridine ring rather than a
quinolinium or pyridinium are typically prepared by either heating
a azabenzazolium or polyazabenzazolium salt that contains a good
leaving group (chloride, methylthio, etc.) at the 2-position, with
a 2- or 4-methyl substituted or unsubstituted quinoline or pyridine
in the presence of acetic anhydride as an activator.
[0124] Alternatively, a corresponding N-methylquinolinium cyanine
dye is dealkylated (typically demethylated) using a good
nucleophile, such as an alkali metal salt of a thiol (e.g. sodium
thiophenoxide) in a polar solvent (e.g. DMF, DMSO). Where R.sup.2
is ethyl (or a higher alkyl), the N-methyl substituent on the
pyridinium or quinolinium ring is removed selectively during
dealkylation. Where the R.sup.2 substituent is also methyl, two
demethylated products may result, but the two products are readily
separated by column chromatography. Where the N-substituent is a
substituted alkyl, such as 2-methoxyethoxymethyl, other reagents,
such as a strong acid, are used to generate the corresponding
pyridine or quinoline end product.
4. Tail
[0125] As described above, TAIL is composed of three parts: LINK,
SPACER and CAP. The chemical composition of SPACER is determined by
the chemistry required to attach the heteroatom in CAP with the dye
core structure via LINK.
[0126] As described above, those dyes of the present invention that
are 4-substituted heterocycles wherein R.sup.4 is a TAIL are most
conveniently synthesized from the 2-halo substituted precursor
either before or after condensation with the azabenzazolium or
polkyazabenzazolium portion of the dye by a nucleophilic
displacement reaction of the halogen by a thiol, alkoxide, or a
primary or secondary amine.
[0127] CAP may be Incorporated directly into TAIL before or after
condensation of the second heterocyclic ring with the
azabenzazolium or polyazabenzazolium salt, or CAP may be added or
further modified at a later stage in the synthesis. For instance,
when CAP is a cyclic or non-cyclic primary, secondary or tertiary
amine, CAP can be alkylated to a quaternary ammonium (Examples 10,
11). This reaction can be used to increase the polarity of the dye
and to thus restrict its penetration through the membrane of living
cells, and to additionally increase the dye's affinity for nucleic
acids.
[0128] Precursors to TAIL include, among other functional groups,
carboxylic acids, halides, alcohols and thiols. Each of these
reactive groups can be used to attach a heteroatom-containing
moiety (i.e., CAP) to the dye's core structure, generally through
the formation of amides, ethers or thioethers, which are
incorporated into SPACER before or after attachment of SPACER to
the dye's core structure.
5. Homo- and Heterodimer Dyes
[0129] The dimeric dyes of the invention that are homodimers are
typically prepared by condensation of two equivalents of a
chemically reactive cyanine dye with one equivalent of a BRIDGE
precursor, by methods known in the art (Example 12, 13).
Heterodimeric dyes are prepared similarly, but the synthesis
typically requires multiple steps in order to achieve the desired
final product (as described in U.S. Pat. No. 5,321,130; U.S. Pat.
No. 5,436,134; U.S. Pat. No. 5,658,751; U.S. Pat. No. 5,869,689,
U.S. Pat. No. 5,582,977; U.S. Pat. No. 5,401,847; U.S. Pat. No.
5,565,554; and U.S. Pat. No. 5,760,201).
C. Reactive Cyanine Dyes and Dye-Conjugates
[0130] In another embodiment of the invention, the cyanine dyes of
the invention are chemically reactive, and are substituted by at
least one group -L-R.sub.x, where R.sub.x is the reactive group
that is attached to the dye by a covalent linkage L. Rx is a
reactive group that functions as the site of attachment for another
moiety wherein the reactive group chemically reacts with an
appropriate reactive or functional group on another substance or
moiety. These reactive groups or reactive precursor are synthesized
during the formation of the present compounds providing present
cyanine compounds that can be covalently attached to another
substance, conjugated substance, facilitated by the reactive group.
In this way, compounds incorporating a reactive group (R.sub.x) can
be covalently attached to a wide variety of biomolecules or
non-biomolecules that contain or are modified to contain functional
groups with suitable reactivity, resulting in chemical attachment
of the conjugated substance (S.sub.c), represented by -L-S.sub.c.
This conjugation typically confers the nucleic acid--and/or
poly(amino acid)--sensing abilities of the cyanine dye on the
conjugated substance. However, the present dye compounds can also
function as reporter molecules for the conjugated substance wherein
the sensing ability of the compounds is not employed. The reactive
group and functional group are typically an electrophile and a
nucleophile that can generate a covalent linkage, Alternatively,
the reactive group is a photoactivatable group, and becomes
chemically reactive only after illumination with light of an
appropriate wavelength. Typically, the conjugation reaction between
the reactive group and the substance to be conjugated results in
one or more atoms of the reactive group Rx to be incorporated into
a new linkage attaching the compound of the invention to the
conjugated substance Sc. Selected examples of functional groups and
linkages are shown in Table 3, where the reaction of an
electrophilic group and a nucleophilic group yields a covalent
linkage.
TABLE-US-00003 TABLE 3 Examples of some routes to useful covalent
linkages with electrophile and nucleophile reactive groups
Electrophilic Group Nucleophilic Group Resulting Covalent Linkage
activated esters* amines/anilines carboxamides acyl azides**
amines/anilines carboxamides acyl halides amines/anilines
carboxamides acyl halides alcohols/phenols esters acyl nitriles
alcohols/phenols esters acyl nitriles amines/anilines carboxamides
aldehydes amines/anilines imines aldehydes or ketones hydrazines
hydrazones aldehydes or ketones hydroxylamines oximes alkyl halides
amines/anilines alkyl amines alkyl halides carboxylic acids esters
alkyl halides thiols thioethers alkyl halides alcohols/phenols
ethers alkyl sulfonates thiols thioethers alkyl sulfonates
carboxylic acids esters alkyl sulfonates alcohols/phenols ethers
anhydrides alcohols/phenols esters anhydrides amines/anilines
carboxamides aryl halides thiols thiophenols aryl halides mines ryl
amines aziridines thiols thioethers boronates glycols boronate
esters carboxylic acids amines/anilines carboxamides carboxylic
acids alcohols esters carboxylic acids hydrazines hydrazides
carbodiimides carboxylic acids N-acylureas or anhydrides
diazoalkanes carboxylic acids esters epoxides thiols thioethers
haloacetamides thiols thioethers halotriazines amines/anilines
aminotriazines halotriazines alcohols/phenols triazinyl ethers
imido esters amines/anilines amidines isocyanates amines/anilines
ureas isocyanates alcohols/phenols urethanes isothiocyanates
amines/anilines thioureas maleimides thiols thioethers
phosphoramidites alcohols phosphite esters silyl halides alcohols
silyl ethers sulfonate esters amines/anilines alkyl amines
sulfonate esters thiols thioethers sulfonate esters carboxylic
acids esters sulfonate esters alcohols ethers sulfonyl halides
amines/anilines sulfonamides sulfonyl halides phenols/alcohols
sulfonate esters *Activated esters, as understood in the art,
generally have the formula --CO.OMEGA., where .OMEGA. is a good
leaving group (e.g. oxysuccinimidyl (--OC.sub.4H.sub.4O.sub.2)
oxysulfosuccinimidyl (--OC.sub.4H.sub.3O.sub.2--SO.sub.3H),
-1-oxybenzotriazolyl (--OC.sub.6H.sub.4N.sub.3); or an aryloxy
group or aryloxy substituted one or more times by electron
withdrawing substituents such as nitro, fluoro, chloro, cyano, or
trifluoromethyl, or combinations thereof, used to form activated
aryl esters; or a carboxylic acid activated by a carbodiimide to
form an anhydride or mixed anhydride --OCOR.sup.a or
--OCNR.sup.aNHR.sup.b, where R.sup.a and R.sup.b, which may be the
same or different, are C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy; or cyclohexyl,
3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can
also rearrange to isocyanates
[0131] The cyanine compounds of the present invention typically
comprise a linker when the dye compound is substituted with a
reactive group or a conjugated substance wherein the linker is used
to covalently attach a conjugated substance or reactive group to
the compound. When present, the linker is a single covalent bond or
a series of stable bonds. Thus, the conjugated substance or
reactive group may be directly attached (where Linker is a single
bond) to the cyanine compounds or attached through a series of
stable bonds. When the linker is a series of stable covalent bonds
the linker typically incorporates 1-20 nonhydrogen atoms selected
from the group consisting of C, N, O, S and P. In addition, the
covalent linkage can Incorporate a platinum atom, such as described
in U.S. Pat. No. 5,714,327. When the linker is not a single
covalent bond, the linker may be any combination of stable chemical
bonds, optionally including, single, double, triple or aromatic
carbon-carbon bonds, as well as carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds,
carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen
bonds, and nitrogen-platinum bonds. Typically the linker
incorporates less than 15 nonhydrogen atoms and are composed of any
combination of ether, thioether, thiourea, amine, ester,
carboxamide, sulfonamide, hydrazide bonds and aromatic or
heteroaromatic bonds. Typically the linker is a single covalent
bond or a combination of single carbon-carbon bonds and
carboxamide, sulfonamide or thioether bonds. The bonds of the
linker typically result in the following moieties that can be found
in the linker: ether, thioether, carboxamide, thiourea,
sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl,
alkoxy, cycloalkyl and amine moieties. The longest linear segment
of the linkage L preferably contains 4-10 nonhydrogen atoms
including one or two heteroatoms. Examples of L include substituted
or unsubstituted polymethylene, arylene, alkylarylene or
arylenealkyl. In one embodiment, L contains 1-6 carbon atoms; in
another, L is a thioether linkage. In yet another embodiment, L has
the formula --(CH.sub.2).sub.a(CONH(CH.sub.2).sub.b).sub.z--, where
a is an integer from 0-5, b is an integer from 1-5 and z is 0 or
1.
[0132] Any combination of linkers may be used to attach the Rx or
Sc and the cyanine compounds together, typically a compound of the
present invention when attached to more than one Rx or Sc will have
one or two linkers attached that may be the same or different. The
linker may also be substituted to alter the physical properties of
the carbocyanine compounds, such as solubility and spectral
properties of the compound.
[0133] The -L-Rx moiety, or precursor to the -L-Rx moiety, is
synthesized on the appropriate precursor during the synthesis of
the cyanine dye compound. The, -L-Rx moieties are typically
prepared from 2-halo substituted heterocyclic precursors, either
before or after condensation with the azabenzazolium or
polyazabenzazolium portion of the dye by a nucleophilic
displacement reaction of the halogen by a thiol, alkoxide, or a
primary or secondary amine, which is then incorporated into the
covalent linkage L.
[0134] The -L-Rx group can be bound to the dye at R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13,
or R.sup.14. Alternatively, the -L-R.sub.x moiety is bound to one
of the aromatic carbons of the aza-benzazole ring system.
Typically, -L-R.sub.x is bound to the dye at R.sup.2, R.sup.4 or
R.sup.5, Preferably -L-R.sub.x is bound to the dye at R.sup.4 or
R.sup.5.
[0135] The selection of covalent linkage to attach the dye to the
conjugated substance typically depends on the chemically reactive
functional group on the substance to be conjugated. The types of
functional groups typically present on the organic or inorganic
substances include, but are not limited to, amines, thiols,
alcohols, phenols, aldehydes, ketones, phosphates, imidazoles,
hydrazines, hydroxylamines, disubstituted amines, halides,
epoxides, sulfonate esters, purines, pyrimidines, carboxylic acids,
or a combination of these groups. A single type of reactive site
may be available on the substance (typical for polysaccharides), or
a variety of sites may occur (e.g. amines, thiols, alcohols,
phenols), as is typical for proteins. A conjugated substance may be
conjugated to more than one dye, which may be the same or
different, or to a substance that is additionally modified by a
hapten. Although some selectivity can be obtained by careful
control of the reaction conditions, selectivity of labeling is best
obtained by selection of an appropriate reactive dye.
[0136] Typically, R.sub.x will react with an amine, a thiol or an
alcohol. In one embodiment, R.sub.x is an acrylamide, an activated
ester of a carboxylic acid, an acyl azide, an acyl nitrile, an
aldehyde, an alkyl halide, an amine, an anhydride, an aniline, an
aryl halide, an azide, an aziridine, a boronate, a carboxylic acid,
a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, an
imido ester, an isocyanate, an isothiocyanate, a maleimide, a
phosphoramidite, a sulfonyl halide, or a thiol group. Preferably,
R.sub.x is a carboxylic acid, a succinimidyl ester, an amine, a
haloacetamide, an alkyl halide, a sulfonyl halide, an
isothiocyanate, a maleimide group or an azidoperfluorobenzamido
group.
[0137] Prior to use in preparation of conjugates to form -L-Sc
moieties, chemically reactive forms of the dyes of the invention
are typically dissolved in water or a water-miscible such as a
lower alcohol, dimethylformamide (DMF), dimethylsulfoxide (DMSO),
acetone, acetonitrile, tetrahydrofuran (THF), dioxane or
acetonitrile. The preparation of dye conjugates using reactive dyes
is well documented, Haugland, MOLECULAR PROBES, INC. HANDBOOK OF
FLUORESCENT PROBES AND RESEARCH CHEMICALS, 1996 and Brinkley,
BIOCONJUGATE CHEM., 3, 2 (1992), Conjugates typically result from
mixing appropriate reactive cyanine dyes and the substance to be
conjugated In a suitable solvent in which both are soluble, using
methods well known in the art, followed by separation of the
conjugate from any unreacted dye and by-products. These dyes are
typically combined with the sample under conditions of
concentration, stoichiometry, pH, temperature and other factors
that affect chemical reactions that are determined by both the
reactive functional groups on the dye and the expected site of
modification on the molecule to be modified. These factors are
generally well known in the art of forming bioconjugates (Haugland
et al. "Coupling of Antibodies with Biotin" THE PROTEIN PROTOCOLS
HANDBOOK, J. M. Walker, ed., Humana Press, (1996); Haugland
"Coupling of Monoclonal Antibodies with Fluorophores" METHODS 1N
MOLECULAR BIOLOGY, VOL. 45: MONOCLONAL ANTIBODY PROTOCOLS, W. C.
Davis, Ed. (1995)). For those reactive dyes that are
photoactivated, conjugation requires illumination of the reaction
mixture to activate the reactive dye. The dye--conjugate is used in
solution or lyophilized and stored for later use.
[0138] A variety of dye-conjugates may be prepared using the
reactive dyes of the invention, including present dye conjugates of
antigens, steroids, vitamins, drugs, haptens, metabolites, toxins,
environmental pollutants, amino acids, peptides, proteins, nucleic
acids, nucleic acid polymers, carbohydrates, lipids, and polymers.
In another embodiment, the conjugated substance is an amino acid,
peptide, protein, polysaccharide, nucleotide, oligonucleotide,
nucleic acid, hapten, drug, lipid, phospholipid, lipoprotein,
lipopolysaccharide, liposome, lipophilic polymer, polymer,
polymeric microparticle, biological cell or virus. In one aspect of
the invention, the conjugated substance is labeled with a plurality
of dyes of the present invention, which may be the same or
different.
[0139] Typically, the conjugated substance (S.sub.c) is an amino
acid, a peptide, a protein, a polysaccharide, a nucleotide, an
oligonucleotide, a nucleic acid, a lipid, a polymeric
microparticle, a biological cell, or a virus. The most preferred
conjugated substances are conjugates of haptens, nucleotides,
oligonucleotides, nucleic acid polymers, proteins, or
polysaccharides. Most preferably, the conjugated substance is a
nucleic acid, or a substance that interacts in a specific fashion
with nucleic acids, such as DNA-binding proteins.
[0140] In one embodiment, the conjugated substance (S.sub.c) is an
amino acid (including those that are protected or are substituted
by phosphates, carbohydrates, or C.sub.1 to C.sub.22 carboxylic
acids), or is a polymer of amino acids such as a peptide or
protein. Preferred conjugates of peptides contain at least five
amino acids, more preferably 5 to 36 amino acids. Preferred
peptides include, but are not limited to, neuropeptides, cytokines,
toxins, protease substrates, and protein kinase substrates. Also
preferred are peptides that serve as organelle localization
peptides, that is, peptides that serve to target the conjugated dye
for localization within a particular cellular substructure by
cellular transport mechanisms. Preferred protein conjugates include
enzymes, antibodies, lectins, glycoproteins, histones, albumins,
lipoproteins, avidin, streptavidin, protein A, protein G,
phycobiliproteins and other fluorescent proteins, hormones, toxins
and growth factors. Typically, the conjugated protein is an
antibody, an antibody fragment, avidin, streptavidin, a toxin, a
lectin, or a growth factor. Preferred haptens include biotin,
digoxigenin and fluorophores.
[0141] In another embodiment, the conjugated substance (S.sub.c) is
a nucleic acid base, nucleoside, nucleotide or a nucleic acid
polymer, optionally containing an additional linker or spacer for
attachment of a fluorophore or other ligand, such as an alkynyl
linkage (U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat.
No. 4,711,955) or other linkage. Preferably, the conjugated
nucleotide is a nucleoside triphosphate or a deoxynucleoside
triphosphate or a dideoxynucleoside triphosphate.
[0142] Preferred nucleic acid polymer conjugates are single- or
multi-stranded, natural or synthetic DNA or RNA oligonucleotides,
or DNA/RNA hybrids, or incorporating an unusual linker such as
morpholine derivatized phosphates (AntiVirals, inc., Corvallis
Oreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine
units, where the nucleic acid contains fewer than 50 nucleotides,
more typically fewer than 25 nucleotides. The dye is optionally
attached via one or more purine or pyrimidine bases through an
amide, ester, ether or thioether bond; or is attached to the
phosphate or carbohydrate by a bond that is an ester, thioester,
amide, ether or thioether. Alternatively, the dye is attached by
formation of a non-covalent association of the nucleic acid and a
photoreactive dye of the invention, followed by illumination,
resulting in covalently bound dye. Nucleotide conjugates of the
invention can be incorporated by some DNA polymerases and can be
used for in situ hybridization and nucleic acid sequencing (e.g.,
U.S. Pat. Nos. 5,332,666; 5,171,534; and 4,997,928, all
incorporated by reference; and WO Appl. 94/05688).
[0143] In another embodiment, the conjugated substance (S.sub.c) is
a carbohydrate or polyol that is typically a polysaccharide, such
as dextran, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,
starch, agarose and cellulose, or is a polymer such as a
poly(ethylene glycol). Preferred polysaccharide conjugates are
dextran or FICOLL conjugates.
[0144] In another embodiment, the conjugated substance (S.sub.c),
is a lipid (typically having 6-25 carbons), including glycolipids,
phospholipids, and sphingolipids. Alternatively, the conjugated
substance is a lipid vesicle, such as a liposome, or is a
lipoprotein (see below). Some ilpophilic substituents are useful
for facilitating transport of the conjugated dye into cells or
cellular organelles.
[0145] Furthermore, the conjugates are optionally dye-conjugates of
polymers, polymeric particles, polymeric microparticles including
magnetic and non-magnetic microspheres, polymeric membranes,
conducting and non-conducting metals and non-metals, and glass and
plastic surfaces and particles. Conjugates are typically prepared
by chemical modification of a polymer that contains functional
groups with suitable chemical reactivity. The conjugated polymer
may be organic or inorganic, natural or synthetic. In a preferred
embodiment, the dye is conjugated to a polymer matrix, such as a
polymeric particle or membrane, including membranes suitable for
blot assays for nucleic acids or proteins. In another embodiment,
the conjugated substance is a glass or silica, which may be formed
into an optical fiber or other structure. In another embodiment,
the conjugated polymer is polyethylene glycol), a poly(acrylate) or
a poly(acrylamide).
[0146] In one embodiment, the conjugated substance is a specific
binding pair members wherein the dye is conjugated to a specific
binding pair member and used to detect nucleic acids or poly(amino
acids). Alternatively, the presence of the labeled specific binding
pair member indicates the location of the complementary member of
that specific binding pair; each specific binding pair member
having an area on the surface or in a cavity which specifically
binds to, and is complementary with, a particular spatial and polar
organization of the other. In this instance, the dye compounds of
the present invention function as a reporter molecule for the
specific binding pair and not a dye selective for poly(amino acids)
or nucleic acids.
TABLE-US-00004 TABLE 4 Representative Specific Binding Pairs
antigen antibody biotin avidin (or streptavidin or anti-biotin)
IgG* protein A or protein G drug drug receptor folate folate
binding protein toxin toxin receptor carbohydrate lectin or
carbohydrate receptor peptide peptide receptor protein protein
receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA).dagger.
hormone hormone receptor ion chelator *IgG is an immunoglobulin
.dagger.cDNA and cRNA are the complementary strands used for
hybridization
D. Methods of Use
[0147] The use of the invention comprises combining a dye (monomer
or dimer) or dye-conjugate of the present invention with a sample
that contains or is thought to contain the desired analyte,
incubating the mixture of dye and sample for a time sufficient for
the dye to combine with the analyte in the sample and to form one
or more dye-analyte complexes having a detectable fluorescent
signal. The characteristics of the dye-analyte complex, including
the presence, location, intensity, excitation and emission spectra,
fluorescence polarization, fluorescence lifetime, photobleaching
rate and other physical properties of the fluorescent signal can be
used to detect, differentiate, sort, quantitate, and/or analyze
aspects or portions of the sample. The dyes of the invention are
optionally used in conjunction with one or more additional
detection reagents (preferably detectably different fluorescent
reagents), including dyes of the same class having different
spectral properties.
[0148] Therefore, a method for determining the presence of an
analyte of interest in a sample, comprises the steps of: [0149] a)
combining said sample with a staining solution, wherein said
staining solution comprises one or more dyes of the present
invention; [0150] b) incubating said sample and said staining
solution for a sufficient amount of time to form a dye-analyte
complex; [0151] c) illuminating said complex with an appropriate
wavelength whereby the presence of said analyte is determined.
[0152] Typically the analyte of interest is a nucleic acid polymer
or a lipid-complexed poly(amino acid). However when a dye-conjugate
is present in the staining solution the analyte may be any compound
the conjugated substance has an affinity for. Thus, in one
embodiment of the invention, the analyte is a nucleic acid polymer,
such as DNA or RNA. In another embodiment of the invention, the
analyte is a lipid-complexed poly(amino acid). By poly(amino acid)
is meant any polymer of amino acid subunits, artificial or natural,
such as peptides and proteins, and including glycoproteins,
lipoproteins and other modified proteins.
[0153] The staining solution is preferably an aqueous or aqueous
miscible solution that is compatible with the sample and the
intended use. For biological samples, where minimal perturbation of
cell morphology or physiology is desired, the staining solution is
selected accordingly. For solution assays, the staining solution
preferably does not perturb the native conformation of the analyte
undergoing evaluation.
[0154] The dyes have greater stability in buffered solutions than
in water alone; and agents that reduce the levels of free oxygen
radicals, such as .beta.-mercaptoethanol, contribute to the
stability of the dyes. When staining nucleic acids, high
concentrations of organic solvents, cations, and oxidizing agents
generally reduce fluorescence of the dye-nucleic acid complex, as
does the ionic detergent sodium dodecyl sulfate (SDS) at
concentrations >0.01%. A number of staining solution additives,
however, do not interfere with the fluorescence of the dye-nucleic
acid complex (e.g. urea up to 8 M; CsCl up to 1 g/mL; formamide up
to 50% of the solution; and sucrose up to 40%). Nonionic detergents
such as Tween 20, NP40 or Triton X-100 at concentration of less
than or equal to 0.1%, DMSO at concentrations less than or equal to
10%, and ethanol at concentrations of less than or equal to about
10% stabilize the dye-buffer staining solution, but can interfere
with staining of poly(amino acids).
[0155] Although typically used in an aqueous or aqueous miscible
solution, the staining solution is typically prepared by first
dissolving the dye in a water-miscible organic solvent such as
dimethylsulfoxide (DMSO), dimethylformamide (DMF), or a lower
alcohol, such as methanol or ethanol. This stock solution is
typically prepared at a concentration of greater than about
100-times that used in the final staining solution, then diluted
one or more times with an aqueous solvent or a buffer solution such
that the dye is present in an effective amount. The buffer solution
may be, for example, a buffered saline (preferably non-phosphate
for some viability discrimination applications), or a
Tris(hydroxymethyl)aminomethane (IRIS) buffer (preferably
containing EDTA). Typically, the dye is first dissolved in 100%
DMSO, and then diluted with buffer.
[0156] The staining solution, containing an effective amount of
dye, is then combined with a sample for a sufficient amount of time
to form a dye-analyte complex. An effective amount of dye is the
amount of dye sufficient to give a detectable fluorescence response
in combination with the desired analyte. The dye concentration in
the staining solution must be sufficient both to contact the
analyte in the sample and to combine with the analyte in an amount
sufficient to give a signal, but too much dye may cause problems
with background fluorescence. The optimal concentration and
composition of the staining solution is determined by the nature of
the sample (including physical, biological, biochemical and
physiological properties), the nature of the dye-analyte
interaction (including the transport rate of the dye to the site of
the analyte), and the nature of the analysis being performed, and
can be determined using standard procedures, similar to those
described in examples below.
[0157] The analyte of interest is optionally enclosed within a
biological structure (i.e. an organism or a discrete unit of an
organism), free in solution (including solutions that contain
biological structures), immobilized in or on a solid or semi-solid
material, or is extracted from a biological structure (e.g. from
lysed cells, tissues, organisms or organelles). Preferably, the
analyte is present in an aqueous environment in order to facilitate
contact with the dye.
[0158] The sample is combined with the staining solution by any
means that facilitates contact between the dye and the analyte.
Typically the contact occurs through simple mixing, as in the case
where the sample is a solution. A staining solution containing the
dye may be added to the analyte solution directly or may contact
the analyte solution in a liquid separation medium such as an
electrophoretic liquid, sieving matrix or running buffer, or in a
sedimentation (e.g. sucrose) or buoyant density gradient (e.g.
containing CsCl), or on an inert matrix, such as a blot or gel, a
testing strip, or any other solid or semi-solid support. Suitable
supports also include, but are not limited to, polymeric
microparticles (including paramagnetic microparticles),
polyacrylamide and agarose gels, nitrocellulose filters, computer
chips (such as silicon chips), natural and synthetic membranes,
liposomes and alginate hydrogels, and glass (including optical
filters), and other silica-based and plastic support. The dye is
optionally combined with the analyte solution prior to undergoing
gel or capillary electrophoresis, gradient centrifugation, or other
separation step, during separation, or after the nucleic acids or
lipid-complexed proteins undergo separation. Alternatively, the dye
is combined with an inert matrix or solution in a capillary prior
to addition of the analyte solution, as in pre-cast gels, capillary
electrophoresis or preformed density or sedimentation
gradients.
[0159] The sample is combined with the dye for a time sufficient to
form the fluorescent dye-analyte complex, preferably the minimum
time required to give a high signal-to-background ratio. Although
all of the present dyes are nucleic acid stains and lipid-complexed
protein stains, detectable fluorescence within biological
structures or in gels requires entry of the dye across the
biological membrane or into gels. Optimal staining with a
particular dye is dependent upon the physical and chemical nature
of the individual sample and the sample medium, as well as the
property being assessed. The optimal time is usually the minimum
time required for the dye, in the concentration being used, to
achieve the highest target-specific signal while avoiding
degradation of the sample over time and minimizing all other
fluorescent signals due to the dye. For example, where the dye is
chosen to be selective for a particular nucleic acid polymer or
type of cell, the optimal time is usually the minimum time required
to achieve the highest signal on that polymer or type of cell, with
little to no signal from other nucleic acids or other cell types.
Over time, undesirable staining may occur as even very low rates of
diffusion may transport small amounts of the very sensitive dyes
into other cell types, or staining selectivity may be lost as the
cell membranes degrade, or as nucleases degrade nucleic acid
polymers in cell-free systems.
[0160] Preferably, the dye is combined with the sample at a
temperature optimal for biological activity of the analyte within
the operating parameters of the dyes (usually between 5.degree. C.
and 50.degree. C., with reduced stability of the dyes at higher
temperatures). For in vitro assays, the dye is typically combined
with the sample at about room temperature (23.degree. C.). At room
temperature, detectable fluorescence in a solution of nucleic acids
is essentially instantaneous depending on the sensitivity of the
instrumentation that is used; fluorescence in solutions is
generally visible by eye within 5 seconds after the dye is added,
and is generally measurable within 2 to 5 minutes, although
reaching equilibrium staining may take longer. In particular,
staining of lipid-complexed proteins in solution may be slower than
equivalent staining of nucleic acids. Where a biological process is
underway during in vitro analysis (e.g. in vitro transcription,
replication, splicing, or recombination), the rapid labeling that
occurs with the subject dyes avoids perturbation of biological
system that is being observed. Gel staining at room temperature
usually takes from 5 minutes to 2 hours depending on the thickness
of the gel and the percentage of agarose or polyacrylamide, as well
as the degree of cross-linking. Typically, post-stained minigels
stain to equilibrium in 20-45 minutes. For cells and other
biological structures, transport of dyes across membranes is
required whether the membranes are intact or disrupted. For
preferred embodiments, visibly detectable fluorescence is obtained
at room temperature within 15-20 minutes of incubation with cells,
commonly within about 5 minutes. Some embodiments give detectable
fluorescence inside cells in less than about 2 minutes. This
property is useful for observing nuclear structure and
rearrangement, for example such as occurs during mitosis or
apoptosis. Some of the dyes are generally not permeant to live
cells with intact membranes; other dyes are generally permeant to
eukaryotes but not to prokaryotes; still other dyes are only
permeant to cells in which the cell membrane integrity has been
disrupted (e.g. some dead cells). The relative permeability of the
cell membrane to the dyes is determined empirically, e.g. by
comparison with staining profiles or staining patterns of killed
cells. The dye with the desired degree of permeability, and a high
absorbance and quantum yield when bound to nucleic acids or
lipid-complexed proteins, is selected to be combined with the
sample.
[0161] The presence, location, and distribution of the analyte is
detected using the spectral properties of the fluorescent
dye-analyte complex. Spectral properties means any parameter that
may be used to characterize the excitation or emission of the
dye-nucleic acid complex including absorption and emission
wavelengths, fluorescence polarization, fluorescence lifetime,
fluorescence intensity, quantum yield, and fluorescence
enhancement. Typically the spectral properties of excitation and
emission wavelength are used to detect the dye complexes. The
wavelengths of the excitation and emission bands of the dyes vary
with dye composition to encompass a wide range of illumination and
detection bands. This allows the selection of individual dyes for
use with a specific excitation source or detection filter. In
particular, complexes formed with dyes having a monomethine moiety
(n=0) generally match their primary excitation band with the 532 nm
excitation line of the frequency-doubled Nd-Yag laser, the 543 nm
excitation line of the green He--Ne laser, the strong 546 nm
emission of the Hg-arc lamp and sometimes the 568 nm excitation
line of the Kr laser; whereas those with dyes with a trimethine
moiety (n=1) primarily tend to match long wavelength excitation
sources such as the orange HeNe laser (594 nm), the red HeNe laser
(633 nm), the Kr laser (568 or 647 nm) and certain laser diodes, in
particular the 635 nm laser diode; and complexes formed with dyes
having a pentamethine moiety (n=2) primarily match very long
excitation sources such as laser diodes or light emitting diodes
(LEDs). In addition to the primary excitation peak in the visible
range, the dye-nucleic acid complexes and dye-lipid-complexed
protein complexes of the invention have a secondary absorption peak
that permits excitation with UV illumination. Dyes with n=1 and n=2
typically form complexes that permit excitation beyond 600 nm.
[0162] Typically, the sample is excited by a light source capable
of producing light at or near the wavelength of maximum absorption
of the fluorescent analyte-complex, such as an ultraviolet or
visible wavelength emission lamp, an arc lamp, a laser, or even
sunlight or ordinary roomlight. Preferably the sample is excited
with a wavelength within 20 nm of the maximum absorption of the
fluorescent complex. Although excitation by a source more
appropriate to the maximum absorption band of the dye-analyte
complex results in higher sensitivity, the equipment commonly
available for excitation of samples can be used to excite the dyes
of the present invention.
[0163] The fluorescence of the complex is detected qualitatively or
quantitatively by detection of the resultant light emission at a
wavelength of greater than about 450 nm, preferably greater than
about 480 nm, more preferably at greater than about 500 nm. The
emission is detected by means that include visual inspection, CCD
cameras, video cameras, photographic film, or the use of
instrumentation such as laser scanning devices, fluorometers,
photodiodes, quantum counters, plate readers, epifluorescence
microscopes, scanning microscopes, confocal microscopes, flow
cytometers, capillary electrophoresis detectors, or by means for
amplifying the signal such as a photomultiplier tube. Many such
instruments are capable of utilizing the fluorescent signal to sort
and quantitate cells or quantitate the nucleic acids. Dyes can be
selected to have emission bands that match commercially available
filter sets such as that for fluorescein, tetramethylrhodamine or
for detecting multiple fluorophores with several excitation and
emission bands.
[0164] The source and type of sample, as well as the use of the
dye, will determine which dye characteristics, and thus which dyes,
will be most useful for staining a particular sample. For most
applications, dyes are selected to give a quantum yield greater
than about 0.1, more usefully 0.2 or 0.3, preferably greater than
0.5, when associated with an analyte; preferably the dyes have a
quantum yield <0.02 and more preferably <0.01 or <0.005
when present in solution and not associated with an analyte.
Preferably, upon associating with an analyte, the dye of the
invention will exhibit a fluorescence enhancement greater than
about 50 fold, preferably greater than 100 fold, more preferably
greater than 200 fold and most preferably greater than 500 fold.
Where the fluorescence of the dye-analyte complex or dye conjugate
is detected utilizing sustained high intensity illumination (e.g.
microscopy), dyes with a rate of photobleaching lower than commonly
used dyes (e.g. fluorescein) are preferred, particularly for use in
live cells. Where the dye must penetrate cell membranes or a gel,
more permeant dyes are preferred, although some cells readily take
up dyes that are shown to be impermeant to other cells by means
other than passive diffusion across cell-membranes, e.g. by
phagocytosis or other types of ingestion. Dyes that rapidly and
readily penetrate cells do not necessarily rapidly penetrate gels.
In applications where the nucleic acids are stained on a gel, the
dye is also selected to have a relatively high binding affinity
(preferably K.sub.d<10.sup.-6 M); whereas in applications where
the nucleic acid will be pre-stained prior to undergoing a
separation step, such as gel or capillary electrophoresis, even
higher binding affinity (preferably K.sub.d<10.sup.-8 M) is
preferred to ensure good separation. In staining nucleic acids in
solution, high binding affinity translates into greater sensitivity
to small amounts of nucleic acid, but dyes with a moderate binding
affinity (preferably 10.sup.-6 M<K.sub.d<10.sup.-8 M) are
more effective over a greater dynamic range. The photostabllity,
binding affinity, quantum yield, and fluorescence enhancement of
dyes are determined according to standard methods known in the
art.
[0165] In a preferred embodiment, the dyes of the present invention
are employed to detect the presence of lipid-poly(amino acids). In
this instance the poly(amino acids) are typically immobilized on a
solid or semi-solid matrix, but alternatively may be present in
solution provided the correct concentration of lipid is present.
Therefore, a method of the present invention for detecting lipid
complexed poly(amino acids) in a sample comprises: [0166] a)
combining said sample with a staining solution, wherein said
staining solution comprises one or more dyes of the present
invention; [0167] b) incubating said sample and said staining
solution for a sufficient amount of time to form a dye-analyte
complex; [0168] c) illuminating said complex with an appropriate
wavelength whereby the presence of said analyte is determined.
[0169] The sample containing a poly(amino acid) is in a solution,
or immobilized on a solid or semi-solid matrix such as a blot or
electrophoretic gel, the dye is preferably combined with the sample
in an effective amount in the presence of a low percentage of a
lipid. The amount of lipid or detergent required is typically a
concentration available from a solution of 0.05-2.0% sodium dodecyl
sulfate (SDS). Although the dyes of the invention may stain some
proteins in the absence of lipid complexation, both the association
of the dye with the protein and the resulting fluorescent signal
are enhanced by the presence of a lipid complexing agent.
Preferably the lipid complexing agent is an amphiphilic lipid, such
as a detergent. Due to the limitation of a detergent for staining
all proteins, the lipid-complexed protein is typically not
associated with a biological cell but is free in solution or is
immobilized on a solid or semi-solid matrix or is in an
electrophoretic matrix or chamber, such as one used for gel or
capillary electrophoresis.
[0170] The detergent is optionally added simultaneously with or as
part of the sample or the staining solution, or is added thereafter
to the combined mixture. The detergent is any amphiphilic surface
active agent or surfactant that serves to coat the poly(amino
acids) (i.e. non-covalently associate with the poly(amino acid)),
or is a mixture of more than one detergent. Useful detergents
Include non-ionic, cationic, anionic, amphoteric and fluorinated
surfactants. While there are a variety of detergents that are
commercially available, including non-ionic, cationic, and anionic
detergents, any detergent that is utilized in protein gel
electrophoresis is a preferred detergent for the present invention.
Typically, the detergent is an anionic detergent, preferably an
alkyl sulfate or alkyl sulfonate salt, More preferably, the
detergent is sodium dodecyl sulfate (SDS), sodium octadecyl
sulfate, or sodium decyl sulfate, or a mixture thereof. Most
preferably, the detergent is sodium dodecyl sulfate.
[0171] The dyes of the invention typically stain micelles, when
present, so it is preferred that any detergent or detergent mixture
present in the sample mixture, staining mixture, or combined
mixture be present below the critical micelle concentration (CMC)
for that detergent.
[0172] Combination of the dye with lipid-complexed proteins is
facilitated when the proteins are first heated in the presence of
detergent, including to temperatures in excess of 90.degree. C.
This heating step optionally occurs in the presence of the dye of
the invention, although exposure to elevated temperatures may
effect the stability of the dye.
[0173] The poly(amino acids) are optionally a synthetic or
naturally occurring poly(amino acid), such as a peptide,
polypeptide or protein. Poly(amino acids) that are stained and
analyzed according to the present method optionally incorporate
non-peptide regions (covalently or non-covalently) including lipid
(lipopeptides and lipoproteins), phosphate (phosphopeptides and
phosphoproteins), and/or carbohydrate (glycopeptides and
glycoproteins) regions; or incorporate metal chelates or other
prosthetic groups or non-standard side chains; or are multi-subunit
complexes, or incorporate other organic or biological substances,
such as nucleic acids. The poly(amino acids) are optionally
relatively homogeneous or heterogeneous mixtures of poly(amino
acids). In one aspect of the invention the poly(amino acids) are
enzymes, antibodies, transcription factors, secreted proteins,
structural proteins, or binding factors, or combinations thereof.
The poly(amino acids) in the sample mixture are optionally
covalently or non-covalently bound to a solid surface, such as a
glass slide, multi-well plate, microliter plate well, plastic pin
or bead, or semiconductor material, or they are unbound. The
staining of a poly(amino acid) that is bound to an analyte on a
solid surface indicates the presence of the analyte as well as that
of the poly(amino acid).
[0174] The poly(amino acids) are optionally unmodified, or have
been treated with a reagent so as to enhance or decrease the
mobility of the poly(amino acid) in an electrophoretic gel. Such
reagents may modify poly(amino acids) by complexing with the
peptide (to decrease migration), by cleaving selected peptide bonds
(to increase migration of the resulting fragments), by changing the
relative charge on the protein (as by phosphorylation or
dephosphorylation) or by covalent coupling of a constituent such as
occurs during glycosylation. The presence or interaction of such a
reagent in the sample mixture is detected by the change in
electrophoretic mobility of the treated poly(amino acids), relative
to untreated poly(amino acids) having the same original
composition, so that the distribution of the dye-poly(amino acid)
complex indicates the presence of another analyte.
[0175] Typically the poly(amino acids) in the sample mixture have a
molecular weight greater than about 500 daltons. More typically the
poly(amino acids) are more than 800 daltons. Smaller polymers of
amino acids (in the <1000 dalton range) are generally difficult
to separate from the detergent front on denaturing gels, and
typically do not adhere to filter membranes, but are still readily
detected in solution. There is no precise upper limit on the size
of the poly(amino acids) that may be stained and detected, except
that they can not be so bulky that they precipitate out of
solution, which also depends in part on the relative hydrophobicity
of the poly(amino acid). Furthermore, poly(amino acids) greater
than about 200,000 daltons are generally not effectively resolved
with current gel technology. The poly(amino acids) present
optionally have essentially the same molecular weight or fall
within a range of molecular weights. In one embodiment of the
invention, the poly(amino acids) present are a mixture of
poly(amino acids) of different molecular weights that are used as
molecular weight standards. A typical such mixture contains equal
mass quantities of myosin, .beta.-galactosidase, phosphorylase B,
bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin
inhibitor, lysozyme and aprotinin.
[0176] Where the sample mixture is an aqueous solution, the
poly(amino acids) of the sample mixture are typically present in a
concentration of 10 ng/mL-50 .mu.g/mL, more preferably in a
concentration of 30 ng/mL-10 .mu.g/mL, most preferably in a
concentration of 50 ng/mL-5 .mu.g/mL. Where the sample mixture is
an electrophoretic gel, the poly(amino acids) of the sample mixture
are typically present in a concentration of 1 ng/band-4
.mu.g/band.
[0177] The poly(amino acids) are obtained from a variety of
sources; such sources include biological fermentation media and
automated protein synthesizers, as well as prokaryotic cells,
eukaryotic cells, virus particles, tissues, and biological fluids.
Suitable biological fluids include, but are not limited to, urine,
cerebrospinal fluid, blood, lymph fluids, interstitial fluid, cell
extracts, mucus, saliva, sputum, stool, physiological or cell
secretions or other similar fluids.
[0178] Depending on the source of the sample mixture, it optionally
contains discrete biological ingredients other than the desired
poly(amino acids), including poly(amino acids) other than those
desired, amino acids, nucleic acids, carbohydrates, and lipids,
which may or may not be removed in the course of, prior to, or
after staining. In one aspect of the invention, the poly(amino
acids) in the sample mixture are separated from each other or from
other ingredients in the sample by mobility (e.g. electrophoretic
gel or capillary) or by size (e.g. centrifugation, pelleting or
density gradient), or by binding affinity (e.g. to a filter
membrane) in the course of the method. In another aspect of the
invention, the sample mixture thought to contain the poly(amino
acids) has undergone separation. In yet another aspect of the
invention, the poly(amino acids) are not separated. Although lipid
assemblies such as intact or fragmented biological membranes (e.g.
membranes of cells and organelles), liposomes, or detergent
micelles, and other lipids are optionally present in the sample
mixture; the presence of large amounts of lipids, particularly
lipid assemblies, increases background labeling due to non-specific
staining. For effective detection of labeled poly(amino acids),
intact or fragmented biological membranes in the sample mixture are
preferably removed, destroyed or dispersed prior to or in the
course of labeling with this method. Typically treatment of the
sample mixture by standard methods to remove some or all of such
lipids, such as ammonium sulfate precipitation, solvent extraction
or trichloroacetic acid precipitation is used. Alternatively or
additionally, lipids are removed in the course of labeling the
poly(amino acids) such as by electrophoretic separation or other
separation techniques (e.g. centrifugation, including gradients),
or are disrupted or dispersed below the concentration at which they
assemble into micelles (critical micelle concentration) by
mechanical means such as sonication. Naturally occurring lipids
that are present below their critical micelle concentration are
optionally used as a detergent for the purposes of the present
invention. Typically, the sample mixture is essentially cell-free.
This method is not effective for detecting proteins that remain in
cells or are associated with biological membranes.
[0179] In another preferred embodiment the present dyes and
staining solution are employed to detect nucleic acids in a sample.
Again, the staining solution is combined with the sample to form a
dye-nucleic acid complex and then illuminated with an appropriate
wavelength whereby the nucleic acid is detected.
[0180] Therefore, when used to stain nucleic acids or to form
conjugates of nucleic acids, the dye is combined with a sample that
contains or is thought to contain a nucleic acid. The nucleic acid
in the sample may be RNA or DNA, or a mixture or a hybrid thereof.
Any nucleic acid is optionally single- or multiple-stranded. The
nucleic acid may be a natural polymer (biological in origin) or a
synthetic polymer (modified or prepared artificially). The nucleic
acid polymer (preferably containing at least 8 bases or base pairs)
may be present as nucleic acid fragments, oligonucleotides, or
larger nucleic acid polymers with secondary or tertiary structure,
the nucleic acid is optionally present in a condensed phase, such
as a chromosome, or in a protein complex, such as a ribosome or
nucleosome. The nucleic acid polymer optionally contains one or
more modified bases or links or contains labels that are
non-covalently or covalently attached. For example, the modified
base is optionally a naturally occurring modified base, a known
minor base, or is synthetically altered to contain an unusual
linker such as morpholine derivatized phosphates, or peptide
nucleic acids, or to contain a simple reactive functional group
(<10 carbons) that is an aliphatic amine, carboxylic acid,
alcohol, thiol or hydrazine, or to contain a fluorescent label or
other hapten. The sensitivity of the dyes for polymers containing
primarily modified bases and links may be diminished by
interference with the binding mode.
[0181] The nucleic acid may be present in solution, immobilized on
a solid or semi-solid matrix or present in a biological structure.
Where the analyte is enclosed in a biological structure, as is
typical for nucleic acids, the sample is typically incubated with
the dye. While permeant dyes of this class have shown an ability to
permeate biological structures rapidly and completely upon addition
of the dye solution, any other technique that is suitable for
transporting the dye into the biological structure is also a valid
method of combining the sample with the subject dye. Some cells
actively transport the dyes across cell membranes (e.g. endocytosis
or ingestion by an organism or other uptake mechanism) regardless
of their cell membrane permeability. Suitable artificial means for
transporting the dyes (or pre-formed dye-nucleic acid complexes)
across cell membranes include, but are not limited to, action of
chemical agents such as detergents, enzymes or adenosine
triphosphate; receptor- or transport protein-mediated uptake;
liposomes or alginate hydrogels; phagocytosis; pore-forming
proteins; microinjection; electroporation; hypo-osmotic shock; or
minimal physical disruption such as scrape loading, patch clamp
methods, or bombardment with solid particles coated with or in the
presence of the dyes. Preferably, where intact structures are
desired, the methods for staining cause minimal disruption of the
viability of the cell and integrity of cell or intracellular
membranes. Alternatively, the cells are fixed and treated with
routine histochemical or cytochemical procedures, particularly
where pathogenic organisms are suspected to be present. The cells
are typically fixed immediately after staining with an aldehyde
fixative that keeps the dye in the cells. In some cases, live or
dead cells may even be fixed prior to staining without
substantially increasing cell membrane permeability of previously
live cells so that only cells that were already dead prior to
fixation stain with the cell-impermeant dye.
[0182] Typically, the biological structure that encloses the
nucleic acid is a cell or tissue, for example where the nucleic
acid is present in a cell or interstitial space as a prokaryote or
eukaryote microorganism, or as a virus, viroid, chromosome or
organelle. Alternatively, the biological structure is not enclosed
in a tissue or cell, and is present either as a virus or as a
microorganism or other cell, or is present as a cellular component
removed from its parent cell (e.g. a plasmid or chromosome, or a
mitochondrion or nucleus or other organelle).
[0183] Typically, the biological structure is an organelle,
chromosome or cell that is optionally inside a eukaryote cell. The
cell that is present inside a eukaryote cell is typically a
parasite or other infective agent such as a bacterium, protozoa,
mycoplasma or mycobacterium. Where the nucleic acid is contained in
a biological structure that is a cell, the cells are viable or dead
cells or a mixture thereof, i.e. the integrity of the cell membrane
is optionally intact or disrupted by natural (autolytic),
mechanical or chemical means or by environmental means such as
changes in temperature or pressure. Alternatively, the cells are
blebbing or undergoing apoptosis or necrosis or are in a cycle of
growth or cell division,
[0184] Cell types for which the dye is an effective nucleic acid
stain include cells with or without nuclei, including but not
limited to, eukaryotes, such as plant and animal cells
(particularly vertebrate cells), including pollen and gamete cells;
prokaryotes, particularly bacteria, including both Gram-negative
and Gram-positive bacteria; as well as yeast and other fungi, and
spores. In one embodiment, the sample comprises reticulocytes. The
dyes are not equally effective in staining all cell types and
certain dyes are generally more permeant than others. Live cells
are less permeable to the dyes than dead cells, and prokaryotes are
typically less permeable than eukaryotes.
[0185] The nucleic acids in the sample, both natural and synthetic,
may be obtained from a wide variety of sources. The presence of the
nucleic acid in the sample may be due to natural biological
processes, or the result of a successful or unsuccessful synthesis
or experimental methodology, undesirable contamination, or a
disease state. The nucleic acid may be endogenous to the natural
source or introduced as foreign material, such as by infection,
transfection, or therapeutic treatment. Nucleic acids may be
present in all, or only part, of a sample, and the presence of
nucleic acids may be used to distinguish between individual
samples, or to differentiate a portion or region within a single
sample, or to identify the sample or characteristics of the
sample.
[0186] Typically, the sample containing nucleic acids is a cell or
is an aqueous or aqueous miscible solution that is obtained
directly from a liquid source or as a wash from a solid material
(organic or inorganic) or a growth medium in which cells have been
introduced for culturing or a buffer solution in which nucleic
acids or biological structures have been placed for evaluation.
Where the nucleic acids are in cells, the cells are optionally
single cells, including microorganisms, or multiple cells
associated with other cells in two or three dimensional layers,
including multicellular organisms, embryos, tissues, biopsies,
filaments, biofilms, etc.
[0187] Alternatively, the sample is a solid, optionally a smear or
scrape or a retentate removed from a liquid or vapor by filtration.
In one aspect of the invention, the sample is obtained from a
biological fluid, including separated or unfiltered biological
fluids such as urine, cerebrospinal fluid, blood, lymph fluids,
tissue homogenate, interstitial fluid, cell extracts, mucus,
saliva, sputum, stool, physiological secretions or other similar
fluids. Alternatively, the sample is obtained from an environmental
source such as soil, water, or air; or from an industrial source
such as taken from a waste stream, a water source, a supply line,
or a production lot. Industrial sources also include fermentation
media, such as from a biological reactor or food fermentation
process such as brewing; or foodstuffs, such as meat, grain,
produce, or dairy products.
[0188] In yet another embodiment, the sample is immobilized in or
on a solid or semi-solid matrix wherein the nucleic acids of
interest are present on or in the matrix. In one aspect of the
invention, the matrix is a membrane. In another aspect, the matrix
is an electrophoretic gel, such as is used for separation and
characterization of nucleic acids. In another aspect, the matrix is
a silicon chip or glass slide, and the nucleic acids of interest
have been immobilized on the chip or slide in an array.
[0189] Where the nucleic acid is present in a solution, the sample
solution can vary from one of purified or synthetic nucleic acids
such as oligonucleotides to crude mixtures such as cell extracts or
homogenates or other biological fluids, or dilute solutions from
biological, industrial, or environmental sources. In some cases it
is desirable to separate the nucleic acids from a mixture of
biomolecules or fluids in the solution prior to combination with
the dye. Numerous techniques exist for separation and purification
of nucleic acids from generally crude mixtures with other proteins
or other biological molecules. These include such means as
chromatographic and electrophoretic techniques, using a variety of
supports or solutions or in a flowing stream. Alternatively,
mixtures of nucleic acids may be treated with RNase or DNase so
that the polymer that is not degraded in the presence of the
nuclease can be discriminated from degradation products using the
subject dyes.
[0190] The fluorescent dye-analyte complex is useful in essentially
any application previously described for other fluorescent nucleic
acid or protein stains (for example as described in U.S. Pat. No.
5,436,134 to Haugland et al. (1995); U.S. Pat. No. 5,445,946 to
Roth et al. (1995); U.S. Pat. No. 5,534,416 to Millard et al.
(1996); U.S. Pat. No. 5,616,502 to Haugland et al. (1997).
[0191] Once the dye-analyte complex is formed, its presence may be
detected and used as an indicator of the presence, location, or
type of analyte in the sample, or as a basis for sorting cells, or
as a key to characterizing the sample or cells in the sample. Such
characterization may be enhanced by the use of additional reagents,
including fluorescent reagents. The amount of analyte in a sample
can also be quantified by comparison with known relationships
between the fluorescence of the dye-analyte complex and
concentration of analyte in the sample.
[0192] In one aspect of the invention, the fluorescence response of
the dye-analyte complex is used as a means for detecting the
presence or location of the analyte in the sample. The fluorescent
signal is detected by eye or by the instrumentation described
above.
[0193] In addition, the staining profile that results from
formation of the dye-analyte complex may be indicative of one or
more characteristics of the sample. By staining profile is meant
the shape, location, distribution, and spectral properties of the
profile of fluorescent signals resulting from excitation of the
fluorescent dye-analyte complexes. The sample can be characterized
simply by staining the sample and detecting the staining profile
that is indicative of a characteristic of the sample. More
effective characterization is achieved by utilizing a dye that is
selective for a certain characteristic of the sample being
evaluated or by utilizing an additional reagent (as described
below), where the additional reagent is selective for the same
characteristic to a greater or lesser extent or where the
additional reagent is selective for a different characteristic of
the same sample.
[0194] In one embodiment of the invention, where the dye is
selected to be membrane permeable or relatively impermeant to cell
membranes, the staining profile that results from the formation of
the dye-analyte complex is indicative of the integrity of the cell
membrane, which in turn is indicative of cell viability.
Alternatively, the more permeant dyes of the invention are used to
stain both cells with intact membranes and cells with disrupted
membranes, which when used in conjunction with a counterstain that
gives a detectably different signal in cells with disrupted
membranes, allows the differentiation of viable cells from dead
cells. The counterstain that gives a detectably different signal in
cells with disrupted membranes is optionally an impermeant dye of
the invention or another reagent that indicates loss of integrity
of the cell membrane or lack of metabolic activity of the dead
cells.
[0195] In a further embodiment of the invention, the shape and
distribution of the staining profile of dye-analyte complexes is
indicative of the type of cell or biological structure that
contains the stained nucleic acids. Cells may be discriminated by
eye based on the visual fluorescent signal or be discriminated by
instrumentation as described above, based on the spectral
properties of the fluorescent signal. Typically the staining
profile used to characterize the sample is indicative of the
presence, shape, or location of organelles or of cells, where the
cells are located in a biological fluid, in a tissue, or in other
cells.
[0196] In another aspect of the invention, the dye-analyte complex
is used as a fluorescent tracer or as a probe for the presence of a
second analyte. In one aspect of the invention, the dye-analyte
complex is used as a size or mobility standard, such as In
electrophoresis or flow cytometry. Alternatively, the fluorescent
signal that results from the interaction of the dye with the first
analyte can be used to detect or quantitate the activity or
presence of other molecules that interact with the first
analyte.
Applications of Chemically Reactive Cyanine Dyes
[0197] The reactive dyes of the invention are particularly useful
for immobilizing the dye on a surface or substrate, such as a
polymeric microparticle, polymeric membrane, fiber, or silicon
chip. In this embodiment, the dye is useful as a capture reagent
for purification or detection of an analyte, particularly nucleic
acids. Alternatively, the immobilized dyes are useful for moving
associated analytes from one environment to another via mechanical
means. Where the subject dyes are attached to a surface, the
surface can also function as a quantitative or qualitative
indicator of an analyte in solution, such as a test strip or
dipstick.
[0198] The reactive dyes of the invention are also useful as
detection reagents during the separation and purification of
nucleic acids. For example the dyes of the invention can be
conjugated to agar or agarose and used to cast plates or gels that
are then useful for spot assays to quantitate nucleic acids. The
ability of the reactive dyes of the invention to form stable
covalent bonds with a variety of substrates allows the attachment
of nucleic acid stains to electrophoretic gel matrices (producing
very stable and uniformly stained pre-prepared gels).
[0199] Alternatively, where nucleic acids are analyzed using
capillary electrophoresis, the dyes of the invention may be
conjugated in a small "band" or region in the capillary, resulting
in detection of nucleic acids with high sensitivity, as the
detecting reagent is concentrated into a small volume, allowing
efficient illumination using a laser or other excitation source.
Where a conjugated dye is used in this fashion, the dye is
optionally utilized only to capture the nucleic acids, with
detection accomplished by another detection reagent, or the
fluorescent enhancement upon binding nucleic acids is utilized to
detect the capture event itself.
[0200] Where the dye is covalently bound to a substrate, such as a
polymeric microparticle or membrane, the nucleic acids that are
associated with the bound dye can be used as templates for
translation and replication. Where the dye of the invention
possesses a particularly high affinity for nucleic acids, passing a
solution containing nucleic acids over a suitable labeled polymer
matrix results in depletion of nucleic acids from the solution. The
complexed nucleic acids can then be utilized in place or extracted
from the matrix for further analysis or utilization.
[0201] Alternatively, a reactive dye of the invention can be
associated with purified nucleic acids and covalently linked to the
nucleic acids, which are then transferred to a membrane by
blotting, and covalently bound to the polymeric membrane, resulting
in a permanently labeled blot.
[0202] The dyes are optionally covalently bound to a molecule, such
as a dextran, which is normally excluded from live cells. When dead
or permeabilized cells then take up these molecules, the bound dye
interacts with intracellular nucleic acids, giving rise to
fluorescence. Similarly, the dye-conjugates of the invention
possess utility for retaining conjugated substances within cells.
This is particularly useful for conjugated substances that are
typically excreted from living cells relatively quickly, such as
small organic molecules or by-products of enzyme activity.
[0203] The dye is optionally conjugated to a substance selected
such that the resulting conjugate has substantially different
physical properties than those of the unbound dye, for example,
conjugation of a dye of the invention to a polysaccharide or a
poly(ethylene glycol) polymer to influence solubility, cellular
retention, or ability to bind analytes.
Applications of Covalently Labeled Nucleic Acids
[0204] The dyes of the invention are useful for labeling unmodified
nucleic acids covalently (typically using a photoreactive dye) so
that the labeled nucleic acid retains its label through capillary
transfer or electrophoretic transfer to filter membranes and
subsequent hybridization (for example as In Southern blotting). The
dyes are also useful in Southwestern analysis, wherein the dye is
first coupled covalently to a nucleic acid, and the nucleic acid is
then used to probe a Western blot containing putative nucleic
acid-binding proteins. Fluorescence enhancement indicates locations
on the blot membrane where the nucleic acid has been captured by
the immobilized protein. Conjugates of proteins that do not
interact specifically with nucleic acids do not exhibit similar
fluorescence enhancement when used at similar concentrations, and
in the absence of detergent. Similar interactions are useful for
detecting immobilized DNA. Alternatively, where the protein is
labeled with a second reagent, colocalization of that reagent and
the dye of the invention indicates an interaction between the dye
and the protein. This type of assay is also useful for analyzing
nucleic acid-binding drugs.
[0205] Where the dyes of the invention are used to covalently label
nucleic acids, the labeled nucleic acid is useful as a probe to
detect interactions between nucleic acid-binding drugs or proteins,
where the drug or protein has been labeled with a second
fluorophore or a fluorescence quenching agent. Binding of the
nucleic acid with the drug or protein results in loss of
fluorescence. In this way, the nucleic acid-binding drug or protein
can be quantitated, and the presence or absence of either
inhibitors or enhancers of nucleic acid binding could be
identified. This application is also useful for in vivo or in vitro
studies, for example in the study of interactions between SSB
(single strand binding protein) and single stranded DNA, histones
and double stranded DNA, sequence specific binding factors and
their cognate sequences, or mismatch repair enzymes and mismatch
regions of DNA hybrids.
[0206] Dye-labeled nucleic acids are useful for monitoring
transfection into cells, either by the labeled nucleic acid itself,
or where another molecule is cotransfected and the labeled nucleic
acid is simply a tracer. The amount of labeled nucleic acid in a
cell could be used to standardize the copy number of transiently
transfected plasmids in reporter gene assays, such as
chloramphenicol acetyltransferase assays (CAT assays).
[0207] Dye-conjugates of nucleic acids possess utility for
detecting hybridization, as the dye-conjugate typically exhibits a
change in fluorescence enhancement upon binding to a complementary
strand of nucleic acid. Dye-labeled nucleic acids are also useful
for following triplex formation, or strand invasion, during DNA
recombination. If two dyes capable of energy transfer are used,
then real-time measurements of hybridization or strand
invasion/displacement may be made. In particular both the
interactions of an antisense oligonucleotides with nucleic acids
and RNA splicing could be followed using this methodology.
[0208] Where the dye is used to covalently label a nucleic acid,
the nucleic acid could then be ligated to another unlabeled nucleic
acid, so that even if the labeled nucleic acid fragment is unable
to hybridize or bind proteins efficiently, the unlabeled portion
retains full biological activity. However, the nucleic acid is
readily detectable due to the presence of the attached dye.
[0209] The dyes of the invention also serve as haptens for
secondary detection. The use of labeled antibodies directed against
the dyes of the invention allows for signal amplification in the
detection of either a conjugated substance, or a labeled nucleic
acid. Alternatively, where the conjugated substance is a specific
binding pair member, the specific binding pair member may be used
to amplify the detectable signal of the cyanine dye, typically by
immunological methods. In this embodiment, the conjugated substance
is typically a hapten, biotin or digoxigenin or a non-nucleic
acid-binding dye. The dye-conjugate forms a nucleic acid-dye
complex, producing enhancement of its fluorescence. The complex is
then labeled with the complement of the specific binding pair
member, which is typically labeled with a fluorophore, producing an
additional fluorescent enhancement.
Additional Detection Reagents
[0210] The dyes of the invention can be used in conjunction with
one or more additional reagents that are separately detectable. The
additional reagents may be separately detectable if they are used
separately, e.g. used to stain different aliquots of the same
sample, or if they stain different parts or components of a sample,
regardless of whether the signal of the additional reagents is
delectably different from the fluorescent signal of the
dye-analyte. Alternatively, the dye of the invention is selected to
give a detectable response that is different from that of other
reagents desired to be used in combination with the subject dyes.
Preferably the additional reagent or reagents are fluorescent and
have different spectral properties from those of the dye-analyte
complex. For example, dyes that form complexes that permit
excitation beyond 600 nm can be used in combination with commonly
used fluorescent antibodies such as those labeled with fluorescein
isothiocyanate or phycoerythrin. Any fluorescence detection system
(including visual inspection) can be used to detect differences in
spectral properties between dyes, with differing levels of
sensitivity. Such differences include, but are not limited to, a
difference in excitation maxima, a difference in emission maxima, a
difference in fluorescence lifetimes, a difference in fluorescence
emission intensity at the same excitation wavelength or at a
different wavelength, a difference in absorptivity, a difference in
fluorescence polarization, a difference in fluorescence enhancement
in combination with target materials, or combinations thereof.
[0211] The detectably different dye is optionally one of the dyes
of the invention having different spectral properties and different
selectivity. In one aspect of the invention, the dye-analyte
complex and the additional detection reagents have the same or
overlapping excitation spectra, but possess visibly different
emission spectra, generally having emission maxima separated by
>10 nm, preferably >20 nm, more preferably >50 nm.
Simultaneous excitation of all fluorescent reagents may require
excitation of the sample at a wavelength that is suboptimal for
each reagent individually, but optimal for the combination of
reagents. Alternatively, the additional reagent(s) can be
simultaneously or sequentially excited at a wavelength that is
different from that used to excite the subject dye-analyte complex.
In yet another alternative, one or more additional reagents are
used to quench or partially quench the fluorescence of the
dye-analyte complex, such as by adding a second reagent to improve
the selectivity for a particular nucleic acid or the AT/GC
selectivity.
[0212] The additional dyes are optionally used to differentiate
cells or cell-free samples containing the desired analyte according
to size, shape, metabolic state, physiological condition, genotype,
or other biological parameters or combinations thereof. The
additional reagent is optionally selective for a particular
characteristic of the sample for use in conjunction with a
non-selective reagent for the same characteristic, or is selective
for one characteristic of the sample for use in conjunction with a
reagent that is selective for another characteristic of the sample.
In one aspect of the invention, the additional dye or dyes are
metabolized intracellularly to give a fluorescent product inside
certain cells but not inside other cells, so that the fluorescence
response of the cyanine dye of the invention predominates only
where such metabolic process is not taking place. Alternatively,
the additional dye or dyes are specific for some external component
of the cell such as cell surface proteins or receptors, e.g.
fluorescent lectins or antibodies. In yet another aspect of the
invention, the additional dye or dyes actively or passively cross
the cell membrane and are used to Indicate the integrity or
functioning of the cell membrane (e.g. calcein AM or BCECF AM). In
another aspect, the additional reagents bind selectively to AT-rich
nucleic acids and are used to indicate chromosome banding. In
another aspect of the invention, the additional reagent is an
organelle stain, i.e. a stain that is selective for a particular
organelle, for example the additional reagent(s) may be selected
for potential sensitive uptake into the mitochondria (for example
as described in U.S. Pat. No. 5,459,268 to Haugland et al. (1995))
or for uptake due to pH gradient in an organelle of a live cell
(U.S. Pat. No. 5,869,689 to Zhang et al. (1999)).
[0213] The additional dyes are added to the sample being analyzed
to be present in an effective amount, with the optimal
concentration of dye determined by standard procedures generally
known in the art. Each dye is optionally prepared in a separate
solution or combined in one solution, depending on the intended
use. After illumination of the dyed cells at a suitable wavelength,
as above, the cells are analyzed according to their fluorescence
response to the illumination. In addition, the differential
fluorescence response can be used as a basis for sorting the cells
or nucleic acids for further analysis or experimentation. For
example, all cells that "survive" a certain procedure are sorted,
or all cells of a certain type in a sample are sorted. The cells
can be sorted manually or using an automated technique such as flow
cytometry, according to the procedures known in the art, such as in
U.S. Pat. No. 4,665,024 to Mansour, et al. (1987).
D. Kits of the Invention
[0214] In one aspect of the invention, the cyanine dyes of the
invention are incorporated into a kit for commercial use. In one
embodiment, the kit comprises a selection of dyes of the invention,
typically present as a concentrated stock solution in a non-aqueous
solvent, preferably DMSO. Where the kit is a "sampler" kit of
cyanine dyes, the kit comprises 2-8 distinct cyanine dyes,
preferably 4-6 distinct cyanine dyes.
[0215] In another embodiment of the invention, the reagent kit
comprises a stock solution of the dye of the invention, typically
present in a DM solution; a buffer suitable for dilution of the
stock solution, and optionally further comprises a standard or an
additional detection reagent, or both. The standard is optionally
one or more nucleic acids (for nucleic acid
detection/quantification), or one or more proteins (for protein
detection/quantification). The additional detection reagent is
typically an organelle stain, a labeled immunoreagent, a drug, or
an enzyme.
[0216] In yet another embodiment of the invention, the reagent kit
comprises a stock solution of the dye of the invention, typically
present in a DMSO solution, a buffer suitable for dilution of the
stock solution, a silicon chip or glass slide, and nucleic acid
standards.
[0217] A detailed description of the invention having been provided
above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
EXAMPLES
Example 1
Preparation of Aza-Benzoxazole Precursors
[0218] The following 4-methyl-2-methylthiooxazolopyridinium
tosylates are prepared by heating the corresponding
2-methylthlooxazolopyridines (M. Y. Chu-Moyer and R. Berger, J.
Org. Chem. 60, 5721-5725 (1995)) with one equivalent of methyl
tosylate at 100-110.degree. C. for one hour:
##STR00040##
(4-methyl-2-methylthiooxazolo[4,5-b]pyridinium tosylate)
##STR00041##
(6-methyl-2-methylthiooxazolo[5,4-c]pyridinium tosylate):
##STR00042##
(1-methyl-2-methylthiooxazolo[5,4-b]pyridinium tosylate):
##STR00043##
(4-methyl-2-methylthiooxazolo[4,5-b]quinolinium tosylate):
Example 2
Preparation of 5-methyl-2-methylthiothiazolo[4,5-b]pyridinium
tosylate
(Compound 5):
##STR00044##
[0220] To 0.626 g of 2-methylthiothiazolo[4,5-b]pyridine (Smith,
K., Lindsay, C., Morris, I. K., Matthews, I. and Pritchard, G. J.,
Sulfur Letters 17, 197-216 (1994)), is added 0.71 g methyl
tosylate. The mixture is heated at 120.degree. C. for one hour.
After cooling, the resulting oily mixture is washed with 10 mL of
ethyl acetate. After the ethyl acetate is decanted, Compound 5 is
isolated as an oil.
Example 3
Preparation of 5-methyl-2-methylthiothiazolo[5,4-c]pyridinium
tosylate (Compound 6)
##STR00045##
[0222] A mixture of 0.127 g of 2-methylthiothiazolo[5,4-c]pyridine
(from methylation of thiazolo[5,4-c]pyridine-2(1H)-thione with
potassium carbonate and methyl iodide) and 0.143 g methyl tosylate
is heated at 120.degree. C. for one hour. Ethyl acetate (8 mL) is
added, the mixture is stirred for 30 minutes, and Compound 6 is
isolated by suction filtration.
Example 4
Preparation of 3-methyl-2-methylthiothiazolo[5,4-b]pyridinium
tosylate
(Compound 7):
##STR00046##
[0224] A mixture of 1.285 g of 3-amino-2-chloropyridine, 0.48 g NaH
(from a hexane-washed 60% dispersion of NaH in mineral oil), 0.6 mL
CS.sub.2 and 10 mL DMF is heated at 110-120.degree. C. for one hour
to yield thiazolo[5,4-b]pyridine-2(1H)-thione. The thione is
methylated with potassium carbonate and methyl iodide in DMF at
room temperature to give the methylthio derivative,
2-methylthiothiazolo[5,4-b]pyridine, which is subsequently heated
with one equivalent of methyl tosylate at 100 for one hour to yield
Compound 7.
[0225] Similarly,
6-chloro-4-methyl-2-methylthiothiazolo[4,5-b]pyridinium tosylate is
prepared from 6-chlorothiazolo[4,5-b]pyridine-2(1H)-thione:
##STR00047##
6-bromo-4-methyl-2-methylthiothiazolo[4,5-b]pyridinium tosylate is
prepared from 2-amino-3,5-dibromopyridine:
##STR00048##
4-methyl-2-methylthio-6-trifluoromethylthiazolo[4,5-b]pyridinium
tosylate is prepared from
2-amino-3-chloro-5-trifluoromethylpyridine:
##STR00049##
and 1-methyl-2,7-bis(methylthio)thiazolo[5,4-d]pyridinium tosylate
is prepared from 5-amino-4,6-dichloropyrimidine:
##STR00050##
Example 5
Preparation of
3-acetoxy-1-methyl-2-acetylimino-1,2-dihydropyridine,
p-toluenesulfonic acid salt (Compound 12)
##STR00051##
[0227] 2-Amino-3-hydroxypyridine (14.48 g) is triacetylated by
heating with 3 equivalents of acetic anhydride at 120-130.degree.
C. for 4 hours to yield, after silica gel column purification, 10.3
g of 3-acetoxy-2-acetimidopyridine. This intermediate compound is
heated for 2 days at 65.degree. C. with 3 equivalents of methyl
tosylate to yield 7 g of Compound 12.
Example 6
Preparation of 5-bromo-3-mercapto-2-methylaminopyridine (Compound
13)
##STR00052##
[0229] Compound 13 is prepared by heating Compound 9 (1.99 g) with
30 mL methanol and 15 mL of 10% NaOH at 80.degree. C. for 8 hours.
The pH is adjusted to 6-7 with acetic acid and the volume is
reduced to -10 mL. The residue is filtered to yield 0.76 g of
Compound 13.
Example 7
Preparation of Compound 14
##STR00053##
[0231] To 0.55 g of Compound 1 and 0.49 g of
1,4-dimethylquinolinium tosylate in 10 mL of CH.sub.2Cl.sub.2 at
room temperature is added 0.24 mL of triethylamine. After 6 hours,
the product is suction filtered to yield 0.42 g of Compound 14.
Example 8
Preparation of Compounds 15, 16, 17, 18, 19, 20, 21, and 44
##STR00054##
[0233] To a solution of 0.235 g of 4-methyl-1-phenyl-2-quinolone in
15 mL THF at -78.degree. C., is added 0.6 mL of a 2.5 M solution of
1-butyl lithium in THF. After 1 hr at -78.degree. C., 1 mL of
acetic acid is added. The reaction mixture is stirred for 1 hr at
room temperature. The volatiles are evaporated and the residue is
dissolved in 10 mL CH.sub.2Cl.sub.2. Compound 1 (0.7 g) and 1 mL
triethylamine are added. After 1 hr at room temperature the solvent
is evaporated, the crude product is dissolved in 10 mL of methanol
and the solution is then added to 2.5 g NaI in 60 mL water. The
product, Compound 15, is filtered and further purified by column
chromatography on silica gel.
[0234] Similarly prepared except using phenyl lithium instead of
butyl lithium is Compound 16, which can be recrystallized from a
mixture of DMF and ethyl acetate.
##STR00055##
[0235] Compound 17 is prepared similarly from
4-methyl-1-phenyl-2-quinolone
##STR00056##
[0236] Compound 18 is prepared similarly from
1-ethyl-6,7-methylenedioxy-4-methyl-2-quinolone
##STR00057##
[0237] Compound 19 is prepared similarly from
6-methoxy-1,4-dimethyl-2-quinolone
##STR00058##
[0238] Compound 20 is prepared similarly from
6,7-dimethoxy-1,4-dimethyl-2-quinolone
##STR00059##
[0239] Compound 21 is prepared similarly from
7-methoxy-1,4-dimethyl-2-quinolone.
##STR00060##
[0240] Compound 44 is prepared similarly, except that Compound 9 is
used instead of Compound 1:
##STR00061##
Example 9
Preparation of Compound 22
##STR00062##
[0242] To 0.605 g of 4'-bromobenzyldiethylamine in 10 mL of THF at
-78.degree. C. under nitrogen is added 0.8 mL of a 2.5 M butyl
lithium in THF, followed by a solution of 0.173 g of
1,4-dimethyl-2-quinolone in 10 mL of THF. After one hour at
-78.degree. C., 1 mL of acetic acid is added. After warming to room
temperature for another hour the volatiles are evaporated and the
residue is dissolved in 10 mL CH.sub.2Cl.sub.2. To this solution is
added 0.67 g of Compound 2, followed by 1 mL triethylamine. After
one hour at room temperature the volatiles are evaporated and the
residue is dissolved in 7 mL of methanol. This solution is added to
a solution of 2.25 g NaI and 0.8 g NaOH in 50 mL water. Compound 22
is filtered, dried and recrystallized from a mixture of DMF and
ethyl acetate.
[0243] Compound 23 is prepared similarly, using
6-methoxy-1,4-dimethyl-2-quinolone.
##STR00063##
Example 10
Preparation of Compound 24
##STR00064##
[0245] The compound is prepared by heating 0.1 g of Compound 22 in
5 mL DMF with 0.5 mL methyl iodide at 60.degree. C. in a sealed
tube for 2 hours. After the reaction mixture is cooled to room
temperature, 5 mL ethyl acetate is added and Compound 24 is
collected by suction filtration.
[0246] Compound 25 is similarly obtained from Compound 23.
##STR00065##
Example 11
Preparation of Compounds 26 and 27
##STR00066##
[0248] A mixture of 1.06 g of Compound 1, 0.88 g of
1-(3'-iodopropyl)-4-methylquinolinium iodide (U.S. Pat. No.
5,321,130 to Yue et al. (1994)), 15 mL CH.sub.2Cl.sub.2 and 0.28 mL
triethylamine is stirred at room temperature for 30 minutes. The
volatiles are evaporated and the residue is dissolved in 10 mL
methanol. The resulting solution is added to 4.5 g NaI in 60 mL
water. Compound 26 is obtained by suction filtration. A solution of
Compound 26 (0.15 g) and 1 mL of a 25% trimethylamine in methanol
solution are heated in a sealed tube at 75-80.degree. C. for 7
hours. Following cooling and filtration, Compound 27 is
recrystallized from a mixture of DMF and CH.sub.2Cl.sub.2.
[0249] Compound 47 is similarly prepared from Compound 9
##STR00067##
Example 12
Preparation of Compound 28
[0250] A mixture of 0.2 g of Compound 26, 1.5 mL DMF and 0.029 mL
N,N,N',N'-tetramethylpropanediamine is heated at 80.degree. C. for
4 days. To the solution is added 6 mL of chloroform and Compound 28
is isolated by suction filtration.
##STR00068##
Example 13
Preparation of Compounds 45 and 46
##STR00069##
[0252] A mixture of 2.1 g of Compound 9, 1.5 g of
1-(3'-iodopropyl)-4-methylquinolinium iodide, 0.7 mL triethylamine
and 30 mL CH.sub.2Cl.sub.2 is stirred at room temperature for 2
hours. Following evaporation, the residue is dissolved in 30 mL DMF
and added to 8 g NaI in 200 mL water. Compound 45 is obtained by
suction filtration. Compound 45 (1 g), 0.125 mL of
N,N,N',N'-tetramethylpropanediamine and 8 mL DMF are heated at
80.degree. C. for 4 days. Following cooling, 24 mL of chloroform is
added and Compound 46 is purified by recrystallization for a
mixture of DMF and methanol.
Example 14
Preparation of Compounds 29, 30, and 41
##STR00070##
[0254] A mixture of 6 g of Compound 1, 3 g of
4-methyl-1-phenyl-2-quinolone, 8.9 mL diisopropylethylamine, and
1.2 mL of trimethylsilyl trifluoromethanesulfonate is heated at
reflux in 100 mL of CH.sub.2Cl.sub.2 for 1 hour. The reaction
mixture is cooled on ice and 100 mL of water is added slowly with
stirring. The layers are separated and the aqueous layer is
extracted with chloroform. The combined organic layers are washed
with brine and dried over Na.sub.2SO.sub.4. The crude
4-oxazolopyridinylmethylidenequinolone thus obtained is heated at
reflux for 3 hours with 5.8 mL of phosphorus oxychloride and 100 mL
of dichloroethane. The reaction mixture is cooled to room
temperature, 100 mL of water is added and after 30 min, the crude
product is recovered by filtration. The crude product is purified
by chromatography on a silica gel column to yield Compound 29. A
mixture of 0.1 g of Compound 29, 10 mL of dichloroethane and 0.27
mL of diethylamine is heated at 60.degree. C. in a sealed tube for
2.5 hours. The volatile components are evaporated and the crude
residue is purified by chromatography on a silica gel column to
yield Compound 30.
[0255] Preparation of Compound 41 is similar to that of Compound
29, except that Compound 9 is used Instead of Compound 1.
##STR00071##
Example 15
Preparation of Compound 31
##STR00072##
[0257] Triethylamine (0.1 mL) is added to 0.176 g of Compound H and
0.165 g of 1,4-dimethylquinolinium tosylate in 10 mL of
CH.sub.2Cl.sub.2 at room temperature. After stirring for one hour,
the organic layer is evaporated and the residue is dissolved in 2
mL of methanol and added dropwise to a solution of 1.5 g NaI in 25
mL of water. Compound 31 is recovered by suction filtration.
Example 16
Preparation of Compound 32
##STR00073##
[0259] A mixture of 0.35 g of 1,4-dimethyl-2-quinolone, 0.56 mL
phosphorus oxychloride, and 0.05 mL DMF is heated in 10 mL toluene
at 70.degree. C. for 2 hours. After cooling, 20 mL CH.sub.2Cl.sub.2
and 1.11 g 2-mercaptopyridine are added and the reaction mixture is
heated overnight at 40.degree. C. The volatiles are evaporated and
to the residue is added 0.84 g of Compound 1, 1.4 mL of
triethylamine and 20 mL CH.sub.2Cl.sub.2, After 2 hours, the
volatiles are evaporated, the residue is dissolved in 3 mL methanol
and the solution is added to 4.5 g NaI in 20 mL water. Compound 32
is purified by recrystallization from a mixture of DMF and ethyl
acetate.
Example 17
Preparation of Compound 33
##STR00074##
[0261] A mixture of 0.244 g of Compound 7, 0.225 g of
1,4-dimethylquinolinium tosylate, 0.14 mL triethylamine and 10 mL
CH.sub.2Cl.sub.2 is stirred at room temperature overnight. The
volatiles are evaporated, residue is dissolved in 4 mL of methanol,
and the resulting solution is added to 1.5 g NaI in 25 mL water.
Compound 33 is obtained by suction filtration.
[0262] Compounds 34, 35, 36, 37, 38, 39, and 40 are similarly
prepared from 1,4-dimethylquinolinium tosylate and Compounds 6, 11,
4, 10, 5, 9, and 8 respectively.
##STR00075## ##STR00076##
Example 18
Preparation of Compound 42
##STR00077##
[0264] A mixture of 0.2 g of Compound 8, 0.065 mL of lepidine, 0.28
mL acetic anhydride and 5 mL DMF is heated at 110-120.degree. C.
for one hour. The reaction mixture is carefully diluted with a
mixture of sodium bicarbonate and water and then extracted with
chloroform. The chloroform is evaporated, and the resulting residue
is purified by silica gel chromatography to yield Compound 42.
[0265] Compound 43 is similarly prepared from Compound 1.
##STR00078##
Example 19
Preparation of Compound 48
##STR00079##
[0267] Compound 1 (0.36 g), 0.28 g of 1,4-dimethylpridinium
tosylate, 0.17 mL of triethylamine and 20 mL of CH.sub.2Cl.sub.2
are stirred at room temperature for one hour, then stirred for an
additional 3 days at -20.degree. C. Compound 48 is isolated by
suction filtration.
Example 20
Preparation of Compound 49
##STR00080##
[0269] A solution of 4.2 mg of
3-methyl-2-methythio-4,5,7-trifluorothiazolo[5,4-c]pyridinium
tosylate (from 4-amino-2,3,5,6-tetrafluoropyridine by a procedure
similar to that used for preparation of Compound 8), 3.3 g of
1,4-dimethylquinolinium tosylate and 1.7 mL of triethylamine is
stirred in 20 mL CH.sub.2Cl.sub.2 at room temperature for 1 hour.
The volatiles are evaporated and the residue is purified by
chromatography on a silica gel column. The product is converted to
Compound 49 with NaI in water.
Example 21
Preparation of Compound 50
##STR00081##
[0271] A mixture of 0.33 g of 1,4-dimethylquinolinium tosylate,
0.24 g of N,N'-diphenylformamidine, and 0.3 mL acetic anhydride is
heated at 130.degree. C. for 2 hours. Ethyl acetate (20 mL) is
added and the solution is heated at reflux for 20 minutes. The
mixture is cooled, and the resulting solid is dissolved in 5 mL
dichloroethane. Compound 12 (0.34 g), 0.35 mL of
diisopropylethylamine and 0.2 mL acetic anhydride are added. After
stirring for 3 hours at room temperature, the volatiles are
evaporated, the residue is dissolved in 3 mL of methanol and the
solution is added dropwise to 1.5 g NaI in 20 mL water. Compound 50
is recovered by filtration and purified by chromatography on a
silica gel column.
Example 22
Preparation of Compound 51
##STR00082##
[0273] A mixture of 1.9 g of Compound 12, 1.18 g of
N,N'-diphenylformamidine, 2.1 mL of triethylamine and 50 mL
dichloroethane is heated at 60-65.degree. C. for 2 hours. After
cooling, one equivalent of 1,4-dimethyl-2-phenylquinolinium salt
(prepared in a manner similar to that for Compound 16) in 10 mL of
dichloroethane is added, followed by 0.7 mL of additional
triethylamine and 1.41 mL of acetic anhydride. After three hours,
the volatiles are evaporated, the residue is dissolved in 10 mL DMF
and the solution is added to 7.5 g of NaI in 120 mL water. Compound
51 is recrystallized from a mixture of methanol and ethyl
acetate.
Example 23
Preparation of Compound 52
##STR00083##
[0275] A mixture of 0.18 g of Compound 2, 0.17 g
1,4-dimethylpyridinium tosylate, 0.09 mL triethylamine and 20 mL
CH.sub.2Cl.sub.2 is stirred at room temperature overnight. The
volatiles are evaporated, the residue is dissolved in 2 mL of MeOH
and added to 1.5 g NaI in 12 mL water. The aqueous layer is
extracted with 1-butanol and Compound 52 is purified by
chromatography on a silica gel column.
Example 24
Preparation of Compound 53
##STR00084##
[0277] Compound 1 (0.35 g), 0.33 g of 1,2-dimethylquinolinium
tosylate, 0.15 mL of triethylamine and 20 mL CH.sub.2Cl.sub.2 are
stirred at room temperature for one hour. The reaction is suction
filtered to yield Compound 53, which is purified by chromatography
on silica gel.
Example 25
Preparation of Compound 54
##STR00085##
[0279] A mixture of 0.173 g of 1,4-dimethyl-2-quinolone, 0.28 mL
phosphorus oxychloride, 0.025 mL DMF and 5 mL toluene is heated at
70.degree. C. for 6 hours. Following cooling, 10 mL of methanol is
added and the solution is heated at 45 for two hours. The volatiles
are evaporated, 0.352 g of Compound 1, 0.7 mL of triethylamine and
20 mL of CH.sub.2Cl.sub.2 are added and the mixture is stirred
overnight at room temperature. The solvent is evaporated, the crude
product is dissolved in 3 mL methanol and added to 2.5 g of sodium
perchlorate in 20 mL water. Compound 64 is collected by filtration,
dried, and recrystallized from a mixture of DMF and ethyl
acetate.
Example 26
Preparation of Compound 55
##STR00086##
[0281] Compound 13 (55 mg) is stirred with 10 mg sodium borohydride
for 1.5 hours. Acetic acid (0.6 mL) is added to quench the
reaction. The volatiles are evaporated. The crude product is
stirred with 0.21 mL triethylamine, 0.19 mL acetic anhydride and
.about.10 mL CH.sub.2Cl.sub.2. After one hour at room temperature
and 30 minutes at 50-60.degree. C., the volatiles are evaporated.
Ethyl acetate (10 mL) is added, followed by 5 mL of hexanes. The
solid is filtered and the salt thus obtained is stirred overnight
at room temperature with a mixture of 65 mg
N,N'-diphenylformamidine and 0.1 mL triethylamine in 4 mL
dichloroethane. To this is added 150 mg of
1,4-dimethyl-2-phenylquinolinium derivative in 10 mL of
acetonitrile, another 0.1 mL of triethylamine and 0.07 mL acetic
anhydride. After 1.5 hr at room temperature the volatiles are
evaporated, the residue is dissolved in 3 mL methanol and the
solution is added to 1 g NaI in 20 mL water. Compound 55 is
isolated by suction filtration followed by purification on a silica
gel column.
Example 27
Preparation of Compound 56
##STR00087##
[0283] A solution of 1.14 g of Compound 12 in 30 mL of
dichloroethane and 1.7 mL triethylamine is stirred at room
temperature for 30 minutes. To this is added 0.7 g of
N,N'-diphenylformamidine and the mixture is heated at 65.degree. C.
for 4 hours. Following cooling, 0.84 g of
1-(4'-sulfobutyl)-4-methylquinolinium inner salt is added, followed
by 0.6 mL of acetic anhydride. The mixture is stirred for 30
minutes and Compound 56 is collected by suction filtration.
Example 28
Preparation of Compound 57
##STR00088##
[0285] To 4-ethyl-1-methyl-2-quinolone in THF at -78.degree. C. is
added 1.5 equivalent of phenyllithium, and the resulting solution
is kept cold and stirred for one hour. Four equivalents of acetic
acid are introduced and the reaction mixture is allowed to stir at
room temperature for 3 hours. All the volatile components are then
removed under reduced pressure. The resulting residue is dissolved
in methylene chloride and one equivalent of Compound 1
(4-methyl-2-methylhiooxazolo[4,5-b]pyridinium tosylate) is
introduced followed by 5 equivalents of triethylamine. The
resulting product is purified by column chromatography on silica
gel.
Example 29
Preparation of Compound 58
##STR00089##
[0287] To 2,4-dimethyl-thiazolo-[4,5-b]pyridinium tosylate in
pyridine at room temperature is added 1.2 equivalent of benzoyl
chloride, and the resulting mixture is stirred at room temperature
overnight. The resulting intermediate is isolated by silica gel
column chromatography. This Intermediate is then heated at reflux
in 3 equivalents of phosphorous oxychloride for 3 hours. Excess
phosphorous oxychloride is removed under reduced pressure and the
residue is stirred in methylene chloride with one equivalent of
1,4-dimethyl-quinolinium tosylate and 4 equivalents of
triethylamine to obtain the desired product.
Example 30
Preparation of Compound 59
##STR00090##
[0289] To a mixture of one equivalent each of
1,4-dimethylquinolinium tosylate and malonaldehyde dianil
hydrochloride in acetic acid, is added one equivalent of acetic
anhydride and the resulting reaction mixture is heated at
150.degree. C. for 30 minutes. All volatile components are removed
under reduced pressure and methylene chloride is introduced
followed by the addition of one equivalent of
2-ethyl-4-methyloxazolo[4,5-b]pyridinium tosylate, 1.5 equivalent
of acetic anhydride, and 3 equivalents of triethylamine to obtain
the desired product.
Example 31
Preparation of Compound 60
##STR00091##
[0291] A mixture of 135 mg of
2-benzoylmethylidene-3-methyl-2,3-dihydrobenzothiazole and 230 mg
of phosphorous oxychloride is heated at reflux in 5 mL of
dichloroethane for 2 hours. Solvent and excess reagents are
evaporated under reduced pressure and 30 mL of ethyl acetate is
added. After stirring for 30 minutes at room temperature, the
resulting solid is collected by filtration and resuspended in 15 mL
of methylene chloride. To the mixture is added 165 mg of
1,4-dimethylquinolinium tosylate and 0.14 mL of triethylamine.
After stirring at room temperature overnight, the desired product
is recovered by recrystallization. When associated with DNA,
Compound 60 exhibits excitation and emission wavelengths of 642 nm
and 659 nm, respectively.
Example 32
Detecting Double-Stranded DNA in Solution
[0292] Samples containing 0.1 ng/mL to 1 microgram/mL
double-stranded DNA are prepared in 10 mM Tris-HCl, 1 mM EDTA, pH
7.5. The selected dye of the invention is dissolved in DMSO at a
concentration of 1-10 mM and then diluted into the same buffer at a
concentration sufficient to achieve final optical densities of 0.1
and 0.01 at 500 nm, 530 nm, 540 nm or 560 nm. Equal volumes (100
microliters each) of DNA sample solution and dye solution at each
concentration are combined and mixed gently at room temperature in
a microplate well.
[0293] Samples are allowed to equilibrate for 5-60 minutes,
protected from light, and the fluorescence of the sample is
measured using a fluorescence microplate reader. DNA concentration
is determined by comparison of the sample fluorescence with a
standard curve obtained with that dye for each dye concentration.
The sensitivity limit of several dyes in this assay is shown (Table
5).
TABLE-US-00005 TABLE 5 Detecting double-stranded DNA in solution.
Sensitivity Limit (ng/ml) Low dye concentration High dye
concentration Dye (0.01 OD) (0.1 OD) Excitation 500 nm/Emission 560
nm Compound 43 10 10 Compound 48 >10 10 Compound 49 >10 20
Compound 53 >10 10 Compound 54 1.0 10 Excitation 530 nm/Emission
560 nm Compound 14 1.0 <10 Compound 15 0.5 10 Compound 17 2.0
<10 Compound 18 0.2-0.5 <10 Compound 20 0.2 10 Compound 21
0.2 <10 Compound 27 0.5 20 Compound 28 0.2 <10 Compound 30
1.0 10 Compound 31 2.0 50 Compound 32 1.0 10 Excitation 540
nm/Emission 570 nm Compound 16 1.0 <10 Compound 19 0.5 <10
Compound 22 1.0 10 Compound 23 0.2 <10 Compound 24 5.0 10
Compound 25 0.5 <10 Compound 29 2.0 100 Compound 33 >10 100
Compound 34 0.2 <10 Compound 35 0.5 10 Excitation 560
nm/Emission 585 nm Compound 37 2.0 10 Compound 38 5.0 200 Compound
39 5.0 10 Compound 40 1.0 10 Compound 41 >10 10 Compound 42
>10 10
Example 33
Detecting Oligonucleotides in Solution
[0294] Selected dyes of the invention are dissolved in DMSO at 1-10
micromolar concentration, then diluted into 10 mM Tris-HCl, 1 mM
EDTA, pH 7.5 to a final optical density of 0.1 or 0.01 at 530 nm or
540 nm. The resulting dye solutions are combined with an equal
volume of an 18-base oligodeoxynucleotide sequencing primer
solution at concentrations of 0.1 ng/mL to 1 microgram/mL in the
same buffer. The resulting mixture is mixed gently and incubated
for 5-60 minutes at room temperature, protected from light, and the
sample fluorescence is measured in a fluorescence microplate
reader. Typical sensitivity limits for some dyes are shown in Table
6.
TABLE-US-00006 TABLE 6 Detection of oligonucleotides in solution.
Sensitivity Limit (ng/mL) Low dye concentration High dye
concentration Dye (0.01 OD) (0.1 OD) Excitation 530 nm/Emission 560
nm Compound 18 >10 <10 Compound 20 10 <10 Compound 28
>10 <10 Excitation 540 nm/Emission 570 nm Compound 23 1.0
<10 Compound 25 >10 <10 Compound 19 >10 <10
Example 34
Detecting RNA in Solution
[0295] Selected dyes of the invention are dissolved in DMSO at 1-10
micromolar concentration, then diluted into 10 mM Tris-HCl, 1 mM
EDTA, pH 7.5 to a final optical density of 0.1 or 0.01 at 530 nm or
540 nm. These dye solutions are combined with an equal volume of
Escherichia coli ribosomal RNA at concentrations ranging from 0.25
ng/mL to 1 microgram/mL in microplate wells. The resulting mixtures
are mixed gently and incubated for 5-60 minutes at room
temperature, protected from light, and the sample fluorescence is
measured. Typical sensitivity limits for selected dyes are shown in
Table 7.
TABLE-US-00007 TABLE 7 Detecting RNA in solution. Sensitivity Limit
(ng/mL) Low dye concentration High dye concentration Dye (0.01 OD)
(0.1 OD) Excitation 530 nm/Emission 560 nm Compound 18 1.2 10
Compound 20 5.0 200 Compound 28 >10 500 Excitation 540
nm/Emission 570 nm Compound 19 10 20 Compound 23 2.5 200 Compound
25 1.2 200
Example 35
Detecting Double-Stranded DNA in the Presence of Single-Stranded
DNA
[0296] Selected dyes of the invention exhibit high sensitivity for
detecting double-stranded DNA, but exhibit little sensitivity for
detecting single-stranded nucleic acids, making them particularly
useful for quantitating double-stranded DNA in the presence of
single-stranded DNA, RNA or oligonucleotide primers (such as might
be present in a PCR reaction). Samples containing nucleic acids at
a concentration of 2 micrograms/mL are combined with Compound 16,
Compound 22, or Compound 24 at a final dye concentration of 1
micromolar in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, and mixed gently.
The resulting mixture is allowed to Incubate for 5-60 minutes at
room temperature, protected from light, and sample fluorescence is
measured using a fluorescence microplate reader, with excitation at
540 nm and emission at 570 nm. Double-stranded DNA is detected
preferentially (FIGS. 1 and 2). If, however, the dye concentration
is reduced relative to the concentration of single-stranded nucleic
acids, the fluorescence upon binding those nucleic acids increases
relative to that of the double-stranded DNA-dye complex. Thus the
assay is always performed in sufficient dye excess.
Example 36
Detecting Nucleic Acids in Gels
[0297] DNA samples are loaded onto agarose or polyacrylamide gels
and electrophoresed using standard conditions. Selected dyes of the
invention are dissolved in DMSO at a concentration of 1-10
micromolar and diluted into 0.5.times.TBE buffer to a final
concentration of 1 micromolar dye. Gels are incubated for 20-90
minutes in this staining solution, with gentle agitation, at room
temperature, protected from light. Nucleic acids are visualized by
transillumination at 300 nm, followed by direct visual detection or
photography using Polaroid black-and-white print film. Nucleic
acids are alternatively visualized by scanning using a FMBIO II
laser scanner with a 532 nm frequency-doubled YAG laser source or a
charge-coupled device camera system coupled with an ultraviolet
(300 nm) light source, or a visible light transilluminator. The
sensitivity of detection obtained using these dyes and various
imaging instruments is shown in Table 8.
TABLE-US-00008 TABLE 8 Sensitivity for detecting double-stranded
bacteriophage .lamda. cl857 DNA that is digested with HindIII
restriction endonuclease, and separated in agarose gels. Imaging
system POLAROID 667 POLAROID 667 BOEHRINGER MANNHEIM HITACHI FMBIO
II B&W film B&W film LUMIIMAGER CCD laser scanner
Excitation FOTODYNE FOTODYNE DARK READER 300 nm trans. 300 nm
trans. 532 nm freq.-doubled source 300 nm trans. 254 nm epi.
visible light box 520 nm em. 545 nm em YAG laser Cpd. 15 240 pg 60
pg 480 pg 210 pg 100 pg 100 pg Cpd. 16 120 pg 30 pg 240 pg 210 pg
50 pg 25 pg Cpd. 18 120 pg 60 pg nd 210 pg 100 pg 50 pg Cpd. 20 480
pg 120 pg nd 830 pg 210 pg 50 pg Cpd. 21 120 pg 60 pg nd 420 pg 50
pg 50 pg Cpd. 22 120 pg 30 pg 60 pg 830 pg 50 pg 7 pg Cpd. 23 120
pg 60 pg 240 pg 830 pg 100 pg 5 pg Cpd. 24 60 pg 30 pg 120 pg 420
pg 50 pg 5 pg Cpd. 25 120 pg 120 pg 240 pg 1.7 ng 100 pg 25 pg nd
indicates that a value was not determined for this dye with this
gel imager.
Example 37
Detecting Nucleic Acids Arrayed on Glass Slides
[0298] Nucleic acid arrays are prepared on glass slides, using an
automated arraying robot. The slide surface is optionally blocked
with a solution of 0.1% Ficoll (Type 400, Amersham Pharmacia) and
0.1% polyvinylpyrrolidone. Arrays are allowed to dry and then
overlaid with a solution containing 1 micromolar dye in 10 mM
Tris-HCl, 1 mM EDTA, pH 7.5. Suitable dyes for this purpose include
Compound 15, Compound 16, Compound 22, Compound 23, Compound 24,
and Compound 25. Slides are incubated with the overlay solution for
5-60 minutes at room temperature, with light protection, and then
are washed briefly in the same buffer without the dye. The amount
of nucleic acid in each spot on the array can be measured by
comparison of the fluorescence of an unknown sample with that of
standards of known concentration, which are applied to the same
slide. Fluorescence measurement is performed using a fluorescence
microscope with a mercury or xenon lamp, equipped with appropriate
optical filters, a charge-coupled device and image analysis
software, or using a dedicated array reader with a 532 nm or 546 nm
excitation source, such as the GENEARRAY 2000 (General
Scanning).
Example 36
Detecting Proteins in Solution
[0299] Compound 51 and Compound 44 are dissolved in DMSO at 1-10 mM
and then diluted into 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, containing
0.05% sodium dodecyl sulfate, to a final concentration of 2
micromolar, This dye solution is combined with samples containing
bovine serum albumin at 100 ng/ml, to 10 micrograms/mL. Samples are
optionally incubated for 10 minutes at 90.degree. C. and then
cooled to room temperature, or are incubated for 5-60 minutes at
room temperature. Fluorescence is measured using a fluorescence
microplate reader with excitation at 540 nm and emission at 590 nm
for Compound 44 and with excitation at 600 nm and emission at 670
nm for Compound 51. The fluorescence signal obtained from Compound
44 or 51 in buffer alone (control) is subtracted from the
fluorescence recorded for the protein-containing samples in order
to determine the intensity of the signal that is due to the
presence of protein. Protein concentrations are then determined by
comparison of the signal intensity thus obtained with the signals
obtained using a dilution series of known concentration prepared
using either the same protein or a protein standard, such as bovine
serum albumin. The sensitivity limit for either dye under either
condition is about 100 ng/mL bovine serum albumin (FIGS. 3 and
4).
Example 39
Detection of Proteins in Sodium Dodecyl Sulfate
(SDS)-Polyacrylamide Gels
[0300] The pure protein or mixture of proteins of interest is
prepared In Loading Buffer (50 mM Tris-HCl, pH 6.8, 2% SOS, 10%
glycerol, and 0.015% bromophenol blue). Dithiothreitol is added to
each sample, to a final concentration of 0.1 M. The samples are
heated for 4-5 minutes at 90-95.degree. C. and loaded onto a 15%
Tris-glycine polyacrylamide mini- or full-sized gel containing
0.05%-0.1% SDS, with a 4% stacking gel. The gel is electrophoresed
under standard conditions, in a standard Tris-glycine buffer
containing 0.05%-0.1% SDS. The resulting gel is transferred to a
small staining dish containing a 1-3 .mu.M solution of Compound 25
in 7.5% acetic acid (in water). The staining solution is then
covered with foil to protect it from room light and gently agitated
for 45 minutes-1 hour. After 10 minutes, the protein bands are
readily apparent, but sensitivity improves over about 30-40
minutes. The gel is then removed from the staining dish, rinsed
briefly in 7.5% acetic acid and transferred directly to a
UV-transilluminator. The gel, with the stained protein bands is
photographed using 300 nm transillumination and black and white
POLAROID 667 print film with a Wratten 9 gelatin filter, The
stained bands appear visually as brightly fluorescent orange bands.
Proteins appear as white bands on a grey to black background in the
POLAROID photograph.
[0301] Although Compound 25 is used in this procedure, a variety of
other dyes of the invention are useful as electrophoretic gel
stains, yielding stained gels that possess bands having
fluorescence emission from yellow to red.
Example 40
Detection of Apoptosis
[0302] A 1 mM solution of Compound 27 in DMSO is prepared. Jurkat
human T-cell leukemia cells are treated with 10 .mu.M camptothecin
to induce apoptosis. A sample of the cells (1.times.10.sup.6
cells/mL) is incubated with enough of the Compound 27 stock
solution to give a final dye concentration of 0.1 .mu.M, and
sufficient propidium iodide to give a final concentration of 1.5
.mu.M. After incubating for 30 minutes on ice, the cell samples are
analyzed on a FacsVanlage flow cytometer using 488-argon laser
excitation, with emission at 530/30 and 675/20. Apoptotic cells are
compared to control cells that were incubated with the same final
concentration of DMSO used in the staining solution. A plot of FL1
versus FL3 shows that Compound 27 is capable of differentiating
between apoptotic and normal cells. Late stage apoptotic and
necrotic cells are stained with propidium iodide.
[0303] It is to be understood that, while the foregoing invention
has been described in detail by way of illustration and example,
numerous modifications, substitutions, and alterations are possible
without departing from the spirit and scope of the invention as
described in the following claims.
[0304] 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.
* * * * *