U.S. patent application number 13/104413 was filed with the patent office on 2011-09-08 for fluorescent chemical compounds having high selectivity for double stranded dna, and methods for their use.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Jolene A. Bradford, Ching-Ying Cheung, Shih Jung Huang, Patrick R. Pinson, Stephen T. Yue.
Application Number | 20110217699 13/104413 |
Document ID | / |
Family ID | 37431576 |
Filed Date | 2011-09-08 |
United States Patent
Application |
20110217699 |
Kind Code |
A1 |
Bradford; Jolene A. ; et
al. |
September 8, 2011 |
Fluorescent Chemical Compounds Having High Selectivity for Double
Stranded DNA, and Methods for Their Use
Abstract
Chemical compounds having a high selectivity for double stranded
DNA over RNA and single stranded DNA are disclosed. The chemical
compounds are stains that become fluorescent upon illumination and
interaction with double stranded DNA, but exhibit reduced or no
fluorescence in the absence of double stranded DNA. The compounds
can be used in a variety of biological applications to
qualitatively or quantitatively assay DNA, even in the presence of
RNA.
Inventors: |
Bradford; Jolene A.;
(Eugene, OR) ; Cheung; Ching-Ying; (San Ramon,
CA) ; Huang; Shih Jung; (Eugene, OR) ; Pinson;
Patrick R.; (Eugene, OR) ; Yue; Stephen T.;
(Eugene, OR) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
37431576 |
Appl. No.: |
13/104413 |
Filed: |
May 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12573809 |
Oct 5, 2009 |
7943777 |
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13104413 |
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11432814 |
May 11, 2006 |
7598390 |
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12573809 |
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60680243 |
May 11, 2005 |
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Current U.S.
Class: |
435/6.1 ;
544/363; 546/157; 546/162; 546/163; 546/176 |
Current CPC
Class: |
C07D 215/227 20130101;
C07D 417/06 20130101; C07D 277/74 20130101; Y10T 436/143333
20150115; G01N 21/6486 20130101; C07D 215/10 20130101; G01N
2201/06113 20130101; C07D 417/04 20130101; C07D 417/14 20130101;
C07D 277/72 20130101; G01N 21/6428 20130101; G01N 33/582 20130101;
G01N 2021/6439 20130101; C12Q 1/6876 20130101 |
Class at
Publication: |
435/6.1 ;
546/157; 546/176; 546/163; 546/162; 544/363 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07D 417/06 20060101 C07D417/06; C07D 417/14 20060101
C07D417/14 |
Claims
1. A chemical compound having the structure: ##STR00115## wherein:
n is a non-negative integer; X is oxygen or sulfur; R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, and R.sup.11 independently comprise hydrogen, a
hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl,
a halogen, an alkyl group, an alkenyl group, an alkynyl group, an
aromatic group, a primary amine group, a secondary amine group, a
tertiary amine group, a reactive group, or combinations thereof;
and R.sup.12 is an alkyl group.
2. The chemical compound of claim 1, wherein: R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, and R.sup.11 are independently hydrogen, a hydroxyl
group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a
halogen, an alkyl group, an alkenyl group, an alkynyl group, an
aromatic group, a primary amine group, a secondary amine group, a
tertiary amine group, a reactive group, or combinations
thereof.
3. The chemical compound of claim 1, wherein at least one of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 comprises an aromatic group
or an alkynyl group.
4. The chemical compound of claim 1, wherein R.sup.9 comprises an
aromatic group, alkyl-aromatic, or an alkynyl group.
5. The chemical compound of claim 1, wherein R.sup.10 is an amine
group.
6. The chemical compound of claim 1, wherein: at least one of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 comprises an aromatic group
or an alkynyl group; and R.sup.9 comprises an aromatic group,
alkyl-aromatic group, or an alkynyl group.
7. The chemical compound of claim 1, wherein: at least one of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 comprises an aromatic group
or an alkynyl group; R.sup.9 comprises an aromatic group,
alkyl-aromatic group, or an alkynyl group; and R.sup.10 comprises
an amine group.
8. The chemical compound of claim 1, wherein R.sup.12 is a
C.sub.1-C.sub.8 alkyl group.
9. The chemical compound of claim 1, wherein R.sup.12 is a methyl
group.
10. The chemical compound of claim 1, further comprising one or
more cations or anions.
11. A kit comprising: DNA, RNA, or both DNA and RNA; and a chemical
compound having the structure: ##STR00116## wherein: n is a
non-negative integer; X is oxygen or sulfur; R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, and R.sup.11 independently comprise hydrogen, a hydroxyl
group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a
halogen, an alkyl group, an alkenyl group, an alkynyl group, an
aromatic group, a primary amine group, a secondary amine group, a
tertiary amine group, a reactive group, or combinations thereof;
and R.sup.12 is an alkyl group.
12. A method of detecting the presence or absence of double
stranded DNA in a sample, the method comprising: providing a sample
suspected of containing double stranded DNA; contacting the sample
with a chemical compound to prepare a test sample; illuminating the
test sample with energy; and detecting emission of energy from the
test sample; wherein the chemical compound has the structure:
##STR00117## wherein: n is a non-negative integer; X is oxygen or
sulfur; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, and R.sup.11 independently
comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a
thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group,
an alkynyl group, an aromatic group, a primary amine group, a
secondary amine group, a tertiary amine group, a reactive group, or
combinations thereof; and R.sup.12 is an alkyl group.
13. The method of claim 12, further comprise calculating the
concentration of double stranded DNA in the sample after the
detecting step.
14. A chemical compound having the structure: ##STR00118## wherein:
n is a non-negative integer; X is oxygen or sulfur; R.sup.13,
R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19,
R.sup.20, and R.sup.21 independently comprise hydrogen, a hydroxyl
group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a
halogen, an alkyl group, an alkenyl group, an alkynyl group, an
aromatic group, a primary amine group, a secondary amine group, a
tertiary amine group, a reactive group, or combinations thereof;
and R.sup.22 is an alkyl group.
15. The chemical compound of claim 14, wherein: R.sup.13.sub.,
R.sup.14.sub., R.sup.15.sub., R.sup.16, R.sup.17, R.sup.18,
R.sup.19, R.sup.20, and R.sup.21 are independently hydrogen, a
hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl,
a halogen, an alkyl group, an alkenyl group, an alkynyl group, an
aromatic group, a primary amine group, a secondary amine group, a
tertiary amine group, a reactive group, or combinations
thereof.
16. The chemical compound of claim 14, wherein at least one of
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 comprises an aromatic
group or an alkynyl group.
17. The chemical compound of claim 14, wherein R.sup.19 comprises
an aromatic group, alkyl-aromatic, or an alkynyl group.
18. The chemical compound of claim 14, wherein R.sup.20 is an amine
group.
19. The chemical compound of claim 14, wherein: at least one of
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 comprises an aromatic
group or an alkynyl group; and R.sup.19 comprises an aromatic
group, alkyl-aromatic group, or an alkynyl group.
20. The chemical compound of claim 14, wherein: at least one of
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 comprises an aromatic
group or an alkynyl group; R.sup.19 comprises an aromatic group,
alkyl-aromatic group, or an alkynyl group; and R.sup.20 comprises
an amine group.
21. The chemical compound of claim 14, wherein R.sup.22 is a
C.sub.1-C.sub.8 alkyl group.
22. The chemical compound of claim 14, wherein R.sup.22 is a methyl
group.
23. The chemical compound of claim 14, further comprising one or
more cations or anions.
24. A kit comprising: DNA, RNA, or both DNA and RNA; and a chemical
compound having the structure: ##STR00119## wherein: n is a
non-negative integer; X is oxygen or sulfur; R.sup.13, R.sup.14,
R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.20, and
R.sup.21 independently comprise hydrogen, a hydroxyl group, an
alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl
group, an alkenyl group, an alkynyl group, an aromatic group, a
primary amine group, a secondary amine group, a tertiary amine
group, a reactive group, or combinations thereof; and R.sup.22 is
an alkyl group.
25. A method of detecting the presence or absence of double
stranded DNA in a sample, the method comprising: providing a sample
suspected of containing double stranded DNA; contacting the sample
with a chemical compound to prepare a test sample; illuminating the
test sample with energy; and detecting emission of energy from the
test sample; wherein the chemical compound has the structure:
##STR00120## wherein: n is a non-negative integer; X is oxygen or
sulfur; R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, R.sup.18,
R.sup.19, R.sup.20, and R.sup.21 independently comprise hydrogen, a
hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl,
a halogen, an alkyl group, an alkenyl group, an alkynyl group, an
aromatic group, a primary amine group, a secondary amine group, a
tertiary amine group, a reactive group, or combinations thereof;
and R.sup.22 is an alkyl group.
26. The method of claim 25, further comprise calculating the
concentration of double stranded DNA in the sample after the
detecting step.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/573,809, filed on Oct. 5, 2009, which is a
continuation of U.S. patent application Ser. No. 11/432,814, filed
May 11, 2006 (now U.S. Pat. No. 7,598,390), which claims priority
to U.S. Provisional Patent Application Ser. No. 60/680,243, filed
May 11, 2005, the contents of which are hereby incorporated herein
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to stains that become fluorescent upon
interaction with DNA. In particular, stains that exhibit higher
fluorescence when contacted with double stranded DNA than when
contacted with RNA and/or single stranded DNA, as well as various
uses for the stains are disclosed.
DESCRIPTION OF RELATED ART
[0003] Stains and dyes are commonly used in chemical,
biotechnological, and biomedical research. These two types of
compounds are different in their properties, and in their intended
uses.
[0004] Stains are chemical compounds that exhibit a detectable
response when contacted with a particular target. In the absence of
the target, a stain does not exhibit the detectable response. These
properties make stains valuable in the detection of the presence or
absence of a particular target in a sample. The detectable response
can be qualitative or quantitative, depending on the compound,
target, and assay parameters.
[0005] In comparison, dyes exhibit a detectable response regardless
of the presence or absence of another material. Dyes are therefore
useful to label a target. For example, an antibody can be labeled
with a fluorescent dye molecule. The localization of the antibody
in a cell or tissue can be monitored by fluorescence.
[0006] The detection and quantitation of DNA is a very common task
in biotechnological research. Early chemical stains such as
ethidium bromide are effective at staining DNA, but also stain RNA.
DNA and RNA are often obtained together when isolated from natural
sources. Stains that are not selective for DNA make quantitation of
the isolated DNA difficult, requiring a purification step to be
performed prior to quantitation. Stains find use in applications
such as gel electrophoresis, PCR, real time PCR quantitation, DNA
solution quantitation, microarrays, and RT-PCR.
[0007] Multiple nucleic acid stains are commercially available. The
following is a representative listing of these materials.
[0008] Ethidium bromide is the most widely used nucleic acid stain,
and is commercially available from a wide array of suppliers.
Ethidium bromide is mutagenic, and its use requires significant
care from the user to avoid contact with staining solutions.
[0009] PicoGreen is a stain selective for double stranded DNA
(commercially available from Molecular Probes, Inc. (Eugene, Oreg.)
since 1994). PicoGreen shows a greater than 1000 fold fluorescence
enhancement upon binding to double stranded DNA, and much less
enhancement upon binding to single stranded DNA or RNA.
[0010] OliGreen is a stain useful for the quantitation of single
stranded DNA such as synthetic oligonucleotides. OliGreen has been
commercially available from Molecular Probes, Inc. (Eugene, Oreg.)
since 1994. Quantitation with OliGreen is about 10,000 times more
sensitive than quantitation with UV absorbance methods, and at
least 500 times more sensitive than detecting oligonucleotides on
electrophoretic gels stained with ethidium bromide. This type of
material is described in U.S. Pat. Nos. 5,436,134 and 5,658,751;
Australian Patent Nos. 676,317 and 714,890; Canadian Patent No.
2,133,765; and European Patent Nos. 0,675,924 and 0,740,689.
[0011] RiboGreen is a stain that is useful for the quantitation of
RNA in solution. RiboGreen has been commercially available from
Molecular Probes, Inc. (Eugene, Oreg.) since 1997. This type of
material is described in U.S. Pat. Nos. 5,658,751 and 5,863,753;
Australian Patent No. 714,890; and European Patent No.
0,740,689.
[0012] SYBR Green I is stain selective for DNA (commercially
available from Molecular Probes, Inc. (Eugene, Oreg.) since 1993).
SYBR Green I has a fluorescence enhancement upon binding to DNA at
least 10 fold greater than that of ethidium bromide, and a
fluorescence quantum yield over five times greater than ethidium
bromide (about 0.8 as compared to about 0.15). This type of
material is described in U.S. Pat. Nos. 5,436,134 and 5,658,751;
Australian Patent Nos. 676,317 and 714,890; Canadian Patent No.
2,133,765; and European Patent Nos. 0,675,924 and 0,740,689.
[0013] SYBR Safe is a nucleic acid stain that is at least twice as
sensitive as ethidium bromide, yet exhibits reduced mutagenicity.
SYBR Safe has been commercially available from Molecular Probes,
Inc. (Eugene, Oreg.) since 2003. This type of material is described
in U.S. Pat. Nos. 4,883,867, 4,957,870, 5,436,134, and 5,658,751;
Australian Patent Nos. 676,317 and 714,890; Canadian Patent No.
2,133,765; and European Patent Nos. 0,675,924 and 0,740,689.
[0014] Hoechst 33258 (CAS 23491-45-4; Phenol,
4-[5-(4-methyl-1-piperazinyl)[2,5'-bi-1H-benzimidazol]-2'-yl]-,
trihydrochloride) is a nuclear counterstain that emits blue
fluorescence when bound to dsDNA. Hoechst 33258 has been
commercially available from Molecular Probes, Inc. (Eugene, Oreg.)
since 1992.
[0015] Dimeric cyanines TOTO-1, YOYO-1, and YO-PRO-1 are useful for
the measurement of double stranded DNA, single stranded DNA, and
RNA in solution. TOTO-1, YOYO-1, and YO-PRO-1 have been
commercially available from Molecular Probes, Inc. (Eugene, Oreg.)
since 1992. These types of materials are described in U.S. Pat.
Nos. 5,321,130 and 5,582,977; Canadian Patent No. 2,119,126; and
European Patent No. 0,605,655 B1.
[0016] Unsymmetrical cyanine dyes having similar spectral
properties to intercalating cyanine dyes, but binding in the minor
groove of DNA were reported in 2003 (Karlsson, H. J. et al.,
Nucleic Acids Res. 31(21): 6227-6234 (2003)). Compounds BEBO, BETO,
and BOXTO were shown, and characterized using a variety of spectral
measurements. Fluorescence quantum yield increased upon binding to
DNA, but RNA binding results were not shown.
[0017] Despite the materials and methods that are currently
available, there still exists a need for stains that are selective
for double stranded DNA in the presence of RNA, single stranded
DNA, or other biological materials.
SUMMARY OF THE INVENTION
[0018] Compounds are disclosed having high selectivity for double
stranded DNA over RNA and single stranded DNA. The compounds act as
fluorescent stains, where they exhibit fluorescent properties when
illuminated in the presence of double stranded DNA, but exhibit
reduced or no fluorescence in the presence of RNA, single stranded
DNA, or in the absence of nucleic acids entirely. The compounds can
contain the core structure of Compound (1A) or Compound (1B).
##STR00001##
[0019] Also disclosed are methods for the preparation of the
compounds, and methods for their use in detecting the presence or
absence of double stranded DNA in a sample. The selectivity of the
compounds for double stranded DNA over RNA and single stranded DNA
enables detection of double stranded DNA in samples containing RNA
and/or single stranded DNA.
DETAILED DESCRIPTION OF THE INVENTION
[0020] While compositions and methods are described in terms of
"comprising" various components or steps (interpreted as meaning
"including, but not limited to"), the compositions and methods can
also "consist essentially of" or "consist of" the various
components and steps, such terminology should be interpreted as
defining essentially closed-member groups.
[0021] Compounds
[0022] A first embodiment of the invention is directed towards
chemical compounds. The chemical compounds can be neutrally
charged, positively charged, or negatively charged. When positively
or negatively charged, the compound can include one or more
counterions.
[0023] One embodiment of the invention is directed towards chemical
compounds containing the core structure of Compound (1A).
##STR00002##
[0024] The double bond(s) in the center of Compound (1A) can be in
either cis or trans configuration. For example, if X is nitrogen,
the two nitrogens can be oriented on the same side of the central
double bond (cis) or can be oriented across the central double bond
(trans). Mixtures of both configurations are also possible in a
sample of a particular compound.
[0025] The value n can be any non-negative integer. For example, n
can be zero, 1, 2, 3, 4, 5, 6, 7, 8, and so on.
[0026] X can be oxygen or sulfur.
[0027] Groups R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10 and R.sup.11 can independently
comprise or be hydrogen (H), hydroxyl group (OH), alkoxy group
(OR), thiol (SH), thioalkyl (SR), thioaryl (SAr), halogen (X),
alkyl group, alkenyl group, alkynyl group, aromatic group, amine
group (primary NH.sub.2, secondary NHR, tertiary NR'R'', or
tertiary NR'.sub.2), a reactive group, or a mixed group having
combinations of two or more of these groups (for example, an alkyl
group having thiol and amino substituents, an alkoxy group having
amino substituents, and so on). Alternatively, one or more of these
groups can be a linker group for covalently attaching Compound (1A)
to another compound. Linking the linker group to another compound
would afford a conjugate of Compound (1A).
[0028] In one embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 comprises or is an aromatic group or an
alkynyl group.
[0029] In one embodiment, R.sup.9 comprises or is an aromatic
group, alkyl-aromatic, or an alkynyl group.
[0030] In one embodiment, R.sup.10 comprises or is an amine
group.
[0031] In one embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 comprises or is an aromatic group or an
alkynyl group; and R.sup.9 comprises or is an aromatic group,
alkyl-aromatic, or an alkynyl group.
[0032] In one embodiment, at least one of R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 comprises or is an aromatic group or an
alkynyl group; R.sup.9 comprises or is an aromatic group,
alkyl-aromatic group, or an alkynyl group; and R.sup.10 comprises
or is an amine group.
[0033] Group R.sup.12 can be an alkyl group such as a
C.sub.1-C.sub.8 alkyl group. The C.sub.1-C.sub.8 alkyl group can be
a straight chain, branched, or cycloalkyl group. Examples of the
C.sub.1-C.sub.8 alkyl group include methyl, ethyl, 1-propyl,
2-propyl, 1-butyl, 2-butyl, 1-pentyl, 1-hexyl, 1-heptyl, and
1-octyl. In a presently preferred embodiment, the C.sub.1-C.sub.8
alkyl group is a methyl group.
[0034] When Compound (1A) is a cationic or anionic structure, it
can further comprise one or more appropriate counterions. For
example, if Compound (1A) is cationic (positively charged), it can
further comprise anions such as chloride, bromide, iodide, sulfate,
and carbonate counterions. Alternatively, if Compound (1A) is
anionic (negatively charged), it can further comprise cations such
as potassium, sodium, ammonium, magnesium, and calcium.
[0035] An additional embodiment of the invention is directed
towards chemical compounds containing the core structure of
Compound (1B).
##STR00003##
[0036] The double bond(s) in the center of Compound (1B) can be in
either cis or trans configuration. For example, if X is nitrogen,
the two nitrogens can be oriented on the same side of the central
double bond (cis) or can be oriented across the central double bond
(trans). Mixtures of both configurations are also possible in a
sample of a particular compound.
[0037] The value n can be any non-negative integer. For example, n
can be zero, 1, 2, 3, 4, 5, 6, 7, 8, and so on.
[0038] X can be oxygen or sulfur.
[0039] Groups R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17,
R.sup.18, R.sup.19, R.sup.20, and R.sup.21 can independently
comprise or be hydrogen (H), hydroxyl group (OH), alkoxy group
(OR), thiol (SH), thioalkyl (SR), thioaryl (SAr), halogen (X),
alkyl group, alkenyl group, alkynyl group, aromatic group, amine
group (primary NH.sub.2, secondary NHR, tertiary NR'R'', or
tertiary NR'.sub.2), a reactive group, or a mixed group having
combinations of two or more of these groups (for example, an alkyl
group having thiol and amino substituents, an alkoxy group having
amino substituents, and so on). Alternatively, one or more of these
groups can be a linker group for covalently attaching Compound (1B)
to another compound. Linking the linker group to another compound
would afford a conjugate of Compound (1B).
[0040] In one embodiment, at least one of R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 comprises or is an aromatic group or an
alkynyl group.
[0041] In one embodiment, R.sup.19 comprises or is an aromatic
group, alkyl-aromatic, or an alkynyl group.
[0042] In one embodiment, R.sup.20 comprises or is an amine
group.
[0043] In one embodiment, at least one of R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 comprises or is an aromatic group or an
alkynyl group; and R.sup.19 comprises or is an aromatic group,
alkyl-aromatic, or an alkynyl group.
[0044] In one embodiment, at least one of R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 comprises or is an aromatic group or an
alkynyl group; R.sup.19 comprises or is an aromatic group,
alkyl-aromatic, or an alkynyl group; and R.sup.20 comprises or is
an amine group.
[0045] Group R.sup.22 can be an alkyl group such as a
C.sub.1-C.sub.8 alkyl group. The C.sub.1-C.sub.8 alkyl group can be
a straight chain, branched, or cycloalkyl group. Examples of the
C.sub.1-C.sub.8 alkyl group include methyl, ethyl, 1-propyl,
2-propyl, 1-butyl, 2-butyl, 1-pentyl, 1-hexyl, 1-heptyl, and
1-octyl. In a presently preferred embodiment, the C.sub.1-C.sub.8
alkyl group is a methyl group.
[0046] When Compound (1B) is a cationic or anionic structure, it
can further comprise one or more appropriate counterions. For
example, if Compound (1A) is cationic (positively charged), it can
further comprise anions such as chloride, bromide, iodide, sulfate,
and carbonate counterions. Alternatively, if Compound (1A) is
anionic (negatively charged), it can further comprise cations such
as potassium, sodium, ammonium, magnesium, and calcium.
[0047] Substituents
[0048] The alkoxy group can generally be any unsubstituted alkoxy
group or substituted alkoxy group. Unsubstituted alkoxy groups
contain an oxygen connected to an alkyl group. Substituted alkoxy
groups contain an oxygen connected to a substituted alkyl group.
Examples of unsubstituted alkoxy groups include methoxy
(OCH.sub.3), ethoxy (OCH.sub.2CH.sub.3), propoxy
(OCH.sub.2CH.sub.2CH.sub.3), and higher straight chain alkoxy
groups. Unsubstituted alkoxy groups also include branched or cyclic
alkoxy groups. Examples of branched alkoxy groups include 2-propoxy
(OCH(CH.sub.3).sub.2), 2-butoxy (OCH(CH.sub.3)CH.sub.2CH.sub.3),
and higher branched alkoxy groups. Cyclic alkoxy groups have an
oxygen connected to a cyclic group. Examples of cyclic alkoxy
groups include cyclopropoxy (oxygen connected to a cyclopropane
ring), cyclobutoxy (oxygen connected to a cyclobutane ring),
cyclopentoxy (oxygen connected to a cyclopentane ring), cyclohexoxy
(oxygen connected to a cyclohexane ring), and higher cyclic alkoxy
groups.
[0049] The halogen can generally be any halogen. Halogen groups
include chloro, fluoro, bromo, and iodo groups.
[0050] Alkyl groups can generally be any unsubstituted or
substituted alkyl group. Unsubstituted alkyl groups contain only
carbon and hydrogen atoms. Substituted alkyl groups can contain one
or more non-carbon and non-hydrogen atoms such as oxygen, nitrogen,
sulfur, halogens, and phosphorous.
[0051] Alkenyl groups can generally be any alkenyl group containing
at least one carbon-carbon double bond. The most simple alkenyl
group is a vinyl group (--CH.dbd.CH.sub.2). Higher alkenyl groups
include 1-propenyl (--CH.dbd.CH.sub.2CH.sub.3), 1-butenyl
(--CH.dbd.CH.sub.2CH.sub.2CH.sub.3), 2-butenyl
(--CH.sub.2--CH.dbd.CHCH.sub.3), and 3-butenyl
(--CH.sub.2CH.sub.2CH.dbd.CH.sub.2). Substituted alkenyl groups can
contain one or more non-carbon and non-hydrogen atoms such as
oxygen, nitrogen, sulfur, halogens, and phosphorous.
[0052] Alkynyl groups can generally be any alkynyl group containing
at least one carbon-carbon triple bond. The most simple alkynyl
group is an ethynyl group (--CCH). Higher alkynyl groups include
propargyl (--CH.sub.2CCH), 2-butynyl (--CH.sub.2CCCH.sub.3), and
3-butynyl (--CH.sub.2CH.sub.2CCH).
[0053] A simple example of an aryl group is a phenyl group. The
aryl group can be a simple unsubstituted aryl group containing
carbon and hydrogen, or it can be a substituted aryl group. Aryl
groups can include one or more aromatic rings. The aryl group can
be a polycyclic aromatic hydrocarbon, or can be a heteroaryl group.
Examples of aryl and heteroaryl groups include phenyl, 1-naphthyl,
2-naphthyl, 4-biphenyl, anthracenyl, acenaphthalenyl,
acenaphthenyl, benzo[a]pyrenyl, benz[.alpha.]anthracenyl,
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2 imidazolyl,
4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5 oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2 furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4 pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5 isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl,
benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl,
benzo[1,3]dioxol-5-yl, and 6-quinolyl. Heteroatoms in the
heteroaryl group can include one or more of nitrogen (N), oxygen
(O), sulfur (S), and phosphorous (P).
[0054] Aryl groups can be connected to the central core structure
Compound (1A) or Compound (1B), either directly by a covalent bond,
or indirectly through one or more atoms. For example, N, O, P, or S
atoms can be used to link the aryl group to Compound (1A) or
Compound (1B). Examples of this include phenylamino
(NHC.sub.6H.sub.5), diphenylamino (N(C.sub.6H.sub.5).sub.2),
phenoxy (OC.sub.6H.sub.5), and thiophenyl (SC.sub.6H.sub.5).
Alternatively, alkyl groups can be used to link the aryl group to
Compound (1A) or Compound (1B). An example of this would be a
benzyl group (CH.sub.2C.sub.6H.sub.5; where a methylene CH.sub.2
group connects the phenyl group to Compound (1A) or Compound (1B)),
or a styrene group (CH.dbd.CH--C.sub.6H.sub.5).
[0055] Amine or amino groups can include NH.sub.2, NHR, NR.sub.2,
and NR'R'' groups. The R, R', and R'' groups can be unsubstituted
alkyl groups, substituted alkyl groups, unsubstituted alkenyl
groups, substituted alkenyl groups, unsubstituted alkynyl groups,
substituted alkynyl groups, unsubstituted aromatic groups, or
substituted aromatic groups.
[0056] The chemical compound can comprise at least one reactive
group capable of reacting with another species, such as an atom or
chemical group to form a covalent bond, that is, a group that is
covalently reactive under suitable reaction conditions, and
generally represents a point of attachment for another substance,
for example, a carrier molecule or a substrate. For example, the
reactive group on a disclosed compound is a moiety, such as
carboxylic acid or succinimidyl ester, on the compounds that can
chemically react with a functional group on a different compound to
form a covalent linkage. Reactive groups generally include
nucleophiles, electrophiles and photoactivatable groups.
[0057] The reactive group can be covalently attached directly to
the Compound (1A) core structure, or can be covalently attached to
at least one of the R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, and R.sup.11 groups.
The reactive group can be covalently attached directly to the
Compound (1B) core structure, or can be covalently attached to at
least one of the R.sup.13, R.sup.14.sub., R.sup.15, R.sup.16,
R.sup.17, R.sup.18, R.sup.19, R.sup.20, and R.sup.21 groups
[0058] Exemplary reactive groups include, but are not limited to,
olefins, acetylenes, alcohols, phenols, ethers, oxides, halides,
aldehydes, ketones, carboxylic acids, esters, amines, amides,
cyanates, isocyanates, thiocyanates, isothiocyanates, hydrazines,
hydrazones, hydrazides, diazo groups, diazonium groups, nitro
groups, nitriles, mercaptans, sulfides, disulfides, sulfoxides,
sulfones, sulfonic acid groups, sulfinic acid groups, acetals,
ketals, anhydrides, sulfates, sulfenic acid groups, isonitriles,
amidines, imides, imidates, nitrones, hydroxylamines, oximes,
hydroxamic acid groups, thiohydroxamic acid groups, allenes, ortho
esters, sulfites, enamines, ynamines, ureas, pseudoureas,
semicarbazides, carbodiimides, carbamates, imines, azides, azo
groups, azoxy groups, and nitroso groups. Reactive functional
groups also include those used to prepare bioconjugates, for
example, N-hydroxysuccinimide esters, maleimides, and the like.
Methods to prepare each of these functional groups are well known
in the art and their application to or modification for a
particular purpose is within the ability of one of skill in the art
(see, for example, Sandler and Karo, eds., Organic Functional Group
Preparations, Academic Press, San Diego, 1989). Reactive groups
include those shown in the following table.
TABLE-US-00001 Electrophilic Group Nucleophilic Group Resulting
Covalent Linkage activated esters* amines/anilines carboxamides
acrylamides thiols thioethers 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 amines aryl amines aziridines thiols
thioethers boronates glycols boronate esters carbodiimides
carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic
acids esters epoxides thiols thioethers haloacetamides thiols
thioethers haloplatinate amino platinum complex haloplatinate
heterocycle platinum complex haloplatinate thiol platinum complex
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers halotriazines thiols triazinyl
thioethers 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. succinimidyloxy (--OC.sub.4H.sub.4O.sub.2)
sulfosuccinimidyloxy (--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.sub.a or
--OCNR.sub.aNHR.sub.b, where R.sub.a and R.sub.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
[0059] Typically, the reactive group will react with an amine, a
thiol, an alcohol, an aldehyde, a ketone, or with silica.
Preferably, reactive groups react with an amine or a thiol
functional group, or with silica. In one embodiment, the reactive
group is an acrylamide, an activated ester of a carboxylic acid, an
acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, a silyl
halide, an anhydride, an aniline, an aryl halide, an azide, an
aziridine, a boronate, a diazoalkane, a haloacetamide, a
halotriazine, a hydrazine (including hydrazides), an imido ester,
an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a
reactive platinum complex, a sulfonyl halide, or a thiol group. By
"reactive platinum complex" is particularly meant chemically
reactive platinum complexes such as described in U.S. Pat. No.
5,714,327 (issued Feb. 3, 1998).
[0060] Where the reactive group is an activated ester of a
carboxylic acid, such as a succinimidyl ester of a carboxylic acid,
a sulfonyl halide, a tetrafluorophenyl ester, a pentafluorophenyl
ester, or an isothiocyanates, the resulting compound is
particularly useful for preparing conjugates of carrier molecules
such as proteins, nucleotides, oligonucleotides, or haptens. Where
the reactive group is a maleimide, haloalkyl or haloacetamide
(including any reactive groups disclosed in U.S. Pat. Nos.
5,362,628; 5,352,803 and 5,573,904) the resulting compound is
particularly useful for conjugation to thiol-containing substances.
Where the reactive group is a hydrazide, the resulting compound is
particularly useful for conjugation to periodate-oxidized
carbohydrates and glycoproteins, and in addition is an
aldehyde-fixable polar tracer for cell microinjection. Where the
reactive group is a silyl halide, the resulting compound is
particularly useful for conjugation to silica surfaces,
particularly where the silica surface is incorporated into a fiber
optic probe subsequently used for remote ion detection or
quantitation.
[0061] The reactive group can be a photoactivatable group such that
the group is only converted to a reactive species after
illumination with an appropriate wavelength. An appropriate
wavelength is generally a UV wavelength that is less than 400 nm.
This method provides for specific attachment to only the target
molecules, either in solution or immobilized on a solid or
semi-solid matrix. Photoactivatable reactive groups include,
without limitation, benzophenones, aryl azides and diazirines.
[0062] The reactive group can be a photoactivatable group,
succinimidyl ester of a carboxylic acid, a haloacetamide,
haloalkyl, a hydrazine, an isothiocyanate, a maleimide group, an
aliphatic amine, a silyl halide, a cadaverine or a psoralen. The
reactive group can be a succinimidyl ester of a carboxylic acid, a
maleimide, an iodoacetamide, or a silyl halide. The reactive group
can be a succinimidyl ester of a carboxylic acid, a sulfonyl
halide, a tetrafluorophenyl ester, an iosothiocyanates or a
maleimide.
[0063] The selection of a covalent linkage to attach the compound
to the carrier molecule or solid support typically depends on the
chemically reactive group on the component to be conjugated.
Examples of reactive groups include 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 component (typical for polysaccharides), or a variety of sites
may occur (e.g. amines, thiols, alcohols, phenols), as is typical
for proteins. A carrier molecule or solid support may be conjugated
to more than one reporter molecule, which may be the same or
different, or to a substance that is additionally modified by a
hapten.
[0064] In an alternative embodiment, the present compound is
covalently bound to a carrier molecule. If the compound has a
reactive group, then the carrier molecule can alternatively be
linked to the compound through the reactive group. The reactive
group may contain both a reactive functional moiety and a linker,
or only the reactive functional moiety.
[0065] A variety of carrier molecules exist. Examples of carrier
molecules include antigens, steroids, vitamins, drugs, haptens,
metabolites, toxins, environmental pollutants, amino acids,
peptides, proteins, nucleic acids, nucleic acid polymers,
carbohydrates, lipids, and polymers.
[0066] The carrier molecule can comprise 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, a virus and
combinations thereof. Alternatively, the carrier molecule can be a
hapten, a nucleotide, an oligonucleotide, a nucleic acid polymer, a
protein, a peptide or a polysaccharide. The carrier molecule can be
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
tyramine, a synthetic polymer, a polymeric microparticle, a
biological cell, cellular components, an ion chelating moiety, an
enzymatic substrate or a virus. Alternatively, the carrier molecule
is an antibody or fragment thereof, an antigen, an avidin or
streptavidin, a biotin, a dextran, an antibody binding protein, a
fluorescent protein, agarose, and a non-biological microparticle.
The carrier molecule can be an antibody, an antibody fragment,
antibody-binding proteins, avidin, streptavidin, a toxin, a lectin,
or a growth factor. Examples of haptens include biotin, digoxigenin
and fluorophores.
[0067] Antibody binding proteins include protein A, protein G,
soluble Fc receptor, protein L, lectins, anti-IgG, anti-IgA,
anti-IgM, anti-IgD, anti-IgE or a fragment thereof.
[0068] The chemical compound can be covalently bonded to another
molecule such as an antibody, protein, peptide, polypeptide, amino
acid, enzyme, nucleic acid, lipid, polysaccharide, drug, a bead, a
solid support (such as glass or plastic), and so on.
[0069] The chemical compounds preferably exhibit little or no
fluorescence when in the absence of nucleic acids. Fluorescence can
be determined by illuminating the chemical compound with an
appropriate wavelength, and monitoring emitted fluorescence. The
chemical compounds preferably exhibit greater fluorescence when in
the presence of DNA than when in the presence of RNA. The
fluorescence in the presence of DNA to the fluorescence in the
presence of RNA is determined using a fixed concentration of
chemical compound, and a fixed concentration of DNA and RNA. Higher
DNA/RNA fluorescence ratios are preferred for the detection of DNA
in the presence of RNA. The DNA/RNA ratio is preferably greater
than about 1. More preferred ratios are greater than about 2,
greater than about 3, greater than about 4, greater than about 5,
greater than about 6, greater than about 7, greater than about 8,
greater than about 9, greater than about 10, greater than about 15,
greater than about 20, greater than about 25, greater than about
30, greater than about 35, greater than about 40, greater than
about 45, greater than about 50, greater than about 55, greater
than about 60, greater than about 65, greater than about 70,
greater than about 75, greater than about 80, greater than about
85, greater than about 90, greater than about 95, greater than
about 100, greater than about 150, greater than about 200, and
ranges between any two of these values.
[0070] The chemical compounds can also be characterized by their
excitation and emission maxima wavelengths. For example, the
excitation maximum can be about 450 nm to about 650 nm. Excitation
maxima between these values can include about 450 nm, about 475 nm,
about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600
nm, about 625 nm, about 650 nm, and ranges between any two of these
values. For example, the emission maximum can be about 500 nm to
about 675 nm. Emission maxima between these values can include
about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600
nm, about 625 nm, about 650 nm, about 675 nm, and ranges between
any two of these values.
[0071] Specific examples of chemical compounds include Compounds
16-35, 38-39, 43-53, 55-58, 60, 62, 64-81, 83, 85-89, 91-97,
99-106, and 109-112.
[0072] Compositions
[0073] An additional embodiment of the invention is directed
towards compositions comprising one or more of the above described
compounds. The compositions can comprise, consist essentially of,
or consist of one or more of the above described compounds.
[0074] The compositions can comprise one, two, three, four, or more
of the above described compounds. The compositions can further
comprise a solvent. The solvent can be aqueous, non-aqueous, or a
mixed aqueous/non-aqueous solvent system. Examples of solvents
include water, methanol, ethanol, dimethylsulfoxide ("DMSO"),
dimethylformamide ("DMF"), dimethylacetamide, and
N-methylpyrrolidinone ("NMP"). The compositions can further
comprise one or more salts or buffers.
[0075] The above described compounds can individually be present in
the composition at a particular concentration. In one embodiment,
the compound is present in a substantially pure form without other
materials present (sometimes referred to as "neat"). Alternatively,
the compounds can be present in a dry mixture or dissolved in a
solvent or solvent system. When dissolved, the compound can
generally be present at any concentration. The compound can be
dissolved in a concentrated solution, or in a final "working"
solution. For example, a compound can be present in a working
solution at about 1 .mu.M to about 1 .mu.M. The concentrated
solution can have the compound at a higher concentration such as
about 10 .mu.M, about 50 .mu.M, about 100 .mu.M, about 500 .mu.M,
about 1 mM, and ranges between any two of these values.
[0076] Kits
[0077] One embodiment of the invention is directed towards kits
comprising one or more of the above described compounds. The kits
can comprise one, two, three, four, or more of the above described
compounds. The kit preferably comprises at least one container
comprising at least one of the above described compounds. The kit
can comprise multiple containers, such as a first container, a
second container, a third container, and so on. The kit can
comprise pipettes, droppers, or other sample handling devices. The
kit can comprise a cuvette, microwell plate, or other test
container suitable for use in an instrument that detects emitted
fluorescent energy.
[0078] The kit can comprise positive and/or negative samples.
Positive samples can comprise DNA, and/or DNA in the presence of
RNA. Negative samples can comprise RNA without DNA, or samples
lacking nucleic acids entirely. The kit can comprise DNA, RNA, or
both DNA and RNA.
[0079] The kit can comprise one or more additional dyes or stains.
For example, the kit can contain a total nucleic acid stain. The
kit can contain a cell impermeant nucleic acid stain to aid in
distinguishing live cells from dead cells.
[0080] The kits can further comprise an instruction protocol for
their use. The kit can further comprise water, a buffer, a buffer
salt, surfactants, detergents, salts, polysaccharides, or other
materials commonly used in assaying biological systems. The kit can
comprise solvents such as aqueous, non-aqueous, or a mixed
aqueous/non-aqueous solvent systems.
[0081] Methods of Preparation
[0082] An additional embodiment of the invention is directed
towards methods for the preparation of the above described
compounds. Illustrative examples of these methods are described in
the Examples below.
[0083] Additional embodiments of the invention include synthetic
intermediates. Many synthetic intermediates are shown in the
Examples section below. Examples of such intermediates include
Compounds 2-15, 36-37, 40-42, 54, 59, 61, 63, 82, 84, 90, and
98.
[0084] Methods of Use
[0085] An additional embodiment of the invention is directed
towards methods of using the above described compounds.
[0086] The above described compounds can be used in methods to
detect the presence or absence of double stranded DNA in a sample.
The method can comprise providing a sample suspected of containing
double stranded DNA; contacting the sample with at least one of the
above described chemical compounds to prepare a test sample; and
illuminating the test sample with energy. The method can further
comprise detecting emission of energy from the test sample after
the illuminating step. The detecting step can be qualitative or
quantitative. The method can further comprise calculating the
concentration of double stranded DNA in the sample after the
detecting step. The calculating step can comprise correlating the
emitted fluorescent energy with the concentration of double
stranded DNA in the sample.
[0087] The presence of an emitted fluorescent energy (or an
increase in emitted fluorescent energy relative to a control) is
indicative of the presence of double stranded DNA in the sample,
while the absence of emitted fluorescent energy (or no increase or
no change relative to a control) is indicative of the absence of
DNA in the sample.
[0088] The methods can also be performed on "blank" or "control"
samples. The blank or control samples can contain RNA but lack
double stranded DNA, or can lack nucleic acids altogether.
[0089] The sample can generally be any type of sample. For example,
the sample can be a cell or group of cells, an organism, cell
lysates, a cell culture medium, a bioreactor sample, and so on.
Alternatively, the sample can be a non-biological sample. The cells
can be any type of cell, such as bacterial cells, fungal cells,
insect cells, and mammalian cells. The sample can be a solid, a
liquid, or a suspension. The sample can be a biological fluid such
as blood, plasma, or urine. The sample can be a material
immobilized in a gel, on a membrane, bound to a bead or beads,
arranged in an array, and so on. The sample can be a partially or
fully purified nucleic acid preparation in a buffer or in
water.
[0090] The contacting step can be performed at any suitable
temperature, and for any suitable length of time. Typically, the
temperature will be ambient or room temperature, or at an elevated
temperature such as 37.degree. C. Examples of temperatures include
about 20.degree. C., about 25.degree. C., about 30.degree. C.,
about 35.degree. C., about 37.degree. C., about 40.degree. C.,
about 42.degree. C., and ranges between any two of these values.
Temperatures higher than about 42.degree. C., and temperatures
lower than about 20.degree. C. are also possible, depending on the
sample tested. The length of time can generally be any length of
time suitable for detection of a change in fluorescence. Examples
of lengths of time include about 10 minutes, about 20 minutes,
about 30 minutes, about 40 minutes, about 50 minutes, about 60
minutes, about 70 minutes, about 80 minutes, about 90 minutes,
about 100 minutes, about 110 minutes, about 120 minutes, about 180
minutes, about 240 minutes, about 300 minutes, about 360 minutes,
about 420 minutes, about 480 minutes, about 540 minutes, about 600
minutes, and ranges between any two of these values. Further
extended lengths of time are also possible, depending on the sample
tested. The contacting step is preferably performed with the test
sample protected from light.
[0091] The compound or compounds can be used at generally any
concentration suitable to produce a detectable emitted fluorescent
energy signal in the presence of double stranded DNA. Example
concentration ranges include about 10 nM to about 1 mM. Examples of
concentrations include about 10 nM, about 100 nM, about 1 .mu.M,
about 2 .mu.M, about 3 .mu.M, about 4 .mu.M, about 5 .mu.M, about 6
.mu.M, about 7 .mu.M, about 8 .mu.M, about 9 .mu.M, about 10 .mu.M,
about 100 .mu.M, about 1 mM, and ranges between any two of these
values.
[0092] The excitation energy can be applied to the test sample in a
variety of ways during the illuminating step. Suitable equipment
includes hand-held ultraviolet lamps, mercury arc lamps, xenon
lamps, lasers (such as argon and YAG lasers), and laser diodes.
These illumination sources are typically optically integrated into
laser scanners, fluorescence microplate readers or standard or
microfluorometers.
[0093] The detecting step can be performed by visual inspection, or
by the use of a variety of instruments. Examples of such
instruments include CCD cameras, video cameras, photographic film,
laser scanning devices, fluorometers, photodiodes, quantum
counters, epifluorescence microscopes, scanning microscopes, flow
cytometers, fluorescence microplate readers, or by amplification
devices such as photomultiplier tubes.
[0094] The detecting step can be performed at a single point in
time, can be performed at multiple points in time, or can be
performed continuously.
[0095] The methods can be used in conjunction with experimental
systems such as DNA minipreps, flow cytometry, fluorescence
microscopy, real time PCR, double stranded DNA quantitation,
microarray hybridizations, double stranded DNA detection in gels,
and so on.
[0096] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor(s) to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1
Preparation of Compound (2)
##STR00004##
[0098] To 10 g of N-methylaniline in 200 mL acetic acid at room
temperature 11.3 mL bromine was added and the mixture was stirred
for several hours. Volatile components were evaporated under
reduced pressure and the residue was dissolved in chloroform and
washed with saturated solutions of NaHCO.sub.3 and
Na.sub.2S.sub.2O.sub.3. The crude was purified on a silica gel
column with ethyl acetate and hexanes.
Example 2
Preparation of Compound (3)
##STR00005##
[0100] To 3.5 g of NaH (60% by weight in dispersion oil and washed
with hexanes) in 200 mL of DMF 18.5 g of Compound (2) was added,
followed by 6.2 mL of CS.sub.2. The mixture was heated at
100.degree. C. for 4 hours. Water was added and the solid was
filtered and purified by silica gel column with ethyl acetate and
hexanes.
Example 3
Preparation of Compound (4)
##STR00006##
[0102] To 9.5 g of Compound (3), 10 g of 2,6-dimethoxyphenylboronic
acid and 1 g of triphenylphosphine in 300 mL of isopropyl alcohol
and 50 mL of toluene was added a solution of 6 g of K.sub.2CO.sub.3
in 40 mL of water and 0.25 g of palladium(II)acetate. The resulting
mixture was heated at 100.degree. C. for 5 hours. The solvent was
removed and the residue was dissolved in CHCl.sub.3 and washed with
water. The crude product was purified on a silica gel column with
ethyl acetate and hexanes.
Example 4
Preparation of Compound (5)
##STR00007##
[0104] Compound (3) was coupled with 2,6-dimethoxyphenylboronic
acid as described in Example 3 (Preparation of compound (4)). This
was followed by quarternization with methyl tosylate at about
130.degree. C. for 1.5 hours.
Example 5
Preparation of Compound (6)
##STR00008##
[0106] Compound (3) was coupled with 5-indolylboronic acid using
the conditions described in Example 3 (Preparation of compound
(4)). This was followed by quarternization with methyl tosylate at
about 130.degree. C. for 1.5 hours.
Example 6
Preparation of Compound (7)
##STR00009##
[0108] Compound (3) was coupled with 2,6-dimethylphenylboronic acid
using the conditions described in Example 3 (Preparation of
compound (4)). This was followed by quarternization with methyl
tosylate at about 130.degree. C. for 1.5 hours.
Example 7
Preparation of Compound (8)
##STR00010##
[0110] Compound (3) was coupled with 4-hydroxyphenylboronic acid
using the conditions described in Example 3 (Preparation of
compound (4)). This was followed by quarternization with methyl
tosylate at about 130.degree. C. for 1.5 hours.
Example 8
Preparation of Compound (9)
##STR00011##
[0112] Compound (3) was coupled with benzothiaphen-2-yl boronic
acid using the conditions described in Example 3 (Preparation of
compound (4)). This was followed by quarternization with methyl
tosylate at about 130.degree. C. for 1.5 hours.
Example 9
Preparation of Compound (10)
##STR00012##
[0114] Compound (3) was coupled with phenylboronic acid using the
conditions described in Example 3 (Preparation of compound (4)).
This was followed by quarternization with methyl tosylate at about
130.degree. C. for 1.5 hours.
Example 10
Preparation of Compound (11)
##STR00013##
[0116] Compound (3) was coupled with thiophene-2-boronic acid using
the conditions described in Example 3 (Preparation of compound
(4)). This was followed by quarternization with methyl tosylate at
about 130.degree. C. for 1.5 hours.
Example 11
Preparation of Compound (12)
##STR00014##
[0118] Compound (3) was coupled with 2-acetamidobenzeneboronic acid
using the conditions described in Example 3 (Preparation of
compound (4)). This was followed by quarternization with methyl
tosylate at about 130.degree. C. for 1.5 hours.
Example 12
Preparation of Compound (13)
##STR00015##
[0120] To 1.5 g NaH (60% in dispersion oil and washed with hexanes)
in 50 ml DMF 1.5 g of 2-hydroxyl-4-methylquinoline was added
followed by 4.5 mL of benzyl bromide. The mixture was stirred at
room temperature for 2 hours. Solvent was removed under reduced
pressure and the residue was dissolved in CHCl.sub.3 and washed
with water. The product was obtained by silica gel column
purification with ethyl acetate and hexanes.
Example 13
Preparation of Compound (14)
##STR00016##
[0122] To 0.5 g of Compound (5), 0.25 g of Compound (13) in 10 mL
of methylene chloride, 0.7 mL of diisopropylethylamine and 0.9 mL
of trimethylsilyl trifluoromethanesulfonate were added, and the
resulting mixture was heated at reflux for 1 hour. The mixture was
washed with water, and the product was purified on a silica gel
column with ethyl acetate and hexanes.
Example 14
Preparation of Compound (15)
##STR00017##
[0124] A mixture of 0.1 g of Compound (14) and 0.06 mL of
phosphorous oxychloride was refluxed in 3 mL of dichloroethane for
5 hours. The mixture was washed with water and the solvent was
removed. The residue was stirred in ethyl acetate and filtered to
obtain the product.
Example 15
Preparation of Compound (16)
##STR00018##
[0126] A mixture of 0.1 g of Compound (15) and 0.3 mL
N,N-dimethyl-N'-propyl-1,3-propanediamine was heated at 55.degree.
C. in 3 mL of 1,2-dichloroethane for 3 hours. The solvent was
removed and the product was purified on a silica gel column with
chloroform and methanol.
Example 16
Preparation of Compound (17)
##STR00019##
[0128] Compound (17) was prepared by following the same procedure
used to prepare Compound (16), substituting
3,3'-iminobis(N,N-dimethyl propylamine) for
N,N-dimethyl-N'-propyl-1,3-propanediamine.
Example 17
Preparation of Compound (18)
##STR00020##
[0130] Compound (18) was prepared by following the same procedure
used to prepare Compound (16), substituting
N,N,N'-trimethylethanediamine for
N,N-dimethyl-N'-propyl-1,3-propanediamine.
Example 18
Preparation of Compound (19)
##STR00021##
[0132] A mixture of 12 mg of Compound (15) and 2 mg of imidazole
was stirred at room temperature in 2 mL of methylene chloride for 2
hours. At the end of the period, 2 mL of ethyl acetate was added
and stirring was continued overnight. The product was obtained by
centrifugation.
Example 19
Preparation of compound (20)
##STR00022##
[0134] Compound (20) was prepared by following the same procedure
used to prepare Compound (16), substituting 1-methyl-piperazine for
N,N-dimethyl-N'-propyl-1,3-propanediamine.
Example 20
Preparation of Compound (21)
##STR00023##
[0136] Compound (21) was prepared by following the same procedure
used to prepare Compound (16), substituting piperazine for
N,N-dimethyl-N'-propyl-1,3-propanediamine.
Example 21
Preparation of Compound (22)
##STR00024##
[0138] Compound (22) was prepared by following the same procedure
used to prepare Compound (16), substituting
N,N'-dimethylethanediamine for
N,N-dimethyl-N'-propyl-1,3-propanediamine.
Example 22
Preparation of Compound (23)
##STR00025##
[0140] Compound (23) was prepared by following the same procedure
used to prepare Compound (16), substituting
N,N'-dimethylpropanediamine for
N,N-dimethyl-N'-propyl-1,3-propanediamine.
Example 23
Preparation of Compound (24)
##STR00026##
[0142] A mixture of 18 mg of Compound (15), 5 mg of
2-N,N-dimethylaminoethanethiol hydrochloride, and 9 uL of
triethylamine in 5 mL of methylene chloride was stirred at room
temperature for 1.5 hours. All volatile components were removed
under reduced pressure and the crude was purified on a silica gel
column using chloroform and methanol.
Example 24
Preparation of Compound (25)
##STR00027##
[0144] To 0.35 g of 4-diethylaminomethyl-bromobenzene in 4 mL dry
THF at -78.degree. C. under nitrogen, 0.32 mL of a 2.5 M
n-butyllithium was introduced followed by 0.1 g of Compound (13) in
2 mL THF. The reaction was stirred at the low temperature for 1
hour before the addition of 1 mL acetic acid. The mixture was
stirred at room temperature for another hour and the solvent was
removed and the residue was further pumped for an hour. To the
residue, 0.2 g of Compound (5), 2 mL of dichloroethane and 0.35 mL
of triethylamine were added and stirred at room temperature for one
hour. The resulting mixture was washed with dilute sodium hydroxide
and purified on a silica gel column chromatography using chloroform
and methanol.
Example 25
Preparation of Compound (26)
##STR00028##
[0146] To 50 mg of Compound (13) in 5 mL of THF at -78.degree. C.
under nitrogen, 0.16 mL of a 2.5 M n-butyllithium was added. After
30 minutes at the low temperature, 0.5 mL of acetic acid was added
and the resulting mixture was stirred at room temperature for 1
hour. Volatile components were evaporated under reduced pressure
and the residue further pumped in vacuo for 30 minutes. To the
resulting residue in 10 mL of methylene chloride, 50 mg of Compound
(5) and 84 uL of triethylamine were added and the mixture was
stirred at room temperature for several hours. The organic layer
was washed with dilute HCl and NaCl and the crude was purified on
silica gel using ethyl acetate, chloroform and methanol.
Example 26
Preparation of Compound (27)
##STR00029##
[0148] Compound (27) was prepared by following the same procedure
used to prepare Compound (15), using
4-methyl-1-phenyl-2(H)-quinolone as the starting material.
Example 27
Preparation of Compound (28)
##STR00030##
[0150] Compound (28) was prepared by following the same procedure
used to prepare Compound (16), using Compound (27) as the starting
material.
Example 28
Preparation of Compound (29)
##STR00031##
[0152] Compound (29) was prepared by following the same procedure
used to prepare Compound (15), using 1,4-dimethyl-2(H)-quinolinone
as the starting material.
Example 29
Preparation of Compound (30)
##STR00032##
[0154] Compound (30) was prepared by following the same procedure
used to prepare Compound (16), using Compound (29) as the starting
material.
Example 30
Preparation of Compound (31)
##STR00033##
[0156] A mixture of 4 mg of Compound (16) and 0.05 mL of methyl
iodide in 0.5 mL DMF was heated at 60.degree. C. overnight. The
solvent was removed and the product was purified on a LH-20 column
with water.
Example 31
Preparation of Compound (32)
##STR00034##
[0158] A mixture of 20 mg of Compound (15) and 0.05 mL
triethylamine in 2 mL methanol was heated at 60.degree. C. for 1
day. The product was precipitated out by the addition of ethyl
acetate.
Example 32
Preparation of Compound (33)
##STR00035##
[0160] Compound (33) was prepared by following the same procedure
used to prepare Compound (32), using 2-dimethylaminoethanol instead
of methanol.
Example 33
Preparation of Compound (34)
##STR00036##
[0162] Compound (34) was prepared by following the same procedure
used to prepare Compound (16) using
4-methyl-1-propargyl-2(H)-quinolinone as the starting material to
generate the intermediate 2-chloro derivative, which in turn was
reacted with N,N-dimethyl-N'-propylpropanediamine to generate the
target.
Example 34
Preparation of Compound (35)
##STR00037##
[0164] Compound (35) was prepared by following the same procedure
used to prepare Compound (16), using Compound (41) as the starting
material.
Example 35
Preparation of Compound (36)
##STR00038##
[0166] To 1 g of N-benzyl-4-methoxyaniline in 10 mL of toluene was
added 0.72 mL of diketene. The mixture was stirred overnight and
the solvent was removed under reduced pressure. To the residue, 8
mL of a 1:1 v/v mix of H.sub.2SO.sub.4:HOAc was added and heated at
50.degree. C. overnight. The mixture was poured onto ice water and
extracted with ethyl acetate. The crude material was purified on
silica gel using ethyl acetate and hexanes.
Example 36
Preparation of Compound (37)
##STR00039##
[0168] Compound (37) was prepared by following the same procedure
used to prepare Compound (36), using N-benzyl-3-methoxyaniline in
place of N-benzyl-4-methoxyaniline.
Example 37
Preparation of Compound (38)
##STR00040##
[0170] Compound (38) was prepared by following the same procedure
used to prepare Compound (16), using Compound (37) as the starting
material.
Example 38
Preparation of Compound (39)
##STR00041##
[0172] Compound (38) was prepared by following the same procedure
used to prepare Compound (16), using Compound (36) as the starting
material.
Example 39
Preparation of Compound (40)
##STR00042##
[0174] Compound (40) was prepared by following the same procedure
used to prepare Compound (13), using bromoethylbenzene as the
starting material.
Example 40
Preparation of Compound (41)
##STR00043##
[0176] Compound (41) was prepared by following the same procedure
used to prepare Compound (13), using bromoethylpyridine as the
starting material.
Example 41
Preparation of Compound (42)
##STR00044##
[0178] A mixture of 0.5 g of lepidine and 2.76 g of p-xylylene
dibromide was refluxed in 10 mL of ethyl acetate for 1 hour. The
product was obtained by filtration.
Example 42
Preparation of Compound (43)
##STR00045##
[0180] A mixture of 0.32 g of Compound (5), 0.26 g of Compound
(42), 0.4 g of N-ethylmaleimde, and 0.16 mL of
diisopropylethylamine was stirred in 5 mL of methylene chloride at
0.degree. C. for 1 hour. Next, 20 mL of ethyl acetate was added,
and the product was collected by filtration.
Example 43
Preparation of Compound (44)
##STR00046##
[0182] The compound was obtained by reacting Compound (43) with an
excess amount of N-methylpiperazine in DMF at room temperature for
3 hours.
Example 44
Preparation of Compound (45)
##STR00047##
[0184] The compound was obtained by reacting Compound (43) with an
excess amount of morpholine in DMF at room temperature for 3
hours.
Example 45
Preparation of Compound (46)
##STR00048##
[0186] The compound was obtained by reacting Compound (43) with an
excess amount of N,N,N',N'-tetramethylpropanediamine in DMF at
50.degree. C. for 4 hours.
Example 46
Preparation of Compound (47)
##STR00049##
[0188] The compound was obtained by reacting Compound (43) with an
excess amount of N,N,N'-trimethylpropanediamine in DMF at
50.degree. C. for 2 hours.
Example 47
Preparation of Compound (48)
##STR00050##
[0190] The compound was obtained by reacting Compound (43) with an
excess amount of trimethylamine in DMF at 50.degree. C. for 4
hours.
Example 48
Preparation of Compound (49)
##STR00051##
[0192] The compound was obtained by reacting Compound (46) with an
excess amount of methyl iodide in DMF at room temperature
overnight.
Example 49
Preparation of Compound (50)
##STR00052##
[0194] The compound was obtained by reacting Compound (46) with an
excess amount of 3,3'-iminobis(N,N-dimethylpropylamine) in DMF at
room temperature for 4 hours.
Example 50
Preparation of Compound (51)
##STR00053##
[0196] The compound was obtained by reacting Compound (46) with an
excess amount of dimethylamine in DMF at 60.degree. C. for 1
hour.
Example 51
Preparation of Compound (52)
##STR00054##
[0198] A mixture of 0.2 g of Compound (5), 0.16 g of Compound (42),
and 0.2 mL of triethylamine was stirred in 10 mL of dichloroethane
at room temperature for 1 hour. The reaction mixture was washed
with water and brine, and the crude material was purified using
HPLC with chloroform and methanol.
Example 52
Preparation of Compound (53)
##STR00055##
[0200] A mixture of 10 mg of Compound (5), 7 mg of
1-benzyl-4-methylquinolinium bromide, and 0.1 mL of triethylamine
was stirred in 1 mL of methanol for 2 hours. Volatile components
were removed under reduced pressure, and the crude was purified
using silica gel chromatography with chloroform and methanol.
Example 53
Preparation of Compound (54)
##STR00056##
[0202] A mixture of 4-bromomethylpyridine HBr and 2 equivalents of
lepidine was heated at 120.degree. C. for one hour, and followed by
stirring in ethyl acetate for several hours. The product was
collected by filtration.
Example 54
Preparation of Compound (55)
##STR00057##
[0204] A mixture of 10 mg of Compound (5), 5 equivalents of
Compound (54) and 0.1 mL of triethylamine was stirred in 0.5 mL of
DMF for 1 hour. Volatile components were removed under reduced
pressure, and the crude material was purified using silica gel
chromatography with chloroform and methanol.
Example 55
Preparation of Compound (56)
##STR00058##
[0206] A mixture of 3 mg of Compound (55) and about 0.2 mL of
iodomethane was stirred at room temperature overnight in 1 mL of
DMF. Ethyl acetate (4 mL) was added and after stirring for an
additional hour, the product was filtered.
Example 56
Preparation of Compound (57)
##STR00059##
[0208] A mixture of 1.7 mg of Compound (34), 1 mg of Cu(I)I, 50 uL
of diisopropylethylamine, and about 5 equivalents of propylazide
was stirred at room temperature in 1 mL of methanol overnight.
Volatile components were removed under reduced pressure, and the
crude material was purified using silica gel chromatography with
chloroform and methanol.
Example 57
Preparation of Compound (58)
##STR00060##
[0210] To 0.1 g of Compound (41) in 5 mL of THF at -78.degree. C.,
0.32 mL of a 2.5 M n-butyllithium was added and stirred at
-78.degree. C. for 1 hour. Next, 0.5 mL of acetic acid was added
and stirred at room temperature for 1 hour. Volatile components
were evaporated and the residue pumped in vacuo. To the dark
residue in several mL of methylene chloride, 243 mg of Compound (5)
and 0.2 mL of triethylamine were added and stirred at room
temperature for 1 hour. The organic layer was washed with water and
brine, and dried over magnesium sulfate. The crude material was
purified using silica gel chromatography with ethyl acetate,
chloroform and methanol.
Example 58
Preparation of Compound (59)
##STR00061##
[0212] A mixture of 1.26 g of
5-(2,6-dimethoxyphenyl)-2-methylbenzothiazole and 0.99 g of methyl
tosylate was heated at 130.degree. C. for 1 hour. The crude
material was stirred in about 30 mL of ethyl acetate and filtered
to obtain the product.
Example 59
Preparation of Compound (60)
##STR00062##
[0214] A mixture of 0.1 g of Compound (59), 32 mg of
4-dimethylaminobenzaldehyde and 21 uL of piperidine was heated at
40.degree. C. in 10 mL of ethanol for 1.5 hours. Volatile
components were removed under reduced pressure, and the residue was
dissolved in chloroform and washed with water and brine. The crude
material was purified using silica gel chromatography with
chloroform and methanol.
Example 60
Preparation of Compound (61)
##STR00063##
[0216] A mixture of 1 g of lepidine and 1 mL of
(2-bromoethyl)benzene was heated at 90.degree. C. for 2 hours.
About 30 mL of ethyl acetate was added and refluxed for 15 minutes.
The product was collected by filtration.
Example 61
Preparation of Compound (62)
##STR00064##
[0218] A mixture of 0.1 g of Compound (5), 67 mg of Compound (61),
and 86 uL of triethylamine in 10 mL of dichloroethane was stirred
at room temperature for 1 hour. Volatile components were evaporated
under reduced pressure, and the residue was stirred in about 30 mL
of ethyl acetate at room temperature overnight. The product was
collected by filtration.
Example 62
Preparation of Compound (63)
##STR00065##
[0220] A mixture of 40 uL of lepidine and 57 mg of
2-(acetamido)-4-(chloromethyl)-thiazole was heated at 90.degree. C.
for 2 hours. The crude material was stirred in about 20 mL of ethyl
acetate for several hours, and filtered to obtain the product.
Example 63
Preparation of Compound (64)
##STR00066##
[0222] A mixture of 0.13 g of Compound (5), 0.26 mmole of Compound
(63), and 0.11 mL of triethylamine was stirred in 10 mL of
dichloroethane at room temperature for 1 hour. The crude product
was purified using silica gel chromatography with chloroform and
methanol.
Example 64
Preparation of Compound (65)
##STR00067##
[0224] A mixture of 0.1 g of Compound (5), 58 mg of
1,4-dimethylquinolinium iodide, and 86 uL of triethylamine was
stirred in 10 mL of dichloroethane at room temperature for 1 hour.
Volatile components were evaporated under reduced pressure, and the
residue was stirred in about 50 mL of ethyl acetate for 30 minutes.
The product was collected by filtration.
Example 65
Preparation of Compound (66)
##STR00068##
[0226] Compound (66) was prepared by following the same procedure
used to prepare Compound (15), using Compounds (7) and (13) as the
starting materials.
Example 66
Preparation of Compound (67)
##STR00069##
[0228] Compound (67) was prepared by following the same procedure
used to prepare Compound (15), using Compound (7) and
4-methyl-1-phenyl-2(H)-quinolone as the starting materials.
Example 67
Preparation of Compound (68)
##STR00070##
[0230] Compound (68) was prepared by following the same procedure
used to prepare Compound (16), using Compound (67) and
N,N-dimethyl-N'-propyl-propanediamine as the starting
materials.
Example 68
Preparation of Compound (69)
##STR00071##
[0232] Compound (69) was prepared by following the same procedure
used to prepare Compound (16), using Compound (67) and
N,N,N'-trimethylpropanediamine as the starting materials.
Example 69
Preparation of Compound (70)
##STR00072##
[0234] Compound (70) was prepared by following the same procedure
used to prepare Compound (16), using Compound (66) and
N,N,N'-trimethylpropanediamine as the starting materials.
Example 70
Preparation of Compound (71)
##STR00073##
[0236] A mixture of 20 mg of Compound (7), 13 mg of
1-benzyl-4-methyl-quinolinium bromide, and 50 uL of triethylamine
was stirred in 1 mL of methanol at room temperature for 1 hour.
Volatile components were evaporated under reduced pressure, and the
crude material was purified using silica gel column chromatography
with chloroform and methanol.
Example 71
Preparation of Compound (72)
##STR00074##
[0238] A mixture of 20 mg of Compound (7), 17 mg of Compound (42),
and 50 uL of triethylamine was stirred at room temperature in 1 mL
of DMF for 1 hour. This was followed by the addition of about 100
uL of 1-methylpiperazine, and the mixture was stirred overnight.
Volatile components were evaporated under reduced pressure, and the
product was purified using silica gel chromatography with
chloroform and methanol.
Example 72
Preparation of Compound (73)
##STR00075##
[0240] A mixture of 8 mg of Compound (7), 8 mg of Compound (42) and
0.5 mL of trimethylamine (25% in methanol) was stirred at room
temperature in 1 mL of DMF for 4 hours. Volatile components were
evaporated under reduced pressure, and then pumped in vacuo. The
crude material was purified using silica gel chromatography with
chloroform and methanol.
Example 73
Preparation of Compound (74)
##STR00076##
[0242] A mixture of 20 mg of Compound (7), 5 equivalents of
Compound (54), and 50 uL of triethylamine was stirred in 1 mL of
DMF at room temperature for 1 hour. Volatile components were
evaporated under reduced pressure. The crude material was purified
using silica gel column chromatography with chloroform and
methanol.
Example 74
Preparation of Compound (75)
##STR00077##
[0244] A mixture of about 3 mg of Compound (74) and 0.2 mL of
iodomethane was stirred at room temperature overnight in 1 mL of
DMF. The product was precipitated by addition of 4 mL of ethyl
acetate.
Example 75
Preparation of Compound (76)
##STR00078##
[0246] Compound (76) was prepared by following the same procedure
used to prepare Compound (15), using Compound (6) and
4-methyl-1-phenyl-2(H)-quinolone as the starting materials.
Example 76
Preparation of Compound (77)
##STR00079##
[0248] Compound (77) was prepared by following the same procedure
used to prepare Compound (16), using Compound (15) and
N,N-dimethyl-N'-propylpropanediamine.
Example 77
Preparation of Compound (78)
##STR00080##
[0250] A mixture of about 35 mg of Compound (6), 30 mg of Compound
(42), and 17 uL of diisopropylethylamine was stirred in 5 mL of a
1:4 v/v DMF/methylene chloride solvent at room temperature
overnight. Volatile components were evaporated under reduced
pressure, and the crude product was purified using silica gel
column chromatography with ethyl acetate, chloroform and
methanol.
Example 78
Preparation of Compound (79)
##STR00081##
[0252] A mixture of about 5 mg of Compound (78) and 3 mL of a 2 M
solution of dimethylamine in THF was heated in 10 mL of methanol at
room temperature for 3 days. Volatile components were removed under
reduced pressure, and the crude product was purified using silica
gel column chromatography with chloroform, methanol and
triethylamine.
Example 79
Preparation of Compound (80)
##STR00082##
[0254] A mixture of about 5 mg of Compound (78) and 30 mg of
1-methylpiperazine was stirred at 35.degree. C. for 4 days.
Volatile components were evaporated under reduced pressure, and the
product was purified on a preparatory TLC plate.
Example 80
Preparation of Compound (81)
##STR00083##
[0256] A mixture of 20 mg of Compound (6), 20 mg of
1-benzyl-4-methylquinolinium bromide, and 0.1 mL triethylamine was
stirred in 0.5 mL of methylene chloride at room temperature for 1
hour. Volatile components were evaporated under reduced pressure,
and the crude material was purified using silica gel column
chromatography with methanol and chloroform.
Example 81
Preparation of Compound (82)
##STR00084##
[0258] To 1.3 g of 1-methylimidazole in 20 mL of THF at -78.degree.
C. under nitrogen, 2.8 mL of a 2.5 M n-butyllithium was introduced.
After 45 minutes at the low temperature, 1.25 g of
1-benzyl-4-methyl-2(H)-quinolone (in 10 mL of THF) was added and
the resulting mixture was further stirred at -78.degree. C. for 1
hour, at 0.degree. C. for 2 hours and room temperature for another
30 minutes. Acetic acid (0.5 mL) was added and stirred for 30
minutes. Volatile components were removed under reduced pressure.
The resulting material was presumably
1-benzyl-4-methyl-2-(1-methylimidazoyl)-quinolinium acetate.
Example 82
Preparation of Compound (83)
##STR00085##
[0260] To a mixture of 20 mg of Compound (6) and 0.01 mmole of
Compound (82) in 1 mL of methanol, 50 uL of triethylamine was added
and stirred at room temperature for 1 hour. Volatile components
were evaporated. The crude material was purified using silica gel
column chromatography with chloroform and methanol, and then on a
LH-20 column with water to obtain the pure product.
Example 83
Preparation of Compound (84)
##STR00086##
[0262] The compound was prepared from the commercially available
5-phenyl-2-mercapto-benzothiazole (Aldrich Chemical; St. Louis,
Mo.) by first converting the mercapto into a methylthio with
potassium carbonate and methyl tosylate, and further
quarternization of the benzothiazole under neat condition with
methyl tosylate to generate the desired compound.
Example 84
Preparation of Compound (85)
##STR00087##
[0264] A mixture of 60 mg of Compound (84), one molar equivalent of
1,4-dimethyl-2-(1-methylimidazoyl)-quinolinium acetate (prepared by
similar protocol to that of Compound (82) using
1,4-dimethyl-2(H)-quinolone as the starting material), and 0.1 mL
of triethylamine was stirred in 1 mL of methanol at room
temperature for 1 hour. The crude material was purified on a LH-20
column eluting with water.
Example 85
Preparation of Compound (86)
##STR00088##
[0266] A mixture of 15 mg of Compound (10) and 9.6 mg of
1,4-dimethylquinolinium iodide in 1 mL of methylene chloride was
stirred at room temperature for 1 hour. The product was obtained by
filtration.
Example 86
Preparation of Compound (87)
##STR00089##
[0268] A mixture of 27 mg of Compound (10), 0.067 mmole of
1,4-dimethyl-2-(1-methylimidazoyl)-quinolinium acetate (prepared by
similar protocol to that of Compound (82) using
1,4-dimethyl-2(H)-quinolone as the starting material), and 0.1 mL
of triethylamine was stirred in 1 mL of methylene chloride for 1
hour. Volatile components were evaporated, and the crude product
was purified on a LH-20 column.
Example 87
Preparation of Compound (88)
##STR00090##
[0270] To a mixture of 8 mg of Compound (84) and one equivalent of
Compound (82) in 1 mL of methanol, 50 uL of triethylamine was added
and stirred at room temperature for 1 hour. Volatile components
were evaporated, and the crude material was purified first by
silica gel column chromatography with chloroform and methanol and
second on a LH-20 column with water to obtain the pure product.
Example 88
Preparation of Compound (89)
##STR00091##
[0272] A mixture of 43 mg of
5-phenyl-3-methyl-2-methylthiobenzoxazolium tosylate, 39 mg of
1-benzyl-4-methylquinolinium bromide, and 0.1 mL of triethylamine
was stirred in 1 mL of methylene chloride for 1 hour. The product
was collected by filtration.
Example 89
Preparation of Compound (90)
##STR00092##
[0274] To 0.36 g of 4-diethylaminomethyl-bromobenzene in 4 mL dry
THF at -78.degree. C. under nitrogen, 0.48 mL of a 2.5 M
n-butyllithium was introduced followed by 0.235 g of
4-methyl-1-phenyl-2(H)-quinolone (in 10 mL THF). The reaction was
stirred at the low temperature for 1 hour before the addition of 1
mL acetic acid. The mixture was stirred at room temperature for
another hour, and the solvent was removed and the residue was
further pumped for an hour. The crude product
2-(4-diethylaminomethyl)-4-methyl-1-phenylquinolinium acetate was
used without further purification.
Example 90
Preparation of Compound (91)
##STR00093##
[0276] A mixture of 20 mg of Compound (10), one equivalent of
Compound (90), and 50 uL of triethylamine was stirred in 1 mL of
methylene chloride at room temperature for 1 hour. Volatile
components were removed, and the crude material was purified using
silica gel column chromatography with chloroform and methanol.
Example 91
Preparation of Compound (92)
##STR00094##
[0278] A mixture of 12 mg of Compound (10), 10 mg of
1-benzyl-4-methylpyridinium bromide, and 0.1 mL of triethylamine in
1 mL of methylene chloride was refluxed for two hours. Volatile
components were removed under reduced pressure, and the crude
material was stirred in about 2 mL of methylene chloride for 1
hour. The product was collected by filtration.
Example 92
Preparation of Compound (93)
##STR00095##
[0280] A mixture of 8 mg of Compound (10), 8.8 mg of
1-benzyl-4-methyl-2-phenylquinolinium bromide, and 50 uL of
triethylamine was refluxed in 2 mL of methylene chloride for 3
hours. The crude material was purified using silica gel
chromatography with chloroform and methanol.
Example 93
Preparation of Compound (94)
##STR00096##
[0282] A mixture of 22 mg of Compound (12), 10 mg of
1-benzyl-4-methylquinolinium bromide, and 50 uL of triethylamine
was stirred in 1 mL of methanol at room temperature for one hour.
The crude material was purified using silica gel chromatography
with chloroform and methanol.
Example 94
Preparation of Compound (95)
##STR00097##
[0284] A mixture of 6.5 mg of Compound (9), 4 mg of
1-benzyl-4-methylquinolinium bromide, and 50 uL of triethylamine
was stirred in 1 mL of methanol at room temperature for 3 hours.
The crude material was purified using silica gel chromatography
with chloroform and methanol.
Example 95
Preparation of Compound (96)
##STR00098##
[0286] A mixture of 6.8 mg of Compound (11), 4.7 mg of
1-benzyl-4-methylquinolinium bromide, and 50 uL of triethylamine
was stirred in 1 mL of methylene chloride at room temperature for 1
hour. The crude material was purified using silica gel column
chromatography with chloroform and methanol.
Example 96
Preparation of Compound (97)
##STR00099##
[0288] A mixture of 18 mg of
3-methyl-6-pyridyl-1,3-benzothiazole-2-thione, 22 mg of
1-benzyl-4-methylquinolinium bromide, 14 mg of methyl tosylate, and
0.1 mL of diisopropylethylamine was heated at 100.degree. C. for 30
minutes. Volatile components were removed under reduced pressure,
and the crude product was purified by preparative TLC plate.
Example 97
Preparation of Compound (98)
##STR00100##
[0290] A mixture of 23 mg of
3-methyl-6-pyridyl-1,3-benzolthiazole-2-thione and 330 mg of methyl
tosylate was heated at 130.degree. C. for 1 hour. Next, 10 mL of
ethyl acetate was added and refluxed for 15 minutes. The product
was collected by filtration.
Example 98
Preparation of Compound (99)
##STR00101##
[0292] A mixture of 27 mg of 1-benzyl-4-methylquinolinium bromide,
one equivalent of Compound (98), and 0.2 mL of triethylamine was
stirred in 2 mL of DMF at room temperature. The product was
collected by filtration.
Example 99
Preparation of Compound (100)
##STR00102##
[0294] A mixture of 50 mg of Compound (8), 1.2 equivalent of
1-benzyl-4-methylquinolinium bromide, and 45 uL of triethylamine
was stirred in a mixed solvent of dichloroethane/DMF (v/v, 1:1, 4
mL) at room temperature for 3 hours. The reaction mixture was
diluted with chloroform, washed with water, and dried over
magnesium sulfate. The product precipitated out from the chloroform
later as the volume was reduced.
Example 100
Preparation of Compound (101)
##STR00103##
[0296] Compound (101) was prepared by following the same procedure
used to prepare Compound (16), using
6-(bis-(1,3-dimethoxy)-prop-2-yl)-3-methyl-2-methylthio-benzothiazolium
tosylate and Compound (13) as the starting materials.
Example 101
Preparation of Compound (102)
##STR00104##
[0298] Compound (102) was prepared by following the same procedure
used to prepare Compound (24), using Compound (5) and
1-benzyl-4-methyl-pyridin-2-one as the starting materials.
Example 102
Preparation of Compound (103)
##STR00105##
[0300] Acetic anhydride (0.1 mL) was added to a mixture of 0.127 mg
of 2-(2-anilinovinyl)-3-methyl-6-phenylquinolinium tosylate, one
equivalent of 1-benzyl-4-methylquinolinium bromide, and 40 uL of
triethylamine in 2 mL of dichloroethane at room temperature. The
mixture was stirred for 2 hours. The reaction was diluted with
chloroform and washed with water and brine. The crude material was
purified by recrystallizing from methanol and ethyl acetate.
Example 103
Preparation of Compound (104)
##STR00106##
[0302] Acetic anhydride (90 uL) was added to a mixture of 0.107 mg
of 2-(2-anilinovinyl)-3-methyl-6-phenylquinolinium tosylate, one
equivalent of
1-((3-ethoxycarbonyl-1-propoxy)phenylmethyl)-4-methylquinolinium
chloride, and 40 uL of triethylamine in 5 mL of dichloroethane at
room temperature. The mixture was stirred for 2 hours. The reaction
mixture was diluted with chloroform and washed with water and
brine. The crude material was purified using silica gel column
chromatography with chloroform and methanol.
Example 104
Preparation of Compound (105)
##STR00107##
[0304] Water (0.5 mL) and 40 uL of 10% sodium hydroxide was added
to 59 mg of Compound (107) in 5 mL of methanol. The mixture was
stirred at room temperature for several hours. The reaction was
diluted with about 30 mL of water, acidified with 1 N HCl, and
filtered to recover the product.
Example 105
Preparation of Compound (106)
##STR00108##
[0306] O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (8.2 mg) was added to 11.3 mg of Compound (105)
in 2 mL of DMF and 8 uL of triethylamine in 2 mL of DMF. The
mixture was stirred overnight at room temperature. About 6 mL of
ethyl acetate was added to precipitate the product, and the product
was obtained by filtration.
Example 106
Preparation of Compound (107)
##STR00109##
[0308] Compound (107) was prepared by following the same procedure
used to prepare Compound (15), using Compound (5) and
1-benzyl-6-(3-ethoxycarbonyl-1-propoxy)-4-methyl-2(H)-quinolone as
the starting materials.
Example 107
Preparation of Compound (108)
##STR00110##
[0310] A mixture of 0.314 g of Compound (107), 0.13 mL of
thiophenol, and 0.3 mL of triethylamine was stirred in 5 mL of
dichloroethane at 60.degree. C. for several hours. The product was
purified using silica gel column chromatography with chloroform and
methane.
Example 108
Preparation of Compound (109)
##STR00111##
[0312] 4-N-methylaminobutyric acid (39 mg) and 107 uL of
triethylamine was dissolved in a mixture of 1.5 mL of isopropyl
alcohol and several drops of water. This mixture was added to a
solution of 30 mg of Compound (15) in 3 mL of dichloroethane and
the resulting mixture was heated at 60.degree. C. for 1 hour. The
crude material was diluted with additional chloroform and washed
with diluted aqueous HCl. The product was purified on a silica gel
column with chloroform and methanol.
Example 109
Preparation of Compound (110)
##STR00112##
[0314] O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (7.4 mg) was added to a mixture of 11 mg of
B36-13-LY and 5 uL of triethylamine in 2 mL of DMF. After 30
minutes stirring at room temperature, the crude material was
purified on a silica gel column eluting with chloroform and
acetone.
Example 110
Preparation of Compound (111)
##STR00113##
[0316] Amino-dPEG4-alcohol (3.0 mg; Quanta Biodesign, Ltd.; Powell,
Ohio) was added to a mixture of about 5 mg of Compound (110) and 2
equivalents of triethylamine. The mixture was stirred for 30
minutes. The mixture was concentrated and several mL of ethyl
acetate was added and stirred briefly and filtered to obtain the
product.
Example 111
Preparation of Compound (112)
##STR00114##
[0318] Triethylamine (61 uL) was added to a mixture of 59 mg of
1-((3-ethoxycarbonyl-1-propoxy)phenylmethyl)-4-methylquinolinium
chloride and 74 mg of Compound (5) in 3 mL of methylene chloride.
The mixture was stirred for 5 minutes. The mixture was then diluted
with chloroform and washed with 1:1 mixture of water/brine to yield
the product.
Example 112
Method for Determination of DNA/RNA Fluorescence Ratios
[0319] The tested compound was dissolved as a stock solution in
DMSO at a concentration of about 0.1-1.0 mg/mL. The exact
concentration is not critical. Three tubes are prepared, each
containing the same 1-20 uL of stock solution. The first tube
contained 10 mM Tris, 1 mM EDTA (pH 7.5) buffer; the second tube
contained buffer and 65 ug/mL calf thymus double stranded DNA; and
the third tube contained buffer and 65 ug/mL ribosomal RNA. The
tubes were incubated at room temperature for 10-15 minutes with
protection from light.
[0320] Fluorescence scans of the three solutions were performed in
disposable cuvettes, with the excitation wavelength corresponding
to the absorption maximum for the compound bound to DNA (or RNA if
the values were significantly different). In some cases, it was
necessary to dilute the sample to keep the fluorescence signal
on-scale. In these cases, all three samples were diluted to the
same degree. The ratio of the fluorescence of the compound in the
presence of DNA and RNA was determined.
Example 113
DNA/RNA Fluorescence Ratios for Prepared Compounds
[0321] The following compounds were selected as representative of
the inventive class of compounds. The fluorescence values in the
presence of DNA and RNA were determined as described in the
previous Example. The following table shows the DNA/RNA
fluorescence ratios, where higher values indicate a selectivity for
DNA. A ratio of 1 would indicate no selectivity. Excitation and
emission values are in nm.
TABLE-US-00002 DNA/RNA Compound ex/em (fluorescence ratio) Thiazole
orange 510/530 1 Compound 16 504/532 15.2 Compound 17 508/536 4.8
Compound 18 505/533 3.5 Compound 19 525/553 7.3 Compound 20 510/536
4 Compound 21 510/534 5.4 Compound 22 505/534 4.7 Compound 23
500/529 3.8 Compound 24 520/570 4.6 Compound 25 518/559 9 Compound
26 505/541 36 Compound 28 497/527 6.5 Compound 30 495/527 5.7
Compound 31 505/534 10.5 Compound 32 498/517 36 Compound 34 503/532
5.8 Compound 38 497522 13 Compound 39 507/536 6.1 Compound 44
510/540 22 Compound 45 511/541 13.2 Compound 46 512/542 11.5
Compound 47 512/541 8.6 Compound 48 513/540 7.3 Compound 49 520/542
18 Compound 50 512/541 9.7 Compound 53 510/540 60 Compound 55
513/541 10.9 Compound 56 515/545 3.7 Compound 57 506/536 3.8
Compound 62 510/539 48 Compound 65 506-536 11.3 Compound 68 498/523
7 Compound 69 494/523 3.2 Compound 70 501/524 2.7 Compound 89
485/512 1.8 Compound 91 522/563 1.5 Compound 92 458/501 1 Compound
93 466/514 1.5 Compound 94 513/530 2.1 Compound 95 550/591 1.9
Compound 96 527/555 8 Compound 97 513/538 3.7 Compound 99 511/534
1.3 Compound 100 513/551 8.1 Compound 101 503/527 3 Compound 102
475/515 3.3 Compound 103 507/537 3.1 Compound 104 652/666 9
Compound 109 496/525 121 Compound 111 497/527 64 Compound 112
500/541 191
Example 114
Evaluation of Compound (24)
[0322] This compound has a 520 nm excitation maximum, and can
effectively be excited with either a 488 nm line (blue laser) or a
532 nm (green laser) line. The compound has an emission maximum of
569 nm (orange).
Example 115
Use of Compound (20) in Flow Cytometry
[0323] Live Jurkat cells (human T-lymphocyte) were suspended at
1.times.10.sup.6 cells/ml in RPMI media with 10% Fetal Bovine Serum
(FBS). 5 .mu.M Compound (20) was added to one mL cell suspension,
and incubated at 37.degree. C. for 60 minutes protected from light.
Cells were processed using a Becton Dickinson (BD) LSRII Flow
Cytometer. A Forward Scatter (FS) vs Side Scatter (SS) dual
parameter plot was used to gate main cell population. On gated
cells, a dual-parameter plot of Fluorescence-Width vs
Fluorescence-Area was used for single cell discrimination gating.
Single color fluorescence was collected at 530/30 bandpass using
the 488 nm excitation laser, collecting 30,000 events at flow rate
of about 200 events/second. The data was further analyzed using
ModFit LT Flow Cytometry Modeling Software from Verity Software
House, Inc. to determine the ratio of G2/G1 and the CV of G1
phase.
[0324] Typical cell cycle histograms were demonstrated showing G0G1
phase, S phase, and G2M phase. This was obtained on the live cell
gate. Further analysis using ModFit Software showed that G1-phase
is 47.08% with peak CV of 6.92%, S-phase is 46.67%, G2-phase is
6.25% and the G2/G1 ratio is 1.83. This demonstrated that the
compound stains live cells for cell cycle where the CV of G1-phase
<8%, and the observed ratio indicated linearity of staining.
Example 116
Use of Compound (24) in Flow Cytometry
[0325] Live Jurkat cells were treated with colcemid for 2 hours, to
arrest cell cycle at mitosis, thus resulting in a larger more
defined G2M-phase. The cells were suspended at 1.times.10.sup.6
cells/ml in RPMI media with 10% Fetal Bovine Serum (FBS). 10 .mu.M
Compound (24) was added to one mL cell suspension, incubated at
37.degree. C. for 30 minutes protected from light. Cells were
processed using the Becton Dickinson (BD) LSRII Flow Cytometer.
Forward Scatter (FS) vs Side Scatter (SS) dual parameter plot was
used to gate main cell population. On gated cells, a dual-parameter
plot of Fluorescence-Width vs Fluorescence-Area was used for single
cell discrimination gating. Single color fluorescence was collected
at 585/42 bandpass using the 488 nm excitation laser, and also
collected at the same 585/42 bandpass using the 532 nm excitation
laser, collecting 30,000 events at flow rate of about 200
events/second. The data was further analyzed using ModFit LT Flow
Cytometry Modeling Software from Verity Software House, Inc. to
determine the ratio of G2/G1 and the CV of G1 phase.
[0326] Typical cell cycle histograms were demonstrated showing G0G1
phase, S phase, and G2M phase. This was obtained on the live cell
gate. Further analysis using ModFit Software showed typical cell
cycle staining. This demonstrated that the compound stains live
cells for cell cycle where the CV of G1-phase <8%, and the
observed ratio indicated linearity of staining.
Example 117
Use of Compound (20) in Flow Cytometry
[0327] Live HL60 cells (human promyeloblasts) were suspended at
1.times.10.sup.6 cells/ml in Iscove's Dulbecco's complete Media
with 20% Fetal Bovine Serum (FBS). 5 .mu.M Compound (20) is added
to one mL cell suspension, and incubated at 37.degree. C. for 30
minutes protected from light. Cells were processed using the Becton
Dickinson (BD) LSRII Flow Cytometer. Forward Scatter (FS) vs Side
Scatter (SS) dual parameter plot was used to gate main cell
population. On gated cells, a dual-parameter plot of
Fluorescence-Width vs Fluorescence-Area was used for single cell
discrimination gating. Single color fluorescence was collected at
530/30 bandpass using the 488 nm excitation laser, collecting
30,000 events at flow rate of about 200 events/second. The data was
further analyzed using ModFit LT Flow Cytometry Modeling Software
from Verity Software House, Inc. to determine the ratio of G2/G1
and the CV of G1 phase.
[0328] Typical cell cycle histograms were demonstrated showing G0G1
phase, S phase, and G2M phase. This was obtained on the live cell
gate. Further analysis using ModFit Software showed typical cell
cycle staining. This demonstrated that the compound stained live
cells for cell cycle where the CV of G1-phase <8%, and the
observed ratio indicated linearity of staining.
Example 118
Use of Compound (24) in Flow Cytometry
[0329] HL60 cells were suspended at 1.times.10.sup.6 cells/ml in
Hanks Balanced Salt Solution (HBSS). 10 .mu.M Compound (24) was
added to one mL cell suspension, and incubated at room temperature
for 30 minutes protected from light. Cells were processed using the
Becton Dickinson (BD) LSRII Flow Cytometer. Forward Scatter (FS) vs
Side Scatter (SS) dual parameter plot was used to gate main cell
population. On gated cells, a dual-parameter plot of
Fluorescence-Width vs Fluorescence-Area was used for single cell
discrimination gating. Single color fluorescence was collected at
585/42 bandpass using the 488 nm excitation laser, and also at the
same 585/42 bandpass using the 532 nm excitation laser, collecting
30,000 events at flow rate of about 200 events/second. The data was
further analyzed using ModFit LT Flow Cytometry Modeling Software
from Verity Software House, Inc. to determine the ratio of G2/G1
and the CV of G1 phase.
[0330] Typical cell cycle histograms were demonstrated showing GOG1
phase, S phase, and G2M phase. This was obtained on the live cell
gate. Further analysis using ModFit Software showed typical cell
cycle staining. This demonstrated that the compound stained live
cells for cell cycle where the CV of G1-phase <8%, and the
observed ratio indicated linearity of staining.
Example 119
Use of Compound (24) in Flow Cytometry with Fixed Cells
[0331] HL60 cells were fixed with 70% ethanol and stored at
-20.degree. C. until use. The fixed cells were washed once in Hanks
Balanced Salt Solution (HBSS) and were then suspended at
1.times.10.sup.6 cells/ml in HBSS. 5 .mu.M Compound (24) was added
to one mL cell suspension, and incubated at 37.degree. C. for 5
minutes protected from light. Cells were processed using the Becton
Dickinson (BD) LSRII Flow Cytometer. Forward Scatter (FS) vs Side
Scatter (SS) dual parameter plot was used to gate main cell
population. On gated cells, a dual-parameter plot of
Fluorescence-Width vs Fluorescence-Area was used for single cell
discrimination gating. Single color fluorescence was collected at
585/42 bandpass using the 488 nm excitation laser, and also
collected at the same 585/42 bandpass using the 532 nm excitation
laser, collecting 30,000 events at flow rate of about 200
events/second. The data was further analyzed using ModFit LT Flow
Cytometry Modeling Software from Verity Software House, Inc. to
determine the ratio of G2/G1 and the CV of G1 phase.
[0332] Typical cell cycle histograms were demonstrated showing G0G1
phase, S phase, and G2M phase. Further analysis using ModFit
Software showed typical cell cycle staining. This demonstrated that
the compound stained live cells for cell cycle where the CV of
G1-phase <8%, and the observed ratio indicated linearity of
staining.
Example 120
Use of Compound (20) for Flow Cytometry with Fixed Cells
[0333] Live Jurkat cells were treated with colcemid for 2 hours, to
arrest cell cycle at mitosis, thus resulting in a larger more
defined G2M-phase. The cells were fixed with 70% ethanol and stored
at -20.degree. C. until use. The fixed cells were washed once in
Hanks Balanced Salt Solution (HBSS) and were then suspended at
1.times.10.sup.6 cells/ml in HBSS. 5 .mu.M Compound (20) was added
to one mL cell suspension, and incubated at 37.degree. C. for 5
minutes protected from light. Cells were processed using the Becton
Dickinson (BD) LSRII Flow Cytometer. Forward Scatter (FS) vs Side
Scatter (SS) dual parameter plot was used to gate main cell
population. On gated cells, a dual-parameter plot of
Fluorescence-Width vs Fluorescence-Area was used for single cell
discrimination gating. Single color fluorescence was collected at
530/30 bandpass using the 488 nm excitation laser, collecting
30,000 events at flow rate of about 200 events/second. The data was
further analyzed using ModFit LT Flow Cytometry Modeling Software
from Verity Software House, Inc. to determine the ratio of G2/G1
and the CV of G1 phase.
[0334] Typical cell cycle histograms were demonstrated showing GOG1
phase, S phase, and G2M phase. Further analysis using ModFit
Software showed typical cell cycle staining. This demonstrated that
the compound stained live cells for cell cycle where the CV of
G1-phase <8%, and the observed ratio indicated linearity of
staining.
Example 121
Use of Compound (24) for Flow Cytometry with Induced Apoptosis
Cells
[0335] Live Jurkat cells were split into 13 samples and each cell
split was suspended in complete RMPI/10% FBS. Samples was treated
with 10 .mu.M Camptothecin in DMSO to induce apoptosis, or treated
with DMSO alone to act as a control. Treatment and control time was
1, 2, 3, 4, 5, and 6 hours, and a time zero point. The cell
suspensions were then incubated at 37.degree. C./5% CO.sub.2 for a
designated time. This type of experiment is sometimes called an
"apoptosis time course". Each split was washed once in complete
RPMI media/10% Fetal Bovine Serum (FBS) and resuspended at
1.times.10.sup.6 cells/mL in complete RPMI media with 10% FBS. Flow
tubes were made up by adding one mL designated cell suspension to
which 10 .mu.M compound (24) was added to one mL cell suspension,
incubated at 37.degree. C. for 30 minutes protected from light, and
the SYTOX.RTM. Blue dead cell stain (SYTOX is a registered
trademark of Molecular Probes, Inc.; Eugene, Oreg.) was added as a
dead cell discriminator. Cells were processed using the Becton
Dickinson LSRII Flow Cytometer. A SYTOX.RTM. Blue stain vs compound
(24) stain plot was made and a gate was made on compound (24) stain
positive and SYTOX.RTM. Blue stain negative cells to gate out dead
cells, and to ensure only live cells were analyzed. Fluorescence
from the SYTOX.RTM. Blue stain was collected using 405 nm
excitation laser with 450/50 bandpass and compound (24) stain
fluorescence was collected at 585/42 bandpass using the 488 nm
excitation laser as well as collected at the same 585/42 bandpass
using the 532 nm excitation laser. Collection of 30,000 events
occurred at a flow rate of about 200 events/second. The data was
further analyzed using ModFit LT Flow Cytometry Modeling Software
from Verity Software House, Inc. to look at ratio of G2/G1 and the
CV of G1 phase and the percent sub-G0 population (apoptotic
population).
[0336] Typical cell cycle histograms were demonstrated showing GOG1
phase, S phase, and G2M phase for each control time point with no
sub-G0 population identified. Cell cycle histograms for the cells
treated with Camptothecin demonstrated a population of cells at the
sub-G0 location which begin to show at 3 hours induction and
continue to increase throughout the time course at each time point
afterwards. Further analysis using ModFit Software with an
apoptotic model, showed typical cell cycle staining for control
cells and growing sub-G0 population with the induced cells. This
demonstrated that compound (24) stains live cells for
identification of a sub-G0 population in apoptotic cells which
increases with time of induction. Similar results were obtained
with 532 nm excitation.
[0337] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the scope and concept of the invention.
* * * * *