U.S. patent application number 14/544359 was filed with the patent office on 2015-09-17 for fluorescent dyes.
The applicant listed for this patent is AnaSpec Incorporated. Invention is credited to Zhenjun Diwu, Xiang Guobing, Yi Tang, Jianheng Zhang.
Application Number | 20150259535 14/544359 |
Document ID | / |
Family ID | 36228348 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259535 |
Kind Code |
A1 |
Diwu; Zhenjun ; et
al. |
September 17, 2015 |
Fluorescent dyes
Abstract
Chemically reactive carbocyanine dyes that are intramolecularly
crosslinked between the 1-position and 3'-position, their
bioconjugates and their uses are described. 1,3'-crosslinked
carbocyanines are superior to those of conjugates of spectrally
similar 1,1'-crosslinked or non-crosslinked dyes. The invention
includes derivative compounds having one or more benzo
nitrogens.
Inventors: |
Diwu; Zhenjun; (Sunnyvale,
CA) ; Zhang; Jianheng; (Santa Clara, CA) ;
Tang; Yi; (Sunnyvale, CA) ; Guobing; Xiang;
(Santa Clara, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AnaSpec Incorporated |
Fremont |
CA |
US |
|
|
Family ID: |
36228348 |
Appl. No.: |
14/544359 |
Filed: |
December 29, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13374729 |
Jan 9, 2012 |
8921543 |
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14544359 |
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12583513 |
Aug 20, 2009 |
8093404 |
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13374729 |
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12287071 |
Oct 6, 2008 |
7820783 |
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12583513 |
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11256581 |
Oct 21, 2005 |
7465810 |
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12287071 |
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60521789 |
Jul 2, 2004 |
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Current U.S.
Class: |
540/460 |
Current CPC
Class: |
C09B 23/02 20130101;
G01N 33/533 20130101; C07D 487/14 20130101; C09B 23/06 20130101;
Y10T 436/143333 20150115; C07D 487/22 20130101; C07D 498/22
20130101; G01N 33/52 20130101; C09B 23/0066 20130101 |
International
Class: |
C09B 23/06 20060101
C09B023/06 |
Claims
1. A compound of the following structure: ##STR00067## and its
biological salts, wherein C is a non-conjugated chain of 10-50
linear atoms selected from a group consisting of carbon, oxygen and
nitrogen and wherein C is substituted with substituents selected
from a group consisting of hydrogen, an alkyl having 1 to 20
carbons, a hydroxyl, a carbonyl, and RGM; R.sub.1-R.sub.11 and
R.sub.13-R.sub.14 are selected from a group consisting of hydrogen,
an alkyl having 1 to 20 carbons, an alkoxy having 1-20 carbons, a
trifluoromethyl, a halogen, a methylthio, a sulfonyl, a carbonyl, a
hydroxyl, an amino, a thiol or a RGM; X is O or S; n is 0 to 3;
wherein RGM is an activated ester, acyl halide, aldehyde,
anhydride, carbodiimide, haloacetamide, isocyanate or
maleimide.
2. The compound according to claim 1, wherein C is a non-conjugated
chain of 10 to 50 linear atoms selected from a group consisting of
carbon and nitrogen.
3. The compound according to claim 1, wherein C is substituted with
substituents selected from a group consisting of hydrogen, a
carbonyl and RGM.
4. The compound according to claim 1, wherein R.sub.1-R.sub.11 and
R.sub.13-R.sub.14 are selected from a group consisting of hydrogen,
a halogen, a sulfonyl, a carbonyl or a RGM.
5. The compound according to claim 2, wherein C is substituted with
substituents selected from a group consisting of hydrogen, a
carbonyl and RGM.
6. The compound according to claim 6, wherein R.sub.1-R.sub.11 and
R.sub.13-R.sub.14 are selected from a group consisting of hydrogen,
a halogen, a carbonyl or a RGM.
7. A compound of the following structure: ##STR00068## and its
biological salts, wherein C is a non-conjugated chain of 10-50
linear atoms selected from a group consisting of carbon, oxygen and
nitrogen and wherein C is substituted with substituents selected
from a group consisting of hydrogen, an alkyl having 1 to 20
carbons, a hydroxyl, a carbonyl, and RGM; R.sub.1-R.sub.11 and
R.sub.13-R.sub.16 are selected from a group consisting of hydrogen,
an alkyl having 1 to 20 carbons, an alkoxy having 1-20 carbons, a
trifluoromethyl, a halogen, a methylthio, a sulfonyl, a carbonyl, a
hydroxyl, an amino, a thiol or a RGM; n is 0 to 3; wherein RGM is
an activated ester, acyl halide, aldehyde, anhydride, carbodiimide,
haloacetamide, isocyanate or maleimide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation application of U.S.
patent application Ser. No. 12/583,513, filed Aug. 20, 2009, which
is a divisional application of U.S. patent application Ser. No.
287,071, filed Oct. 6, 2008, issued as U.S. Pat. No. 7,820,783,
which is a divisional application of U.S. patent application Ser.
No. 11/256,581, filed Oct. 21, 2005, issued as U.S. Pat. No.
7,465,810 on Dec. 16, 2008, which claims priority to U.S.
Provisional Patent Application Ser. No. 60/621,789, filed Oct. 25,
2004, the entire disclosures of which are incorporated by reference
into this document.
FIELD OF THE INVENTION
[0002] The invention relates to fluorescent chemicals, including
reactive dyes and dye-conjugates; and to their uses.
BACKGROUND OF THE INVENTION
[0003] Luminescent probes are valuable reagents for the analysis
and separation of molecules and cells and for the detection and
quantification of other materials. A very small number of
luminescent molecules can be detected under optimal circumstances.
Barak and Webb visualized fewer than 50 fluorescent lipid analogs
associated with the LDL reception of cells using a SIT camera, J.
CELL BIOL., 90, 595-604 (1981). Flow cytometry can be used to
detect fewer than 10,000 fluorescein molecules associated with
particles or certain cells (Muirhead, Horan and Poste,
BIOTECHNOLOGY, 3, 337-356 (1985)). Some specific examples of the
application of fluorescent probes are (1) identification and
separation of subpopulations of cells in a mixture of cells by the
techniques of fluorescence flow cytometry, fluorescence-activated
cell sorting and fluorescence microscopy; (2) determination of the
concentration of a substance that binds to a second species (e.g.,
antigen-antibody reactions) in the technique of fluorescence
immunoassay; (3) localization of substances in gels and other
insoluble supports by the techniques of fluorescence staining.
These techniques are described by Herzenberg, et al., "CELLULAR
IMMUNOLOGY" 3rd ed., Chapter 22; Blackwell Scientific
Publications
[0004] (1978); and by Goldman, "FLUORESCENCE ANTIBODY METHODS",
Academic Press, New York, (1968); and by Taylor, et al.,
APPLICATIONS OF FLUORESCENCE IN THE BIOMEDICAL SCIENCES, Alan Liss
Inc., (1986).
[0005] When employing fluorescent dyes for the above purposes,
there are many constraints on the choice of the fluorescent dye.
One constraint is the absorption and emission characteristics of
the fluorescent dye, since many ligands, receptors, and materials
in the sample under test, e.g. blood, urine, cerebrospinal fluid,
will fluoresce and interfere with an accurate determination of the
fluorescence of the fluorescent label. This phenomenon is called
autofluorescence or background fluorescence. Another consideration
is the ability to conjugate the fluorescent dye to ligands and
receptors and other biological and non-biological materials and the
effect of such conjugation on the fluorescent dye. In many
situations, conjugation to another molecule may result in a
substantial change in the fluorescent characteristics of the
fluorescent dye and, in some cases, substantially destroy or reduce
the quantum efficiency of the fluorescent dye. It is also possible
that conjugation with the fluorescent dye will inactivate the
function of the molecule that is labeled. A third consideration is
the quantum efficiency of the fluorescent dyes which should be high
for sensitive detection. A fourth consideration is the light
absorbing capability, or extinction coefficient, of the fluorescent
dyes, which should also be as large as possible. Also of concern is
whether the fluorescent molecules will interact with each other
when in close proximity, resulting in self-quenching. An additional
concern is whether there is non-specific binding of the fluorescent
dyes to other compounds or container walls, either by themselves or
in conjunction with the compound to which the fluorescent dye is
conjugated.
[0006] The applicability and value of the methods indicated above
are closely tied to the availability of suitable fluorescent
compounds. In particular, there is a need for fluorescent
substances that emit in the longer wavelength region (yellow to
near infrared), since excitation of these chromophores produces
less autofluorescence and also multiple chromophores fluorescing at
different wavelengths can be analyzed simultaneously if the full
visible and near infrared regions of the spectrum can be utilized.
Fluorescein, a widely used fluorescent compound, is a useful
emitter in the green region although in certain immunoassays and
cell analysis systems background autofluorescence generated by
excitation at fluorescein absorption wavelengths limits the
detection sensitivity. However, the conventional red fluorescent
label rhodamine has proved to be less effective than
fluorescein.
[0007] Phycobiliproteins have made an important contribution
because of their high extinction coefficient and high quantum
yield. These chromophore-containing proteins can be covalently
linked to many proteins and are used in fluorescence antibody
assays in microscopy and flow cytometry. The phycobiliproteins have
the disadvantages that (1) the protein labeling procedure is
relatively complex; (2) the protein labeling efficiency is not
usually high (typically an average of 0.5 phycobiliprotein
molecules per protein); (3) the phycobiliproteins are natural
products and their preparation and purification are complex; (4)
the phycobiliproteins are expensive; (5) there are at present no
phycobiliproteins available as labeling reagents that fluoresce
further to the red region of the spectrum than allophycocyanine,
which fluoresces maximally at 680 nrn; (6) the phycobiliproteins
are large proteins with molecular weights ranging from 33,000 to
240,000 and are larger than many materials that are desirable to
label, such as metabolites, drugs, hormones, derivatized
nucleotides, and many proteins including antibodies. The latter
disadvantage is of particular importance because antibodies,
avidin, DNA-hybridization probes, hormones, and small molecules
labeled with the large phycobiliproteins may not be able to bind to
their targets because of steric limitations imposed by the size of
the conjugated complex.
[0008] Other techniques involving histology, cytology, immunoassays
would also enjoy substantial benefits from the use of a fluorescent
dye with a high quantum efficiency, absorption and emission
characteristics at longer wavelengths, having simple means for
conjugation and being substantially free of nonspecific
interference.
[0009] Fluorescent compounds are covalently or noncovalently
attached to other materials to impart color and fluorescence.
Brightly fluorescent dyes permit detection or location of the
attached materials with great sensitivity. Certain carbocyanine
dyes have demonstrated utility as labeling reagents for a variety
of biological applications, e.g. U.S. Pat. No. 4,981,977 to
Southwick, et al. (1991); U.S. Pat. No. 5,268,486 to Waggoner, et
al. (1993); U.S. Pat. No. 5,569,587 to Waggoner (1996); U.S. Pat.
No. 5,569,766 to Waggoner, et al. (1996); U.S. Pat. No. 5,486,616
to Waggoner, et al. (1996); U.S. Pat. No. 5,627,027 to Waggoner
(1997); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S. Pat.
No. 5,877,310 to Reddington, et al. (1999); U.S. Pat. No. 6,002,003
to Shen, et al. (1999); U.S. Pat. No. 6,004,536 to Leung, et al.
(1999); U.S. Pat. No. 6,008,373 to Waggoner, et al. (1999); U.S.
Pat. No. 6,043,025 to Minden, et al. (2000); U.S. Pat. No.
6,127,134 to Minden, et al. (2000); U.S. Pat. No. 6,130,094 to
Waggoner, et al. (2000); U.S. Pat. No. 6,133,445 to Waggoner, et
al. (2000); also WO 97/40104, WO 99/51702, WO 01/21624, and EP 1
065 250 A1; and TETRAHEDRON LETT., 41, 9185-88 (2000).
Nevertheless, many carbocyanine dyes are known to share certain
disadvantages, e.g. severe quenching of the fluorescence of
carbocyanine dyes in biopolymer conjugates, e.g. quenching of Cy5
and Cy7 dye variants on conjugates, as discussed by Gruber, et al.,
BIOCONJUGATE CHEM., 11, 696 (2000), and in EP 1 065 250 A1, 0004.
In addition, certain desired sulfoalkyl derivatives of the reactive
carbocyanine dyes are difficult to prepare, as indicated for Cy3
and Cy5 variants by Waggoner and colleagues in BIOCONJUGATE CHEM.,
4, 105, 109 (1993). Cyanine dyes also have a very strong tendency
to self-aggregate (i.e. stack), which can significantly reduce the
fluorescence quantum yields, as described in the extensive review
by Mishra, et al., CHEM. REV., 100, 1973 (2000).
[0010] Another problem with the existing carbocyanine labeling dyes
is the free rotation/vibration of two indolium (or benzothiazolium,
or benzoimidazolium) heads around the middle conjugated double
bonds that significantly reduce their fluorescence intensities (see
Scheme 1). This phenomenon is called `loose belt effect` that is
described in "MODERN MOLECULAR PHOTOCHEMISTRY", Chapters 5 and 6,
University Science Books, Sausalito, Calif., authored by Nicholas
J. Turro (1991).
##STR00001##
This so-called `loose belt effect` can be eliminated by the
crosslinking of the two heads. 1,1'-crosslinking of cyanines is
disclosed by R. Singh, et al. WO 01/02374 (2001), which is supposed
to eliminate the `loose belt effect` described above. However, we
observe that the 1,1'-crosslinking actually causes the decreased
fluorescence quantum yield of dye-protein conjugates compared to
that of non-crosslinked carbocycanineprotein conjugates at the
similar ratios of dye/protein (see FIG. 3). This unfavorable
fluorescence quantum decrease might be caused by the inappropriate
stereochemistry of 1,1'-crosslinking.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Absorption spectra of Cy5 free acid (from Amersham
Biosciences) and Compound 13 in PBS buffer (pH=7.4). Absorption
characteristics of the Compound 13 are similar to those of Cy5,
when present as the free-acid.
[0012] FIG. 2. Fluorescence spectra of Cy5 free acid (from Amersham
Biosciences) and Compound 13 in PBS buffer (pH=7.4, excited at 630
run). Fluorescence characteristics of the Compound 13 are similar
to those of Cy5, when present as the free-acid.
[0013] FIG. 3. Comparison of fluorescence quantum yields of Cy5 SE
and Compound 38 when conjugated to goat anti-rabbit IgG (GAR). The
conjugates are prepared and characterized as described in Examples
58.
[0014] FIG. 4. Comparison of fluorescence quantum yields of Cy5 SE
and Compounds 14 and 38 when conjugated to goat anti-rabbit IgG
(GAR). The conjugates are prepared and characterized as described
in Examples 58.
[0015] FIG. 5. Photostability comparison of Compound 13 (solid
circles) with Cy5 free acid (squares) in PBS buffer (pH 7.4). The
detailed experimental conditions are described in Example 64.
[0016] FIG. 6. Synthesis of a cyanine that has a RGM at
1-position.
[0017] FIG. 7. Synthesis of a cyanine that has a RGM at
3'-position.
[0018] FIG. 8. Synthesis of a cyanine that has a RGM at the
non-conjugated Chain C.
[0019] FIG. 9. Synthesis of a cyanine that has a RGM at the
conjugated double bondbridge.
[0020] FIG. 10. Synthesis of a cyanine that has a RGM at Ring A or
Ring B.
[0021] FIG. 11. Synthesis of a cyanine through intramolecular
coupling.
SUMMARY OF INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS
[0022] We discovered that 1,3'-crosslinking of an carbocyanine dye
unexpectedly mitigates problems discussed in the background section
and results in dye-polymer conjugates that are substantially more
fluorescent on proteins, nucleic acids and other biopolymers, than
conjugates labeled with structurally similar 1,1'-crosslinked
carbocyanine or noncrosslinked dyes (see FIG. 4). The enhanced
fluorescence intensity of dye-biomolecule conjugates of the
invention results in greater assay sensitivity. The increase in
fluorescence quantum yields may result from the reduction of the
ground state aggregation caused by the sterohindrance of 1,3
`-crosslinking of an carbocyanine dye. This intramolecular
1,3`-crosslinking might also reduce the oxidative dimerization of
carbocyanines, and thus decrease their sensitivity to ozone (see T.
Katoh, et al. BULL. CHEM. SOC. JPN., 70, 1109-1114 (1997)). The
increased ozone resistance provides a great advantage for their
applications of the claimed dyes in microarrays. The ozone
sensitivity of carbocyanines has been a serious problem for the
microarray applications of Cy3, Cy5 and their analogs.
##STR00002##
In addition to having more intense fluorescence emission than
structurally similar cyanine dyes at similar wavelengths, and
decreased artifacts in their absorption spectra upon conjugation to
biopolymers, certain embodiments of the invention also have greater
photostability (see FIG. 5) and higher absorbance (extinction
coefficients) at the wavelength(s) of peak absorbance than such
structurally similar dyes. The enhanced photostability might also
be related to the reduction of oxidative dimerization. These
improvements result in significantly greater sensitivity in assays
that use these dyes and their conjugates, while utilizing existing
filters and instrumentation already commercially available for use
with similar dyes such as Cy3, Cy5, Cy5.5 and Cy7.
[0023] Furthermore, the dyes of the invention typically exhibit
absorbance maxima between about 530 nm and about 800 nm, so these
dyes can be selected to match the principal emission lines of the
mercury arc lamp (546 nm), frequency-doubled Nd-Yag laser (532 nm),
Kr-ion laser (568 nm and 647 nm). HeNe laser (543 nm, 594 nm, and
633 nm) or long-wavelength laser diodes (especially 635 nm and
longer). Some dyes of the invention exhibit very long wavelength
excitation (at least 640 nm, but some greater than about 730 nm)
and emission bands (at least 665 nm, and some greater than about
750 nm), so they are particularly useful for samples that are
transparent to infrared wavelengths.
[0024] The present invention comprises reactive 1,3'-crosslinked
carbocyanine dyes and their conjugates. The dyes and dye conjugates
are used to locate or detect the interaction or presence of
analytes or ligands in a sample. Kits incorporating such dyes or
dye conjugates facilitate their use in such methods.
[0025] The dyes of the invention typically have Formula I:
##STR00003##
wherein rings A and B represent the atoms necessary to form a
nitrogen-containing five-membered heterocyclic ring that has zero
to three fused aromatic rings; and each said fused aromatic ring
selected from the group consisting of C, CH, C(alkyl), O, S,
N(aryl) and N(alkyl), and said five-membered ring contains=N(alkyl)
coupled to the bridged and conjugated double bonds, and said
aromatic rings are optionally substituted one or more times by
substituents selected from the group consisting of a hydrogen, an
alkyl having 1-20 carbons, a hydroxy, an alkoxy having 1-20
carbons, a trifluoromethyl, a halogen, a methylthio, a sulfonyl, a
carbonyl, a hydroxy, an amino, a thiol, a sulfate, a phosphonate or
a RGM
[0026] C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, a
hydroxy, an alkoxy having 1-20 carbons, a trifluoromethyl, a
halogen, a methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino,
an alkylamino, an arylamino, a thiol, a sulfite, a phosphonate or a
RGM.
[0027] n is 0 to 3.
[0028] R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are
independently-selected from the group consisting of a hydrogen, an
alkyl having 1-20 carbons, a cycloalkyl having 3-20 carbons, an
aryl, a heteroaryl, an amino, an alkylamino, an arylamino, a thiol
and a RGM.
[0029] RGM is a chemically reactive group described below.
[0030] The dyes of the invention comprise a cyanine dye that
contains: 1) a RGM group; and 2) a bridged and non-conjugated chain
C that intramolecularlly crosslinks position 1 (ring A) with
position 3' (ring B). In one embodiment of the invention, the first
or second ring system is substituted by aside chain at position 1
that contains a RGM group. In another embodiment, the first or
second ring contains a RGM group directly located on the aromatic
rings (A or B). In another embodiment, the bridged methine is
substituted by a side chain that contains a RGM group. In another
embodiment, the bridged and non-conjugated chain C is substituted
by a side chain that contains a RGM group. In another embodiment,
the carbon atom at position 3 or 3' is substituted by a side chain
that contains a RGM group.
[0031] Preferred compounds have at least one substituted indolium
ring system wherein the substituent contains a RGM and a
non-conjugated bridged chain. Other preferred compounds incorporate
at least a charged group (e.g., sulfonate and ammonium moieties) to
increase water solubility. By "sulfo" is meant sulfonic acid, or
salt of sulfonic acid (sulfonate). Similarly, by "carboxy" is meant
carboxylic acid or salt of carboxylic acid. "phosphate", as used
herein, is an ester of phosphoric acid, and includes salts of
phosphate. "phosphonate", as used herein, means phosphoric acid and
includes salts of phosphonate. As used herein, unless otherwise
specified, the alkyl portions of substituents such as alkyl,
alkoxy, arylalkyl, alkylamino, dialkylamino, trialkylammonium, or
perfluoroalkyl are optionally saturated, unsaturated, linear or
branched, and all alkyl alkoxy, alkylamino, and dialkylamino
substituents are themselves optionally further substituted by
carboxy, sulfo, amino, or hydroxy.
[0032] A preferred embodiment is a compound of Formula II:
##STR00004##
wherein C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, an
alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, a
methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol or
a RGM. R.sub.1 to R.sub.16 are a hydrogen, an alkyl having 1-20
carbons, an alkoxy having 1-20 carbons, a trifluoromethyl, a
halogen, a methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino,
a thiol or a RGM; X is O, S, Se, NR.sub.15 or CR.sub.15R.sub.16; n
is 0 to 3.
[0033] Another preferred embodiment is a compound of Formula
III:
##STR00005##
wherein C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, an
alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, a
methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol or
a RGM. R.sub.1 to R.sub.16 are a hydrogen, an alkyl having 1-20
carbons, an alkoxy having 1-20 carbons, a trifluoromethyl, a
halogen, a methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino,
a thiol or a RGM; X is O, S, Se, NR.sub.15, CR.sub.15R.sub.16; n is
0 to 3.
[0034] Another preferred embodiment is a compound of Formula
IV:
##STR00006##
wherein C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, an
alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, a
methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol or
a RGM. R.sub.1 to R.sub.16 are a hydrogen, an alkyl having 1-20
carbons, an alkoxy having 1-20 carbons, trifluoromethyl, a halogen,
a methylthio, a sulfonyl, a carbonyl, a hydroxyl, an amino, a thiol
or a RGM; X is 0, S, Se, NR.sub.15, CR.sub.15R.sub.16; n is 0 to
3.
[0035] Another preferred embodiment is a compound of Formula V:
##STR00007##
wherein C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, an
alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, a
methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol or
a RGM. R.sub.1 to R.sub.16 are a hydrogen, an alkyl having 1-20
carbons, an alkoxy having 1-20 carbons, a trifluoromethyl, a
halogen, a methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino,
a thiol or a RGM; X is O, S, Se, NR.sub.15, CR.sub.15R.sub.16; n is
0 to 3.
[0036] The length of the conjugated polymethine bridge between the
two ring systems greatly affects the dye's absorption and emission
properties. Each of R.sub.1, R.sub.2, R.sub.3, when present, is
independently a hydrogen, a fluoro, a chloro, an alkyl having 1-6
carbons, an alkoxy having 1-6 carbons, an aryloxy, a
N-heteroaromatic moiety, or an iminium ion. Alternatively, two
substituents R.sub.1/R.sub.2, R.sub.2/R.sub.3, when taken in
combination, form a 4-, 5-, or 6-membered saturated or unsaturated
hydrocarbon ring that is unsubstituted or is optionally substituted
one or more times by a saturated or unsaturated alkyl having 1-6
carbons, a halogen, or a carbonyl oxygen. Typically, each of
R.sub.1, R.sub.2 and R.sub.3, when present, is a hydrogen. Where
one of R.sub.1, R.sub.2 and R.sub.3 is a nonhydrogen, it is
typically the substituent on the center carbon of bridged and
conjugated double bonds. Similarly, where bridged and conjugated
double bonds incorporate a 4-, 5-, or 6-membered ring, it typically
occurs at the center of the conjugated bridge moiety.
[0037] Additionally, selection of the A, B and X moieties may also
significantly affect the dye's absorption and fluorescence emission
properties. A and B optionally the same or different, and spectral
properties of the resulting dye may be tuned by careful selection
of A and B. In one embodiment, X is CR.sub.15R.sub.16 where
R.sub.15 and R.sub.16 are a hydrogen or an alkyl group having 1-30
carbons, that is optionally substituted one or more times by a
hydroxy, a carboxy, a sulfo, an amino, an alkylamino having 1-6
carbons or dialkylarnino having 2-20 carbons. Alternatively,
R.sub.15 and R.sub.16 in combination complete a five or six
membered saturated or unsaturated ring that is optionally
substituted by a RGM. Preferably R.sub.15 and R.sub.16 are
independently an alkyl with 1-6 carbon atoms that are unsubstituted
or are substituted once by a hydroxy, a sulfo, a carboxy or an
amino. In one aspect of the invention, R.sub.15 and R.sub.16 are
alkyls having 1-6 carbons, preferably methyls. In another aspect of
the invention, one of R.sub.15 and R.sub.16 is a methyl, and the
other is an alkyl having 1-10 carbons that is substituted by a
carboxy or by a sulfo or by a hydroxy, or by a RGM.
[0038] Incorporation of one or more non-hydrogen substituents on
the fused rings can be used to fine tune the absorption and
emission spectrum of the resulting dye.
[0039] Another preferred embodiment of the invention is a compound
of Formula VI
##STR00008##
wherein C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, an
alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, a
methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol or
a RGM. R.sub.1 to R.sub.27 are a hydrogen, an alkyl having 1-20
carbons, an alkoxy having 1-20 carbons, a trifluoromethyl, a
halogen, a methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino,
a thiol or a RGM; X is 0, S, Se, NR.sub.15 or CR.sub.15R.sub.16; n
is 0 to 3.
[0040] Another preferred embodiment of the invention is a compound
of Formula VII
##STR00009##
wherein C is a non-conjugated chain of 10-50 linear atoms selected
from carbon, nitrogen, oxygen, phosphorus and sulfur that are
further substituted by a hydrogen, an alkyl having 1-20 carbons, an
alkoxy having 1-20 carbons, a trifluoromethyl, a halogen, a
methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino, a thiol or
a RGM. R.sub.1 to R.sub.23 are a hydrogen, an alkyl having 1-20
carbons, an alkoxy having 1-20 carbons, a trifluoromethyl, a
halogen, a methylthio, a sulfonyl, a carbonyl, a hydroxy, an amino,
a thiol or a RGM; X is O, S, Se, NR.sub.15 or CR.sub.15R.sub.16; n
is 0 to 3.
[0041] In one aspect of the invention, one or two or more of
R.sub.1 to R.sub.27 is an amino, a carboxy and a thiol according to
Formula I. In one aspect of the invention, the carbocyanine dyes of
the invention are sulfonated one or more times.
[0042] In addition, the dyes of the invention are substituted by
one or more chemically reactive groups (RGM) or conjugated
substances as described below. In a preferred embodiment, the dye
of the invention is substituted by only one RGM.
[0043] Many embodiments of the compounds of the invention possess
an overall electronic charge. It is to be understood that when such
electronic charges are shown to be present, they are balanced by
the presence of appropriate counterions, which may or may not be
explicitly identified. A biologically compatible counterion, which
is preferred for some applications, is not toxic in biological
applications, and does not have a substantially deleterious effect
on biomolecules. Where the compound of the invention is positively
charged, the counterion is typically selected from, but not limited
to, chloride, bromide, iodide, sulfate, alkanesulfonate,
arylsulfonate, phosphate, perchlorate, tetrafluoroborate,
tetraarylboride, nitrate and anions of aromatic or
aliphatic-carboxylic acids. Where the. compound of the invention is
negatively charged, the counterion is typically selected from, but
not limited to, alkali metal ions, alkaline earth metal ions,
transition metal ions, ammonium or substituted ammonium or
pyridinium ions. Preferably, any necessary counterion is
biologically compatible, is not toxic as used, and does not have a
substantially deleterious effect on biomolecules. Counterions are
readily changed by methods well known in the art, such as
ion-exchange chromatography, or selective precipitation.
[0044] It is to be understood that--the dyes of the invention have
been drawn in one or another particular electronic resonance
structure. Every aspect of the instant invention applies equally to
dyes that are formally drawn with other permitted resonance
structures, as the electronic charge on the subject dyes is
delocalized throughout the dye itself.
[0045] In one embodiment of the invention, the dye contains at
least one L-RGM, where RGM is the reactive group that is attached
to the dye by a covalent linkage L. In certain embodiments, the
covalent linkage attaching the dye to RGM contains multiple
intervening atoms that serve as a spacer. The dyes with a RGM label
a wide variety of organic or inorganic substances that contain or
are modified to contain functional groups with suitable reactivity,
resulting in chemical attachment of the conjugated substance. As
used herein, "reactive group moiety (RGM)" means moiety on the
compound that is capable of chemically reacting with a functional
group on a different compound to form a covalent linkage. Typically
the reactive group is an electrophile or nucleophile that can form
a covalent linkage through exposure to the corresponding functional
group that is a nucleophile or electrophile, respectively.
Alternatively, the reactive group is a photoactivatable group, and
becomes chemically reactive only after illumination with light of
an appropriate wavelength. Typically, the conjugation reaction
between the reactive dye and the substance to be conjugated results
in one or more atoms of the reactive group RGM to be incorporated
into a new linkage L attaching the dye to the conjugated substance.
Selected examples of reactive groups and linkages are shown in
Table 1 where the reaction of an electrophilic group and a
nucleophilic group yields a covalent linkage.
TABLE-US-00001 TABLE 1 Examples of RGM groups that are used for
preparing covalent linkages: Electrophilic Group Nucleophilic Group
Resulting Conjugate 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 carbo 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
thioethers 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 haloacetarnides thiols
thioethers haloplatinate amino platinum complex haloplatinate
heterocycle platinum complex haloplatinate thiol platinum complex
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers imido esters amines/anilines
amidines isocyanates amines/anilines ureas isocyanates
alcohols/phenols urethanes isothiocyanates amines/anilines
thioureas maleimides thiols thioethers phosphoramidites alcohols
phosphite esters silyl halides alcohols silyl ethers sulfonate
esters amines/anilines alkyl amines sulfonate esters thiols.
thioethers sulfonate esters carboxylic acids esters sulfonate
esters alcohols ethers sulfonyl halides amines/anilines
sulfonamides sulfonyl halides phenols/alcohols sulfonate esters
*Activated esters, as understood in the art, generally have the
formula -COL, where L is a good leaving group (e.g. succinimidyloxy
(-ONC.sub.4H.sub.40.sub.2) sulfosuccinimidyloxy
(-ONC.sub.4H.sub.3O.sub.2, -SO.sub.3H), -1-oxybenzotriazoly1
(-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 foim an anhydride or
mixed anhydride -OCOAlk or -OCN(Alk.sub.1)NH(Alk.sub.2), where
Alk.sub.1 and Alk.sub.2 , which may be the same or different, are
C.sub.1-C.sub.20 alkyl , C.sub.1-C.sub.20 perfluoroalkyl, or
C.sub.1-C.sub.20 alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or
N-morpholinoethyl). **Acyl azides can also rearrange to
isocyanates.
[0046] Choice of the reactive group used to attach the dye to the
substance to be conjugated typically depends on the functional
group on the substance to be conjugated and the type or length of
covalent linkage desired. The types of functional groups typically
present on the organic or inorganic substances include, but are not
limited to, amines, amides, thiols, alcohols, phenols, aldehydes,
ketones, phosphonates, imidazoles, hydrazines, hydroxylamines,
disubstituted amines, halides, epoxides, carboxylate esters,
sulfonate esters, purines, pyrimidines, carboxylic acids, olefinic
bonds, or a combination of these groups. A single type of reactive
site may be available on the substance (typical for
polysaccharides), or a variety of sites may occur (e.g. amines,
thiols, alcohols, phenols), as is typical for proteins. A
conjugated substance may be conjugated to more than one dye, which
may be the same or different, or to a substance that is
additionally modified by a hapten, such as biotin. Although some
selectivity can be obtained by careful control of the reaction
conditions, selectivity of labeling is best obtained by selection
of an appropriate reactive dye.
[0047] Typically, RGM will react with an amine, a thiol, an
alcohol, an aldehyde or a ketone. Preferably RGM reacts with an
amine or a thiol functional group. In one embodiment, RGM is an
acrylamide, a reactive amine (including a cadaverine or
ethylenediamine), an activated ester of a carboxylic acid
(typically a succinimidyl ester of a carboxylic acid), an acyl
azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride,
an aniline, an aryl halide, an azide, an aziridine, a boronate, a
carboxylic acid, 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. Nos. 5,580,990; 5,714,327;
5,985,566.
[0048] Where the reactive group is a photoactivatable group, such
as an azide, diazirinyl, azidoaryl, or psoralen derivative, the dye
becomes chemically reactive only after illumination with light of
an appropriate wavelength. Where RGM is an activated ester of a
carboxylic acid, the reactive dye is particularly useful for
preparing dye-conjugates of proteins, nucleotides,
oligonucleotides, or haptens. Where RGM is a maleimide or
haloacetamide the reactive dye is particularly useful for
conjugation to thiol-containing substances. Where RGM is a
hydrazide, the reactive dye is particularly useful for conjugation
to periodate-oxidized carbohydrates and glycoproteins, and in
addition is an aldehyde-fixable polar tracer for cell
microinjection. Preferably, RGM is a carboxylic acid, a
succinimidyl ester of a carboxylic acid, a haloacetamide, a
hydrazine, an isothiocyanate, a maleimide group, an aliphatic
amine, a perfluorobenzamido, an azidoperfluorobenzamido group, or a
psoralen. More preferably, RGM is a succinimidyl ester of a
carboxylic acid, a maleimide, an iodoacetamide, or a reactive
platinum complex. Based on the above-mentioned attributes, the
appropriate reactive dyes of the invention are selected for the
preparation of the desired dye-conjugates, whose advantageous
properties make them useful for a wide variety of applications.
Particularly useful dye conjugates include, among others,
conjugates where substrate is a peptide, a nucleotide, an antigen,
a steroid, a vitamin, a drug, a hapten, a metabolite, a toxin, an
environmental pollutant, an amino acid, a protein, a nucleic acid,
a nucleic acid polymer, a carbohydrate, a lipid, an ion-complexing
moiety, a glass or a non-biological polymer. Alternatively,
substrate is a cell, a cellular system, a cellular fragment, or a
subcellular particle (e.g. inter alia), a virus particle, a
bacterial particle, a virus component, a biological cell (such as
animal cell, plant cell, bacteria, yeast, or protist), or a
cellular component. Reactive dyes typically label functional groups
at the cell surface, in cell membranes, organelles, or
cytoplasm.
[0049] Typically substrate is an amino acid, a peptide, a protein,
a tyrainine, a polysaccharide, an ion-complexing moiety, a
nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a
hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a
polymer, a polymeric microparticle, a biological cell or virus.
More typically, substrate is a peptide, a protein, a nucleotide, an
oligonucleotide, or a nucleic acid. When conjugating dyes of the
invention to such biopolymers, it is possible to incorporate more
dyes per molecule to increase the fluorescent signal. For example,
it is possible to incorporate at least three molecules of such dyes
per molecule of antibody without loss of total fluorescence,
whereas fluorescence of the spectrally comparable Cy5 (wherein n=2)
is strongly quenched when greater than approximately two Cy5 dyes
are incorporated per antibody. These results confirm problems with
Cy5 conjugates reported by others, e.g. BIOCONJUGATE CHEM., 11, 696
(2000). The optimally labeled conjugates of the invention are
typically much more fluorescent than conjugates of the Cy5 dye or
1,1'-crosslinked Cy5 at the same antibody concentration.
[0050] In one embodiment, substrate is an amino acid (including
those that are protected or are substituted by phosphonates,
carbohydrates, or C.sub.1 to C.sub.25 carboxylic acids), or is a
polymer of amino acids such as a peptide or protein. Preferred
conjugates of peptides contain at least five amino acids, more
preferably 5 to 36 amino acids. Preferred peptides include, but are
not limited to, neuropeptides, cytokines, toxins, protease
substrates, and protein kinase substrates. Preferred protein
conjugates include enzymes, antibodies, lectins, glycoproteins,
histones, albumins, lipoproteins, avidin, streptavidin, protein A,
protein G, phycobiliproteins and other fluorescent proteins,
hormones, toxins, chemokines and growth factors. In one preferred
aspect, the conjugated protein is a phycobiliprotein, such as
allophycocyanin, phycocyanin, phycoerythrin, allophycocyanin B,
B-phycoerythrin, and phycoerythrocyanin, (for example, see U.S.
Pat. No. 5,714,386 to Roederer (1998)). Particularly preferred are
conjugates of R-phycoerythrin and of allophycocyanin with selected
dyes of the invention that serve as excited-state energy acceptors
or donors. In these conjugates, excited state energy transfer
results in long wavelength fluorescence emission when excited at
relatively short wavelengths.
[0051] In one aspect of the invention, substrate is a conjugated
substance that is an antibody (including intact antibodies,
antibody fragments, and antibody sera, etc.), an amino acid, an
angiostatin or endostatin, an avidin or streptavidin, a biotin
(e.g. an amidobiotin, a biocytin, a desthiobiotin, etc.), a blood
component protein (e.g. an albumin, a fibrinogen, a plasminogen,
etc.), a dextran, an enzyme, an enzyme inhibitor, an IgG-binding
protein (e.g. a protein A, protein G, protein A/G, etc.), a
fluorescent protein (e.g. a phycobiliprotein, an aequorin, a green
fluorescent protein, etc.), a growth factor, a hormone, a lectin
(e.g. a wheat germ agglutinin, a conconavalin A, etc.), a
lipopolysaccharide, a metal-binding protein (e.g. a calmodulin,
etc.), a microorganism or portion thereof (e.g. a bacteria, a
virus, a yeast, etc.), a neuropeptide and other biologically active
factors (e.g. a dermorphin, a deltropin, an endomorphin, an
endorphin, a tumor necrosis factor etc.), a non-biological
microparticle (e.g. of ferrofluid, gold, polystyrene, etc.), a
nucleotide, an oligonucleotide, a peptide toxin (e.g. an apamin, a
bungarotoxin, a phalloidin, etc.), a phospholipid-binding protein
(e.g. an annexin, etc.), a small-molecule drug (e.g. a
methotrexate, etc.), a structural protein (e.g. an actin, a
fibronectin, a laminin, a microtubule-associated protein, a tublin,
etc.), or a tyramide.
[0052] In another embodiment, substrate is a nucleic acid base,
nucleoside, nucleotide or a nucleic acid polymer, including those
that are modified to possess an additional linker or spacer for
attachment of the dyes of the invention, such as an alkynyl linkage
(U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat. No.
4,711,955), or a heteroatomsubstituted linker (U.S. Pat. No.
5,684,142) or other linkage. In another embodiment, the conjugated
substance is a nucleoside or nucleotide analog that links a purine
or pyrimidine base to a phosphate or polyphosphate moiety through a
noncyclic spacer. In another embodiment, the dye is conjugated to
the carbohydrate portion of a nucleotide or nucleoside, typically
through a hydroxyl group but additionally through a thiol or amino
group (U.S. Pat. Nos. 5,659,025; 5,668,268; 5,679,785). Typically,
the conjugated nucleotide is a nucleoside triphosphate or a
deoxynucleoside triphosphate or a dideoxynucleoside triphosphate.
Incorporation of methylene moieties or nitrogen or sulfur
heteroatoms into the phosphate or polyphosphate moiety is also
useful. Nonpurine and nonpyrimidine bases such as 7-deazapurines
(U.S. Pat. No. 6,150,510) and nucleic acids containing such bases
can also be coupled to dyes of the invention. Nucleic acid adducts
prepared by reaction of depurinated nucleic acids with amine,
hydrazide or hydroxylamine derivatives provide an additional means
of labeling and detecting nucleic acids, e.g. "A method for
detecting abasic sites in living cells: age-dependent changes in
base excision repair." Atamna H, Cheung I, Ames B N. PROC. NATL.
ACAD. SCI. U.S.A. 97, 686-691 (2000).
[0053] Preferred nucleic acid polymer conjugates are labeled,
single- or multi-stranded, natural or synthetic DNA or RNA, DNA or
RNA oligonucleotides, or DNA/RNA hybrids, or incorporate an unusual
linker such as morpholine derivatized phosphates, or peptide
nucleic acids such as N-(2-aminoethyl)glycine units. When the
nucleic acid is a synthetic oligonucleotide, it typically contains
fewer than 50 nucleotides, more typically fewer than 25
nucleotides. Conjugates of peptide nucleic acids (PNA) (Nielsen, et
al. U.S. Pat. No. 5,539,082) may be preferred for some applications
because of their generally faster hybridization rates.
[0054] In one embodiment, the conjugated oligonucleotides of the
invention are aptamers for a particular target molecule, such as a
metabolite, dye, hapten, or protein. That is, the oligonucleotides
have been selected to bind preferentially to the target molecule.
Methods of preparing and screening aptamers for a given target
molecule have been previously described and are known in the art
[for example, U.S. Pat. No. 5,567,588 to Gold (1996)].
[0055] In another embodiment, substrate is a carbohydrate that is
typically a polysaccharide, such as a dextran, heparin, glycogen,
amylopectin, mannan, inulin, starch, agarose and cellulose.
Alternatively, the carbohydrate is a polysaccharide that is a
lipopolysaccharide. Preferred polysaccharide conjugates are
dextran, or lipopolysaccharide conjugates.
[0056] Conjugates having an ion-complexing moiety serve as
indicators for calcium, sodium, magnesium, zinc, potassium, or
other biologically important metal ions. Preferred ion-complexing
moieties are crown ethers (U.S. Pat. No. 5,405,975); derivatives of
1,2-bis(2-aminophenoxyethane)-N,N,M,N.sup.t-tetraacetic acid (BAPTA
chelators; U.S. Pat. Nos. 5,453,517; 5,516,911 and 5,049,673);
derivatives of 2-carboxymethoxyaniline-N,N-diacetic acid (APTRA
chelators; AM. J. PHYSIOL., 256, C540 (1989)); or pyridine- and
phenanthroline-based metal ion chelators (U.S. Pat. No. 5,648,270);
or derivatives of nitrilotriacetic acid, see e.g. "Single-step
synthesis and characterization of biotinylated nitrilotriacetic
acid, a unique reagent for the detection of histidine-tagged
proteins immobilized on nitrocellulose", McMahan S A and Burgess R
R, ANAL. BIOCHEM., 236, 101-106 (1996). Preferably, the
ion-complexing moiety is a crown ether chelator, a BAPTA chelator,
an APTRA chelator or a derivative of nitrilotriacetic acid.
[0057] Other conjugates of non-biological materials include
dye-conjugates of organic or inorganic polymers, polymeric films,
polymeric wafers, polymeric membranes, polymeric particles, or
polymeric microparticles (magnetic and non-magnetic microspheres);
iron, gold or silver particles; conducting and non-conducting
metals and non-metals; and glass and plastic surfaces and
particles. Conjugates are optionally prepared by copolymerization
of a dye that contains an appropriate functionality while preparing
the polymer, or by chemical modification of a polymer that contains
functional groups with suitable chemical reactivity. Other types of
reactions that are useful for preparing dye-conjugates of polymers
include catalyzed polymerizations or copolymerization of alkenes
and reactions of dienes with dienophiles, transesterifications or
transaminations. In another embodiment, the conjugated substance is
a glass or silica, which may be formed into an optical fiber or
other structure. In one embodiment, conjugates of biological
polymers Such as peptides, proteins, oligonucleotides, nucleic acid
polymers are also labeled with at least a second luminescent dye,
which is optionally an additional dye of the present invention, to
form an energy-transfer pair. In some aspects of the invention, the
labeled conjugate functions as an enzyme substrate, and enzymatic
hydrolysis disrupts the energy transfer. In another embodiment of
the invention, the energy-transfer pair that incorporates a dye of
the invention is conjugated to an oligonucleotide that displays
efficient fluorescence quenching in its hairpin conformation [the
so-called "molecular beacons" of Tyagi, et al., NATURE
BIOTECHNOLOGY, 16, 49 (1998)] or fluorescence energy transfer.
[0058] The preparation of dye conjugates using reactive dyes is
well documented, e.g. Hermanson G T, BIOCONJUGATE TECHNIQUES,
Academic Press, New York (1996); Haugland R P, METHODS MOL. BIOL.,
45; 205-21 (1995); and Brinkley, BI00014.TUGATE CHEM., 3, 2 (1992).
Conjugates typically result from mixing appropriate reactive dyes
and the substance to be conjugated in a suitable solvent in which
both are soluble. The majority of the dyes of the invention are
readily soluble in aqueous solutions, facilitating conjugation
reactions with most biological materials. For those reactive dyes
that are photoactivated, conjugation requires illumination of the
reaction mixture to activate the reactive dyes.
[0059] Synthesis
[0060] Synthesis of the cyanine dyes of the invention depends on
initial preparation of certain key intermediates. The intermediates
have the following general structures (for simplicity, all but a
few of the possible substituents are shown as hydrogen):
##STR00010##
[0061] These basic structures are optionally further substituted,
during or after synthesis, to give the corresponding dye
substituents as defined above. For carbocyanines, the novel key
intermediates are readily synthesized by a reaction that is
analogous to a Fischer indole synthesis (see Sundberg R J, THE
CHEMISTRY OF INDOLES, Organic chemistry, a series of monographs,
1970, Academic Press). The typical synthesis of different
substituted carbocyanines is illustrated in FIGS. 6-11.
[0062] Synthesis of the cyanine dyes of the invention, where RGM is
at the 3-position of the indolium and imidazolium, depends on
initial preparation of key inteiniediate IM 2, Licha, et al., U.S.
Pat. No. 6,083,485 (2000) described a typical synthesis of
intermediate IM 2. These basic structures are optionally further
substituted, during or after synthesis, to give the corresponding
dye substituents as defined above. The novel key intermediates are
readily synthesized by a reaction that is analogous to a Fischer
indole synthesis or through the condensations of phenylendiamine
with a carbonyl compound. The typical total synthesis of
3-RGM-substituted carbocyanines is illustrated in FIG. 7.
[0063] Synthesis of the cyanine dyes of the invention, where
attachment is at the bridged and non-conjugated chain C, is either
through the initial preparation of key intermediate IM 4 or through
the modification of the disclosed procedures described for the
synthesis of 1,1'-crosslinked carbocyanines (WO 01/02374 to Singh,
et al). The typical total synthesis of carbocyanines with RGM on
the non-conjugated chain C is illustrated in FIGS. 8 and 11.
[0064] Synthesis of the cyanine dyes of the invention, where
attachment is at the bridged and conjugated double bonds, depends
on initial preparation of certain key bridged intermediates such as
IM 5. For example, N,N'-diphenylformamidine, triethylorthoformate
malonaldehyde bis(phenylimine) hydrochloride,
1,1,3-trimethoxypropane, 1,1,3,3-tetramethoxypropane and
glutaconaldehyde dianil monochloride are the well-known bridged
intermediates used in the synthesis of carbocycanines. More
examples of appropriate carbocyanines that have bridged and
conjugated double bonds have been previously described in the
literature of U.S. Pat. No. 5,831,098 to Ollmann, Jr (1998); U.S.
Pat. No. 6,086,737 to Patonay, et al. (2000); U.S. Pat. No.
6,048,982 to Waggoner (2000); and U.S. Pat. No. 5,453,505 to Lee,
et al. (1995); U.S. Pat. No. 5,639,874 to Middendorf, et al.
(1997); U.S. Pat. No. 3,864,644 to Lincoln, et al. (1975); U.S.
Pat. No. 4,011,086 to Simson (1977). Typically, each of R.sub.1,
R.sub.2 and R.sub.3 in Formula I, when present, is hydrogen. Where
one of R.sub.1, R.sub.2 and R.sub.3 is nonhydrogen, it is typically
the substituent on the center carbon of BRIDGE. Similarly, where
bridged incorporates a 4-, 5-, or 6-membered ring, it typically
occurs at the center of the bridged moiety. The typical total
synthesis of carbocyanines substituted at the bridged and
conjugated carbon atoms with RGM is illustrated in FIG. 9.
##STR00011##
[0065] For the synthesis of carbocyanines, an appropriately
substituted aryl hydrazine (for simplicity, all but a few of the
possible substituents are shown as hydrogen), which is typically an
appropriately substituted phenylhydrazine, is reacted with an
appropriately substituted methyl ketone to yield a
3,3-disubstituted 2-methylindole derivative (see Scheme 3). It is
particularly suitable to utilize a sulfonated phenylhydrazine
derivative or a sulfonated naphthylhydrazine derivative to increase
the solubility of the final dye. The
3,3-disubstituted-2-methylindole is then quatemized on the nitrogen
atom to an indolium derivative with an alkylating agent that is
typically an alkyl halide such as ethyl iodide, an alkylsulfonate
such as methyl p-toluenesulfonate or a cyclic sulfonate such as
propanesultone or butanesultone. Typically, the key indolium or
benzoindolium intermediates are sulfonated one or more times before
or after quaternization and subsequent condensation with the
benzazolium moiety and polymethine moiety to form the subject dyes.
Variations on these methods are well known in the art that yield
substituents on the polymethine bridge or on the indolium or
benzolium portion of the dye precursor.
[0066] The azacarbocyanine dyes of the present invention can be
analogously synthesized. [for example, see Leung W, et al., WO
02/26891; Brooker, et at, J. AM. CHEM. SOC., 64,199 (1942); Heravi,
et al., INDIAN J. CHEM., 36B, 1025 (1997); Smith, et al. SULFUR
LETTERS, 17, 197 (1994); Chu-Moyer, et al. J. ORG. CHEM., 60, 5721
(1995); Turner, J. ORG. CHEM., 48, 3401 (1983); Couture, et al. J.
HETEROCYCLIC CHEM., 24, 1765 (1987); Petrie, et al. J. HETEROCYCLIC
CHEM, 14, 1045, (1977); Barlin, et al. AUST. J. CHEM., 37, 1729
(1984); Saikachi et al. CHEM. & PHARM. BULL., 9, 941 (1961);
Barlin, AUST. J. CHEM., 36, 983 (1983); Foye, et al., J. PHARM.
SCI., 64, 1371 (1975); Khanna, et al. J. ORG. CHEM., 60, 960
(1995)); British Patent No. 870,753 to Ficken, et al. (1961);
Ficken, et al., "DIAZAINDENES AND THEIR QUANTERNARY SALTS-Part I",
pp 3202-3212 (1959); Ficken, et al., "DIAZAINDENES AND THEIR
QUANTERNARY SALTS-Part II", pp 584-588 (1961)1. In general, the
synthesis of these dyes requires three precursors: the appropriate
benzazolium or azabenzazolium salt (the "A" and "B" moieties), and
a source for the polymethine spacer. Typically each component is
selected so as to incorporate the appropriate chemical
substituents, or functional groups (e.g. RGM) that can be converted
to the appropriate substituents. The chemistry that is required to
prepare and combine these precursors so as to yield any of the
subject derivatives is generally well understood by one skilled in
the art.
[0067] It is recognized that there are many possible variations
that may yield equivalent results. The substituents on the aromatic
carbons of the azabenzazolium moiety are typically incorporated in
the parent aza- or polyazabenzazole molecule prior to
quaternization with an alkylating agent. However, such substituents
may also be incorporated during the synthesis of the azabenzazole
moiety. Alkyl, alkoxy, carboxyl, and halogen substituents at
aromatic carbons are typically already present as substituents on
the benzazole or azabenzazole precursors, or on compounds that are
readily converted to such precursors using methods well-known in
the art. Sulfonic acid groups are typically introduced on the
precursors prior to condensation of the cyanine dye [for example,
see U.S. Pat. No. 5,767,287 to Bobrow, et al. (1998)]. Aminoalkyl
groups typically contain by a protecting group when they are first'
introduced, typically by substitution onto the benzazole or
azabenzazole precursor. The protecting group is then removed after
condensation of the cyanine dye. Aromatic amino groups are
typically preparedvia the reduction of a nitro substituted
benzazolium precursor, which in turn is prepared by the nitration
of the benzazole precursor.
[0068] The methods for synthesis of dyes that contain a variety of
reactive groups such as those described in Table 1 are well
documented in the art. Particularly useful are amine-reactive dyes
such as "activated esters" of carboxylic acids, which are typically
synthesized by coupling a carboxylic acid to a relatively acidic
"leaving group". Other preferred amine-reactive groups include
sulfonyl halides, which are prepared from sulfonic acids using a
halogenating agent such as PCl.sub.5 or POCl.sub.3; halotriazines,
which are prepared by the reaction of cyanuric halides with amines;
and isocyanates or isothiocyanates, which are prepared from amines
and phosgene or thiophosgene, respectively.
[0069] Dyes containing amines and hydrazides are particularly
useful for conjugation to carboxylic acids, aldehydes and ketones.
Most often these are synthesized by reaction of an activated ester
of a carboxylic acid or a sulfonyl halide with a diamine, such as
cadaverine, or with a hydrazine. Alternatively, aromatic amines are
commonly synthesized by chemical reduction of a nitroaromatic
compound. Amines and hydrazines are particularly useful precursors
for synthesis of thiol-reactive haloacetamides or maleimides by
standard methods.
[0070] Nucleosides and nucleotides labeled with dyes of the
invention are particularly useful for some applications of nucleic
acid labeling. The use of carbocyanine-amidites for labeling
nucleotides and nucleosides have been previously described [U.S.
Pat. No. 5,986,086 to Brush, et al. (1999); U.S. Pat. No. 5,808,044
to Brush, et al. (1998); U.S. Pat. No. 5,556,959 to Brush, et al.
(1996)].
[0071] Applications and Methods of Use
[0072] In one aspect of the invention, the dye compounds of the
invention are used to directly stain or label a sample so that the
sample can be identified or quantitated. For instance, such dyes
may be added as part of an assay for a biological target analyte,
as a detectable tracer element in a biological or non-biological
fluid; or for such purposes as photodynamic therapy of tumors, in
which a dyed sample is irradiated to selectively destroy tumor
cells and tissues; or to photoablate arterial plaque or cells,
usually through the photosensitized production of singlet oxygen.
In one preferred embodiment, dye conjugate is used to stain a
sample that comprises a ligand for which the conjugated substance
is a complementary member of a specific binding pair (e.g. Table
2).
[0073] Typically, the sample is obtained directly from a liquid
source or as a wash from a solid material (organic or inorganic) or
a growth medium in which cells have been introduced for culturing,
or a buffer solution in which cells have been placed for
evaluation. Where the sample comprises cells, the cells are
optionally single cells, including microorganisms, or multiple
cells associated with other cells in two or three dimensional
layers, including multicellular organisms, embryos, tissues,
biopsies, filaments, biofilms, etc.
[0074] Alternatively, the sample is a solid, optionally a smear or
scrape or a retentate removed from a liquid or vapor by filtration.
In one aspect of the invention, the sample is obtained from a
biological fluid, including separated or unfiltered biological
fluids such as urine, cerebrospinal fluid, blood, lymph fluids,
tissue homogenate, interstitial fluid, cell extracts, mucus,
saliva, sputum, stool, physiological secretions or other similar
fluids. Alternatively, the sample is obtained from an environmental
source such as soil, water, or air; or from an industrial source
such as taken from a waste stream, a water source, a supply line,
or a production lot.
TABLE-US-00002 TABLE 2 Representative specific binding pairs
Antigen Antibody Biotin Anti-biotin or avidin or streptavidin or
neutravidin IgG* Protein A or protein G or anti-IgG antibody Drug
Drug receptor Toxin Toxin Carbohydrate Lectin.or carbohydrate
receptor Peptide Peptide receptor Nucleotide Complimentary
nucleotide Protein Protein receptor Enzyme substrate Enzyme DNA
(RNA) aDNA (aRNA)** Hormone Hormone receptor Psoralen Nucleic acid
Target molecule RNA or DNA aptamer Ion Ion chelator *IgG is an
immunoglobulin; **aDNA and aRNA are the antisense (complementary)
strands used for hybridization
[0075] In yet another embodiment, the sample is present on or in
solid or semi-solid matrix. In one aspect of the invention, the
matrix is a membrane. In another aspect, the matrix is an
electrophoretic gel, such as is used for separating and
characterizing nucleic acids or proteins, or is a blot prepared by
transfer from an electrophoretic gel to a membrane. In another
aspect, the matrix is a silicon chip or glass slide, and the
analyze of interest has been immobilized on the chip or slide in an
array (e.g. the sample comprises proteins or nucleic acid polymers
in a microarray). In yet another aspect, the matrix is a microwell
plate or microfluidic chip, and the sample is analyzed by automated
methods, typically by various methods of high-throughput screening,
such as drug screening.
[0076] The dye compounds of the invention are generally utilized by
combining a dye compound of the invention as described above with
the sample of interest under conditions selected to yield a
detectable optical response. The term "dye compound" is used herein
to refer to all aspects of the claimed dyes, including both
reactive dyes and dye conjugates. The dye compound typically forms
a covalent or non-covalent association or complex with an element
of the sample, or is simply present within the bounds of the sample
or portion of the sample. The sample is then illuminated at a
wavelength selected to elicit the optical response. Typically,
staining the sample is used to determine a specified characteristic
of the sample by further comparing the optical response with a
standard or expected response.
[0077] A detectable optical response means a change in, or
occurrence of, an Opticarsignal that is detectable either by
observation or instrumentally. Typically the detectable response is
a change in fluorescence, such as a change in the intensity,
excitation or emission wavelength distribution of fluorescence,
fluorescence lifetime, fluorescence polarization, or a combination
thereof. The degree and/or location of staining, compared with a
standard or expected response, indicates whether and to what degree
the sample possesses a given characteristic. Some dyes of the
invention may exhibit little fluorescence emission, but are still
useful as chromophoric dyes. Such chromophores are useful as energy
acceptors in FRET applications, or to simply impart the desired
color to a sample or portion of a sample.
[0078] For biological applications, the dye compounds of the
invention are typically used in an aqueous, mostly aqueous or
aqueous-miscible solution prepared according to methods generally
known in the art. The exact concentration of dye compound is
dependent upon the experimental conditions and the desired results,
but typically ranges from about one nanomolar to one millimolar or
higher. The optimal concentration is determined by systematic
variation until satisfactory results with minimal background
fluorescence are accomplished.
[0079] The dye compounds are most advantageously used to stain
samples with biological components. The sample may comprise
heterogeneous mixtures of components (including intact cells, cell
extracts, bacteria, viruses, organelles, and mixtures thereof), or
a single component or homogeneous group of components (e.g. natural
or synthetic amino acids, nucleic acids or carbohydrate polymers,
or lipid membrane complexes). These dyes are generally non-toxic to
living cells and other biological components, within the
concentrations of use.
[0080] The dye compound is combined with the sample in any way that
facilitates contact between the dye compound and the sample
components of interest. Typically, the dye compound or a solution
containing the dye compound is simply added to the sample. Certain
dyes of the invention, particularly those that are substituted by
one or more sulfonic acid moieties, tend to be impermeant to
membranes of biological cells, and once inside viable cells are
typically well retained. Treatments that peinteabilize the plasma
membrane, such as electroporation, shock treatments or high
extracellular ATP can be used to introduce selected dye compounds
into cells. Alternatively, selected dye compounds can be physically
inserted into cells, e.g. by pressure microinjection, scrape
loading, patch clamp methods, or phagocytosis.
[0081] Dyes that incorporate an aliphatic amine or a hydrazine
residue can be microinjected into cells, where they can be fixed in
place by aldehyde fixatives such as foinialdehyde or
glutaraldehyde. This fixability makes such dyes useful for
intracellular applications such as neuronal tracing.
[0082] Dye compounds that possess a lipophilic substituent, such as
phospholipids, will noncovalently incorporate into lipid
assemblies, e.g. for use as probes for membrane structure; or for
incorporation in liposomes, lipoproteins, films, plastics,
lipophilic microspheres or similar materials; or for tracing.
Lipophilic dyes are useful as fluorescent probes of membrane
structure.
[0083] Chemically reactive dye compounds will covalently attach to
a corresponding functional group on a wide variety of materials,
forming dye conjugates as described above. Using dye compounds to
label reactive sites on the surface of cells, in cell membranes or
in intracellular compartments such as organelles, or in the cell's
cytoplasm, permits the determination of their presence or quantity,
accessibility, or their spatial and temporal distribution in the
sample. Photoreactive dyes can be used similarly to photolabel
components of the outer membrane of biological cells or
as-photo-fixable polar tracers for cells.
[0084] Optionally, the sample is washed after staining to remove
residual, excess or unbound dye compound. The sample is optionally
combined with one or more other solutions in the course of
staining, including wash solutions, peimeabilization and/or
fixation solutions, and solutions containing additional detection
reagents. An additional detection reagent typically produces a
detectable response due to the presence of a specific cell
component, intracellular substance, or cellular condition,
according to methods generally known in the art. Where the
additional detection reagent has, or yields a product with,
spectral properties that differ from those of the subject dye
compounds, multi-color applications are possible. This is
particularly useful where the additional detection reagent is a dye
or dye-conjugate of the present invention having spectral
properties that are detectably distinct from those of the staining
dye.
[0085] The dye conjugates of the invention are used according to
methods extensively known in the art; e.g. use of antibody
conjugates in microscopy and immunofluorescent assays; and
nucleotide or oligonucleotide conjugates for nucleic acid
hybridization assays and nucleic acid sequencing (e.g., U.S. Pat.
No. 5,332,666 to Prober, et al. (1994); U.S. Pat. No. 5,171,534 to
Smith, et al. (1992); U.S. Pat. No. 4,997,928 to Hobbs (1991); and
WO Appl. 94/05688 to Menchen, et al.). Dye-conjugates of multiple
independent dyes of the invention possess utility for multi-color
applications.
[0086] At any time after or during staining, the sample is
illuminated with a wavelength of light selected to give a
detectable optical response, and observed with a means for
detecting the optical response. Equipment that is useful for
illuminating the dye compounds of the invention includes, but is
not limited to, hand-held ultraviolet lamps, mercury arc lamps,
xenon lamps, lasers and laser diodes. These illumination sources
are optionally integrated into laser scanners, fluorescence
microplate readers, standard or minifluorometers, or
chromatographic detectors. Preferred embodiments of the invention
are dyes that are be excitable at or near the wavelengths 633-636
nm, 647 nm, 660 nm, 680 nm and beyond 700 nm, as these regions
closely match the output of relatively inexpensive excitation
sources.
[0087] The optical response is optionally detected by visual
inspection, or by use of any of the following devices: CCD cameras,
video cameras, photographic films, laser-scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample is examined using a flow
cytometer, examination of the sample optionally includes sorting
portions of the sample according to their fluorescence
response.
[0088] One aspect of the instant invention is the formulation of
kits that facilitate the practice of various assays using any of
the dyes of the invention, as described above. The kits of the
invention typically comprise a colored or fluorescent dye of the
invention, either present as a chemically reactive label useful for
preparing dye-conjugates, or present as a dye-conjugate where the
conjugated substance is a specific binding pair member, or a
nucleoside, a nucleotide, an oligonucleotide, a nucleic acid
polymer, a peptide, or a protein. The kit optionally further
comprises one or more buffering agents, typically present as an
aqueous solution. The kits of the invention optionally further
comprise additional detection reagents, a purification medium for
purifying the resulting labeled substance, luminescence standards,
enzymes, enzyme inhibitors, organic solvent, or instructions for
carrying out an assay of the invention.
EXAMPLES
[0089] Examples of some synthetic strategies for selected dyes of
the invention, as well as their characterization, synthetic
precursors, conjugates and method of use are provided in the
examples below. Further modifications and permutations will be
obvious to one skilled in the art. The examples below are given so
as to illustrate the practice of this invention. They are not
intended to limit or define the entire scope of this invention.
##STR00012##
Example 1
Preparation of Compound 1
[0090] The potassium salt of 2,3,3-trimetylindolinium-5-sulfonate
is synthesized by Fisher indole synthesis through the reaction of
4-hydrazinobenzenesulfonic acid and 3-methyl-2-butanone, followed
by neutralizing the indolinyl sulfonic acid with saturated solution
of potassium hydroxide in 2-propanol. The mixture of the potassium
salt of 2,3,3-trimetylindolinium-5-sulfonate (11 g, 39.7 mmol) and
6-bromohexanoic acid (9.68 g, 49.6 mmol) in 1,2-dichlorobenzene
(100 mL) is heated at 120.degree. C. for 10 hours under nitrogen.
The crude product is triturated with 2-propanol. The solid is
filtered and washed with 2-propanol and ether, and dried under
vacuum to give Compound 1 (9.2 g).
Example 2
Preparation of Compound 2
##STR00013##
[0092] To the solution of sodium ethoxide (173.4 mmol, prepared
from 4.0 g sodium in 200 mL dry ethanol) is added ethyl
2-methylacetoacetate (25.0 g, 173.4 mmol), followed by ethyl
6-bromohexanonate (44.5 g, 190.7 mmol). The mixture is heated to
reflux for 12 hours. After cooling to room temperature, the mixture
is filtered and the filtrate is concentrated. The residue is
treated with 1M HCl to pH 1 and the aqueous solution is extracted
with chloroform twice. The organic layer is washed with brine and
dried over Na.sub.2SO.sub.4. After removal of solvent, the residue
is purified on silica gel to afford 15 g ethyl
2-(5-ethoxycarbonyl)pentyl-2-methylacetoacetate.
[0093] The above acetoacetate (13.6 g) in methanol (130 mL) is
mixed with a solution of NaOH (6.6 g) in water (60 mL). The mixture
is stirred at 50.degree. C. for 3 hours. After removal of methanol,
the residue is acidified with 1M HCl to pH 2. The aqueous solution
is extracted with EtOAc (2.times.100 mL). The organic layer is
washed with brine and dried over Na.sub.2SO.sub.4. The crude
product is purified with silica gel chromatography to yield
7-methyl-8-oxo-nonanoic acid Compound 2 (6.4 g).
Example 3
Preparation of Compound 3
##STR00014##
[0095] To the solution of Compound 2 (6.4 g) in methanol (50 mL) is
added H.sub.2SO.sub.4 (1.0 mL) dropwisely. The mixture is refluxed
30 min. After cooling to room temperature, the reaction mixture is
concentrated and the residue is diluted with ethyl acetate (100
mL). The solution is washed with saturated NaHCO.sub.3 and brine.
The organic layer is dried over Na.sub.2SO.sub.4. After removal of
solvent, the methyl 7-methyl-8-oxo-nonanoate Compound 3 is obtained
and used without further purification.
##STR00015##
Example 4
Preparation of Compound 4
[0096] The mixture of 7-methyl-8-oxo-nonanoic acid (Compound 2, 4.2
g, 21.5 mmol) and 4-hydrazinobenzenesulfonic acid (4.23 g, 22.5
mol) in acetic acid (30 mL) is heated to reflux for 8 hours. After
removal of the solvent, the residue is purified on silica gel to
give Compound 4 (3.1 g).
Example 5
Preparation of Compound 5
##STR00016##
[0098] The mixture of methyl 7-methyl-8-oxo-nonanoate (Compound 3,
6.9 g, 34.4 mmol) and 4-hydrazinobenzenesulfonic acid (6.45 g, 32.7
rnol) in acetic acid (50 mL) is heated to reflux for 8 hours. After
removal of the solvent, the residue is purified on silica gel to
give Compound 5 (9.7 g).
Example 6
Preparation of Compound 6
##STR00017##
[0100] A solution of Compound 4 (3.1 g) and potassium acetate (1.1
g) in methanol (20 mL) is stirred at room temperature for 15 min.
After removal of methanol, the resulting potassium salt is heated
with 1,3-propanesultone (2.0 g) in 1,2-dichlorobenzene (5 mL) at
110.degree. C. for 1.5 hour. The mixture is cooled to room
temperature and 1,2-dichlorobenzene is decanted. The solid is
triturated with 2-propanol and the free powder is filtered and
washed with 2-propanol and ether and dried under vacuum to yield
Compound 6.
Example 7
Preparation of Compound 7
##STR00018##
[0102] A solution of Compound 5 (3.3 g) and potassium acetate (1.0
g) in methanol (20 mL) is stirred at room temperature for 15 min.
After removal of methanol; the resulting potassium salt is heated
with 6-bromohexanoic acid (3.4 g) in 1,2-dichlorobenzene (10 mL) at
110.degree. C. overnight. The mixture is cooled to room temperature
and 1,2-dichlorobenzene is decanted. The solid is triturated with
ethyl ether and the free powder is filtered and washed with ether
and dried under vacuum to yield Compound 7.
Example 8
Preparation of Compound 8
##STR00019##
[0104] A solution of Compound 4 (3.3 g) and potassium acetate (1.0
g) in methanol (20 mL) is stirred at room temperature for 15 min.
After removal of methanol, the resulting potassium salt is heated
with ethyl 6-bromohexanonate (8.01 g) in 1,2-dichlorobenzene (10
mL) at 110.degree. C. overnight. The mixture is cooled to room
temperature and 1,2-dichlorobenzene is decanted. The solid is
triturated with ethyl ether and the free powder is filtered and
washed with ether and dried under vacuum to yield Compound 8.
Example 9
Preparation of Compound 9
##STR00020##
[0106] The mixture of Boc-Lys(Boc)-OH (1.0 g, 2.9 mmol),
N-hydroxysuccinimide (0.33 g, 2.9 mmol) and DCC (0.63 g, 3.03 mmol)
in THF (25 mL) is stirred at room temperature overnight. After
removal of solid, the filtrate [Boc-Lys(Boc)-OSu] is added to a
solution of 6-aminocaprioic acid (0.38 g, 2.9 mmol) in water (20
mL), followed by addition of 2N Na.sub.2CO.sub.3 to adjust pH to
8-9. The mixture is stirred at room temperature overnight. After
diluted with water (150 mL), the mixture is acidified with 4%
aqueous HCl to pH 3 and extracted with ethyl acetate (2.times.50
mL). The combined extract is washed with brine and dried over
Na.sub.2SO.sub.4. After removal of solvent, the residue
[Boc-Lys(Boc)-NH(CH.sub.2).sub.5COOH] is dissolved in 1,4-dioxane
(15 mL), followed by addition of 4M HCl in dioxane (10 mL). The
mixture is stirred for 1 hour. The solvent is decanted and the
solid is washed with ethyl acetate (3.times.20 mL) and ether
(3.times.20 mL). The HCl salt of Compound 9 is dried under
vacuum.
Example 10
Preparation of Compound 10
##STR00021##
[0108] Compound 10 is prepared starting from
DL-2,3-diaminopropionic acid analogously to the preparation of
Compound 9.
Example 11
Preparation of Compound 11
[0109] 5-Ethoxycarbonyl-2,3,3-trimethyl-3H-indole is synthesized
through the reaction of ethyl 4-hydrazinobenzoate and
3-methyl-2-butanone. Compound 11 is synthesized by the similar
procedure described for the synthesis of Compound 1.
##STR00022##
Example 12
Preparation of Compound 12
##STR00023##
[0111] A solution of Compound 1 (100 mg, 0.283 mmol) and
malonaldehyde bis(phenylimine)monohydrochloride (77 mg, 0.297 mmol)
in acetic acid (0.5 mL) and acetic anhydride (0.sub.--5 mL) is
heated at 120.degree. C. for 1 hour. The completion of the reaction
is monitored by absorption spectra in methanol. The solution of
anyl intermediate is mixed with Compound 6 (131 mg, 0.283 mol),
then more acetic anhydride (0.5 mL) and pyridine (1.0 mL) is added.
The mixture is heated for 30 min until the anyl intermediate
disappears (monitored by absorption spectra). The reaction mixture
is cooled and poured into ethyl acetate (50 mL). The crude product
is collected by centrifugation and washed with ethyl acetate twice.
Preparative HPLC purification give Compound 12 as blue powder (35
mg).
Example 13
Preparation of Compound 13
[0112] To a solution of Compound 12 (28.4 mg, 0.0334 mmol) and
O--(N-succinimidyl)-N,N,N,N'-tetramethyluronium tetrafluoroborate
(26 mg, 0.0864 mmol) in DMF (0.65 mL) is added triethylamine (0.04
mL) The mixture is stirred at room temperature for 1 h. The
reaction mixture is poured into EtOAc (15 mL) The di-succinimidyl
ester of Compound 12 is collected by centrifugation and washed with
EtOAc (2.times.10 mL), EtOEt (1.times.10 mL) and dried under
vacuum.
[0113] The above di-succinimidyl ester of Compound 12 is dissolved
in water (50 mL) and a solution of Compound 9 (22.2 mg, 0.0667
mmol) in water (25 mL) [neutralized with Na.sub.2CO.sub.3 (7.1 mg,
0.0667 mmol)] is added slowly during the period of 30 minutes. The
mixture is stirred at room temperature overnight. After removal of
solvent, the residue is purified by preparative HPLC to give
Compound 13 as blue powder (20 mg).
##STR00024##
Example 14
Preparation of Compound 14
##STR00025##
[0115] To a solution of Compound 13 (10 mg, 0.0093 mmol) in DMF
(0.4 mL) is added O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (3.64 mg, 0.0121 mmol), followed by triethylamine
(0.03 mL). The mixture is stirred at room temperature for 1 h. The
solution is poured into EtOAc (15 mL). The solid is centrifuged and
washed with EtOAc (3.times.10 mL), ether (1.times.10 mL) and dried
under vacuum to give Compound 14 as bright blue powder (11 mg).
Example 15
Preparation of Compound 15
##STR00026##
[0117] A solution of Compound 6 (100 mg, 0.217 rnmol) and
malonaldehyde bis(phenylimine)monohydrochloride (56 mg, 0.217 mmol)
in acetic acid (0.5 mL) and acetic anhydride (0.5 mL) is heated at
120.degree. C. for 1 hour. The completion of the reaction is
monitored by absorption spectra. The solution of anyl intermediate
is mixed with Compound 7 (101 mg, 0.217 mol), then more acetic
anhydride (0.5 mL) and pyridine (1.0 mL) is added. The mixture is
heated for 30 min until the anyl intermediate disappears (monitored
by absorption spectra). The reaction mixture is cooled and poured
into ethyl acetate (50 mL). The crude product is collected by
centrifugation and washed with ethyl acetate twice. Preparative
HPLC purification gives Compound 15 as bright blue powder (15
mg).
Example 16
Preparation of Compound 16
[0118] To a solution of Compound 15 (6.0 mg, 0.0060 mmol) and
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
(5.4 mg, 0.018 mmol) in DMF (0.40 mL) is added triethylamine (0.04
mL). The mixture is stirred at room temperature for 1 h. The
resulting solution of di-succinimidyl ester of Compound 15 is
diluted with DMF (30 mL), followed by addition of a solution of
ethylenediamine (0.71 mg, 0.012 mmol) in DMF (20 mL) during the
period of 30 minutes. The mixture is stirred at room temperature
overnight. After removal of solvent, the residue is treated with 1N
NaOH (2 mL). After the hydrolysis reaction is completing (monitored
by HPLC), the reaction mixture is diluted with water (5 mL) and
neutralized with 1N HCl. Preparative HPLC purification gives
Compound 16 as blue powder (2 mg).
##STR00027##
Example 17
Preparation of Compound 17
##STR00028##
[0120] To a solution Compound 16 (2 mg, 0.002 mm91) in MAT (0.4 mL)
is added 0-(N succinimidyl)-NAN';N'-fetramethyluronium
tetrafluoroborate (0.8 mg, 0.0027 mmol), followed by triethylamine
(0.02 mL). The mixture is stirred at room temperature for 1 h. The
solution is poured into EtOAc (15 mL). The solid is centrifuged and
washed with EtOAc (3.times.10 mL), ether (1.times.10 mL) and dried
under vacuum to give Compound 17 as bright blue powder (2 mg).
Example 18
Preparation of Compound 18
##STR00029##
[0122] A solution of Compound 1 (100 mg, 0.283 mmol) and
N,N.sup.--diphenylformamidine (58 mg, 0.297 mmol) in acetic acid
(0.5 mL) and acetic anhydride (0.5 mL) is heated at 120.degree. C.
for 1 hour. The completion of the reaction is monitored by
absorption spectra in methanol. The solution of anyl intermediate
is mixed with Compound 6 (131 mg, 0.283. mol), then more acetic
anhydride (0.5 mL) and pyridine (1.0 mL) is added. The mixture is
heated for 30 min until the anyl intermediate disappears (monitored
by absorption spectra). The reaction mixture is cooled and poured
into ethyl acetate (50 mL). The crude product is collected by
centrifugation and washed with ethyl acetate twice. Preparative
HPLC purification gives Compound 18 (33 mg).
Example 19
Preparation of Compound 19
##STR00030##
[0124] To a solution of Compound 18 (25 mg, 0.0303 mmol) and
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
(24 mg, 0.0788 mmol) in DMF (0.5 mL) is added triethylamine (0.04
mL). The mixture is stirred at room temperature for 1 h. The
reaction mixture is poured into EtOAc (15 mL). The di-succinimidyl
ester of Compound 18 is collected by centrifugation and washed with
EtOAc (2.times.10 mL), EtOEt (1.times.10 mL) and dried under
vacuum.
[0125] The above di-succinimidyl ester of Compound 18 is dissolved
in water (50 mL) and a solution of Compound 10 (17.6 mg, 0.0606
mmol) in water (25 mL) [neutralized with. Na.sub.2CO.sub.3 (7.1 mg,
0.0606 mmol)] is added slowly during the period of 30 minutes. The
mixture is stirred at room temperature overnight. After removal of
solvent, the residue is purified by preparative HPLC to give
Compound 19 (20 mg).
Example 20
Preparation of Compound 20
##STR00031##
[0127] To a solution Compound 19 (10 mg, 0.0099 mmol) in DMF (0.4
mL) is added O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (3.64 mg, 0.0119 mmol), followed by triethylamine
(0.03 mL). The mixture is stirred at room temperature for 1 h. The
solution is poured into EtOAc (15 mL). The solid is centrifuged and
washed with EtOAc (3.times.10 mL), ether (1.times.10 mL) and dried
under vacuum to give Compound 20 (10 mg).
Example 21
Preparation of Compound 21
[0128] A solution of Compound 1 (100 mg, 0.283 mmol) and
glutaconaldehyde dianil hydrochoride (85 mg, 0.297 mmol) in acetic
acid (0.5 mL) and acetic anhydride (0.5 mL) is heated at
120.degree. C. for 1.5 hour. The completion of the reaction is
monitored by absorption spectra in methanol. The solution of anyl
intermediate is mixed with Compound 6 (130 mg, 0.283 mol), then
more acetic anhydride (0.5 mL) and pyridine (1.0 mL) is added. The
mixture is heated for 30 min until the anyl intermediate disappears
(monitored by absorption spectra). The reaction mixture is cooled
and poured into ethyl acetate (50 rriL). The crude product is
collected by centrifugation and washed. with ethyl acetate twice.
Preparative HPLC purification gives Compound 21 (20 mg).
##STR00032##
Example 22
Preparation of Compound 22
##STR00033##
[0130] To a solution of Compound 21 (20 mg, 0.0228 mmol) and
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
(17.2 mg, 0.0570 mmol) in DMF (0.5 mL) is added triethylamine (0.03
mL). The mixture is stirred at room temperature for 1 h. The
reaction mixture is poured into EtOAc (15 mL). The di-succinimidyl
ester of Compound 21 is collected by centrifugation and washed with
EtOAc (2.times.10 mL), EtOEt (1.times.10 mL) and dried under
vacuum.
[0131] The above di-succinimidyl ester of Compound 21 is dissolved
in water (40 mL) and a solution of Compound 9 (151 mg, 0.0456 mmol)
in water (25 mL) (neutralized with Na.sub.2CO.sub.3 (4.8 mg, 0.0456
mmol)) is added slowly during the period of 30 minutes. The mixture
is stirred at room temperature overnight. After removal of solvent,
the residue is purified by preparative HPLC to give Compound 22 (20
mg).
Example 23
Preparation of Compound 23
##STR00034##
[0133] A solution of Compound 1 (100 mg, 0.283 mmol) and
malonaldehyde bis(phenylimine)monohydrochloride (77 mg, 0.297 mmol)
in acetic acid (0.5 mL) and acetic anhydride (0.5 mL) is heated at
120.degree. C. for 1 hour. The completion of the reaction is
monitored by absorption spectra in methanol. The solution of anyl
intermediate is mixed with Compound 8 (136 mg, 0.283 mol), then
more acetic anhydride (0.5 mL) and pyridine (1.0 mL) is added. The
mixture is heated for 30 min until the anyl intermediate disappears
(monitored by absorption spectra). The reaction mixture is cooled
and poured into ethyl acetate (50 mL). The crude product is
collected' by centrifugation and washed with. ethyl acetate twice.
Preparative HPLC purification gives Compound 23 as bright blue
powder (30 mg).
Example 24
Preparation of Compound 24
##STR00035##
[0135] To a solution of Compound 23 (30.0 mg, 0.0344 mmol) and
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
(26.0 mg, 0.0861 mmol) in DMF (0.60 mL) is added triethylamine
(0.04 mL). The mixture is stirred at room temperature for 1 h. The
resulting solution of di-succinimidyl ester of Compound 23 is
diluted with DMF (50 mL), followed by addition of a solution of
ethylenediamine (4.1 mg, 0.0688 mmol) in DMF (30 mL) during the
period of 30 minutes. The mixture is stirred at room temperature
overnight. After removal of solvent, the residue is treated with 1N
NaOH (3 mL). After the hydrolysis reaction is completing (monitored
by HPLC), the reaction mixture is diluted with water (10 mL) and
neutralized with IN HCl. Preparative HPLC purification gives
Compound 24 as blue powder (22 mg).
Example 25
Preparation of Compound 25
##STR00036##
[0137] A solution of Compound 11 (100 mg, 0.292 mmol) and.
malonaldehyde bis(phenylimine)monohydrochloride (79 mg, 0.306 mmol)
in acetic acid (0.5 mL) and acetic anhydride (0.5 mL) is heated at
120.degree. C. for 1 hour. The completion of the reaction is
monitored by absorption spectra in methanol. The solution of anyl
intelinediate is mixed with Compound 6 (135 mg, 0.292 mol), then
more acetic anhydride (0.5 mL) and pyridine (1.0 mL) is added. The
Mixture is heated for 30 Min until the anyl intermediate disappears
(monitored by absorption spectra). The reaction mixture is cooled
and poured into ethyl acetate (50 mL) The crude product is
collected by centrifugation and washed with ethyl acetate twice.
Preparative HPLC purification gives Compound 25 as bright blue
powder (33 mg).
Example 26
Preparation of Compound 26
##STR00037##
[0139] To a solution of Compound 25 (30.0 mg, 0.0356 mmol) and
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
(26.8 mg, 0.0890 mmol) in DMF (0.60 mL) is added triethylamine
(0.04 mL). The Mixture is stirred at room temperature for 1 h. The
resulting solution of di-succinimidyl ester of Compound 25 is
diluted with DMF (50 mL), followed by addition of a solution of
ethylenediamine (4.3 mg, 0.0712 mmol) in DMF (30 mL) during the
period of 30 minutes. The mixture is stirred at room temperature
overnight. After removal of solvent, the residue is treated with 1N
NaOH (3 mL) After the hydrolysis reaction is completing (monitored
by HPLC), the reaction mixture is diluted with water (10 mL) and
neutralized with 1N HCl. Preparative HPLC purification gives
Compound 26 as blue powder (20 mg).
Example 27
Preparation of Compound 27
[0140] The reaction of di-potassium salt of
1,1,2-trimethylbenzindolenium-6,8-disulfonic acid [BIOCONJUGATE
CHEM., 356-362 (1996)] (5.0 g, 0.011 mmol) and 6-bromohexanoic acid
(5.3 g, 0.027 mmol) in dichlorobenzene at 120.degree. C. overnight,
followed by the same work-up procedure as described for the
synthesis of Compound 1, affords Compound 27 (4.5 g).
##STR00038##
Example 28
Preparation of Compound 28
##STR00039##
[0142] The Compound 28 is analogously synthesized by the same
procedure described for the synthesis of Compound 4 and Compound 6,
starting from the reaction of 6-hydrazinonaphthalene
1,3-disulfonate [BIOCONJUGATE CHEM., 356-362 (1996)] with
7-methyl-8-oxo-nonanoic acid Compound 2, followed by quaternization
with 1,3-propanesultone.
Example 29
Preparation of Compound 29
[0143] A solution of Compound 27 (100 mg, 0.207 mmol) and
malonaldehyde bis(phenylimine)monohydrochloride (56 mg, 0.217 mmol)
in acetic acid (0.5 mL) and acetic anhydride (0.5 mL) is heated at
120.degree. C. for 1 hour. The completion of the reaction is
monitored by absorption spectra in methanol. The solution of anyl
intermediate is mixed with Compound 28 (123 mg, 0.207 mol), then
more acetic anhydride (0.5 mL) and pyridine (1.0 mL) is added. The
mixture is heated for 30 min until the anyl intermediate disappears
(monitored by absorption spectra). The reaction mixture is cooled
and poured into ethyl acetate (50 mL). The crude product is
collected by centrifugation and washed with ethyl acetate twice.
Preparative HPLC purification gives Compound 29 as bright blue
powder (30 mg).
##STR00040##
Example 30
Preparation of Compound 30
##STR00041##
[0145] To a solution of Compound 29 (25 mg, 0.0225 mmol) and
O--(N-succinimidyl)-N,N,N,N'-tetramethyluronium tetrafluoroborate
(17 mg, 0.0562 mmol) in DMF (0.6 mL) is added triethylamine (0.03
mL). The mixture is stirred at room temperature for 1 h. The
reaction mixture is poured into EtOAc (15 mL). The di-succinimidyl
ester of Compound 29 is collected by centrifugation and washed with
EtOAc (2.times.10 mL), EtOEt (1.times.10 mL) and dried under
vacuum.
[0146] The above di-succinimidyl ester of Compound 29 is dissolved
in water (50 mL) and a solution of Compound 9 (15 mg, 0.0450 mmol)
in water (25 mL) [neutralized with Na.sub.2CO.sub.3 (4.8 mg, 0.0450
mmol)] is added slowly during the period of 30 minutes. The mixture
is stirred at room temperature overnight. After removal of solvent,
the residue is purified by preparative HPLC to give Compound 30 as
blue powder (20 mg).
Example 31
Preparation of Compound 31
##STR00042##
[0148] To a solution Compound 30 (10 mg, 0.0075 rrunol) in DMF (0.4
mL) is added O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (2.82 mg, 0.0094 mmol), followed by triethylamine
(0.03 mL) The mixture is stirred at room temperature for 1 h. The
solution is poured into EtOAc (15 mL). The solid is centrifuged and
washed with EtOAc (3.times.10 mL), ether (1.times.10 mL) and dried
under vacuum to give Compound 31 as bright blue powder (11 mg).
Example 32
Preparation of Compound 32
[0149] A solution of Compound 1 (353 mg, 1 mmol) and
2-chloro-1-formyl-3-(hydroxymethylene)-cyclohex-1-ene (173 mg, 1
mmol) in 1-butanol (48 mL) and benzene (12 mL) is heated to reflux
for 2 h. After the reaction mixture is cooled to room temperature,
a suspension of Compound 6 (462 mg, 1 mmol) in 1-butanol (7 mL) and
benzene (3 mL) is added. The mixture is continued to reflux for 10
h with removal of water by a Dean-Stark condenser. After removal of
solvent, the residue is purified by preparative HPLC to give
Compound 32.
##STR00043##
Example 33
Preparation of Compound 33
##STR00044##
[0151] To a solution of Compound 32 (50.0 mg, 0.0574 mmol) and
O--(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate
(43 mg, 0.143 mmol) in DMF (1.0 mL) is added triethylamine (0.05
mL). The mixture is stirred at room temperature for 1 h. The
resulting solution of di-succinimidyl ester of Compound 32 is
diluted with DMF (50 mL), followed by addition of a solution of
ethylenediamine (6.9 mg, 0.115 mmol) in DMF (30 mL) during the
period of 30 minutes. The mixture is stirred at room temperature
overnight. After removal of solvent, the chloro dye is converted to
Compound 33 by 4-hydroxybenoic acid and-sodium hydride in DMF
according to the procedure of N. Narayanan and G. Patonary (J. ORG.
CHEM, 60, 2391 (1995)). Preparative HPLC purification gives pure
Compound 33 (20 mg).
Example 34
Preparation of Compound 34
##STR00045##
[0153] To a solution of Compound 33 (10.0 mg, 0.01 mmol) and
O--(N-succinimidyl)-N,N,N,N'-tetramethyluronium tetrafluoroborate
(8 mg, 0.05 mmol) in DMF (0.5 mL) is added triethylamine (5 .mu.L).
The mixture is stirred at room temperature for 1 h, and
precipitated with ether to give the blue powder.
Example 35
Preparation of Compound 35
[0154] To Compound 20 in DMF is added 5 equivalents of anhydrous
hydrazine. The mixture is stirred at ambient temperature for 15
minutes. The product is precipitated with 4 20 volumes of ethyl
acetate and purified by HPLC.
##STR00046##
Example 36
Preparation of Compound 36
##STR00047##
[0156] To Compound 14 in DMF at room temperature is added 4
equivalents of triethylamine and 1.2 equivalents of
N-(2-arninoethyl)maleimide, trifluoroacetic acid salt. The mixture
is stirred at ambient temperature for 15 minutes. The product is
precipitated with 4 volumes of ethyl acetate and purified by
HPLC.
Example 37
Preparation of Compound 37 (1,1'-Crosslinked Cyanine)
[0157] Compound 37 is prepared from Compound 9 by modification of
WO 01/02374 (to R. Singh, et al.).
##STR00048##
Example 38
Preparation of Compound 38 (1,1'-Crosslinked Cyanine, SE)
[0158] Compound 37 is converted to Compound 38 analogous to the
procedure of Compound 14 as described in Example 14.
##STR00049##
Example 39
Preparation of Compound 39
##STR00050##
[0160] To a solution Compound 1 (5.0 g, 14.14 mmol) in DMF (20 mL)
is added di(N-succinimidyl) carbonate (3.81 g, 14.85 mmol),
followed by triethylamine (3.9 mL, 228.29 mmol). The mixture is
stirred at room temperature for 1 h. The solution is poured into
EtOAc (150 mL). The solid is centrifuged and washed with EtOAc
(3.times.100 mL), ether 10 (1.times.100 mL) and dried under vacuum
to give Compound 39, 6.0 g.
Example 40
Preparation of Compound 40
##STR00051##
[0162] Compound 40 is prepared starting from Compound 6 analogously
to the preparation of Compound 39.
Example 41
Preparation of Compound 41
##STR00052##
[0164] Compound is prepared starting from Compound 7 analogously to
the preparation of Compound 39.
Example 42
Preparation of Compound 42
##STR00053##
[0166] Compound 42 is prepared starting from Compound 8 analogously
to the preparation of Compound 39.
Example 43
Preparation of Compound 43
##STR00054##
[0168] Compound 43 is prepared starting from Compound 27
analogously to the preparation of Compound 39.
Example 44
Preparation of Compound 44
##STR00055##
[0170] Compound 44 is prepared starting form Compound 28
analogously to the preparation of Compound 39.
Example 45
Preparation of Compound 45
##STR00056##
[0172] To a solution of Compound 39 (1.5 g, 3.33 mmol) in DMF (15
mL) is added and
t-BuOCONHCH.sub.2CH(CH.sub.2CH.sub.2CH.sub.2CH9CH.sub.2COOH)CH.-
sub.2NH.sub.2.HCl (product of AnaSpec, Inc.) (1.1 g, 3.33 mmol),
followed by addition of triethylamine (0.34 g, 0.46 mL, 3.33 mmol).
The reaction mixture is stirred at room temperature and the
reaction is monitored by HPLC. After reaction is complete, the
solvent is removed and the residue is used for the next reaction
without further purification.
Example 46
Preparation of Compound 46
##STR00057##
[0174] The above Compound 45 is dissolved in TFA (10 mL) at
0.degree. C. and the solution is stirred at room temperature for 30
minutes. After removal of TFA, the residue is treated with ethyl
ether. The solid is collected by filtration and washed with ether
twice. After dried under vacuum, the solid is dissolved in DMF (15
mL) and the solution is neutralized with triethylamine. Then a
solution of Compound 40 (1.86 g, 3.33 mmol) in DMF (10 mL) is
added. The reaction mixture is stirred at room temperature. After
the reaction is complete (monitored by HPLC), the solvent is
removed and the residue is treated with ethyl acetate to give
Compound 46.
Example 47
Preparation of Compound 47
##STR00058##
[0176] Compound 47 is prepared starting from Compound 43 and
Compound 44 analogously to the preparation of Compound 46.
Example 48
Preparation of Compound 48
##STR00059##
[0178] Compound 48 is prepared starting from Compound 40,
BocNHCH.sub.2CH.sub.2NH.sub.2 and Compound 41 analogously to the
preparation of Compound 46.
Example 49
Preparation of Compound 49
##STR00060##
[0180] Compound 49 is prepared starting from Compound 39,
BocNHCH2CH2NH2 and compound 42 analogously to the preparation of
Compound 46.
Example 50
Preparation of Compound 50
##STR00061##
[0182] Compound 46 (500 mg, 0.517 mmol) and malonaldehyde
bis(phenylimine)monohydrochloride (67 mg, 0.258 mmol) are dissolved
in acetic anhydride (3 mL), followed by addition of pyridine (3
mL). The mixture is heated to 120.degree. C. for 1 h. After cooling
to room temperature, the mixture is dropped into ethyl acetate. The
crude dye is collected by centrifugation and washed with ethyl
acetate twice. Preparative HPLC purification gives Compound 50 as
bright blue powder (200 mg).
Example 51
Preparation of Compound 51
##STR00062##
[0184] Compound 46 (500 mg, 0.517 mmol) and N,N-diphenylformamidine
(51 mg, 0.258 mmol) are dissolved in acetic anhydride (3 mL),
followed by addition of pyridine (3 mL). The mixture is heated to
120.degree. C. for 1 h. After cooling to room temperature, the
mixture is dropped into ethyl acetate. The crude dye is collected
by centrifugation and washed with ethyl acetate twice. Preparative
HPLC purification gives Compound 51 (220 mg).
Example 52
Preparation of Compound 52
##STR00063##
[0186] Compound 46 (500 mg, 0.517 mmol) and glutaconaldehyde dianil
hydrochoride (74 mg, 0.258 mmol) are dissolved in acetic anhydride
(3 mL), followed by-addition of pyridine (3 mL). The mixture is
heated to 120.degree. C. for 1 h. After Cooling to room
temperature, the mixture is dropped into ethyl acetate. The crude
dye is collected by centrifugation and washed with ethyl acetate
twice. Preparative HPLC purification gives Compound 52 (190
mg).
Example 53
Preparation of Compound 53
##STR00064##
[0188] Compound 47 (500 mg, 0.407 mmol) and malonaldehyde
bis(phenylimine)monohydrochloride (53 mg, 0.204 mmol) are dissolved
in acetic anhydride (3 mL), followed by addition of pyridine (3
mL). The mixture is heated to 120.degree. C. for 1 h. After cooling
to room temperature, the mixture is dropped into ethyl acetate. The
crude dye is collected by centrifugation and washed with ethyl
acetate twice. Preparative HPLC purification gives Compound 53 as
bright blue powder (180 mg).
Example 54
Preparation of Compound 54
##STR00065##
[0190] Compound 48 (500 mg, 0.525 mmol) and N,N-diphenylformamidine
(51.5 mg, 0.262 mmol) are dissolved in acetic anhydride (3 mL),
followed by addition of pyridine (3 mL). The mixture is heated to
120.degree. C. for 1 h. After cooling to room temperature, the
mixture is dropped into ethyl acetate. The crude dye is collected
by centrifugation and washed with ethyl acetate twice and then
dissolved in 1N NaOH (10 mL). After the hydrolysis reaction is
completing (monitored by HPLC), the reaction mixture is diluted
with water (10 mL) and neutralized with 1N HCl. Preparative HPLC
purification gives Compound 54 (180 mg).
Example 55
Preparation of Compound 55
##STR00066##
[0192] Compound 49 (500 mg, 0.582 mmol) and
N,N-diphenylfoiniamidine (57 mg, 0191 mmol) are dissolved in acetic
anhydride (3 mL), followed by addition of pyridine (3 mL). The
mixture is heated to 120.degree. C. for 1 h. After cooling to room
temperature, the mixture is dropped into ethyl acetate. The crude
dye is collected by centrifugation and washed with ethyl acetate
twice and then dissolved in 1N NaOH (10 mL). After the hydrolysis
reaction is completing (monitored by HPLC), the reaction mixture is
diluted with water (10 mL) and neutralized with IN HCl. Preparative
HPLC purification gives Compound 55 (180 mg).
Example 56
Preparation of a Peptide-Dye Conjugate
[0193] To aminophalloidin (3.5 mg, 4 urnol, Alexis Corp.) and the
succinimidyl ester derivative Compound 14 (6.0 mg, 5 pmol) in DMF
is added N,N-diisopropylethylamine (2 pL, 11 .mu.mol. The mixture
is stirred at room temperature for 3 hours. To this solution is
added 7 mL of diethyl ether. The solid is collected by
centrifugation. The crude product is purified on SEPHADEX LH-20,
eluting with water, followed by preparative HPLC to give the pure
phaltoidin conjugate. The product is art effective stain for
F-actin filaments in fixed-cell preparations.
Example 57
Preparation of a Drug-Dye Conjugate
[0194] A fluorescent dopamine ID, antagonist is prepared as
follows: 10 mg of N-(p-aminophenethyl)spiperone (Amlaiky, et al.,
FEBS LETT., 176, 436 (1984)), and 10 N,N-diisopropylethylamine in 1
mL of DMF is added 15 mg of Compound 14 or 20. After 3 hours, the
reaction mixture is poured into 5 mL ether. The precipitate is
centrifuged, then purified by chromatography on silica gel using
10-30% methanol in chloroform.
Example 58
Preparation of Protein-Dye Conjugates
[0195] A series of dye conjugates of goat anti-mouse IgG (GAM),
goat anti-rabbit IgG (GAR), streptavidin, transferrin and other
proteins, including R-phycoerythrin (R-PE) and allophycocyanin
(APC) are prepared by standard means (Haugland, et al., METH. MOL.
BIOL., 45, 205 (1995); Haugland, METH: MOL. BIOL., 45, 223 (1995);
Haugland, METH. MOL. BIOL., 45,235 (1995); Haugland, CURRENT
PROTOCOLS IN CELL BIOLOGY, 16.5.1-16.5.22 (2000)) using Compound 14
or 20 and a mono-succinimidyl ester derivative of the Cy5 dye
(Amersham Biosciences).
[0196] The typical method for protein conjugation with succinimidyl
esters of the invention is as follows. Variations in ratios of dye
to protein, protein concentration, time, temperature, buffer
composition and other variables that are well known in the art are
possible that still yield useful conjugates. A solution of the
protein is prepared at about 10 mg/mL in 0.1 M sodium bicarbonate.
The labeling reagents are dissolved in a suitable solvent such as
DMF or DMSO at about 10 mg/mL. Water is a suitable solvent for many
dyes of the invention. Predetermined amounts of the labeling
reagents are added to the protein solutions with stirring. A molar
ratio of 10 equivalents of dye to 1 equivalent of protein is
typical, though the optimal amount varies with the particular
labeling reagent, the protein being labeled and the protein's
concentration, and is determined empirically.
[0197] When optimizing the fluorescence yield and determining the
effect of degree of substitution (DOS) on this brightness, it is
typical to vary the ratio of reactive dye to protein over a
several-fold range. The reaction mixture is incubated at room
temperature for one hour or on ice for several hours. The
dye-protein conjugate is typically separated from free unreacted
reagent by size-exclusion chromatography, such as on Amersham PD-10
resin equilibrated with phosphate-buffered saline (PBS). The
initial, protein-containing colored band is collected and the
degree of substitution is determined from the absorbance at the
absorbance maximum of each fluorophore, using the extinction
coefficient of the free fluorophore. The dye-protein conjugate thus
obtained can be subfractionated to yield conjugates with higher,
lower or more uniform DOS.
[0198] Following is a specific example of using Compound 14 to
prepare IgG-dye conjugate:
[0199] Step 1. Preparing Protein Solution (Solution A):
[0200] Mix 50 .mu.L of 1 M NaHCO.sub.3 with 450 .mu.L of IgG
protein solution (4 mg/mL) to give 0.5 mL protein sample solution.
The resulted solution should have pH 8.5.+-.0.5.
[0201] Step 2. Preparing Dye Solution (Solution B):
[0202] To 50 .mu.L of DMSO add 1 mg of Compound 14, and stir until
the compound is completely dissolved.
[0203] Step 3. Running Conjugation Reaction:
[0204] Add the protein solution (A) to the dye solution (B) with
effective stirring or shaking, and keep the reaction mixture
stirred or shaken for 1-3 hrs.
[0205] Step 4. Purifying the Conjugate:
[0206] a) Dilute 10.times. elution buffer with de-ionized water to
give 1.times. elution buffer (Solution C) that is used to elute the
protein conjugate from PD-10 column;
[0207] b) Load the column with the reaction mixture (from step 3,
filtrated if necessary) or supernatant as soon as the liquid in the
pre-packed column rims just below the top surface;
[0208] c) Add 1 mL of the IX elution buffer as soon as the sample
runs just below the top resin surface; Repeat this `sample washing`
process twice; Add more IX elution buffer solution to elute the
desired sample;
[0209] d) Collect the faster-running band that is usually the
desired labeled protein. Keep the slower-running band that is
usually free or hydrolyzed dye until the desired product is
identified.
[0210] Step 5. Characterizing the Desired Dye-Protein
Conjugate:
[0211] a). Measure OD (absorbance) at 280 nm and 650 nm (Note: for
most spectrophotometers, the sample (from the column fractions)
need be diluted with de-ionized water so that the OD values are in
the range 0.1 to 0.9). The O.D. (absorbance) 280 nm is the maximum
absorption of protein while 650 nn is the maximum absorption of
Compound 14 amide (Note: to obtain accurate DOS, you must make sure
that the conjugate is free of the non-conjugated dye); b).
Calculating DOS using the following equation:
DOS=[dye]/[protein]=A650.times..epsilon..sub.p/250000(A280-0.05A650)
[0212] [dye] is the dye concentration, and can be readily
calculated from the Beer-Lambert Law: A=s.sub.dyeC.times.L;
[protein] is the target protein concentration. This value can be
either estimated by the weight (added to the reaction) if the
conjugation efficiency is high enough (preferably >70%) or more
accurately calculated by the Beer-Lambert Law:
A=.epsilon..sub.proteinC.times.L. For example, IgG has the s value
to be 203,000 cm.sup.-1M.sup.-1. For effective labeling, the degree
of substitution should fall between 2-6 moles of Compound 14 to one
mole of antibody.
Example 59
Fluorescent Labeling of Periodate-Oxidized Proteins
[0213] Two samples of 5 mg each of goat IgG antibody in 1 mL of 0.1
M acetate, 0.135 M NaCl, pH 5.5, are treated with 2.1 mg of sodium
metaperiodate on ice, for 1 and 2 hours, respectively. The
reactions are stopped by addition of 301.1 L ethylene glycol. The
antibodies are purified on a Sephadex G25 column packed in PBS pH
7.2. One-tenth volume of 1 M sodium bicarbonate is added to raise
the pH and Compound 35 is added at a molar ratio of dye to protein
of 50:1. The reaction is stirred for 2 hours at room temperature.
Sodium cyanoborohydride is added to a final concentration of 10 mM
and the reaction is stirred for 4 hours at room temperature. The
antibody conjugates are purified by dialysis and on Sephadex G25
columns as described above. Antibodies that are oxidized for 1 hour
typically yield a degree of substitution of 1 mole of dye per mole
of IgG. Antibodies that are oxidized for 2 hours typically yield a
DOS of approximately 2 mole of dye per mole of IgG.
Periodate-oxidized proteins in gels and on blots can also be
labeled, essentially as described in Estep T N and Miller T J,
(ANAL. BIOCHEM., 157, 100-105 (1986)). The conjugates of Compound
35 exhibit greater fluorescence than the conjugates of Cy3 dye at
similar DOS when conjugated to a wide variety of proteins.
Example 60
Labeling Beta-Galactosidase with a Thiol-Reactive Dye
[0214] A solution of beta-galactosidase, a protein rich in free
thiol groups, is prepared in PBS (2.0 mg in 400 .mu.L). The protein
solution is then treated with a 20 mg/L solution of the maleimide
derivative Compound 36 in DMF. Unreacted dye is removed on a spin
column. The degree of substitution by the dye is estimated using
the extinction coefficient of the free dye as described in Example
58. The protein concentration is estimated from the absorbance at
280 nm, corrected for the absorbance of Compound 36 at that
wavelength.
Example 61
Total Fluorescence of Selected Dye Protein Conjugates Compared with
Cy5
[0215] In general, the higher the DOS, the brighter the Compounds
14 and 17 bioconjugates relative to the Cy5 bioconjugates,
although, Compound 14 and 17 bioconjugates are brighter at all DOS
tested. The decrease in the RQY of the Cy5 bioconjugates is found
to be accompanied by an increase in the 600-nm absorbance band
relative to the 650-nm absorbance band. The increase in extinction
of the 600 nm band is always associated with a large quenching of
the fluorescence. This result is completely supportive of the work
of Gruber, et al. (BIOCONJUGATE CHEM., 11, 696 (2000)) who observed
a similar correlation of an increased absorbance at 600 nm and a
large decrease in fluorescence intensity. FIG. 4 shows a direct
comparison of the fluorescence emission of the Compound 14
conjugate of GAR IgG at nearly equivalent DOS. The 600 nm
absorbance band is always much lower in extinction for Compound 14
than for an equivalently labeled Cy5 derivative. This general
observation has now been confirmed with several other proteins.
Example 62
Comparison of the Protein Conjugates Prepared from 1,1'-Crosslinked
and Non-Crosslinked "Cy5-Like" Isomers with Compound 14
[0216] 1,1'-Crosslinked Cy5 isomer is synthesized as described in
Example 37 and conjugated to GAR at various DOS. FIG. 4 is a direct
comparison of fluorescence properties of GAR conjugates prepared
from Cy5 SE, Compounds 14 and 38. One can see that the
1,3'-intramolecular crosslinking has resulted in a drastic
improvement of fluorescence performance of Compound 14 GAR
conjugates over those of Cy5 (non-crosslinked cyanine) and Compound
38 (1,1'-crosslinked cyanine). Compound 14 GAR conjugate also has
much weaker absorbance around 600 nm (non-fluorescent excitation).
The brighter fluorescence emission of compound 14 GAR conjugate
(than Cy5 and Compound 38) is observed at all of the tested
DOS's.
Example 63
Comparison of the Fluorescence of Goat Anti-Mouse IgG (GAM)
[0217] Conjugates of Cy3 and Compound 20 are prepared analogously
to the procedure of Example 58 with Compound 20 and the Cy3
reactive dyes at a variety of degrees of substitution ranging from
1.0-12. The conjugates are characterized using excitation
wavelength=532 nm analogously to Example 58.
Example 64
The Photostability of Compound 13 is Greater than that of Cy5 Free
Acid
[0218] Photobleaching experiments are performed at 0.111 M
concentrations of Compound 13 and commercially available Cy5 free
acid. Both of the compounds are irradiated with A100 W Mercury lamp
in PBS (pH 7.0), where both of the dyes receive the same amount of
irradiation as determined by photometric measurements. As shown in
FIG. 5, Compound 13 remains about 3 times brighter than the Cy5
free acid after 500 minutes of illumination.
Example 65
Fluorescence Energy Transfer in Conjugates of R-Phycoerythrin and
Allophycocyanin
[0219] R-phycoerythrin (R-PE) conjugate of Compound 14 or 17 is
prepared as in Example 58 with a DOS sufficiently high to quench
the donor fluorescence almost completely (DOS about 4-8). The
resulting phycobiliprotein conjugate is excited at 488 um and the
fluorescence emission is compared to that of unmodified
R-phycoerythrin excited at the same wavelength. Highly efficient
energy transfer (>99%) occurs from the protein to the
fluorescent dye. A conjugate of these complexes with streptavidin
is prepared essentially as described by Haugland (METH. MOL. BIOL.,
45, 205 (1995)). This streptavidin conjugate retains the energy
transfer properties and is useful for cell staining in flow
cytometers that utilize the argon-ion laser for excitation. Tandem
conjugates of allophycocyanin can also be made, with longer
wavelength dyes of the invention such as Compound 34 yield emission
well beyond 700 um when excited near 633 urn.
Example 66
Labeling of Actin in Cultured Mammalian Cells
[0220] Bovine pulmonary artery cells (BPAEC) are grown to 30-50% of
confluence on glass. The cells are fixed with 3.7% formaldehyde,
permeabilized with 0.2% Triton X-100, and blocked with 6% BSA. The
cells are incubated with the phalloidin dye-conjugate of Example
56. The cells are rinsed with blocking buffer and mounted in PBS pH
7.4. The stained cells display actin filaments decorated with red
fluorescence.
Example 67
Preparation and Use of a Fluorescent Alpha-Bungarotoxin
Dye-Conjugate
[0221] Alpha-Bungarotoxin (1 mg) in 25 .mu.L 0.1 M NaHCO.sub.3 is
treated with 1.5 equivalents of Compound 14 or 20 at room
temperature for 2 hours. The product is purified by size exclusion,
by ion exchange chromatography, and finally by reverse-phase HPLC.
The conjugate is used for staining of acetylcholine receptors.
Example 68
Preparation and Use of a Fluorescent Tyramide
[0222] A 2-fold molar excess of tyramine hydrochloride is added to
Compound 20 in aqueous solution at room temperature followed by an
excess of triethylamine. After 30 minutes the red solid is
precipitated with acetone, washed with ether and purified by
preparative HPLC. Bovine pulmonary artery cells (BPAEC) are grown
to 30-50% of confluence on glass. The cells are fixed with 3.7%
formaldehyde, permeabilized with 0.2% Triton X100, and blocked with
1 mg/mL streptavidin and 1 mM biotin. After washing, cells are
exposed to about 0.05 pg/mL of biotinylated anti-cytochrome C
oxidase (anti-COX) then incubated with Streptavidin-HRP conjugate
at room temperature. Cells are rinsed again. The sample is then
incubated with Compound 20 tyramide and examined using fluorescence
microscopy.
Example 69
Preparation of Aminodextran Dye-Conjugates
[0223] 70,000 MW aminodextran (50 mg) derivatized with an average
of 13 amino groups is dissolved at 10 mg/mt, in 0.1 M NaHCO.sub.3.
Compound 14 or 20 or 31 is added so as to give a dye/dextran ratio
of about 10-15. After 6-12 hours the conjugate is purified on
SEPHADEX G-50, eluting with water. Typically 4-6 moles of dye are
conjugated to 70,000 MW dextran.
Example 70
Preparation Offluorescent-Dye Labeled Microspheres
[0224] Uniform microspheres are chemically modified to have
functional groups such as amino or carboxyl or aldehydes. These
functionalized microspheres are covalently conjugated with the
corresponding reactive dyes as listed in Table 1. For example, the
amine-modified microspheres are readily conjugated to the dyes of
the invention through succinimidyl esters such as Compounds 14, 17,
20 and 31. A dye-labeled protein is covalently coupled through its
amine residues to the carboxylate groups of the polymer using ethyl
3-(dimethylaminopropyl)carbodiimide (EDAC).
[0225] The dyes of invention can also be physically adsorbed on
microspheres. For example, carboxylate-modified microspheres are
suspended in a solution of a protein that has been conjugated to a
dye of the invention. The protein is passively adsorbed on the
microspheres, and excess protein is removed by centrifugation and
washing. Microparticles of a size that cannot be centrifuged are
separated from excess protein by dialysis through a semi-permeable
membrane with a high MW cutoff or by gel filtration chromatography.
Another example is that biotinylated microspheres are treated with
a streptavidin, avidin or anti-biotin conjugate of a dye of the
invention.
Example 71
Preparation Offluorescent Liposoines Using Dyes of the
Invention
[0226] Selected dyes of the invention (such as Compound 13 and 19)
are sufficiently water soluble to be incorporated into the interior
of liposomes by methods well known in the art (S. BIOL. CHEM., 257,
13892 (1982) and PROC. NATL. ACAD. SCI., USA 75,4194 (1978)).
Alternatively, liposomes containing dyes of the invention having a
lipophilic substituent (e.g. alkyl having 11-22 carbons), within
their membranes are prepared by co-dissolving the fluorescent lipid
and the unlabeled lipids. hospholipid(s) that make up the liposome
before forming the liposome dispersion essentially as described by
Szoka Jr., et al. (ANN. REV. BIOPHYS. BIOENG., 9, 467 (1980)).
Example 72
Preparation of Dye-Bacteria Conjugates
[0227] Heat-killed Escherichia coli are suspended at 10 mg/mL in pH
8-9 buffer then incubated with 0.5-1.0 mg/mL of an amine-reactive
dye, typically a succinimidyl ester derivative (such as Compound 14
or 20 or 31). After 30-60 minutes the labeled bacteria are
centrifuged and washed several times with buffer to remove any
unconjugated dye. Labeled bacteria is analyzed by flow
cytometry.
Example 73
Preparation of Nucleotide-Dye Conjugates
[0228] To 2 mg of 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate
(Sigma Chemical) in 100 water is added Compound 14 or 20 in 100
.mu.L DMF and 5 .mu.L triethylamine. After 3 hours, the solution is
evaporated and the residue is purified by HPLC. The product
fractions are lyophilized to give the red-fluorescent nucleotide
conjugate. Alternatively, fluorescent dye-conjugates of
deoxyuridine 5'-triphosphate are prepared from
5-(3-amino-1-propynyl)-2'-deoxyuridine 5'-triphosphate, or by
treating a thiolated nucleotide or a thiophosphate nucleotide with
a thiol-reactive dye of the invention (such as the maleimide
Compound 36). Additionally, 2'- (or
3')-2-amninoethylaminocarbonyladenosine 5'-triphosphate is reacted
with a slight excess of Compound 14 and, following precipitation
with ethanol, the ribose-modified product is purified by
preparative HPLC. Additional nucleotides conjugated with the dyes
of invention can be readily prepared by someone skilled in the art
following the published procedures such as Nimmakayalu M, et al.,
BIOTECHNIQUES, 28, 518-522 (2000); Muhlegger K, et al., BIOL. CHEM.
HOPPE SEYLER, 371, 953-965 (1990); Giaid A, et al. HISTOCHEMISTRY,
93, 191-196 (1989).
Example 74
Preparation of an Oligonucleotide Dye-Conjugate
[0229] A 5'-amine-modified, 18-base M13 primer sequence (about 100
.mu.L is dissolved in 4, water. To this is added 250 .mu.g of
Compound 14 or 20 in 100 .mu.L 0.1 M sodium borate, pH 8.5. After
16 hours, 10 .mu.L of 5 M NaCl and 3 volumes of cold ethanol are
added. The mixture is cooled to -20.degree. C., centrifuged, the
supernatant is decanted the pellet is rinsed with ethanol and then
dissolved in 100 .mu.L water. The labeled oligonucleotide is
purified by HPLC. The desired peak is collected and evaporated to
give the fluorescent oligonucleotide.
Example 75
In Situ Hybridization of an RNA Probe
[0230] Mouse fibroblasts are fixed and prepared for mRNA in-situ
hybridization using standard procedures. A dye-labeled RNA probe is
prepared by in vitro transcription of a plasmid containing the
mouse actin structural gene cloned downstream of a phage T3 RNA
polymerase promoter. Labeling reactions comprise combining 2 .mu.L
DNA template (1 .mu.g DNA), 1 .mu.L each of 10 mM ATP, CTP and GTP,
0.75 .mu.L 10 mM UTP, 2.5 .mu.L 1 mM aminoallyl-labeled UTP, 2
.mu.L10.times. transcription buffer (400 mM Tris, pH 8.0, 100 mM
MgCl.sub.2, 20 mM spermidine, 100 mM NaCl), 1 .mu.LT3 RNA
polymerase (40 units/.mu.L), 1 .mu.L 2 mg/mL BSA, and 8.75 .mu.L
water. Reactions are incubated at 37.degree. C. for two hours. The
DNA template is removed by treatment with 20 units DNase I for 15
minutes, at 37.degree. C. The RNA transcript is purified by
extraction with an equal volume of phenol:chloroform, 1:1, then by
chromatography on SEPHADEX G50. Labeled RNA is denatured for 5
minutes at 50.degree. C., then hybridized to cellular preparations
using standard procedures. The long-wavelength fluorescence of the
labeled cells is detected by excitation through an optical filter
optimized for Cy5-Iike dyes.
Example 76
Preparing DNA Hybridisation Probes Using Amine-Modified DNA and an
Amine-Reactive Dye of the Invention
[0231] Nick translation is performed using pUC 1.77 plasmid DNA
containing a chromosome 1 human alpha-satellite probe. To a
microcentrifuge tube is added, in the following order: 23.5 .mu.L
water, 5 .mu.L 10.times. Nick Translation buffer (0.5 M Tris-HCl,
50 mM MgCl.sub.2, 0.5 mg/mL BSA, pH 7.8), 5 .mu.L 0.1 M DTT, 4
.mu.L d(GAC)TP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP), 1 .mu.L
0.5 mM-dTTP, 4 .mu.L 0:5 mM aminoallyl-dUTP, 1 .mu.L1 .mu.g/.mu.L
template DNA, 5 .mu.L DNase I(1 .mu.g/mL, 2000 Kunitz units/mg),
1.5 .mu.L DNA polymerase I(10 U/.mu.L). The tube is incubated 2
hours at 15.degree. C., then brought to a final volume of 100 .mu.L
with water. The amine-modified DNA is purified using a QIAQUICK PCR
purification Kit (Qiagen). The amine-modified DNA is resuspended in
5 .mu.L water. To the solution is added 3 .mu.L 25 mg/mL sodium
bicarbonate and 50 .mu.g of Compound 14 or 20 in 5 .mu.L DMF. The
reaction is incubated for 1 hour at room temperature in the dark,
to the reaction is added 90 .mu.L water, and it is purified using a
QIAQUICK PCR purification kit (Qiagen). The labeled DNA products
are suitable for in situ hybridization experiments, use on
microarrays and as fluorescence donors or acceptors in
hybridization-based assays.
Example 77
Staining Cells with Tandem Dye-Labeled Streptavidin
[0232] Jurkat cells are washed twice with 1% BSA/PBS and
resuspended at a concentration of 1.times.10.sup.7 cells/mL The
Jurkat cells are then incubated on ice for 60 minutes with mouse
anti human CD4 biotin (Biosource International) at the recommended
concentration of 10 .mu.L for 1.times.10.sup.7 cells. After
incubation with the primary antibody, the cells are washed with 1%
BSA/PBS and incubated on ice for 30 minutes with 1 .mu.g of either
the fluorescent streptavidin-phycoerythrin conjugate of Example 58,
or a streptavidin conjugate of GII3CO'S RED 670. The cells are
washed with 1% BSA/PBS, centrifuged, and resuspended with 400 .mu.L
of 1% BSA/PBS. The samples are analyzed on a FacsVantage flow
cytometer exciting with the 488-nm line of an argon laser,
collecting the emission by a 700-nm long pass filter (XF-48). Using
a FSC versus SSC dot plot the live cells are gated and the
geometric mean of the fluorescence for FL3 is measured. The data is
analyzed for both fluorescence and signal/noise ratio.
* * * * *