U.S. patent application number 11/466085 was filed with the patent office on 2007-05-03 for methods of labelling polynucleotides with dibenzorhodamine dyes.
This patent application is currently assigned to Applera Corporation. Invention is credited to Scott C. Benson, Joe Y. L. Lam, Steven Michael Menchen.
Application Number | 20070099210 11/466085 |
Document ID | / |
Family ID | 25526378 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070099210 |
Kind Code |
A1 |
Benson; Scott C. ; et
al. |
May 3, 2007 |
Methods of Labelling Polynucleotides with Dibenzorhodamine Dyes
Abstract
Dibenzorhodamine compounds having the structure ##STR1## are
disclosed, including nitrogen- and aryl-substituted forms thereof.
In addition, two intermediates useful for synthesizing such
compounds are disclosed, a first intermediate having the structure
##STR2## including nitrogen- and aryl-substituted forms thereof,
and a second intermediate having the structure ##STR3## including
nitrogen- and aryl-substituted forms thereof, wherein substituents
at positions C14 to C18 taken separately are selected from the
group consisting of hydrogen, chlorine, fluorine, lower alkyl,
carboxylic acid, sulfonic acid, --CH.sub.2OH, alkoxy, phenoxy,
linking group, and substituted forms thereof. The invention further
includes energy transfer dyes comprising the dibenzorhodamine
compounds, nucleosides labeled with the dibenzorhodamine compounds,
and nucleic acid analysis methods employing the dibenzorhodamine
compounds.
Inventors: |
Benson; Scott C.; (Alameda,
CA) ; Lam; Joe Y. L.; (Castro Valley, CA) ;
Menchen; Steven Michael; (Fremont, CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.;APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
25526378 |
Appl. No.: |
11/466085 |
Filed: |
August 21, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11177233 |
Jul 7, 2005 |
|
|
|
11466085 |
Aug 21, 2006 |
|
|
|
10441950 |
May 20, 2003 |
6919445 |
|
|
11177233 |
Jul 7, 2005 |
|
|
|
09969430 |
Oct 2, 2001 |
6566071 |
|
|
10441950 |
May 20, 2003 |
|
|
|
09784943 |
Feb 14, 2001 |
6326153 |
|
|
09969430 |
Oct 2, 2001 |
|
|
|
09556040 |
Apr 20, 2000 |
6221606 |
|
|
09784943 |
Feb 14, 2001 |
|
|
|
09199402 |
Nov 24, 1998 |
6111116 |
|
|
09556040 |
Apr 20, 2000 |
|
|
|
08978775 |
Nov 25, 1997 |
5936087 |
|
|
09199402 |
Nov 24, 1998 |
|
|
|
Current U.S.
Class: |
435/6.16 ;
536/25.32; 546/36; 548/417; 549/224 |
Current CPC
Class: |
C09B 11/02 20130101;
C07D 221/10 20130101; C07D 209/60 20130101; C07H 21/04 20130101;
C07H 19/10 20130101; C07D 491/147 20130101; C07H 19/20 20130101;
C07D 491/14 20130101; C09B 11/24 20130101; C07D 311/78 20130101;
C07H 21/00 20130101; Y10T 436/143333 20150115; C12Q 1/6869
20130101; C12Q 1/6869 20130101; C12Q 2563/107 20130101; C12Q 1/6869
20130101; C12Q 2563/107 20130101; C12Q 2527/125 20130101 |
Class at
Publication: |
435/006 ;
536/025.32; 546/036; 548/417; 549/224 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C07D 491/22 20060101
C07D491/22 |
Claims
1. A dibenzorhodamine compound having the structure ##STR21##
including nitrogen- and aryl-substituted forms thereof.
2-40. (canceled)
41. An intermediate useful for the synthesis of dibenzorhodamine
compounds having the structure ##STR22## including aryl- and
nitrogen-substituted forms thereof.
42-69. (canceled)
70. An intermediate useful for the synthesis of dibenzorhodamine
compounds having the structure ##STR23## including nitrogen- and
aryl-substituted forms thereof wherein: R.sub.1 taken together with
the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a
first ring structure having from 4 to 7 members; and/or R.sub.1
taken together with the C-12-bonded nitrogen and the C-11 and C-12
carbons forms a second ring structure having from 5 to 7
members.
71-100. (canceled)
101. A labeled nucleoside/tide having the formula: NUC-D wherein
NUC is a nucleoside/tide or nucleoside/tide analog; D is a
dibenzorhodamine dye compound of claim 1, NUC and D being connected
by a linkage; wherein if NUC comprises a purine base, the linkage
is attached to the 8-position of the purine, if NUC comprises a
7-deazapurine base, the linkage is attached to the 7-position of
the 7-deazapurine, and if NUC comprises a pyrimidine base, the
linkage is attached to the 5-position of the pyrimidine.
102-103. (canceled)
104. A method of polynucleotide sequencing comprising the steps of:
forming a mixture of a first, a second, a third, and a forth class
of polynucleotides such that: each polynucleotide in the first
class includes a 3'-terminal dideoxyadenosine and is labeled with a
first dye; each polynucleotide in the second class includes a
3'-terminal dideoxycytidine and is labeled with a second dye; each
polynucleotide in the third class includes a 3'-terminal
dideoxyguanosine and is labeled with a third dye; and each
polynucleotide in the forth class includes a 3'-terminal
dideoxythymidine and is labeled with a forth dye; wherein at least
one of the first, second, third, or forth dyes is a
dibenzorhodamine compound of claim 1; the other of the dyes being
spectrally resolvable from the dibenzorhodamine dye(s) and from
each other; electrophoretically separating the polynucleotides
thereby forming bands of similarly sized polynucleotides;
illuminating the bands with an illumination beam capable of causing
the dyes to fluoresce; and identifying the classes of the
polynucleotides in the bands by the fluorescence spectrum of the
dyes.
105. A method of fragment analysis comprising: forming labeled
polynucleotide fragments, the fragments being labeled with a
dibenzorhodamine compound of claim 1; subjecting the labeled
polynucleotide fragments to a size-dependent separation process;
and detecting the labeled polynucleotide fragments subsequent to
the separation process.
106. (canceled)
Description
[0001] This application is a divisional of application Ser. No.
11/177,233, which is a continuation of application Ser. No.
10/441,950, filed May 20, 2003, which is a continuation of
application Ser. No. 09/969,430 filed Oct. 2, 2001 which is a
division of application Ser. No. 09/784,943, filed Feb. 14, 2001,
which is a continuation of application Ser. No. 09/556,040, filed
Apr. 20, 2000, now U.S. Pat. No. 6,221,606, which is a division of
application Ser. No. 09/199,402, filed Nov. 24, 1998, now U.S. Pat.
No. 6,111,116, which is a division of application Ser. No.
08/978,775, filed Nov. 25, 1997, now U.S. Pat. No. 5,936,087, which
are all incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to fluorescent dye
compounds. More specifically, this invention relates to modified
rhodamine dyes useful as fluorescent labeling reagents.
BACKGROUND
[0003] The non-radioactive detection of biological analytes
utilizing fluorescent labels is an important technology in modern
molecular biology. By eliminating the need for radioactive labels,
safety is enhanced and the environmental impact and costs
associated with reagent disposal is greatly reduced. Examples of
methods utilizing such non-radioactive fluorescent detection
include 4-color automated DNA sequencing, oligonucleotide
hybridization methods, detection of polymerase-chain-reaction
products, immunoassays, and the like.
[0004] In many applications it is advantageous to employ multiple
spectrally distinguishable fluorescent labels in order to achieve
independent detection of a plurality of spatially overlapping
analytes, e.g., single-tube multiplex DNA probe assays and 4-color
automated DNA sequencing methods. In the case of multiplex DNA
probe assays, by employing spectrally distinguishable fluorescent
labels, the number of reaction tubes may be reduced thereby
simplifying experimental protocols and facilitating the production
of application-specific reagent kits. In the case of 4-color
automated DNA sequencing, multicolor fluorescent labeling allows
for the analysis of multiple bases in a single lane thereby
increasing throughput over single-color methods and reducing
uncertainties associated with inter-lane electrophoretic mobility
variations.
[0005] Assembling a set of multiple spectrally distinguishable
fluorescent labels is problematic. Multi-color fluorescent
detection imposes at least six severe constraints on the selection
of dye labels, particularly for applications requiring a single
excitation light source, an electrophoretic separation, and/or
treatment with enzymes, e.g., automated DNA sequencing. First, it
is difficult to find a set of structurally similar dyes whose
emission spectra are spectrally resolved, since the typical
emission band half-width for organic fluorescent dyes is about
40-80 nanometers (nm). Second, even if dyes with non-overlapping
emission spectra are identified, the set may still riot be suitable
if the respective fluorescent quantum efficiencies are too low.
Third, when several fluorescent dyes are used concurrently,
simultaneous excitation becomes difficult because the absorption
bands of the dyes are usually widely separated. Fourth, the charge,
molecular size, and conformation of the dyes must not adversely
affect the electrophoretic mobilities of the analyte. Fifth, the
fluorescent dyes must be compatible with the chemistry used to
create or manipulate the analyte, e.g., DNA synthesis solvents and
reagents, buffers, polymerase enzymes, ligase enzymes, and the
like. Sixth, the dye must have sufficient photostability to
withstand laser excitation.
[0006] Currently available multiplex dye sets suitable in 4-color
automated DNA sequencing applications require blue or blue-green
laser light to adequately excite fluorescence emissions from all of
the dyes making up the set, e.g., argon-ion lasers. Use of Blue or
blue-green lasers in commercial automated DNA sequencing systems is
disadvantageous because of the high cost and limited lifetime of
such lasers.
SUMMARY
[0007] The present invention is directed towards our discovery of a
class of dibenzorhodamine dye compounds suitable for the creation
of sets of spectrally-resolvable fluorescent labels useful for
multi-color fluorescent detection. The subject dye compounds are
particularly well suited for use in automated 4-color DNA
sequencing systems using an excitation light source having a
wavelength greater than about 630 nm, e.g., a helium-neon gas laser
or a solid state diode laser.
[0008] In a first aspect, the invention comprises dibenzorhodamine
dye compounds having the structure ##STR4## including nitrogen- and
aryl-substituted forms thereof.
[0009] In a second aspect, the invention comprises intermediates
useful for the synthesis of dibenzorhodamine compounds having the
structure ##STR5## including nitrogen- and aryl-substituted forms
thereof.
[0010] In a third aspect, the invention comprises intermediates
useful for the synthesis of dibenzorhodamine compounds having the
structure ##STR6## including nitrogen- and aryl-substituted forms
thereof, wherein R.sub.1 taken together with the C-12-bonded
nitrogen and the C-12 and C-13 carbons forms a first ring structure
having from 4 to 7 members; and/or R.sub.1 taken together with the
C-12-bonded nitrogen and the C-11and C-12 carbons forms a second
ring structure having from 5 to 7 members.
[0011] In a fourth aspect, the invention includes energy transfer
dye compounds comprising a donor dye, an acceptor dye, and a linker
linking the donor and acceptor dyes. The donor dye is capable of
absorbing light at a first wavelength and emitting excitation
energy in response, and the acceptor dye is capable of absorbing
the excitation energy emitted by the donor dye and fluorescing at a
second wavelength in response. The linker serves to facilitate the
efficient transfer of energy between the donor dye and the acceptor
dye. According to the present invention, at least one of the donor
and acceptor dyes is a dibenzorhodamine dye having the structure
set forth above.
[0012] In a fifth aspect, the present invention includes labeled
nucleoside/tides having the structure NUC-D wherein NUC is a
nucleoside/tide or nucleoside/tide analog and D is a
dibenzorhodainine dye compound having the structure set forth
above. According to the invention, NUC and D are connected by a
linkage wherein the linkage is attached to D at one of the
substituent positions. Furthermore, if NUC comprises a purine base,
the linkage is attached to the 8-postition of the purine, if NUC
comprises a 7-deazapurine base, the linkage is attached to the
7-position of the 7-deazapurine, and if NUC comprises a pyrimidine
base, the linkage is attached to the 5-position of the
pyrimidine.
[0013] In a sixth aspect, the invention includes polynucleotide
analysis methods comprising the steps of forming a set of labeled
polynucleotide fragments labeled with a dibenzorhodamine dye having
the structure set forth above, subjecting the labeled
polynucleotide fragments to a size-dependent separation process,
e.g., electrophoresis, and detecting the labeled polynucleotide
fragments subsequent to the separation process.
[0014] Various aspects of the above-described invention achieve one
or more of the following important advantages over known
fluorescent dye compounds useful for multiplex fluorescent
detection: (1) the subject dye compounds may be efficiently excited
by a low-cost red laser using wavelengths at or above 630 nm; (2)
the emission spectra of the subject dye compounds can be modulated
by minor variations in the type and location of nitrogen and/or
aryl-substituents, allowing for the creation of dye sets having
similar absorption characteristics yet spectrally resolvable
fluorescence emission spectra; (3) the subject dye compounds may be
easily attached to nucleosides/tides or polynucleotides without
compromising their favorable fluorescence properties; (4) the
subject dye compounds have narrow emission bandwidths, i.e., the
emission bandwidth has a full-width at half the maximum emission
intensity of below about 50 nm; (5) the subject dye compounds are
highly soluble in buffered aqueous solution while retaining a high
quantum yield; (6) the subject dye compounds are relatively
photostable; and (7) the subject dye compounds have relatively
large extinction coefficients, i.e., greater than about 50,000.
[0015] These and other features and advantages of the present
invention will become better understood with reference to the
following description, figures, and appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIGS. 1-3 show exemplary synthetic pathways for the
synthesis of the 1 -amino-3-hydroxynapthalene intermediates of the
invention.
[0017] FIG. 4 shows a generalized synthetic pathway for the
synthesis of the dibenzorhodamine dye compounds of the
invention.
[0018] FIGS. 5 and 6 show exemplary synthetic pathways for the
synthesis of the dibenzorhodamine dye compounds of the
invention.
[0019] FIG. 7 shows the structures of several exemplary
dibenzorhodamine dye compounds of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] Reference will now be made in detail to the preferred
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. While the invention will be described in
conjunction with the preferred embodiments, it will be understood
that they are not intended to limit the invention to those
embodiments. On the contrary, the invention is intended to cover
all alternatives, modifications, and equivalents, which may be
included within the invention as defined by the appended
claims.
[0021] Generally, the present invention comprises a novel class of
dibenzorhodamine dye compounds useful as fluorescent labels,
methods and intermediates for synthesis of such dyes, reagents
employing such dyes, and methods utilizing such dyes and reagents
in the area of analytical biotechnology. The compounds of the
present invention find particular application in the area of
fluorescent nucleic acid analysis, e.g., automated DNA sequencing
and fragment analysis, detection of probe hybridization in
hybridization arrays, detection of nucleic acid amplification
products, and the like.
I. Definitions
[0022] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings:
[0023] "Spectral resolution" in reference to a set of dyes means
that the fluorescent emission bands of the dyes are sufficiently
distinct, i.e., sufficiently non-overlapping, that reagents to
which the respective dyes are attached, e.g. polynucleotides, can
be distinguished on the basis of a fluorescent signal generated by
the respective dyes using standard photodetection systems, e.g.
employing a system of band pass filters and photomultiplier tubes,
charged-coupled devices and spectrographs, or the like, as
exemplified by the systems described in U.S. Pat. Nos. 4,230,558,
4,811,218, or in Wheeless et al, pgs. 21-76, in Flow Cytometry:
Instrumentation and Data Analysis (Academic Press, New York,
1985).
[0024] "Electron-rich heterocycle" means cyclic compounds in which
one or more ring atoms are not carbon, i.e., are hetero atoms, and
the heteroatoms have unpaired electrons which contribute to a
6-.pi. electronic system. Exemplary electron-rich heterocycles
include but are not limited to pyrrole, indole, furan, benzofuran,
thiophene, benzothiophene and other like structures.
[0025] "Linking group" means a moiety capable of reacting with a
"complementary functionality" attached to a reagent or member of an
energy transfer dye pair, such reaction forming a "linkage"
connecting the dye to the reagent or member of the energy transfer
dye pair. Preferred linking groups include isothiocyanate, sulfonyl
chloride, 4,6-dichlorotriazinyl, succinimidyl ester, or other
active carboxylate whenever the complementary functionality is
amine. Preferably the linking group is maleimide, halo acetyl, or
iodoacetainide whenever the complementary functionality is
sulfhydryl. See R. Haugland, Molecular Probes Handbook of
Fluorescent Probes and Research Chemicals, Molecular probes, Inc.
(1992). In a particularly preferred embodiment, the linking group
is a N-hydroxysuccinimidyl (NHS) ester and the complementary
functionality is an amine, where to form the NHS ester, a dye of
the invention including a carboxylate linking group is reacted with
dicyclohexylcarbodiimide and N-hydroxysuccinimide.
[0026] "Substituted" as used herein refers to a molecule wherein
one or more hydrogen atoms are replaced with one or more
non-hydrogen atoms, functional groups or moieties. For example, an
unsubstituted nitrogen is --NH.sub.2, while a substituted nitrogen
is --NHCH.sub.3. Exemplary substituents include but are not limited
to halo, e.g., fluorine and chlorine, lower alkyl, lower alkene,
lower alkyne, sulfate, sulfonate, sulfone, amino, ammonium, amido,
nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic
aromatic, electron-rich heterocycle water-solubilizing group, and
linking group.
[0027] "Polycyclic aromatic" means aromatic hydrocarbons having
multiple ring structures including biaryls and condensed benzenoid
hydrocarbons. The biaryls are benzenoid compounds where two or more
rings are linked together by a single bond. The parent system of
this class is biphenyl. The condensed benzenoid compounds are
characterized by two or more benzene rings fused together at ortho
positions in such a way that each pair of rings shares two carbons.
The simplest members of this group are napthalene, with two rings,
and anthracene and phenanthrene, each with three rings.
[0028] "Lower alkyl" denotes straight-chain and branched
hydrocarbon moieties containing from 1 to 8 carbon atoms, e.g.,
methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl,
neopentyl, tert-pentyl, and the like.
[0029] "Lower alkene" denotes a hydrocarbon containing from 1 to 8
carbon atoms wherein one or more of the carbon-carbon bonds are
double bonds.
[0030] "Lower alkyne" denotes a hydrocarbon containing from 1 to 8
carbon atoms wherein one or more of the carbons are bonded with a
triple bond.
[0031] "Nucleoside" refers to a compound consisting of a purine,
deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine,
cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the
like, linked to a pentose at the 1' position. When the nucleoside
base is purine or 7-deazapurine, the sugar moiety is attached at
the 9-position of the purine or deazapurine, and when the
micleoside base is pyrimidine, the sugar moiety is attached at the
1-position of the pyrimidine, e.g., Kornberg and Baker, DNA
Replication, 2nd Ed. (Freeman, San Francisco, 1992). The term
"nucleotide" as used herein refers to a phosphate ester of a
nucleoside, e.g., triphosphate esters, wherein the most common site
of esterification is the hydroxyl group attached to the C-5
position of the pentose. The tern "nucleoside/tide" as used herein
refers to a set of compounds including both nucleosides and
nucleotides. "Analogs" in reference to nucleosides/tides include
synthetic analogs having modified base moieties, modified sugar
moieties and/or modified phosphate moieties, e.g. described
generally by Scheit, Nucleotide analogs (John Wiley, New York,
1980). Phosphate analogs comprise analogs of phosphate wherein the
phosphorous atom is in the +5 oxidation state and one or more of
the oxygen atoms is replaced with a non-oxygen moiety. Exemplary
analogs include but are not limited to phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate,
boronophosphates, including associated counterions, e.g., H.sup.+,
NH.sub.4.sup.+, Na.sup.+, if such counterions are present.
Exemplary base analogs include but are not limited to
2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne,
isocytosine, isoguanine, 2-thiopyrimidine, and other like analogs.
Exemplary sugar analogs include but are not limited to 2'- or
3'-modifications where the 2'- or 3'-position is hydrogen, hydroxy,
alkoxy, e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy,
isobutoxy and phenoxy, amino or alkylamino, fluoro, chloro and
bromo. The term "labeled nucleoside/tide" refers to
nucleosides/tides which are covalently attached to the dye
compounds of Formula I through a linkage.
[0032] "Water solubilizing group" means a substituent which
increases the solubility of the compounds of the invention in
aqueous solution. Exemplary water-solubilizing groups include but
are not limited to quaternary amine, sulfate, sulfonate,
carboxylate, phosphate, polyether, polyhydroxyl, and boronate.
[0033] "Polynucleotide" or "oligonucleotide" means polymers of
natural nucleotide monomers or analogs thereof, including double
and single stranded deoxyribonucleotides, ribonucleotides,
.alpha.-anomeric forms thereof, and the like. Usually the
nucleoside monomers are linked by phosphodiester linkages, where as
used herein, the term "phosphodiester linkage" refers to
phosphodiester bonds or bonds including phosphate analogs thereof,
including associated counterions, e.g., H.sup.+, NH.sub.4.sup.+,
Na.sup.+, if such counterions are present. Polynucleotides
typically range in size from a few monomeric units, e.g. 5-40, to
several thousands of monomeric units. Whenever a polynucleotide is
represented by a sequence of letters, such as "ATGCCTG," it will be
understood that the nucleotides are in 5'->3' order from left to
right and that "A" denotes deoxyadenosine, "C" denotes
deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes
deoxythymidine, unless otherwise noted.
[0034] "Rhodamine dye" refers to dyes including the general
polycyclic structure ##STR7## including any and all substituted
versions thereof. II. 1-AMINO-3-HYDROXYNAPTHALENE INTERMEDIATES
[0035] A. Structure
[0036] In a first aspect, the present invention comprises a novel
class of 1-amino-3-hydroxynapthalene compounds useful as
intermediates in the synthesis of dibenzorhodamine dyes. These
compounds have the general structure shown in Formula I immediately
below, including substituted forms thereof, where R.sub.1 taken
together with the C-12-bonded nitrogen and the C-12 and C-13
carbons forms a first ring structure having from 4 to 7 members;
and/or R.sub.1 taken together with the C-12-bonded nitrogen and the
C-11 and C-12 carbons forms a second ring structure having from 5
to 7 members. (Note that all molecular structures provided herein
are intended to encompass not only the exact electronic structures
presented, but also include all resonant structures, protonation
states and associated counterions thereof.) ##STR8##
[0037] In the compound of Formula I, preferably the first ring
structure has five members. More preferably, the five membered
first ring structure includes one gem disubstituted carbon, e.g.,
wherein the gem substituents are lower alkyl, e.g., methyl. In an
alternative preferred embodiment, the five membered ring is
substituted with linking group or water-solubilizing group.
[0038] In another preferred embodiment of the intermediate of
Formula I, the second ring structure has six members. More
preferably, the six-membered second ring structure includes one gem
disubstituted carbon, e.g., wherein the gem substituents are lower
alkyl, e.g., methyl. In an alternative preferred embodiment, the
five membered ring is substituted with linking group.
[0039] Preferably, the compound of Formula I includes one or more
nitrogen substituents. Exemplary nitrogen substituents include but
are not limited to lower alkyl, lower alkene, lower alkyne, phenyl,
aromatic, electron-rich heterocycle, polycyclic aromatic,
water-solubilizing group, and linking group, including substituted
forms thereof. In a particularly preferred embodiment, the nitrogen
substituents are lower alkyl and/or phenyl, including substituted
forms thereof. More preferably, the nitrogen substituents are
substituted lower alkyl or substituted phenyl, wherein the
substituent is linking group, or water-solubilizing group.
[0040] In an additional preferred embodiment, one or more of
carbons at positions C-8 to C-11 are substituted. Exemplary
substituents include but are not limited to fluorine, chlorine,
lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate,
sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile,
lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic,
electron-rich heterocycle, water-solubilizing group, and linking
group, including substituted forms thereof. Preferably, one or more
of the substituents is sulfonate.
[0041] Several representative 1-amino-3-hydroxynapthalene compounds
of the invention are shown in FIGS. 1-3, i.e., compounds 4, 9, 15,
17, 22, 27 and 29.
[0042] B. Synthetic Methods
[0043] Several synthetic methods are available for the synthesis of
the 1-amino-3-hydroxynapthalene compounds described above,
different methods being preferred depending on the nature of the
ring structure and the nitrogen substituents of the particular
compound to be synthesized.
[0044] A first preferred synthesis method suitable for the
synthesis of 1-substituted-amino-3hydroxynapthalene compounds,
e.g., 1-diethylamino-3-hydroxynapthalene 4, is shown in FIG. 1. In
this first method, a 3-methoxy-1-hydroxy napthalene 1 is reacted
with dry triethylamine and trifluoromethanesulfonic anhydride to
form a crude 3-methoxynapthalene-1-triflate 2. The triflate 2 is
then reacted with an amine, e.g., a secondary amine, e.g.,
diethylamine, using palladium catalyzed triflate/amine coupling to
form the substituted amine compound 3. Compound 3 is then
deprotected using a boron tribromide deprotection procedure to
produce the 1-amino-3-hydroxynapthalene product, e.g.,
1-diethylamino-3-hydroxynapthalene 4. An example of this synthesis
is provided in Example 1 below.
[0045] A second preferred synthesis method suitable for the
synthesis of benzoindoline compounds, e.g.,
N-phenyl-3,3-dimethyl-4-hydroxy-benzoindoline 9, is also shown in
FIG. 1. In this method, the 3-methoxynapthalene-1-triflate 2 is
derivatized with a primary amine, e.g., aniline, using a palladium
catalyzed triflate coupling reaction to give a secondary amine,
e.g., 1-anilino-3-methoxynapthalene 5. The secondary amine 5 is
acetylated using an acid chloride, e.g., an haloacetylchloride, to
give a disubstituted amide, e.g., 1-amido-3-methoxynapthalene 6.
The tertiary amide 6 is cyclized using a Lewis-acid-catalyzed
Friedel-Crafts cyclization procedure to give compound 7, e.g.,
using AlCl.sub.3. Compound 7 is than reduced, e.g., using LAH, to
give compound 8. Subsequent methoxy group deprotection by a boron
tribromide deprotection procedure gives the benzoindoline, e.g.,
N-phenyl-3,3-dimethyl-4-hydroxy-benzoindoline 9. An example of this
synthesis is provided in Example 2 below.
[0046] A third preferred synthesis method suitable for the
synthesis of N-substituted-5-hydroxy-(tetrahydro)benzoquinoline
compounds, e.g., N-methyl-5-hydroxy -(tetrahydro)benzoquinoline 15,
is shown in FIG. 2. In this method, compound 10 is synthesized from
methoxy-napthaldehyde by condensation with malonic acid using a
piperidine catalyst in pyridine. Compound 10 is then reduced with
hydrogen, followed by LAH reduction, and reacted with
trifluoromethanesulfonic anhydride to give the triflate 11. The
triflate 11 is reacted with NaN.sub.3 to give compound 12. Compound
12 is complexed with a Lewis acid, e.g., AlCl.sub.3, and refluxed
yielding the cyclized benzoquinoline derivative 13. Next, a
nitrogen substituent is added, e.g., the nitrogen is alkylated
using a conventional alkylation procedure, e.g., the benzoquinoline
derivative 13 is reacted with n-butyl lithium and an alkylating
agent, e.g., MeI to give compound 14 or propane sultone to give
compound 16. The methoxy group is then removed by a boron
tribromide procedure giving a N-alkylbenzoquinoline derivative,
e.g., compound 15 or 17. An example of this synthesis is provided
in Example 3 below.
[0047] A fourth preferred synthesis method suitable for the
synthesis of N-substituted-2,2,4-trimethyl-5-hydroxy-benzoquinoline
compounds, e.g. N-methyl-2,2,4-trimethyl-5-hydroxy
-(tetrahydro)benzoquinoline 22, is shown in FIG. 3. In this method,
following the procedure of A. Rosowsky and E. J. Modest (J.O.C. 30
1832 1965, and references therein), 1-amino-3-methoxynapthalene 18
is reacted with acetone catalyzed by iodine and then quenched with
saturated Na.sub.2S.sub.2O.sub.3 to give the benzoquinoline
compound 19. Compound 19 is then alkylated with an alkylating
agent, e.g., MeI, according to a general alkylation procedure to
give compound 20. The alkylated compound 20 is reduced with H.sub.2
catalyzed by Pd/C to give a N-methyl-methoxyquinoline intermediate
21, and subsequent methoxy group deprotection by a general boron
tribromide procedure yields the
N-substituted-2,2,4-trimethyl-5-hydroxy-benzoquinoline compound,
e.g., N-methyl-2,2,4-trimethyl-5 -hydroxy
-(tetrahydro)benzoquinoline 22. An example of this synthesis is
provided in Example 4 below.
[0048] A fifth preferred general synthesis method suitable for the
synthesis of N-substituted-3,3-dimethyl-4-hydroxy-benzoindoline
compounds, e.g N-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27, is
also shown in FIG. 3. In this method, a 1-amino-3-methoxynapthlene
18 is acetylated with an acid chloride, e.g.,
2-bromo-2-methylpropionyl chloride, to give compound 23. Compound
23 is cyclized by reaction with AlCl.sub.3 to give compound 24.
Compound 24 is then reduced with LAH to give the
3,3-dimethyl-4-methoxybenzoindoline 25. Compound 25 is then
alkylated with an alkylating agent, e.g., methyl iodide, to give a
N-methyl-3,3-dimethyl-4-methoxybenzoindoline, e.g., compound 26.
Subsequent methoxy group deprotection by with boron tribromide
gives compound 27. An example of this synthesis is provided in
Example 5 below.
III. Dibenzorhodamine Dye Compounds
[0049] A. Structure
[0050] In a second aspect, the present invention comprises a novel
class of dibenzorhodamine dye compounds useful as molecular labels
having the general structure shown in Formula II immediately below,
including aryl- and nitrogen-substituted fonns thereof.
##STR9##
[0051] In one preferred embodiment of the compound of Formula II,
the compound includes a first bridging group which when taken
together with the C-12-bonded nitrogen and the C-12 and C-13
carbons forms a first ring structure having from 4 to 7 members,
and/or a second bridging group which when taken together with the
C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring
structure having from 4 to 7 members. More preferably, one or both
of the first and second ring structures has five members. In yet a
more preferred embodiment, the five membered ring structure
includes one gem disubstituted carbon, wherein the gem substituents
are lower alkyl, e.g., methyl. In an alternative preferred
embodiment, the five membered ring is substituted with linking
group.
[0052] In another preferred embodiment, the compound of Formula II
includes a C-7 substituent selected from the group consisting of
acetylene, lower alkyl, lower alkene, cyano, phenyl, heterocyclic
aromatic, electron-rich heterocycle, and substituted forms thereof.
In a more preferred embodiment, the C-7 substituent is a phenyl or
substituted phenyl having the structure ##STR10## wherein aryl
substituents at positions C-14 to C-18 taken separately may be
selected from the group consisting of hydrogen, chlorine, fluorine,
lower alkyl, carboxylic acid, sulfonic acid, --CH.sub.2OH, alkoxy,
phenoxy, linking group, and substituted forms thereof. Preferably,
the phenyl substituent at C-18 is selected from the group
consisting of carboxylic acid and sulfonate, and is most preferably
carboxylic acid. In another preferred embodiment, substituents at
positions C-14 and C-17 are chlorine. In yet another preferred
embodiment, substituents at positions C-14 to C-17 are all chlorine
or all fluorine. In a particularly preferred embodiment,
substituents at one of positions C-15 and C-16 is linking group and
the other is hydrogen, substituents at positions C-14 and C-17 are
chlorine, and a substituent at position C-18 is carboxy.
[0053] In yet another preferred embodiment of the invention, the
compound of Formula II includes one or more nitrogen substituents.
Preferably, such substituents are selected from the group
consisting of lower alkyl, lower alkene, lower alkyne, phenyl,
aromatic, electron-rich heterocycle, polycyclic aromatic,
water-solubilizing group, linking group, and substituted fonns
thereof. More preferably, the nitrogen substituents are selected
from the group consisting of lower alkyl, phenyl, and substituted
forms thereof, where exemplary substituents include linking group,
and water-solubilizing group.
[0054] In another preferred embodiment of this second aspect of the
invention, the compound of Formula II includes a third bridging
group which when taken together with the C-12-bonded nitrogen and
the C-11 and C-12 carbons forms a third ring structure having from
5 to 7 members, and/or a fourth bridging group which when taken
together with the C-2-bonded nitrogen and the C-2 and C-3 carbons
forms a fourth ring structure having from 5 to 7 members.
Preferably, one or both of the third and fourth ring structures has
six members. More preferably, the six membered ring structure
includes one gem disubstituted carbon, wherein the gem substituents
are lower alkyl, e.g., methyl.
[0055] In another preferred embodiment of the invention, the
compound of Formula II includes aryl substituents at one or more of
carbons C-1, C-3 through C-6, C-8 through C-11, and C-13. Exemplary
aryl substituents include but are not limited to fluorine,
chlorine, lower alkyl, lower alkene, lower alkyne, sulfate,
sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido,
nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic
aromatic, water-solubilizing group, electron-rich heterocycle, and
linking group, including substituted forms thereof. In a
particularly preferred embodiment, at least one substituent is
sulfonate.
[0056] Several exemplary dye compounds according to this second
aspect of the invention are shown in FIG. 7, i.e., compounds
41-47.
[0057] B. Synthetic Methods
[0058] Generally, the dibenzorhodamine dyes of the present
invention are synthesized as follows. See FIG. 4. An anhydride
derivative 30, e.g., a phthalic anhydride, is mixed with
1-amino-3-methoxy internediates 31 and 32, and Lewis acid, e.g.,
ZnCl.sub.2, where the R-substituents in compound 30 may be the same
or different, but are preferably the same. Exemplary R-substituents
include but are not limited to acetylene, lower alkyl, lower
alkene, phenyl, heterocyclic aromatic, electron-rich heterocycle,
and substituted forms thereof. The mixture is heated briefly until
melting is observed. A solvent, e.g., 1,2-dichlorobenzene, is added
to the reaction mixture, and the heterogeneous mixture is heated to
about 130.degree. C. to about 180.degree. C. The crude reaction
mixture is cooled and purified by normal phase flash chromatography
to yield dye compound 33. When the anhydride is part of a
substituted phthalic anhydride, e.g., compound 34, two isomers are
formed. See FIG. 5. The isomers 35 and 36 are separated by PTLC.
The isomerically pure dyes are identified by single spots on normal
and reverse phase TLC and by their UV/Visible absorption spectra
and their long wavelength fluorescent excitation and emission
spectra.
[0059] An alternative procedure for the synthesis of asymmetrical
dye compounds is shown in FIG. 6. In this process, an anhydride
derivative, e.g., phthalic anhydride 34, is mixed with dry
nitrobenzene and heated. The mixture is cooled to room temperature
and anhydrous AlCl.sub.3 is added with stirring. Subsequently a
1-amino-3-methoxy intermediate 31 is added with stirring and the
reaction is heated. The reaction is cooled and suspended in EtOAc.
The organic layer is washed with saturated NH.sub.4Cl, brine, dried
over Na.sub.2SO.sub.4, filtered, and the solvent removed in vacuo.
The resulting ketone intermediates 37/38 are purified and separated
into distinct isomers 37 and 38 (except where substituents at C-14
and C-17 are the same and substituents at C-15 and C-16 are the
same) by flash chromatography or recrystallization. The methoxy
group of the isomerically pure ketone intermediate 37 or 38 is
removed according to a general boron tribromide deprotection
procedure to give the amino-hydroxynapthalene ketone intermediate
39. Amino-hydroxynapthalene ketone intermediate 39 is then reacted
with a 1-amino-3-methoxy intermediate 32. The reaction is cooled,
giving isomerically pure and asymmetrically substituted product 40
that may be further purified by PTLC.
V. Energy Transfer Dyes Incorporating the Dibenzorhodamine Dyes
[0060] In another aspect, the present invention comprises energy
transfer dye compounds incorporating the dibenzorhodamine dye
compounds of Formula I. Generally, the energy transfer dyes of the
present invention include a donor dye which absorbs light at a
first wavelength and emits excitation energy in response, an
acceptor dye which is capable of absorbing the excitation energy
emitted by the donor dye and fluorescing at a second wavelength in
response, and a linker which attaches the donor dye to the acceptor
dye, the linker being effective to facilitate efficient energy
transfer between the donor and acceptor dyes. A through discussion
of the structure, synthesis and use of such energy transfer dyes is
provided by Lee et al., U.S. patent application Ser. No.
08/726,462, and Mathies et al., U.S. Pat. No. 5,654,419.
[0061] One linker according to the present invention for linking a
donor dye to an acceptor dye in an energy transfer fluorescent dye
has the general structure ##STR11## where R.sub.21 is a lower alkyl
attached to the donor dye, Z.sub.1 is either NH, sulfur or oxygen,
R.sub.22 is a substituent which includes an alkene, diene, alkyne,
a five and six membered ring having at least one unsaturated bond
or a fused ring structure which is attached to the carbonyl carbon,
and R.sub.28 includes a functional group which attaches the linker
to the acceptor dye.
[0062] In one embodiment of this linker, illustrated below, the
linker has the general structure ##STR12## where R.sub.21 and
R.sub.22 are as detailed above, Z.sub.1 and Z.sub.2 are each
independently either NH, sulfur or oxygen, R.sub.29 is a lower
alkyl, and the terminal carbonyl group is attached to a ring
structure of the acceptor dye. In the variation where Z.sub.2 is
nitrogen, the C(O)R.sub.22R.sub.29Z.sub.2 subunit forms an amino
acid subunit. Particular examples of five or six membered rings
which may be used as R.sub.22 in the linker include, but are not
limited to cyclopentene, cyclohexene, cyclopentadiene,
cyclohexadiene, furan, thiofuran, pyrrole, pyrazole, isoimidazole,
pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, triazine,
pyrazine and oxazine. Examples of fused ring structures include,
but are not limited to indene, benzofuran, thionaphthene, indole
and naphthalene. A preferred embodiment of this linker is where
R.sub.21 and R.sub.29 are methylene, Z.sub.1 and Z.sub.2 are NH,
and R.sub.22 is benzene, as shown below. ##STR13##
[0063] In another preferred embodiment of the energy-transfer-dye
aspect of the present invention, the linker attaches to the
dibenzorhodamine dye component of the energy transfer dye at the
C-1 or 13 positions, or, alternatively, where the C-7 substituent
is phenyl or substituted phenyl, at one of the C-15 or C-16
positions. In a particularly preferred embodiment, both members of
the energy transfer pair are dibenzorhodamine dyes, and the first
member is linked through the C-1 position and the second member is
linked through one of the C-15 or C-16 positions.
[0064] In yet another preferred embodiment of the
energy-transfer-dye aspect of the present invention, a first member
the dye pair is a dibenzorhodamine dye, and a second member of the
dye pair is cyanine, phthalocyanine, squaraine, bodipy,
fluorescein, or dibenzorhodamine dye having different substitutions
than the first member.
VI. Reagents Incorporating the Dibenzorhodamine Dyes
[0065] In another aspect, the present invention comprises reagents
labeled with the dibenzorhodamine dye compounds of Formula I.
Reagents of the invention can be virtually anything to which the
dyes of the invention can be attached. Preferably, the dye is
covalently attached to the reagent. Reagents may include but are
not limited to proteins, polypeptides, polysaccharides,
nucleotides, nucleosides, polynucleotides, lipids, solid supports,
organic and inorganic polymers, and combinations and assemblages
thereof, such as chromosomes, nuclei, living cells, such as
bacteria or other microorganisms, mammalian cells, tissues, and the
like.
[0066] A. Nucleoside/tide Reagents
[0067] A prefrred class of labeled reagents comprise
nucleoside/tides that incorporate the dibenzorhodamine dyes of the
invention. Such nucleoside/tide reagents are particularly useful in
the context of labeling polynucleotides formed by enzymatic
synthesis, e.g., nucleotide triphosphates used in the context of
PCR amplification, Sanger-type polynucleotide sequencing, and
nick-translation reactions.
[0068] Generally, the structure of the labeled nucleoside/tide
reagent is NUC-D FORMULA III where NUC is a nucleoside/tide or
nucleoside/tide analog and D is a dibenzorhodamine dye compound of
Formula II.
[0069] The linkage linking the nucleoside/tide and the dye may be
attached to the dye at any one of substituent positions C-1 to C-18
or at a C-2 bonded nitrogen or a C-12 bonded nitrogen. Preferably,
the dye includes a phenyl or substituted phenyl substituent at the
C-7 position and is attached to the nucleoside/tide at one of the
C-15 or C-16 substituent positions, the other position being a
hydrogen atom.
[0070] When NUC includes a purine base, the linkage between NUC and
D is attached to the N.sup.8-position of the purine, when NUC
includes a 7-deazapurine base, the linkage is attached to the
N.sup.7-position of the 7-deazapurine, and when NUC includes a
pyrimidine base, the linkage is attached to the N.sup.5-position of
the pyrimidine.
[0071] Nucleoside labeling can be accomplished using any one of a
large number of known nucleoside/tide labeling techniques employing
known linkages, linking groups, and associated complementary
functionalities. Generally, the linkage linking the dye and
nucleoside should (i) not interfere with oligonucleotide-target
hybridization, (ii) be compatible with relevant enzymes, e.g.,
polymerases, ligases, and the like, and (iii) not adversely affect
the fluorescence properties of the dye. Exemplary base labeling
procedures suitable for use in connection with the present
invention include the following: Gibson et al, Nucleic Acids
Research, 15:6455-6467 (1987); Gebeyehu et al, Nucleic Acids
Research, 15: 4513-4535 (1987); Haralambidis et al, Nucleic Acids
Research, 15: 4856-4876 (1987); Nelson et al., Nucleosides and
Nucleotides, 5(3): 233-241 (1986); Bergstrom, et al., JACS, 111:
374-375 (1989); and U.S. Pat. Nos. 4,855,225, 5,231,191, and
5,449,767.
[0072] Preferably, the linkages are acetylenic amido or alkenic
amido linkages, the linkage between the dye and the nicleoside/tide
base being formed by reacting an activated N-hydroxysuccinimide
(NHS) ester of the dye with an alkynylamino- or
alkenylamino-derivatized base of a nucleoside/tide. More
preferably, the resulting linkage is 3-(carboxy)amino-1-propyn-1-yl
having the structure ##STR14##
[0073] Alternative preferred linkages include substituted
propargylethoxyamido linkages having the structure
NUC-C.ident.-C-CH.sub.2OCH.sub.2CH.sub.2NR.sub.3X-D wherein X is
selected from the group consisting of ##STR15## where n ranges from
1 to 5, ##STR16## where n ranges from 1 to 5, ##STR17## R.sub.1 is
selected from the group consisting of --H, lower alkyl and
protecting group; and R.sub.3 is selected from the group consisting
of --H and lower alkyl. See Khan et al., U.S. patent application
Ser. No. 08/833,854 filed Apr. 10, 1997.
[0074] The synthesis of alkynylamino-derivatized nucleosides is
taught by Hobbs et al. in European Patent Application No.
87305844.0, and Hobbs et al., J. Org. Chem., 54: 3420 (1989).
Briefly, the alkynylamino-derivatized nucleotides are formed by
placing the appropriate halodideoxynucleoside (usually
5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides as
taught by Hobbs et al. (cited above)) and Cu(I) in a flask,
flushing with argon to remove air, adding dry DMF, followed by
addition of an alkynylamine, triethylamine and Pd(0). The reaction
mixture is stirred for several hours, or until thin layer
chromatography indicates consumption of the halodideoxynucleoside.
When an unprotected alkynylamine is used, the
alkynylamino-nucleoside can be isolated by concentrating the
reaction mixture and chromatographing on silica gel using an
eluting solvent which contains ammonium hydroxide to neutralize the
hydrohalide generated in the coupling reaction. When a protected
alkynylamine is used, methanol/methylene chloride can be added to
the reaction mixture, followed by the bicarbonate form of a
strongly basic anion exchange resin. The slurry can then be stirred
for about 45 minutes, filtered, and the resin rinsed with
additional methanol/methylene chloride. The combined filtrates can
be concentrated and purified by flash-chromatography on silica gel
using a methanol-methylene chloride gradient. The triphosphates are
obtained by standard techniques.
[0075] Particularly preferred nucleosides/tides of the present
invention are shown below in Formula IV wherein ##STR18## B is a
nucleoside/tide base, e.g., uracil, cytosine, deazaadenine, or
deazaguanosine; W.sub.1 and W.sub.2 taken separately are OH or a
group capable of blocking polymerase-mediated template-directed
polymerization, e.g., H, fluorine and the like; W.sub.3 is OH, or
mono-, di- or triphosphate or phosphate analog; and D is a dye
compound of Formula I. In one particularly preferred embodiment,
the nucleotides of the present invention are dideoxynucleotide
triphosphates having the structure shown in Formula IV.1 below,
including associated counterions if present. ##STR19## Labeled
dideoxy nucleotides such as that shown in Formula IV.1 find
particular application as chain terminating agents in Sanger-type
DNA sequencing methods utilizing fluorescent detection.
[0076] In a second particularly preferred embodiment, the
nucleotides of the present invention are deoxynucleotide
triphosphates having the structure shown in Formula IV.2 below.
##STR20## Labeled deoxynucleotides such as that shown in Formula
IV.2 find particular application as reagents for labeling
polymerase extension products, e.g., in the polymerase chain
reaction or nick-translation. B. Polynucleotide Reagents
[0077] Yet another preferred class of reagents of the present
invention comprise polynucleotides labeled with the
dibenzorhodamine dyes of the invention. Such labeled
polynucleotides are useful in a number of important contexts
including as DNA sequencing primers, PCR primers, oligonucleotide
hybridization probes, oligonucleotide ligation probes, and the
like.
[0078] In one preferred embodiment, the labeled polynucleotides of
the present invention include multiple dyes located such that
fluorescence energy transfer takes place between a donor dye and an
acceptor dye. Such multi-dye energy-transfer polynucleotides find
application as spectrally-tunable sequencing primers, e.g., Ju et
al., Proc. Natl. Acad. Sci. USA 92: 4347-4351 (1995), and as
hybridization probes, e.g., Lee et al. Nucleic Acids Research, 21:
3761-3766 (1993).
[0079] Labeled polynucleotides may be synthesized either
enzymatically, e.g., using a DNA polymerase or ligase, e.g.,
Stryer, Biochemistry, Chapter 24, W.H. Freeman and Company (1981),
or by chemical synthesis, e.g., by the phosphoramidite method, the
phosphite-triester method, and the like, e.g., Gait,
Oligonucleotide Synthesis, IRL Press (1990). Labels may be
introduced during enzymatic synthesis utilizing labeled nucleotide
triphosphate monomers as described above, or introduced during
chemical synthesis using labeled non-nucleotide or nucleotide
phosphoramidites as described above, or may be introduced
subsequent to synthesis.
[0080] Generally, if the labeled polynucleotide is made using
enzymatic synthesis, the following procedure may be used. A
template DNA is denatured and an oligonucleotide primer is annealed
to the template DNA. A mixture of deoxynucleotide triphosphates is
added to the mixture including dGTP, dATP, dCTP, and dTTP where at
least a fraction of the deoxynucleotides is labeled with a dye
compound of the invention as described above. Next, a polymerase
enzyme is added under conditions where the polyinerase enzyme is
active. A labeled polynucleotide is formed by the incorporation of
the labeled deoxynucleotides during polymerase-mediated strand
synthesis. In an alternative enzymatic synthesis method, two
primers are used instead of one, one primer complementary to the +
strand and the other complementary to the - strand of the target,
the polymerase is a thermostable polymerase, and the reaction
temperature is cycled between a denaturation temperature and an
extension temperature, thereby exponentially synthesizing a labeled
complement to the target sequence by PCR, e.g., PCR Piotocols,
Innis et al. eds., Academic Press (1990).
[0081] Labeled polynucleotides may be chemically synthesized using
the phosphoramidite method. Detailed descriptions of the chemistry
used to form polynucleotides by the phosphoramidite method are
provided elsewhere, e.g., Caruthers et al., U.S. Pat. Nos.
4,458,066 and 4,415,732; Caruthers et al., Genetic Engineering, 4:
1-17 (1982); Users Manual Model 392 and 394 Polynucleotide
Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No.
901237 (1991).
[0082] The phosphoramidite method of polynucleotide synthesis is
the preferred method because of its efficient and rapid coupling
and the stability of the starting materials. The synthesis is
performed with the growing polynucleotide chain attached to a solid
support, so that excess reagents, which are in the liquid phase,
can be easily removed by filtration, thereby eliminating the need
for purification steps between synthesis cycles.
[0083] The following briefly describes the steps of a typical
polynucleotide synthesis cycle using the phosphoramidite method.
First, a solid support including a protected nucleotide monomer is
treated with acid, e.g., trichloroacetic acid, to remove a
5'-hydroxyl protecting group, freeing the hydroxyl for a subsequent
coupling reaction. An activated intermediate is then formed by
simultaneously adding a protected phosphoramidite nucleoside
monomer and a weak acid, e.g., tetrazole, to the reaction. The weak
acid protonates the nitrogen of the phosphoramidite forming a
reactive intermediate. Nucleoside addition is complete within 30 s.
Next, a capping step is performed which terminates any
polynucleotide chains that did not undergo nucleoside addition.
Capping is preferably done with acetic anhydride and
1-methylimidazole. The internucleotide linkage is then converted
from the phosphite to the more stable phosphotriester by oxidation
using iodine as the preferred oxidizing agent and water as the
oxygen donor. After oxidation, the hydroxyl protecting group is
removed with a protic acid, e.g., trichloroacetic acid or
dichloroacetic acid, and the cycle is repeated until chain
elongation is complete. After synthesis, the polynucleotide chain
is cleaved from the support using a base, e.g., ammonium hydroxide
or t-butyl amine. The cleavage reaction also removes any phosphate
protecting groups, e.g., cyanoethyl. Finally, the protecting groups
on the exocyclic amines of the bases and the hydroxyl protecting
groups on the dyes are removed by treating the polynucleotide
solution in base at an elevated temperature, e.g., 55.degree.
C.
[0084] Any of the phosphoramidite nucleoside monomers may be
dye-labeled phosphoramidites as described above. If the 5'-terminal
position of the nucleotide is labeled, a labeled non-nucleotidic
phosphoramidite of the invention may be used during the final
condensation step. If an internal position of the oligonucleotide
is labeled, a labeled nucleotidic phosphoramidite of the invention
may be used during any of the condensation steps.
[0085] Subsequent to synthesis, the polynucleotide may be labeled
at a number of positions including the 5'-terminus, e.g.,
Oligonucleotides and Analogs, Eckstein ed., Chapter 8, IRL Press
(1991) and Orgel et al., Nucleic Acids Research 11(18): 6513
(1983); U.S. Pat. No. 5,118,800; the phosphodiester backbone, e.g.,
ibid., Chapter 9; or at the 3'-terminus, e.g., Nelson, Nucleic
Acids Research 20(23): 6253-6259, and U.S. Pat. Nos. 5,401,837 and
5,141,813. For a through review of oligonucleotide labeling
procedures see R. Haugland in Excited States of Biopolymers,
Steiner ed., Plenum Press, NY (1983).
[0086] In one preferred post-synthesis chemical labeling method an
oligonucleotide is labeled as follows. A dye including a carboxy
linking group is converted to the N-hydroxysuccinimide ester by
reacting with approximately 1 equivalent of
1,3-dicyclohexylcarbodiimide and approximately 3 equivalents of
N-hydroxysuccinimide in dry ethyl acetate for 3 hours at room
temperature. The reaction mixture is washed with 5% HCI, dried over
magnesium sulfate, filtered, and concentrated to a solid which is
resuspended in DMSO. The DMSO dye stock is then added in excess
(10-20.times.) to an aminohexyl derivatized oligonucleotide in 0.25
M bicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6
hours, e.g., U.S. Pat. No. 4,757,141. The dye labeled
oligonucleotide is separated from unreacted dye by passage through
a size-exclusion chromatography column eluting with buffer, e.g.,
0.1 molar triethylamine acetate (TEAA). The fraction containing the
crude labeled oligonucleotide is further purified by reverse phase
HPLC employing gradient elution.
VII. Methods Utilizing the Dibenzorhodamine Dyes
[0087] The dyes and reagents of the present invention are well
suited to any method utilizing fluorescent detection, particularly
methods requiring the simultaneous detection of multiple
spatially-overlapping analytes. Dyes and reagents of the invention
are particularly well suited for identifying classes of
polynucleotides that have been subjected to a biochemical
separation procedure, such as electrophoresis, or that have been
distributed among locations in a spatially-addressable
hybridization array.
[0088] In a preferred category of methods referred to herein as
"fragment analysis" or "genetic analysis" methods, labeled
polynucleotide fragments are generated through template-directed
enzymatic synthesis using labeled primers or nucleotides, e.g., by
ligation or polymerase-directed primer extension; the fragments are
subjected to a size-dependent separation process, e.g.,
electrophoresis or chromatography; and, the separated fragments are
detected subsequent to the separation, e.g., by laser-induced
fluorescence. In a particularly preferred embodiment, multiple
classes of polynucleotides are separated simultaneously and the
different classes are distinguished by spectrally resolvable
labels.
[0089] One such fragment analysis method known as amplified
fragment length polymorphism detection (AmpFLP) is based on
amplified fragment length polymorphisms, i.e., restriction fragment
length polymorphisms that are amplified by PCR. These amplified
fragments of varying size serve as linked markers for following
mutant genes through families. The closer the amplified fragment is
to the mutant gene on the chromosome, the higher the linkage
correlation. Because genes for many genetic disorders have not been
identified, these linkage markers serve to help evaluate disease
risk or paternity. In the AmpFLPs technique, the polynucleotides
may be labeled by using a labeled polynucleotide PCR primer, or by
utilizing labeled nucleotide triphosphates in the PCR.
[0090] In another such fragment analysis method known as nick
translation, a reaction is used to replace unlabeled nucleoside
triphosphates in a double-stranded DNA molecule with labeled ones.
Free 3'-hydroxyl groups are created within the unlabeled DNA by
"nicks" caused by deoxyribonuclease I (DNAase I) treatment. DNA
polymerase I then catalyzes the addition of a labeled nucleotide to
the 3'-hydroxyl terminus of the nick. At the same time, the 5' to
3'-exonuclease activity of this enzyme eliminates the nucleotide
unit from the 5'-phosphoryl terminus of the nick. A new nucleotide
with a free 3'--OH group is incorporated at the position of the
original excised nucleotide, and the nick is shifted along by one
nucleotide unit in the 3' direction. This 3' shift will result in
the sequential addition of new labeled nucleotides to the DNA with
the removal of existing unlabeled nucleotides. The nick-translated
polynucleotide is then analyzed using a separation process, e.g.,
electrophoresis.
[0091] Another exemplary fragment analysis method is based on
variable number of tandem repeats, or VNTRs. VNTRs are regions of
double-stranded DNA that contain adjacent multiple copies of a
particular sequence, with the number of repeating units being
variable. Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A
subset of VNTR methods are those methods based on the detection of
microsatellite repeats, or short tandem repeats (STRs), i.e.,
tandem repeats of DNA characterized by a short (2-4 bases) repeated
sequence. One of the most abundant interspersed repetitive DNA
families in humans is the (dC-dA)n-(dG-dT)n dinucleotide repeat
family (also called the (CA)n dinucleotide repeat family). There
are thought to be as many as 50,000 to 100,000 (CA)n repeat regions
in the human genome, typically with 15-30 repeats per block. Many
of these repeat regions are polymorphic in length and can therefore
serve as useful genetic markers. Preferably, in VNTR or STR
methods, label is introduced into the polynucleotide fragments by
using a dye-labeled PCR primer.
[0092] In a particularly preferred fragment analysis method,
classes identified in accordance with the invention are defined in
terms of terminal nucleotides so that a conespondence is
established between the four possible terminal bases and the
members of a set of spectrally resolvable dyes. Such sets are
readily assembled from the dyes of the invention by measuring
emission and absorption bandwidths with commercially available
spectrophotometers. More preferably, the classes arise in the
context of the chemical or chain tennination methods of DNA
sequencing, and most preferably the classes arise in the context of
the chain termination methods, i.e., dideoxy DNA sequencing, or
Sanger-type sequencing.
[0093] Sanger-type sequencing involves the synthesis of a DNA
strand by a DNA polymerase in vitro using a single-stranded or
double-stranded DNA template whose sequence is to be determined.
Synthesis is initiated at a defined site based on where an
oligonucleotide primer anneals to the template. The synthesis
reaction is terminated by incorporation of a nucleotide analog that
will not support continued DNA elongation. Exemplary
chain-terminating nucleotide analogs include the
2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs) which lack the
3'-OH group necessary for 3' to 5' DNA chain elongation. When
proper proportions of dNTPs (2'-deoxynucleoside 5'-triphosphates)
and one of the four ddNTPs are used, enzyme-catalyzed
polymerization will be terminated in a fraction of the population
of chains at each site where the ddNTP is incorporated. If labeled
primers or labeled ddNTPs are used for each reaction, the sequence
information can be detected by fluorescence after separation by
high-resolution electrophoresis. In the chain termination method,
dyes of the invention can be attached to either sequencing primers
or dideoxynucleotides. Dyes can be linked to a complementary
functionality on the 5'-end of the primer, e.g. following the
teaching in Fung et al, U.S. Pat. No. 4,757,141; on the base of a
primer; or on the base of a dideoxynucleotide, e.g. via the
alkynylamino linking groups disclosed by Hobbs et al, supra.
[0094] In each of the above fragment analysis methods labeled
polynucleotides are preferably separated by electrophoretic
procedures, e.g. Gould and Matthews, cited above; Rickwood and
Hames, Eds., Gel Electrophoresis of Nucleic Acids: A Practical
Approach, IRL Press Limited, London, 1981; Osterman, Methods of
Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag, Berlin,
1984; or U.S. Pat. Nos. 5,374,527, 5,624,800 and/or 5,552,028.
Preferably the type of electrophoretic matrix is crosslinked or
uncrosslinked polyacrylamide having a concentration (weight to
volume) of between about 2-20 weight percent. More preferably, the
polyacrylamide concentration is between about 4-8 percent.
Preferably in the context of DNA sequencing in particular, the
electrophoresis matrix includes a denaturing agent, e.g., urea,
formamide, and the like. Detailed procedures for constructing such
matrices are given by Maniatis et al., "Fractionation of Low
Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98%
Formamide or 7 M Urea," in Methods in Enzymology, 65: 299-305
(1980); Maniatis et al., "Chain Length Determination of Small
Double- and Single-Stranded DNA Molecules by Polyacrylamide Gel
Electrophoresis," Biochemistry, 14: 3787-3794 (1975); Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York, pgs. 179-185 (1982); and ABI PRISM.TM. 377
DNA Sequencer User's Manual, Rev. A, January 1995, Chapter 2 (p/n
903433, The Perkin-Elmer Corporation, Foster City, Calif.). The
optimal electrophoresis conditions, e.g., polymer concentration,
pH, temperature, concentration of denaturing agent, employed in a
particular separation depends on many factors, including the size
range of the nucleic acids to be separated, their base
compositions, whether they are single stranded or double stranded,
and the nature of the classes for which information is sought by
electrophoresis. Accordingly application of the invention may
require standard preliminary testing to optimize conditions for
particular separations.
[0095] Subsequent to electrophoretic separation, the
dye-polynucleotide conjugates are detected by measuring the
fluorescence emission from the dye labeled polynucleotides. To
perform such detection, the labeled polynucleotides are illuminated
by standard means, e.g. high intensity mercury vapor lamps, lasers,
or the like. Preferably the illumination means is a laser having an
illumination beam at a wavelength above about 600 nm. More
preferably, the dye-polynucleotides are illuminated by laser light
generated by a He-Ne gas laser or a solid-state diode laser. The
fluorescence is then detected by a light-sensitive detector, e.g.,
a photomultiplier tube, a charged coupled device, or the like.
Exemplary electrophoresis detection systems are described
elsewhere, e.g., U.S. Pat. Nos. 5,543,026; 5,274,240; 4,879,012;
5,091,652 and 4,811,218.
VIII. EXAMPLES
[0096] The invention will be further clarified by a consideration
of the following examples, which are intended to be purely
exemplary of the invention and not to in any way limit its
scope.
Materials and Methods
[0097] All chemicals were purchased from Aldrich Chemical Company
unless otherwise noted. Martius yellow was purchased from Fluka.
Acetone was dried over CaSO.sub.4 and distilled. Dichloromethane
(CH.sub.2Cl.sub.2) and nitrobenzene were dried over CaH.sub.2 and
distilled. Tetrahydrofuran (THF) was dried over lithium aluminum
hydride (LAH) and distilled as needed. Triethylamine (Et.sub.3N)
was dried over sodium and distilled. DMSO (99.9%) and
N,N-diisopropylethylamine (99.5%) were dried and stored over
activated molecular sieves. Silica gel (220-400 mesh) from VWR was
used for normal phase flash chromatography. Reverse phase
chromatography was performed on octadecyl functionalized silica gel
from Aldrich. Preparative thin layer chromatography (PTLC) was
performed on 1 and 2 mm pre-made silica gel plates from EM science
(VWR). TLC was performed on aluminum back silica gel 60 plates from
EM science (VWR). Developed spots were visualized with both long
and short wavelength UV irradiation.
[0098] Absorption spectroscopy was performed on a Hewlett Packard
model 8451A UV/Vis diode array spectrophotometer. Fluorescence
measurements were made on a Perkin-Elmer LS-50B luminescence
spectrophotometer. NMR spectra were determined on a Varian 300 MHz
NMR referenced relative to a solvent peak at 7.26 ppm in CD.sub.3Cl
or 3.31 ppm in CD.sub.3OD. HPLC purification of oligomer labeled
dye fragments was performed on a Perkin-Elmer Series 200 pump,
employing a reverse phase C-18 column, with both UV and
fluorescence emission detection. Fluorescence detection was
performed by a Perkin-Elmer LC 240 fluorescence detector equipped
with a red sensitive PMT, and UV detection was performed with a
Model LC 295 UV/Vis detector. Pump and detectors were all
interfaced with a Perkin-Elmer Model 1022 computer run in
two-channel mode. Buffers were made up fresh from the following
concentrated stock: 10.times.TBE (0.89 M
tris-(hydroxymethyl)aminomethane, 0.89 M borate, 0.02 M
ethylenediaminetetraacetic acid disodium salt), and 0.1 M TEAA
(triethylamine acetate).
[0099] All reactions were run in an oven-dried round bottom flask,
under argon atmosphere, and capped with a rubber septum. Anhydrous
solvents were manipulated under an argon atmosphere with oven-dried
syringes. As used herein, the term "aqueous workup" refers to a
purification method comprising the following steps: (i) adding a
reaction mixture to a saturated aqueous NH.sub.4Cl solution, a 5%
HCI solution, or a saturated Na.sub.2S.sub.2O.sub.3 solution, (ii)
extracting the solution three times with an organic solvent, e.g.,
EtOAc, or CH.sub.2Cl.sub.2, (iii) washing the combined organic
layer once with saturated NaCl, (iv) drying the solution with
Na.sub.2SO.sub.4, (v) filtering the drying agent, and (vi) removing
the solvent in vacuo. 3-Methoxy-1-hydroxynapthalene 1 was
synthesized from 1,3-dihydroxynapthalene by the method of K. H.
Bell and L. F. McCaffery (Aust. J. Chem. 46: 731 (1993)).
1-Amino-3-methoxynapthalene 18 was synthesized according the
procedure of G. T. Morgan and E. D. Evans (J. Chem. Soc. 115: 1126
(1919)).
Example 1
Synthesis of 1-Diethylamino-3-Hydroxynapthalene 4 (FIG. 1)
[0100] 3-methoxy-1-hydroxy napthalene 1 (1 gm) was suspended in dry
CH.sub.2Cl.sub.2 (30 mL). Dry triethylamine (1.2 equivalents) was
added and the reaction was cooled to -5.degree. C.
Trifluoromethanesulfonic anhydride (1.1 equivalents) suspended in
CH.sub.2Cl.sub.2 (15 mL) was added dropwise with vigorous stirring
over a period of 2 hours. The reaction was allowed to come to room
temperature and subjected to aqueous work up using 5% HCl and
CH.sub.2Cl.sub.2. The resulting crude
3-methoxynapthalene-1-triflate 2 was purified by normal phase flash
chromatography employing an EtOAc/Hexane (1:10) mobile phase.
[0101] The purified 3-methoxynapthalene-1-triflate 2 was converted
to the 1-diethylamino -3-methoxynapthalene 3 using the
palladium-catalyzed triflate/amine coupling procedure of Wolfe as
follows (J. P. Wolfe and S. L. Buchwald, JOC, 61: 1133 (1996)). The
3-methoxy napthalene-1-triflate 2 (1 gram) was suspended in 100 mL
of dry toluene with 0.015 equivalents of
(S)-(-)-2,2'-bis(diphenylphosphino)-1,1'-binapthyl (BINAP), 0.005
equivalents of tris(dibenzylideneacetone)dipalladium
(Pd.sub.2(dba).sub.3), and 3 equivalents of dry diethyl amine. The
reaction was purged with argon, and 3.3 equivalents of solid sodium
t-butoxide was added with stirring. The reaction was then heated,
and stirred for 16 hours at 80.degree. C. in an oil bath. The
reaction was allowed to come to room temperature and subjected to
aqueous work up using 5% HCl and CH.sub.2Cl.sub.2 resulting in a
crude 1-diethylamino-3-methoxynapthalene 3, which was purified by
normal phase flash chromatography employing EtOAc:hexane (1:49) as
the mobile phase (.sup.1HNMR: CD.sub.3Cl d 8.20 (broad d, 1 H, J=9
Hz), 7.72(broad d, 1 H, J=7.8 Hz), 7.43 (dt, 1 H, J=7.2, 1.2 Hz),
7.34 (dt, 1 H, J=7.7, 1.2 Hz), 6.88 (d, 1 H, J=2.4 Hz), ), 6.82 (d,
1 H, J=2.4 Hz), 3.93 (s, 3 H), 3.21 (q, 4 H, J=7.2 Hz0, 1.08 (t, 6
H, J=7.2 Hz)).
[0102] Next, the methyl group of the
1-diethyl-amino-3-methoxy-napthalene 3 was removed by boron
tribromide deprotection as follows. The
1-amino-3-methoxy-napthalene (100 mg) was suspended in dry
CH.sub.2Cl.sub.2 (5 mL,) and the mixture was cooled to -70.degree.
C. in a dry ice/acetone bath. Boron tribromide (10 equivalents) was
added dropwise and the reaction was stirred for 30 minutes, then
placed in a refrigerator (0.degree. C.) overnight. The reaction was
quenched at -70.degree. C. by careful addition of MeOH (10 mL).
Solid NaHCO.sub.3 (30 equivalents) was added and the reaction was
warmed to room temperature, then briefly heated to reflux. The
mixture was cooled and filtered, the filtrate was acidified with
AcOH, and the solvent was removed in vacuo to give the crude
1-diethylamino-3-hydroxynapthalene 4, which was purified by normal
phase flash chromatography employing EtOAc:hexane (1:4) as the
mobile phase.
Example 2
Synthesis of N-Phenyl-3,3-Dimethyl-Hydroxy-Benzoindoline 9 (FIG.
1)
[0103] The 3-methoxynapthalene-1-triflate 2 was derivatized with
aniline according to the palladium catalyzed triflate/amine
coupling reaction described above in Example 1 to give the
1-amino-3-methoxynapthalene 5.
[0104] The 1-anilino-3-methoxynapthalene 5 was acetylated by an
amino group acetylation procedure as follows. The
1-amino-3-methoxynapthalene 5 (500 mg) and 1.2 equivalents of dry
Et.sub.3N were suspended in 10 mL of dry CH.sub.2Cl.sub.2 and
cooled to -5.degree. C. using an ice/NaCl bath. 1.1 equivalent of
2-bromo-2-methylpropionylchloride was added dropwise and the
reaction was stirred for 1 hour at -5.degree. C. and stirred at
room temperature for an additional 1 hour. The reaction was allowed
to come to room temperature and subjected to aqueous work up using
5% HCl and EtOAc resulting in the crude intermediate
1-(bromoalkyl)amido-3-methoxy-napthalene 6, which was purified by
normal phase flash chromatography employing EtOAc: hexane (1:9) as
the mobile phase.
[0105] The 1-(bromoalkyl)amido-3-methoxy-napthalene 6 was cyclized
using an AlCl.sub.3 catalyzed Friedel-Crafts cyclization procedure
as follows. 1 to 3 equivalents of AlCl.sub.3 in nitrobenzene was
added to the 1-(bromoalkyl)amido-3-hydroxy-napthalene 6. The
reaction was heated to 130.degree. C. and reacted for 1 hour.
Aqueous work-up using NH.sub.4Cl and EtOAc gave the crude
N-phenyl-benzoindolinone intermediate 7, which was purified by
normal phase flash chromatography employing EtOAc: hexane (1:4) as
the mobile phase. The amide carbonyl group of the
N-phenyl-benzoindolione intermediate 7 was then reduced with LAH to
give compound 8 (.sup.1HNMR: CD.sub.3Cl d 7.71 (d, 1 H, J=7.8 Hz),
7.32 (m, 2 H), 7.24 (m, 2 H), 7.07 (bt, 1 H, J=6.6 Hz), 6.96 (m, 3
H), 6.84 (s, 1 H), 3.97 (s, 3 H), 3.92 (s, 2 H), 1.44 (s, 6 H).
[0106] Methoxy group deprotection of compound 8 was effected using
the boron tribromide deprotection procedure described in Example 1,
resulting in the N-phenyl-3,3-dimethyl-hydroxy-benzoindoline 9.
Example 3
Synthesis of N-Methyl-5-Hydroxy-(Tetrahydro)benzoquinoline 15 (FIG.
2)
[0107] Compound 10 was synthesized by condensation of
methoxy-napthaldehyde and malonic acid employing piperidine
catalysis in pyridine. Compound 10 was reduced with hydrogen over
10 % Pd/carbon, followed by LAH reduction, and reacted as outlined
for the synthesis of compound 2 above with trifluoromethanesulfonic
anhydride to give the triflate 11. Triflate 11 was then reacted
with NaN.sub.3 (3 equiv.) in DMF at 100.degree. C. for 6 hours.
Then, the reaction was allowed to come to room temperature and
subjected to aqueous work up using pure water and EtOAc resulting
in pure compound 12. Compound 12 was suspended in dry
CH.sub.2Cl.sub.2, complexed with 3 to 5 equivalents of solid
AlCl.sub.3, and refluxed for 2 hours yielding compound 13.
[0108] Compound 13 was alkylated with MeI according to a general
amino group alkylation procedure as follows. The
3-methoxybenzoquinoline derivative (100 mg) 13 was suspended in 5
mL of dry THF and cooled to -5.degree. C. (ice/NaCl). 1.1
equivalents of n-butyl lithium (1 M) was added dropwise, and the
reaction was stirred for 1 hour. 3 equivalents of the MeI
alkylating agent was added slowly and the reaction was allowed to
stir at room temperature for 2 hours. Aqueous work-up using
NH.sub.4Cl and EtOAc gave a crude alkylated 3-methoxybenzoquinoline
intermediate 14. Intermediate 14 was then purified by normal phase
flash chromatography employing EtOAc:hexane (1:19) as the mobile
phase (.sup.1HNMR: CD.sub.3Cl d 8.1 (broad d, 1 H, J=8.1 Hz),
7.68(dd, 1 H, J=8.1, 1.8 Hz), 7.34 (m, 2 H), 6.8 (s, 1 H), 3.92 (s,
3 H), 3.21 (m, 2 H), 2.94 (s, 3 H), 2.77 (t, 2 H, J=6.6 Hz), 1.92
(m, 2 H)). Subsequent methoxy group deprotection by the general
boron tribromide procedure described above in Example 1 resulted in
the N-methyl-hydroxybenzoquinoline derivative 15.
Example 4
Synthesis of 3-(5-Hydroxybenzoquinolin-1-yl) propanesulfonic acid
17 (FIG. 2)
[0109] Compound 13 was synthesized according to the procedure
outlined above in Example 3 for the synthesis of the
N-methyl-hydroxybenzoquinoline derivative 15. Compound 13 was then
alkylated according to the general amino group alkylation procedure
described above in Example 3, this time using 1,3-propane sultone
as the alkylating agent rather than MeI, to give a
5-methoxybenzoquinoline-N-propanesulfonic acid intermediate 16
(.sup.1HNMR: CD.sub.3OD d 7.94 (d, 1 H, J=8.7 Hz), 7.65 (d, 1 H,
J=8.4 Hz), 7.32 (t, 1 H), 7.27 (t, 1 H), 6.85 (s, 1 H), 4.89 (s, 3
H), 3.20 (m, 2 H), 3.08 (bt, 2 H, J=6 Hz), 2.91 (m, 2 H), 2.72 (t,
2 H, J=6.6 Hz), 2.33 (m, 2 H), 1.89 (m, 2 H). Subsequent methoxy
group deprotection of compound 16 by the general boron tribromide
procedure described above in Example 1 resulted in the
3-(5-hydroxybenzoquinolin-1-yl) propanesulfonic acid 17.
Example 5
Synthesis of
N-Methyl-2,2,4-Trimethyl-5-Hydroxy-(Tetrahydro)benzoquinoline 22
(FIG.3)
[0110] Following the procedure of A. Rosowsky and E. J. Modest
(J.O.C., 30: 1832 (1965)), 1-amino-3-methoxynapthalene 18 (1 gm)
was dissolved in dry acetone (50 mL,), and 0.01 equivalent of
iodine was added to the solution. The reaction was heated and
stirred for 16 hours, cooled, and then quenched with saturated
Na.sub.2S.sub.2O.sub.3. The reaction mixture was then subjected to
aqueous work up using saturated Na.sub.2S.sub.2O.sub.3 and EtOAc
resulting in the crude methoxybenzoquinoline 19. The
methoxybenzoquinoline 19 was purified by flash chromatography using
an EtOAc/hexane 1:9 mobile phase. Compound 19 was then alkylated
with MeI according to the general amino group alkylation procedure
described above in Example 3 to give compound 20. Compound 20 was
reduced with H.sub.2 in a Parr hydrogenator at 70 psi and 10% Pd/C
catalysis to give a
N-methyl-2,2,4-trimethyl-5-methoxybenzoquinoline intermediate 21
(.sup.1HNMR: CD.sub.3Cl d 8.20 (bd, 1 H, J=7.5 Hz), 7.65 (bd, 1 H,
J=7.5 Hz), 7.33 (m, 2 H), 6.89 (s, 1 H), 3.94 (s, 3 H), 3.14 (b
sextet, 1 H, J=6.6 Hz), 2.80 (3, 3 H), 1.89 (d, 2 H, J=8.7), 1.42
(d, 3 H, J=6.9 Hz), 1.34 (s, 3 H), 1.05 (s, 3 H). Subsequent
methoxy group deprotection of compound 21 by the general boron
tribromide procedure described above in Example 1 gave the
N-methyl-5-hydroxy-(tetrahydro)benzoquinoline 22.
Example 6
Synthesis of N-Methyl-3,3-Dimethyl-4-Hydroxy-Benzoindoline 27 (FIG.
3)
[0111] 1-Amino-3-methoxynapthalene 18 was acetylated with
2-bromo-2-methylpropionyl chloride according to the general amino
group acylation procedure described above in Example 2 to give
compound 23. Compound 23 was cyclized by the Friedel-Crafts
cyclization procedure described above in Example 2 to give compound
24. Next, compound 24 was reduced with 3 equivalents LAH in THF to
give the 4-methoxybenzoindoline 25. Compound 25 was alkylated using
the general amino group alkylation procedure described above in
Example 3 using methyl iodide as the alkylating agent to give a
N-methyl-3,3-dimethyl-4-methoxybenzoindoline intermediate 26
(.sup.1HNMR: CD.sub.3Cl d 8.07 (bd, 1 H, J=8.4 Hz), 7.69 (bd, 1 H,
J=8.1 Hz), 7.33 (bt, 1 H, J=7.8 Hz), 7.22 (bt, 1 H, J=8.1 Hz), 6.70
(s, 1 H), 3.92 (s, 3 H), 3.32 (s, 2 H), 3.32 (s, 3 H), 1.44 (s, 6
H). Subsequent methoxy group deprotection of compound 26 by the
general boron tribromide procedure described in Example 1 resulted
in the N-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27.
Example 7
Synthesis of N-Ethyl-3,3-Dimethyl-4-Hydroxy-Benzoindoline 29 (FIG.
3)
[0112] The 4-methoxybenzoindoline 25 was synthesized as described
above in Example 6. Compound 25 was alkylated by the general amino
group alkylation procedure described in Example 3 employing ethyl
iodide as the alkylating agent to give the
N-ethyl-3,3-dimethyl-4-methoxybenzoindoline intermediate 28
(.sup.1HNMR: CD.sub.3Cl d 7.90 (d, 1 H, J=8.7 Hz), 7.68 (d, 1 H,
J=8.1 Hz), 7.32 (bt, 1 H, J=7.5 Hz), 7.22 (bt, 1 H, J=6.9 Hz), 6.69
(s, 1 H), 3.83 (s, 3 H), 3.52 (q, 2 H J=7.5 Hz), 3.38 (s, 2 H),
1.46 (s, 6 H), 1.27 (t, 3 H, J=7.5 Hz). Subsequent methoxy group
deprotection of compound 28 by the general boron tribromide
procedure described in Example 1 yielded the
N-ethyl-3,3-dimethyl-4-hydroxy-benzoindoline 29.
Example 8
Synthesis of Selected Dibenzorhodamine Dye Compounds
[0113] General Procedure A (FIG. 5). A solid phthalic anhydride
derivative 34 was mixed with 1.4 equivalents of an aminohydroxy
intermediate 31 and 2.8 equivalents of ZnCl.sub.2. The oven dried
reaction vessel was capped with a rubber septa and purged with
Argon. The solid mixture was heated briefly at 130.degree. C. until
melting was observed, e.g., after approximately 15 minutes.
1,2-Dichlorobenzene (approximately 10 equivalents) was added by
syringe to the reaction mixture, and the heterogeneous mixture was
heated to 130.degree. C. to 170.degree. C. for 4 hours. The crude
reaction mixture was cooled, suspended in a minimal amount of MeOH:
CH.sub.2Cl.sub.2 (1:19), loaded directly onto a normal phase flash
chromatography column, and the crude dye was eluted with an MeOH:
CH.sub.2Cl.sub.2 (1:19) mobile phase. When necessary, the dye was
purified and separated into distinct isomers 35 and 36 by PTLC
developed with MeOH: CH.sub.2Cl.sub.2 (1:9). The isomerically pure
dye, which migrated as a single spot on silica TLC eluting with 1:9
MeOH:CH.sub.2Cl.sub.2, was identified by its UV/Visible absorption
spectra and its long wavelength fluorescent excitation and emission
spectra.
[0114] General Procedure B (FIG. 6). In the general procedure
outlined in FIG. 6, a solid phthalic anhydride derivative 34 (100
mg) was placed in a round bottom flask capped with a rubber septa
and purged with dry argon. Dry nitrobenzene (2 mL) was added and
heated to dissolve the anhydride. The mixture was cooled to room
temperature and 3 to 6 equivalents of anhydrous AlCl.sub.3 was
added with stirring to dissolve the solid. Subsequently, 1
equivalent of a 1-amino-3-methoxynapthalene intermediate 31 was
added with stirring and the reaction was heated to 130.degree. C.
for 1 hour. The reaction was then cooled and suspended in EtOAc.
The organic layer was washed with saturated NH.sub.4Cl and brine,
dried over Na.sub.2SO.sub.4, filtered, and the solvent was removed
in vacuo. When necessary, the resulting ketone intermediate 37/38
was purified and separated into distinct isomers 37 and 38 using
normal phase flash chromatography using (MeOH: CH.sub.2Cl.sub.2,
1:19) as the mobile phase, or by recrystallization. The methoxy
group of the isomerically pure intermediate 37 or 38 was removed
according to the general boron tribromide deprotection procedure
described in Example 1 to give amino-hydroxynapthalene ketone 39.
The amino-hydroxynapthalene ketone 39 (100 mg) was then reacted at
130.degree. C. with 1 equivalent of a 1-amino-3-napthalene
intermediate 32 in dry 1,2-dichlorobenzene (2 mL) for 2 hours. The
reaction was cooled, giving isomerically pure and asymmetrically
substituted product 40 that was purified as in General Procedure A
above.
[0115] Synthesis of Dibenzorhodamine Dye 41 (FIG. 7). General
procedure A was followed employing dichlorotriinellitic anhydride
as the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-14 and C-17 are Cl and the substituent at C-15 is
CO.sub.2H, and 1-diethylamino-3-hydroxynapthalene 4 as the
aminohydroxy intermediate 31.
[0116] Synthesis of Dibenzorhodamine Dye 42 (FIG. 7). General
procedure A was followed employing dichlorotrimellitic anhydride as
the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-1 4 and C-17 are Cl and the substituent at C-15
is CO.sub.2H, and N-methyl-5-hydroxy-benzoquinoline 15 as the
aminohydroxy intermediate 31.
[0117] Synthesis of Dibenzorhodamine Dve 43 (FIG. 7). General
procedure A was followed employing dichlorotrimellitic anhydride as
the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-14 and C-17 are Cl and the substituent at C-15 is
CO.sub.2H, and 5-hydroxy-benzoquinoline 17 as the aminohydroxy
intermediate 31.
[0118] Synthesis of Dibenzorhodamine Dye 44 (FIG. 7). General
procedure A was followed employing dichlorotrimellitic anhydride as
the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-14 and C-17 are Cl and the substituent at C-15 is
CO.sub.2H, the N-methyl-2,2,4-trimethyl-5-hydroxy-benzoquinoline 22
as the aminohydroxy intermediate 31.
[0119] Synthesis of Dibenzorhodamine Dye 45 (FIG. 7). General
procedure A was followed employing dichlorotrimellitic anhydride as
the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-14 and C-17 are Cl and the substituent at C-15 is
CO.sub.2H, and N-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27 as
the aminohydroxy intermediate 31.
[0120] Synthesis of Dibenzorhodamine Dye 46 (FIG. 7). General
procedure A was followed employing tetrafluorophthalic anhydride as
the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-14 to C-17 are F, and
N-ethyl-3,3-dimethyl-4-hydroxy-benzoindoline 29 as the aminohydroxy
intermediate 31.
[0121] Synthesis of Dibenzorhodamine Dye 47 (FIG. 7). General
procedure A was followed employing dichlorotrimellitic anhydride as
the phthalic anhydride derivative, i.e., compound 34 where the
substituents at C-14 and C-17 are Cl and the substituent at C-15 is
CO.sub.2H, and N-phenyl-3,3-dimethyl-4-hydroxy-benzoindoline 9 as
the aminohydroxy intermediate 31.
Example 9
Spectral Properties of Selected Dibenzorhodamine Dye Compounds
[0122] The following table presents important spectral properties
of several representative dibenzorhodamine dye compounds of the
invention. All spectra were recorded at room temperature, in
1.times.TBE buffer and 8 M urea, for the free dye having 0.05
absorption at the dye's .lamda..sub.max, abs. Dye concentration was
approximately 10.sup.-6 M. TABLE-US-00001 Absorption Emission Full
Width at Dye Maximum (nm) Maximum (nm) Half Max (nm) 41 585 614 59
42 609 634 42 43 597 637 47 44 598 640 50 45 639 650 31 46 639 652
33 47 632 676 66
[0123] All publications, patents, and patent applications referred
to herein are hereby incorporated by reference to the same extent
as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference.
[0124] Although only a few embodiments have been described in
detail above, those having ordinary skill in the chemical arts will
clearly understand that many modifications are possible in these
embodiments without departing from the teachings thereof. All such
modifications are intended to be encompassed within the scope
following claims.
* * * * *