U.S. patent application number 11/381342 was filed with the patent office on 2006-08-24 for atropisomers of asymmetric xanthene fluorescent dyes and methods of dna sequencing and fragment analysis.
This patent application is currently assigned to Applera Corporation. Invention is credited to Linda G. LEE, Barnett B. ROSENBLUM, Meng C. TAING.
Application Number | 20060188915 11/381342 |
Document ID | / |
Family ID | 24831567 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188915 |
Kind Code |
A1 |
LEE; Linda G. ; et
al. |
August 24, 2006 |
ATROPISOMERS OF ASYMMETRIC XANTHENE FLUORESCENT DYES AND METHODS OF
DNA SEQUENCING AND FRAGMENT ANALYSIS
Abstract
Atropisomeric energy-transfer dye compounds are disclosed. A
variety of molecular biology applications utilize atropisomeric
xanthene fluorescent dyes as labels for substrates such as
nucleotides, nucleosides, polynucleotides, polypeptides and
carbohydrates. Methods include DNA sequencing, DNA fragment
analysis, PCR, SNP analysis, oligonucleotide ligation,
amplification, minisequencing, and primer extension.
Inventors: |
LEE; Linda G.; (Palo Alto,
CA) ; TAING; Meng C.; (Hayward, CA) ;
ROSENBLUM; Barnett B.; (San Jose, 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: |
24831567 |
Appl. No.: |
11/381342 |
Filed: |
May 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10716165 |
Nov 18, 2003 |
7038063 |
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11381342 |
May 2, 2006 |
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10227058 |
Aug 21, 2002 |
6649769 |
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11381342 |
May 2, 2006 |
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09704966 |
Nov 1, 2000 |
6448407 |
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11381342 |
May 2, 2006 |
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Current U.S.
Class: |
435/6.12 ;
536/25.32; 540/123; 549/227 |
Current CPC
Class: |
C07H 21/04 20130101;
C07H 19/10 20130101; C09B 11/08 20130101; G01N 33/533 20130101;
C09B 11/24 20130101; C07H 21/00 20130101; C07H 19/20 20130101 |
Class at
Publication: |
435/006 ;
536/025.32; 549/227; 540/123 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07D 487/22 20060101 C07D487/22; C07D 405/02 20060101
C07D405/02; C07H 21/04 20060101 C07H021/04 |
Claims
1. An energy-transfer dye comprising: a donor dye capable of
absorbing light at a first wavelength and emitting excitation
energy in response thereto; an acceptor dye capable of absorbing
the excitation energy emitted by the donor dye and fluorescing at a
second wavelength in response; and a linker for linking the donor
dye and the acceptor dye; wherein at least one of the donor dye and
acceptor dye is an atropisomerically enriched xanthene compound
having the structure: ##STR21## Z.sup.1 is OH, NH.sub.2, NHR, or
NR.sub.2, wherein each R is independently hydrogen,
C.sub.1-C.sub.12 alkyl, phenyl, benzyl, aryl, heterocycle, or a
linking moiety; Z.sup.2 is O, .sup.+NH.sub.2, .sup.+NHR, or
.sup.+NR.sub.2, wherein each R is independently hydrogen,
C.sub.1-C.sub.12 alkyl, phenyl, benzyl, aryl, heterocycle, or a
linking moiety; X is carboxylate or sulfonate; and said structure
includes aryl-substituted forms thereof.
2. The energy-transfer dye of claim 1 wherein the xanthene compound
is a substantially pure atropisomer.
3. The energy-transfer dye of claim 1 wherein the donor dye is an
enriched atropisomer of a xanthene compound and the acceptor dye is
a cyanine, a phthalocyanine, a squaraine, a bodipy, a
benzophenoxazine, a fluorescein, a dibenzorhodamine, or a rhodamine
dye.
4. The energy-transfer dye of claim 1 wherein the acceptor dye is
an enriched atropisomer of a xanthene compound and the donor dye is
linked to the xanthene compound and to a polynucleotide.
5. The energy-transfer dye of claim 3 wherein the donor dye is
linked to the 5'-terminus of the polynucleotide.
6. The energy-transfer dye of claim 3 wherein the donor dye is
linked to the 3'-terminus of the polynucleotide.
7. The energy-transfer dye of claim 3 wherein the donor dye is
linked to a nucleobase of the polynucleotide, wherein if the
nucleobase is a purine, the linker is attached at the 8-position,
if the nucleobase is a 7-deazapurine, the linker is attached at the
7-position or 8-position, and if the nucleobase is a pyrimidine,
the linker is attached at the 5-position.
8. The energy-transfer dye of claim 1 wherein the linker has the
structures: ##STR22## wherein Z is selected from the group
consisting of NH, S and O; R.sup.21 is a C.sub.1-C.sub.12 alkyl
attached to the donor dye; R.sup.22 is a substituent selected from
the group consisting of a C.sub.1-C.sub.12 alkyldiyl, a five and
six membered ring having at least one unsaturated bond and a fused
ring structure which is attached to the carbonyl carbon; and
R.sup.23 includes a functional group which attaches the linker to
the acceptor dye.
9. The energy-transfer dye of claim 8 wherein the linker has the
structure: ##STR23## and n ranges from 2 to 10.
10. The energy-transfer dye of claim 8 wherein R.sup.23 has the
structure ##STR24## wherein R.sup.24 is a C.sub.1-C.sub.12
alkyl.
11. The energy-transfer dye of claim 8 wherein R.sup.22 is a five
or six membered ring selected from the group consisting of
cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan,
thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole,
pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine
oxazine, indene, benzofuran, thionaphthene, indole and
naphthalene.
12. The energy-transfer dye of claim 1 wherein the linker has the
structure ##STR25##
13. The energy-transfer dye of claim 1 wherein the linker has the
structure ##STR26##
14. The energy-transfer dye of claim 1 wherein the linker has the
structure ##STR27##
15. The energy-transfer dye of claim 1 in which the linker has the
structure: ##STR28## wherein D is a donor dye, A is an acceptor dye
and n is 1 or 2.
16. The energy-transfer dye of claim 1 wherein the linker is
attached at R.sup.1, R.sup.11, R.sup.18 or R.sup.19 of the xanthene
compound having the structure: ##STR29##
17. The energy-transfer dye of claim 16 wherein a linker to the
donor or the acceptor is attached to R.sup.1 or R.sup.11, and a
linker to a polynucleotide is attached to R.sup.18 or R.sup.19.
18. The energy-transfer dye of claim 16 wherein the linker is
attached at one of positions Z.sup.1 or Z.sup.2 of the
substantially pure atropisomeric xanthene compound.
19. The energy-transfer dye of claim 1 wherein a linking moiety is
selected from azido, monosubstituted primary amine, disubstituted
secondary amine, thiol, hydroxyl, halide, epoxide,
N-hydroxysuccinimidyl ester, carboxyl, isothiocyanate, sulfonyl
chloride, sulfonate ester, silyl halide, chlorotriazinyl,
succinimidyl ester, pentafluorophenyl ester, maleimide, haloacetyl,
epoxide, alkylhalide, allyl halide, aldehyde, ketone, acylazide,
anhydride, and iodoacetamide.
20. The energy-transfer dye of claim 1 wherein an aryl substituent
is independently selected from fluorine, chlorine, C.sub.1-C.sub.8
alkyl, carboxylate, sulfate, sulfonate (--SO.sub.3.sup.-),
alkylsulfonate (--R--SO.sub.3.sup.-), aminomethyl
(--CH.sub.2NH.sub.2), aminoalkyl, 4-dialkylaminopyridinium,
hydroxymethyl (--CH.sub.2OH), methoxy (--OCH.sub.3), hydroxyalkyl
(--ROH), thiomethyl (--CH.sub.2SH), thioalkyl (--RSH), alkylsulfone
(--SO.sub.2R), arylthio (--SAr), arylsulfone (--SO.sub.2Ar),
sulfonamide (--SO.sub.2NR.sub.2), alkylsulfoxide (--SOR),
arylsulfoxide (--SOAr), amino (--NH.sub.2), ammonium
(--NH.sub.3.sup.+), amido (--CONR.sub.2), nitrile (--CN),
C.sub.1-C.sub.8 alkoxy (--OR), phenoxy, phenolic, tolyl, phenyl,
aryl, benzyl, heterocycle, phosphonate, phosphate, quaternary
amine, sulfate, polyethyleneoxy, and linking moiety.
21. The energy-transfer dye of claim 20 wherein a heterocycle is
selected from 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl,
3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole,
4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline,
pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl,
5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O, 3-N)-oxazole, 5-(1-O,
3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S, 3-N)-thiazole,
2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, and
benzimidazole.
22. A labelled nucleoside or nucleotide having the formula:
##STR30## wherein DYE is the energy-transfer dye of claim 1; B is a
nucleobase; L is a linker; R.sup.25 is H, monophosphate,
diphosphate, triphosphate, thiophosphate, or phosphate analog; and
R.sup.26 and R.sup.27, when taken alone, are each independently H,
HO, F, or a moiety which blocks polymerase-mediated target-directed
polymerization, or when taken together form
2'-3'-didehydroribose.
23. The labelled nucleoside or nucleotide of claim 22 wherein the
xanthene compound of the energy-transfer dye is a substantially
pure atropisomer.
24. The labelled nucleoside or nucleotide of claim 22 wherein B is
selected from the group consisting of uracil, thymine, cytosine,
adenine, 7-deazaadenine, guanine, and 7-deazaguanosme.
25. The labelled nucleoside or nucleotide of claim 22 in which L
is: ##STR31## wherein n is 0, 1, or 2.
26. The labelled nucleoside or nucleotide of claim 22 which is
enzymatically incorporatable.
27. The labelled nucleoside or nucleotide of claim 22 which is a
terminator.
28. The labelled nucleoside or nucleotide of claim 27 wherein
R.sup.26 and R.sup.27, when taken alone, are each independently H,
F, or a moiety which blocks polymerase-mediated target-directed
polymerization, or when taken together form
2'-3'-didehydroribose.
29. The labelled nucleoside or nucleotide of claim 22 which is
enzymatically extendable.
30. A labelled polynucleotide comprising a polynucleotide
covalently attached to the energy-transfer dye of claim 1.
31. The labelled polynucleotide of claim 30 wherein the xanthene
compound of the energy-transfer dye is a substantially pure
atropisomer.
32. The labelled polynucleotide of claim 30 comprising the formula:
##STR32## wherein DYE is the energy-transfer dye; B is a
nucleobase; L is a linker; R.sup.27 is H, OH, halide, azide, amine,
alkylamine, C.sub.1-C.sub.6 alkyl, allyl, C.sub.1-C.sub.6 alkoxy,
OCH.sub.3, or OCH.sub.2CH.dbd.CH.sub.2; and R.sup.28 and R.sup.29
when taken alone, are each independently H, phosphate,
internucleotide phosphodiester, or internucleotide analog; wherein
the polynucleotide comprises 2 to 100 nucleotides.
33. The labelled polynucleotide of claim 32 wherein B is selected
from the group consisting of uracil, thymine, cytosine, adenine,
7-deazaadenine, guanine, and 7-deazaguanosine.
34. The labelled polynucleotide of claim 30 comprising the formula:
##STR33## wherein DYE is the energy-transfer dye; B is a
nucleobase; X is O, NH, or S; L is a linker; R.sup.27 is H, OH,
halide, azide, amine, alkylamine, C.sub.1-C.sub.6 alkyl, allyl,
C.sub.1-C.sub.6 alkoxy, OCH.sub.3, or OCH.sub.2CH.dbd.CH.sub.2; and
R.sup.28 is internucleotide phosphodiester or internucleotide
analog; wherein the polynucleotide comprises 2 to 100
nucleotides.
35. The labelled polynucleotide of claim 34 wherein B is selected
from the group consisting of uracil, thymine, cytosine, adenine,
7-deazaadenine, guanine, and 7-deazaguanosine.
36. The labelled polynucleotide of claim 34 in which L is
C.sub.1-C.sub.12 alkyldiyl.
37. The labelled polynucleotide of claim 34 in which L comprises
--(CH.sub.2CH.sub.2O).sub.n--, where n is 1 to 100.
38. A labelled polypeptide comprising a polypeptide covalently
attached to an energy-transfer dye of claim 1.
39. The labelled polypeptide of claim 38 wherein the xanthene
compound of the energy-transfer dye is a substantially pure
atropisomer.
40. A phosphoramidite compound having the formula: ##STR34##
wherein DYE is the energy-transfer dye of claim 1; L is a linker;
R.sup.30 and R.sup.31 taken separately are selected from the group
consisting of C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 cycloalkyl,
and aryl; or R.sup.30 and R.sup.31 taken together with the nitrogen
atom form a saturated nitrogen heterocycle; and R.sup.32 is a
phosphite ester protecting group.
41. The phosphoramidite compound of claim 40 wherein the xanthene
compound of the energy-transfer dye is a substantially pure
atropisomer.
42. The phosphoramidite compound of claim 40 wherein R.sup.32 is
selected from the group consisting of methyl, 2-cyanoethyl, and
2-(4-nitrophenyl)ethyl.
43. The phosphoramidite compound of claim 40 wherein R.sup.30 and
R.sup.31 are each isopropyl.
44. The phosphoramidite compound of claim 40 wherein R.sup.30 and
R.sup.31 taken together is morpholino.
45. The phosphoramidite compound of claim 40 wherein L is
C.sub.1-C.sub.12 alkyldiyl.
46. The phosphoramidite compound of claim 40 wherein L is attached
at R.sup.18 or R.sup.19 of DYE having the structure: ##STR35##
47. The phosphoramidite compound of claim 46 having the structure:
##STR36##
48. The phosphoramidite compound of claim 40 wherein L is ##STR37##
and n ranges from 1 to 10.
49. A kit for labelling a polynucleotide, comprising an
energy-transfer dye according to claim 1 and a polynucleotide.
50. A kit for labelling a polynucleotide, comprising a
phosphoramidite compound according to claim 40 and a
polynucleotide.
51. A kit for labelling a polypeptide, comprising an
energy-transfer dye according to claim 38 and a polypeptide.
52. A kit for generating a labelled primer extension product,
comprising one or more enzymatically-incorporatable nucleotides and
a primer, wherein said primer is a labelled polynucleotide
according to claim 30.
53. A kit for generating a labelled primer extension product,
comprising one or more enzymatically-incorporatable nucleotides and
a primer, wherein at least one nucleotide is a labelled nucleotide
according to claim 22.
54. The kit of claim 53 wherein the labelled nucleotide is a
terminator.
55. The kit of claim 54 which comprises four different terminators,
one which terminates at a target A, one which terminates at a
target G, one which terminates at a target C and one which
terminates at a target T or U.
Description
I. CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
10/716,165, filed Nov. 18, 2003, now U.S. Pat. No. 7,038,063, which
is a divisional of application Ser. No. 10/227,058, filed Aug. 21,
2002, now U.S. Pat. No. 6,649,769, which is a divisional of
application Ser. No. 09/704,966 filed Nov. 1, 2000, now U.S. Pat.
No. 6,448,407, all of which are incorporated herein by
reference.
II. FIELD OF THE INVENTION
[0002] The invention relates to certain atropisomer forms of
asymmetric xanthene fluorescent dyes and the field of nucleic acid
sequencing and analysis with fluorescent dye-labelled reagents.
III. BACKGROUND OF THE INVENTION
[0003] Methods of analyzing fluorescent-labelled biomolecules after
separating based on size- or charge are central to molecular
biology. Examples of methods utilizing fluorescent-labelled nucleic
acids include automated DNA sequencing, oligonucleotide probe
methods, detection of polymerase-chain-reaction products,
immunoassays, and the like. In the case of multi-color automated
DNA sequencing, labelled nucleic acid fragments of varying size are
separated by electrophoresis, typically in a single electrophoresis
lane, channel, or capillary. Employing these methods, automated
four-color Sanger-type DNA sequencing has enabled entire genome
characterization at the molecular level.
[0004] Stereochemical purity is of importance in the field of
pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit
chirality (U.S. Pat. No. 6,075,024). A case in point is provided by
the L-form of the beta-adrenergic blocking agent, R(-) albuterol,
which is known to be 100 times more potent than the D-enantiomer
(U.S. Pat. No. 5,760,090). Furthermore, optical purity is important
since certain isomers may actually be deleterious rather than
simply inert.
[0005] Atropisomers are stereoisomeric conformations of a molecule
whose interconversion is slow enough to allow separation and
isolation under predetermined conditions (McGraw-Hill Dictionary of
Chemical Terms, (1984), S. Parker, Ed., p. 36). The energy barrier
to thermal racemization may be determined by the steric hindrance
to free rotation of one or more bonds forming a chiral axis.
Certain biaryl compounds exhibit atropisomerism where rotation
around an intraannular bond lacking C.sub.2 symmetry is restricted.
The free energy barrier for enantiomerization is a measure of the
stability of the intraannular bond with respect to rotation.
Optical and thermal excitation can promote racemization, dependent
on electronic and steric factors (Tetreau (1982) Nouv. Jour. de
Chimie, 6:461-65).
[0006] Ortho-substituted biphenyl compounds may exhibit this type
of conformational, rotational isomerism known as atropisomerism
(Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic
Compounds, John Wiley & Sons, Inc., pp. 1142-55). Such
biphenyls are enantiomeric, chiral atropisomers where the sp2-sp2
carbon-carbon, intraannular bond between the phenyl rings has a
sufficiently high energy barrier to free rotation, and where
substituents X.noteq.Y and U.noteq.V render the molecule
asymmetric. The steric interaction of X-U, X-V, and/or Y-V, Y-U is
large enough to make the planar conformation an energy maximum. Two
nonplanar, axially chiral enantiomers, shown below, then exist as
atropisomers when their interconversion is slow enough such that
they can be isolated free of each other. By one definition,
atropisomerism is defined to exist where the isomers have a
half-life t1/2 of at least 1000 seconds, which is a free energy
barrier of 22.3 kcal mol.sup.-1 (93.3 kJ mol.sup.-1) at 300K (Oki,
M. (1983) "Recent Advances in Atropisomerism," Topics in
Stereochemistry, 14:1). Bold lines and dashed lines in the figures
shown below indicate those moieties, or portions of the molecule,
which are sterically restricted due to a rotational energy barrier.
Bolded moieties exist orthoganally above the plane and dashed
moieties exist orthogonally below the plane of the rest of the
molecule. ##STR1##
[0007] Xanthene dyes have important applications as detectable
fluorescent labels of nucleic acids (U.S. Pat. Nos. 5,188,934;
5,654,442; 5,885,778; 6,096,723; 6,020,481; 5,863,727; 5,800,996;
5,945,526; 5,847,162; 6,025,505; 6,008,379; 5,936,087; 6,015,719).
Xanthene compounds containing an asymmetric biannular bond can
exist in stable atropisomeric forms. Conjugates of atropisomeric
xanthene compounds and chiral substrates, such as nucleotides,
polynucleotides, polypeptides, and carbohydrates, form
diastereomers. These diastereomeric conjugates can separate under
certain conditions, such as electrophoresis, chromatography, and
other methods. Separation of diastereomers can hinder detection by
display of double peaks or bands, i.e. "peak doubling". Thus,
atropisomerically enriched or purified forms of xanthene dyes are
important as labels for methods based on separation and detection
of analytes.
IV. SUMMARY
[0008] The present invention is directed towards
atropisomerically-enriched and substantially pure atropisomers of
asymmetric xanthene compounds as novel compositions. The invention
also includes methods for isolation, labelling, and detecting
labelled compositions.
[0009] In a first aspect, the invention includes substantially pure
atropisomer compounds having the structure II: ##STR2## wherein
positions R.sup.1, R.sup.4, R.sup.5, R.sup.11, R.sup.13, R.sup.14,
R.sup.17, R.sup.18, R.sup.19, R.sup.20, Z.sup.1, or Z.sup.2 may be
substituted with substituents. At least one substituent may be a
linking moiety. One or more rings may be fused on the ring
structure II.
[0010] Another aspect of the invention includes energy-transfer dye
compounds comprising a donor dye capable of absorbing light at a
first wavelength and emitting excitation energy in response
thereto; an acceptor dye capable of absorbing the excitation energy
emitted by the donor dye and fluorescing at a second wavelength in
response; and a linker for linking the donor dye and the acceptor
dye; wherein at least one of the donor dye and acceptor dye is a
substantially pure atropisomer of a xanthene compound.
[0011] Another aspect of the invention is labelled substrates,
including nucleoside, nucleotides, polynucleotides, and
polypeptides wherein the label is a substantially pure atropisomer
of a xanthene compound or an energy-transfer dye comprising a
substantially pure atropisomer of a xanthene compound.
[0012] Another aspect of the invention is labelling reagents,
including phosphoramidite and active ester linking moieties of a
substantially pure atropisomer of a xanthene compound, which form
covalent attachments with substrates and methods of labelling
substrates with the reagents.
[0013] Another aspect of the invention is methods for forming a
labelled substrate comprising the step of reacting a substrate with
the linking moiety of a substantially pure atropisomer of a
xanthene compound or an energy-transfer dye comprising a
substantially pure atropisomer of a xanthene compound.
[0014] Another aspect of the invention is methods for separating
atropisomers of xanthene compounds by forming diastereomers with
substantially enantiomerically pure compounds, and separating the
diastereomers. The diastereomers may be converted to substantially
pure atropisomers of xanthene compounds.
[0015] Another aspect of the invention is methods for separating a
mixture of labelled substrates wherein the labels are comprised of
a substantially pure atropisomer of a xanthene compound or an
energy-transfer dye comprising a substantially pure atropisomer of
a xanthene compound. The labelled substrates may be primer
extension polynucleotide fragments. The labelled substrates may be
separated by electrophoresis, chromatography, or other separation
technique. The mixture of labelled polynucleotides may be formed
from a labelled primer or a labelled terminator. The labelled
substrates may be detected by fluorescence detection.
[0016] Another aspect of the invention is methods of generating a
labelled primer extension product by extending a primer-target
hybrid with an enzymatically-incorporatable nucleotide. The primer
or the nucleotide may be labelled with a substantially pure
atropisomer of a xanthene compound or an energy-transfer dye
comprising a substantially pure atropisomer of a xanthene
compound.
[0017] Another aspect of the invention is methods of polynucleotide
sequencing by forming a mixture of four classes of polynucleotides
where each class is labelled at the 3' terminal nucleotide with a
substantially pure atropisomer of a xanthene compound or an
energy-transfer dye comprising a substantially pure atropisomer of
a xanthene compound, and the labels are spectrally resolvable. The
polynucleotides are separated by size.
[0018] Another aspect of the invention is methods of
oligonucleotide ligation by annealing two probes to a target
sequence and forming a phosphodiester bond between the 5' terminus
of one probe and the 3' terminus of the other probe wherein one or
both probes are labelled with a substantially pure atropisomer of a
xanthene compound or an energy-transfer dye comprising a
substantially pure atropisomer of a xanthene compound.
[0019] Another aspect of the invention is methods of amplification
by annealing two or more primers to a target polynucleotide and
extending the primers by a polymerase and a mixture of
enzymatically-extendable nucleotides wherein at least one of the
primers or one of the nucleotides is labelled with a substantially
pure atropisomer of a xanthene compound or an energy-transfer dye
comprising a substantially pure atropisomer of a xanthene
compound.
[0020] Another aspect of the invention is kits of reagents
including a substantially pure atropisomer of a xanthene compound
or an energy-transfer dye comprising a substantially pure
atropisomer of a xanthene compound.
V. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1a shows the reaction of C-1 aminomethyl, C-19 carboxy
fluorescein and (-) Menthyl chloroformate to give menthyl carbamate
diastereomers 1a and 1b.
[0022] FIG. 1b shows reverse phase HPLC analysis of preparative
HPLC fractions in the separation of menthyl carbamate diastereomers
1a and 1b. Fractions #1 and #2 show pure 1a. Fraction #3 shows a
mixture of 1a and 1b. Fractions #4 and #5 show pure 1b.
[0023] FIG. 2a shows the reactions of: (i) menthyl carbamate
diastereomer 1a with sulfuric acid to hydrolyze the menthyl
carbamate group to give atropisomer amine 2a, (ii) amidation of 2a
with ethyl trifluoroacetate to give atropisomer trifluoroacetamide
3a, (iii) menthyl carbamate diastereomer 1b with sulfuric acid to
hydrolyze the menthyl carbamate group to give atropisomer amine 2b,
and (iv) amidation of 2b with ethyl trifluoroacetate to give
atropisomer trifluoroacetamide 3b.
[0024] FIG. 2b shows an HPLC chromatogram of the racemic mixture of
atropisomers 2a and 2b under chiral HPLC conditions.
[0025] FIG. 2c shows an HPLC chromatogram of the purified
atropisomer 2a under chiral HPLC conditions.
[0026] FIG. 3 shows the reactions of: (i) atropisomer
trifluoroacetamide 3a with N-hydroxysuccinimide and DAE
carbodiimide HCl salt to give atropisomeric NHS ester 4a, and (ii)
atropisomer trifluoroacetamide 3b with N-hydroxysuccinimide and DAE
carbodiimide HCl salt to give atropisomeric NHS ester 4b.
[0027] FIG. 4 shows the synthesis of
2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acid NHS
5.
[0028] FIG. 5 shows the synthesis of propargylphosphorylamino-ddATP
11.
[0029] FIG. 6 shows the synthesis of
aminomethyl-FAM-propargylphosphorylamino-ddATP 15.
[0030] FIG. 7 shows the synthesis of tricyclic amine 20.
[0031] FIG. 8 shows the synthesis of NHS-rhodamine dye 17.
[0032] FIG. 9 shows the structure of energy-transfer ddATP
terminator 25.
[0033] FIG. 10 shows the synthesis of bis-trifluoroacetamide
rhodamine NHS 27.
[0034] FIG. 11 shows the synthesis of
aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP
32.
[0035] FIG. 12 shows the structure of energy-transfer ddTTP
terminator 26.
[0036] FIG. 13a shows DNA sequencing of pGEM with phosphate-linker,
energy-transfer terminator ddATP 25, fragment size 119-242 bp.
[0037] FIG. 13b shows DNA sequencing of pGEM with phosphate-linker,
energy-transfer terminator ddATP 25, fragment size 148-205 bp.
[0038] FIG. 13c shows DNA sequencing of pGEM with sulfonate-linker,
energy-transfer terminator ddATP 33, fragment size 148-242 bp.
[0039] FIG. 13d shows DNA sequencing of pGEM with energy-transfer
terminator ddGTP 34, fragment size 24-99 bp.
[0040] FIG. 14 shows the structure of sulfonate-linker,
energy-transfer terminator ddATP 33.
[0041] FIG. 15 shows the structure of energy-transfer terminator
ddGTP 34.
VI. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0042] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying drawings. While the invention will be described in
conjunction with the illustrated embodiments, it will be understood
that they are not intended to limit the invention to those
embodiments. On the contrary, the invention is intended to cover
all alternatives, modifications, and equivalents, which may be
included within the scope of the claimed invention.
VI.1 Definitions
[0043] Stereochemical definitions and conventions used herein
generally follow McGraw-Hill Dictionary of Chemical Terms (1984) S.
P. Parker, Ed., McGraw-Hill Book Company, New York and
Stereochemistry of Organic Compounds (1994); and Eliel, E. and
Wilen, S., John Wiley & Sons, Inc., New York. Many organic
compounds exist in optically active forms, i.e., they have the
ability to rotate the plane of plane-polarized light. In describing
an optically active compound, the prefixes D and L or R and S are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and l or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or l meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these compounds, called stereoisomers, are
identical except that they are mirror images of one another. A
specific stereoisomer may also be referred to as an enantiomer, and
a mixture of such isomers is often called an enantiomeric mixture.
A 50:50 mixture of enantiomers is referred to as a racemic mixture
or a racemate. The terms "racemic mixture" and "racemate" refer to
an equimolar mixture of two enantiomeric species, devoid of optical
activity.
[0044] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0045] The term "stereoisomers" refers to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0046] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties, e.g.
melting points, boiling points, spectral properties, and
reactivities. Mixtures of diastereomers may separate under high
resolution analytical procedures such as electrophoresis and
chromatography.
[0047] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0048] The term "atropisomer" refers to a stereoisomer resulting
from restricted rotation about single bonds where the rotation
barrier is high enough to permit isolation of the isomeric species.
Atropisomers are enantiomers without a single asymmetric atom.
Atropisomers are conformational stereoisomers which occur when
rotation about a single bond in the molecule is prevented, or
greatly slowed, as a result of steric interactions with other parts
of the molecule and the substituents at both ends of the single
bond are unsymmetrical.
[0049] The terms "substantially pure atropisomer" and
"substantially free of its stereoisomer" mean that the composition
contains at least 90% by weight of one atropisomer, and 10% by
weight or less of the stereoisomeric atropisomer.
[0050] The term "atropisomerically enriched" means that the
composition is a greater proportion or percentage of one of the
atropisomers of the xanthene compound, in relation to the other
atropisomer.
[0051] "Nucleobase" means a nitrogen-containing heterocyclic moiety
capable of forming Watson-Crick hydrogen bonds in pairing with a
complementary nucleobase or nucleobase analog, e.g. a purine, a
7-deazapurine, or a pyrimidine. Typical nucleobases are the
naturally occurring nucleobases adenine, guanine, cytosine, uracil,
thymine, and analogs of the naturally occurring nucleobases, e.g.
7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,
7-deaza-8-azaadenine (Kutyavin, U.S. Pat. No. 5,912,340), inosine,
nebularine, nitropyrrole, nitroindole, 2-aminopurine,
2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine,
pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine,
7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,
4-thiouracil, O.sup.6-methylguanine, N.sup.6-methyladenine,
O.sup.4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil,
4-methyl-indole, and ethenoadenine (Fasman (1989) Practical
Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC
Press, Boca Raton, Fla.).
[0052] "Nucleoside" means a compound consisting of a nucleobase
linked to the C-1' carbon of a ribose sugar. The ribose may be
substituted or unsubstituted. Substituted ribose sugars include,
but are not limited to, those riboses in which one or more of the
carbon atoms, e.g., the 3'-carbon atom, is substituted with one or
more of the same or different --R, --OR, --NRR or halogen groups,
where each R is independently hydrogen, C.sub.1-C.sub.6 alkyl or
C.sub.5-C.sub.14 aryl. Riboses include ribose, 2'-deoxyribose,
2',3'-dideoxyribose, 3'-haloribose, 3'-fluororibose,
3'chlororibose, 3'-alkylribose, e.g. 2'-O-methyl,
4'-.alpha.-anomeric nucleotides, 1'-.alpha.-anomeric nucleotides,
and 2'-4'-linked and other "locked", bicyclic sugar modifications
(Imanishi WO 98/22489; Imanishi WO 98/39352; Wengel WO 99/14226).
When the nucleobase is purine, e.g. A or G, the ribose sugar is
attached to the N.sup.9-position of the nucleobase. When the
nucleobase is pyrimidine, e.g. C, T or U, the pentose sugar is
attached to the N.sup.1-position of the nucleobase (Kornberg and
Baker, (1992) DNA Replication, 2.sup.nd Ed., Freeman, San
Francisco, Calif.).
[0053] "Nucleotide" means a phosphate ester of a nucleoside, as a
monomer unit or within a nucleic acid. Nucleotides are sometimes
denoted as "NTP", or "dNTP" and "ddNTP" to particularly point out
the structural features of the ribose sugar. "Nucleotide
5'-triphosphate" refers to a nucleotide with a triphosphate ester
group at the 5' position. The triphosphate ester group may include
sulfur substitutions for the various oxygens, e.g.
.alpha.-thio-nucleotide 5'-triphosphates.
[0054] As used herein, the terms "oligonucleotide" and
"polynucleotide" are used interchangeably and mean single-stranded
and double-stranded polymers of nucleotide monomers, including
2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotide phosphodiester bond linkages, or internucleotide
analogs, and associated counter ions, e.g., H.sup.+,
NH.sub.4.sup.+, trialkylammonium, Mg.sup.2+, Na.sup.+ and the like.
A polynucleotide may be composed entirely of deoxyribonucleotides,
entirely of ribonucleotides, or chimeric mixtures thereof.
Polynucleotides may be comprised of internucleotide, nucleobase and
sugar analogs. Polynucleotides typically range in size from a few
monomeric units, e.g. 5-40, when they are frequently referred to as
oligonucleotides, to several thousands of monomeric nucleotide
units. Unless denoted otherwise, whenever a polynucleotide sequence
is represented, it will be understood that the nucleotides are in
5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise
noted.
[0055] "Attachment site" means a site on a label or a substrate,
such as an oligonucleotide, which is covalently attached to a
linker.
[0056] "Linker" means a chemical moiety comprising a covalent bond
or a chain of atoms that covalently attaches a label to a
polynucleotide, or one label to another.
[0057] "Linking moiety" means a chemically reactive group,
substituent or moiety, e.g. a nucleophile or electrophile, capable
of reacting with another molecule to form a covalent bond, or
linkage.
[0058] "Alkyl" means a saturated or unsaturated, branched,
straight-chain, branched, or cyclic hydrocarbon radical derived by
the removal of one hydrogen atom from a single carbon atom of a
parent alkane, alkene, or alkyne. Typical alkyl groups consist of
1-12 saturated and/or unsaturated carbons, including, but not
limited to, methyl, ethyl, propyl, butyl, and the like.
[0059] "Alkoxy" means --OR where R is (C.sub.1-C.sub.6) alkyl.
[0060] "Alkyldiyl" means a saturated or unsaturated, branched,
straight chain or cyclic hydrocarbon radical of 1-20 carbon atoms,
and having two monovalent radical centers derived by the removal of
two hydrogen atoms from the same or two different carbon atoms of a
parent alkane, alkene or alkyne. Typical alkyldiyl radicals
include, but are not limited to, 1,2-ethyldiyl, 1,3-propyldiyl,
1,4-butyldiyl, and the like.
[0061] "Aryl" means a monovalent aromatic hydrocarbon radical of
6-20 carbon atoms derived by the removal of one hydrogen atom from
a single carbon atom of a parent aromatic ring system. Typical aryl
groups include, but are not limited to, radicals derived from
benzene, substituted benzene, naphthalene, anthracene, biphenyl,
and the like.
[0062] "Aryldiyl" means an unsaturated cyclic or polycyclic
hydrocarbon radical of 6-20 carbon atoms having a conjugated
resonance electron system and at least two monovalent radical
centers derived by the removal of two hydrogen atoms from two
different carbon atoms of a parent aryl compound.
[0063] "Substituted alkyl", "substituted alkyldiyl", "substituted
aryl" and "substituted aryldiyl" mean alkyl, alkyldiyl, aryl and
aryldiyl respectively, in which one or more hydrogen atoms are each
independently replaced with another substituent. Typical
substituents include, but are not limited to, --X, --R, --O.sup.-,
--OR, --SR, --S.sup.-, --NR.sub.2, --NR.sub.3, .dbd.NR, --CX.sub.3,
--CN, --OCN, --SCN, --NCO, --NCS, --NO, --NO.sub.2, .dbd.N.sub.2,
--N.sub.3, NC(O)R, --C(O)R, --C(O)NRR--S(O).sub.2O.sup.-,
--S(O).sub.2OH, --S(O).sub.2R, --OS(O).sub.2OR, --S(O).sub.2NR,
--S(O)R, --OP(O)O.sub.2RR, --P(O)O.sub.2RR, --P(O)(O.sup.-).sub.2,
--P(O)(OH).sub.2, --C(O)R, --C(O)X, --C(S)R, --C(O)OR,
--C(O)O.sup.-, --C(S)OR, --C(O)SR, --C(S)SR, --C(O)NRR, --C(S)NRR,
--C(NR)NRR, where each X is independently a halogen and each R is
independently --H, alkyl, aryl, heterocycle, or linking group.
[0064] "Internucleotide analog" means a phosphate ester analog of
an oligonucleotide such as: (i) alkylphosphonate, e.g.
C.sub.1-C.sub.4 alkylphosphonate, especially methylphosphonate;
(ii) phosphoramidate; (iii) alkylphosphotriester, e.g.
C.sub.1-C.sub.4 alkylphosphotriester; (iv) phosphorothioate; and
(v) phosphorodithioate. Internucleotide analogs also include
non-phosphate analogs wherein the sugar/phosphate subunit is
replaced by an a non-phosphate containing backbone structure. One
type of non-phosphate oligonucleotide analogs has an amide linkage,
such as a 2-aminoethylglycine unit, commonly referred to as PNA
(Nielsen (1991) "Sequence-selective recognition of DNA by strand
displacement with a thymidine-substituted polyamide", Science
254:1497-1500).
[0065] The terms "target sequence" and "target polynucleotide" mean
a polynucleotide sequence that is the subject of hybridization with
a complementary polynucleotide, e.g., a primer or probe. The
sequence can be composed of DNA, RNA, an analog thereof, including
combinations thereof.
[0066] The term "label", as used herein, means any moiety which can
be attached to a substrate, e.g., an oligonucleotide, nucleotide or
nucleotide 5'-triphosphate, and that functions to: (i) provide a
detectable signal; (ii) interact with a second label to modify the
detectable signal provided by the first or second label, e.g. FRET;
(iii) stabilize hybridization, i.e. duplex formation; (iv) affect
mobility, e.g. electrophoretic mobility or cell-permeability, by
charge, hydrophobicity, shape, or other physical parameters, or (v)
provide a capture moiety, e.g., affinity, antibody/antigen, or
ionic complexation.
[0067] "Heterocycle" means a molecule with a ring system in which
one or more ring atoms have been replaced with a heteroatom, e.g.
nitrogen, oxygen, and sulfur.
[0068] "Electron-deficient nitrogen heterocycle" is a monovalent
electron-deficient nitrogen heterocycle derived by the removal of
one hydrogen atom from a single atom of the ring system to join the
heterocycle as a substituent to the fluorescein dyes of the
invention (Joule, Heterocyclic Chemistry, 3rd Ed., Stanley Thornes
Publisher, Ltd., Cheltenham, U.K. (1998); Acheson, R., An
Introduction to the Chemistry of Heterocyclic Compounds, 2nd Ed.
Interscience Publishers, division of John Wiley & Sons, New
York (1967)).
[0069] "Substrate" is an entity to which dye compounds of the
present invention are attached. Substrates include, but are not
limited to a (i) polynucleotide, (ii) nucleoside and nucleotide,
(iii) polypeptide, (iv) carbohydrate, (v) ligand, and (vi) any
analog of the preceding (i) to (v).
[0070] "Enzymatically incorporatable" is a property of a nucleotide
in which it is capable of being enzymatically incorporated onto the
terminus, e.g. 3', of a nascent polynucleotide chain through the
action of a polymerase enzyme.
[0071] "Terminator" means an enzymatically incorporatable
nucleotide which prevents subsequent incorporations of nucleotides
to the resulting polynucleotide chain and thereby halt polymerase
extension. Typical terminators lack a 3'-hydroxyl substituent and
include 2',3'-dideoxyribose, 2',3'-didehydroribose, and
2',3'-dideoxy, 3'-haloribose, e.g. 3'-fluoro. Alternatively, a
ribofuranose analog could be used, such as arabinose. Exemplary
nucleotide terminators include
2',3'-dideoxy-.beta.-D-ribofuranosyl, .beta.-D-arabinofuranosyl,
3'-deoxy-.beta.-D-arabinofuranosyl,
3'-amino-2',3'-dideoxy-.beta.-D-ribofuranosyl, and
2',3'-dideoxy-3'-fluoro-.beta.-D-ribofuranosyl (Chidgeavadze (1984)
Nucleic Acids Res., 12: 1671-1686; and Chidgeavadze (1985) FEB.
Lett., 183: 275-278). Nucleotide terminators also include
reversible nucleotide terminators (Metzker (1994) Nucleic Acids
Res., 22(20): 4259).
[0072] "Enzymatically extendable" is a property of a nucleotide in
which it is enzymatically incorporatable at the terminus of a
polynucleotide and the resulting extended polynucleotide can
undergo subsequent incorporations of nucleotides or nucleotide
analogs.
VI.2 Atropisomer Compounds
[0073] The compositions of the invention are asymmetric xanthene
compounds that exist in stable atropisomeric forms. Aryl
substituents can restrict rotation around the biannular bond
between C-10 and C-15 (noted by an arrow) in the following
structure I: ##STR3##
[0074] Asymmetric compounds of the invention include xanthene dyes
characterized by the general structure II: ##STR4## and include
asymmetric fluorescent dye classes such as fluorescein (Z.sup.1,
Z.sup.2=O), rhodol (Z.sup.1=O, Z.sup.2=NR.sub.2), and rhodamine
(Z.sup.1, Z.sup.2=NR.sub.2). Where Z.sup.1 or Z.sup.2 is NR.sub.2,
R may independently be hydrogen, C.sub.1-C.sub.12 alkyl, phenyl,
benzyl, aryl, heterocycle, or a linking moiety.
[0075] Substituents R.sup.1, R.sup.4, R.sup.5, R.sup.11, R.sup.13,
R.sup.14, R.sup.17, R.sup.18, R.sup.19, and R.sup.20 may be
independently fluorine, chlorine, C.sub.1-C.sub.8 alkyl,
carboxylate, sulfate, sulfonate (--SO.sub.3.sup.-), alkylsulfonate
(--R--SO.sub.3.sup.-), aminomethyl (--CH.sub.2NH.sub.2),
aminoalkyl, 4-dialkylaminopyridinium, hydroxymethyl (--CH.sub.2OH),
methoxy (--OCH.sub.3), hydroxyalkyl (--ROH), thiomethyl
(--CH.sub.2SH), thioalkyl (--RSH), alkylsulfone (--SO.sub.2R),
arylthio (--SAr), arylsulfone (--SO.sub.2Ar), sulfonamide
(--SO.sub.2NR.sub.2), alkylsulfoxide (--SOR), arylsulfoxide
(--SOAr), amino (--NH.sub.2), ammonium (--NH.sub.3.sup.+), amido
(--CONR.sub.2), nitrile (--CN), C.sub.1-C.sub.8 alkoxy (--OR),
phenoxy, phenolic, tolyl, phenyl, aryl, benzyl, heterocycle,
phosphonate, phosphate, quaternary amine, sulfate, polyethyleneoxy,
and linking moiety.
[0076] The compounds of the invention include fused benzo rings
where R.sup.13 and R.sup.14, or R.sup.4 and R.sup.5, taken together
form benzo, and where the fused benzo groups are substituted with
substituents.
[0077] Substituents R.sup.1, R.sup.4, R.sup.5, R.sup.11, R.sup.13,
R.sup.14, R.sup.17, R.sup.18, R.sup.19, and R.sup.20 may also be
independently an electron-deficient heterocycle, including
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl,
4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole,
pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine,
quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl,
5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O, 3-N)-oxazole, 5-(1-O,
3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S, 3-N)-thiazole,
2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, or
benzimidazole.
[0078] Examples of asymmetric fluorescein dyes include the
structures: ##STR5##
[0079] The compounds of the invention include atropisomeric,
asymmetric rhodamines with ring structures formed by the Z.sup.1
nitrogen, the Z.sup.1-bonded carbon, and the R.sup.1-bonded carbon,
to make a first ring structure having from 4 to 7 members.
Optionally, the compounds may have a second ring structure formed
by the Z.sup.2 nitrogen, the Z.sup.2-bonded carbon, and the
R.sup.11-bonded carbon, also having from 4 to 7 members. An example
includes the structure IIa: ##STR6##
[0080] Asymmetry results where either: (1) R.sup.1.noteq.R.sup.11,
R.sup.4.noteq.R.sup.14, R.sup.5.noteq.R.sup.13, or
Z.sup.1.noteq.Z.sup.2, and (2) R.sup.17.noteq.R.sup.19, or
R.sup.20.noteq.X. In other words, both aryl substituents on the
biannular sp2-sp2 bond are asymmetric and the compound lacks a
C.sub.2 axis of symmetry along the biannular bond axis. C.sub.2
symmetry is defined by taking the biannular bond in I or II as the
axis such that rotation of 180.degree. around the axis, gives the
same molecule. An example of a symmetric xanthene compound with
C.sub.2 symmetry is fluorescein (R.sup.1, R.sup.4, R.sup.5,
R.sup.11, R.sup.13, R.sup.14, R.sup.17, R.sup.18, R.sup.19,
R.sup.20=H; X=CO.sub.2H). An example of an asymmetric xanthene
compound without C.sub.2 symmetry is C-11 aminomethyl, C-19
carboxyfluorescein (R.sup.1, R.sup.4, R.sup.5, R.sup.13, R.sup.14,
R.sup.17, R.sup.18, R.sup.20=H; R.sup.11=CH.sub.2NH.sub.2;
R.sup.19, X=CO.sub.2H). This compound is atropisomeric because the
substituents adjacent to the C-10 to C-15 biannular bond are
sufficiently bulky that rotation is hindered. The energy barrier to
rotation is sufficiently high that stable, non-superimposable,
mirror image atropisomeric forms 2a and 2b result, as shown:
##STR7## Rotation around the biannular bond results in racemization
and loss of atropisomerism. Typically, racemization of the
asymmetric xanthene compounds occurs upon heating.
[0081] The compounds of the present invention can be prepared by
any suitable method available in the art. Exemplary methods for
preparing a variety of different asymmetric xanthene compounds can
be found in the Example section below, and as discussed in greater
detail below.
[0082] As a specific example, reference is made throughout the
specification to Z.sup.1 and Z.sup.2 substituents. As this
nomenclature corresponds to the illustrated structural formulae,
which represent only one of several possible tautomeric forms (or
resonance structures) of the compounds, it will be understood that
these references are for convenience only, and that any such
references are not intended to limit the scope of the compounds
described herein.
[0083] Those of skill in the art will also recognize that the
compounds of the invention may exist in many different protonation
states, depending on, among other things, the pH of their
environment. While the structural formulae provided herein depict
the compounds in only one of several possible protonation states,
it will be understood that these structures are illustrative only,
and that the invention is not limited to any particular protonation
state--any and all protonated forms of the compounds are intended
to fall within the scope of the invention.
[0084] The compounds of the invention may bear multiple positive or
negative charges. The net charge of the dyes of the invention may
be either positive or negative. The counter ions associated with
the dyes are typically dictated by the synthesis and/or isolation
methods by which the compounds are obtained. Typical counter ions
include, but are not limited to ammonium, sodium, potassium,
lithium, halides, acetate, trifluoroacetate, etc., and mixtures
thereof. It will be understood that the identity of any associated
counter ion is not a critical feature of the invention, and that
the invention encompasses the dyes in association with any type of
counter ion. Moreover, as the compounds can exists in a variety of
different forms, the invention is intended to encompass not only
forms of the dyes that are in association with counter ions (e.g.,
dry salts), but also forms that are not in association with counter
ions (e.g., aqueous or organic solutions).
[0085] Asymmetric xanthene compounds can be conveniently
synthesized from precursors (U.S. Pat. Nos. 5,188,934; 5,654,442;
5,885,778; 6,096,723; 6,020,481; 5,863,727; 5,800,996; 5,945,526;
5,847,162; 6,025,505; 6,008,379; 5,936,087; 6,015,719). An
exemplary synthetic route starts by aminomethylation of C-19
carboxyfluorescein (Shipchandler (1987) Anal. Biochem. 162:89-101;
U.S. Pat. No. 4,510,251; EP 232736; EP110186) to give C-1 (C-11)
aminomethyl, C-19 carboxyfluorescein.
[0086] An atropisomer substantially free of its stereoisomer may be
obtained by resolution of the mixture of stereoisomers of a
xanthene compound using a method such as formation of diastereomers
using optically active resolving agents ("Stereochemistry of Carbon
Compounds," (1962) by E. L. Eliel, McGraw Hill; Lochmuller, C. H.,
(1975) J. Chromatogr., 113:(3) 283-302). Atropisomers of xanthene
dyes can be separated and isolated, prior to, or after,
derivatization to give reactive labelling reagents. Separation of
the atropisomer xanthene compounds of the invention from the
racemic mixture can be accomplished by any suitable method,
including: (1) formation of ionic, diastereomeric salts with chiral
compounds and separation by fractional crystallization or other
methods, (2) formation of diastereomeric compounds with chiral
derivatizing reagents, separation of the diastereomers, and
conversion to the pure atropisomers, and (3) separation of the
atropisomers directly under chiral conditions.
[0087] Under method (1), diastereomeric salts can be formed by
reaction of enantiomerically pure chiral bases such as brucine,
quinine, ephedrine, strychnine,
.alpha.-methyl-.beta.-phenylethylamine (amphetamine), and the like
with asymmetric xanthene compounds bearing acidic functionality,
such as carboxylic acid and sulfonic acid. The diastereomeric salts
may be induced to separate by fractional crystallization or ionic
chromatography. For separation of the optical isomers of amino
compounds, addition of chiral carboxylic or sulfonic acids, such as
camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid
can result in formation of the diastereomeric salts.
[0088] Alternatively, by method (2), the substrate to be resolved
is reacted with one enantiomer of a chiral compound to form a
diastereomeric pair (Eliel, E. and Wilen, S. (1994) Stereochemistry
of Organic Compounds, John Wiley & Sons, Inc., p. 322).
Diastereomeric compounds can be formed by reacting asymmetric
xanthene compounds with enantiomerically pure chiral derivatizing
reagents, such as menthyl derivatives, followed by separation of
the diastereomers and hydrolysis to yield the free,
enantiomerically enriched xanthene. A method of determining optical
purity involves making chiral esters, such as a menthyl ester or
Mosher ester, .alpha.-methoxy-.alpha.-(trifluoromethyl)phenyl
acetate (Jacob III. (1982) J. Org. Chem. 47:4165), of the racemic
mixture, and analyzing the NMR spectrum for the presence of the two
atropisomeric diastereomers. For example, C-1 aminomethyl, C-19
carboxy fluorescein, an asymmetric xanthene compound useful for
attaching to nucleotides, polynucleotides, and other fluorescent
dyes was reacted with (-) menthyl chloroformate in the presence of
base to form the diastereomeric mixture of menthyl carbamates 1a
and 1b (Example 1, FIG. 1a). Stable diastereomers of atropisomeric
xanthene compounds can be separated and isolated by normal- and
reverse-phase chromatography following methods for separation of
atropisomeric naphthyl-isoquinolines (Hoye, T., WO 96/15111).
Diastereomers 1a and 1b were separated by preparative reverse-phase
HPLC (Example 2, FIG. 1b).
[0089] By method (3), a racemic mixture of two asymmetric
enantiomers can be separated by chromatography using a chiral
stationary phase ("Chiral Liquid Chromatography" (1989) W. J.
Lough, Ed. Chapman and Hall, New York; Okamoto, (1990) "Optical
resolution of dihydropyridine enantiomers by High-performance
liquid chromatography using phenylcarbamates of polysaccharides as
a chiral stationary phase", J. of Chromatogr. 513:375-378).
Enantiomeric atropisomers of xanthene compounds can be separated
and isolated by chromatography on chiral stationary phase. A sample
of racemic, C-1 aminomethyl, C-19 carboxy fluorescein gave two
peaks, resolving the atropisomeric stereoisomers, by HPLC analysis
on a chiral adsorbent column (FIG. 2b). When the atropisomers are
separated, for example, by the chiral derivatization method
(Example 1), preparative HPLC separation (Example 2) and hydrolysis
of the chiral menthyl auxiliaries (Example 3, FIG. 2a), the
separated atropisomer 2a showed a single peak when analyzed by HPLC
on the chiral adsorbent column (FIG. 2c).
[0090] Atropisomers can be distinguished by methods used to
distinguish other chiral molecules with asymmetric carbon atoms,
such as optical rotation and circular dichroism.
VI.3 Energy-Transfer Dyes
[0091] In another aspect, the present invention comprises
energy-transfer dye compounds containing atropisomeric xanthene
compounds such as those defined by structure II. 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. The donor dye may be attached to the
acceptor dye through a linker, the linker being effective to
facilitate efficient energy transfer between the donor and acceptor
dyes (Lee, "Energy-transfer dyes with enhanced fluorescence", U.S.
Pat. No. 5,800,996; Lee "Energy-transfer dyes with enhanced
fluorescence", U.S. Pat. No. 5,945,526; Mathies, "Fluorescent
labels and their use in separations", U.S. Pat. No. 5,654,419; Lee
(1997) Nucleic Acids Res. 25:2816-22). Alternatively, the donor dye
and the acceptor dye may be labelled at different attachment sites
on the substrate. For example, an oligonucleotide may be labelled
with a donor dye at the 5' terminus and an acceptor dye at the 3'
terminus. A polypeptide may be labelled with a donor dye at the
carboxyl terminus and an acceptor dye at an internal cysteine or
lysine sidechain (Komoriya, "Compositions for the detection of
proteases in biological samples and methods of use thereof", U.S.
Pat. No. 5,605,809). In the energy-transfer dye of the invention,
at least one of the donor or acceptor dyes which label a substrate
is an atropisomeric xanthene compounds. Other dyes comprising the
energy-transfer dye may be any fluorescent moiety which undergoes
the energy transfer process with an atropisomeric xanthene
compound, including a fluorescein, rhodol, and a rhodamine. Other
dyes include classes of fluorescent dyes such as cyanine,
phthalocyanine, squaraine, bodipy, benzophenoxazine, fluorescein,
dibenzorhodamine, or rhodamine.
[0092] Energy-transfer dyes have advantages for use in the
simultaneous detection of multiple labelled substrates in a
mixture, such as DNA sequencing. A single donor dye can be used in
a set of energy-transfer dyes so that each dye has strong
absorption at a common wavelength. By then varying the acceptor dye
in the energy-transfer set, the acceptor dyes can be spectrally
resolved by their respective emission maxima. Energy-transfer dyes
also provide a larger effective Stokes shift than
non-energy-transfer dyes. The Stokes shift is the difference
between the excitation maximum, the wavelength at which the donor
dye maximally absorbs light, and the emission maximum, the
wavelength at which the acceptor maximally emits light.
[0093] Generally the linker between the donor dye and acceptor dye
has the structures: ##STR8## wherein Z is selected from the group
consisting of NH, S and O; R.sup.21 is a C.sub.1-C.sub.12 alkyl
attached to the donor dye; R.sup.22 is a substituent selected from
the group consisting of a C.sub.1-C.sub.12 alkyldiyl, a five and
six membered ring having at least one unsaturated bond and a fused
ring structure which is attached to the carbonyl carbon; and
R.sup.23 includes a functional group which attaches the linker to
the acceptor dye. R.sup.22 may be a five or six membered ring such
as cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene,
furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole,
isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine,
pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene,
indole and naphthalene. Specifically, the linker may have the
structure: ##STR9## where n ranges from 2 to 10.
[0094] Generally also, R.sup.23 may have the structure: ##STR10##
wherein R.sup.24 is a C.sub.1-C.sub.12 alkyl and
[0095] In one embodiment, the linker between the donor dye and
acceptor dye includes a functional group which gives the linker
some degree of structural rigidity, such as an alkene, diene, an
alkyne, a five and six membered ring having at least one
unsaturated bond or a fused ring structure. The donor dye and the
acceptor dye of the energy-transfer dye may be attached by linkers
which have the exemplary structures: ##STR11## wherein D is a donor
dye, A is an acceptor dye and n is 1 or 2. The phenyl rings may be
substituted with groups such as sulfonate, phosphonate, and other
charged groups.
[0096] The attachment sites of the linker between the donor dye and
acceptor dye of an energy-transfer dye may be at any position where
one or both of the donor dye and acceptor dye is a compound of the
present invention. Exemplary attachment sites include R.sup.1,
R.sup.11, R.sup.18, R.sup.19, Z.sup.1 and Z.sup.2. Examples of
linkers and attachment sites are shown in terminator nucleotides
25, 26, 33, and 34 where the linkers attach at R.sup.1 or R.sup.11
of the donor fluorescein dye and at R.sup.18 or R.sup.19 of the
acceptor rhodamine dye. An alternative embodiment is where the
donor dye and the acceptor dye are attached by a linker through the
R.sup.18 or R.sup.19 sites, and either the donor dye or the
acceptor dye is attached to a substrate through the R.sup.1,
R.sup.11, Z.sup.1 or Z.sup.2 site. Another alternative embodiment
is where the donor dye and the acceptor dye are attached by a
linker through the R.sup.1, R.sup.11, Z.sup.1 or Z.sup.2 sites, and
either the donor dye or the acceptor dye is attached to a substrate
through the R.sup.18 or R.sup.19 site.
[0097] The energy-transfer dye compound is covalently attached to a
substrate through a linker. The linker may be a bond,
C.sub.1-C.sub.12 alkyldiyl or C.sub.6-C.sub.20 aryldiyl. The linker
may bear functional groups including amide, carbamate, urea,
thiourea, phosphate, phosphonate, sulfonate, phosphorothioate, and
the like. Preferred linkers include 1,2-ethyldiyl and
1,6-hexyldiyl. The attachment sites of the linker between the
energy-transfer dye and the substrate may be at any position on the
energy-transfer dye, where one or both of the donor dye and
acceptor dye is a dye of the present invention. Where the substrate
is a nucleoside or nucleotide, a preferred attachment site on the
substrate is on the nucleobase. If the nucleobase is a purine, the
linker may be attached at the 8-position. If the nucleobase is a
7-deazapurine, the linker may be attached at the 7-position or
8-position. If the nucleobase is a pyrimidine, the linker may be
attached at the 5-position. As examples, in terminator nucleotide
examples 25 and 26, the energy-transfer dye is attached to the
nucleobase at R.sup.19. Where the substrate is an oligonucleotide,
preferred attachment sites include the 3' and 5' terminii. Other
oligonucleotide attachment sites include the internucleotide
phosphate, or phosphate-analog linkage, or at a position on the
sugar, e.g. 2' or 4'. Where the substrate is a polypeptide (peptide
or protein), preferred attachment sites include the amino and
carboxyl termini, and lysine residue amino substituents.
VI.4 Methods of Labelling
[0098] The present invention comprises labelling reagents wherein
atropisomeric xanthene compounds are in reactive form to react with
substrates. In another aspect, the present invention comprises
substrates labelled, i.e. conjugated, with the compounds of the
invention, formula I. Substrates can be virtually any molecule or
substance to which the dyes of the invention can be conjugated,
including by way of example and not limitation, a polynucleotide, a
nucleotide, a nucleoside, a polypeptide, a carbohydrate, a ligand,
a substantially enantiomerically pure compound, a particle, a
surface, a lipid, a solid support, organic and inorganic polymers,
and combinations and assemblages thereof, such as chromosomes,
nuclei, living cells (e.g., bacteria or other microorganisms,
mammalian cells, tissues, etc.), and the like. A particle may
include a nanoparticle, a microsphere, a bead, or a liposome. A
surface may be glass or other non-porous planar material. The
compounds of the invention are conjugated with the substrate via an
optional linker by a variety of means, including hydrophobic
attraction, ionic attraction, and covalent attachment.
[0099] Labelling typically results from mixing an appropriate
reactive atropisomeric xanthene and a substrate to be conjugated in
a suitable solvent, using methods well-known in the art (Hermanson,
Bioconjugate Techniques, (1996) Academic Press, San Diego, Calif.
pp. 40-55, 643-71), followed by separation of the labelled
substrate, conjugate, from any unconjugated starting materials or
unwanted by-products. The conjugate can be stored dry or in
solution for later use.
[0100] A racemic mixture of atropisomeric xanthenes may be
separated to isolate substantially pure atropisomers at any
intermediate stage in the synthesis of the labelling reagents,
according to the aforementioned separation and isolation methods
(1), (2), and (3).
[0101] The atropisomeric xanthene may include a linking moiety at
one of the substituent positions or covalent attachment of the dye
to another molecule. A linking moiety is typically an electrophilic
functional group, capable of forming a covalent bond by reacting
with nucleophilic functionality on a substrate. Nucleophilic
functionality may include, for example, alcohols, alkoxides,
amines, hydroxylamines, and thiols. Alternatively, a linking moiety
may include nucleophilic functionality that reacts with an
electrophilic group on a substrate. Examples of linking moieties
include azido, monosubstituted primary amine, disubstituted
secondary amine, thiol, hydroxyl, halide, epoxide,
N-hydroxysuccinimidyl ester, carboxyl, isothiocyanate, sulfonyl
chloride, sulfonate ester, silyl halide, chlorotriazinyl,
succinimidyl ester, pentafluorophenyl ester, maleimide, haloacetyl,
epoxide, alkylhalide, allyl halide, aldehyde, ketone, acylazide,
anhydride, iodoacetamide and an activated ester.
[0102] One linking moiety is N-hydroxysuccinimidyl ester (NHS) of a
carboxyl group substituent of the atropisomeric xanthene compound
(FIGS. 3, 6, 10, 11). The NHS ester form of the compound is a
labelling reagent. The NHS ester of the dye may be preformed,
isolated, purified, and/or characterized, or it may be formed in
situ and reacted with a nucleophilic group of a substrate, such as
an oligonucleotide, a nucleotide, a polypeptide, or the like
(Brinkley, M. (1992) Bioconjugate Chem. 3:2-13). Typically, the
carboxyl form of the dye is activated by reacting with some
combination of: (1) a carbodiimide reagent, e.g.
dicyclohexylcarbodiimide, diisopropylcarbodiimide, or a uronium
reagent, e.g. TSTU (O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate, HBTU
(O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate), HATU
(O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate); (2) an activator, such as
1-hydroxybenzotriazole (HOBt); and (3) N-hydroxysuccinimide to give
the NHS ester of the dye. A representative example of an NHS ester
are structures 4a and 4b (FIG. 3), 8 (FIG. 4), and 13 (FIG. 5).
[0103] Functional groups on an atropisomeric xanthene compound may
be protected prior to derivatization and reaction at other
functional groups on the compound. For example, the amino group of
atropisomers 2a and 2b were trifluoroacetylated to give 3a and 3b,
separately (FIG. 2a, Examples 5 and 6). The carboxyl groups of were
then converted to the active ester, NHS with N-hydroxysuccinimide
and a carbodiimide reagent, e.g. DAE to give 4a and 4b, separately
(FIG. 3, Examples 7 and 8).
[0104] In some cases, the atropisomeric xanthene compound and the
substrate may be coupled by in situ activation of the compound and
reaction with the substrate to form the atropisomeric
xanthene-substrate conjugate in one step. Other activating and
coupling reagents include TBTU
(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluronium
hexafluorophosphate), TFFH(N,N',N'',N'''-tetramethyluronium
2-fluoro-hexafluorophosphate), PyBOP
(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate, EEDQ
(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC
(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT
(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl
sulfonyl halides, e.g. triisopropylbenzenesulfonyl chloride.
[0105] Another preferred reactive linking group is a
phosphoramidite form of asymmetric xanthene compounds.
Phosphoramidite dye reagents are particularly useful for the
automated synthesis of oligonucleotides labelled with the dyes of
the invention. Most conveniently, phosphoramidite dye reagents may
be coupled to oligonucleotides bound to a solid support during the
normal course of solid-phase synthesis. Oligonucleotides are
commonly synthesized on solid supports by the phosphoramidite
method (Caruthers, M. and Beaucage, S. "Phosphoramidite compounds
and processes", U.S. Pat. No. 4,415,732; Caruthers, M. and
Matteucci, M. "Process for preparing polynucleotides", U.S. Pat.
No. 4,458,066; Beaucage, S. and Iyer, R. (1992) "Advances in the
synthesis of oligonucleotides by the phosphoramidite approach",
Tetrahedron 48:2223-2311).
[0106] Phosphoramidite atropisomeric xanthene reagents can be
nucleosidic or non-nucleosidic. Non-nucleosidic forms of the
phosphoramidite reagents have the general formula III: ##STR12##
where DYE is a protected or unprotected form of atropisomer
xanthene II, including energy-transfer dye; L is a linker; R.sup.30
and R.sup.31 taken separately are C.sub.1-C.sub.12 alkyl,
C.sub.4-C.sub.10 aryl, and cycloalkyl containing up to 10 carbon
atoms, or R.sup.30 and R.sup.31 taken together with the
phosphoramidite nitrogen atom form a saturated nitrogen
heterocycle; and R.sup.32 is a phosphite ester protecting group
which prevents unwanted extension of the oligonucleotide.
Generally, R.sup.32 is stable to oligonucleotide synthesis
conditions yet is able to be removed from a synthetic
oligonucleotide product with a reagent that does not adversely
affect the integrity of the oligonucleotide or the dye. R.sup.32
may be: (i) methyl, (ii) 2-cyanoethyl; --CH.sub.2CH.sub.2CN, or
(iii) 2-(4-nitrophenyl)ethyl; --CH.sub.2CH.sub.2(p-NO.sub.2Ph).
Embodiments of phosphoramidite reagents include where: (i) R.sup.30
and R.sup.31 are each isopropyl, (ii) R.sup.30 and R.sup.31 taken
together is morpholino, (iii) L is C.sub.1-C.sub.12 alkyl, (iv)
R.sup.32 is 2-cyanoethyl, and (v) DYE is attached at R.sup.18 or
R.sup.19 by a linker. The linker, L, may alternatively be:
##STR13## where n ranges from 1 to 10. An example of
phosphoramidite reagent III has the structure: ##STR14##
[0107] Phosphoramidite dye reagents III effect labelling of a
substrate with a single, substantially pure, atropisomeric xanthene
of the invention. Where the substrate is an oligonucleotide, the
dye will be attached at the 5' terminus of the oligonucleotide, as
a consequence of the typical 3' to 5' direction of synthesis, or at
the 3' terminus of the oligonucleotide when the 5' to 3' direction
synthesis method is practiced (Wagner (1997) Nucleosides &
Nucleotides 16:1657-60). Reagent III may be coupled to a
polynucleotide which is bound to a solid support, e.g. through the
3' terminus. Other phosphoramidite dye reagents, nucleosidic and
non-nucleosidic allow for labelling at other sites of an
oligonucleotide, e.g. 3' terminus, nucleobase, internucleotide
linkage, sugar. Labelling at the nucleobase, internucleotide
linkage, and sugar sites allows for internal and multiple labelling
with fluorescent dyes.
[0108] An atropisomeric xanthene compound of the invention may be
converted to a non-nucleosidic, phosphoramidite labelling reagent
by any known method of phosphitylation of nucleophilic
functionality with trivalent phosphitylating reagents. For example,
when the xanthene contains a carboxyl group, e.g.
R.sup.19=CO.sub.2H, the carboxyl may be activated, e.g. to the NHS,
and amidated with 6-amino-1-hexanol. The resulting hydroxyl may be
phosphitylated with bis(diisopropylamino)cyanoethylphosphite or
chloro-diisopropylamino-cyanoethylphosphine to give the
phosphoramidite dye-labelling reagent (Theisen (1992) "Fluorescent
dye phosphoramidite labelling of oligonucleotides", in Nucleic Acid
Symposium Series No. 27, Oxford University Press, Oxford, pp.
99-100). Alternatively, the carboxyl group of the compound may be
reduced to the hydroxyl, to be phosphitylated.
[0109] The phosphoramidite reagent III reacts with a hydroxyl
group, e.g. 5' terminal OH of an oligonucleotide bound to a solid
support, under mild acid activation, to form an internucleotide
phosphite group which is then oxidized to an internucleotide
phosphate group. In some instances, the xanthene compound may
contain functional groups, e.g. Z.sup.1 and Z.sup.2 oxygens as in
structure I, that require protection either during the synthesis of
the phosphoramidite reagent or during its subsequent use to label
molecules such as oligonucleotides. The protecting group(s) used
will depend upon the nature of the functional groups, and will be
apparent to those having skill in the art (Greene, T. and Wuts, P.
Protective Groups in Organic Synthesis, 2nd Ed., John Wiley &
Sons, New York, 1991). Generally, the protecting groups used should
be stable under the acidic conditions (e.g. trichloroacetic acid,
dichloroacetic acid) commonly employed in oligonucleotide synthesis
to remove 5'-hydroxylprotecting groups (e.g., dimethoxytrityl) and
labile under the basic conditions (ammonium hydroxide, aqueous
methylamine) used to deprotect and/or cleave synthetic
oligonucleotides from solid supports.
[0110] Polypeptides, antibodies, and other biopolymers comprised of
amino acids and amino acid analogs may be covalently labelled by
conjugation with the atropisomeric xanthene compounds of the
invention. Typically, the compound is in electrophilic form, e.g.
NHS reactive linking group, which reacts with a nucleophilic group
of the peptide, e.g. amino terminus, or amino side chain of an
amino acid such as lysine. Alternatively, the dye may be in
nucleophilic form, e.g. amino- or thiol-reactive linking group,
which may react with an electrophilic group of the peptide, e.g.
NHS of the carboxyl terminus or carboxyl side chain of an amino
acid. Labelled polypeptides may retain their specific binding and
recognition properties in interacting with cell surface and
intracellular components. The xanthene compound, acting as a dye,
provides a detection element for localizing, visualizing, and
quantitating the binding or recognition event. Polypeptides can
also be labelled with two moieties, a fluorescent reporter and
quencher, which together undergo fluorescence resonance energy
transfer (FRET). The fluorescent reporter may be partially or
significantly quenched by the quencher moiety in an intact
polypeptide. Upon cleavage of the polypeptide by a peptidase or
protease, a detectable increase in fluorescence may be measured
(Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes",
Methods in Enzymology, Academic Press, 248:18-34).
VI.4A Labelled Nucleotides
[0111] A preferred class of labelled substrates include conjugates
of nucleosides and nucleotides that are labelled with the dyes of
the invention. Such labelled nucleosides and nucleotides are
particularly useful for labelling polynucleotides formed by
enzymatic synthesis, e.g., labelled nucleotide 5'-triphosphates
used in the context of PCR amplification, Sanger-type
polynucleotide sequencing, and nick-translation reactions.
[0112] Nucleosides and nucleotides can be labelled at sites on the
sugar or nucleobase moieties. Preferred nucleobase labelling sites
include the 8-C of a purine nucleobase, the 7-C or 8-C of a
7-deazapurine nucleobase, and the 5-position of a pyrimidine
nucleobase. Between a nucleoside or nucleotide and a dye, a linker
may attach to an atropisomeric xanthene compound at any
position.
[0113] The labelled nucleoside or nucleotide may be enzymatically
incorporatable and enzymatically extendable. Nucleosides or
nucleotides labelled with compounds of the invention may have
formula IV: ##STR15## where DYE is a protected or unprotected form
of compounds I or II, including energy-transfer dye. B may be any
nucleobase, e.g. uracil, thymine, cytosine, adenine,
7-deazaadenine, guanine, and 8-deazaguanosine. R.sup.25 is H,
monophosphate, diphosphate, triphosphate, thiophosphate, or
phosphate ester analog. R.sup.26 and R.sup.27, when taken alone,
are each independently H, HO, F and a phosphoramidite. Where
R.sup.26 or R.sup.27 is phosphoramidite, R.sup.25 is an
acid-cleavable hydroxyl protecting group, e.g. dimethoxytrityl,
which allows subsequent monomer coupling under automated synthesis
conditions (Caruthers, "Phosphoramidite compounds and processes",
U.S. Pat. No. 4,415,732; Caruthers, "Process for preparing
polynucleotides", U.S. Pat. No. 4,458,066; Beaucage, S. and Iyer,
R. (1992) "Advances in the synthesis of oligonucleotides by the
phosphoramidite approach", Tetrahedron 48:2223-2311).
[0114] Where the labelled nucleoside or nucleotide is a terminator,
R.sup.26 and R.sup.27 are selected to block polymerase-mediated
template-directed polymerization. In terminator nucleotides,
R.sup.26 and R.sup.27, when taken alone, are each independently H,
F, and a moiety which blocks polymerase-mediated template-directed
polymerization, or when taken together form 2'-3'-didehydroribose.
In formula IV, when both R.sup.26 and R.sup.27 are hydroxyl, the
resultant compounds are labelled ribonucleosides and
ribonucleotides (NTP). When R.sup.27 is hydrogen and R.sup.26 is
hydroxyl, the resultant compounds are labelled
2'-deoxyribonucleosides and nucleotides (dNTP). When R.sup.26 and
R.sup.27 are each hydrogen, the resultant compounds are
2',3'-dideoxyribonucleosides and nucleotides (ddNTP). Labelled
ddNTP find particular use as terminators in Sanger-type DNA
sequencing methods utilizing fluorescent detection. Labelled
2'-deoxyribonucleoside-5'-triphosphates (dNTP) find particular use
as reagents for labelling DNA polymerase extension products, e.g.,
in the polymerase chain reaction or nick-translation. Labelled
ribonucleoside-5'-triphosphates (NTP) find particular use as
reagents for labelling RNA polymerase extension products.
##STR16##
[0115] Alkynylamino-linked compounds IV, where L includes an
alkyndiyl group, are useful for conjugating atropisomeric xanthene
compounds to nucleosides, nucleotides and analogs therein. Their
synthesis is taught in EP 87305844.0 and Hobbs, (1989) J. Org.
Chem. 54:3420. The corresponding nucleoside mono-, di- and
triphosphates are obtained by standard techniques (for example, the
methods described in U.S. Pat. Nos. 5,821,356; 5,770,716;
5,948,648; 6,096,875). Methods for synthesizing compounds IV with
modified propargylethoxyamido linkers L can also be found in these
patents. Additional synthesis procedures suitable for use in
synthesizing compounds according to structural formula IV are
described, for example, in Gibson (1987) Nucl. Acids Res.
15:6455-6467; Gebeyehu (1987) Nucl. Acids Res. 15:4513-4535;
Haralambidis (1987) Nucl. Acids Res. 15:4856-4876; Nelson (1986)
Nucleosides and Nucleotides. 5(3):233-241; Bergstrom (1989) J. Am.
Chem. Soc. 111:374-375; U.S. Pat. No. 4,855,225, U.S. Pat. No.
5,231,191 and U.S. Pat. No. 5,449,767, which are incorporated
herein by reference. Any of these methods can be routinely adapted
or modified as necessary to synthesize the full range of labelled
nucleosides, nucleotides, and analogs described herein.
[0116] One embodiment of the alkynyl linker L may be: ##STR17##
wherein n is 0, 1, or 2.
[0117] Energy-transfer dye pairs can be conjugated to a nucleotide
5'-triphosphate by linking through a nucleobase amino group to: (i)
an activated ester of a energy-transfer dye pair, or (ii) stepwise
coupling to one dye, e.g. R.sup.11-protected aminomethyl,
R.sup.18-carboxyl fluorescein, then coupling the unprotected
R.sup.11-aminomethyl to the second dye of the pair.
[0118] Linker reagents may be prepared by known synthetic methods.
For example, phosphate linker reagent 5 is synthesized starting
from the cyclic phosphoramidite 7. Phosphitylation of 7 with methyl
glycolate 6 was followed by in situ oxidation to the pentavalent
phosphate 8. Hydrolysis of the methyl ester, the trifluoroacetate
group, and demethylation gave 9. Protection of the amino group with
Fmoc gave 10 which was activated as the N-hydroxysuccinimide ester,
linker reagent 5 (FIG. 4, Example 9).
[0119] An alkynylamino-linked nucleotide can be prepared by first
coupling NHS linker reagent 5 with 7-deaza-7-propargylamino-ddATP
12 to give 13, followed by hydrolysis of the Fmoc group to give 11
(FIG. 5, Example 10). The amino atropisomeric xanthene 1a is
coupled with the N-Fmoc, NHS ester of p-aminomethylbenzoic acid
(Example 11) and then activated as the NHS ester to give 14 (FIG.
6). Reaction of 11 and 14 gave the atropisomeric xanthene ddATP
compound 16. The Fmoc group was removed with ammonium hydroxide to
give 15 (FIG. 6, Example 12).
[0120] The NHS-rhodamine dye 17 was synthesized from bicyclic amine
18. Cyclization with 1-bromo-3-chloropropane gave tricyclic ester
19, which was hydrolyzed to tricyclic amine 20 (FIG. 7, Example
13). Friedel-Crafts acylation of 20 with anhydride 21 gave the
ketone 22 which was reacted with another equivalent of 20 to give
symmetric rhodamine isopropyl ester 23. The ester of 23 was cleaved
and the carboxylic acid 24 was converted to NHS-rhodamine dye 17
(FIG. 8, Example 13).
[0121] The substantially pure atropisomer xanthene energy transfer
ddATP terminator 25 was formed by coupling 15 with 17, followed by
anion-exchange HPLC purification (FIG. 9, Example 14).
[0122] Alternative synthetic routes to energy-transfer nucleotides
and polynucleotides, with different convergent schemes may be
practiced. The substrate, dye, and linker subunits, or synthons,
may be assembled for coupling in any order. For example, the
energy-transfer pair of donor dye and acceptor dye may be
covalently attached through a linker and then coupled to the
nucleotide or polynucleotide. Many different synthetic routes can
be practiced which result in the labelling of nucleotides with the
dyes of the invention. Reactive functionality, such as carboxylic
acid, amino, hydroxyl groups, may require protection, utilizing the
vast art of organic synthesis methodology.
[0123] Another rhodamine dye 28 was protected as the
bis-trifluoroacetamide 29 and converted to the NHS compound 27
(FIG. 10, Example 15). Propargylethoxyamino ddTTP 30 was coupled
with atropisomer xanthene compound 14 to give Fmoc atropisomer
xanthene ddTTP 31 which was hydrolyzed to 32 (FIG. 11, Example 16).
Reaction of 27 and 32 gave atropisomer, energy-transfer terminator
ddTTP 26, purified by anion-exchange HPLC (FIG. 12, Example
17).
VI.4B Labelled Oligonucleotides
[0124] Oligonucleotides are commonly synthesized on solid supports
by the phosphoramidite method (U.S. Pat. Nos. 4,415,732; 4,973,679;
4,458,066; Beaucage, S. and Iyer, R. (1992) Tetrahedron
48:2223-2311) using commercially available phosphoramidite
nucleosides, supports e.g. silica, controlled-pore-glass (U.S. Pat.
No. 4,458,066) and polystyrene (U.S. Pat. Nos. 5,047,524 and
5,262,530) and automated synthesizers (Models 392, 394, 3948
DNA/RNA Synthesizers, Applied Biosystems).
[0125] Another preferred class of labelled substrates include
conjugates of oligonucleotides and the compounds of the invention.
Such conjugates may find utility as DNA sequencing primers, PCR
primers, oligonucleotide hybridization probes, oligonucleotide
ligation probes, double-labelled 5'-exonuclease (TaqMan.TM.)
probes, and the like (Fung, U.S. Pat. No. 4,757,141; Andrus,
"Chemical methods for 5' non-isotopic labelling of PCR probes and
primers" (1995) in PCR 2: A Practical Approach, Oxford University
Press, Oxford, pp. 39-54; Hermanson, Bioconjugate Techniques,
(1996) Academic Press, San Diego, Calif. pp. 40-55, 643-71; Mullah
(1998) "Efficient synthesis of double dye-labelled
oligodeoxyribonucleotide probes and their application in a real
time PCR assay", Nucl. Acids Res. 26:1026-1031). A labelled
oligonucleotide may have formula V: ##STR18## where the
oligonucleotide comprises 2 to 100 nucleotides. DYE is a protected
or unprotected form of compounds I or II, including energy-transfer
dye. B is any nucleobase, e.g. uracil, thymine, cytosine, adenine,
7-deazaadenine, guanine, and 8-deazaguanosine. L is a linker.
R.sup.27 is H, OH, halide, azide, amine, C.sub.1-C.sub.6
aminoalkyl, C.sub.1-C.sub.6 alkyl, allyl, C.sub.1-C.sub.6 alkoxy,
OCH.sub.3, or OCH.sub.2CH.dbd.CH.sub.2. R.sup.22 is H, phosphate,
internucleotide phosphodiester, or internucleotide analog. R.sup.29
is H, phosphate, internucleotide phosphodiester, or internucleotide
analog. In this embodiment, structure V, the nucleobase-labelled
oligonucleotide may bear multiple dyes of the invention attached
through the nucleobases. Nucleobase-labelled oligonucleotide V may
be formed by: (i) enzymatic incorporation of enzymatically
incorporatable nucleotide reagents IV where R.sup.25 is
triphosphate, by a DNA polymerase or ligase, and (ii) coupling of a
nucleoside phosphoramidite reagent by automated synthesis. Whereas,
nucleobase-labelled oligonucleotides V may be multiply labelled by
incorporation of more than one incorporatable nucleotide IV,
labelling with a dye label reagent such as III leads to singly
5'-labelled oligonucleotides, according to formula VI: ##STR19##
where X is O, NH, or S; R.sup.27 is H, OH, halide, azide, amine,
C.sub.1-C.sub.6 aminoalkyl, C.sub.1-C.sub.6 alkyl, allyl,
C.sub.1-C.sub.6 alkoxy, OCH.sub.3, or OCH.sub.2CH.dbd.CH.sub.2;
R.sup.28 is H, phosphate, internucleotide phosphodiester, or
internucleotide analog; and L is C.sub.1-C.sub.12 alkyl, aryl, or
polyethyleneoxy of up to 100 ethyleneoxy units.
[0126] The linker L in formulas V or VI may be attached at any site
on the atropisomeric xanthene compound of the invention, DYE,
including R.sup.1, R.sup.11, R.sup.18, R.sup.19, Z.sup.1 and
Z.sup.2 of structure I.
[0127] In a first method for labelling synthetic oligonucleotides,
a nucleophilic functionality, e.g. a primary aliphatic amine, is
introduced at a labelling attachment site on an oligonucleotide,
e.g. a 5' terminus. After automated, solid-support synthesis is
complete, the oligonucleotide is cleaved from the support and all
protecting groups are removed. The nucleophile-oligonucleotide is
reacted with an excess of a label reagent containing an
electrophilic moiety, e.g. isothiocyanate or activated ester, e.g.
N-hydroxysuccinimide (NHS), under homogeneous solution conditions
(Hermanson, Bioconjugate Techniques, (1996) Academic Press, San
Diego, Calif. pp. 40-55, 643-71; Andrus, A. "Chemical methods for
5' non-isotopic labelling of PCR probes and primers" (1995) in PCR
2: A Practical Approach, Oxford University Press, Oxford, pp.
39-54). Labelled oligonucleotides VI may be formed by reacting a
reactive linking group form, e.g. NHS, of a dye, with a
5'-aminoalkyl oligonucleotide.
[0128] In a second method, a label is directly incorporated into
the oligonucleotide during or prior to automated synthesis, for
example as a support reagent (Mullah, "Solid support reagents for
the direct synthesis of 3'-labelled polynucleotides", U.S. Pat. No.
5,736,626; Nelson, "Multifunctional controlled pore glass reagent
for solid phase oligonucleotide synthesis", U.S. Pat. No.
5,141,813) or as a phosphoramidite reagent III. Certain fluorescent
dyes and other labels have been functionalized as phosphoramidite
reagents for 5' labelling (Theisen (1992) Nucleic Acid Symposium
Series No. 27, Oxford University Press, Oxford, pp. 99-100).
[0129] Generally, if the labelled oligonucleotide is made by
enzymatic synthesis, the following procedure may be used. A target
DNA is denatured and an oligonucleotide primer is annealed to the
template DNA. A mixture of enzymatically-incorporatable nucleotides
or nucleotide analogs capable of supporting continuous
template-directed enzymatic extension of the primed target (e.g., a
mixture including dGTP, dATP, dCTP and dTTP or dUTP) is added to
the primed target. At least a fraction of the nucleotides are
labelled terminators IV, labelled with an atropisomer xanthene dye
II. A polymerase enzyme is next added to the mixture under
conditions where the polymerase enzyme is active. A labelled
oligonucleotide is formed by the incorporation of the labelled
nucleotides or terminators during polymerase-mediated strand
synthesis. In an alternative enzymatic synthesis method, two
primers are used instead of one: one complementary to the (+)
strand of the target and another 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
labelled complement to the target sequence by PCR (Innis (1990) PCR
Protocols, Eds., Academic Press).
[0130] In one preferred post-synthesis chemical labelling method an
oligonucleotide is labelled as follows. An NHS form of a dye
according to structure I is dissolved or suspended in DMSO and
added in excess (5-20 equivalents) to a 5'-aminohexyl
oligonucleotide in 0.25 M bicarbonate/carbonate buffer at about pH
9 and allowed to react for 6 hours, e.g., U.S. Pat. No. 4,757,141.
The atropisomer xanthene labelled oligonucleotide can be 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
labelled oligonucleotide is further purified by reverse phase HPLC
employing gradient elution.
[0131] Polynucleotides labelled with the atropisomer xanthene
compounds of the present invention may be additionally labelled
with moieties that affect the rate of electrophoretic migration,
i.e. mobility-modifying labels. Mobility-modifying labels include
polyethyleneoxy units, --(CH.sub.2CH.sub.2O).sub.n-- where n may be
1 to 100 (Grossman, U.S. Pat. No. 5,624,800). Preferably, n is from
2 to 20. The polyethyleneoxy units may be interspersed with
phosphate groups. Specifically labelling atropisomer
xanthene-labelled polynucleotides with additional labels of
polyethyleneoxy of discrete and known size allows for separation by
electrophoresis, substantially independent of the number of
nucleotides in the polynucleotide. That is, polynucleotides of the
same length may be discriminated upon by the presence of spectrally
resolvable dye labels and mobility-modifying labels.
Polynucleotides bearing both dye labels and mobility-modifying
labels may be formed enzymatically by ligation or polymerase
extension of the single-labelled polynucleotide or nucleotide
constituents.
VI.5 Methods
[0132] Methods requiring simultaneous detection of multiple
spatially-overlapping analytes may benefit from substantially pure
atropisomers of asymmetric xanthene dyes as labels. The atropisomer
xanthene compounds of the present invention are well suited for any
method utilizing fluorescent detection, such as polymerase chain
reaction (PCR) amplification, DNA sequencing, antisense
transcriptional and translational control of gene expression,
genetic analysis, and DNA probe-based diagnostic testing (Kricka,
L. (1992) Nonisotopic DNA Probe Techniques, Academic Press, San
Diego, pp. 3-28). Fluorescence detection of fluorescent
dye-labelled oligonucleotides is the basis for nucleic acid
sequence detection assays such as 5' exonuclease assay (Livak, U.S.
Pat. No. 5,723,591), FRET hybridization (Tyagi, S. and Kramer, F.
(1996) "Molecular Beacons: Probes that fluoresce upon
hybridization", Nature Biotechnology, 14:303-08), genetic linkage
mapping (Dib (1996) "A comprehensive genetic map of the human
genome based on 5,264 microsatellites", Nature 380:152-54) and
oligonucleotide-ligation assay (Grossman (1994) "High-density
multiplex detection of nucleic acid sequences: oligonucleotide
ligation assay and sequence-coded separation", Nucl. Acids Res.
22:4527-34).
[0133] The present invention is particularly well suited for
detecting classes of differently-labelled polynucleotides that have
been subjected to a biochemical separation procedure, such as
electrophoresis (Rickwood and Hames, Eds., Gel Electrophoresis of
Nucleic Acids: A Practical Approach, IRL Press Limited, London,
1981). The electrophoretic matrix may be a sieving polymer, e.g.
crosslinked or uncrosslinked polyacrylamide, or other
amide-containing polymer, having a concentration (weight to volume)
of between about 2-20 weight percent (Madabhushi, U.S. Pat. Nos.
5,552,028; 5,567,292; 5,916,426). The electrophoretic matrix may be
configured in a slab gel or capillary format (Rosenblum, (1997)
Nucleic Acids Res. 25:3925-29; Mathies, U.S. Pat. No.
5,274,240).
VI.5A Primer Extension
[0134] In a preferred category of methods referred to herein as
"fragment analysis" or "genetic analysis" methods, polynucleotide
fragments labelled with fluorescent dyes, including substantially
pure atropisomeric xanthene compounds, are generated through
template-directed enzymatic synthesis using labelled primers or
nucleotides, e.g. by ligation or polymerase-directed primer
extension. The polynucleotide fragments may be subjected to a
size-dependent separation process, e.g., electrophoresis or
chromatography, and the separated fragments are detected subsequent
to the separation, e.g., by laser-induced fluorescence
(Hunkapiller, U.S. Pat. No. 4,811,218). Multiple classes of
polynucleotides may be separated simultaneously and the different
classes are distinguished by spectrally resolvable labels,
including dyes of the invention. In electrophoresis, the classes
separate on the basis of electrophoretic migration rate.
VI.5B DNA Sequencing
[0135] Preferably, the chain termination methods of DNA sequencing,
i.e. dideoxyu DNA sequencing, or Sanger-type sequencing, and
fragment analysis is employed (Sanger (1977) "DNA sequencing with
chain-terminating inhibitors", Proc. Natl. Acad. Sci. USA
74:5463-5467). Exemplary chain-terminating nucleotide analogs
include the 2',3'-dideoxynucleoside 5'-triphosphates (ddNTP) which
lack the 3'-OH group necessary for 3' to 5' DNA chain elongation.
Primers or ddNTP may be labelled with the substantially pure
atropisomer xanthene dyes of the invention and detected by
fluorescence after separation of the fragments by high-resolution
electrophoresis. Dyes can be linked to functionality on the 5'
terminus of the primer, e.g. amino (Fung, U.S. Pat. No. 4,757,141),
on the nucleobase of a primer; or on the nucleobase of a
dideoxynucleotide, e.g. via alkynylamino linking groups (Khan, U.S.
Pat. Nos. 5,770,716; and 5,821,356; Hobbs, U.S. Pat. No.
5,151,507).
[0136] Each of the terminators bears a different fluorescent dye
and collectively the terminators of the experiment bear a set of
dyes including one or more from the dyes of the invention. In a
preferred fragment analysis method, fragments labelled with dyes
are identified by relative size, i.e. sequence length.
Correspondence between fragment size and sequence is established by
incorporation of the four possible terminating nucleotides
("terminators") and the members of a set of spectrally resolvable
dyes (Bergot, U.S. Pat. No. 5,366,860). The set of spectrally
resolvable dyes may include at least one substantially pure
atropisomeric xanthene compound.
VI.6 Ligation
[0137] The covalent joining of nucleic acid probes by ligase
enzymes is one of the most useful tools available to molecular
biologists. When two probes are annealed to a template nucleic acid
where the two probes are adjacent and without intervening gaps, a
phosphodiester bond can be formed between a 5' terminus of one
probe and the 3' terminus of the other probe by a ligase enzyme,
(Whiteley, U.S. Pat. No. 4,883,750; Landegren, (1988) "A ligase
mediated gene detection technique", Science 241:1077-80; Nickerson,
"Automated DNA diagnostics using an ELISA-based oligonucleotide
assay" (1990) Proc. Natl. Acad. Sci USA 87:8923-27).
Oligonucleotide ligation assays detect the presence of specific
sequences in target DNA sample. Where one or both probes are
labelled with a dye, the ligation product may be detected by
fluorescence. One or both probes may be labelled with a
substantially pure atropisomeric xanthene dye. Ligation products
may be detected by electrophoresis, chromatography, or other size-
or charge-based separation method.
VI.7 Amplification
[0138] The atropisomer xanthene compounds of the invention find
applications as labels on 5'-labelled oligonucleotide primers for
the polymerase chain reaction (PCR) and other nucleic acid
amplification and selection methods. PCR applications include the
use of labelled oligonucleotides for genotyping by variable number
tandem repeat (VNTR), short tandem repeat (STR), and microsatellite
methods of amplification of repeat regions of double-stranded DNA
that contain adjacent multiple copies of a particular sequence,
with the number of repeating units being variable. Preferably, in
such PCR genotyping methods, the PCR primer is labelled with an
atropisomer xanthene of the invention.
[0139] In a particularly preferred embodiment, atropisomer xanthene
compounds may be used in quantitative methods and reagents that
provide real time or end-point measurements of amplification
products during PCR (U.S. Pat. Nos. 5,210,015; 5,538,848). The
exonuclease assay (Taqman.RTM.) employing fluorescent dye-quencher
probes (U.S. Pat. No. 5,723,591; Mullah, (1998) "Efficient
synthesis of double dye-labelled oligodeoxyribonucleotide probes
and their application in a real time PCR assay", Nucl. Acids Res.
26:1026-1031) gives direct detection of polymerase chain reaction
(PCR) products in a closed-tube system, with no sample processing
beyond that required to perform the PCR. In the Taqman assay, the
polymerase that conducts primer extension and amplifies the
polynucleotide also displaces and cleaves a probe annealed to
target sequence by 5' to 3' exonuclease activity. In a Taqman-type
assay, the probe is self-quenching, labelled with fluorescent dye
and quencher moieties, either of which may be dyes of the
invention. Spectral overlap allows for efficient energy transfer
(FRET) when the probe is intact (Clegg, (1992) "Fluorescence
resonance energy transfer and nucleic acids", Meth. Enzymol.
211:353-388). When hybridized to a target sequence, the probe is
cleaved during PCR to release a fluorescent signal that is
proportional to the amount of target-probe hybrid present (U.S.
Pat. Nos. 5,538,848; 5,723,591).
[0140] The progress of amplification can be monitored continuously,
i.e. real-time detection. Spectrally-resolvable atropisomer
xanthene dyes of the invention are useful in genotyping experiments
after PCR amplification of target. In particular, a set of primer
oligonucleotides, labelled at the 5' terminus, each with different
dyes, can amplify multiple loci and discriminate single nucleotide
polymorphisms (SNP) and alleles. Electrophoretic separation of the
dye-labelled amplification products, with size standards,
establishes a profile or characteristic data set indicating a
certain genotype dependent on the set of primer sequences.
VI.7A Hybridization Assays
[0141] Certain fluorescent dye-quencher probes which hybridize to
target nucleic acids are useful in hybridization assays. When the
probe is not hybridized to target, the probe may attain
conformations that allow spatial proximity between the fluorescent
dye and the quencher moieties resulting in fluorescence quenching.
Upon hybridization to target, the moieties are physically
separated, quenching ceases or diminishes, and fluorescence
increases. Where the fluorescence is detectable or quantitated, the
presence of target sequence in the sample is deduced. The
atropisomeric dyes of the invention can also be employed as the
fluorescent dye or the quencher moiety. Fluorescent dye-quencher
probes with self-complementary sequences that form a "hairpin"
region, so called "Molecular beacons" (Tyagi and Kramer) undergo
the fluorescent change upon hybridization to their complementary
target sequence, e.g. in situ quantitation of mRNA in living cells.
Hybridization probes labelled with different fluorescent dyes,
including the atropisomeric dyes of the invention, enable
multiplex, homogeneous hybridization assays to be carried out in
sealed reaction tubes.
VI.8 Chromatography
[0142] The aforementioned methods employing substrates labelled
with substantially pure atropisomer xanthene compounds may also be
conducted where the labelled substrates are detected by
chromatography (HPLC of Macromolecules, A Practical Approach,
Second Edition, R. W. A. Oliver, Ed. (1997) Oxford University
Press). The well established techniques of HPLC enable the
separation of large substrates such as polynucleotides under
reverse phase conditions where the sample substrate is dissolved
and eluted in aqueous organic mobile phase from sorptive
ion-exchange or hydrophobic interactions with an immobilized solid
phase. When a chiral substrate such as a polynucleotide,
polypeptide, or polysaccharide is labelled with a racemic mixture
of atropisomeric xanthene compounds, diastereomers result.
Essentially a redundant set of analytes are created which may
obscure the analytical result. Analysis of the resulting
diastereomeric mixture may lead to double peaks, broad peaks, and
other limiting artifacts under the high-resolution conditions of
HPLC. This problem is especially exacerbated where the chiral
substrate is a mixture of closely related compounds, such as the
nested set of polynucleotide fragments generated by the Sanger
sequencing method. Use of a substantially pure atropisomeric form
of xanthene compounds as labels for chiral substrates prevents this
unwanted hindrance to analysis by removing one of the
diastereomers. The surprising and unexpected benefit of the
invention may be exemplified by sharper peaks, less split peaks,
and better resolution in general.
VI.9 Kits
[0143] The invention includes kits comprising the substantially
pure atropisomer xanthene compounds of the invention and/or their
labelled conjugates. In one embodiment, the kits are useful for
conjugating an atropisomer xanthene compound with a linking moiety
to another molecule, i.e. a substrate. Such kits generally comprise
an atropisomer xanthene of the invention including an optional
linking moiety and reagents, enzymes, buffers, solvents, etc.
suitable for conjugating the dye to another molecule or substance.
The atropisomer xanthene may be an acceptor or donor of an
energy-transfer dye.
[0144] In one embodiment, the kits are useful for labelling
enzymatically synthesized oligonucleotides and polynucleotides with
the atropisomer xanthenes of the invention. Such kits generally
comprise a labelled enzymatically-incorporatable nucleotide or
nucleotide analog according to the invention, a mixture of
enzymatically-incorporatable nucleotides or nucleotide analogs
capable of supporting continuous primer extension and a polymerase
enzyme. Preferably, the labelled enzymatically-incorporatable
nucleotide or nucleotide analog is a compound according to
structure IV, most preferably a labelled terminator. Preferred
polymerases are thermostable, such as AMPLITAQ DNA polymerase FS
(Applied Biosystems, Foster City, Calif.).
[0145] Alternatively, the kit may include one or more primers. The
primers may be labelled with atropisomer xanthenes and
energy-transfer dyes including atropisomer xanthenes.
VI.10 Examples
[0146] The invention will be further clarified by a consideration
of the following examples, which are intended to be purely
exemplary of the present invention and not to in any way limit its
scope.
EXAMPLE 1
[0147] Preparation of menthyl carbamate diastereomers of C-1
aminomethyl, C-19 carboxy fluorescein, 1a and 1b.
[0148] The hydrochloride salt of C-1 aminomethyl, C-19 carboxy
fluorescein (5.16 gm, 11.6 mMol, 441.8 MW; Shipchandler (1987)
Anal. Biochem. 162:89-101) was dissolved in 50 ml of deionized
formamide and 10.2 ml diisopropylethylamine. (-) Menthyl
chloroformate (3.06 gm, 3.0 ml, 14 mMol, 219 MW; Aldrich Chemical,
Milwaukee, Wis.; Jour. Chem. Soc., Chem. Commun. (1987) 470; Yodo
(1988) Chem. Pharm. Bull. 36:902) was added dropwise with stirring
at room temperature under argon. After 1.5 hours, TLC analysis
(ethyl acetate/hexane:4/1) showed partial conversion of reactant to
a higher Rf spot. Another 1 ml (-) Menthyl chloroformate was added
and stirring was continued for another 0.5 hour. TLC analysis
showed complete conversion to the higher Rf product. Dilution of
the reaction mixture with saturated aqueous NaHCO.sub.3 was
followed by extraction with 500 ml ethyl acetate. The aqueous
fraction was acidified to pH 3 and extracted with ethyl acetate.
The combined organic fractions were dried over Na.sub.2SO.sub.4,
filtered and concentrated under vacuum to give 4.5 gm, 66% yield of
a mixture of 1a and 1b as a yellow powder (FIG. 1a).
EXAMPLE 2
[0149] Separation and isolation of diastereomers 1a and 1b by
HPLC.
[0150] Crude diastereomers 1a and 1b were separated and purified by
a two stage chromatography process on an open column, flash reverse
phase column rough separation, followed by preparative reverse
phase HPLC.
[0151] The mixture of 1a and 1b was dissolved in ethyl acetate and
adsorbed on C-18 reverse phase silica gel. The solvent was removed
under vacuum and the solid was loaded on the top of a
pre-equilibrated C-18 reverse phase column (21 cm length.times.6 cm
diameter). The diastereomers were separated and eluted with 25%
CH.sub.3CN in 100 mM TEAA (triethylammonium acetate) by collecting
fractions. The fractions were analyzed by analytical reverse phase
HPLC on a C-18 column (Metachem ODS3, 25 cm length.times.4.6 mm
inner diameter) with a linear gradient of 25% to 35% CH.sub.3CN in
100 mM TEAA from 0 to 30 minutes at 1.0 ml/min flow rate and 260 nm
UV detection. The fractions that contained the first eluting
diastereomer of at least 75% purity were combined and concentrated
under vacuum to an orange oil. The first eluting diastereomer was
arbitrarily assigned structure 1a. The fractions that contained the
second eluting diastereomer of at least 75% purity were combined
and concentrated under vacuum to an orange oil. The second eluting
diastereomer was arbitrarily assigned structure 1b.
[0152] Diastereomer 1a was purified to 99% isomeric purity by
preparative reverse phase HPLC by loading 600-800 mg of 1a purified
to 75% purity by the flash process, dissolved in 500 ml of 100 mM
TEAA on to a Metachem ODS3 8.mu. column (Waters Prep LC 2000
System) and eluting under a gradient of 0 to 10% CH.sub.3CN in 100
mM TEAA over 16 min., 10 to 35% CH3CN over 80 min., then hold at
35% CH.sub.3CN for 32 min., at a flow rate of 40 ml/min., with UV
detection at 260 nm. Fractions were collected and analyzed by the
analytical reverse phase HPLC conditions above (FIG. 1b). Fractions
with isomeric purity of at least 99% were combined, acidified to pH
2 with 6N HCl and extracted with ethyl acetate. The ethyl acetate
fraction was washed with saturated NaCl, dried over anhydrous
Na.sub.2SO.sub.4, concentrated under vacuum, precipitated with
hexane, filtered, and dried to yield 300 to 500 mg of 1a as a
bright yellow solid. .sup.1H NMR (Acetone-d6) .delta. 9.85, 2H, br;
9.10, 1H, br; 8.35, 1H, d; 8.15, 1H, d; 7.83, 1H, s; 7.43, 1H, br;
6.95, 1H, s; 6.70, 4H, m; 4.60, 3H, m; 1.90, 2H, m; 1.65, 2H, m;
1.45, 1H, m; 1.30, 2H, m; 0.89, 3H, d; 0.82, 3H, d; 0.78, 3H, d.
Electrospray Mass Spectroscopy: 610 (M+Na), 588.5 (M+H),
[0153] Diastereomer 1b is purified by the same preparative reverse
phase HPLC process.
EXAMPLE 3
Synthesis of atropisomer amine 2a
[0154] Diastereomer 1a (1.1 gm, 1.87 mmoles, 587.6 MW) was
dissolved in 100 ml water and cooled to 0.degree. C. Concentrated
sulfuric acid (15 ml) was added dropwise to give a brownish
solution (FIG. 2a). The temperature was allowed to rise to room
temperature and the mixture was stirred overnight. The mixture was
added slowly to 1.5 ml of ice water and then adsorbed on
pre-equilibrated C-18 silica gel (4 cm length.times.3 cm diameter).
The support was washed with water until the pH of the eluent was
neutral. The crude product was eluted with 200 ml CH.sub.3OH which
was concentrated under vacuum and dried to yield atropisomer 2a C-1
aminomethyl, C-19 carboxy fluorescein sulfate salt as an orange
solid (0.93 gm, 95% yield, 503.4 MW). .sup.1H NMR (methanol-d4)
.delta. 8.43, 1H, d; 8.34, 1H, d; 7.92, 1H, s; 7.23, 3H, m; 7.06,
1H, d; 6.98, 1H, d; 4.58, 2H, s.
[0155] The enantiomeric purity of hydrolyzed and purified 2a was
analyzed by chiral column HPLC (Regies (S,S) Whelk-01 10-100
Kromasil FEC column, 25 cm length.times.4.6 mm ID). The sample 2a
was dissolved in water and eluted with a gradient of 0 to 35%
ethanol in water containing 0.1% acetic acid over 30 minutes at 1
ml/min. with 254 nm UW detection (FIG. 2c) and distinguished from
the racemic mixture (FIG. 2b).
EXAMPLE 4
Synthesis of atropisomer amine 2b
[0156] Diastereomer 1b is hydrolyzed, purified, and analyzed to
give atropisomer 2b by the same processes as Example 3 (FIG.
2a).
EXAMPLE 5
Synthesis of atropisomeric trifluoroacetamide 3a
[0157] Atropisomer 2a as the sulfate salt (0.93 gm, 1.84 mmoles,
503.4 MW) was dissolved in 15 ml ethanol. Triethylamine (1.8 ml, 13
mmoles) and ethyl trifluoroacetate (2.2 ml, 18 mmoles) were slowly
added (FIG. 2a). The mixture was stirred at room temperature under
argon for 2.5 hours. Volatiles were removed under vacuum and the
resulting residue was dissolved in 300 ml ethyl acetate and washed
with 2.times.50 ml of 5% HCl. The ethyl acetate fraction was dried
over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under
vacuum to yield atropisomeric trifluoroacetamide 3a as an orange
solid (0.92 gm, 100% yield, 501.4 MW). .sup.1H NMR (methanol-d4)
.delta. 8.35, 1H, d; 8.15, 1H, d; 7.80, 1H, s; 6.82, 1H, s; 6.65,
4H, m; 4.82, 2H, s.
EXAMPLE 6
Synthesis of atropisomeric trifluoroacetamide 3b
[0158] Atropisomer 2b is converted to atropisomeric
trifluoroacetamide 3b by the same process and analyzed by the same
methods as Example 5 (FIG. 2a).
EXAMPLE 7
Synthesis of atropisomeric NHS ester 4a
[0159] Atropisomeric trifluoroacetamide 3a (0.92 gm, 1.83 mmoles,
501.4), N-hydroxysuccinimide (0.85 gm, 7.3 mmoles), and
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DAE)
(1.05 gm, 5.5 mmoles) were dissolved in 24 ml ethyl acetate and 12
ml of 1-methyl-2-pyrrolidinone (NMP) and stirred at room
temperature under argon for 2.5 hours (FIG. 3). The mixture was
diluted with 300 ml ethyl acetate and washed with 2.times.80 ml of
5% HCl, dried over anhydrous Na.sub.2SO.sub.4, filtered,
concentrated under vacuum, and adsorbed on silica gel. The silica
gel with adsorbed product was loaded on the top of a dry-packed
column of silica gel (12 cm length.times.3 cm ID) and eluted with
ethyl acetate:hexane/2:1. Fractions containing atropisomeric NHS
ester 4a were collected and combined, concentrated under vacuum and
precipitated from hexane to yield 4a as a bright yellow solid (0.74
gm, 67% yield, 598.4 MW). .sup.1H NMR (methanol-d4) .delta. 8.42,
1H, d; 8.22, 1H, d; 7.95, 1H, s; 6.81, 1H, s; 6.68, 4H, m; 4.82,
2H, s; 2.85, 4H, s.
EXAMPLE 8
Synthesis of Atropisomeric NHS Ester 4b
[0160] Atropisomeric trifluoroacetamide 3b is converted to
atropisomeric NHS ester 4b by the same process and analyzed by the
same methods as Example 7 (FIG. 3).
EXAMPLE 9
Synthesis of 2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic
acid NHS 5
[0161] Protected phosphodiester linker synthon 5 was prepared by
reacting methyl glycolate 6 (4.5 eq.) with cyclic phosphoramidite
Amino-Link.TM. 7 (1 eq.) (Connell (1987) BioTechniques 5:342-348;
U.S. Pat. No. 4,757,151) and 4-N,N-dimethylaminopyridine (DMAP)
(0.1 eq.). The mixture was stirred at ambient temperature for 1
hour. After the reaction was complete (TLC analysis), the solution
was cooled with ice-bath and then treated with a solution of
3-chloroperoxybenzoic acid (4 eq.) in methylene chloride. The
ice-bath was removed. After 30 minutes, an aqueous solution of
NaHSO.sub.3 (10%) was added. The mixture was diluted with ethyl
acetate. The organic layer was washed with NaHSO.sub.3 (10%),
saturated solution of NaHCO.sub.3, and dried with Na.sub.2SO.sub.4.
The crude product was purified by flash chromatography to afford
ester 8, which was heated at reflux for 3 hours (36 mM, 1 eq.) in
methylethylketone and NaI (10 eq.). The crude demethylated
phosphodiester was dissolved in 0.3 M solution of LiOH (5 eq.) in
H.sub.2O/CH.sub.3OH:1/3) and stirred overnight to cleave the methyl
ester. Solvent was removed to afford crude compound 9 which was
then dissolved in aqueous Na.sub.2CO.sub.3 (5%).
N-(9-Fluorenylmethoxy-carbonyloxy)succinimide (FmocOSu, 1.5 eq.) in
THF was added in one portion and stirred at ambient temperature for
3 hours. The crude product was diluted with ethyl acetate and
washed with 10% aqueous HCl. The organic layer was dried with
Na.sub.2SO.sub.4, filtered, concentrated under vacuum, and purified
by flash chromatography to afford Fmoc-acid 10 as a yellow oil.
[0162] Fmoc-acid 10 was dissolved in anhydrous CH.sub.2Cl.sub.2 (1
eq.). N-hydroxysuccinimide (4 eq.) was added. The solution was
cooled with an ice-bath and then treated with dicyclohexyl
carbodiimide (DCC, 2 eq.). The ice-bath was then removed, and
stirring was continued for 2 hours (with TLC analysis). When the
reaction was complete, ethyl acetate was added and the solution was
washed with 5% aqueous HCl. Removal of solvent gave
2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acid NHS 5
(FIG. 4).
EXAMPLE 10
Synthesis of 7-propargylphosphorylamino-7-deaza-ddATP 11
[0163] 7-Deaza-7-propargylamino-ddATP 12
(7-(3-amino-1-propynyl)-2',3'-dideoxy-7-deazaadenosine-5'-triphosphate;
U.S. Pat. Nos. 5,047,519 and 5,151,507) was suspended in 250 mM
bicarbonate (pH 9.0) and a solution of
2-[(2-Fmoc-aminoethoxy)(hydroxyphosphoryl)oxy]acetic acid NHS 5 in
DMSO was added. After 1 hour, the reaction mixture was purified by
HPLC (AX-300 anion exchange). The product fractions were collected,
concentrated to dryness, and purified by RP HPLC (C-18 reverse
phase) to afford Fmoc-linker ddATP 13. Concentrated ammonium
hydroxide (28-30%) was added to Fmoc-linker ddATP 13 and the
solution was heated to 55.degree. C. for 20 minutes. Concentration
under vacuum gave crude 7-propargylphosphorylamino-7-deaza-ddATP 11
which was purified by C-18 reverse phase HPLC (FIG. 5).
EXAMPLE 11
Synthesis of Fmoc-aminomethyl-NHS-FAM 14
[0164] Fmoc-aminomethyl-NHS-FAM 14 was prepared by reacting the
fluorenylmethoxy-carbonyloxy ester of N-hydroxysuccinimide
(Fmoc-OSu) with the HCl salt of p-aminomethylbenzoic acid (both
commercially available) in the presence of base to form the
expected N-Fmoc derivative. This product was then reacted with
N-hydroxysuccinimide in the presence of DCC to form the NHS ester
of the benzoic acid carboxyl group. This NHS-ester, N-Fmoc
derivative ofp-aminomethylbenzoic acid having the structure:
##STR20## was then reacted with atropisomer C-1 aminomethyl, C-19
carboxy fluorescein 2a, purified by the method of Example 2,
followed by reaction with N-hydroxysuccinimide in the presence of
DCC to produce substantially pure atropisomer,
Fmoc-aminomethyl-NHS-FAM 14 (FIG. 6).
EXAMPLE 12
Synthesis of
Aminomethylbenzamide-aminomethyl-FAM-propargylphosphorylamino-ddATP
15
[0165] A solution of Fmoc-aminomethyl-NHS-FAM 14 in DMSO was added
to 7-propargylphosphorylamino-7-deaza-ddATP 11 suspended in 250 mM
bicarbonate (pH 9.0). The reaction mixture was placed in the dark
at ambient temperature for 2 hours. The Fmoc-amino protected
product 16 was purified by HPLC (AX-300 anion exchange), then
heated at 55.degree. C. in concentrated ammonium hydroxide (28-30%)
for 20 minutes to hydrolyze the Fmoc group. Concentration under
vacuum gave crude, substantially pure atropisomer,
aminomethylbenzamide-aminomethyl-FAM-propargylphosphorylamino-ddATP
15 which was purified by C-18 reverse phase HPLC (FIG. 6).
EXAMPLE 13
Synthesis of NHS-rhodamine dye 17
[0166] Bicyclic amine 18 (12.8 gm, 47 mmole, U.S. Pat. No.
5,688,808), 1-bromo-3-chloropropane (29.3 gm, 187 mmole), sodium
iodide (56.4 gm, 376 mmole) and sodium bicarbonate (7.9 gm, 94
mmole) was refluxed in 150 ml CH.sub.3CN for 18 hours. The mixture
was cooled to room temperature, filtered, and evaporated. The
filter cake was washed with 300 ml hexane which was combined with
the filtrate and washed with 2.times.50 ml water and 50 ml
saturated NaCl, dried over MgSO.sub.4, filtered, and concentrated
under vacuum. The product was purified by chromatography on silica
gel, eluting with hexane/ethyl acetate:20/1, to give tricyclic
amine pivalate ester 19 as a pale yellow oil (9.5 gm, 30 mmole, 64%
yield). The ester of 19 was hydrolyzed in a solution of lithium
hydroxide monohydrate (2.6 gm, 60 mmole) in 15 ml water and 120 ml
methanol. After stirring for one hour at room temperature, the
mixture was concentrated under vacuum and dissolved in 30 ml 1M HCl
which was extracted with 3.times.100 ml of diethylether. The
combined ether extracts were washed with 50 ml of 200 mM pH 7
phosphate buffer, dried over MgSO.sub.4, filtered and concentrated
under vacuum to give tricyclic amine 20 as a brown solid (FIG. 7).
Tricyclic amine 20 and 3,6-dichloro, 4-isopropylcarboxylate
phthalic anhydride 21 were refluxed in toluene to give
Friedel-Craft acylation product ketone 22 (Abs. max 400 nm) (FIG.
8).
[0167] Cyclization of 22 with 20 in phosphoryl trichloride and
chloroform at reflux gave 23 as a mixture of isopropylcarboxylate
regioisomers. After cleavage of the isopropyl group, the rhodamine
carboxylic acid 24 was converted to NHS-rhodamine dye 17 (FIG.
8).
EXAMPLE 14
Synthesis of phosphate-linker, energy-transfer terminator ddATP
25
[0168]
Aminomethylbenzamide-aminomethyl-FAM-propargylphosphorylamino-ddAT-
P 15 from Example 12 was suspended in a solution of 250 mM
bicarbonate (pH 9.0). A solution of NHS ester 17 (U.S. Pat. No.
5,847,162 for synthesis) in DMSO was added. The reaction mixture
was placed in the dark at ambient temperature for 2 hours.
Purification was done by HPLC, AX-300 anion exchange and then C-18
reverse phase to afford pure energy-transfer ddATP terminator 25
(FIG. 9).
EXAMPLE 15
Synthesis of bis-trifluoroacetamide rhodamine NHS 27
[0169] Rhodamine dye 28 was converted to the bis-trifluoroacetamide
29 by treatment with trifluoroacetic anhydride and triethylamine in
diethylether at room temperature. The carboxylic acid was converted
to the NHS ester with dicyclohexylcarbodiimide and
N-hydroxysuccinimide to give bis-trifluoroacetamide rhodamine NHS
27 (FIG. 10).
EXAMPLE 16
Synthesis of
Aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP
32
[0170]
5-(3-Aminoethoxy-1-propynyl)-2',3'-dideoxythymidine-5'-triphosphat-
e 30 (U.S. Pat. No. 5,821,356) was reacted with substantially pure
atropisomer, Fmoc-aminomethyl-NHS-FAM 14 under the same conditions
as Example 12 to give
Fmoc-aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP
31 which was purified by anion-exchange HPLC. The Fmoc group of 31
was cleaved to give substantially pure atropisomer,
aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 32
(FIG. 11).
EXAMPLE 17
Synthesis of energy-transfer terminator ddTTP 26
[0171] Following the conditions of Example 14, substantially pure
atropisomer,
aminomethylbenzamide-aminomethyl-FAM-propargylethoxyamino-ddTTP 32
was suspended in a solution of 250 mM bicarbonate (pH 9.0). A
solution of bis-trifluoroacetamide rhodamine NHS ester 27 in DMSO
was added. The reaction mixture was placed in the dark at ambient
temperature for 2 hours. Ammonium hydroxide was added to cleave the
trifluoroacetamide groups. Purification was done by HPLC, AX-300
anion exchange and then C-18 reverse phase to afford pure
energy-transfer ddTTP terminator 26 (FIG. 12).
EXAMPLE 18
Sequencing of pGEM with phosphate-linker, energy-transfer
terminator ddATP 25
[0172] Following the conditions of U.S. Pat. Nos. 5,770,716;
5,948,648; and 6096875, the energy-transfer ddATP terminator 25 was
used with other standard reagents in a Sanger-type, one-color
automated DNA sequencing experiment. The terminator nucleotide 25
was tested as the racemic mixture of atropisomers (top
electropherogram) and as a substantially pure atropisomer (bottom
electropherogram) shown in FIGS. 13a and 13b.
[0173] The dye-terminator sequencing reactions were performed using
AmpliTaq DNA Polymerase, FS following protocols provided in the ABI
PRISM.TM. Dye Terminator Cycle Sequencing Core Kit Manual (Applied
Biosystems p/n 402116). Sequencing of the pGEM-3Zf(+) template was
conducted with unlabelled -21 M13 sequencing primer (forward).
Reagents, including buffer, unlabelled primer, AmpliTaq DNA
Polymerase, FS, were from an ABI PRISM.TM. Dye Terminator Core Kit
(Applied Biosystems p/n 402117). The dNTP mix consisted of 2 mM
each of dATP, dCTP, dITP, and dUTP or dTTP. A premix of reaction
components was prepared including: 5.times. Buffer 4.0 .mu.L; dNTP
mix 1.0 .mu.L; Template: pGEM.RTM. -3Zf(+), 0.2 .mu.g/.mu.L, 2.0
.mu.L; Primer: -21 M13 (forward), 0.8 pmol/.mu.L, 4.0 .mu.L;
AmpliTaq DNA Polymerase, FS, 0.5 .mu.L; and H.sub.2O 3.5 .mu.L,
wherein all quantities are given on a per reaction basis.
[0174] Reactions were assembled in 0.5 ml tubes adapted for the
Perkin-Elmer 480 DNA Thermal Cycler (Applied Biosystems p/n
N801-100 or 0.2 ml tubes for the Applied Biosystems Gene Amp PCR
System 9700). From 1 to 250 pmol of the dye terminator was added to
each reaction. 30 .mu.L of mineral oil was added to the top of each
reaction to prevent evaporation (when using the Applied Biosystems
480 Thermal Cycler). Reaction volumes were 20 .mu.L, including 15
.mu.L of the above reaction premix, a variable amount of dye
labelled terminator, and a sufficient volume of water to bring the
total reaction volume up to 20 .mu.L. Reactions were thermocycled
as follows: 96.degree. C. for 30 sec, 50.degree. C. for 15 sec, and
60.degree. C. for 4 min, for 25 cycles; followed by a 4.degree. C.
hold cycle.
[0175] All reactions were purified by spin-column purification on
Centri-Sep spin columns according to manufacturer's instructions
(Princeton Separations p/n CS-901). Gel material in the column was
hydrated with 0.8 mL deionized water for at least 30 minutes at
room temperature. After the column was hydrated and it was
determined that no bubbles were trapped in the gel material, the
upper and lower end caps of the column were removed, and the column
was allowed to drain by gravity. The column was then inserted into
the wash tubes provided in the kit and centrifuged in a variable
speed microcentrifuge at 1300 g for 2 minutes, removed from the
wash tube, and inserted into a sample collection tube. The reaction
mixture was carefully removed from under the oil and loaded onto
the gel material and the tube re-centrifuged. Eluted samples were
then dried in a vacuum centrifuge.
[0176] Prior to loading onto a sequencing gel, the dried samples
were resuspended in 25 .mu.L of Template Suppression Reagent
(Applied Biosystems p/n 401674), vortexed, heated to 95.degree. C.
for 2 minutes, cooled on ice, vortexed again, and centrifuged
(13,000.times.g). 10 .mu.L of the resuspended sample was aliquoted
into sample vials (Applied Biosystems p/n 401957) adapted for the
ABI PRISM.TM. 310 Genetic Analyzer (Applied Biosystems p/n
310-00-100/120). Electrophoresis on the 310 Genetic Analyzer was
performed with sieving polymers and capillaries specially adapted
for DNA sequencing analysis (PE Applied Biosystems p/n 402837 or
4313087 (polymer) and p/n 402840 (capillary)). In each case, the
sieving polymer included nucleic acid denaturants. Samples were
electrokinetically injected onto the capillary for 30 sec at 2.5
kV, and run for up to 2 hr at 10 to 12.2 kV with the outside wall
of the capillary maintained at 50C to generate electropherograms as
sequencing data (FIGS. 13a-d).
[0177] The electropherograms in FIGS. 13a and 13b show the specific
incorporation of the energy-transfer terminator 25 onto the 3'
terminus of primer extension, polynucleotide fragments during
single color sequencing reactions. The electropherograms plot the
fluorescence intensity emitted (Emission maxima about 650 nm) by
the acceptor rhodamine dye of the labelled fragments between about
20 to about 600 nucleotides in length as a function of time during
an electrophoresis run on the ABI PRISM.TM. 310 Genetic
Analyzer.
[0178] Eluting fragments from 119 to 242 base pairs are plotted in
FIG. 13a. Each of the fragments was 3' terminated by
energy-transfer terminator 25. FIG. 13b shows a more magnified view
of fragments 148 to 205 base pairs. The three regions under the
arrows in FIGS. 13a and 13b illustrate the surprising and
unexpected improvement in separating fragments labelled with the
substantially pure atropisomer form of 25 (bottom panels) relative
to the separation of fragments labelled with the racemic mixture of
25. Better resolution of the fragments was observed in the bottom
electropherogram with the substantially pure atropisomer than with
the racemic mixture of atropisomers in 25. The locations marked
with arrows are particular loci where substantially pure
atropisomer form of 25 provided the unexpected benefit of better
resolution. By contrast, use of the racemic mixture of
atropisomeric form of 25 in labelling the chiral primer extension
products led to diastereomeric populations of fragments which
migrate electrophoretically at different rates, as exemplified by
the broad, overlapping, and split peaks under the arrows in the top
electropherogram.
[0179] Additionally, the bottom electropherogram shows more even
peak heights throughout the sequencing ladder than was observed in
the top electropherogram with the racemic mixture of atropisomers
in 25.
EXAMPLE 19
Sequencing of pGEM with sulfonate-linker, energy-transfer
terminator ddATP 33
[0180] Following the general synthesis routes and conditions of the
previous Examples, substantially pure atropisomer,
sulfonate-linker, energy-transfer terminator ddATP 33 (FIG. 14) was
synthesized. Following the protocol and conditions of Example 18,
33 was used in single-color sequencing the pGEM target. Separately,
the racemic mixture of 33 was also used in the same single-color
sequencing experiment. Eluting fragments from 148 to 242 base pairs
are plotted in FIG. 13c. Each of the fragments was 3' terminated by
energy-transfer terminator 33. The three regions under the arrows
in FIG. 13c illustrate the surprising and unexpected improvement in
separating fragments labelled with the substantially pure
atropisomer form of 33 (bottom panel) relative to the separation of
fragments labelled with the racemic mixture of 33 (top panel).
Better resolution of the fragments was observed in the bottom
electropherogram with the substantially pure atropisomer than with
the racemic mixture of atropisomers in 33.
EXAMPLE 20
Sequencing of pGEM with energy-transfer terminator ddGTP 34
[0181] Following the general synthesis routes and reaction
conditions of the previous Examples, energy-transfer terminator
ddGTP 34 (FIG. 15) was synthesized. The atropisomer forms were
separated at the final stage of synthesis, i.e. compound 34, by
reverse-phase HPLC. Following the protocol and conditions of
Example 18, a substantially pure atropisomer of 34 was used in
single-color sequencing the pGEM target. Separately, the racemic
mixture of 34 was also used in the same single-color sequencing
experiment. Eluting fragments from 24 to 99 base pairs, detected at
about 535 nm, are plotted in FIG. 13d. Each of the fragments was 3'
terminated by energy-transfer terminator 34. The regions under the
arrows in FIG. 13d illustrates the surprising and unexpected
improvement in separating fragments labelled with the substantially
pure atropisomer form of 34 (bottom panel) relative to the
separation of fragments labelled with the racemic mixture of 34
(top panel). Better resolution of virtually every fragment was
observed in the bottom electropherogram with the substantially pure
atropisomer than with the racemic mixture of atropisomers in 34.
The locations marked with arrows are particular loci where
substantially pure atropisomer form of 34 provided the unexpected
benefit of better resolution. Every fragment in the bottom panel,
labelled with racemic 34, separates into two peaks, detracting from
the utility of the data.
[0182] All publications cited herein are incorporated by reference,
and to the same extent as if each individual publication was
specifically and individually indicated to be incorporated by
reference.
[0183] Although only a few embodiments have been described in
detail above, those having ordinary skill in the chemical and
molecular biology arts will clearly understand that many
modifications are possible in the illustrated embodiments without
departing from the teachings thereof. All such modifications are
intended to be encompassed within the following claims.
* * * * *