ENERGY TRANSFER DYES

COLORANTS A TRANSFERT ENERGETIQUE

English Abstract

A novel class of energy transfer dyes, their preparation, and their use as
labels in biological systems is disclosed. The dyes are preferably in the form
of cassettes which enable their attachment to a variety of biological
materials. The dyes and the reagents that can be made from them offer a wide
variety of fluorescent labels with large Stokes' shifts enabling their use in
a variety of fluorescence applications over a wide range of the visible
spectrum.

French Abstract

L'invention concerne une nouvelle classe de colorants à transfert énergétique, leur préparation et leur utilisation comme marqueurs dans les biosystèmes. Ces colorants sont présentés de préférence sous forme de cassettes, ce qui permet de les fixer sur différentes matières biologiques. Les colorants et les réactifs pouvant être préparés à partir de ces derniers comprennent différents marqueurs fluorescents présentant des décalages de Stokes importants, permettant leur utilisation dans une grande variété d'applications utilisant la fluorescence, couvrant une plage importante du spectre visible.

Claims

15
Claims
What we claim is:
1. An energy transfer dye of the formula:
<IMG>
wherein
R1 is a first dye suitable as an acceptor or donor in an energy transfer
arrangement;
R5 is a second dye suitable as a donor or acceptor in an energy transfer
arrangement
with said first dye;
A comprises a chain of 5 to 20 linearly linked atoms, wherein each atom is
independently selected from the group consisting of carbon, nitrogen and
oxygen;
R3 is a hydrogen or a reactive or functional group suitable for attaching the
energy
transfer dye to a target material; and
D comprises an atom or group for attaching said R5 to said A, wherein the
covalent
linkage between said A and said R5 does not include a sulphur atom.
2. The energy transfer dye of claim 1, wherein
said A-D is (CHR2)m-CH(R3)-NH-CO-(CHR4)n-NH-CO,
wherein
R2 is hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
R3 does not consist of thiol;
R4 is hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
m is 1,2, or 3; and
n is 1,2,3,4,5,6,7,8, or 9.
3. The energy transfer dye of claim 1, wherein said A comprises a C6-C17
hydrocarbon
chain.

16
4. The energy transfer dye of claim 1, wherein said R1 is selected from the
group
consisting of a fluorescein dye and a cyanine dye, and said R5 is selected
from the group
consisting of a rhodamine dye and a cyanine dye.
5. The energy transfer dye of claim 4, wherein said R1 is selected from the
group
consisting of:
5-carboxyfluorescein, 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-
dimethoxyfluorescein, CyA (3-( -carboxypentyl)-3'-ethyl-5,5'-dimethyl
oxacarbocyanine),
and Cy3(3-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
carbocyanine).
6. The energy transfer dye of claim 4, wherein said R5 is selected from the
group
consisting of:
6-carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G (R6G-5 or REG-5),
6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-
carboxyrhodamine
(TAMRA or TMR), 6-carboxy-X-rhodamine (ROX), Cy3.5 (3-(-carboxypentyl)-1'-
ethyl-
3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-disulphonato)dibenzo-carbocyanine), Cy5
(1-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
dicarbocyanine, Cy5.5
(1-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-
disulphonato)-dibenzo-
dicarbocyanine, and Cy7(1-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-
disulphonato-tricarbocyanine.
7. The energy transfer dye of claim 1, wherein said target material comprises
a biological
material.
8. The energy transfer dye of claim 1, wherein said R3 is selected from the
group
consisting of carboxyl, succinimidyl ester, sulpho-succinimidyl ester,
isothiocyanate,
maleimide and phosphoranudite, and groups covalently reactive with carboxyl,
succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, and
phosphoramidite.

17
9. The energy transfer dye of claim 4, wherein said R3 is selected from the
group
consisting of carboxyl, succinimidyl ester, sulpho-succinimidyl ester,
isothiocyanate,
maleimide and phosphoramidite, and groups covalently reactive with carboxyl,
succinimidyl ester, sulpho-succinimidyl ester, isothiocyanate, maleimide, and
phosphoramidite.
10. The energy transfer dye of claim 1, further comprising a third dye
attached to said
R5 through a linker group, wherein said attachment results in an energy
transfer
arrangement of said third dye with said R5.
11. The energy transfer dye of claim 4, further comprising a third dye
attached to said
R5 through a linker group, wherein said attachment results in an energy
transfer
arrangement or said third dye with said R5.
12. The energy transfer dye of claim 10, wherein said third dye is a cyanine
dye.
13. The energy transfer dye of claim 11, wherein said third dye is a cyanine
dye.
14. A method for producing an energy transfer dye of the formula:
<IMG>
wherein
R1 is a first dye suitable as an acceptor or donor in an energy transfer
arrangement;
R5 is a second dye suitable as a donor or acceptor in an energy transfer
arrangement
with said first dye;
A comprises a chain of 5 to 20 linked atoms, wherein each atom is
independently
selected from the group consisting of carbon, nitrogen and oxygen;
R3 comprises a hydrogen or a reactive or functional group suitable for
attaching the
energy transfer dye to a target material; and

18
D comprises an atom or group for attaching said R5 to said A, wherein the
covalent
linkage between said A and said R5 does not include a sulphur atom;
said method comprising:
(a) coupling said R1 to a thiol containing component of said A, wherein said A
also
comprises said R3;
(b) coupling the product of part (a) with the remaining part of said A,
wherein said
remaining part of A is substituted by a reactive group suitable for forming
the
attachment group D, and
(c) coupling the product of (b) with said R5 thereby forming said attachment
group D.
15. A method for fluorescently labeling a biological material comprising:
a) adding to a liquid which contains said biological material an energy
transfer dye of
the formula:
<IMG>
wherein R1 comprises a first dye suitable as an acceptor or donor in an energy
transfer
arrangement;
R5 comprises a second dye suitable as a donor or acceptor in an energy
transfer
arrangement with said first dye;
A comprises a chain of 5 to 20 linked atoms, wherein each atom is
independently
selected from the group consisting of carbon, nitrogen and oxygen;
R3 comprises a hydrogen or a reactive or functional group suitable for
attaching the
energy transfer dye to a target material; and
D comprises an atom or group for attaching said R5 to said A, wherein the
covalent
linkage between said A and said R5 does include a sulphur atom; and
b) reacting said dye so that said dye covalently reacts with and labels said
biological
material.
16. The method of claim 15, wherein said biological material is selected from
the group
consisting of antibodies, antigens, peptides, proteins, carbohydrates, lipids,
nucleotides,

19
oxy or deoxy polynucleic acids and cells which are optionally derivatised so
that they
contain one or more amino, hydroxy, thiophosphoryl, sulphydryl or carboxy
groups.
17. The method of claim 15, wherein said energy transfer dye comprises a
compound,
wherein
said A-D is (CHR2)m-CH(R3)-NH-CO-(CHR4)n-NH-CO;
R2 comprises hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
R3 does not consist of thiol;
R4 comprises hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
m is 1,2, or 3; and
n is 1,2,3,4,5,6,7,8, or 9.
18. The method of claim 15, wherein said energy transfer dye comprises a
compound
wherein said A comprises a C6-C17 hydrocarbon chain.
19. The method of claim 15, wherein said energy transfer dye comprises a
compound
wherein said R1 is selected from the group consisting of a fluorescein dye and
a cyanine
dye, and said R5 is selected from the group consisting of a rhodamine dye and
a cyanine
dye.
20. The method of claim 19, wherein said energy transfer dye comprises a
compound
wherein said R1 is selected from the group consisting of:
5-carboxyfluorescein, 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-
dimethoxyfluorescein, CyA(3-(-carboxypentyl)-3'-ethyl-5,5'-dimethyl
oxacarbocyanine),
and Cy3(3-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
carbocyanine).
21. The method of claim 19, wherein said energy transfer dye comprises a
compound
wherein said R5 is selected from the group consisting of:
6-carboxyrhodamine(Rhodamine 110),5-carboxyrhodamine-6G(R6G-5 or REG-5),
6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-
carboxyrhodamine

20
(TAMRA or TMR), 6-carboxy-X-rhodamine (ROX), Cy3.5 (3-(-carboxypentyl)-1'-
ethyl-
3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-disulphonato)dibenzo-carbocyanine), Cy5(1-
(-
carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
dicarbocyanine, Cy5.5(1-
(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-disulphonato)-
dibenzo-
dicarbocyanine, and Cy7(1-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-
disulphonato-tricarbocyanine.
22. The method of claim 15, wherein said energy transfer dye comprises a
compound
wherein said R3 is selected from the group consisting of carboxyl,
succinimidyl ester,
sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite, and
groups
covalently reactive with carboxyl, succinimidyl ester, sulpho-succinimidyl
ester,
isothiocyanate, maleimide, and phosphoramidite.
23. The method of claim 19, wherein said energy transfer dye comprises a
compound
wherein said R3 is selected from the group consisting of carboxyl,
succinimidyl ester,
sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite, and
groups
covalently reactive with carboxyl, succinimidyl ester, sulpho-succinimidyl
ester,
isothiocyanate, maleimide, and phosphoramidite.
24. The method of claim 15, wherein said energy transfer dye comprises a
compound
further comprising a third dye attached to said R5 through a linker group,
wherein said
attachment results in an energy transfer arrangement of said third dye with
said R5.
25. The method of claim 19, wherein said energy transfer dye comprises a
compound
further comprising a third dye attached to said R5 through a linker group,
wherein said
attachment results in an energy transfer arrangement of said third dye with
said R5.
26. The method of claim 25, wherein said third dye is a cyanine dye.
27. The method of claim 24, wherein said third dye is a cyanine dye.

21
28. A method for fluorescently labeling a first component and then using said
labeled first
component to detect the presence of a second component in a sample,
comprising:
a) adding to a liquid which contains said first component an energy transfer
dye of the
formula:
<IMG>
wherein R1 comprises a first dye suitable as an acceptor or donor in an energy
transfer
arrangement;
R5 comprises a second dye suitable as a donor or acceptor in an energy
transfer
arrangement with said first dye;
A comprises a chain of 5 to 20 linked atoms, wherein each atom is
independently
selected from the group consisting of carbon, nitrogen and oxygen;
R3 comprises a hydrogen or a reactive or functional group suitable for
attaching the
energy transfer dye to a target material; and
D comprises an atom or group for attaching said R5 to said A, wherein the
covalent
linkage between said A and said R5 does include a sulphur atom;
b) reacting said dye with said first component such that said dye covalently
reacts with
and thereby labels said first component;
c) providing said labeled first component to said sample to permit binding of
said labeled
first component to said second component if present; and
d) detecting said second component if present by detecting the fluorescent
label by an
optical method.
29. The method of claim 28, wherein said energy transfer dye comprises a
compound
wherein
said A-D is (CHR2)m-CH(R3)-NH-CO-(CHR4)n-NH-CO, wherein
R2 comprises hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
R3 does not consist of thiol;
R4 comprises hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
m is 1,2, or 3; and

22
n is 1,2,3,4,5,6,7,8, or 9.
30. The method of claim 28, wherein said energy transfer dye comprises a
compound
wherein said A comprises a C6-C17 hydrocarbon chain.
31. The method of claim 28, wherein said energy transfer dye comprises a
compound
wherein said R1 is selected from the group comprising a fluorescein dye and a
cyanine dye,
and said R5 is selected from the group comprising a rhodamine dye and a
cyanine dye.
32. The method of claim 31, wherein said energy transfer dye comprises a
compound
wherein said R1 is selected from the group consisting of:
5-carboxyfluorescein, 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-
dimethoxyfluorescein, CyA (3-(-carboxypentyl)-3'-ethyl-5,5'-dimethyl
oxacarbocyanine),
and Cy3 (3-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-
disulphonatocarbocyanine).
33. The method of claim 31, wherein said energy transfer dye comprises a
compound
wherein said R5 is selected from the group consisting of:
6-carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G (R6G-5 or REG-5),
6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-
carboxyrhodamine
(TAMRA or TMR), 6-carboxy-X-rhodamine (ROX), Cy3.5 (3-(-carboxypentyl)-1'-
ethyl-
3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-disulphonato)dibenzo-carbocyanine), Cy5
(1-(-
carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
dicarbocyanine,
Cy5.5 (1-(-carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-
disulphonato)-dibenzo-
dicarbocyanine, and Cy7 (1-( -carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-
5,5'-
disulphonato-tricarbocyanine.
34. The method of claim 28, wherein said target material comprises a
biological material.
35. The method of claim 28, wherein said energy transfer dye comprises a
compound
wherein said R3 is selected from the group consisting of carboxyl,
succinimidyl ester,

23
sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite, and
groups
covalently reactive with carboxyl, succinimidyl ester, sulpho-succinimidyl
ester,
isothiocyanate, maleimide, and phosphoramidite.
36. The method of claim 31, wherein said energy transfer dye comprises a
compound
wherein said R3 is selected from the group consisting of carboxyl,
succinimidyl ester,
sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite, and
groups
covalently reactive with carboxyl, succinimidyl ester, sulpho-succinimidyl
ester,
isothiocyanate, maleimide, and phosphoramidite.
37. The method of claim 28, wherein said energy transfer dye comprises a
compound
further comprising a third dye attached to said R5 through a linker group,
wherein said
attachment results in an energy transfer arrangement of said third dye with
said R5.
38. The method of claim 31, wherein said energy transfer dye comprises a
compound
further comprising a third dye attached to said R5 through a linker group,
wherein said
attachment results in an energy transfer arrangement of said third dye with
said R5.
39. The method of claim 37, wherein said third dye is a cyanine dye.
40. The method of claim 38, wherein said third dye is a cyanine dye.
41. A reagent comprising:
a) an energy transfer dye of the formula:
<IMG>
wherein R1 comprises a first dye suitable as an acceptor or donor in an energy
transfer
arrangement;
R5 comprises a second dye suitable as a donor or acceptor in an energy
transfer
arrangement with said first dye;

24
A comprises a chain of 5 to 20 linked atoms, wherein each atom is
independently
selected from the group consisting of carbon, nitrogen and oxygen;
R3 comprises a hydrogen or a reactive or functional group suitable for
attaching the
energy transfer dye to a target material; and
D comprises an atom or group for attaching said R5 to said A, wherein the
covalent
linkage between said A and said R5 does include a sulphur atom; and
b) a carrier material which contains or has been derivatised to include at
least one first
reactive group capable of forming a covalent bond with a functional group, or
functional
group capable of forming a covalent bond with a reactive group on the energy
transfer dye
and is covalently bonded thereto.
42. The reagent of claim 41, wherein said carrier material is selected from
the group
consisting of polymer particles, cells, glass beads, antibodies, proteins,
peptides, enzymes,
carbohydrates, lipids and nucleic acids.
43. The reagent of claim 42, wherein said energy transfer dye comprises a
compound,
wherein
said A-D is (CHR2)m-CH(R3)-NH-CO-(CHR4)n-NH-CO, wherein
R2 comprises hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
R3 does not consist of thiol;
R4 comprises hydrogen, a 1,2,3, or 4 carbon containing alkyl or phenyl;
m is 1,2, or 3; and
n is 1,2,3,4,5,6,7,8, or 9.
44. The reagent of claim 42, wherein said energy transfer dye comprises a
compound
wherein said A comprises a C6-C17 hydrocarbon chain.
45. The reagent of claim 41, wherein said energy transfer dye comprises a
compound
wherein said R1 is selected from the group comprising a fluorescein dye and a
cyanine dye,
and said R5 is selected from the group comprising a rhodamine dye and a
cyanine dye.

25
46. The reagent of claim 45, wherein said energy transfer dye comprises a
compound,
wherein said R1 is selected from the group consisting of:
5-carboxyfluorescein, 6-carboxyfluorescein, 6-carboxy-4',5'-dichloro-2',7'-
dimethoxyfluorescein, CyA (3-( -carboxypentyl)-3'-ethyl-5,5'-dimethyl
oxacarbocyanine),
and Cy3 (3-( -carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
carbocyanine).
47. The reagent of claim 45, wherein said energy transfer dye comprises a
compound
wherein said R5 is selected from the group consisting of:
6-carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G (R6G-5 or REG-5),
6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-
carboxyrhodamine
(TAMRA or TMR), 6-carboxy-X-rhodamine (ROX), Cy3.5 (3-( -carboxypentyl)-1'-
ethyl-
3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-disulphonato)dibenzo-carbocyanine), Cy5
(1-( -
carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-
dicarbocyanine, Cy5.5 (1-
( -carboxypentyl}-1'-ethyl-3,3,3',3'-tetramethyl-4,5,4',5'-(1,3-disulphonato)-
dibenzo-
dicarbocyanine, and Cy7 (1-( -carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-
5,5'-
disulphonato-tricarbocyanine.
48. The reagent of claim 41, wherein said target material comprises biological
material.
49. The reagent of claim 41, wherein said energy transfer dye comprises a
compound
wherein said R3 is selected from the group consisting of carboxyl,
succinimidyl ester,
sulpho-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite, and
groups
covalently reactive with carboxyl, succinimidyl ester, sulpho-succinimidyl
ester,
isothiocyanate, maleimide, and phosphoramidite.
50. The reagent of claim 41, wherein said energy transfer dye comprises a
compound
further comprising a third dye attached to said R5 through a linker group,
wherein said
attachment results in an energy transfer arrangement of said third dye with
said R5.
51. The reagent of claim 50, wherein said third dye is a cyanine dye.

26
52. A biological material with an attached energy transfer dye, comprising:
a) an energy transfer dye of the formula:
<IMG>
wherein R1 comprises a first dye suitable as an acceptor or donor in an energy
transfer
arrangement;
R5 comprises a second dye suitable as a donor or acceptor in an energy
transfer
arrangement with said first dye;
A comprises a chain of 5 to 20 linked atoms, wherein each atom is
independently
selected from the group consisting of carbon, nitrogen and oxygen;
R3 comprises a hydrogen or a reactive or functional group suitable for
attaching the
energy transfer dye to a target material; and
D comprises an atom or group for attaching said R5 to said A, wherein the
covalent
linkage between said A and said R5 does include a sulphur atom; and
b) a biological material which includes or has been derivatised to include at
least one
first reactive group capable of forming a covalent bond with a functional
group, or a
functional group capable of forming a covalent bond with a reactive group on
the energy
transfer dye and which is covalently bonded thereto.

Description

CA 02319490 2000-08-02
WO 99/39203 PCT/US99/02105
1
DESCRIPTION
ENERGY TRANSFER DYES
Field of the Invention
The present invention relates to a novel class of energy transfer dyes, their
preparation and their use as labels in biological systems.
Background of the Invention
l0 The following describes certain relevant art, none of which is admitted to
be prior
art to the appended claims.
UK Patent No. 2301 833 B discloses, inter alia, that complexes including:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the
wavelength of the emission maximum of the second fluorochrome being longer
than the
wavelength of the emission maximum of the first fluorochrome, and a portion of
the
absorption spectrum of the second fluorochrome overlapping a portion of the
emission
spectrum of the first fluorochrome;
iii) at least one linker group chosen from the group consisting of alkyl
chains
2 o containing from 1 to 20 carbon atoms, which may optionally include from 1
to 8 oxygen
atoms as polyether linkages, or from 1 to 8 nitrogen atoms as polyamine
linkages, or from
1 to 4 CO-NH groups as polyamide linkages and up to 2 bicyclo[2,2,2]octyl
groups, for
covalently attaching the first and second fluorochromes for transfer of
resonance energy
transfer between the first and second fluorochromes; and
2 5 iv) at least one target bonding group capable of forming a covalent bond
with a
target compound;
wherein at least one of the first or second fluorochromes is a cyanine dye and
the
combined molecular weight of the first and second fluorochromes and the linker
group is
less than about 20,000 Daltons, are energy transfer dyes which may be attached
to
3 o biological systems. The fluorescent nature of the dyes enables them to
monitor processes
in which the biological systems are involved. They are of use in sequencing
and in nucleic

CA 02319490 2000-08-02
WO 99/39203 PCT/US99/02105
2
acid detection.
Summary of the Invention
It has now been found that a novel class of energy transfer dyes are of use in
labeling materials involved in sequencing reactions and other applications.
The dyes are
preferably in the form of "cassettes" which enable their attachment to a
variety of
biological materials. A cassette includes a covalently linked structure or
complex with at
least two fluorescent dye moieties, a linker group, and preferably a reactive
group for
attaching the complex to a biological material or other target material. The
reactive group
is chosen to be suitable for forming a covalent linkage with a functional
group on a
particular target material. The dyes are selected so that the emission
spectrum of one dye
overlaps the absorption spectrum of a second dye, so that energy transfer can
occur
between the dyes.
Accordingly, the present invention provides an energy transfer dye of the
formula
(I):
R3
R1-S-A-D-RS
(I)
where R' is a first dye suitable as an acceptor or donor in an energy transfer
arrangement;
R5 is a second dye that is suitable as a donor or acceptor in an energy
transfer
2 5 arrangement with the first dye;
A comprises a chain that contains 5,6,7,8,9,10, I 1,12, I 3, I 4, I 5,16, I
7,18, I 9 or 20
linearly linked atoms selected from carbon, nitrogen and oxygen. The chain may
optionally be substituted, if desired, with groups as known to those skilled
in the art which
do not prevent energy transfer, for example, by C,,2,3 ~4 linear or branched
alkyl or phenyl,
3 0 optionally substituted with 1,2,3, or 4 substituents independently
selected from OH, halo,
methyl or ethyl) groups). Preferably A is a chain of linearly linked atoms;
R3 is a hydrogen or a reactive or functional group suitable for attaching the
energy
transfer dye to a target material, e.g., a biological material as noted above;
and

CA 02319490 2000-08-02
WO 99/39203 PCT/US99/02105
3
D comprises an atom or group for attaching RS to the linker chain A, in which
the
covalent linkage between A and RS does not include a sulphur atom. Also,
preferably no
sulphur atom is present in D, for example, as a side group not in the direct
covalent linkage
between RS and A.
When R' and RS are a fluorescein/rhodamine pair there are preferably
9,10,11,12,13,14,15,16, or 17 linker atoms in A, and more preferably
9,10,11,12,13,14, or
linker atoms. When R' and RS are cyanine dyes, there are preferably
6,7,8,9,10,1 l,or
12 linker atoms, more preferably 8,9 or 10. Preferably A is a C6 ~ 8 9
~0,11,12,I3,14,15,16, or 17
hydrocarbon chain.
10 'The specification of a range of values for the number of atoms in a chain
or group,
whether an express listing of each integer within the range as above, or a
description of the
range by specifying the end points of the range, includes the specific
description of each
integer value within that range, including the endpoints. It further includes
the specific
description of each subrange within the larger range. For example, the range 1-
6 includes
15 the subranges 1-4 and 3-6, along with the other included subranges.
The reactive or functional group, R3, may be any group suitable for attaching
the
energy transfer dye to a target material, preferably a target biological
material and, as such,
will be well known to those skilled in the art. Preferably R3 is selected from
the group
consisting of carboxyl, succinimidyl ester, sulpho-succinimidyl ester,
isothiocyanate,
2 0 maleimide and phosphoramidite, and groups covalently reactive with
carboxyl, succinimidyl
ester, sulpho-succinimidyl ester, isothiocyanate, maleimide and
phosphoramidite.
Suitable dyes for R' may be dyes which contain reactive or functional groups
capable of linking with S. The attachment group D may be any group, other than
sulphur,
suitable for connecting RS with A. Preferably D is P03 or NH-CO.
2 5 The dye moieties, e.g., R' and R5, of the present energy transfer dyes are
fluorophores which are selected, as further indicated herein, to be able to
participate in an
energy transfer arrangement.
Preferably, the energy transfer dyes of this invention have a total molecular
weight
of less than 10,000 or 5,000 daltons, more preferably less than 3,000 or 2000
daltons, still
30 more preferably less than 1,500 or 1,200 daltons.
In connection with the energy transfer dyes of the present invention, by
"energy

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4
transfer arrangement" is meant that two fluorescent dyes are selected having
absorption
and emission spectra suitable for energy transfer between the dyes, and
located with
sufficient physical proximity and linkage such that photoexitation of a first
dye (the donor)
results in the transfer of energy from the first dye to the second dye (the
acceptor).
Additional energy transfers involving one or more additional dye moieties can
also be
created.
Thus, an "energy transfer dye" refers to a fluroescent dye complex having at
least
two dye moieties which can participate in energy transfer between those two
dye moieties.
By "acceptor" in an energy transfer arrangement is meant a dye moiety which
1 o absorbs energy at a wavelength emitted by a donor dye moiety, i.e., the
absorption
spectrum of the acceptor overlaps the emission spectrum of the donor.
By "donor" in an energy transfer arrangement is meant a dye moiety which
absorbs
energy from light, and emits light at frequencies at least partially within
the absorption
spectrum of an acceptor dye moiety.
By "linear or branched alkyl" is meant a straight-chain or branched saturated
aliphatic hydrocarbon. Typical alkyl groups include methyl, ethyl, propyl,
isopropyl,
butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like. By halo is meant
fluoro, chloro,
bromo or iodo.
In the context of this invention, the term "target material" refers to a
compound or
2 0 structure to which a energy transfer dye is to be covalently attached or
to which such a dye
is attached.
By "biological material" is meant a compound produced by or present in an
organism, including but not limited to polypeptides, nucleic acid molecules,
carbohydrates, and lipids. Such compounds may be derivatised to include a
group suitable
2 5 for covalent attachment of an energy transfer dye. The term does not mean
that the dyes of
the present invention must be used with intact organisms, as often the dyes
will be used
with extracts, such as nucleic acid extracts, or samples, including preserved
samples such
as tissue sections, or in nucleic acid sequencing reactions.
Preferably, the energy transfer dye is of the formula (II):
R'-S-(CHR2)m-CH(R3)-NH-CO-(CHR4)~ NH-CO-R5

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~I)
wherein R' is a first dye suitable as an acceptor or donor in an energy
transfer
5 arrangement;
RZ is hydrogen, C,~,3 or4 lineal' or branched alkyl or phenyl (optionally
substituted as
above);
R3 is hydrogen or a reactive or functional group other than thiol;
R4 is hydrogen, C~ 2 3 or 4 linear or branched alkyl, or substituted or. .
ullsubstituted phenyl;
RS is a second dye that is suitable as a donor or acceptor in an energy
transfer arrangement
with the first dye;
m is 1,2 or 3;
n is 1,2,3,4,5,6,7,8, or 9.
Preferably, the donor dye is a fluorescein or cyanine dye. Preferably R'
contains a
reactive or functional group suitable for covalent attachment of the dye to a
thiol-
containing component of A. In the case of attachment to thiol groups,
preferred reactive
groups include iodoacetamido- and maleimido- groups.
Preferably the acceptor is a rhodamine or cyanine dye. Preferably RS contains
a
reactive or functional group suitable for attachment of the dye to a
corresponding
2 0 functional or reactive group component of A. For example, for attachment
of RS to linker
chain A which terminates in amino, dyes which contain a carboxyl or activated
carboxyl
group are preferred. The choice of reactive and functional group-containing
dyes which
are suitable for forming covalent linkages with the linker chain will be well
known to
those skilled in the art.
2 5 Suitable fluorescein donor dyes include but are not limited to 5- and 6-
carboxyfluorescein and 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein.
Suitable cyanine donor dyes include but are not limited to CyA (3-( -
carboxypentyl)-3'-ethyl-S,5'-dimethyl oxacarbocyanine), Cy3 (3-( -
carboxypentyl)-1'-
ethyl-3,3,3',3'-tetramethyl-5,5'-disulphonato-carbocyanine).
3 0 Suitable rhodamine acceptor dyes include, but are not limited to 6-
carboxyrhodamine (Rhodamine 110), 5-carboxyrhodamine-6G (R6G-5 or REG-S), 6-

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6
carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N',N'-tetramethyl-6-carboxyrhodamine
(TAMRA or TMR), 6-carboxy-X-rhodamine (ROX).
Suitable cyanine acceptor dyes include but are not limited to, Cy3.5 (3-( -
carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-4, 5,4',5' -( 1,3-
disulphonato)dibenzo-
carbocyanine), Cy5 (1-( -carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-5,5'-
disulphonato-
dicarbocyanine, Cy5.5 (1-( -carboxypentyl)-1'-ethyl-3,3,3',3'-tetramethyl-
4,5,4',5'-(1,3-
disulphonato)-dibenzo-dicarbocyanine, Cy7 (1-( -carboxypentyl)-1'-ethyl-
3,3,3',3'-
tetramethyl-5,5'-disulphonato-tricarbocyanine. Cyanine dyes suitable for use
in the
energy transfer dyes of the present invention are disclosed in US Patent No.
4,268,486
(Waggoner et al; incorporated herein by reference in its totality including
any drawings).
The above and additional dyes are described, for example, in Southwick et al.,
1990, Cytometry 11:418-430; Mujumdar et al., 1993, Bioconjugate Chemistry
4:105-111;
and Waggoner and Ernst, Fluorescent Reagents for Flow Cytometry, Part 1:
Principles of
Clinical Flow Cytometry (1993).
Optionally the complexes may contain a third dye, e.g. a cyanine dye, attached
to RS
through a suitable linker group and being in an energy transfer arrangement
with R5.
Suitably RZ is hydrogen or methyl and preferably hydrogen.
It will be appreciated by those skilled in the art that when m is other than
1, there
will be several Rz groups present. In such a situation the Rz groups may be
the same or
2 o different. Preferably RZ is hydrogen.
Similarly, R4 is preferably hydrogen or methyl, preferably hydrogen. When n is
other than 1 then the R4 groups may be the same or different.
R3 is as hereinbefore defined and is preferably a carboxyl or activated
carboxyl
group such as succinimidyl ester or sulpho-succinimidyl ester.
2 5 Suitably m + n is 7,8,9 or 10 when R'/RS is a fluorescein/rhodamine pair
and 3,4 or
S when R' and RS are cyanine dyes.
Suitably m is 1,2 or 3, preferably 1. Suitably n is 1,2,3,4,5,6,7,8, or 9 and
preferably 5 for a fluorescein/rhodamine pair.
Preferred compounds of the present invention are:

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7
6-carboxyfluorescein-S-CHZ-CH-NH-CO-(CHz)5-NH-CO-ROX
COON
and
6-carboxyfluorescein-S-CHZ-CH-NH-CO-(CH2)5-NH-CO-ROX
COSu
where Su = N-hydroxysuccinimidyl
In a further aspect, the present invention relates to a biological material
containing
an energy transfer dye of the formula (I) or (II).
Suitable biological materials include, but are not limited to, antibodies,
antigens,
peptides, proteins, carbohydrates, lipids, nucleotides, oxy or deoxy
polynucleic acids and
cells which may be derivatised, if necessary so that they contain one or more
groups
suitable for attachment of an energy transfer dye, e.g., amino, hydroxy,
thiophosphoryl,
sulphydryl or carboxy groups.
2 0 In a further aspect, the present invention provides a method for the
preparation of an
energy transfer dye of the present invention using at least three coupling
reactions:
(i) coupling the dye R' to a thiol containing component of A, which will also
contain
R3, where A, R' and R3 are as hereinbefore defined;
(ii) coupling the product of reaction (i) with the remaining part of A which
will be
substituted by a reactive group suitable for forming the attachment group,
said D, and
(iii) coupling the product of reaction (ii) with the dye RS thereby forming
the attachment
3 0 group, said D.
In connection with the present energy transfer dyes and the attachment of the

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8
various moieties of those dyes, the term "coupling" refers to the formation of
a covalent
bonds) linking two components, for example, linking a dye moiety with the A
portion of
the energy transfer dye.
As there may be several reactive groups present in any component taking part
in
one of the coupling reactions, it may be necessary for those not taking part
in that reaction
to be blocked or protected and then deprotected as appropriate later in the
reaction
sequence.
The dye R' will normally contain a substituent suitable for reaction with a
thiol
group or will be modified to contain such a group. For example, iodoacetamide
is a
1 o suitable substituent for fluorescein dyes, maleimido is a suitable
substituent for cyanine
dyes.
The fluorescent labeling energy transfer dyes may be used to form reagents by
covalently binding the dyes to carrier materials such as polymer particles,
cells, glass
beads, antibodies, proteins, peptides, enzymes, carbohydrates, lipids and
nucleotides or
nucleic acids (DNA and RNA) and analogues which contain or have been
derivatised to
include at least one first reactive group capable of forming a~covalent bond
with the
functional group on the labeling complex (or functional group capable of
forming a
covalent bond with a reactive group on the complex, as described above) and at
least one
second reactive group (or functional group, as the case may be), having
specificity for, and
2 0 being capable of forming a covalent bond with, a target biological
compound, such as
antibodies, cells, drugs, antigens, bacteria, viruses and other micro-
organisms.
When the carrier has functional groups, said functional groups may be antibody
or
DNA suited for attachment to antigen or a complementary DNA sequence,
respectively.
When the carrier material has reactive groups, the carrier may be a polymer
particle or an
2 5 antigen suitable for attachment to DNA or an antibody for example.
Techniques for
covalently binding fluorochromes to carrier materials such as those mentioned
are well
known in the art and readily available in the literature.
The Garner material can further include nucleotides derivatised to contain one
of
amino, sulphydryl, carboxyl, carbonyl or hydroxyl groups, and oxy or deoxy
polynucleic
3 0 acids derivatised to contain one of amino, thiophosphoryl, sulphydryl,
carboxyl, carbonyl
or hydroxyl groups.

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9
The functional groups on the carrier material which are complementary to i.e.
capable of forming covalent bonds with, the reactive groups of the labeling
complexes of
the invention include amino, sulphydryl, carboxyl, carbonyl and hydroxyl
groups.
The present invention also relates to labeling processes in which, in a first
step, an
energy transfer dye of the present invention covalently reacts with and
thereby labels a
first component and then uses the labeled first component to bind with a
second
component to form a labeled second component. Suitably, the first component
may be one
member of a specific binding pair, (a specific binding partner). In the second
step of the
procedure, the fluorescently labeled specific binding partner is then used as
a probe for
l0 binding to a second member of the specific binding pair (the second
component) for which
it has specific affinity.
The specific binding pairs may include a wide variety of molecules which are
arbitrarily termed ligands and receptors. An example of such ligand-receptor
pairs
includes an antibody and the corresponding antigen for which the antibody is
specific.
When the target of the so-labeled antibody is a cell, the second step of the
procedure may
be used to determine the amount of labeled antibodies which are attached to
that type of
cell by determining the intensity of the fluorescence of the cells. By this
procedure,
monoclonal antibodies and other components covalently labeled in a first step
with the
fluorescent compounds of the present invention could be used as antigen
probes.
2 0 Numerous other examples are known to those skilled in the art. Thus,
additional
ligand-receptor pairs include, for example, biotin-(strept)avidin, hormone
receptor-
hormone, DNA-complementary DNA, DNA-RNA, DNA-binding protein, enzyme-
substrate, and the like. It is to be understood that any two molecules which
possess a
specific binding affinity may be employed, so that the energy transfer dyes of
the present
2 5 invention may be used for labeling one member of a specific binding pair
which in turn
may be used in the detection of the complementary member.
The energy transfer dyes of the present invention provide a valuable set of
fluorescent labels which are particularly useful for multiparameter analysis
and
importantly, are sufficiently low in molecular weight to permit materials
labeled with the
3 0 fluorescent complexes to penetrate cell structures. As such, the dyes are
well suited for
use with DNA probes. Multiparameter analysis can be performed on multiple
samples to

CA 02319490 2000-08-02
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detect the presence of target biological compounds. Each sample is labeled by
well known
labeling methods with a different dye or energy transfer dye.
For example, one sample suspected of containing a target biological compound
is
incubated with a single fluorochrome, such as fluorescein, Cascade Blue, a
BODIPY dye,
5 or one of the monomethine rigidized dyes, or CY30(S03)2, or CY3(S03)Z, all
emitting in
the S00-575nm wavelength range (green to orange). A second sample suspected of
containing the target biological compound (the same compound or a different
compound
as that in sample 1 ), is incubated with an energy transfer dye of the
invention, for example
fluorescein-CY3NH2, which will absorb light at 488nm and emits fluorescence at
574nm
10 (orange). Additional samples suspected of containing another target
compound are
incubated with other dyes of the invention, such as fluorescein-CY3-CYS and
fluorescein-
CY3-CY7, both of which absorb light at 488nm, but emit fluorescence at 672nm
and
782nm respectively (red to near infra-red). After a suitable period to permit
the
fluorescent labels to bind with the target compounds, unbound label is removed
by
washing and the labeled samples are mixed.
Detection is possible with a single wavelength excitation source, i.e. at
laser line
488nm. Each differently labeled sample will fluoresce a different color at the
emission
wavelength of its particular label, allowing the individual labels to be
distinguished from
each other.
2 o Those skilled in the art will recognize that the fluorescent energy
transfer labeling
dyes of the present invention can be used for a variety of immunofluorescent
techniques,
including direct and indirect immunoassays, and other known fluorescent
detection
methods. The conditions of each labeling reaction, e.g. pH, temperature and
time are
known in the art, but generally room temperature is preferred. If reacting
with an amine,
pH 9.4 is preferred. The pH is adjusted depending on the optimum reaction
conditions for
the particular reactive groups according to known techniques.
The energy transfer dyes of the present invention and the reagents that can be
made
from them offer a wide variety of fluorescent labels with large Stokes'
shifts. Those in the
art will recognize that the dyes of the invention can be used in a variety of
fluorescence
3 0 applications over a wide range of the visible spectrum.
Other features and advantages of the invention will be apparent from the
following

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11
description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments
The following examples serve to illustrate the preparation of the energy
transfer
dyes of the present invention. These examples are in no way intended to limit
the scope of
the invention.
Example 1
1 o Preparation of FAM-Cvsteine-Linker-ROX Ener,~v Transfer Dye
i) FAM-Cysteine Coupling Reaction
The following mixture was prepared in a microcentrifuge tube: 3001 of 0.25M L-
cysteine (free base), 2001 1M potassium phosphate, pH 8, 8001 of a solution of
SOmg/ml
5-iodoacetamido-fluorescein (Molecular Probes) in DMF, and 7001 of water. The
reaction
mixture was incubated at room temperature for 1 hour protected from light.
ii) FAM-Cysteine-Linker Coupling Reaction
To the crude reaction mixture from i) was added 6001 water, 4001 1 M sodium
2 0 carbonate buffer, pH 8.3, and lml of a solution of 75mg/ml of the
trifluoroacetyl-protected
NHS ester of 6-aminocaproic acid in DMF. The reaction mixture was incubated at
room
temperature for 1 hour protected from light.
iii) Deprotection of the FAM-Cysteine-Linker
The reaction mixture from ii) was dried in a Speed-Vac apparatus and lOml of
concentrated ammonium hydroxide was added to the tube. After thorough mixing,
the
reaction was incubated at room temperature for 2 hours and then dried in the
Speed-Vac
apparatus.
3 o iv) Purification of FAM-Cysteine Linker
Purification was accomplished by HPLC using a DeltaPak column (15 micron, C18

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12
reverse phase, 7.8 x 300mm) and a gradient of triethylammonium acetate, pH 7.0
and SO%
acetonitrile in triethylammonium acetate, pH 7Ø Fractions with absorbance at
496nm
were collected and dried overnight in a SpeedVac apparatus.
v) FAM-Cvsteine-Linker-ROX Coupling Reaction
To a microcentrifuge tube was added the following:
1501 HPLC purified FAM-Cysteine-Linker in DMF equivalent to 3mg (as measured
by
absorbance at 496nm), 1001 water, 501 1M sodium carbonate buffer, pH 8.3, and
200p1
5'-ROX-NHS ester (Molecular Probes) (25mg/ml in DMF). The reaction mixture was
incubated overnight at room temperature.
Note: Other rhodamine acceptor dyes may be substituted for ROX in the above
reaction to generate four different energy transfer dye cassettes for use in
sequencing
applications.
vi) Purification of FAM-Cysteine-Linker-ROX
The product energy transfer dye from v) was purified by reverse phase HPLC
using
a DeltaPak column (15 micron, 300A, C18 reverse phase, 7.8 x 300mm) and a
gradient of
triethylammonium acetate, pH 7.0 and 50% acetonitrile in triethylammonium
acetate, pH
2 0 7Ø Fractions having absorbance at both 496nm and 576nm were collected,
pooled and
dried overnight in a SpeedVac apparatus. The product was redissolved in water
and
evaluated spectroscopically using a Perkin Elmer LS-SOB Luminescence
Spectrometer.
With excitation at 488nm, a strong peak was observed at 603nm, characteristic
of the ROX
emission and indicating excellent energy transfer. The absorbance spectrum
showed bands
2 5 characteristic of both fluorescein and ROX.
vii) Conversion of the Carboxylic Acid Derivative to its NHS Ester
The cysteine carboxyl of the above energy transfer dye may be converted to an
NHS ester derivative by the following method.
3 o The above acid derivative is dissolved in DMF at a concentration of l
Omg/ml. To
the stirred solution in a round bottomed flask is added 1.5 mole equivalents
of

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13
dicyclohexylcarbodiimide (DCC} and 1.5 mole equivalents of N-
hydroxysuccinimide.
The flask containing the mixture is sealed and covered with foil. The reaction
is stirred
under argon at room temperature for 4 hours. It is then checked by TLC using
the
appropriate standards to monitor that the free acid has been converted into
the NHS ester
(TLC using dichloromethane-methanol-acetic acid, 4:1:1 ). The activated dye is
then
precipitated from solution by adding four volumes of ethyl acetate. The
resulting pellet is
rinsed 2 times with ethyl acetate and finally dried under vacuum. The NHS
ester may now
be coupled to a suitable target material (e.g. an amino-link oligonucleotide)
using standard
conditions.
l0
All patents and publications mentioned in the specification are indicative of
the levels of
skill of those skilled in the art to which the invention pertains. All
references cited in this
disclosure are incorporated by reference to the same extent as if each
reference had been
incorporated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is
well adapted to
carry out the objects and obtain the ends and advantages mentioned, as well as
those
inherent therein. The dyes, substituents, and target materials described
herein as presently
representative of preferred embodiments are exemplary and are not intended as
limitations
2 0 on the scope of the invention. Changes therein and other uses will occur
to those skilled in
the art, which are encompassed within the spirit of the invention, are defined
by the scope
of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions and
modifications may be made to the invention disclosed herein without departing
from the
scope and spirit of the invention. For example, those skilled in the art will
readily
recognize that the present energy transfer dyes can incorporate a variety of
different dye
moieties, linkers; attachment groups, and reactive groups, and can be attached
to a variety
of different target materials. Thus, such additional embodiments are within
the scope of
3 o the present invention and the following claims.

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14
The invention illustratively described herein suitably may be practiced in the
absence of
any element or elements, limitation or limitations which is not specifically
disclosed
herein. Thus, for example, in each instance herein any of the terms
"comprising",
"consisting essentially off' and "consisting of may be replaced with either of
the other two
terms. The terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that in the use
of such terms and
expressions of excluding any equivalents of the features shown and described
or portions
thereof, but it is recognized that various modifications are possible within
the scope of the
invention claimed. Thus, it should be understood that although the present
invention has
been specifically disclosed by preferred embodiments and optional features,
modification
and variation of the concepts herein disclosed may be resorted to by those
skilled in the
art, and that such modifications and variations are considered to be within
the scope of this
invention as defined by the appended claims.
In addition, where features or aspects of the invention are described in terms
of Markush
groups or other grouping of alternatives, those skilled in the art will
recognize that the
invention is also thereby described in terms of any individual member or
subgroup of
members of the Markush group or other group.
2 0 Thus, additional embodiments are within the scope of the invention and
within the
following claims.