U.S. patent application number 15/694279 was filed with the patent office on 2018-03-01 for reagents useful for synthesizing rhodamine-labeled oligonucleotides.
This patent application is currently assigned to Applied Biosystems, LLC. The applicant listed for this patent is Applied Biosystems, LLC. Invention is credited to Scott C. Benson, Jonathan M. Cassel, Paul M. Kenney, Krishna G. Upadhya, Ruiming N. Zou.
Application Number | 20180057516 15/694279 |
Document ID | / |
Family ID | 38564295 |
Filed Date | 2018-03-01 |
United States Patent
Application |
20180057516 |
Kind Code |
A1 |
Benson; Scott C. ; et
al. |
March 1, 2018 |
REAGENTS USEFUL FOR SYNTHESIZING RHODAMINE-LABELED
OLIGONUCLEOTIDES
Abstract
The present disclosure provides reagents that can be used to
label synthetic oligonucleotides with rhodamine dyes or dye
networks that contain rhodamine dyes.
Inventors: |
Benson; Scott C.; (Alameda,
CA) ; Zou; Ruiming N.; (Foster City, CA) ;
Upadhya; Krishna G.; (Union City, CA) ; Kenney; Paul
M.; (Sunnyvale, CA) ; Cassel; Jonathan M.;
(Half Moon Bay, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Applied Biosystems, LLC |
Carlsbad |
CA |
US |
|
|
Assignee: |
Applied Biosystems, LLC
Carlsbad
CA
|
Family ID: |
38564295 |
Appl. No.: |
15/694279 |
Filed: |
September 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14702499 |
May 1, 2015 |
9783560 |
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15694279 |
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11695548 |
Apr 2, 2007 |
9040674 |
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14702499 |
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60787777 |
Mar 31, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09B 11/24 20130101;
C07F 9/650952 20130101; C07D 311/96 20130101; C07F 9/65515
20130101; C07F 9/242 20130101; C07H 19/04 20130101; C07F 9/65312
20130101; C07F 9/6561 20130101; C07H 21/04 20130101; C07F 9/2408
20130101 |
International
Class: |
C07F 9/6561 20060101
C07F009/6561; C07H 21/04 20060101 C07H021/04; C07D 311/96 20060101
C07D311/96; C07H 19/04 20060101 C07H019/04 |
Claims
1-66. (canceled)
67. An oligonucleotide comprising a label moiety that comprises an
N-protected NH-rhodamine moiety.
68. The oligonucleotide of claim 67 in which the N-protected
NH-rhodamine moiety comprises a structure selected from:
##STR00037## wherein LM represents a label moiety, PEP represents
the phosphate ester precursor group, B represents a suitably
protected nucleobase, L.sup.2 represents a linker linking label
moiety LM to nucleobase B and, in structure (IX.4), R.sup.11
represents a protecting group.
69. The oligonucleotide of claim 68 in which L.sup.2 is selected
from --C.ident.C--CH.sub.2--NH--, --C.ident.C--C(O)--,
--CH.dbd.CH--NH--, --CH.dbd.CH--C(O)--,
--C.ident.C--CH.sub.2--NH--C(O)--(CH.sub.2).sub.1-6--NH--,
--CH.dbd.CH--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--,
--C.ident.CH--CH.sub.2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub.2].sub.0--
6--NH--,
--C.ident.C--C.ident.C--CH.sub.2--O--CH.sub.2CH.sub.2--[O--CH.sub-
.2CH.sub.2].sub.0-6--NH--,
--C.ident.C--(Ar).sub.1-2--C.ident.C--CH.sub.2--O--CH.sub.2CH.sub.2--[O---
CH.sub.2CH.sub.2].sub.0-6--NH--,
--C.ident.C--(Ar).sub.1-2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub.2].sub-
.0-6--NH-- and
--C.ident.C--(Ar).sub.1-2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub.2].sub-
.0-6--NH--, where each Ar represents, independently of the others,
an optionally substituted monocyclic or polycyclic cycloalkylene,
cycloheteroalkynene, arylene or heteroarylene group.
69. The oligonucleotide of claim 68 in which --B-L.sup.2- is
selected from ##STR00038## ##STR00039##
70. The oligonucleotide of claim 68 in which the N-protected
NH-rhodamine moiety comprises a structure selected from structural
formulae (IIIc), (IIIb) and (IIIc): ##STR00040## wherein: R' is
selected from R.sup.3' and hydrogen; R'' is selected from R.sup.6'
and hydrogen; R.sup.9 is an acyl protecting group; R.sup.1',
R.sup.2', R.sup.2'', R.sup.4', R.sup.4'', R.sup.5', R.sup.5'',
R.sup.7', R.sup.7'', R.sup.8', R.sup.4, R.sup.5, R.sup.6, and
R.sup.7, when taken alone, are each, independently of one another,
selected from hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20)
arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl,
--R.sup.b and --(CH.sub.2).sub.x--R.sup.b, where x is an integer
ranging from 1 to 10 and R.sup.b is selected from --X, --OH,
--OR.sup.a, --SH, --SR.sup.a, --NH.sub.2, --NHR.sup.a,
--NR.sup.cR.sup.c, --N.sup.+R.sup.cR.sup.cR.sup.c, perhalo lower
alkyl, trihalomethyl, trifluoromethyl, --B(OH).sub.3,
--B(OR.sup.a).sub.3, --B(OH)O.sup.-, --B(OR.sup.a).sub.2O.sup.-,
--B(OH)(O.sup.-).sub.2, --B(OR.sup.a)(O.sup.-).sub.2,
--P(OH).sub.2, --P(OH)O.sup.-, --P(OR.sup.a).sub.2,
--P(OR.sup.a)O.sup.-, --P(O)(OH).sub.2, --P(O)(OH)O.sup.-,
--P(O)(O.sup.-).sub.2, --P(O)(OR.sup.a).sub.2,
--P(O)(OR.sup.a)O.sup.-, --P(O)(OH)(OR.sup.a), --OP(OH).sub.2,
--OP(OH)O.sup.-, --OP(OR.sup.a).sub.2, --OP(OR.sup.a)O.sup.-,
--OP(O)(OH).sub.2, --OP(O)(OH)O.sup.-, --OP(O)(O.sup.-).sub.2,
-OP(O)(OR.sup.a).sub.2, --OP(O)(OR.sup.a)O.sup.-,
--OP(O)(OR.sup.a)(OH), --S(O).sub.2O.sup.-, --S(O).sub.2OH,
--S(O).sub.2R.sup.a, --C(O)H, --C(O)R.sup.a, --C(S)X,
--C(O)O.sup.-, --C(O)OH, --C(O)NH.sub.2, --C(O)NHR.sup.a,
--C(O)NR.sup.cR.sup.c, --C(S)NH.sub.2, --C(O)NHR.sup.a,
--C(O)NR.sup.cR.sup.c, --C(NH)NH.sub.2, --C(NH)NHR.sup.a, and
--C(NH)NR.sup.cR.sup.c, where X is halo, each R.sup.a is,
independently of the others, selected from lower alkyl, (C6-C14)
aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20
membered heteroarylalkyl, and each R.sup.c is, independently of the
others, an R.sup.a, or, alternatively, two R.sup.c bonded to the
same nitrogen atom may be taken together with that nitrogen atom to
form a 5- to 8-membered saturated or unsaturated ring that may
optionally include one or more of the same or different ring
heteroatoms, which are typically selected from O, N and S, or,
alternatively, R.sup.1' and R.sup.2' or R.sup.7' and R.sup.8' are
taken together with the carbon atoms to which they are bonded to
form an optionally substituted (C6-C14) aryl bridge and/or R.sup.4'
and R.sup.4'' and/or R.sup.5' and R.sup.5'' are taken together with
the carbon atoms to which they are bonded to form a benzo group;
and R.sup.3' and R.sup.6', when taken alone, are each,
independently of one another, selected from lower alkyl, (C6-C14)
aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20
membered heteroarylalkyl, or alternatively, R.sup.3' and R.sup.2'
or R.sup.4' and/or R.sup.6' and R.sup.5' or R.sup.7' in the
compounds of structural formula (IIIa), R.sup.3' and R.sup.2' or
R.sup.4' and/or R.sup.6' and R.sup.5' or R.sup.7'' in the compounds
of structural formula (III6), or R.sup.3' and R.sup.2'' or R.sup.4'
and/or R.sup.6' and R.sup.5' or R.sup.7'' in the compounds of
structural formula (IIIc) are taken together with the atoms to
which they are bonded to form a 5- or 6-membered saturated or
unsaturated ring which is optionally sutstituted with one or more
of the same or different lower alkyl, benzo or pyrido groups, with
the proviso that at least one of R.sup.2', R.sup.4', R.sup.5',
R.sup.7', R.sup.5 or R.sup.6 in the compounds structural formula
(IIIa), at least one of R.sup.2', R.sup.4', R.sup.5', R.sup.7'',
R.sup.5 or R.sup.6 in the compounds of structural formula (IIIb)
and at least one of R.sup.2'', R.sup.4', R.sup.5', R.sup.7'',
R.sup.5 or R.sup.6 in the compounds of structural formula (IIIc)
comprises a group of the formula --Y--, where Y represents a
portion of a linkage contributed by a functional group F.sup.y.
71. The oligonucleotide of claim 70, in which the N-protected
NH-rhodamine moiety comprises a structure selected from structural
formulae (IIIa.1), (IIIa.2), (IIIb.1), (IIIb.2), (IIIc.1) and
(IIIc.2): ##STR00041## ##STR00042## wherein R', R'', R.sup.1',
R.sup.2', R.sup.2'', R.sup.3', R.sup.4', R.sup.4'', R.sup.5',
R.sup.5'', R.sup.6', R.sup.7', R.sup.7'', R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8', R.sup.9 and Y are as previously defined
in claim 70.
72. The oligonucleotide of claim 70 in which the N-protected
NH-rhodamine moiety has one or more applicable features selected
from: (i) Y is selected from --C(O)--, --S(O).sub.2--, --S-- and
--NH--; (ii) R.sup.4 and R.sup.7 are each chloro; (iii) R.sup.1'
and R.sup.8' are each hydrogen; (iv) R.sup.1' and R.sup.2' or
R.sup.7' and R.sup.8' are taken together to form a benzo group; (v)
R.sup.2' and R.sup.7' are each hydrogen or lower alkyl; (vi) R' is
R.sup.3' and R'' is R.sup.6'; and (vii) R' is R.sup.3', R'' is
R.sup.6', and R.sup.3' and R.sup.6' are taken together with a
substituent group on an adjacent carbon atom to form a group
selected from --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--C(CH.sub.3).sub.2CH.dbd.C(CH.sub.3)--,
--C(CH.sub.3).sub.2CH.dbd.CH--, --CH.sub.2--C(CH.sub.3).sub.2-- and
##STR00043##
73. The oligonucleotide of claim 67 in which the label moiety is
linked to the 3'- or 5'-hydroxyl of the oligonucleotide.
74. The oligonucleotide of claim 67 in which the label moiety is
linked to a nucleobase of the oligonucleotide.
75. The oligonucleotide of claim 67 in which the oligonucleotide is
further labeled with a donor and/or acceptor moiety for the
N-protected NH-rhodamine moiety.
76. The oligonucleotide of claim 75 in which the label moiety
comprises structural formula (VI): A-Z.sup.1-Sp-Z.sup.2-D (VI)
wherein A represents the N-protected NH-rhodamine moiety, D
represents the donor moitey, Z.sup.1 and Z.sup.2, which may be the
same or different, represent portions of linkages provided by
linking moieties comprising a functional group F.sup.z, and Sp
represents a spacing moiety.
77. The oligonucleotide of claim 76 in which A is selected from
structural formulae A.1, A.2, A.3, A.4, A.5 and A.6 and D is
selected from structural formulae D.1, D.2, D.3, D.4, D.5 and D.6,
or A is selected from structural formulae A.7, A.8, A.9, A.10, A.11
and A.12 and D is selected from structural formulae D.7, D.8, D.9,
D.10, D.11 and D.12; ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051##
wherein: E.sup.1 is selected from --NHR.sup.9, --NR.sup.3'R.sup.9
and --OR.sup.9b; E.sup.2 is selected from --NHR.sup.9,
--NR.sup.6'R.sup.9 and --OR.sup.9b; R.sup.9b is R.sup.9; Y.sup.1a,
Y.sup.1b, Y.sup.2a, Y.sup.2b, Y.sup.3a, and Y.sup.3b are each,
independently of one another, selected from --O--, --S--, --NH--,
--C(O--) and --S(O).sub.2--; and R', R'', R.sup.1', R.sup.2'',
R.sup.3', R.sup.4', R.sup.4'', R.sup.5', R.sup.5'', R6', R.sup.7',
R.sup.7'', R.sup.8', R.sup.4, R.sup.5, R.sup.6, R.sup.7, and
R.sup.9 are as previously defined in claim 34, with the proviso
that when E.sup.1 and E.sup.2 are --OR.sup.9b, then R.sup.1' and
R.sup.2' and/or R.sup.7' and R.sup.8' may only be taken together
with the carbon atoms to which they are bound to form an optionally
substituted (C6-C14) aryl bridge.
78. The oligonucleotide of claim 67 in which the oligonucleotide is
further labeled with a quencher moiety.
79. The oligonucleotide of claim 67 in which the oligonucleotide is
further labeled with a minor groove binding moiety.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/702,499, filed May 1, 2015, which is a divisional of U.S.
non-provisional application Ser. No. 11/695,548, filed Apr. 2,
2007, now U.S. Pat. No. 9,040,674, which claims benefit under 35
U.S.C. .sctn. 119(e) to provisional application No. 60/787,777,
filed Mar. 31, 2006, which disclosures are herein incorporated by
reference in their entirety.
2. BACKGROUND
[0002] The use of fluorescent dyes as detection labels has found
widespread use in molecular biology, cell biology and molecular
genetics. For example, the use of fluorescently-labeled
oligonucleotides is now widespread in a variety of different
assays, including polynucleotide sequencing, fluorescence in situ
hybridization (FISH), hybridization assays on nucleic acid arrays,
fluorescence polarization studies, and nucleic acid amplification
assays, including polymerase chain amplification assays carried out
with fluorescent probes and/or primers.
[0003] Some fluorescent labels can be attached to nascent or
completed oligonucleotide chains synthesized in situ using
fluorescent phosphoramidite reagents. For example, fluorescein
phosphoramidite reagents are available commercially (see, e.g.,
2006 product catalog of Glen Research Corporation, Sterling, Va.).
In such reagents, the 3'- and 6'-exocyclic oxygen atoms of the
fluorescein ring are protected with pivaloyl groups to prevent side
reactions. Modification of the fluorescein ring with these groups
also holds the carboxylate group at the 3-position in the closed,
spiro lactone form, preventing proton donation from the carboxylate
to the phosphoramidite group, which would convert this
phosphoramidite group into a good leaving group, leading to
decomposition of the reagent. The fluorescein ring is also stable
to the conditions used to oxidize the nascent oligonucleotide and
to treatment with aqueous ammonium, the standard method by which
the nucleobase protecting groups are removed and the synthetic
oligonucleotide is cleaved from the synthesis resin.
[0004] Unfortunately, many rhodamine dyes are susceptible to
chemical modification when treated with the reagents commonly
employed to oxidize and deprotect/cleave synthetic oligonucleotides
negatively impacting their fluorescent properties. As a
consequence, rhodamine dyes are commonly attached to
oligonucleotides following synthesis, deprotection and cleavage
from the synthesis resin. This adds additional steps and manual
labor, resulting in greater cost and inconvenience in the overall
synthesis of rhodamine-labeled oligonucleotides.
[0005] Owing to these and other limitations, there are currently
only two rhodamine dyes that are commercially available as
phosphoramidite reagents: tetramethyl rhodamine ("TAMRA") and
rhodamine X ("ROX"). Additional reagents that permit labeling of
oligonucleotides with myriad different rhodamine dyes during solid
phase chemical synthesis would be desirable.
3. SUMMARY
[0006] In one aspect, the present disclosure provides reagents
useful for labeling synthetic oligonucleotides with labels
comprising rhodamine dyes that fluoresce when irradiated with
incident light of an appropriate wavelength. The labels can
comprise a single rhodamine dye, or they can comprise a dye network
in which at least one of the dyes is a rhodamine dye. The reagents
can be used to label synthetic oligonucleotides with rhodamine
dye-containing labels directly during the step-wise synthesis of
the oligonucleotides, thereby reducing the manipulation steps
necessary to obtain oligonucleotides that are labeled with
rhodamine dyes. Moreover, because the labels are attached to the
oligonucleotide directly during step-wise synthesis, HPLC
separation of uncoupled label from the labeled oligonucleotide,
which is necessary when using currently available post-synthesis
rhodamine labeling reagents such as rhodamine NHS esters, is
unnecessary.
[0007] The reagents can be used to label an oligonucleotide at its
3'-terminus, at its 5-terminus, and/or at one or more internal
positions. The resultant label can be attached to the terminal
hydroxyl(s) of the oligonucleotide, to one or more nucleobases
comprising the oligonucleotide, or it can be disposed between two
nucleotides comprising the oligonucleotide chain Thus, the reagents
can take the form of non-nucleosidic synthesis reagents (see, e.g.,
FIGS. 3 and 5), nucleosidic synthesis reagents (see, e.g., FIGS. 4
and 6), non-nucleosidic solid supports (see, e.g., FIG. 7) and/or
nucleosidic solid supports (see, e.g., FIG. 8).
[0008] The synthesis reagents generally comprise a label moiety, a
phosphate ester precursor ("PEP") group and an optional linker
linking the phosphate ester precursor group to the label moiety.
The phosphate ester precursor group generally comprises a
functional group that, when used in the step-wise synthesis of
oligonucleotides, ultimately yields, after optional deprotection
and/or oxidation, the internucleotide phosphate ester linkage.
Several types of chemistries and functional groups suitable for
synthesizing internucleotide phosphate ester linkages are known in
the art, and include, by way of example and not limitation,
phosphite triester chemistry, which utilizes phosphoramidite PEP
groups (see, e.g., Letsinger et al., 1969, J. Am. Chem. Soc.
91:3350-3355; Letsinger et al., 1975, J. Am. Chem. Soc. 97:3278;
Matteucci & Caruthers, 1981, J. Am. Chem. Soc. 103:3185;
Beaucage & Caruthers, 1981, Tetrahedron Lett. 22:1859),
phosphotriester chemistry, which utilizes 2-chlorophenyl- or
2,5-dichlorophenyl-phosphate PEP groups (see, e.g., Sproat &
Gait, "Solid Phase Synthesis of Oligonucleotides by the
Phosphotriester Method," In: Oligonucleotide Synthesis, A Practical
Approach, Gait, Ed., 1984, IRL Press, pages 83-115) and
H-phosphonate chemistry, which utilizes H-phosphonate PEP groups
(see, e.g., Garegg et al., 1985, Chem. Scr. 25:280-282; Garegg et
al., 1986, Tet. Lett. 27:4051-4054; Garegg et al. 1986, Tet. Lett.
27:4055-4058; Garegg et al., 1986, Chem. Scr. 26:59-62; Froehler
& Matteucci, 1986, Tet. Lett. 27:469-472; Froehler et al.,
1986, Nucl. Acid Res. 14:5399-5407). All of these various PEP
groups, as well as later-discovered PEP groups, can comprise the
phosphate ester precursor group of the synthesis reagents described
herein. The identity of the PEP group is not critical for success,
and will depend upon the desired chemistry for synthesizing the
labeled oligonucleotides. In some embodiments, the phosphate ester
precursor group comprises a phosphoramidite group.
[0009] The optional linker linking the label moiety and phosphate
ester precursor group can comprise virtually any combination of
atoms or functional groups stable to the synthetic conditions used
for the synthesis of the labeled oligonucleotides, and can be
linear, branched, or cyclic in structure, or can include
combinations of linear, branched and/or cyclic structures. The
linker can be designed to have specified properties, such as the
ability to be cleaved under desired conditions.
[0010] The synthesis reagents may optionally further comprise one
or more synthesis handles to which nucleosides or other groups or
moieties can be attached. The synthesis handles can include
protecting groups that can be selectively removed during the
step-wise synthesis of the labeled oligonucleotide, permitting
attachment of moieties to the synthetic oligonucleotide prior to
cleavage from the resin, or, alternatively, the synthesis handles
can include protecting groups that are stable to the conditions
used to deprotect and/or cleave the synthesized oliognucloetide
from the synthesis resin, permitting attachment of moieties to the
synthetic labeled oligonucleotide following synthesis, deprotection
and cleavage from the synthesis resin. The synthesis handles
comprising a synthesis reagent that includes more than one
synthesis handle may be the same or different.
[0011] In some embodiments, the synthesis reagents comprise a
single optional synthesis handle that comprises a protected
hydroxyl of the formula --OR.sup.e, where F.sup.e represents an
acid-labile protecting group.
[0012] The synthesis handle can be linked to the label moiety, or
it can be included in the optional linker linking the label moiety
and phosphate ester precursor group. Embodiments in which a
synthesis handle of the formula --OR.sup.e is included in the
optional linker can be non-nucleosidic in nature or nucleosidic in
nature, in which latter case the linker comprises a nucleoside and
the synthesis handle is provided by a hydroxyl group on the sugar
moiety of a nucleoside, typically the 5'-hydroxyl of the nucleoside
sugar moiety. Specific, non-limiting embodiments of non-nucleosidic
synthesis reagents in which the linker includes a synthesis handle
are illustrated in FIG. 5. Specific, non-limiting embodiments of
nucleosidic synthesis reagents in which the linker includes a
synthesis handle are illustrated in FIG. 6.
[0013] The solid support reagents generally comprise a label
moiety, a synthesis handle of the formula --OR.sup.e where R.sup.e
is as defined above, a solid support and a linker linking the label
moiety and the synthesis handle to the solid support. The synthesis
handle provides a group to which nucleoside monomer reagents can be
coupled. The solid support reagents can optionally include one or
more additional synthesis handles, which can be the same or
different.
[0014] A wide variety of materials suitable for use as solid
supports in the solid-phase synthesis of oligonucleotides that
either include appropriate functional groups, or that can be
derivatized to include appropriate functional groups, are known in
the art, and include, by way of example and not limitation,
controlled pore glass (CPG), polystyrene, and various graft
co-polymers. All of these various materials are suitable for use as
the solid support in the solid support reagents described
herein.
[0015] The shape of the solid support is not critical. Virtually
any shape can be utilized. For example, the solid support can be in
the form of spherical or irregularly shaped beads, cubes,
rectangles, cylinders, cylindrical tubes, or even sheets. The solid
support may be porous or non-porous. In some embodiments, the solid
support is a CPG or polystyrene bead.
[0016] The linker linking the label moiety and synthesis handle to
the solid support can comprise virtually any combination of atoms
or functional groups stable to the synthesis conditions typically
used for the solid phase synthesis of oligonucleotides, and can be
linear, branched, or cyclic in structure, or can include
combinations of linear, branched and/or cyclic structures. The
linker can be designed to have specified properties, such as the
ability to be cleaved under desired conditions. In some
embodiments, the linker includes a linkage that can be cleaved
under specified conditions to release the solid support from the
remainder of the reagent. For example, the linker can include
linkages that are stable under oligonucleotides synthesis
conditions and labile to the conditions used to deprotect the
synthesized oligonucleotides (for example, incubation in ammonium
hydroxide at 55.degree. C. or room temperature). Such specifically
cleavable linkages are well-known in the art, and include by way of
example and not limitation, esters, carbonate esters,
diisopropylsiloxy ethers, modified phosphate esters, etc.
[0017] As described above for the synthesis reagents, the linker of
the solid support reagents can include the synthesis handle, and
can be nucleosidic or non-nucleosidic in nature. Specific,
non-limiting embodiments of non-nucleosidic solid support reagents
are illustrated in FIG. 7. Specific, non-limiting embodiments of
nucleosidic solid support reagents are illustrated in FIG. 8.
[0018] The label moiety of the synthesis and solid support reagents
described herein comprises a rhodamine dye. The exocyclic nitrogen
atoms at the 3'- and 6'-positions of the rhodamine dye are either
unsubstituted or mono-substituted such they are included in a
primary or secondary amine, and are further substituted with a
protecting group (unprotected rhodamine dyes having primary or
secondary amine groups at their 3'- and 6'-positions are referred
to herein as "NH-rhodamines" and rhodamine dyes having protecting
groups at their 3'- and 6'-positions are referred to herein as
"N-protected NH-rhodamines").
[0019] The protecting group can be virtually any specifically
removable group that is stable to the synthesis conditions that
will be used to synthesize the labeled oligonucleotide, for example
the phosphite triester chemistry conditions typically used for
solid-phase synthesis of oligonucleotides. It has been discovered
that protecting the 3'- and 6'-secondary or primary amines of
NH-rhodamines with groups that form amides, such as, for example
carboxamides, sulfonamides, phorphoramides, etc., permits the
N-protected NH-rhodamine to exist in the closed, lactone form,
thereby permitting the rhodamines to be used in the step-wise
synthesis of oligonucleotides to conveniently synthesize
oligonucleotides including rhodamine dyes without the need for
post-synthesis manipulation or purification. As demonstrated in the
working examples, phosphoramidite reagents including such
N-protected NH-rhodamines are soluble in the solvents commonly
employed in the step-wise synthesis of oligonucleotides, are stable
to multiple rounds of DMT deprotection, coupling, oxidation and
capping, and also to treatment with concentrated ammonium
hydroxide, conditions which are commonly used to deprotect any
exocyclic amine protecting groups and cleave the synthetic
oligonucleotide from the synthesis resin.
[0020] The protecting groups can be labile, and thus removable,
under the conditions used to remove the nucleobase protecting
groups of the synthesized labeled oligonucleotide, or,
alternatively, the protecting groups can be stable to these
conditions and labile to other conditions. In most instances, it
will likely be desirable to utilize protecting groups that are
labile to the conditions used to remove the nucleobase protecting
groups of the synthesized labeled oligonucleotide and/or to cleave
the labeled synthetic oligonucleotodie from the synthesis
resin.
[0021] In some embodiments, the protecting groups are acyl groups
of the formula --C(O)F.sup.10, where R.sup.10 is selected from
lower alkyl, methyl, --CX.sub.3, --CHX.sub.2, --CH.sub.2X,
--CH.sub.2--OR.sup.b and phenyl optionally mono-substituted with a
lower alkyl, methyl, X, --OR.sup.b, cyano or nitro group, where
R.sup.b is selected from lower alkyl, pyridyl and phenyl and each X
is a halo group, typically fluoro, chloro or bromo. In a specific,
non-limiting embodiment, R.sup.10 is t-butyl or
trifluoromethyl.
[0022] The label moiety may further comprise additional protected
fluorophores, such that the N-protected NH-rhodamine dye is a
member of a larger, energy-transfer dye network. Such
energy-transfer dye networks are well-known in the art, and include
combinations of fluorophores whose spectral properties are matched,
or whose relative distances to one another are adjusted, so that
one fluorophore in the network, when excitated with incident
irradiation of an appropriate wavelength, transfers its excitation
energy to another fluorophore in the network, which in turn
transfers its excitation energy to yet another fluorophore in the
network, and so forth, resulting in fluorescence by the ultimate
acceptor fluorophore in the network. Such networks give rise to
labels having long Stokes shifts. In such networks, fluorophores
that transfer, or donate, their excitation to another fluorophore
in the network are referred to as "donors." Fluorophore that
receive, or accept, excitation energy from another fluorophore and
fluorescein reagents thereto are referred to as "acceptors." In dye
networks containing only two dyes, one dye typically acts as the
donor and the other as the acceptor. In dye networks containing
three or more different dyes, at least one dye acts as both a donor
and acceptor.
[0023] Energy transfer dye networks containing two, three, four, or
even more dyes are well-known in the art (see, e.g., US
2006/057565). Any of the dyes used in these networks that can be
suitably protected for use in the solid phase synthesis
oligonucleotides can be included in the label moieties described
herein.
[0024] In some embodiments of the synthesis and solid support
reagents described herein, the label moiety further comprises a
suitably protected donor for the N-protected NH-rhodamine. When
deprotected, such donors transfer their excitation energy to the
NH-rhodamine such that the NH-rhodamine emits fluorescence upon
excitation of the donor.
[0025] In some embodiments of the synthesis and solid support
reagents described herein, the label moiety further comprises a
suitably protected acceptor for the N-protected NH-rhodamine. When
deprotected, such acceptors accept excitation energy from the donor
NH-rhodamine such that the acceptor fluoresces upon excitation of
the donor NH-rhodamine.
[0026] The identities of the donor or acceptor will depend upon the
identity of the NH-rhodamine comprising the label moiety. Examples
of fluorophores capable of acting as a donor for a wide variety of
rhodamine dyes are well-known in the art. Non-limiting examples of
such donors include xanthene dyes (such as, for example,)
fluoresceins, rhodamines and rhodols), pyrene dyes, coumarin dyes
(for example hydroxy and amino coumarins), cyanine dyes,
phthalocyanine dyes, and lanthenide complexes. Examples of
fluorophores capable of acting as acceptors for rhodamine dyes are
also well-known in the art. Non-limiting examples of such acceptors
include rhodamines dyes and cyanines dyes. Any of these dyes that
can be suitably protected for use under the conditions used to
synthesize oligonucleotides, such as the phosphite triester
chemistry conditions typically used for the solid-phase synthesis
of oligonucleotides, can be used as the donor or acceptor in the
synthesis and solid support reagents described herein.
[0027] The mechanism by which energy is transferred from a donor to
the acceptor is not critical. All that is necessary for such
donor-acceptor pairs to be operable is that the acceptor fluoresce
in response to excitation of the donor.
[0028] The label moiety may also include acceptors that are
non-fluorescent. Such non-fluorescent acceptors can be used to
quench, either in whole or in part, the fluorescence of the
NH-rhodamine or other fluorescent dye(s) comprising the label
moiety. Examples of such non-fluorescent moieties that can act as
quenchers for rhodamine dyes such as the NH-rhodamines described
herein include, but are not limited to, dabcyl, the various
non-fluorescent quenchers described in WO 01/86001and the various
non-fluorescent quenchers described in US 2005/0164225, the
disclosures of which are incorporated herein by reference.
[0029] In some embodiments, the label moiety further comprises a
donor dye that in turn comprises an N-protected NH-rhodamine dye as
described herein or a fluorescein dye in which the exocylic 3'-and
6'-oxygen atoms are protected with protecting groups that are
stable to oligonucleotide synthesis conditions, such as the
phosphite triester chemistry conditions typically used for the
solid phase synthesis of oligonuclotides, and labile to the
conditions used to deprotect the synthesized oligonucleotide.
Suitable protecting groups are well-known in the art and include,
by way of example and not limitation, acyl groups carbonates and
carbamates. Fluorescein dyes including protecting groups at the 3'-
and 6'-exocyclic oxygen atoms are referred to herein as
"O-protected fluoresceins." In a specific, non-limiting embodiment,
the protecting groups on the O-protected fluorescein are acyl
groups.
[0030] The N-protected NH-rhodamine or O-protected fluorescein
donor dye and the N-protected NH-rhodamine acceptor dye can be
linked to one another in a variety of orientations, either directly
or with the aid of a linker. In some embodiments, the donor is
linked to the 2'-, 2''-, 4'-, 5'-, 7'-, 7''-, 5- or 6-position of
the N-protected NH-rhodamine acceptor via its 2'-, 2''-, 4'-, 5'-,
7'-, 7''-, 5- or 6-position, optionally with the aid of a linker.
In some embodiments, the donor and acceptor are linked to one
another in a head-to-head, head-to-tail, tail-to-tail or
side-to-side orientation, as will be described in more detail in a
later section.
[0031] The optional linker linking the dyes may comprise virtually
any combination of atoms and/or functional groups stable to
oligonucleotide synthesis conditions, such as the phosphite
triester chemistry conditions typically used for the solid phase
synthesis of oligonucleotides. Combinations of atoms and/or
functional groups can be selected to tailor the properties and/or
length of the linker as desired. A variety of linkers useful for
linking fluorescein and rhodamine dyes to one another in the
context of energy transfer dye networks are known in the art (see,
e.g., US 2006/057565, U.S. Pat. No. 7,015,000, U.S. Pat. No.
6,627,748, U.S. Pat. No. 6,544,744, U.S. Pat. No. 6,177,247, U.S.
Pat. No. 6,150,107, U.S. Pat. No. 6,028,190, U.S. Pat. No.
5,958,180, U.S. Pat. No. 5,869,255, U.S. Pat. No. 5,853,992, U.S.
Pat. No. 5,814,454, U.S. Pat. No. 5,804,386, U.S. Pat. No.
5,728,528, U.S. Pat. No. 5,707,804 and U.S. Pat. No. 5,688,648, the
disclosures of which are incorporated herein by reference.) All of
these linkers can be used to link O-protected fluoresceins to
N-protected NH-rhodamines in the reagents described herein. In some
embodiments, the linker is rigid in nature and is about 8 to 16
.ANG. in length.
[0032] The label moiety, phosphate ester precursor group and
optional synthesis handle of the synthesis reagents, and the label
moiety, solid support and synthesis handle of the solid support
reagents can be linked to one another in any fashion or orientation
that does not interfere with the abilities of the various groups
and moieties to carry out their respective functions. The label
moiety is typically linked to one of the other moieties or groups
comprising the particular reagent via a linker linked to a
functional group on one of the dyes comprising the label moiety,
or, alternatively, to a linker linking two or more dyes of a dye
network. In embodiments in which the label moiety includes only a
single N-protected NH-rhodamine fluorophore, the label moiety can
be linked to the reagent via any position of the NH-rhodamine ring
that does not interfere with the ability of the NH-rhodamine ring
to exist in the closed, spiro lactone form. Suitable positions
include, but are not limited to, carbon atoms that are adjacent to
an exocylic nitrogen atom, or atoms on the phenyl moiety, such as,
for example, the 2'-, 2''-, 4'-, 5'-, 7'-, 7''-, 5- or 6-position
of the rhodamine dye. In some embodiments, the label moiety is
linked to a group or moiety of the reagent via the 5- or 6-position
of the N-protected NH-rhodamine dye.
[0033] In embodiments in which the label moiety comprises a dye
network, the label moiety may be linked to any group or moiety of
the reagent at any position on any one of the dyes comprising the
network that does not interfere with the desired function of the
reagent, or to a linker linking two or more dyes of the network. In
some specific embodiments in which the label moiety comprises an
O-protected fluorescein or N-protected NH-rhodamine donor in
addition to the N-protected NH-rhodamine acceptor, the label moiety
can be linked to a group or moiety of the reagent at any available
position on the donor or acceptor, such as, for example, the 2'-,
2''-, 4'-, 5 7'-, 7''-, 5- or 6-position of the donor or acceptor.
In some embodiments, the label moiety is linked to a group or
moiety of the regent via the 5-or 6-position of the donor or
acceptor.
[0034] In some embodiments, the label moiety is linked to a group
or moiety of the reagent via the linker linking the donor and
N-protected NH-rhodamine acceptor.
[0035] In other aspects, the disclosure provides intermediate
molecules useful for synthesizing the reagents described herein,
methods of making the reagents described herein, compounds labeled
with the reagents described herein, such as, for example, labled
oligo or polynucleotides, and methods of using the labeled
compounds in a variety of contexts. All of the various aspects of
the disclosure are described in more detail below.
4. BRIEF DESCRIPTION OF THE FIGURES
[0036] FIGS. 1A and 1B provide exemplary embodiments of parent
NH-rhodamine dyes that can be incorporated into the reagents
described herein;
[0037] FIG. 1C provides exemplary embodiments of parent fluorescein
dyes that can act as donors for NH-rhodamines, and that can be
incorporated into the reagents described herein in embodiments in
which the label moiety comprises a dye network;
[0038] FIG. 2 provides exemplary linkers that can be used to link
the various different moieties comprising the reagents described
herein to one another;
[0039] FIG. 3 provides exemplary embodiments of non-nucleosidic
synthesis reagents that do not include synthesis handles;
[0040] FIG. 4 provides exemplary embodiments of nucleosidic
synthesis reagents that do not include synthesis handles;
[0041] FIG. 5 provides exemplary embodiments of non-nucleosidic
synthesis reagents that include a synthesis handle;
[0042] FIG. 6 provides exemplary embodiments of nucleosidic
synthesis reagents that include synthesis handles;
[0043] FIG. 7 provides exemplary embodiments of non-nucleosidic
solid support reagents;
[0044] FIG. 8 provides exemplary embodiments of nucleosidic solid
support reagents;
[0045] FIG. 9 illustrates the use of a specific embodiment of a
synthesis reagent to synthesize an oligonucleotide labled at its
5'-hydroxyl with an NH-rhodamine dye;
[0046] FIG. 10 illustrates the use of a specific embodiment of a
synthesis reagent to synthesize an oligonucleotide labeled at its
3-hydroxyl with an energy-transfer dye;
[0047] FIG. 11A illustrates the use of a specific embodiment of a
synthesis reagent to synthesize in situ an oligonucleotide labeled
at its 5'-hydroxyl with an energy-transfer dye; and
[0048] FIG. 11B illustrates the use of a linker phosphoramidite and
a specific embodiment of a synthesis reagent to synthesize in situ
an oligonucleotide labeled at its 5'-terminus with an energy
transfer dye.
5. DETAILED DESCRIPTION
[0049] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only, and are not intended to be restrictive of the
compositions and methods described herein. In this disclosure, the
use of "or" means "and/or" unless stated otherwise. Similarly, the
expressions "comprise," "comprises," "comprising," "include,"
"includes" and "including" are not intended to be limiting.
5.1 Definitions
[0050] As used herein, the following terms and phrases are intended
to have the following meanings:
[0051] Alkyl," by itself or as part of another substituent, refers
to a saturated or unsaturated branched, straight-chain or cyclic,
monovalent hydrocarbon radical having the stated number of carbon
atoms (i.e., C1-C6 means one to six carbon atoms) that is derived
by the removal of one hydrogen atom from a single carbon atom of a
parent alkane, alkene or alkyne. Typical alkyl groups include, but
are not limited to, methyl; ethyls such as ethanyl, ethenyl,
ethynyl; propyls such as propan-1-yl, propan-2-yl,
cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl,
cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,
prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,
2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,
but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,
but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,
buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,
cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl,
but-3-yn-1-yl, etc.; and the like. Where specific levels of
saturation are intended, the nomenclature "alkanyl," "alkenyl"
and/or "alkynyl" is used, as defined below. As used herein, "lower
alkyl" means (C1-C8) alkyl.
[0052] "Alkanyl," by itself or as part of another substituent,
refers to a saturated branched, straight-chain or cyclic alkyl
derived by the removal of one hydrogen atom from a single carbon
atom of a parent alkane. Typical alkanyl groups include, but are
not limited to, methanyl; ethanyl; propanyls such as propan-1-yl,
propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as
butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl
(isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.;
and the like. As used herein, "lower alkanyl" means (C1-C8)
alkanyl.
[0053] "Alkenyl," by itself or as part of another substituent
refers, to an unsaturated branched, straight-chain or cyclic alkyl
having at least one carbon-carbon double bond derived by the
removal of one hydrogen atom from a single carbon atom of a parent
alkene. The group may be in either the cis or trans conformation
about the double bond(s). Typical alkenyl groups include, but are
not limited to, ethenyl; propenyls such as prop-1-en-1-yl,
prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,
cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such as
but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,
but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,
buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,
cyclobuta-1,3-dien-1-yl, etc.; and the like. As used herein, "lower
alkenyl" means (C2-C8) alkenyl.
[0054] "Alkynyl," by itself or as part of another substituent,
refers to an unsaturated branched, straight-chain or cyclic alkyl
having at least one carbon-carbon triple bond derived by the
removal of one hydrogen atom from a single carbon atom of a parent
alkyne. Typical alkynyl groups include, but are not limited to,
ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.;
butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.;
and the like. As used herein, "lower alkynyl" means (C2-C8)
alkynyl.
[0055] "Alkyldiyl," by itself or as part of another substituent,
refers to a saturated or unsaturated, branched, straight-chain or
cyclic divalent hydrocarbon group having the stated number of
carbon atoms (i.e., C1-C6 means from one to six carbon atoms)
derived by the removal of one hydrogen atom from each of two
different carbon atoms of a parent alkane, alkene or alkyne, or by
the removal of two hydrogen atoms from a single carbon atom of a
parent alkane, alkene or alkyne. The two monovalent radical centers
or each valency of the divalent radical center can form bonds with
the same or different atoms. Typical alkyldiyl groups include, but
are not limited to, methandiyl; ethyldiyls such as ethan-1,1-diyl,
ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as
propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3
-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,
prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,
prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl,
cycloprop-2-en-1,2-diyl, cycloprop-2-en-1,1-diyl,
prop-1-yn-1,3-diyl, etc.; butyldiyls such as, butan-1,1-diyl,
butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl, butan-2,2-diyl,
2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,
cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,
but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,
but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,
2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,
buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl,
buta-1,3-dien-1,4-diyl, cyclobut-1-en-1,2-diyl,
cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,
cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,
but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.;
and the like. Where specific levels of saturation are intended, the
nomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used.
Where it is specifically intended that the two valencies are on the
same carbon atom, the nomenclature "alkylidene" is used. In some
embodiments, the alkyldiyl group is (C1-C8) alkyldiyl. Specific
embodiments include saturated acyclic alkanyldiyl groups in which
the radical centers are at the terminal carbons, e.g., methandiyl
(methano); ethan-1,2-diyl (ethano); propan-1,3-diyl (propano);
butan-1,4-diyl (butano); and the like (also referred to as
alkylenos, defined infra). As used herein, "lower alkyldiyl" means
(C1-C8) alkyldiyl.
[0056] "Alkylene," by itself or as part of another substituent,
refers to a straight-chain saturated or unsaturated alkyldiyl group
having two terminal monovalent radical centers derived by the
removal of one hydrogen atom from each of two terminal carbon atoms
of straight-chain or branched parent alkane, alkene or alkyne, or
by the removal of one hydrogen atom from each of two different ring
atoms of a parent cycloalkyl. The locant of a double bond or triple
bond, if present, in a particular alkylene is indicated in square
brackets. Typical alkylene groups include, but are not limited to,
methylene (methano); ethylenes such as ethano, etheno, ethyno;
propylenes such as propano, prop[1]eno, propa[1,2]dieno,
prop[1]yno, etc.; butylenes such as butano, but[1] eno, but[2]eno,
buta[1,3]dieno, but[1]yno, but[2]yno, buta[1,3]diyno, etc.; and the
like. Where specific levels of saturation are intended, the
nomenclature alkano, alkeno and/or alkyno is used. In some
embodiments, the alkylene group is (C1-C8) or (C1-C3) alkylene.
Specific embodiments include straight-chain saturated alkano
groups, e.g., methano, ethano, propano, butano, and the like. As
used herein, "lower alkylene" means (C1-C8) alkylene.
[0057] "Heteroalkyl," Heteroalkanyl," Heteroalkenyl,"
Heteroalkynyl," Heteroalkyldiyl" and "Heteroalkylene," by
themselves or as part of another substituent, refer to alkyl,
alkanyl, alkenyl, alkynyl, alkyldiyl and alkylene groups,
respectively, in which one or more of the carbon atoms are each
independently replaced with the same or different heteroatoms or
heteroatomic groups. Typical heteroatoms and/or heteroatomic groups
which can replace the carbon atoms include, but are not limited to,
--O--, --S--, --S--O--, --NR'--, --PH--, --S(O)--, --SO.sub.2--,
--S(O) NR'--, --SO.sub.2NR'--, and the like, including combinations
thereof, where R' is hydrogen or a substitutents, such as, for
example, (C1-C8) alkyl, (C6-C14) aryl or (C7-C20) arylalkyl.
[0058] "Cycloalkyl" and "Heterocycloalkyl," by themselves or as
part of another substituent, refer to cyclic versions of "alkyl"
and "heteroalkyl" groups, respectively. For heteroalkyl groups, a
heteroatom can occupy the position that is attached to the
remainder of the molecule. Typical cycloalkyl groups include, but
are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyl
and cyclobutenyl; cyclopentyls such as cyclopentanyl and
cyclopentenyl; cyclohexyls such as cyclohexanyl and cyclohexenyl;
and the like. Typical heterocycloalkyl groups include, but are not
limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,
piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,
morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,
piperazin-2-yl, etc.), and the like.
[0059] "Parent Aromatic Ring System" refers to an unsaturated
cyclic or polycyclic ring system having a conjugated 7C electron
system. Specifically included within the definition of "parent
aromatic ring system" are fused ring systems in which one or more
of the rings are aromatic and one or more of the rings are
saturated or unsaturated, such as, for example, fluorene, indane,
indene, phenalene, tetrahydronaphthalene, etc. Typical parent
aromatic ring systems include, but are not limited to,
aceanthrylene, acenaphthylene, acephenanthrylene, anthracene,
azulene, benzene, chrysene, coronene, fluoranthene, fluorene,
hexacene, hexaphene, hexalene, indacene, s-indacene, indane,
indene, naphthalene, octacene, octaphene, octalene, ovalene,
pentacene, pentalene, pentaphene, perylene, phenalene,
phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,
tetrahydronaphthalene, triphenylene, trinaphthalene, and the
like.
[0060] "Aryl," by itself or as part of another substituent, refers
to a monovalent aromatic hydrocarbon group having the stated number
of carbon atoms (i.e., C6-C14 means from 6 to 14 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, groups derived from aceanthrylene,
acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,
chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,
hexalene, as-indacene, s-indacene, indane, indene, naphthalene,
octacene, octaphene, octalene, ovalene, pentacene, pentalene,
pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene,
pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and
the like, as well as the various hydro isomers thereof. Specific
exemplary aryls include phenyl and naphthyl.
[0061] "Arylalkyl," by itself or as part of another substituent,
refers to an acyclic alkyl group in which one of the hydrogen atoms
bonded to a carbon atom, in some embodiments a terminal or sp.sup.3
carbon atom, is replaced with an aryl group. Typical arylalkyl
groups include, but are not limited to, benzyl, 2-phenylethan-1-yl,
2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,
2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and
the like. Where alkyl moieties having a specified degree of
saturation are intended, the nomenclature arylalkanyl, arylalkenyl
and/or arylalkynyl is used. When a defined number of carbon atoms
are stated, for example, (C7-C20) arylalkyl, the number refers to
the total number of carbon atoms comprising the arylalkyl
group.
[0062] "Parent Heteroaromatic Ring System" refers to a parent
aromatic ring system in which one or more carbon atoms are each
independently replaced with the same or different heteroatoms or
heteroatomic groups. Typical heteroatoms or heteroatomic groups to
replace the carbon atoms include, but are not limited to, N, NH, P,
O, S, S(O), SO.sub.2, Si, etc. Specifically included within the
definition of "parent heteroaromatic ring systems" are fused ring
systems in which one or more of the rings are aromatic and one or
more of the rings are saturated or unsaturated, such as, for
example, benzodioxan, benzofuran, chromane, chromene, indole,
indoline, xanthene, etc. Also included in the definition of "parent
heteroaromatic ring system" are those recognized rings that include
common substituents, such as, for example, benzopyrone and
1-methyl-1,2,3,4-tetrazole. Typical parent heteroaromatic ring
systems include, but are not limited to, acridine, benzimidazole,
benzisoxazole, benzodioxan, benzodioxole, benzofuran, benzopyrone,
benzothiadiazole, benzothiazole, benzotriazole, benzoxaxine,
benzoxazole, benzoxazoline, carbazole, .beta.-carboline, chromane,
chromene, cinnoline, furan, imidazole, indazole, indole, indoline,
indolizine, isobenzofuran, isochromene, isoindole, isoindoline,
isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,
oxazole, perimidine, phenanthridine, phenanthroline, phenazine,
phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,
pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine,
quinazoline, quinoline, quinolizine, quinoxaline, tetrazole,
thiadiazole, thiazole, thiophene, triazole, xanthene, and the
like.
[0063] "Heteroaryl," by itself or as part of another substituent,
refers to a monovalent heteroaromatic group having the stated
number of ring atoms (e.g., "5-14 membered" means from 5 to 14 ring
atoms) derived by the removal of one hydrogen atom from a single
atom of a parent heteroaromatic ring system. Typical heteroaryl
groups include, but are not limited to, groups derived from
acridine, benzimidazole, benzisoxazole, benzodioxan, benzodiaxole,
benzofuran, benzopyrone, benzothiadiazole, benzothiazole,
benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole,
.beta.-carboline, chromane, chromene, cinnoline, furan, imidazole,
indazole, indole, indoline, indolizine, isobenzofuran, isochromene,
isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,
naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,
phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,
pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,
pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,
tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,
and the like, as well as the various hydro isomers thereof.
[0064] "Heteroarylalkyl," by itself or as part of another
substituent, refers to an acyclic alkyl group in which one of the
hydrogen atoms bonded to a carbon atom, in some embodiments a
terminal or sp.sup.3 carbon atom, is replaced with a heteroaryl
group. Where alkyl moieties having a specified degree of saturation
are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl
and/or heteroarylalkynyl is used. When a defined number of atoms
are stated, for example, 6-20-membered hetoerarylalkyl, the number
refers to the total number of atoms comprising the arylalkyl
group.
[0065] "Haloalkyl," by itself or as part of another substituent,
refers to an alkyl group in which one or more of the hydrogen atoms
is replaced with a halogen. Thus, the term "haloalkyl" is meant to
include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to
perhaloalkyls. For example, the expression "(C1-C2) haloalkyl"
includes fluoromethyl, difluoromethyl, trifluoromethyl,
1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl,
1,1,1-trifluoroethyl, perfluoroethyl, etc.
[0066] The above-defined groups may include prefixes and/or
suffixes that are commonly used in the art to create additional
well-recognized substituent groups. As non-limiting specific
examples, "alkyloxy" and/or "alkoxy" refer to a group of the
formula --OR'', "alkylamine" refers to a group of the formula
--NHR'' and "dialkylamine" refers to a group of the formula
--NR''R'', where each R'' is an alkyl.
5.2 Exemplary Embodiments
[0067] The present disclosure provides reagents that can be used to
chemically synthesize oligonucleotides bearing label moieties that
comprise rhodamine dyes. Traditionally, it has been difficult to
chemically synthesize rhodamine-labeled oligonucleotides owing, in
part, to the lack of availability of rhodamine-containing synthesis
reagents that are stable to the synthesis and/or deprotection
conditions commonly employed in the step-wise chemical synthesis of
oligonucleotides. It has now been discovered that protecting the
exocyclic amine groups of NH-rhodamine dyes with base-labile
protecting groups, such as acetyl groups, provides N-protected
NH-rhodamine dyes that are stable to the chemical synthesis and
deprotection conditions commonly employed in the solid-phase
synthesis of oligonucleotides. As a consequence, the N-protected
NH-rhodamines can be incorporated into reagents that can be used to
synthesize oligonucleotides labeled with label moieties that
comprise rhodamine dyes, thereby obviating the need to attach the
labels post-synthesis. Because the labels are attached during
synthesis, the resultant labeled oligonucleotide can be purified
for use without the use of HPLC.
[0068] The reagents take advantage of various features of reagents
and chemistries that are well-known for the step-wise solid phase
synthesis of oligonucleotides, and can be in the form of synthesis
reagents that are coupled to a hydroxyl group during the step-wise
solid phase synthesis of an oligonucleotide chain, or in the form
of solid support reagents to which nucleoside monomer reagents,
such as nucleoside phosphoramidite reagents, and/or optionally
other reagents, are coupled in a step-wise fashion to yield a
synthetic oligonucleotide.
[0069] The synthesis and solid support reagents can be nucleosidic
in nature in that they can include a nucleoside moiety, or they can
be non-nucleosidic in nature.
[0070] All of the reagents described herein include a label moiety
that comprises an N-protected NH-rhodamine dye or moiety. The
N-protected NH-rhodamine dye can be the only dye comprising the
label moiety or, alternatively, it can be one of two or more dyes
comprising a larger dye network. The solid support reagents
additionally include a solid support and one or more synthesis
handles to which additional groups can be coupled. The synthesis
reagents additionally include a phosphate ester precursor group
useful for coupling the reagent to a primary hydroxyl group, and
may optionally include one or more synthesis handles. The various
moieties and groups comprising the reagents can be linked together
in any fashion and/or orientation that permits them to carry out
their respective functions. They can be linked to one another
through linking groups included on the moieties, or they can be
linked to one another with the aid of linkers.
[0071] The various moieties, groups and linkers comprising the
reagents described herein are described in more detail below.
5.3 Linkers and Linking Groups
[0072] The various groups and moieties comprising the reagents
described herein are typically connected to one another with
linkers. The identity of any particular linker will depend, in
part, upon the identities of the moieties being linked to one
another. In general, the linkers include a spacing moiety that can
comprise virtually any combination of atoms or functional groups
stable to the synthetic conditions used for the synthesis of
labeled oligonucleotides, such as the conditions commonly used to
synthesize oligonucleotides by the phosphite triester method, and
can be linear, branched, or cyclic in structure, or can include
combinations of linear, branched and/or cyclic structures. The
spacing moiety can be monomeric in nature, or it can be or include
regions that are polymeric in nature. The spacing moiety can be
designed to have specified properties, such as the ability to be
cleaved under specified conditions, or specified degrees of
rigidity, flexibility, hydrophobicity and/or hydrophilicity.
[0073] As will be described in more detail below, many embodiments
of the reagents described herein are synthesized by condensing
synthons to one another in specified fashions to yield the desired
reagents. Each synthon typically includes one or more linking
groups suitable for forming the desired linkages. Generally, the
linking group comprises a functional group F.sup.y that is capable
of reacting with, or that is capable of being activated so as to be
able to react with, another functional group F.sup.z to yield a
covalent linkage Y--Z, where Y represents the portion of the
linkage contributed by F.sup.y and Z the portion contributed by
F.sup.z. Such groups F.sup.y and F.sup.z are referred to herein as
"complementary functional groups."
[0074] Pairs of complementary functional groups capable of forming
covalent linkages with one another are well-known in the art. In
some embodiments, one of F.sup.y or F.sup.z comprises a
nucleophilic group and the other one of F.sup.y or F.sup.z
comprises an electrophilic group. Complementary nucleophilic and
electrophilic groups useful for forming linkages (or precursors
thereof that are or that can be suitably activated so as to form
linkages) that are stable to a variety of synthesis and other
conditions are well-known in the art. Examples of suitable
complementary nucleophilic and electrophilic groups that can be
used to effect linkages in the various reagents described herein,
as well as the resultant linkages formed therefrom, are provided in
Table 1, below:
TABLE-US-00001 TABLE 1 Electrophilic Group Nucleophilic Group
Resultant Covalent Linkage activated esters* amines/anilines
carboxamides acyl azides** amines/anilines carboxamides acyl
halides amines/anilines carboxamides acyl halides alcohols/phenols
esters acyl nitriles alcohols/phenols esters acyl nitriles
amines/anilines carboxamides aldehydes amines/anilines imines
aldehydes or ketones hydrazines hydrazones aldehydes or ketones
hydroxylamines oximes Alkyl halides amines/anilines alkyl amines
Alkyl halides carboxylic acids esters Alkyl halides thiols
thioethers Alkyl halides alcohols/phenols ethers Alkyl sulfonates
thiols thioethers Alkyl sulfonates carboxylic acids esters Alkyl
sulfonates alcohols/phenols esters anhydrides alcohols/phenols
esters anhydrides amines/anilines caroboxamides aryl halides thiols
thiophenols aryl halides amines aryl amines aziridines thiols
thioethers boronates glycols boronate esters carboxylic acids
amines/anilines carboxamides carboxylic acids alcohols esters
carboxylic acids hydrazines hydrazides carbodiimides carboxylic
acids N-acylureas or anhydrides diazoalkanes carboxylic acids
esters epoxides thiols thioethers haloacetamides thiols thioethers
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers imido esters amines/anilines
amidines isocyanates amines/anilines ureas isocyanates
alcohols/phenols urethanes isothiocyanates amines/anilines
thioureas maleimides Thiols thioethers phosphoramidites Alcohols
phosphate esters silyl halides Alcohols silyl ethers sulfonate
esters amines/anilines alkyl amines sulfonate esters Thiols
thioethers sulfonate esters carboxylic acids esters sulfonate
esters Alcohols esters sulfonyl halides amines/anilines
sulfonamides sulfonyl halides phenols/alcohols sulfonate esters
Diazonium salt aryl azo *Activated esters, as understood in the
art, generally have the formula --C(O).OMEGA., where .OMEGA. is a
good leaving group (e.g., oxysuccinimidyl, oxysulfosuccinimidyl,
1-oxybenzotriazolyl, etc.). **Acyl azides can rearrange to
isocyanates.
[0075] Thus, linker synthons can generally be described by the
formula LG-Sp-LG, where each LG represents, independently of the
other, a linking group, and Sp represents the spacing moiety. In
some embodiments, linker synthons can be described by the formula
F.sup.z-Sp-F.sup.z, where each F.sup.z represents, independently of
the other, one member of a pair of complementary nucleophilic or
electrophilic functional groups as described above. In specific
embodiments, each F.sup.z is, independently of the other, selected
from the groups listed in Table 1, supra. Linker synthons of this
type form linker moieties of the formula --Z-Sp-Z--, where each Z
represents, independently of the other, a portion of a linkage as
described above.
[0076] Specific linkers suitable for linking specified groups and
moieties to one another in the reagents described herein will be
discussed in more detail in connection with exemplary embodiments
of the reagents. Non-limiting exemplary embodiments of linkers that
can be used to link the various groups and moieties comprising the
reagents described herein to one another are illustrated in FIG. 2.
In FIG. 2, Z.sup.1 and Z.sup.2 each represent, independently of one
another, a portion of a linkage contributed by a functional group
F.sup.z, as previously described, and K is selected from --CH-- and
--N--. In some specific embodiments of the linkers illustrated in
FIG. 2, one of Z.sup.1 or Z.sup.2 is --NH-- and the other is
selected from --O--, --C(O)-- and --S(O).sub.2--.
5.4 Label Moiety
[0077] All of the reagents described herein include a label moiety
that comprises an NH-rhodamine dye that is protected at the
exocyclic amine groups with a protecting group having specified
properties. Generally, rhodamine dyes are characterized by four
main features: (1) a parent xanthene ring; (2) an exocyclic amine
substituent; (3) an exocyclic imminium substituent; and (4) a
phenyl group substituted at the ortho position with a carboxyl
group. The exocyclic amine and/or imminium groups are typically
positioned at the C3 and C6 carbon atoms of the parent xanthene
ring, although "extended" rhodamines in which the parent xanthene
ring comprises a benzo group fused to the C3 and C4 carbons and/or
the C5 and C6 carbons are also known. In these extended rhodamines,
the characteristic exocyclic amine and imminium groups are
positioned at the corresponding positions of the extended xanthene
ring.
[0078] The carboxyl-substituted phenyl group is attached to the C9
carbon of the parent xanthene ring. As a consequence of the ortho
carboxyl substituent, rhodamine dyes can exist in two different
forms: (1) the open, acid form; and (2) the closed, lactone form.
While not intending to be bound by any theory of operation, because
NMR spectra of exemplary N-protected NH-rhodamine dyes described
herein are consistent with the closed spiro lactone form of the
dye, it is believed that the N-protected NH-rhodamine dyes
comprising the label moiety of the reagents described herein are in
the closed, spiro lactone form. Thus, the various rhodamines, as
well as their unprotected counterparts, are illustrated herein in
their closed, spiro lactone form. However, it is to be noted that
this is for convenience only and is not intended to limit the
various reagents described herein to the lactone form of the
dyes.
[0079] In the closed, spiro lactone form, the A and C rings of the
parent xanthene ring are aromatic, and both the C3' and C6'
substituents are amines The exocyclic amine groups of the rhodamine
dyes included in the label moieties described herein are either
unsubstituted or mono-substituted such that these amine groups are
primary or secondary amines Such rhodamine dyes are referred to
herein as "NH-rhodamines." Thus, as used herein, an "NH-rhodamine"
generally comprises one of the following parent NH-rhodamine ring
structures:
##STR00001##
[0080] In the parent NH-rhodamine rings depicted above, the various
carbon atoms are numbered using an arbitrary numbering convention
adopted from a numbering convention commonly used for the closed,
spiro lactone form of rhodamine dyes. This numbering system is
being used for convenience only, and is not intended to be limiting
in any way.
[0081] In the parent NH-rhodamines rings of structural formula
(Ia), (Ib) and (Ic), R.sup.3' and R.sup.6' represent hydrogen or
substituent groups substituting the exocyclic amines The R.sup.3'
and/or R.sup.6' substituents can be the same or different, and can
comprise groups such as substituted or unsubstituted alkyl, aryl or
arylalkyl groups. Alternatively, the R.sup.3' and/or R.sup.6'
groups can comprise substituents that are bridged to an adjacent
carbon atom such that the illustrated nitrogen atom is included in
a ring that contains 5- or 6-ring atoms. The ring may be saturated
or unsaturated, and one or more of the ring atoms can be
substituted. When the ring atom(s) are substituted, the
substituents are typically, independently of one another, selected
from lower alkyl, C6-C10 aryl and C7-C16 arylalkyl groups.
Alternatively, two adjacent ring atoms may be included in an aryl
bridge, such as a benzo or naphtho group. Non-limiting exemplary
embodiments of rhodamine dyes that include a parent NH-rhodamine
ring according to structural formula (Ia) in which the R.sup.3'
and/or R.sup.6' groups are hydrogen or lower alkyl groups or are
included in optionally substituted rings with adjacent carbon atoms
are illustrated in FIG. 1A. Non-limiting exemplary embodiments of
rhodamine dyes that include a parent NH-rhodamine ring according to
structural formula (Ic) in which the R.sup.3' and R.sup.6' groups
are hydrogen or lower alkyl groups or are included in optionally
substituted rings with adjacent carbon atoms are illustrated in
FIG. 1B.
[0082] One or more of the carbon atoms at positions C1', C2', C2'',
C4', C4'', C5', C5'', C7', C7'' and C8' of the parent NH-rhodamine
rings according to structural formulae (Ia), (Ib) and (Ic) can be,
independently of one another, substituted with substituent groups.
Groups useful for substituting rhodamine dyes at these positions
are well known in the art, and are described, for example, in U.S.
Pat. No. 4,622, 400, U.S. Pat. No. 5,750,409, U.S. Pat. No.
5,847,162, U.S. Pat. No. 6,017,712, U.S. Pat. No. 6,080,852, U.S.
Pat. No. 6,184,379 and U.S. Pat. No. 6,248,884, the disclosures of
which are incorporated herein by reference. All of these
substituent groups can be used to substitute the parent
NH-rhodamine rings described herein.
[0083] In some embodiments, the substituent groups are,
independently of one another, selected from lower alkyl, (C6-C14)
aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered
heteroarylalkyl, --R.sup.b and --(CH.sub.2).sub.x--R.sup.b, where x
is an integer ranging from 1 to 10 and R.sup.b is selected from
--X, --OH, --OR.sup.a, --SH, --SR.sup.a, --NH.sub.2, --NHR.sup.a,
--NR.sup.cR.sup.c, --N.sup.+R.sup.cR.sup.cR.sup.c, perhalo lower
alkyl, trihalomethyl, trifluoromethyl, --B(OH).sub.3,
--B(OR.sup.a).sub.3, --B(OH)O.sup.-, --B(OR.sup.a).sub.2O.sup.-,
--B(OH)(O.sup.-).sub.2, --B(OR.sup.a)(O.sup.-).sub.2,
--P(OH).sub.2, --P(OH)O.sup.-, --P(OR.sup.a).sub.2,
--P(OR.sup.a)O.sup.-, --P(O)(OH).sub.2, --P(O)(OH)O.sup.-,
--P(O)(O.sup.-).sub.2, --P(O)(OR.sup.a).sub.2,
--P(O)(OR.sup.a)O.sup.-, --P(O)(OH)(OR.sup.a), --OP(OH).sub.2,
--OP(OH)O.sup.-, --OP(OR.sup.a).sub.2, --OP(OR.sup.a)O.sup.-,
--OP(O)(OH).sub.2, --OP(O)(OH)O.sup.--, --OP(O)(O.sup.-).sub.2,
--OP(O)(OR.sup.a).sub.2, --OP(O)(OR.sup.a)O.sup.-,
--OP(O)(OR.sup.a)(OH), --S(O).sub.2O.sup.-, --S(O).sub.2OH,
--S(O).sub.2R.sup.a, --C(O)H, --C(O)R.sup.a, --C(S)X,
--C(O)O.sup.-, --C(O)OH, --C(O)NH.sub.2, --C(O)NHR.sup.a,
--C(O)NR.sup.cR.sup.c, --C(S)NH.sub.2, --C(O)NHR.sup.a,
--C(O)NR.sup.cR.sup.c, --C(NH)NH.sub.2, --C(NH)NHR.sup.a, and
--C(NH)NR.sup.cR.sup.c, where X is a halo (preferably fluoro or
chloro), each R.sup.a is, independently of the others, selected
from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered
heteroaryl and 6-20 membered heteroarylalkyl, and each R.sup.c is,
independently of the others, an R.sup.a, or, alternatively, two
R.sup.c bonded to the same nitrogen atom may be taken together with
that nitrogen atom to form a 5- to 8-membered saturated or
unsaturated ring that may optionally include one or more of the
same or different ring heteroatoms, which are typically selected
from O, N and S.
[0084] Alternatively, the C1' and C2' substituents, the C7' and C8'
substituents, the C5' and C5'' substituents and/or the C4' and C4''
substituents can be taken together to form substituted or
unsubstituted aryl bridges, such as benzo bridges, with the proviso
that the C1' and C2' substituents, and C7' and C8' substituents are
not simultaneously included in an aryl bridge.
[0085] In general, the groups used to substitute the C1', C2',
C2'', C4', C4'', C5', C5'', C7', C7'' and C8' carbons should not
promote quenching of the rhodamine dye, although in some
embodiments quenching substituents may be desirable. Substituents
that tend to quench rhodamine dyes include carbonyl, carboxylate,
heavy metals, nitro, bromo and iodo. Phenyl groups positioned at
R.sup.3' and/or R.sup.6' also tend to cause quenching.
[0086] The carbon atoms at positions C4, C5, C6 and C7 of the
parent NH-rhodamine rings of structural formulae (Ia), (Ib) and
(Ic) can also, independently of one another, include optional
substituents. These substituents can be selected from the various
substituents described above. In some embodiments, the carbon atoms
at positions C4 and C7 are substituted with chloro groups such that
the parent NH-rhodamine dye is an NH-4,7-dichlororhodamine dye.
[0087] A vast number of rhodamine dyes that include parent
NH-rhodamine rings according to structural formulae (Ia), (Ib) and
(Ic) that can be included in the label moiety of the reagents
described herein are known in the art, and are described, for
example, in U.S. Pat. No. 6,248,884; U.S. Pat. No. 6,111,116; U.S.
Pat. No. 6,080,852; U.S. Pat. No. 6,051,719; U.S. Pat. No.
6,025,505; U.S. Pat. No. 6,017,712; U.S. Pat. No. 5,936,087; U.S.
Pat. No. 5,847,162; U.S. Pat. No. 5,840,999; U.S. Pat. No.
5,750,409; U.S. Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S.
Pat. No. 5,227,487; WO 97/36960; WO 99/27020; Lee et al., 1992,
Nucl. Acids Res. 20:2471-2483; Arden-Jacob, "Neue Lanwellige
Xanthen-Farbstoffe fur Fluoreszenzsonden and Farbstoff Lauer,
Springer-Verlag, Germany, 1993; Sauer et al., 1995, Fluorescence
5:247-261; Lee et al., 1997, Nucl. Acids Res. 25:2816-2822; and
Rosenblum et al., 1997, Nucl. Acids Res. 25:4500-4504, the
disclosures of which are incorporated herein by reference. Any of
the dyes described in these references in which the exocyclic
amines are primary or secondary amines as described herein, or
4,7-dichloro analogues of such NH-rhodamine dyes, can be included
in the label moiety of the reagents described herein.
[0088] When included in a label moiety, the exocyclic amines of the
parent NH-rhodamine ring are protected with protecting groups
having specified properties. Such protected NH-rhodamines are
referred to herein as "N-protected NH-rhodamines." The N-protected
NH-rhodamines that correspond to the parent NH-rhodamine rings of
structural formulae (Ia), (Ib) and (Ic) and are illustrated below
as structural formulae (IIa), (IIb) and (IIc):
##STR00002##
[0089] In structural formulae (IIa), (IIb) and (IIc), R' is
hydrogen or R.sup.3' and R'' is hydrogen or R.sup.6', where
R.sup.3' and R.sup.6' re as defined for structural formulae (Ia),
(Ib) and (Ic), supra, and R.sup.9 represents a protecting group.
The N-protected NH-rhodamines can include substituents at one or
more of positions C1', C2', C2'', C4', C4'', C5', C5'', C7', C7'',
C8', C4, C5, C6, and C7, as previously described in connection with
the parent NH-rhodamine rings according to structural formulae
(Ia), (Ib) and (Ic).
[0090] Since the reagents described herein will be used to
chemically synthesize labeled oligonucleotides, protecting groups
R.sup.9 that are stable to the organic synthesis conditions used to
synthesize oligonucleotides should be used. As mentioned above,
protecting groups R.sup.9 that protect the amine in the form of an
amide, for example, a carboxamide, a sufonamide or a phosphoramide,
should be selected, as protecting the exocyclic amines in this
matter is believed to "lock" the protected NH-rhodamine in the
closed, lactone, form, contributing to the stability of the
reagents described herein. Although not required, it is convenient
to utilize protecting groups R.sup.9 that are labile under the
conditions used to remove the groups protecting the exocyclic
amines of the nucleobases of the synthetic oligonucleotide, so that
all protecting groups can be removed in a single step.
[0091] The conditions used to synthesize and deprotect synthetic
oligonucleotides are well-known in the art, and are described, for
example, in Current Protocols in Nucleic Acid Chemistry, Vol. I,
Beancage et al., Eds., John Wiley & Sons, 2002, the disclosure
of which are incorporated herein by reference. Briefly, synthesis
methods that employ phosphoramidite reagents involve multiple
rounds of:(i) DMT deprotection to reveal a free hydroxyl, which can
be effected by treatment with 2.5% or 3% di- or tri-chloroacetic
acid in dichloromethane; (ii) coupling of nucleoside or other
phosphoramidite reagents to the free hydroxyl, which can be carried
out in acetonitrile containing 0.45 M or 0.5 M tetrazole; (iii)
oxidation, which can be carried out by treatment with
I.sub.2/2,6-lutidine/H.sub.2O; and capping, which can be carried
out by treatment with 6.5% acetic anhydride in tetrahydrofuran
(THF) followed by treatment with 10% 1-methylimidazole (NMI) in
THF.
[0092] Other conditions for carrying out the various steps in the
synthesis are also known and used. For example, phosphoramidite
coupling can be carried out in acetonitrile containing 0.25 M
5-ethylthio-1H-tetrazole, 0.25 M4 ,5-dicyanoimidazole (DCI) or 0.25
M 5-benzylthio-1H-tetrazole (BTT). Oxidation an be carried out in
0.1 M, 0.05 M or 0.02 M I.sub.2 in THF/H.sub.2O/pyridine (7:2:1).
Capping can be carried out by treatment with THF/lutidine/acetic
anhydride followed by treatment with 16% NMI in THF; by treatment
with 6.5% DMAP in THF followed by treatment with 10% Melm in THF;
or by treatment with 10% Melm in THF followed by treatment with 16%
Melm in THF.
[0093] Removal of any protecting groups and cleavage from the
synthesis reagent is typically effected by treatment with
concentrated ammonium hydroxide at 60.degree. C. for 1-12 hr,
although nucleoside phosphoramidite reagents protected with groups
that can be removed under milder conditions, such as by treatment
with concentrated ammonium hydroxide at room temperature for 4-17
hrs or treatment with 0.05 M potassium carbonate in methanol, or
treatment with 25% t-butylamine in H.sub.2O/EtOH, are also known
and used.
[0094] Skilled artisans will be readily able to select protecting
groups having properties suitable for use under specific synthesis
and deprotection and/or cleavage conditions. A wide variety of
amine protecting groups are taught, for example in, Greene &
Wuts, "Protective Groups In Organic Chemistry," 3d Edition, John
Wiley & Sons, 1999 (hereinafter "Green & Wuts") at for
example, pages 309-405. Skilled artisans can readily select
protecting groups R.sup.9 having suitable properties from amongst
those taught in Green & Wuts.
[0095] In some embodiments, the protecting groups R.sup.9 are acyl
groups of the formula --C(O)R.sup.10, where R.sup.10 is selected
from hydrogen, lower alkyl, methyl, --CX.sub.3, --CHX.sub.2,
--CH.sub.2X, --CH.sub.2--OR.sup.d and phenyl optionally
mono-substituted with a lower alkyl, methyl, --X, --OR.sup.d, cyano
or nitro group, where R.sup.d is selected from lower alkyl, phenyl
and pyridyl, and each X is a halo group, typically fluoro, or
chloro. In some embodiments, R.sup.10 is methyl. In some
embodiments, R.sup.10 is trifluoromethyl.
[0096] Acyl protecting groups such as those defined by
--C(O)R.sup.10 can be removed under a variety of basic conditions,
including the mild conditions used to remove protecting groups from
oligos synthesized with "base labile" phosphoramidite reagents, as
are well-known in the art. Exemplary conditions that can be used
are specified above.
[0097] As will be described in more detail in later sections, the
N-protected NH-rhodamine moiety comprising the label moiety may be
linked to other groups or moieties. For example, the N-protected
NH-rhodamine may be linked to another dye comprising the label
moiety, to a phosphate ester precursor group, to a linker, to a
synthesis handle, to a quenching moiety, to a moiety that functions
to stabilize base-pairing interactions (such as, for example an
intercalating dye or a minor-groove-binding molecule), or to other
moieties. Such linkages are typically effected via linking groups
LG (described above in connection with the linkers) attached to the
N-protected NH-rhodamine synthons used to synthesize the
reagents.
[0098] The linking group LG can be attached to any available carbon
atom of the N-protected NH-rhodamine synthon, or to a substituent
group attached to one of these carbon atoms. The positions of the
linking groups may depend, in part, on the group or moiety to which
the N-protected NH-rhodamine snython will be attached. In some
embodiments, the linking group is attached at the C2', C2'', C4',
C5', C7', C7'', C5, or C6 position of the N-protected NH-rhodamine
synthon. In a specific embodiment, the linking group is attached at
the C4', C5', C5 or C6 position.
[0099] The N-protected NH-rhodamine snython can include a single
linking group LG, or it can include more than one linking group LG.
In embodiments that employ more than one linking group, the linking
groups may be the same, or they may be different. N-protected
NH-rhodamine synthons that include multiple linking groups LG that
are different from one another can have different groups or
moieties attached to different positions of the parent NH-rhodamine
ring using orthogonal chemistries.
[0100] The identity of a linking group may, in some instances,
depend upon its location on the parent NH-rhodamine ring. In some
embodiments in which the linking group LG is attached at the C4'-or
C5'-position of the parent NH-rhodamine ring, the linking group LG
is a group of the formula --(CH.sub.2).sub.n-F.sup.y, where n is an
integer ranging from 0 to 10 and F.sup.y is as described above. In
a specific embodiment, n is 1 and F.sup.y is --NH.sub.2.
[0101] In some embodiments in which the linking group LG is
attached at the 5- or 6-position of the parent NH-rhodamine ring,
the linking group LG is a group of the formula
--(CH.sub.2).sub.n--C(O)OR.sup.f, where R.sup.f is selected from
hydrogen and a good leaving group and n is as previously defined.
In some specific embodiments, the linking group LG comprises an NHS
ester. In some specific embodiments, n is 0 and R.sup.f is NHS.
[0102] In some embodiments, the N-protected NH-rhodamine comprising
the label moiety of the various reagents described herein is
described by structural formulae (IIIc), (IIIb) or (IIIc);
below:
##STR00003##
[0103] wherein: [0104] R' is selected from R.sup.3' and hydrogen;
[0105] R'' is selected from R.sup.6' and hydrogen; [0106] R.sup.9
is an acyl protecting group, optionally of the formula
--C(O)R.sup.10 , where R.sup.10 is as previously defined; [0107]
R.sup.1', R.sup.2', R.sup.2'', R.sup.4', R.sup.4'', R.sup.5',
R.sup.5'', R.sup.7', R.sup.7'' and R.sup.8', when taken alone, are
each, independently of one another, selected from hydrogen lower
alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl,
6-20 membered heteroarylalkyl, --R.sup.b and
--(CH.sub.2).sub.x--R.sup.b, where x and R.sup.b are as previously
defined, or, alternatively, R.sup.1' and R.sup.2' or R.sup.7'and
R.sup.8' are taken together with the carbon atoms to which they are
bonded to form a benzo group and/or R.sup.4' and R.sup.4'' and/or
R.sup.5' and R.sup.5'' are taken together with the carbon atoms to
which they are bonded to form a benzo group; [0108] R.sup.3' and
R.sup.6', when taken alone, are each, independently of one another,
selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14
membered heteroaryl and 6-20 membered heteroarylalkyl, or
alternatively, R.sup.3' and R.sup.2' or R.sup.4' and/or R.sup.6'
and R.sup.5' or R.sup.7 in the compounds of structural formula
(Ma), R.sup.3' and R.sup.2' or R.sup.4' and/or R.sup.6' and
R.sup.5' or R.sup.7'' in the compounds of structural formula
(IIIb), or R.sup.3' and R.sup.2'' or R.sup.4' and/or R.sup.6' and
R.sup.5' or R.sup.7'' in the compounds of structural formula (IIIc)
are taken together with the atoms to which they are bonded to form
a 5- or 6-membered saturated or unsaturated ring that may
optionally include from 1 to 4 additional heteroatoms (typically
selected from O, N and S) and that is optionally sutstituted with
one or more of the same or different lower alkyl, benzo or pyrido
groups; [0109] and R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are each,
independently of one another, selected from hydrogen, lower alkyl,
(C6-C14) aryl, (C7-C20) arylalkyl, 6-14 membered heteroaryl, 7-20
membered heteroarylalkyl, --R.sup.b and
--(CH.sub.2).sub.x--R.sup.b, where x and R.sup.b are as previously
defined,
[0110] with the proviso that at least one of R.sup.2', R.sup.4',
R.sup.5', R.sup.7', R.sup.5 or R.sup.6 in the compounds structural
formula (IIIa), at least one of R.sup.2', R.sup.4', R.sup.5',
R.sup.7', R.sup.5 or R.sup.6 in the compounds of structural formula
(IIIb) and at least one of R.sup.2'', R.sup.4', R.sup.5',
R.sup.7'', R.sup.5 or R.sup.6 in the compounds of structural
formula (IIIc) comprises a group of the formula --Y--, where Y
represents a portion of a linkage contributed by a linking group
comprising a functional group F.sup.y, as described above.
[0111] In some embodiments, the N-protected NH-rhodamine comprising
the label moiety of the various reagents described herein excludes
4,7-dichloro R6G (5- and/or 6-isomers) and/or rhodamines described
by structural formula (IIIa) in which: [0112] R' and R'' are each
ethyl; [0113] R.sup.1', R.sup.4', R.sup.5' and R.sup.6' are each
hydrogen; [0114] R.sup.2' and R.sup.7' are each methyl; [0115]
R.sup.4 and R.sup.7 are each chloro; and [0116] one of R.sup.5 or
R.sup.6 is hydrogen and the other is --C(O)--.
[0117] In some embodiments, the N-protected NH-rhodamine moieties
according to structural formulae (IIIa), (IIIb) and (IIIc), are,
respectively, selected from moieties defined by structural formulae
(IIIa.1), (IIIa.2), (IIIb.1), (IIIb.2), (IIIc.1) and (IIIc.2),
below:
##STR00004## ##STR00005##
[0118] wherein R', R'', R.sup.1', R.sup.2', R.sup.2'', R.sup.3',
R.sup.4', R.sup.4'', R.sup.5', R.sup.5'', R.sup.6', R.sup.7',
R.sup.7'', R.sup.8', R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9 and Y are as previously defined for structural formulae
(IIIa), (IIIb) and (IIIc).
[0119] Specific exemplary embodiments of moieties defined by
structural formulae (IIIa), (IIIa.1), (IIIa.2), (IIIb), (IIIb.1),
(IIIb.2), (IIIc), (IIIc.1) and (IIIc.2) include structures that
have one or more applicable features selected from:
[0120] (i) Y is selected from --C(O)--, --S(O).sub.2--, --S-- and
--NH--;
[0121] (ii) R.sup.4 and R.sup.7 are each chloro;
[0122] (iii) R.sup.1' and R.sup.8' are each hydrogen;
[0123] (iv) R.sup.1' and R.sup.2' or R.sup.7' and R.sup.8' are
taken together to form a benzo group;
[0124] (v) R.sup.2' and R.sup.7' are each hydrogen or lower
alkyl;
[0125] (vi) R' is R.sup.3' and R'' is R.sup.6';
[0126] (vii) R' is R.sup.3', R'' is R.sup.6', and R.sup.3' and
R.sup.6' are taken together with a substituent group on an adjacent
carbon atom to form a group selected from --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--.
--C(CH.sub.3).sub.2CH.dbd.C(CH.sub.3)--,
--C(CH.sub.3).sub.2CH.dbd.CH--, --CH.sub.2--C(CH.sub.3).sub.2--
and
##STR00006##
[0127] As discussed previously, the label moiety can comprise one
or more additional dyes such that the N-protected NH-rhodamine,
once deprotected, is a member of a larger, energy transfer dye
network. Such energy transfer dye networks are well-known in the
art, and include combinations of fluorescent dyes whose spectral
properties are matched, and/or whose relative distances to one
another are adjusted, so that one fluorescent dye in the network,
when excited by incident irradiation of an appropriate wavelength,
transfers its excitation energy to another fluorescent dyes in the
network, which then transfers its excitation energy to yet another
fluorescent dye in the network, and so forth, resulting in
fluorescence by the ultimate acceptor dye in the network. Dye
networks provide label moieties having long Stoke's shifts. In such
networks, fluorophores that transfer, or donate, their excitation
energy to another fluorphore in the network are referred to as
"donors." Fluorophores that receive, or accept, excitation energy
from another fluorophore are referred to as "acceptors." In dye
networks containing only two fluorescent dyes, one acts as the
donor and the other as the acceptor. In dye networks containing
three or more fluorescent dyes, at least one dye acts as both a
donor and acceptor. The principles of how dye networks work, as
well as the criteria for selecting and linking individual dyes
suitable for creating such networks are well known, and are
described, for example, in Hung et al., 1997, Anal. Biochem.
252:78-88.
[0128] In the label moieties described herein that comprise dye
networks, the N-protected NH-rhodamine dye, once deprotected, may
act as a donor or an acceptor, or as both a donor and acceptor,
depending upon the identities of the other dyes comprising the
network and the desired incident and fluorescent wavelengths. A
vast number of dyes suitable for use as donors and/or acceptors for
NH-rhodamine dyes are known in the art, and include by way of
example and not limitation, xanthene dyes (such as, for example,
fluorescein, rhodamine and rhodol dyes), pyrene dyes, coumarin dyes
(for example, hydroxy- and amino-coumarins), cyanine dyes,
phthalocyanine dyes and lanthenide complexes. Specific,
non-limiting examples of these dyes in the context of energy
transfer dye networks are described in Hung et al., 1996, Anal.
Biochem. 238:165-170; Medintz et al., 2004, Proc. Nat'l Acad. Sci.
USA 101(26):9612-9617; U.S. Pat. No. 5,800,996; Sudhaker et al.,
2003, Nucleosides, Nucleotides & Nucleic Acids 22:1443-1445;
U.S. Pat. No. 6,358,684; Majumdar et al., 2005, J. Mol. Biol.
351:1123-1145; Dietrich et al., 2002, Reviews Mol. Biotechnology
82(3):211-231; Tsuji et al., 2001, Biophysical J. 81(1):501-515;
Dickson et al., 1995, J. Photochemistry & Photobiology
27(1):3-19; and Kumar et al., 2004, Developments in Nucl. Acid Res.
1:251-274, the disclosures of which are incorporated herein by
references. Any of these dyes that can be suitably protected in
accordance with the principles desribed herein can be used as donor
and acceptor dyes in label moieties that comprise dye networks. In
some embodiments, one or more of the donor and/or acceptor dyes
comprising the network can be an N-protected NH-rhodamine dye as
described herein. Specific positions for attaching donor and/or
acceptor dyes to rhodamine dyes to form dye networks, as well as
specific linkages and linkers useful for attaching such dyes, are
well-known in the art. Specific examples are described, for
example, in U.S. Pat. No. 6,811,979; U.S. Pat. No. 6,008,379; U.S.
Pat. No. 5,945,526; U.S. Pat. No. 5,863,727; and U.S. Pat. No.
5,800,996, the disclosures of which are incorporated herein by
reference.
[0129] In some embodiments, the linker linking the donor and
acceptor dyes is an anionic linker as described in U.S. Pat. No.
6,811,979, the disclosure of which is incorporated herein by
reference (see, e.g., the disclosure at Col. 17, line 25 through
Col. 18, line 37 and FIGS. 1-17).
[0130] In some embodiments of the reagents described herein, the
label moiety includes a donor dye for the NH-rhodamine dye. In some
embodiments, the donor dye is a fluorescein or rhodamine dye, such
as, for example, one of the NH-rhodamine dyes described herein. In
a specific embodiment, the donor dye is a fluorescein dye.
Fluorescein dyes are similar in structure to rhodamine dyes, with
the exception that the 3- and 6-positions of the parent xanthene
ring (corresponding to the 3'- and 6'-positions of the NH-rhodamine
rings of structural formulae (Ia), (Ib) and (Ic)), are substituted
with a hydroxyl groups. Like the rhodamines, the fluoresceins can
also have extended ring structures in which the carbon atoms at
positions C3 and C4 and/or C5 and C6 of the parent xanthene ring
are included in aryl bridges such as benzo groups. Thus, the
fluoresceins generally include compounds according to structural
formulae (IVa), (IVb) and (IVc), below:
##STR00007##
[0131] Like the NH-rhodamines, the carbons at positions C1', C2',
C2'', C4', C4'', C5', C5'', C7', C7'', C8', C4, C5, C6 and C7 of
the fluorescein rings of structural formulae (IVa), (IVb) and (IVc)
can be substituted with a variety of different substituents, such
as those described previously for the NH-rhodamines.
[0132] When included in the label moieties described herein, the
hydroxyls at the C3' and C6' positions should be protected with
protecting groups having the same general properties as the groups
protecting the exocyclic amines of the NH-rhodamines, discussed
above. Thus, in specific embodiments the protecting groups are
stable to the conditions used to synthesize oligonucleotides, such
as the conditions used to synthesize and oxidize oligonucleotides
via the phosphite triester method, and are labile under the
conditions typically used to deprotect and/or cleave synthetic
oligonucleotides from the synthesis resin, such as, for example,
incubation in concentrated ammonium hydroxide at room temperature
or 55.degree. C.
[0133] A wide variety of protecting groups having suitable
properties are known in the art, and include by way of example and
not limitation, the acyl groups described above in connection with
N-protected NH-rhodamine dyes. In a specific embodiment, the
protecting group is of the formula --C(O)--R.sup.10, where R.sup.10
is as previously defined. In some embodiments, R.sup.10 is t-butyl.
Fluoresceins in which the C3' and C6' exocyclic hydroxyls include
protecting groups are referred to herein as "O-protected
fluoresceins." O-protected fluoresceins corresponding to the
fluoresceins of structural formulae (IVa), (IVb) and (IVc),
respectively, are illustrated as structural formulae (Va), (Vb) and
(Vc), below:
##STR00008##
[0134] wherein R.sup.9 represents the protecting group.
[0135] A vast variety of different fluorescein dyes that can be
suitably protected and incorporated into label moieties for use as
a donors for the NH-rhodamine moiety are known in the art. Specific
exemplary fluorescein dyes are described, for example, in U.S. Pat.
No. 6,221,604; U.S. Pat. No. 6,008,379; U.S. Pat. No. 5,840,999;
U.S. Pat. No. 5,750,409; U.S. Pat. No. 5,654,441; U.S. Pat. No.
5,188,934; U.S. Pat. No. 5,066,580; U.S. Pat. No. 4,481,136; U.S.
Pat. No. 4,439,356; WO 99/16832; and EP 0 050 684, the disclosures
of which are incorporated herein by reference. Skilled artisans
will be able to select a fluorescein having spectral properties
suitable for use as a donor for a specific NH-rhodamine. Specific
embodiments of parent fluoroescein dyes that may be incorporated in
the label moieties of the reagents described herein are illustrated
in FIG. 1C.
[0136] The donor and N-protected NH-rhodamine acceptor can be
linked to one another in a variety of orientations, either directly
or with the aid of a linker. In some embodiments in which the donor
is an O-protected fluorescein or an N-protected NH-rhodamine, the
donor is linked to the 2'-, 2''-, 4'-, 5'-, 7'-, 7''-, 5- or
6-position of the N-protected NH-rhodamine acceptor via its 2'-,
2''-, 4'-, 5'-, 7'-, 7''-, 5- or 6-position.
[0137] Specific exemplary linkage orientations are provided in
Table 2, below:
TABLE-US-00002 TABLE 2 donor/acceptor acceptor/donor nickname 4'-
or 5'- 4'- or 5'- head-to-head 4'- or 5'- 5- or 6- head-to-tail 5-
or 6' 5- or 6- tail-to-tail 2'-, 2''-, 7'- or 7''- 2'-, 2''-, 7'-
or 7''- side-to-side 2'-, 2''-, 7'- or 7''- 4'- or 5'- side-to-head
2'-, 2''-, 7'- or 7''- 5- or 6- side-to-tail
[0138] Label moieties comprising dye networks, such as the
donor-acceptor dye networks of Table 2, can be linked to the
remainder of the reagent at any available position. In some
embodiments, label moieties comprising head-to-head linked
acceptor/donor pairs are attached to the remainder of the reagent
via the 5- or 6-position of the donor or acceptor moiety. In some
embodiments, label moieties comprising head-to-tail linked
acceptor/donor pairs are attached to the remainder of the reagent
via an available 4'-, 5'-, 5- or 6-position of the donor or
acceptor moiety. In some embodiments, label moieties comprising
tail-to-tail linked acceptor/donor pairs are attached to the
remainder of the reagent via the 4'- or 5'-position of the donor or
acceptor. In some embodiments, label moieties comprising
side-to-side linked acceptor/donor pairs are attached to the
remainder of the reagent via the 4'-, 5'-, 5- or 6-position of the
donor or acceptor. In some embodiments, label moieties comprising
side-to-head linked acceptor/donor pairs are attached to the
remainder of the reagent via an available 4'-, 5'-, 5- or
6-position of the donor or acceptor. In some embodiments, label
moieties comprising side-to-tail linked acceptor/donor pairs are
attached to the remainder of the reagent via an available 4'-, 5'-,
5- or 6-position of the donor or acceptor.
[0139] Regardless of their orientation, the O-protected fluorescein
or N-protected NH-rhodamine donor and the N-protected NH-rhodamine
acceptor are typically linked to one another via a linker. It has
been discovered previously that it may be advantageous to link such
donor and acceptor dyes via linkers that are rigid in nature and/or
that are relatively long, for example, in the range of
approximately 12-20 .ANG. in length (as used herein, the "length"
of a linker refers to the distance between the linked moieties as
determined by calculating the sum of the lengths of the chemical
bonds defining the shortest continuous path between the moieties).
Without intending to be bound by any theory of operation, it is
believed that linkers that tend to hold the donor and acceptor in
close proximity to one another without permitting their
chromophores to touch one another yield suitably efficient energy
transfer. In this regard, the rigidity and length of the linker are
coupled parameters. Generally, shorter linkers (for example linkers
having a length of about 5 to 12 .ANG.) should include a greater
degree of rigidity. Longer linkers (for example linkers having a
length in the range of about 15 to 30 .ANG.) can include a lesser
degree of rigidity, or even no rigidity. Short, non-rigid (floppy)
linkers should be avoided.
[0140] Rigidity can be achieved through the use of groups that have
restricted angles of rotation about their bonds, for example,
through the use of arylene or heteroarylene moieties, and/or
alkylene moieties that comprise double and/or triple bonds. A
variety of linkers useful for linking rhodamine and fluorescein
dyes to one another in the context of energy transfer dyes are
known in the art, and are described, for example, in U.S. Pat. No.
5,800,996, the disclosure of which is incorporated herein by
reference. Specific examples of linkers useful for linking
O-protected fluorescein or N-protected NH-rhodamine donors to
N-protected NH-rhodamine acceptors in the label moieties described
herein include, by way of example and not limitation, groups of the
formula:
--Z--(CH.sub.2).sub.a--[(Ar).sub.b--(CH.sub.2).sub.a].sub.c--Z--;
(L.1)
--Z--(CH.sub.2).sub.a--[C.ident.C--(CH.sub.2).sub.a].sub.c--Z--;
(L.2)
--Z--(CH.sub.2).sub.a--[C.ident.C--(Ar).sub.b].sub.c--(CH.sub.2).sub.a---
Z--; (L.3)
--Z--(CH.sub.2).sub.d--NH--C(O)--[(CH.sub.2).sub.a--(Ar)--(CH.sub.2).sub-
.a--C(O)--NH].sub.c--(CH.sub.2).sub.d--Z--; and (L.4)
--Z--[CH.sub.2(CH.sub.2).sub.eO].sub.f--CH.sub.2(CH.sub.2).sub.eO--,
(L.5)
where each Z represents, independently of the others, a portion of
a linkage contributed by a linking group F.sup.z, as previously
described, each a represents, independently of the others, an
integer ranging from 0 to 4; each b represents, independently of
the others, an integer ranging from 1 to 2; each c represents,
independently of the others, an integer ranging from 1 to 5; each d
represents, independently of the others, an integer ranging from 1
to 10; each e represents, independently of the others, an integer
ranging from 1 to 4; each f represents, independently of the
others, an integer ranging from 1 to 10; and each Ar represents,
independently of the others, an optionally substituted monocyclic
or polycyclic cycloalkylene, cycloheteroalkynene, arylene or
heteroarylene group. Non-limiting exemplary embodiments of Ar
include groups derived from lower cycloalkanes, lower
cycloheteroalkanes, parent aromatic ring systems and parent
heteroaromatic ring systems, as described previously. Specific,
non-limiting exemplary embodiments of Ar include cyclohexane,
piperazine, benzene, napthalene, phenol, furan, pyridine,
piperidine, imidazole, pyrrolidine and oxadizole. Specific,
non-limiting exemplary embodiments of linkers are illustrated in
FIG. 2. In FIG. 2, Z.sup.1 and Z.sup.2 each represent,
independently of one another, a portion of a linkage contributed by
a functional group F.sup.z, as previously described, and K is
selected from --CH-- and --N--. In some specific embodiments of the
linkers illustrated in FIG. 2, one of Z.sup.1 or Z.sup.2 is --NH--
and the other is selected from --O--, --C(O)-- and
--S(O).sub.2--.
[0141] In some embodiments, the linker linking the donor and
acceptor dyes is an anionic linker as described in U.S. Pat. No.
6,811,979, the disclosure of which is incorporated herein by
reference (see, e.g., the disclosure at Col. 17, line 25 through
Col. 18, line 37 and FIGS. 1-17). Specific, non-limiting exemplary
embodiments of suitable anionic linkers include the linkers of
formulae (L.1) through (L.4), above, in which one or more of the Ar
groups are substituted with one or more substitent groups having a
negative charge under the conditions of use, such as, for example,
at a pH in the range of about pH 7 to about pH 9. Specific,
non-limiting examples of suitable substituent groups include
phosphate esters, sulfate esters, sulfonate and carboxylate
groups.
[0142] In some embodiements, the label moiety is of the formula
(VI):
A-Z.sup.1-Sp-Z.sup.2-D (VI)
[0143] where A represents the N-protected NH-rhodamine acceptor, D
represents the donor, for example, an N-protected NH-rhodamine or
O-protected fluorescein donor, Z.sup.1 and Z.sup.2 represent
portions of linkages provided by linking moieties comprising a
functional group F.sup.z, as previously described, and Sp
represents a spacing moiety, as previously described. In some
specific embodiments, A is selected from structural formulae A.1,
A.2, A.3, A.4, A.5 and A.6 and D is selected from structural
formulae D.1, D.2, D.3, D.4, D.5 and D.6, illustrated below. In
some specific embodiments, A is selected from structural formulae
A.7, A.8, A.9, A.10, A.11 and A.12 and D is selected from
structural formulae D.7, D.8, D.9, D.10, D.11 and D.12, illustrated
below.
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015## ##STR00016##
[0144] In structural formulae A.1-A.12 and D.1-D.12:
[0145] E.sup.1 is selected from --NHR.sup.9, --NR.sup.3'R.sup.9 and
--OR.sup.9b;
[0146] E.sup.2 is selected from --NHR.sup.9, --NR.sup.6'R.sup.9 and
--OR.sup.9b;
[0147] R.sup.9b is R.sup.9;
[0148] Y.sup.1a, Y.sup.1b, Y.sup.2a, Y.sup.2b, Y.sup.3a, and
Y.sup.3b are each, independently of one another, selected from
--O--, --S--, --NH--, --C(O--) and --S(O).sub.2--; and
[0149] R', R'', R.sup.1', R.sup.2', R.sup.2'', R.sup.3', R.sup.4',
R.sup.4'', R.sup.5', R.sup.5'', R.sup.6', R.sup.7', R.sup.7'',
R.sup.8', R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are as previously
defined for structural formulae (IIIa), (IIIb) and (IIIc), with the
proviso that when E.sup.1 and E.sup.2 are --OR.sup.9b, then
R.sup.1' and R.sup.2' and R.sup.7' and R.sup.8' may both include
benzo and/or pyrido groups simultanously.
[0150] In some specific embodiments of label moieties according to
structural formula (VI), Y.sup.1a, Y.sup.2a and Y.sup.3a are
--NH--; Y.sup.1b, Y.sup.2b and Y.sup.3b are selected from --C(O)--
and --S(O).sub.2--; Z.sup.1 is selected from --C(O)-- and
--S(O).sub.2--; Z.sup.2 is --NH-- and Sp is a group selected
from:
[0151] (Sp.1)
--(CH.sub.2).sub.a--[(Ar).sub.b--(CH.sub.2).sub.a].sub.c--;
[0152] (Sp.2)
--(CH.sub.2).sub.a--[C.ident.C--(CH.sub.2).sub.a].sub.c--;
[0153] (Sp.3)
--(CH.sub.2).sub.a--[C.ident.C--(Ar).sub.b].sub.c--(CH.sub.2).sub.a--;
[0154] (Sp.4)
--(CH.sub.2).sub.d--NH--C(O)--[(CH.sub.2).sub.a--(Ar)--(CH.sub.2).sub.a---
C(O)--NH].sub.c--(CH.sub.2).sub.d--; and
[0155] (Sp.5)
--[CH.sub.2(CH.sub.2).sub.eO].sub.f--CH.sub.2(CH.sub.2)--, where a,
b, c, d, e, f and Ar are as previously defined.
[0156] In some specific embodiments of label moieties according to
structural formula (VI), R.sup.9 is selected from --C(O)CH.sub.3
and C(O)CF.sub.3 and R.sup.9a is --C(O)C(CH.sub.3).sub.3.
5.5 Phosphate Ester Precursor Group
[0157] Many embodiments of the reagents described herein include a
phosphate ester precursor group ("PEP"). When used in a step-wise
synthesis to synthesize a labeled oligonucleotide, the PEP group is
coupled to any available hydroxyl group, which may be the
5'-hydroxyl group of a nascent synthetic oligonucleotide,
ultimately contributing, after any required oxidation and/or
deprotection steps, a linkage linking the label moiety to the
synthetic oligonucleotide. The linkage formed may be a phosphate
ester linkage or a modified phosphate ester linkage as is know in
the art.
[0158] A variety of different groups suitable for coupling reagents
to primary hydroxyl groups to yield phosphate ester or modified
phosphate ester linkages are well-known in the art. Specific
examples include, by way of example and not limitation,
phosphoramidite groups (see, e.g., Letsinger et al., 1969, J. Am.
Chem. Soc. 91:3350-3355; Letsinger et al., 1975 J. Am. Chem. Soc.
97:3278; Matteucci & Caruthers, 1981, J. Am. Chem. Soc.
103:3185; Beaucage & Caruthers, 1981, Tetrahedron Lett.
22:1859; the disclosures of which are incorporated herein by
reference), 2-chlorophenyl- or 2,5-dichlorophenyl-phosphate groups
(see, e.g., Sproat & Gait, "Solid Phase Synthesis of
Oligonucleotides by the Phosphotriester Method," In:
Oligonucleotide Synthesis, A Practical Approach, Gait, Ed., 1984,
IRL Press, pages 83-115), the disclosures of which are incorporated
herein by reference), and H-phosphonate groups (see, e.g., Garegg
et al., 1985, Chem. Scr. 25:280-282; Garegg et al., 1986, Tet.
Lett. 27:4051-4054; Garegg et al. 1986, Tet. Lett. 27:4055-4058;
Garegg et al., 1986, Chem. Scr. 26:59-62; Froehler & Matteucci,
1986, Tet. Lett. 27:469-472; Froehler et al., 1986, Nucl. Acid Res.
14:5399-5407, the disclosures of which are incorporated herein by
reference). In a specific embodiment, the PEP group is a
phosphoramidite group of the formula (P.1):
##STR00017##
[0159] wherein: [0160] R.sup.20 is selected from a linear, branched
or cyclic saturated or unsaturated alkyl containing from 1 to 10
carbon atoms, 2-cyanoethyl, an aryl containing from 6 to 10 ring
carbon atoms and an arylalkyl containing from 6 to 10 ring carbon
atoms and from 1 to 10 alkylene carbon atoms; and [0161] R.sup.21
and R.sup.22 are each, independently of one another, selected from
a linear, branched or cyclic, saturated or unsaturated alkyl
containing from 1 to 10 carbon atoms, an aryl containing from 6 to
10 ring carbon atoms and an arylalkyl containing from 6 to 10 ring
carbon atoms and from 1 to 10 aklylene carbon atoms, or,
alternatively, R.sup.21 and R.sup.22 are taken together with the
nitrogen atom to which they are bonded to form a saturated or
unsaturated ring that contains from 5 to 6 ring atoms, one or two
of which, in addition to the illustrated nitrogen atom, can be
heteroatom selected from O, N and S.
[0162] In a specific embodiment, R.sup.20 is 2-cyanoethyl and
R.sup.21 and R.sup.22 are each isopropyl.
5.6 Synthesis Handles
[0163] Many embodiments of the reagents described herein include
one or more synthesis handles that provide, after suitable
deprotection, if necessary, sites that can be used for the
attachment of additional groups or moieties to the synthetic
labeled oligonucleotide. The groups can be attached to a synthesis
handle during the course of synthesizing the labeled
oligonucleotide, or, alternatively, the synthesis handle can be
deprotected post-synthesis to reveal a functional group to which
additional groups or moieties can be attached. For example, a
synthesis handle could comprise a primary amine group that is
protected with a protecting group that is stable to the conditions
used to carry out the synthesis of the labeled oligonucleotide.
Removal of the protecting group following synthesis, either
concurrently with, or separately from, the removal of the various
other protecting groups on the synthetic oligonucleotide, provides
a primary amino group to which additional groups and/or moieties
can be attached.
[0164] A variety of different types of reactive groups protected
with protecting groups suitable for use in oligonucleotide
synthesis are known in the art, and include by way of example and
not limitation, amino groups (protected with, for example,
trifluroacetyl or 4-monomethoxytrityl groups), hydroxyl groups
(protected with, for example, 4,4'-dimethoxytrityl groups), thiol
groups (protected with, for example, trityl or alkylthiol groups)
and aldehyde groups (protected with, for example, an acetal
protecting group). All of these protected reactive groups can
comprise the synthesis handle of the reagents described herein.
[0165] In some embodiments, the synthesis handle comprises a
protected primary hydroxyl of the formula --OR.sup.e, where R.sup.e
represents an acid-labile protecting group that can be selectively
removed during the course of synthesizing an oligonucleotide. Acid
labile protecting groups suitable for protecting primary hydroxyl
groups in the context of oligonucleotide synthesis are known in the
art, and include, by way of example and not limitation,
triphenylmethyl (trityl), 4-monomethoxytrityl,
4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, bis
(p-anisyl)phenylmethyl, naphthyldiphenylmethyl,
p-(p'-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl and 9-(9-phenyl-10-oxo)anthryl. All of these
groups can be removed by treatment with mild acid, such as by
treatment with 2.5% or 3% di- or trichloro acid and in
dichoromethane. Methods of protecting primary hydroxyl groups with
the above-listed acid-labile protecting groups are well-known.
5.7 Solid Supports
[0166] Many embodiments of the reagents described herein comprise
solid supports to which the other moieties and/or groups are
attached. The solid supports are typically activated with
functional groups, such as amino or hydroxyl groups, to which
linkers bearing linking groups suitable for attachment of the other
moieties are attached.
[0167] A variety of materials that can be activated with functional
groups suitable for attachment to a variety of moieties and
linkers, as well as methods of activating the materials to include
the functional groups, are known in the art, and include by way of
example, controlled pore glass, polystyrene and graft co-polymers.
Any of these materials be used as solid supports in the reagents
described herein.
5.8 Synthesis Regents Useful for Terminal Hydroxyl Labeling
[0168] Some embodiments of the synthesis reagents described herein
are described by stuctural formula (VII):
LM-L-PEP (VII)
[0169] where LM represents a label moiety as described herein, L
represents an optional linker as described herein and PEP
represents a phosphate ester precursor group as described herein.
The reagents can include additional groups or moieties, such as
synthesis handles. In some embodiments, the synthesis reagents
comprise a label moiety and a PEP group, and do not include
additional moieties or groups. Such synthesis reagents can be
coupled to a hydroxyl group during the step-wise synthesis of an
oligonucleotide, and are useful for, among other things, attaching
a label moiety to a terminal hydroxyl group of a synthetic
oligonucleotide, which is commonly the 5'-hydroxyl.
[0170] The PEP group can be attached directly to the label moiety,
or it may be attached to the label moiety with the aid of a linker.
As PEP groups are generally linked to molecules by coupling
suitable reagents to primary hydroxyl groups, in embodiments in
which the PEP group is attached directly to the label moiety, the
label moitey should include a substituent group that comprises a
primary hydroxyl group. In embodiments in which the PEP group is
linked to the label moiety with the aid of a linker, the linker
synthon should include a linking group suitable for forming a
linkage with a linking group on the label moiety synthon and a
primary hydroxyl group suitable for attachment to the PEP group.
Suitable linker synthons include, but are not limited to, synthons
of the formula F.sup.z-Sp-OH, where F.sup.z is a functional group
complementary to a functional group on the label moiety synthon and
Sp represents a spacing moiety. The spacing moiety can comprise any
combination of atoms and/or functional groups stable to the
conditions that will be used to synthesize and deprotect the
labeled synthetic oligonucleotide. Non-limiting exemplary linkers
are illustrated in FIG. 2, where Z.sup.2 is O. In some embodiments,
Sp is an optionally substituted alkylene chain that contains from 1
to 10 chain atoms. In a specific embodiment, Sp is an unsubstitued
alkylene chain containing from 1 to 9 carbon chain atoms.
[0171] In some embodiments, the synthesis reagents are compounds
according to structural formula (VII) in which: [0172] LM is one of
the embodiments of label moieties specifically exemplified above;
[0173] L is selected from --Z--(CH.sub.2).sub.3-6--O--,
--Z--(CH.sub.2).sub.a--[(Ar).sub.b--(CH.sub.2).sub.a].sub.c--O--,
--Z--(CH.sub.2).sub.a--[C.ident.C--(CH.sub.2).sub.a].sub.c--O--,
--Z--(CH.sub.2).sub.a--[C.ident.C--(Ar).sub.b].sub.c--(CH.sub.2).sub.a--O-
--,
--Z--(CH.sub.2).sub.d--NH--C(O)--[(CH.sub.2).sub.a--(Ar)--(CH.sub.2).s-
ub.a--C(O)--NH].sub.c--(CH.sub.2).sub.d--O--,
--Z--[CH.sub.2(CH.sub.2).sub.eO].sub.f--CH.sub.2(CH.sub.2).sub.eO--
and one of the linkers illustrated in FIG. 2 in which Z.sup.2 is O;
and [0174] PEP is a phosphoramidite group, such as for example, a
phosphoramidite group of structural formula P.1, as described
above. In some specific embodiments, Z in linker L is --NH--.
[0175] In some embodiments, the linker in synthesis reagents
according to structural formula (VII) comprises a nucleoside, such
that the synthesis reagent is nucleosidic. In some embodiments,
nucleosidic synthesis reagents are compounds according to
structural formula (VII.1):
##STR00018##
[0176] where PEP represents the phosphate ester precursor group, B
represents a nucleobase, LM represents the label moiety and L.sup.2
represents a linker linking nucleobase B to linker LM. The features
and properties of nucleobase B and linker L are described in more
detail, below. Non-limiting exemplary nucleosidic synthesis
reagents according to structural formula (VII.1) are illustrated in
FIG. 4.
[0177] An exemplary scheme for synthesizing embodiments of
synthesis reagents in which the PEP group is linked to the label
moiety via an optional linker is provided in Scheme (I), below,
where the various R, F.sup.y, F.sup.z, Y, Z and S.sub.p groups are
as previously defined:
##STR00019##
[0178] In Scheme (I) parent NH-rhodamine synthon 100, which
includes a linking group that comprises functional group F.sup.y,
is acetylated with anhydride 101 to yield N-acetyl-protected
NH-rhodamine synthon 102. Synthon 102 is then coupled to linker
synthon 103 to yield compound 104. Depending upon the identity of
P, synthon 102 may require activation prior to coupling. For
example, if F.sup.y is a carboxyl group, it can be activated as an
ester, such as an NHS ester, prior to coupling. In compound 104,
--Y--Z-- represents the linkage formed by complementary functional
groups F.sup.y and F.sup.z, where Y represents the portion
contributed by F.sup.y and Z represents the portion contributed by
F.sup.z, as previously described. Compound 104 is then reacted with
PEP synthon 105, which in the specific embodiment illustrated is a
phosphine, to yield phosphoramidite synthesis reagent 106.
5.9 Synthesis Reagents Useful for Internal or 3'-End Labeling
[0179] The synthesis reagents described herein may optionally
include one or more synthesis handles useful for the attachment of
additional groups and/or moieties. Synthesis reagents that include
a synthesis handle of the formula --OR.sup.e, where R.sup.e
represents an acid-labile protecting group as previously described,
provide a primary hydroxyl group to which additional nucleotides
can be attached. As a consequence, synthesis reagents that include
such a synthesis handle can be used to label synthetic
oligonucleotides at the 5'-hydroxyl, the 3'-hydroxyl or at one or
more internal positions. They can also be coupled to one another,
or to other phosphoramidite labeling reagents, permitting the
synthesis of oligonucleotides containing a plurality of label
moieties.
[0180] The label moiety, PEP group and synthesis handle --OR.sup.e
comprising the synthesis reagent can be linked together in any
fashion and/or orientation that permits them to perform their
respective functions. As a specific example, the PEP group and
synthesis handle can each be linked to the label moiety, optionally
via linkers. In some embodiments, such synthesis reagents are
compounds according to structural formula (VIII):
R.sup.eO-L-LM-L-PEP (VIII)
[0181] where each L represents, independently of the other, an
optional linker, LM represents the label moiety and PEP represents
the phosphate ester precursor group. Non-limiting examples of
suitable protecting groups R.sup.e, linkers L, label moieties LM
and phosphate ester precursor groups include those specifically
exemplifed above.
[0182] As another specific example, the PEP group and synthesis
handle --OR.sup.e may be attached to a branched linker that is
attached to the label moiety. In some embodiments, such synthesis
reagents are compounds according to structural formula (IX):
##STR00020##
[0183] where L represent a linker, LM represents the label moiety
and PEP represents the phosphate ester precursor group.
[0184] In a specific embodiment, synthesis reagents according to
structural formula (IX) are compounds according to structural
formula (IX.1):
##STR00021##
[0185] where LM represents the label moiety, --Z-- represents a
portion of a linkage contributed by a functional F on the linker,
Sp.sup.1, Sp.sup.2 and Sp.sup.3, which can be the same or
different, each represent spacing moieties, G represents CH, N, or
a group comprising and arylene, phenylene, heteroarylene, lower
cycloalkylene, cyclohexylene, and/or lower cycloheteroalkylene, and
PEP represents the phosphate ester precursor group. In some
embodiments, LM is one of the embodiments of label moities
specifically exemplified above, Sp.sup.1, Sp.sup.2 and Sp.sup.3 are
each, independently of one another, selected from an alkylene chain
containing from 1 to 9 carbon atoms, Sp.1, Sp.2, Sp.3, Sp.4 and
Sp.5 (defined above), and/or PEP is a phosphoramidite group
according to structural formula P.1, supra. Non-limiting specific
embodiments of exemplary synthesis reagents according to structural
formula (IX.1) are illustrated in FIGS. 3 and 4.
[0186] In some embodiments, the synthesis handle --OR.sup.e is
provided by a nucleoside, such that the synthesis reagent is
nucleosidic. In such nucleosidic synthesis reagents, the label
moiety is typically linked to the nucleobase of the nucleoside by
way of a linker, and any exocyclic functional groups on the
nucleobase that are reactive under the conditions used to
synthesize the labeled oligonucleotide, such as, for example,
exocyclic amines, are protected.
[0187] The nucleoside can be any nucleoside that can be suitably
protected for use in the synthesis of oligonucleotides, and may
comprise a 2'-deoxyribose sugar moiety, a 3'-deoxyribose sugar
moiety (useful for synthesizing labeled oligonucleotides including
a 2'-5' internucleotide linkage), a suitably protected ribose
moiety, a substituted version of any of these ribose moieties, or
even a non-ribose sugar moiety.
[0188] In some embodiments, such nucleosidic synthesis reagents are
compounds according to structural formulae (IX.2), (IX.3), (IX.4)
and (IX.5):
##STR00022##
[0189] wherein LM represents the label moiety, B represents a
suitably protected nucleobase, L.sup.2 represents a linker linking
the label moiety to the nucleobase, R.sup.e represents the
acid-labile protecting group, PEP represents the phosphate ester
precursor group, O is an oxygen atom and, in structural formula
(IX.4), R.sup.11 represents a 2'-hydroxyl protecting group.
[0190] In the synthesis reagents according to structural formulae
(VII.1), (IX.2), (IX.3), (IX.4) and (IX.5), the nucleobase B can be
virtually any heterocycle useful for incorporation into
oligonucleotides. For example, the nucleobase may be one of the
genetically encoding purines (adenine or guanine), one of the
genetically encoding pyrimidines (cytosine, uracil or thymine),
anologs and/or derivatives of the genetically encoding purines
and/or pyrimidines (e.g., 7-deazadenine, 7-deazaguanine,
5-methylcytosine), non-genetically encoding purines and/or
pyrimidines (e.g., inosine, xanthene and hypoxanthene) or other
types of heterocycles. A wide variety of heterocycles useful for
incorporating into oligonucleotides are known in the art and are
described, for example, in Practical Handbook of Biochemistry and
Molecular Biology, Fasman, Ed., 1989, CRC Press (see, e.g., pages
385-393 and the references cited therein), the disclosures of which
are incorporated herein by reference. All of these various
heterocycles, as well as those that are later discovered, can be
included in the nucleosidic synthesis reagents described
herein.
[0191] When B is a purine in the synthesis reagents according to
structural formulae (VII.1), (IX.2), (IX.3), (IX.4) and (IX.5), the
illustrated sugar moiety is typically attached to the N9 position
of the purine, and when B is a pyrimidine, the illustrated sugar
moiety is typically attached at the N1 position of the pyrimidine.
Attachment sites for other nucleobases will be apparent to those of
skill in the art.
[0192] Any exocyclic amine or other reactive group(s) on the
nucleobase are protected with protecting groups that are stable to
the synthesis conditions used to synthesize the labeled
oligonucleotide. A variety of groups that are suitable for
protecting the exocyclic amine groups of nucleoside nucleobases in
the context of oligonucleotide synthesis are well-known in the art,
as are methods of preparing such protected nucleosides.
[0193] For example, groups that have been used to protect the
exocyclic amine of adenine include benzyol (Bz), phenoxyacetyl
(Pac) and isobutyryl (iBu). Groups that have been used to protect
the exocyclic amine of cystosine include acetyl (Ac) and Bz. Groups
that have been used to protect the exocyclic amine of guanine
include iBu, dimethylformamide (Dmf) and 4-isopropyl-phenoxyacetyl
(iPr-Pac). All of these protecting groups can be removed by
treatment with ammonium hydroxide at 55-65.degree. C. for 2-3 hr.
However, certain of these protecting groups can be removed under
milder conditions. For example, cleavage of the protecting groups
from A.sup.iBu, A.sup.Pac, C.sup.Ac and G.sup.iPr-Pac can be
effected in 4-17 hrs at room temperature with ammonium hydroxide,
or with 0.05M potassium carbonate in methanol, or treatment with
25% t-butylamine in H.sub.2O/EtOH. As some of the NH-rhodamine
and/or other dyes comprising the reagents described herein may not
be stable to the harsher deprotection conditions required by other
protecting groups, nucleosidic reagents which utilize protecting
groups that can be removed under these milder deprotection
conditions are preferred.
[0194] The linker L.sup.2 linking the label moiety LM to the
nucleobase B may be attached to any position of the nucleobase. In
some embodiments, when B is a purine, the linker is attached to the
8-position of the purine, when B is a 7-deazapurine, the linker is
attached to the 7-position of the 7-deazapurine, and when B is a
pyrimidine, the linker is attached to the 5-position of the
pyrimidine.
[0195] In some embodiments, linkers L.sup.2 useful for attaching LM
to a nucleobase comprise an acetylenic or alkenic amino linkage,
such as, for example, a linkage selected from
--C.ident.C--CH.sub.2--NH--, --C.ident.C--C(O)--,
--CH.dbd.CH--NH--, --CH.dbd.CH--C(O)--,
--C.ident.C--CH.sub.2--NH--C(O)--(CH.sub.2).sub.1-6--NH--, and
--CH.dbd.CH--C(O)--NH--(CH.sub.2).sub.1-6--NH--C(O)--, a
propargyl-1-ethoxyamino linkage, such as, for example, a linkage
having the formula
--C.ident.CH--CH.sub.2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub.2].sub.0--
6--NH-- or a rigid linkage, such as for example, a linkage selected
from
--C.ident.C--C.ident.C--CH.sub.2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub-
.2].sub.0-6--NH--,
--C.ident.C--(Ar).sub.1-22--C.ident.C--CH.sub.2--O--CH.sub.2CH.sub.2--[O--
-CH.sub.2CH.sub.2].sub.0-6--NH--,
--C.ident.C--(Ar).sub.1-2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub.2].sub-
.0-6--NH-- and
--C.ident.C--(Ar).sub.1-2--O--CH.sub.2CH.sub.2--[O--CH.sub.2CH.sub.2].sub-
.0-6--NH--, where Ar is as defined previously.
[0196] In some embodiments, linkers L.sup.2 useful for attaching LM
to a purine nucleobase comprise an alkylamine, such as, for
example, a linkage of the formula
--NH--(CH.sub.2).sub.1-6--NH--.
[0197] In some embodiments, linkers L.sup.2 useful for attaching LM
to a purine or pyrimidine nucleobse are anionic linkers as
described in U.S. Pat. No. 6,811,979, the disclosure of which is
incorporated herein by reference (see, e.g., the disclosure at Col.
17, line 25 through Col. 18, line 37 and FIGS. 1-17).
[0198] Methods of synthesizing nucleosides derivatized with linkers
such as those described above that are suitable for incorporating
into the reagents described herein are described, for example, in
Hobbs et al., 1989, J. Org. Chem. 54:3420; U.S. Pat. No. 5,151,507
to Hobbs et al., U.S. Pat. No.5,948,648 to Khan et al.; and U.S.
Pat. No. 5,821,356 to Khan et al, the disclosures of which are
incorporated herein by reference. The derivatized nucleosides can
be used as synthons to synthesize nucleosidic synthesis reagents as
will be described in more detail, below.
[0199] Specific exemplary embodiments of linker-dervatized
nucleobases that may comprise the nucleosidic reagents described
herein are illustrated below:
##STR00023## ##STR00024##
[0200] Nucleosidic synthesis reagents can be prepared from
linker-derivatized nucleoside synthons as illustrated in Scheme
(II), below:
##STR00025##
[0201] In Scheme (II), linker-derivatized nucleoside synthon 110 is
protected at the 5'-hydroxyl with an acid-labile protecting group,
which is illustrated in the Scheme with exemplary chloride reagent
R.sup.eCl, where R.sup.e is as previously defined. Treatment with
base to remove the trifluoroacetyl protecting group yields synthon
112. Reaction of synthon 112 with label moiety synthon 102 (see
Scheme (I), supra, followed by treatment with PEP synthon 105,
which in this specific example illustrated is a phosphine (see
Scheme (I), supra) yields nucleosidic synthesis reagent 114.
Specific conditions for carrying out the various synthetic steps
illustrated above are well known. Non-nucleosidic synthesis
reagents that include a synthesis handle, such as a synthesis
handle of the formula --OR.sup.e, can be prepared by routine
adaptation of Scheme (II).
5.10 Solid Support Reagents
[0202] Many embodiments of the reagents described herein include
solid supports. Such reagents generally comprise a solid support, a
label moiety as described herein and a synthesis handle, and may
include additional groups or moieties, such as additional label
moieties, quenching moieties, synthesis handles and/or groups
useful for, among other things, stabilizing oligonucleotide
duplexes, such as, for example, agents that intercalate between
base paris (intercalating agents) and agent that bind the duplex
minor groove (minor groove binding, or MGB, agents). The solid
support, label moiety, synthesis handle and any optional additional
moieties may be linked to one another in any fashion or orientation
that permits them to perform their respective functions.
[0203] In some embodiments, the solid support is attached to the
remainder of the reagent via a linker. Linkers attaching solid
supports to the remainder of the reagent typically include linkages
that are selectively cleavable under specified conditions such
that, following synthesis, the synthesized labeled oligonucleotide
can be released from the solid support. In some embodiments, the
linkages are labile to the conditions used to deprotect the
synthetic labeled oligonucleotide, such that the oligonucleotide is
deprotected and cleaved from the solid support in a single step.
Such linkers typically include ester linkages, but may include
other linkages, such as, for example, carbonate esters,
diisopropylsiloxy ethers, modified phosphates esters, etc.
[0204] Myriad selectively cleavable linkers useful in the context
of oligonucleotide synthesis are known in the art, as are methods
of derivatizing solid supports with such linkers. All of these
various linkers can be adapted for use in the solid support
reagents described herein. Non limiting examples of solid support
reagents comprising exemplary linkers that are cleavable under the
basic conditions used to deprotect synthetic oligonucleotides are
are illustrated in FIG. 7.
[0205] Like the synthesis reagents, the solid support reagents can
be non-nucleosidic or nucleosidic in nature. Exemplary embodiments
of non-nucleosidic solid support reagents include reagents
according to structural formula (X):
##STR00026##
[0206] where LM represents the label moiety, L represents an
optional selectively cleavable linker and --OR.sup.e represents the
synthesis handle, where R.sup.e is an acid-labile protecting group,
as previously described.
[0207] In some embodiments, the solid support synthesis reagents of
structural formula (X) are non-nucleosidic reagents according to
structural formula (X.1):
##STR00027##
[0208] where Z, LM, G, Sp.sup.1, Sp.sup.2 and R.sup.e are as
previously defined in connection with structural formula (IX.1) and
Sp.sup.4 represents a selectively cleavable spacing moiety. In some
specific embodiments, selectively cleavable spacing moiety Sp.sup.4
comprises an ester linkage.
[0209] In some embodiments, the solid support synthesis reagents of
structural formula (X) are nucleosidic reagents according to
structural formuale (X.2), (X.3), (X.4) or (X.5):
##STR00028##
[0210] wherein LM, R.sup.e, B. L.sup.2 are as previously defined
for structural formulae (X.2), (X.3), (X.4) and/or (X.5), R.sup.11
is as previously defined for structural formula (IX.4) and Sp.sup.4
represents a selectively cleavable spacing moiety, as described
above, which in some embodiments comprises an ester linkage.
5.11 Additional Exemplary Embodiments
[0211] It is to be understood that the specific embodiments of the
various moieties, groups and linkers described throughout the
disclosure can be included in all of the reagents described herein.
Moreover, the various specific embodiments can be combined with one
another in any combination as though the specific combination had
been specifically exemplified. As a specific example, any one of
the specific embodiments of label moiety LM described herein can be
included in any of the specifically exemplified embodiements of
non-nucleosidic and nucleosidic solid support and synthesis
reagents described herein. As another specific example, any one of
the specific embodiments of phosphate ester precursor group PEP,
such as the phosphoramidite group of structural formula (P.1),
supra, can be included in any of the synthesis reagents described
herein.
5.12 Uses of the Reagents
[0212] The various reagents described herein can be used in the
step-wise synthesis of oligonucleotides to synthesize
oligonucleotides labeled with rhodamine dyes directly on the
synthesis resin. Thus, the various reagents make available the
ability to synthetically label oligonucleotides with myriad
different rhodamines, obviating the need for laborious
post-synthesis modifications. The use of an exemplary synthesis
reagents to synthesize an oligonucleotide labeled with an NH
rhodamine dye is illustrated in FIG. 9.
[0213] As will be appreciated by skilled artisans, owing to the
availability of phosphoramidite reagents that can act as donors,
acceptors, or even quenchers for NH-rhodamine dyes, the reagents
described herein permit the ability to synthesize oligonucleotides
labeled with energy transfer dyes and/or NH-rhodamine-quencher dye
pairs, that are synthesized in situ. Exemplary syntheses of
oligonucleotides labeled with NH-rhodamine-fluorescein energy
transfer dye pairs that illustrate the versatility provided by the
reagents described herein are illustrated in FIGS. 10 and 11.
Because the reagents described herein permit virtually any
NH-rhodamine dye to be included in a solid support and/or synthesis
reagent, oligonucleotides labeled with energy transfer dye pairs
having spectral properties that are adjusted for specified
applications can be conveniently synthesized in situ, without the
need for post synthesis modification. Moreover, oligonucleotides
labeled with myriad different energy transfer dye pair combinations
can be synthesized from individual monomer reagents, obviating the
need to make synthesis reagents containing specified dye pairs.
Each member of the dye pair can be attached to the nascent
oligonucleotide in a step-wise fashion, with or without the
addition of intervening linking moieties.
[0214] Referring to FIG. 9, support-bound synthetic oligoncleotide
200 is treated with acid to remove the DMT group protecting its
5'-hydroxyl, yielding 5'-deprotected support-bound oligonucleotide
202 Coupling of N-protected NH-rhodamine phosphoramidite reagent
204 followed by oxidation yields support-bound NH-rhodamine-labeled
oliognucleotide 206. Treatment with concentrated ammonium hydroxide
to remove any protecting groups and cleave the synthesized
oligonucleotide from the solid support (resin) yields an
oligonucleotide 208 that is labeled with an NH-rhodamine dye.
[0215] Referring to FIG. 10, solid support reagent 210, which
includes a protected NH-rhodamine-fluorescein energy transfer dye
pair as the label moiety, can undergo three cycles of synthesis to
yield labeled support-bound oligonucleotide 212. Cleavage from the
solid support yields deprotected, labeled oligonucleotide 214.
[0216] Referring to FIG. 11A, nascent support-bound oligonucleotide
220 can be labeled with an NH-rhodamine-fluorescein dye pair
synthesized in situ by coupling N-protected NH-rhodamine
phosphoramidite synthesis reagent 222 to the 5'-hydroxyl of
oligonucleotide 220, which, after oxidation, yields
NH-rhodamine-labeled oligonucleotide 224. Removal of the DMT group
followed by coupling with an O-protected phosphoramidite (which in
the specific example illustrated is FAM-phosphoramidite) yields
labeled, support-bound oligonucleotide 226. Cleavage and
deprotection yields oligonucleotide 228, which is labeled with an
NH-rhodamine-FAM energy transfer dye pair.
[0217] The length and character of the linkage linking the donor
and acceptor dyes can also be manipulated through the use of
phosphoramidite linker reagents. This aspect is illustrated in FIG.
11B, where linker phosphoramidite 230 is coupled to
rhodamine-labeled oligonucleotide 225, yielding reagent 232.
Coupling with FAM-phosphoramidite followed by oxidation,
deprotection and cleavage yields oligonucleotide 234, which is
labeled with an NH-rhodamine-FAM energy transfer dye pair. In
linker phosphoramidite 230, "Sp" is a spacer, as previously
defined. For example, "Sp" could represent (Sp.1), (Sp.2), (Sp.3),
(Sp.4) or (Sp.5), as previously defined.
[0218] In the scheme illustrated in FIG. 11B, the length and
properties of the linker linking the NH-rhodamine and FAM dyes can
be adjusted by coupling additional linker phosphoramidites to
reagent 232 prior to coupling with the FAM-phosphoramidite. The
linker phosphosphoramites could be the same, or they could be
different. In this way, oligonucleotides labeled with energy
transfer dye pairs in which the donor and acceptor dyes, as well as
the linker linking the donor and acceptors, are tailored for
specific purposes can be readily synthesized in situ.
[0219] While FIGS. 11A & B exemplify the use of a specific
N-protected NH-rhodamine reagent, skilled artisan will appreciate
that any N-protected NH-rhodamine reagent that acts as an acceptor
for FAM could be used. Moreover, other O-protected fluoresceins
could be used, as could other types of phosphormidite dyes. Since
the dyes are added as monomers, the number of energy transfer dye
labels available is greater than the number of phosphoramidite
reagents necessary to synthesize them. For example,
oligonucleotides labeled with 9 different energy-transfer dye pairs
can be synthesized from 3 different N-protected NH-rhodamine
phosphoramidite reagents (reagents A, B and C) and 3 different
O-protected fluorescein phosphoramidite reagents (reagents 1, 2 and
3): oligo-Al, oligo-A2, oligo-A3, oligo-B1, oligo-B2, oligo-B3,
oligo-C1, oligo-C2 and oligo-C3.
6. EXAMPLES
Example 1
Synthesis of N-Protected NH-Rhodamine Phosphoramidite Synthesis
Reagents
[0220] Parent NH-rhodamine dyes including a carboxyl substitutent
at the C5- or C6-position were synthesized as described in U.S.
Pat. No. 4,622,400, U.S. Pat. No. 5,750,409, U.S. Pat. No.
5,847,162, U.S. Pat. No. 6,017,712, U.S. Pat. No. 6,080,852, U.S.
Pat. No. 6,184,379 or U.S. Pat. No. 6,248,884. The parent
NH-rhodamine dyes were then protected at the exocylcic amines with
either acetyl or trifluoroacetyl protecting groups, the resultant
N-protected NH-rhodamine dyes were converted to hydroxyl-amide
derivatives via the corresponding NHS ester derivative and the
hydroxyl functionality converted to a phosphoramidite using
standard procedures. The overall scheme is illustrated below:
##STR00029## ##STR00030##
[0221] Protection with acetyl groups. NH-rhodamine dye acid 6 (mono
TEA salt, 1.676 mmol) was suspended in DCM (40 mL) and TEA (3.67
mL). Acetic anhydride (3.13 mL) was added, and the reaction mixture
was stirred at room temperature for 3 days. H.sub.2O (10 mL) was
added and stirring was continued for 30 min. The mixture was
diluted with DCM (200 mL), washed with NaHCO.sub.3 solution (200
mL=2), dried (Na.sub.2SO.sub.4), filtered, and evaporated. The
residue was purified by flush chromatography on silica (using a
gradient of MeOH/EtOAc/DCM as eluent, from 5:20:75 to 20:20:60).
Evaporation of the appropriate fractions gave 881 mg (87%) of
colorless lactonized bis acetyl N-protected NH-rhodamine dye 8.
[0222] Protection with trifluoroacetyl groups. NH-rhodamine dye
acid 6 (mono TEA salt, 1.5 g, 2.402 mmol) was suspended in DCM (30
mL) and TEA (6.696 mL). The suspension was cooled to 0.degree. C.
and trifluoroacetic anhydride (2.0 mL) added drop wise through a
syringe. After the addition was complete, the reaction mixture was
stirred at room temperature for 10 min (and sonicated to break up
dye particles). The resultant brownish solution was evaporated,
re-dissolved in DCM (50 mL), and stirred with 5% HCl (40 mL) at
room temperature for 1 h. The reaction mixture was transferred to a
separatory funnel and the two layers separated. The DCM layer was
washed with a Brine solution (40 mL), dried (Na.sub.2SO.sub.4),
filtered, and evaporated. The residue was co-evaporated with MeCN
(2.times.) to give 1.79 g of crude N-protected NH-rhodamine dye
7.
[0223] Synthesis of N-Protected NH-Rhodamine NHS Esters. To a
solution of bis-acetyl dye 8 (588 mg, 0.968 mmol) and NHS (334 mg,
2.904 mmol) in DCM (20 mL), was added DCC (599 mg, 2.904 mmol). The
reaction mixture was stirred at room temperature for 2 h, and then
the reaction mixture was filtered. The filtrate was diluted with
DCM (80 mL), washed with H.sub.2O (50 mL x 2), dried
(Na.sub.2SO.sub.4), filtered, and evaporated. The residue was
purified by flash chromatography on silica (using a gradient of
AcOH/MeOH/EtOAc/DCM as eluent, from 1:1:20:78 to 1:5:20:74).
Evaporation of the appropriate fractions gave 680 mg (100%) of bis
acetyl N-protected NH-rhodamine NHS ester 10.
[0224] Following the procedure described above for bis acetyl
N-protected NH-rhodamine NHS ester 10, bis-TFA N-protected
NH-rhodamine Dye 7 (0.322 mmol), gave bis-TFA N-protected
NH-rhodamine NHS ester 9 in 80-90% yield.
[0225] Synthesis of N-Protected NH-Rhodamine Phosphoramidites. NHS
ester 9 (0.4 mmol) was dissolved in DCM (8 mL). To this stirred
solution, a mixture of 6-amino-1-hexanol/DIPEA/DCM (56 mg/0.07 mL/2
mL) was added. The reaction was stirred at room temperature for 30
min. The solid byproduct was removed by filtration and the filtrate
was purified by flush chromatography on silica (using a gradient of
Et0Ac/DCM as eluent, from 30% to 60%). Evaporation of the
appropriate fractions gave 0.344 mmol (86%) of bis-TFA-hexanolamide
rhodamine dye 11.
[0226] NHS ester 10 was converted to the bis-acetyl-hexanolamide
NH-rhodamine dye 12 using a similar procedure.
[0227] To a solution of bis-TFA-hexanolamide rhodamine dye 11
(0.338 mmol) and 2-Cyanoethyl
N,N,N',N'-tetraisopropylphosphorodiamidite (0.214 mL, 0.674 mmol, 2
equiv.) in DCM (10 mL), was added tetrazole amine (4 mg) in one
portion. The reaction mixture was stirred at room temperature for
20 h Volatile materials were removed by evaporation and the residue
was purified by precipitation three times from DCM/hexane. The
solid product was dried in vacuo to give 0.256 mmol (76%)
bis-TFA-N-protected NH-rhodamine dye phosphoramidite 13.
[0228] Similarly, bis-acetyl dye 12 was converted to phospharmidite
14.
Example 2
Synthesis of Heterodimeric Dye Networks
[0229] A dye network comprising an O-protected fluorescein linked
to an N-protected NH-rhodamine was synthesized as illustrated
below:
##STR00031##
[0230] Bis-acetyl N-protected NH-rhodamine NHS ester 16 (324 mg,
0.460 mmol) was dissolved in a solution of DMF (8 mL) and DIPEA
(0.3 mL). Fluoresecin derivative 17 (239 mg, 0.32 mmol; synthesized
as described in U.S. Pat. No. 5,800,996) was added and the reaction
mixture stirred at room temperature for 1 h. The mixture was
evaporated and then co-evaporated with MeOH (2.times.). The residue
was dissolved in 10% MeOH/DCM (100 mL) and washed with Brine
solution (100 mL). The aqueous layer was extracted with 10%
MeOH/DCM (50 mL.times.3), and the combined organic layer was dried
(Na.sub.2SO.sub.4), filtered, and evaporated. The residue was
purified by flush chromatography on silica (using a gradient of
MeOH/DCM as eluent, from 10% to 30%). Evaporation of the
appropriate fractions gave 270 mg (63%) of heterodimeric dye
network 19 (as the DIPEA salt).
[0231] The corresponding bis-TFA protected dye network 18 was
synthesized by a similar procedure.
Example 3
Synthesis of Heterodimeric Dye Network Phosphoramidite
[0232] A phosphoramidite synthesis reagent comprising the
heterodimeric dye network as the label moiety was synthesized as
illustrated below:
##STR00032## ##STR00033##
[0233] A solution of heterodimeric dye network 19 (0.173 mmol),
DIPEA (1.028 mL) and pivalic anhydride (0.702 mL) in DCM (10 mL)
was stirred at room temperature for 1 day. H.sub.2O (5 ml) was
added and stirring was continued for 1 h. The reaction mixture was
diluted with DCM (50 mL) and washed with H.sub.2O 40 mL). The
aqueous layer was extracted with DCM (40 mL). The combined organic
layer was dried (Na.sub.2SO.sub.4), filtered, and evaporated. The
residue was purified by flush chromatography on silica (using a
gradient of MeOH/DCM as eluent, from 3% to 15%). Evaporation of the
appropriate fractions gave 0.145 mmol (84%) of bis-acetyl
bis-pivaloyl heterodimeric dye derivative 21 (as the DIPEA
salt).
[0234] Dye derivative 21 (0.146 mmol) was suspended in a dissolved
of DIPEA (0.2 mL) and DCM (8 mL). Solid N-hydroxysuccinimide
tetramethyluronium tetrafluoroborate (88 mg) was added and the
reaction stirred at room temperature for 1 h. 6-Amino-1-hexanol (51
mg) was added and stirring was continued for 1 h. The reaction
mixture was filtered and the filtrate was diluted with DCM (50 mL).
The DCM solution was washed with brine solution (40 mL), dried
(Na.sub.2SO.sub.4), filtered, and evaporated. The residue was
purified by flush chromatography on silica gel (using a gradient of
MeOH:EtOAc:DCM as eluent, from 5:20:75 to 15:20:65). Evaporation of
the appropriate fractions gave 0.124 mmol (85%) of Dye hexanolamide
derivative 23 (as the DIPEA salt).
[0235] Dye derivative 23 (91 mg, 0.057 mmol) and tetrazole amine (2
mg) were dissolved in DCM (5 mL).
2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite (CE diamidite)
(0.04 mL), and was added and the reaction stirred at room
temperature for 16 h. The reaction mixture was diluted with a
solution of TEA:EtOAc:DCM (3:20:77, 3 mL) and purified by silica
gel chromatography (using a gradient of TEA:MeOH:EtOAc:DCM as
eluent, from 3:0:20:77 to 3:5:20:72). Evaporation of the
appropriate fractions and precipitation from DCM/hexane gave 75 mg
(74%) of heterodimeric dye phosphoramidite 25.
Example 4
Synthesis of a Non-Nucleosidic Phosphoramidite Synthesis Reagent
Including an Optional Synthesis Handle
[0236] A non-nucleosidic phosphoramidite synthesis reagent that
includes an optional synthesis handle of the formula --OR.sup.e was
synthesized as illustrated below:
##STR00034##
[0237] Bis-TFA derivative 9 (1.354 mmol) was dissolved in a
solution of DIPEA (0.236 mL) in DCM (30 mL). To the stirred
solution, linker synthon 29 and DCM (0.609 g, 1.355 mmol;
synthesized as described in Nelson et al., 1992, Nucl. Acids Res.
20:6253-6259) was added stirring continued at room temperature for
2 h. EtOAc was added and the mixture was loaded on a silica column
Pure product was isolated by elution using a gradient of EtOAc:DCM
from 5:95 to 1:5. Evaporation of the appropriate fractions gave
1.25 g (80%) of bis-TFA-rhodamine-hydroxy amide 30.
[0238] A solution of bis-TFA-rhodamine-hydroxy amide 30 (1.081
mmol) and tetrazole amine (18.5 mg) were dissolved in DCM (35 mL).
CE diamidite was added (0.685 mL) and the reaction stirred at room
temperature for 18 h. The solvent was removed under reduced
pressure and the residue precipitated from DCM/hexane (3.times.) to
give 1.081 mmol (100%) of pure bis-TFA-rhodamine DMT
phosphoramidite 31.
Example 5
Solid Phase Synthesis of a Labeled Oligonucleotide
[0239] Oligonucleotides labeled with the N-protected NH-rhodamine
phosphoramidite synthesis reagents were synthesized on polystyrene
solid supports using the standard operating conditions on an AB
3900 automated DNA synthesizer. The N-protected NH-rhodamine
phosphoramidites were soluble in the acetonitrile solvent for the
coupling reactions, and the N-protected Nh-rhodamine dye adducts
were stable to repeated synthesis cycles which employed removal of
DMT with dichloroacetic acid, addition of nucleoside
phosphoramidite monomers, capping with acetic anhydride and
oxidation with iodine to generate the internucleotide phosphate
linkages. This class of NH-rhodamine was also found to be stable to
the conditions used to deprotect and cleave the synthesized labeled
oligonucleotide from the solid support (treatment with ammonium
hydroxide at 60.degree. C. for 1-2 h). The overall scheme used to
synthesize the labeled oligonucleotide is illustrated below:
##STR00035## ##STR00036##
[0240] By this process, TFA-rhodamine DMT phosphoramidite 31 was
coupled to the 5'-hydroxyl of a support-bound oligonucleotide to
give the phosphite intermediate 32. Fluorescein phosphoramidite
(Glenn Research) was coupled to the free hydroxyl of intermediate
32. The resultant labeled oligo was oxidized, cleaved from the
support with concentrated ammonia hydroxide for 1 to 2 hours at
60.degree. C., washed with aceonitrile/water and dried under
reduced pressure to yield labeled oligonucleoide 33. Oligo 33 was
re-precipitated using a standard sodium acetate/EtOH precipitation
protocol. Labeled oligo 33 was produced in greater then 90% purity
and in greater than 85% yield (170,000 pM from 0.2 uM support) and
used without further purification.
Example 6
Labeled Oligonucleotides Synthesized with the Synthesis Reagents
Exhibit Good Spectral Properties
[0241] Poly(dT).sub.10 oligonucleotides labeled with NH-rhodamines
or NH-rhodamine-fluorescein dye pairs were synthesized as
illustrated above. Following cleavage, fluorescence spectra were
recorded. All labeled oligos synthesized exhibited good
fluorescence properties.
[0242] Although the foregoing inventions have been described in
some detail to facilitate understanding, it will be apparent that
certain changes and modifications may be practiced within the scope
of the appended claims. Accordingly, the described embodiments are
to be considered as illustrative and not restrictive, and the
various inventions described herein are not to be limited to the
details provided herein.
[0243] All literature and patent references cited throughout the
disclosure are incorporated into the application by reference for
all purposes.
* * * * *