U.S. patent application number 09/941986 was filed with the patent office on 2002-11-28 for nucleic acid probes and methods.
This patent application is currently assigned to Duke University. Invention is credited to Beilstein, Amy E., Grinstaff, Mark W., Khan, Shoeb I..
Application Number | 20020177695 09/941986 |
Document ID | / |
Family ID | 26793119 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177695 |
Kind Code |
A1 |
Grinstaff, Mark W. ; et
al. |
November 28, 2002 |
Nucleic acid probes and methods
Abstract
The present invention provides metal-containing purines,
pyrimidines, nucleosides, nucleotides and oligonucleotides;
including phosphoramidite and photolabile derivatives thereof,
including methods of making and method of using same. The present
invention provides a method for detection of nucleic acid sequences
via electrochemical or photochemical means.
Inventors: |
Grinstaff, Mark W.; (Durham,
NC) ; Beilstein, Amy E.; (Durham, NC) ; Khan,
Shoeb I.; (Durham, NC) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Assignee: |
Duke University
|
Family ID: |
26793119 |
Appl. No.: |
09/941986 |
Filed: |
August 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09941986 |
Aug 30, 2001 |
|
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09377612 |
Aug 19, 1999 |
|
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60097327 |
Aug 20, 1998 |
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Current U.S.
Class: |
536/23.1 ;
536/26.6; 544/225 |
Current CPC
Class: |
C07H 19/10 20130101;
C07H 19/06 20130101; C07H 21/00 20130101 |
Class at
Publication: |
536/23.1 ;
536/26.6; 544/225 |
International
Class: |
C07H 021/04; C07H
019/04; C07F 015/00 |
Claims
We claim:
1. A nucleoside, nucleotide, purine or pyrimidine derivative
comprising a detectable marker which is a metal complex containing
at least one of a M(diimine).sub.x.sup.Y+ complex,
M(terpyridine).sub.x.sup.Y+ complex or a metallocene wherein M is a
transition metal, said diimine is selected from a bipyridine,
phenanthroline or terpyridine derivative; x is 1, 2, or 3 and y is
0, 1, 2, 3, or 4, said bipyridine, phenanthroline, terpyridine and
metallocene being optionally substituted by any one of alkyl,
alkene, alkyne, aryl, alkylaryl, and carboxyalkyl, amide, ester or
ether.
2. A nucleoside, nucleotide, purine or pyrimidine derivative
according to claim 1 wherein said detectable marker contains at
least one of a M(diimine).sub.2(diimine).sup.2+ complex wherein M
is a metal selected from Fe.sup.+2, Ru.sup.+2, Os.sup.+2,
Co.sup.+2, Rh.sup.+2, and Cr.sup.+2, or a
M(diimine).sub.2(diimine).sup.3+ complex wherein M is a metal
selected from Fe.sup.+3, Ru.sup.+3, Os.sup.+3, Co.sup.+3,
Rh.sup.+3, and Cr.sup.+3, or a M(terpyridine).sub.2.sup.3+ complex
wherein M a metal selected from Fe.sup.+3, Ru.sup.+3, Os.sup.+3,
Co.sup.+3, Rh.sup.+3, and Cr.sup.+3, or a M(diimine).sub.2.sup.3+
complex wherein M is a metal selected from Fe.sup.+2, Ru.sup.+2,
Os.sup.+2, CO.sup.+2, Rh.sup.-2, and Cr.sup.+2.
3. An oligonucleotide comprising at least one nucleoside,
nucleotide or nucleic acid derivative of claim 1.
4. A nucleoside, nucleotide, purine or pynimidine derivative of
claim 1 which comprises a sugar selected from the group consisting
of a ribose, a deoxyribose and a dideoxyribose.
5. A nucleoside nucleotide, purine or pyrimidine derivative of
claim 1 which fuirther comprises a dimethoxytritylchloride,
.alpha.-methyl-6-nitropipronyloxy carbonyl or 2-cyanoethyl
N,N'-diisopropylcholoro phosphoramidite derivative.
6. A nucleoside, nucleotide, purine or pyrimidine derivative of
claim 1 which further comprises a compound of the following
structure: 7wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4
independently are a hydrogen atom, a lower alkyl, aryl, benzyl,
halogen, hydroxyl, alkoxyl. thio, thioether, amino, nitro,
carboxyl, formate, foramino or phosphido group, or adjacent
substituents R.sub.1-R.sub.4 are substituted oxygen groups that
together form a cyclic acetal or ketal; R.sub.5 is hydrogen,
alkoxyl, alkyl, halo, aryl or alkenyl group, n=0, 1, 2 or 3; and Y
is a hydroxyl group of the nucleoside or nucleotide.
7. A nucleotide, or nucleoside, purine or pyrimidine derivative of
claim 6, wherein Y is a 5'-hydroxyl or a 3'-hvdroxyl group of said
nucleoside or nucleotide.
8. A double-stranded nucleic acid sequence comprising the
oligonucleotide of claim 3 and an electron acceptor moiety which
contains a metal selected from the group consisting of Cr, Cu, Co,
Fe, Ru and Os.
9. A nucleoside, nucleotide, purine or pvrimidine derivative of
claim 1 which contains a uricil, adenine, thymine, cytosine,
guanine or natural or synthetic analogs or derivatives thereof.
10. A nucleoside, nucleotide, purine or pyrimidine derivative of
claim 1 which contains a di- or tri-phosphate optionally
substituted by a sulfur, NH or BH group.
11. A nucleoside, nucleotide, purine or pyrimidine according to
claim 1, or an oligonucleotide of claim 3 attached to a solid
surface through one of a covalent bond, an electrostatic
association or hydrogen bond.
12. A nucleoside, nucleotide, purine or pyrimidine according to
claim 11 wherein said solid surface is one of a nanoparticle,
glass, ITO, TiO.sub.2, SnO.sub.2, Au, Pt, silicon, porous silicon,
plastic or graphite.
13. A nucleoside, nucleotide, purine or pyrimidine derivative
according to claim 1 wherein said detectable marker is selected
from a bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carbonyl
propargyl amine) ruthenium (II) substituent, a ferrocene
substituent; and a bis(2,2'-bipyridine)
(4'-methyl-2,2'-bipyridine-4-carbonyl propargyl amine) osmium (II)
substituent.
14. A nucleoside, purine or pyrimidine nucleotide of claim 1
wherein said complex is chiral wherein said chirality is fac or
ras, R or S, or D or L.
15. A nucleoside, nucleotide, purine or pyrimidine derivative
according to claim 1 wherein said diimine ligands around said metal
are all the same.
16. A nucleoside, nucleotide, purine or pyrimidine derivative
according to claim 1 wherein at least two of said diimine ligands
around said metal are not the same.
17. A method of identifying a nucleic acid molecule comprising
contacting said nucleic acid molecule with an oligonucleotide of
claim 3 under conditions where said oligonucleotide can
specifically bind to complementary regions of said nucleic acid to
form an identifiable complex, and detecting said binding.
18. A method according to claim 17, wherein said method is an in
vivo, ex vivo or in vitro method.
19. A method according to claim 17, wherein said detecting
comprises measuring a photophysical alteration or an
electrochemical event of said complex.
20. A method according to claim 19 wherein said photophysical
alteration is an energy or electron transfer event.
21. A method according to claim 17 wherein said identifiable
complex is a double or triple helix.
22. A method according to claim 17 wherein said nucleic acid
molecule is of human, animal, viral, bacteria, or plant origin.
23. A method according to claim 19 wherein said electron transfer
event occurs due to interaction between two metal complexes on a
single nucleic acid strand.
24. A method according to claim 19 wherein said electron transfer
event occurs due to interaction between two metal complexes on a
separate nucleic acid strand.
25. A method according to claim 19 wherein said electron transfer
event occurs due to interaction between a metal complex on a single
nucleic acid strand and said nucleoside or nucleotide.
26. A method according to claim 19 wherein said electron transfer
event occurs due to an interaction between a metal complex and an
organic donor/acceptor on a single nucleic acid strand wherein said
organic donor/acceptor is selected from the group comprising
acridine, fluorescein, anthracene, metylphenothiazine,
methylviolgen, phenothiazine and quione.
27. A method according to claim 19 wherein said detection is
between a metal complex on a single strand and an organic
donor/acceptor on a second single nucleic acid strand wherein said
organic donor/acceptor is selected from the group comprising
acridine, fluorescein, anthracene, metylphenothiazine,
methylviolgen, and quione.
28. A method according to claim 19 wherein the electron transfer
event is a photoinduced electron transfer event, electrochemical
event, or an electroluminescence event.
29. A method according to claim 28 wherein the electron-transfer
event is performed electrochemically.
30. A method according to claim 17 wherein said oligonucleotide is
attached to a surface and said metal complex donates an electron or
energy to the surface or said metal complex accepts and electron
energy from the surface, and said detecting comprising measuring a
transfer of said electron or energy.
31. A method according to claim 17 wherein said oligonucleotide
binds directly or indirectly to a nucleic acid molecule attached to
a surface wherein said metal complex donates an electron or energy
to the surface or said metal complex accepts and electron or energy
from the surface and said detecting comprising measuring a transfer
of said electron or energy.
32. An improved method of synthesizing a labeled oligonucleotide
comprising: attaching a nucleoside of the oligonucleotide to be
synthesized to a solid support through a labile linker arm; said
nucleoside containing a removable protecting group attached to a
5'hyroxyl of a sugar group of said nucleoside; removing said
protecting group; adding a further nucleoside containing a labile
linker arm and a protecting group to said solid support under
conditions where said further nucleoside is coupled to said
5'hydroxyl of said sugar group through said linker arm; optionally
capping any uncoupled nucleoside; removing the protecting group of
said further nucleoside; repeating the above with further
nucleoside derivatives wherein at least one of said further
nucleoside contains a detectable label, until a desired
oligonucleotide is formed; and cleaving said oligonucleotide from
said solid support; wherein the improvement comprises adding at
least one dimethoxytrityl, phosphoramidite halodonucleoside
derivative in place of one of said at least one nucleoside
containing a detectable label; and adding a detectable label to
said halodonucleoside derivative subsequent to formation of said
oligonucleotide.
33. The method of claim 32 wherein said cleaving comprising adding
concentrated ammonium hydroxide.
34. The method of claim 32, further comprising heating said cleaved
oligonucleotide to remove any protecting groups.
35. An oligonucleotide comprising at least one nucleoside,
nucleotide or nucleic acid of claim 2.
36. An improved method of oligonucleotide synthesis comprising
combining phosphoramidite nucleoside derivatives in combination
with a solid support wherein the improvement comprises adding at
least one phosphoramidite halonucleoside during the synthesis and
subsequently labeling said halonucleoside with a marker in a metal
catalyzed reaction.
37. The improved method of claim 36 wherein said metal is
paladium.
38. The improved method of claim 36 wherein said halonucleoside is
selected from the group consisting of fluorouridine,
fluoroadenosine, fluoroguanosine, fluorocytidine, fluorothymidine,
chlorouridine, chloroadenosine, chloroguanosine, chlorocytidine,
chlorothymidine, iodouridine, iodoadenosine, jodoguanosine,
iodocytidine, iodothymidine, bromouridine, bromoadenosine,
bromoguanosine, bromocytidine and bromothymidine.
39. The improved method of claim 36 wherein said oligonucleotide
synthesis progresses in the 3' to 5' or the 5' to 3' direction.
40. The improved method of claim 36 wherein the marker is a
metal-containing alkylene derivative.
Description
[0001] The present application is based on application Ser. No.
60/097,327, filed Aug. 20, 1998, the entire contents of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention provides metal-containing purines,
pyrimidines, nucleosides, nucleotides and oligonucleotides;
including phosphoramidite and photolabile derivatives thereof,
including methods of making and methods of using same. The present
invention also provides a method for detection of nucleic acid
sequences via electrochemical or photochemical means. The present
invention provides an improved method of making labeled nucleic
acid polymers.
BACKGROUND
[0003] Modifying a nucleoside, nucleotide or oligonucleotide with a
specific chemical functionality, such as a photo-, redox- or
chemically-active metal complex, is of wide-spread interest for
analytical applications (sequencing, hybridization assays),
therapeutic uses (anticancer, antiviral pharmaceuticals) and
mechanistic studies (electron transfer, structure-function). U.
Englisch, D. D. Gauss, Angew. Chem. Int. Ed. Engl. 30 (1991)
613-629; G. H. Keller, M. M. Manak, DNA Probes, Stockton Press, New
York 20 1993; P. G. Sammes, and G. Yahioglu, Natural Product
Reports (1996) 1-28. Synthetic strategies toward these
supra-molecular bioassemblies focus primarily on post-modification
of the synthesized nucleic acid single strand or complementary
duplex. A number of researchers, (C. J. Murphy, M. R. Arkin, J. K.
Barton, Science 262 (1993) 1025-1029; R. E. Holmlin, P .J.
Dandliker, J. K. Barton, Angew, Chem. Int. Ed. Engl. 36 (1997)
2714-2730 and references therein; D. Magda, R. A. Miller, J. L.
Sessler, B. L. Iverson, J. Am. Chem. Soc. 119, (1994) 7439-7440; J.
Telser, K. A. Cruickshank. K. S. Schanze, T. L. Netzel. J. Am Chem.
Soc. 111 (1989) 7221-7226; W. Bannwarth, D. Schmidt. R. L.
Stallard. C. Homung. R. Knorr, F. Muller, Helv. Chim. Acta 71
(1988) 2085-2099), have used this strategy to link a metal complex,
usually as the activated succinimide ester, to the terminus of the
nucleic acid single strand previously modified to contain an alkyl
amine. In another approach, an amino- or diimine-modified (e.g.,
phenanthroline) nucleoside is synthesized, and subsequently reacted
with a metal to form the desired complex. G. B. Dreyer, P. B.
Dervin, Biochemistry 82 (1985) 968-972; C. B. Chen. D. S. Sigman,
J. Am. Chem. Soc. 110 (1988) 6570-6572; M. Matsumura. M. Endo, M.
Korniyama, J. Chem. Soc., Chem. Commun. (1994) 2019-2020; J. K.
Bashkin. E. I. Frolova. U. Sampath, J. Am. Chem. Soc. 116 (1994)
5981-5982; T. J. Meade, J. F. Kayyem Angew. Chem. Int. Ed. Engl. 34
(1995) 352-354. An alternative strategy that offers clear synthetic
advantages over the previous systems is to use DNA/RNA solid-phase
synthetic methodologies for the site-specific labeling of an
oligonucleotide with a transition metal complex. A number of
investigations are currently exploring this approach. J. Schliepe,
U. Berghoff, B. Lippert, D. Cech, Angew. Chem. Int. Ed. Engl. 35
(1996) 646-648; R. Manchanda. S. U. Dunham, S. J. Lippard. J. Am.
Chem. Soc. 118 (1996) 5144-5145; W. Bannwarth. D. Schmidt,
Tetrahedron Letters 30 (1989) 1513-1516; E. Meggers. D. Kusch. B.
Giese, Helvetica Chim. Acta 80 (1997) 640-652; D. J. Hurley. Y.
Tor. J. Am. Chem. Soc. 120 (1998) 2194-2195).
[0004] The present invention provides novel metal-containing
purines, pyrimidines, nucleosides and nucleotides, and derivatives
thereof, including those which are useful in nucleic acid
synthesis, and identification, such as in detection and sequencing.
The present invention also provides a new method of labeling
purines, pyrimidines, nucleosides, nucleotides and their
derivatives during automated or manual synthesis of nucleic acid
polymers, such as oligonucleotides, DNA or RNA molecules.
SUMMARY OF THE INVENTION
[0005] It is an object of the invention to provide metal-containing
purines, pyrimidines, nucleosides, nucleotides, and
oligonucleotides, including derivatives and intermediates
thereof.
[0006] Another object of the present invention is to provide a
novel method of synthesizing labeled nucleic acid polymers, such as
oligonucleotides, DNA or RNA molecules, preferably by automated
methods, which preferably, include use of known iodonucleoside
derivatives.
[0007] It is another object of the invention to provide a method of
making the purine, pyrimidine, nucleoside and nucleotide
derivatives of the present invention.
[0008] It is yet another object of the invention to provide a
method of conducting a polymerase chain reaction or other
primer-directed reaction to make and/or detect a nucleic acid of
interest in an amplification product of the reaction by
incorporating in the reaction product a metal-containing derivative
or iodonucleosides, (which may be subsequently labeled) according
to the present invention.
[0009] It is also an object of the invention to provide a method of
detecting a nucleic acid analyte, such as DNA or RNA or dideoxy
residues of same, of interest in the product of a polymerase chain
reaction or other primer-directed reaction by incorporating at
least one purine, pyrimidine, nucleoside or nucleotide derivative
of the present invention in the reaction.
[0010] Yet another object of the present invention is to provide a
solid-phase method of making a nucleic acid polymer, such as an
oligonucleotide, which incorporates the metal-containing derivative
or halonucleoside, such as iodonucleoside, derivatives of the
present invention.
[0011] An object of the present invention is to provide a solid
surface, including, but not limited to, a bead, plate, or
electrode, with a metal-containing derivative of the present
invention; and a method for using same.
[0012] Specifically, the present invention provides ferrocene;
bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carbornyl
propargyl amine) ruthenium (II); and bis(2,2'-bipyridine)
(4'-methyl-2,2'-bipyridin- e-4-carbonyl propargyl amine) osmium
(II), and corresponding Rh, Cr and Co, nucleoside derivatives.
Dimethoxytrityl; 2-cyanoethyl-N,N-diisopropyl- -phosphoramidite;
phosphotriester; phosphoramidite; and 1-(2-nitro4-,5-methylene
dioxyphenyl) ethyl-choloroformate derivatives are also
provided.
[0013] The metal complexes described in the present invention are
linked to the nucleoside via an amide bond through either the 5, 3
or 2 carbon of the sugar (ribose, deoxyribose or dideoxyribose, for
example) or the 5 carbon of a pyrimidine or the 8 carbon of a
purine. For example, the amide bond will allow for delocalization
of the excited electron on the metal diimine and this will
facilitate the electron transfer into the nucleic acid. Preferred
amide bonds include alkynylamines. This is a preferred bonding
motif, however other bonding configurations are possible such as,
for example, ethers and esters.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A and 1B. Synthesis of ruthenium-nucleoside
phosphoramidite wherein (a) is propargyl amine HCl, DCC,
(dicyclohexylcarbodiimide) HOBt, DIPEA, DMF producing an 82% yield
of 2; (b) is Ru(bp).sub.2Cl.sub.2 70% ag. C.sub.2H.sub.5OH
providing an 82% yield of 3; (c) is 2'deoxy-3',
5-dibenzoyloxy-5-iodouridine, Pd (PPh.sub.3).sub.4, CuI, TEA, DMF
giving a 79% yield; (d) NH.sub.3/CH.sub.3OH, 90% yield; (e) DMT-Cl,
C.sub.6H.sub.5N, 81% yield 3'R.dbd.H and 5'R.dbd.DMT, and (f) CIP
(iPr.sub.2N)(OCH.sub.2CH.sub.2CN), CH.sub.3 CN, 90% yield where
HOBt is 1-hydroxybenzotriazole; DIPEA is N,N-diisopropylethylamine;
DMF is N,N-dimethylformamide; TEA is triethylamine; and DMT is
dimethoxytrityl.
[0015] FIG. 2. Automated oligonucleotide synthesis with
phosphoramidite derivative of ruthenium nucleoside of the present
invention.
[0016] FIG. 3. DNA melting curve for ruthenium modified
oligonucleotides.
[0017] FIG. 4A-4C. Synthesis of DMT-FPAU.
[0018] FIG. 5A-5D. Synthesis of an osmium derivative of the present
invention.
[0019] FIG. 6A-6C. Schematics of metallo-nucleic acid incorporated
into a double stranded DNA wherein x represents known types of
linkages found in nucleic acids and nucleic acid derivatives.
[0020] FIG. 7. Synthesis scheme for nucleotide derivative according
to the present invention wherein the following conditions apply: a)
MsCl/pyridine, 0.degree. C., 12 h, 77% yield; b) LiN.sub.3/DMF,
90.degree. C., 3h, 73% yield; c) PPh.sub.3/dioxane, NH.sub.4OH,
25.degree. C., 12 h, 72% yield; d) CDI/DMF, LH-20/THF,
Ru(bpy).sub.2(4m-4'-ca-bpy), 25.degree. C., 12 h, 80% yield; e)
2-cyanoethylchloro-N,N-diisopropylphosphoramidite, DIPEA,
CH.sub.3CN, 25.degree. C., 2 h (yield>95% TLC).
[0021] FIG. 8. Exemplification of automated synthesis with a
nucleotide derivative of the present invention wherein the
following conditions apply: (a) normal synthesis; (b) 30% NH.sub.3,
55.degree. C., 16 h. B=nucleotide bases A, C, G or T.
[0022] FIG. 9. Thermal denaturation curve of oligonucleotides
according to Table 2.
[0023] FIG. 10. Spectrum of B-DNA.
[0024] FIG. 11. Absorption spectrum, of Ru(bpy).sub.3.sup.2+.
[0025] FIG. 12. Excitation and emission spectrum of
Ru(bpy).sub.3.sup.2+.
[0026] FIG. 13. FTIR scan of compound 5 of FIG. 7.
[0027] FIG. 14. The emission lifetime spectrum of oligonucleotide
duplex, 12.cndot.19 (586 ns) at 25.degree. C. in phosphate
buffer.
DETAILED DESCRIPTION OF THE INVENTION
[0028] A number of requirements, both synthetic and photophysical,
must be met to ensure successful incorporation of a functional
photoactive and/or electroactive metal complex into an
oligonucleotide. Synthesis of a metallo-oligonucleotide using a
metallo-nucleoside phosphoramidite approach, for example, requires:
1) straightforward reactions and purification steps to the
precursor metal complex and metallo-nucleoside phosphoramidite, 2)
sufficient solubility of the metallo-nucleoside phosphoramidite in
acetonitrile for the solid-phase reactions, 3) high stability of
the metal complex during oligonucleotide synthesis and deprotection
reactions to prevent undesired side reactions and decomposition,
and finally 4) efficient coupling of the metallo-nucleoside
phosphoramidite using standard automated synthesis. M. J. Gait,
Oligonucleotide Synthesis: A Practical Approach, IRL Press,
Washington, D.C. 1984. Favorable physical properties of the metal
complex include: 1) spectroscopically distinguishable metal redox
states, 2) tunable electronic structures, 3) energetic excited
states, 4) long lifetimes, 5) near unity quantum yield, and 6)
photochemical stability.
[0029] With these issues in mind, a ruthenium-nucleoside
phosphoramidite, 5 (FIGS. 1A-1B) was synthesized. Tris-diimine
metal complexes have been found to possess a number of favorable
properties including high thermal and photochemical stability,
inertness to ligand exchange reactions, tunable electronic
structures, long lifetimes in fluid solution (.tau.>1 .mu.s),
and high quantum yields, V. Balzani, A.
[0030] Juris, M. Venturi, S. Campagne, S. Serroni, Chem. Rev. 96
(1996) 759-833; N. H. Damrauer, G. Cerullo, A. Yeh, T. R. Boussie,
C. V. Shank, J. M. McCusker, Science 275 (1997) 54-57. Metal
chromophores of this class are currently used to study a number of
photophysical processes including electron-transfer reactions in
supramolecular assemblies (A. Juris, V. Balzani, F. Barigelletti,
S. Campagna, P. Belser, A. von Zelewsky, Coord. Chem. Rev. 84
(1988) 85-277; V. Balzani, S. Campagna. G. Denti. A. Juris, S.
Serroni, M. Venturi. Acc. Chem. Res. 31 (1998) 26-34; V. Balzani,
F. Barigelletti. L. Decola. T. Curr. Chem. 158 (1990) 31-71; M.
Gratzel, K. Kalyanasundaram, Current. Sci. 66 (1994) 706-714) and
biological systems (H. B. Gray, J. R. Winkler, Ann. Rev. Biochem.
65 (1996) 537-561; J. R. Winkler, H. B. Gray, Chem. Rev. 92 (1992)
369-379; G. McLendon, Acc. Chem. Res. 21 (1988) 160-167).
Ru(bpy.sub.3).sup.2+ has been selected as an exemplification of the
present invention, rather than a phenanthroline analogue, since
these complexes, in general, are more potent oxidants in their
excited state. Phenanthroline analogues are expected however to be
useful as well. The formation of an amide bond with the
4'-carboxylic acid of bipyridine and propargylamine favors
localization of the excited state electron on that particular
bipyridine, and has been previously shown to enhance the electronic
coupling between adjacent M(bpy.sub.3).sup.2+ (where M=Ru or Os) in
a covalently crosslinked metallo-bipyridine polymeric Vh system. L.
M. Dupray, M. Devenney, D. R. Striplin, T. J. Meyer, J. Am. Chem.
Soc. 119 (1997) 10243-10244. An alkynyl group was introduced on the
bipyridine to efficiently cross-couple the metal complex to a
halonucleoside using a Pd(0) Heck catalyst. R. F. Heck, Acc. Chem.
Res. (1978) 146-151; A. Meijere, F. E. Meyer, Angew. Chem. Int. Ed.
Engl. 33 (1994) 2379-2411. To ensure sufficient solubility in
organic solvents and to aid in purification and recrystallization
steps a large counter ion such a PF.sub.6.sup.-, was used. Other
large counter ions can be used, such as BF.sub.4.sup.- and
ClO.sub.4.sup.-. One skilled in the art will recognize the large
number of large positive counter ions which may also be used.
[0031] As exemplified in FIGS. 1A-1B,
4'methyl-2-2'-bipyridine4-carboxylic acid, 1, was coupled to
propargyl amine in DMF using dicyclohexylcarbodiimide (DCC). The
resulting modified bipyridine, 2, was reacted with
Ru(bpy).sub.2Cl.sub.2 to form the tris-bypyridine complex, 3. A
Pd(PPh.sub.3).sub.4 cross-coupling reaction between the Ru complex,
3, and 2'-deoxy-3',5'-dibenzoyloxy-5-iodouridine afforded the
metallo-nucleoside, 4. Benzoyl deprotection in methanolic ammonia,
followed by reaction with 4,4'-dimethoxytrityl chloride and
2-cyanoethylchloro-N,N-diisopropylphosphoramidite yielded the
ruthenium-nucleoside phosphoramidite, 5, ready for automated
solid-phase synthesis.
[0032] In an analogous manner, the hydroxyl group of the
phenothiazine derivative, 6, was treated with
2-cyanoethylchloro-N,N'-diisopropylphosph- oramidite in the
presence of diisopropylethylamine, to yield the phenothiazine
phosphoramidite, 7.
[0033] An HPLC trace of the ruthenium-modified 2-deoxyuridine, 5,
showed one peak, and its elution time was greater than that of
2-deoxyuridine. The MH.sup.+-PF.sub.6.sup.-(1036) and
-2PF.sub.6.sup.-(891) peaks were observed in the FAB-MS, confirming
formation of the product.
[0034] The site-specific incorporation of the ruthenium-nucleoside
in a 16-mer oligonucleotide was performed using an automated ABI
392 DNA/RNA synthesizer (FIG. 2). All syntheses were accomplished
at both the 0.2 and 1 .mu.mol scales (Table 1).
1TABLE 1 Oligonucleotides Synthesized 6 (SEQ ID NO: 1) 5'-TCA ACA
GU*T TGT AGC A-3' 7 (SEQ ID NO: 2) 5'-U*CA ACA GTT TGT AGC A-3' 8
(SEQ ID NO: 3) 5'-TCA ACA GTT TGU* AGC A-3' 9 (SEQ ID NO: 4) 5'-TCA
ACA GTT TGT AGC A-3' 10 (SEQ ID NO: 5) 5'-TGC TAC AAA CTG TTG A-3'
11 (SEQ ID NO: 6) 5'-TCG TAC AAA CTG TTG A-NH.sub.2-3' * =
ruthenium-modified uridine
[0035] Using the standard coupling protocol, the metallo-nucleobase
phosphoramidite was incorporated at different positions within an
oligonucleotide. The coupling efficiencies were high (>95%) for
all standard bases except for the ruthenium modified analogue which
had a coupling efficiency of 40% as determined by measuring the
release of DMT during synthesis (498 nm). Others have also observed
less than 98% coupling efficiencies when using phosphoramidites
that were sterically crowded, of large molecular weight, or of
nonstandard nucleobases (M. Grotli et al. Tetrahedron 53, (1997),
11317-11346). Optimization of the automated solid-phase synthesis
by varying, for example, the capping times, coupling times,
phosphoramidite concentrations, tetrazole concentration, and
waiting times are expected to increase coupling efficiencies. Once
the ruthenium-modified oligonucleotide was synthesized, the 5'DMT
was left on the oligonucleotide for purification ease. The
nitrogenous bases and phosphate groups were subsequently
deprotected in 30% ammonium hydroxide at 55.degree. C. for 12
hours. Finally, the ruthenium-labeled oligonucleotide was purified
using a standard Poly-Pak.TM. cartridge (Glen Research) and
reverse-phase HPLC methods (C 18; 0.1 M TEAA/CH.sub.3CN; 10-50%
gradient over 50 minutes; monitoring at 254 and/or 450 nm). The
ruthenium-modified oligonucleotides exhibit one peak in an HPLC
trace, with retention times greater than the corresponding
unmodified oligonucleotide. MALDI, Matrix Assisted Laser Desorption
Ionization-time of flight, mass spectrometry of the
metallo-oligonucleotide also confirms formation [e.g., ruthenium
modified oligonucleotide 8, MALDI (5058.25; 5055.6; calculated:
found.+-.3)].
[0036] Thermal denaturation experiments on the unmodified and
modified oligonucleotide duplexes showed a moderate decrease in
thermal stability when the ruthenium was introduced into the middle
of the oligonucleotide sequence compared to an unmodified duplex
(150 mM sodium phosphate; pH 7;
[0037] monitoring at 260 nm). (FIG. 3) The melting temperature (Tm)
of the unmodified duplex, duplex. 9.cndot.10 (see Table 1), was
60.degree. C., compared to 51.degree. C. for the ruthenium-labeled
duplex, duplex 6.cndot.10. This effect was diminished when the
metal complex was on the terminal nucleotide of the oligonucleotide
sequence all (duplex 7.cndot.10), Tm=60). This decrease in Tm with
the ruthenium-modified oligonucleotide duplex, 6.cndot.10, was of
lesser magnitude than that observed for a single mismatch in a
duplex. These data illustrate that the metallo-oligonucleotides
form stable duplexes at room temperature, and are amenable to
photophysical characterization and further study.
[0038] The ruthenium-uridine described herein is a suitable
chromophore for reductive quenching studies since it is
photochemically stable, inert to ligand substitution reactions,
possesses an energetic excited state (0.84 eV), and a long lifetime
in fluid solution. Moreover, the excited-state electron is
localized on the bipyridine attached to the uridine. The
electron-transfer quencher, phenothiazine is known to be a very
efficient electron donor for quenching
*Ru(bpy).sub.3.sup.2+.(Meckle- nburg, S. L.; Peek, B. M.;
Schoonover, J. R.; McCafferty, D. G.; Wall, C. G.; Erickson, B. W.;
Meyer, T. J. J. Am. Chem. Soc. 1993, 115, 5479-5495. Slate, C. A.;
Striplin, D. R.; Moss, J. A.; Chen, P.; Erickson, B. W.; Meyer, T.
J. J. Am. Chem. Soc. 1998, 120, 4885-4886.) The biomolecular
electron-transfer reaction between compounds 4 and 7 of FIGS. 1A-1B
was studied in solution by varying the quencher concentration.
[0039] Stern-Volmer analysis yielded a quenching rate constant (kq)
of 1.3.times.10.sup.9 M.sup.-1 s.sup.-1. Based on the reduction
potential of PTZ+/0 (0.76 eV), the driving force for this
electron-transfer reaction was estimated to be approximately 0.1
eV.
[0040] In this DNA-mediated electron-transfer system, the electron
donor and acceptor were covalently attached to different
oligonucleotide strands and separated by about 30 .ANG.. First, the
complimentary duplex containing only the ruthenium acceptor (5'-TCA
ACA GU*T TGT AGC A-3' (SEQ ID NO: 1); 5'-TGC TAC AAA CTG TTG
A-3'(SEQ ID NO: 5)) was synthesized (U*=Ru(diimine).sub.3.sup.2+
linked uridine). The emission maximum for this ruthenium-labeled
oligonucleotide duplex was centered at 660 nm and the emission
lifetime was measured to be 540 ns at 20.degree. C. in phosphate
buffer (monitoring at 640 nm after 455 nm pulse excitation). Next,
phenothiazine (PTZ) was attached to the 5'-terminal of the
complimentary sequence of the ruthenium labeled oligonucleotide
(5'-TCA ACA GU*T TGT AGC A-3' (SEQ ID NO: 1); 5'-PTZ-TGC TAC AAA
CTG TTG A-3' SEQ ID NO: 5)). Reductive quenching of the excited
state was observed, and the rate constant was determined to be
2.6.times.10.sup.5 s.sup.-1. The lifetime and electron-transfer
rates were measured using a Laser Photonics LN1000 Nitrogen
Laser-LN102 dye laser (coumarin 460 dye). The emission was
monitored at right angle with a Macpherson 272 monochromator and
Hammamatsu R666-10 PMT at 22.degree. C. The signal was processed by
a LeCroy 7200A transient digitizer interfaced with an IBM-PC. The
excitation wavelength was 455 nm and the monitoring wavelength was
640 nm. Power at the sample was 60 W/pulse.mm3 as measured by a
Molectron J3-09 power meter. The measured instrument response
function is 10 ns (FWHM). The acquired emission decay curves were
analyzed by a locally written software based on the Marquardt
algorithm. The data were fit to a single exponential. The residuals
between the experimental and fitted curves were less than 2%. The
electron-transfer rate constant was determined using the following
equation: k=1/t-1/t0.
[0041] Over the temperature range of 5 to 30.degree. C., the rate
constant increased slightly from 2.5 to 2.8.times.10.sup.5
S.sup.-1.
[0042] The synthetic strategy of the present invention will be
recognized to be a general one which is amenable to attaching
electron donors and acceptors at site-specific locations; and these
modified duplexes are suitable for electron-transfer measurements.
The microsecond electron-transfer rate was measured over this
extended distance indicates that long-range electron transfer from
phenothiazine to *Ru(diimine).sub.3.sup.2+ occurs. Importantly, the
Ru(diimine).sub.3.sup.2+ modified nucleoside and phenothiazine
phosphoramidites expand the current repertoire of available DNA
electron-transfer probes.
[0043] In a further embodiment, the present invention provides
labeled or detectable moieties which are attached to the 5', 3' or
2' carbon of a nucleoside or nucleotide sugar. As an
exemplification of this embodiment, DNA was labeled at the
5'-amino-ribose of an oligonucleotide with a substitutinally inert
transition metal complex, by automated solid phase synthesis.
Specifically, a Ru(diimine).sub.3.sup.2+-thymidine derivative,
5'-[Ru(bpy).sub.2(4-m-4'ca-bpy).sup.2+]-thymidine, was synthesized
and subsequently incorporated in an oligonucleotide as an active
phosphoramidite complex.
[0044] The synthesis of thymidine derivative is described with
reference to FIG. 7 The Ru(diimine).sub.3.sup.2+
derivatized-phosphoramidite, 6, for automated DNA synthesis was
synthesized in five steps starting from thymidine. As shown in FIG.
7, the 5'-position of thymidine was first converted from a hydroxyl
to an amine. Thymidine was dissolved in pyridine and reacted with
methanesulfonyl chloride to form compound 2. Next, the mesyl group
was substitute with an azide, and subsequently reduced with
PPh.sub.3 to yield the 5'-amino-thymidine, 4. The mono-carboxylic
acid derivatized Ru(diimine).sub.3.sup.2+ complex,
Ru(bpy).sub.3(4-m-4'-ca-bpy).sup.2+, was then coupled as the
activated ester to 5-amino-thymidine, 4, in DMF using CDI. Finally,
5 was reacted with
2-cyanoethylchloro-N,N-diisopropylphosphoramidite in dry CH.sub.3CN
to afford the metallo-phosphoramidite, 6. The ruthenium-thymidine
phosphoramidite was used in an oligonucleotide solid-phase
synthesizer whereby, in the last coupling step, the
ruthenium-modified thymidine phosphoramidite was introduced, as
shown in FIG. 8. All syntheses were performed at the 1.0 tmol scale
using the standard coupling protocol except that the final step
proceeded for 15 minutes to ensure sufficient time for the
Ru(diimine).sub.3.sup.2+-thymidine phosphoramidite to react with
the 5'-terminal alcohol of the oligonucleotide sequence. Once the
ruthenium-labeled oligonucleotide was synthesized, the
oligonucleotide was cleaved from the column. Next, the nitrogenous
bases and phosphate groups were deprotected in 30% ammonium
hydroxide at 55.degree. C. for 16 hours (see Table 2).
2TABLE 2 Oligonucleotides Synthesized 12 (SEQ ID NO: 7)
5'-*TTCAACAGTTTGT-3' 13 (SEQ ID NO: 8) 5'-*TCAACAGTTTGTAGCA-3' 14
(SEQ ID NO: 9) 5'-*TGCTACCCTCTGTTGA-3' 15 (SEQ ID NO: 10)
5'-*TTTCAACAGTTTGTAGCA-3' 16 (SEQ ID NO: 11) 5'-TTCAACAGTTTGT-3' 17
(SEQ ID NO: 12) 5'-TGCTACCCTCTGTTGA-3' 18 (SEQ ID NO: 13)
5'-TTCAACAGTTTGTAGCA-3' 19 (SEQ ID NO: 14) 5'-ACAAACTGTTGAA-3' 20
(SEQ ID NO: 15) 5'-TCAACAGAGGGTAGCA-3' 21 (SEQ ID NO: 16)
5'-TGCTACAAACTGTTGAA-3' * = ruthenium-modified thymidine
[0045] As shown in FIG. 9, the thermal denaturation curve for the
unlabeled (11.cndot.15) and Ru(diimine).sub.3.sup.2+ labeled
(12.cndot.19) 13-mer oligonucleotide duplexes are similar. The
decrease in the melting temperature from 42.degree. C. to
39.degree. C., respectively, is small and this magnitude of change
is also observed with larger duplexes (e.g., 18.cndot.21,
51.degree. C.; 15.cndot.21, 52.degree. C.). This relatively small
change indicates labeling the 5'-terminal nucleotide of the
oligonucleotide does not dramatically alter the duplex structure of
DNA. These results are further supported by circular dichroism (CD)
spectroscopy experiments. CD spectra of the unlabeled (16.cndot.19)
and Ru(diimine).sub.3.sup.2+ labeled oligonucleotides (12.cndot.19)
are similar, and the characteristic spectral features for B-DNA are
present (FIG. 10). In comparison to other labeling sites in an
oligonucleotide, attaching a Ru(diimine).sub.3.sup.2- + to the
terminal nucleobase of an oligonucleotide does not alter the
melting temperature nor the CD spectrum compared to the unlabeled
analog, (16.cndot.19). On the other hand, the melting temperature
decreases by approximately 15.degree. C. when the
Ru(diimine).sub.3.sup.2+ label is linked to the 5'-terminal
phosphate. The 5'-position of thymidine is a suitable site for
introducing labels such as transition metal complexes.
[0046] The absorption spectrum of the ruthenium-labeled
oligonucleotide single strand exhibits the characteristic
metal-to-ligand charge-transfer band (.sup.1MCT-.sup.1A.sub.1)
centered at 450 nm analogous to Ru(bpy.sub.3).sup.2+. Excitation of
the MLCT band of the ruthenium modified single strand
oligonucleotide produces an emission centered at 610 nm
corresponding to the .sup.3MLCT excited state. This emission is
essentially unchanged when a duplex is formed. This emissive
metallo-oligonucleotide can be attached to a surface and visualized
with a confocal microscope (excitation at 488 nm, emission
monitored at 600 nm).
[0047] The Ru(diimine).sub.3.sup.2+ derivatized thymidine complex,
5 (FIG. 7), exhibits the characteristic metal-to-ligand charge
transfer band (.sup.1MLCT-.sup.1A.sub.1), centered at 450 nm in the
absorption spectrum, analogous to Ru(bpy).sub.3.sup.2+ (FIG. 11).
At higher energy the absorbances at 260 and 280 nm are the n-.pi.*
and .pi.-.pi.* transitions of thymidine and bipyridine,
respectively. Excitation of the MLCT band at 450 nm produces an
emission centered at 666 nm in phosphate buffer (see FIG. 12), and
slightly red-shifted from Ru(bpy).sub.3.sup.2+ (625 nm) . The
emission lifetime of compound 5 is 430 ns in phosphate buffer
(25.degree. C.). These absorption and emission results are also
consistent with similar Ru(diimine).sub.3.sup.2+ complexes designed
for labeling the 5-terminal phosphate or the nucleobase of an
oligonucleotide.
[0048] To gain further insight into the excited-state electronic
structure of compound 5 (FIG. 7), time-resolved step-scan FTIR
spectroscopy (S.sup.2FTIR TRS) with 10 ns time resolution probed
the excited state of 5 in CD.sub.3CN at room temperature. The
ground- and excited-state infrared .upsilon.(CO) band energies for
5 are 1679 and 1647 cm.sup.-1, respectively, as shown in FIG. 13.
This substantial negative shift in .upsilon. of -31 cm.sup.-1is
similar in magnitude to other mono-amide derivatized
tris-bipyridine complexes such as those containing a propargylamide
and ethanolamide. The frequency shift to lower energy for
.upsilon.(CO) indicates that the MLCT excited-state electron
resides on the asymmetrically substituted bipyridine and that
significant C.dbd.O character is present in the lowest .pi.*
excited-state. The relatively large shift in .upsilon.(CO) suggests
considerable metal-ligand polarization of the MLCT excited state
with the receiving orbital localized primarily on the
amide-substituted pyridine ring. The metal d.pi.*ligand .pi.*
orbital overlap is also substantial in this complex. Finally, these
data are in agreement with previous work with related
asymmetrically and symmetrically amide and ester substituted
analogs of Ru(bpy).sub.3.sup.2+, indicating that on the nanosecond
time scale the excited-state electron is localized on the modified
bipyridine rather than delocalized over all three ligands or
exchanging between the ligands.
[0049] Once compound 5 of FIG. 7 is incorporated in the
oligonucleotide, 12, the emission maximum shifts to higher energy
(677 nm). The emission maximum is essentially unchanged after
hybridization, and the labeled duplex, 12.cndot.19, emission is
centered at 674 nm (FIG. 12). The emission lifetime of this
ruthenium-labeled oligonucleotide, 12, is 572 ns, and that of the
modified oligonucleotide duplex, 12.cndot.19, is 586 ns at
25.degree. C. in phosphate buffer (FIG. 14). The
Ru(diimine).sub.3.sup.2+ excited state is not quenched in the
presence of DNA and this observation is consistent with the redox
potentials of A, C, G, and T.
[0050] A ruthenium-oligonucleotide modified surface according to
the present invention may be synthesized by first constructing a
complementary oligonucleotide single strand, such as 11, to 6 that
contains an alkyl amine linker at the 3-terminus. The resulting
ruthenium modified duplex (6.cndot.11) containing the amino linker
may then be annealed, and then anchored to a cyclohexane epoxide
derivatized glass slide under basic conditions. Finally, the slide
is rinsed with copious amounts of buffer (HEPES 100 mM; pH=7), to
afford the surface bound ruthenium-oligonucleotide duplex.
[0051] While the above and the following exemplifies the use of
uridine, one of ordinary skill will appreciate that other
nucleosides, including modified nucleosides, and their
corresponding nucleotides (DNA and RNA) may be made and are
included within the present invention.
[0052] The present invention provides a purine, pyrimidine,
nucleoside or nucleotide derivative mi comprising a detectable
marker which is a metal complex containing at least one of a
M(diimine).sub.x.sup.y+ complex or a metallocene wherein M is a
transition metal, and the diimine is selected, for example, from a
bipyridine, phenanthroline or terpyridine derivative; x is 1, 2, or
3 and y is 0, 1, 2, 3, or 4, and the bipyridine, phenanthroline,
terpyridine or metallocene may be optionally substituted by any one
of alkyl (such as C.sub.1--C.sub.8, linear or branched alkyl),
alkene, alkyne, aryl, alkylaryl, carboxyalkyl, amide, ester or
ether. The diimines surrounding the metal of the complex may be the
same or different. The nucleoside or nucleotide derivatives
preferably contain the purine or pyrimidine. The purines of the
present invention are selected from natural or synthetic adenine
and guanine, or analogs and derivatives thereof which may also
include 8-aza-7-deazapurines, and 7-deazapurines. The pyrimidines
of the present invention are selected from natural or synthetic
cytosine, uracil and thymine, or analogs and derivatives thereof,
including 6-azapyrimidines.
[0053] In one embodiment, the purine, pyrimidine, nucleoside or
nucleotide derivative of the present invention contains a
detectable marker containing at least one of a
M(diiriine).sub.2(diimine).sup.2+ complex wherein M is a metal
selected from, for example, Fe.sup.+2, Ru+.sup.2, Os.sup.+2,
Co.sup.+2, Rh.sup.+2, and Cr.sup.+2 or a
M(diimine).sub.2(diimine).sup.3+ complex wherein M is a metal
selected from, for example, Fe.sup.+3, Ru.sup.+3, OS.sup.+3,
Co.sup.+3, Rh.sup.+3, and Cr.sup.30 3 or a M(terpyridine).sub.2
.sup.3+ complex wherein M a metal selected from, for example,
Fe.sup.+3, Ru.sup.+3, OS.sup.+3, Co.sup.+3, Rh.sup.+3, and
Cr.sup.+3 or a M(terpyridine).sub.2.sup.2+ complex wherein M is
metal selected from, for example, Fe.sup.+2, Ru.sup.+2, Os.sup.+2,
Co.sup.+2, Rh.sup.+2, and Cr.sup.+2.
[0054] The nucleoside, nucleotide or oligonucleotide, derivative of
the present invention is preferably linked to the detectable label
or marker through at least one of the 5, 3 or 2 carbon atom of a
sugar (ribose, deoxyribose or dideoxyribose) or the 5 carbon of a
pyrimidine or the 8 carbon of a purine. Preferably, the marker or
label is linked through a purine or pyrimidine.
[0055] The nucleoside or nucleotide of the present invention is
preferably optically active such that the complex is chiral wherein
said chirality is fac or ras, R or S, or D or L. Racemic mixtures
are also included.
[0056] The detectable marker of the present invention is preferably
selected from a bis
(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carbony- l propargyl
amine) ruthenium (II) substituent; a ferrocen substituent; a
bis(2,2'-bipyridine) (4'-methyl-2,2'-bipyridine-4-carbonyl
propargyl amine) osmium (II) substituent; and a
bis(2,2'-bipyridine)(4'-methyl-2,2'- -bipyridine-4-carbonyl
propargyl amine) rhodium (II) substituent.
[0057] An oligonucleotide containing at least one purine,
pyrimidine, nucleoside or nucleotide derivative of the present
invention is also provided. The oligonucleotide of the present
invention optionally contains substitutions, such as sulphur (S),
selenium (Se), imino (NH) or boron (B), in the phosphate
backbone.
[0058] The purine, pyrimidine, nucleoside or nucleotide derivative
of the present invention may include a sugar selected from, for
example, a ribose, a deoxyribose or a dideoxyribose. The purine,
pyrimidine, nucleoside or nucleotide derivative of the present
invention may further include any of a dimethoxytritylchloride,
oc-methyl-6-nitropipronyloxy carbonyl or 2-cyanoethyl
N,N'-diisopropylcholoro phosphoramidite derivative and/or a
compound of the following structure: 1
[0059] wherein n is either 0 or 1;
[0060] Y is an oxygen from a hydroxyl group, such as the 3' or 5'
hydroxyl group, of the nucleoside or nucleotide;
[0061] R.sub.1 , R.sub.2, R.sub.3, and R.sub.4 are selected
independently from hydrogen, a lower alkyl, alkyl, aryl, benzl,
halogen, hydroxyl, alkoxyl, thio, thioether, amino, nitro,
carboxyl, formate, formamido, or phosphido or adjacent substituents
are substituted oxygen groups that together form a cyclic acetal or
ketal; and R.sub.5 is selected from hydrogen, alkoxy, alkyl, aryl,
halo or alkenyl.
[0062] The metal complex of the present invention is preferably
covalently bound to a purine or pyrimidine derivative, or
nucleobase, in the nucleoside or nucleotide of the present
invention. The metal complex is preferably bound through an
alkynylamine formed from a terminal alkyne wherein the triple bond
may be attached to an amine by a linker moiety of 1-20 atoms. The
linker moiety can be straight-chain alkylene, (C.sub.1--C.sub.20
e.g., -C.sub.3H.sub.6-), or can contain double bonds (e.g., as in
-CCH .dbd.CHCH.sub.2-), triple bonds (e.g., as in
-C.dbd.C--CH.sub.2-) or aryl groups (e.g., para -C.sub.6H.sub.4-,
or para-CH.sub.2--CH.sub.2--C.sub.6H.sub.3-). The linker moiety can
also contain heteroatoms such as N, O, or S in the chain as part of
ether, ester, amine, or amido groups. Suitable substituents on the
linker moiety can include C.sub.1--C.sub.6 alkyl, aryl, ester,
ether, amine, amide or chloro groups. Preferably, the linker moiety
is a straight-chain alkylene (C.sub.1--C.sub.10); most preferably,
the linker moiety is -CH.sub.2-. Suitable substituents on the amine
are lower alkyl (C.sub.1--C.sub.4) and protecting groups such as
trifluoroacetyl. In general, the amine of the alkynylamine can be
primary, secondary or tertiary. For use as a linker, however, the
alkynylamine is preferably a primary amine.
[0063] In another embodiment, the present invention provides a
double-stranded nucleic acid sequence comprising the purine,
pyrimidine, nucleoside, nucleotide or oligonucleotide of the
present invention and an electron acceptor moiety such as one of
Cd, Mg, Cu, Co, Pd, Zn, Fe, Ru and Os. Methods for detecting the
transfer of electrons or energy between the electron acceptor and
the purine, pyrimidine, nucleoside, nucleotide or oligonucleotide
of the present invention are also provided. Detection of the
transfer of energy or electrons according to the methods of the
present invention may be used to detect the presence or absence of
binding of the purine, pyrimidine, nucleoside nucleotide,
oligonucleotides, nucleic acids, DNA or RNA of the present
invention to a complementary purine, pyrimidine, nucleoside,
nucleotide, oligonucleotide, nucleic acid, DNA or RNA in a sample
or population. This detection can, in turn, be used to detect,
diagnose or treat diseases by means known in the art.
[0064] The oligonucleotide, nucleoside or nucleotide of the present
invention may be attached to a solid surface which may include or
constitute an electrode, nonoparticle, glass, Au, Pt, TiO, ITO,
SnO.sub.2, silicon, porous silicon, plastic, such as polystyrene,
or graphite, for example, attached through one of a covalent bond,
an electrostatic association or a hydrogen bond.
[0065] The oligonucleotide, nucleoside or nucleotide of the present
invention may also contain a further reported group. A reporter can
include a chemical group which has a physical or chemical
characteristic which can be readily measured or detected by
appropriate physical or chemical detector systems or procedures.
Ready detectability can be provided by such characteristics as
color change, luminescence, fluorescence or radioactivity; or it
may be provided by the ability of the reporter to serve as a ligand
recognition site to form specific ligand-ligand complexes which
contain groups detectable by conventional (e.g., calorimetric,
spectrophotometric, fluorometric or radioactive) detection
procedures. The ligand-ligand complexes can be in the form of
protein-ligand, enzyme-substrate, antibody-antigen,
carbohydrate-lectin, protein-cofactor, protein-effector, nucleic
acid-nucleic acid or nucleic acid-ligand complexes.
[0066] The present invention provides an in vivo, in vitro, or ex
vivo method of identifying a nucleic acid molecule or detecting the
presence or absence of a nucleic acid molecule which method
includes contacting a sample containing or suspected of containing
the nucleic acid with a nucleotide, nucleoside, or oligonucleotide
of the present invention or a composition containing same, under
conditions where said nucleotide, nucleoside, or oligonucleotide
can specifically bind to a complementary region of said nucleic
acid to form an identifiable complex which may include a double or
triple helix, and detecting the binding, preferably by measuring a
photophysical, electrochemical, or electron transfer event or
alteration, such as an energy or electron transfer to or from the
detectable marker of the present invention. The nucleic acid
molecule of this method may be synthetic or of human, animal,
viral, bacteria, or plant origin.
[0067] The electron transfer event detected in the method of the
present invention may occur, for example, due to an interaction
between two metal complexes on a single nucleic acid strand, an
interaction between two metal complexes on separate nucleic acid
strands, or an interaction between a metal complex on a single
nucleic acid strand and a nucleoside or nucleotide of the present
invention.
[0068] Alternatively, the photophysical alteration or event
detected in the method of the present invention may occur due to an
interaction between a metal complex and an organic donor/acceptor
on a single nucleic acid strand, or an interaction between a metal
complex on a single strand and an organic donor/acceptor on a
second single nucleic acid strand, wherein the organic
donor/acceptor may be selected from, for example, acridine,
fluorescein, anthracene, metylphenothiazine, methylviolgen,
anthroquione, benzoquione, phenothiazine and quione. The electron
transfer event may be, for example, photochemically or
electrochemically induced, such as by electrogenerated
luminescence.
[0069] A method of the present invention includes use of a solid
surface containing the oligonucleotide, nucleotide or nucleoside of
the present invention attached to the surface and the metal complex
donates an electron or energy to the surface or the metal complex
accepts an electron or energy from the surface, whereby the
detection or identification involves measuring the transfer of the
electron or energy.
[0070] Alternatively, the method of the present invention involves
binding the oligonucleotide, nucleotide or nucleoside directly or
indirectly to a nucleic acid molecule attached to a surface wherein
the metal complex donates an electron or energy to the surface or
the metal complex accepts an electron or energy from the surface
such that the detection or identification involves measuring the
transfer of the electron or energy.
[0071] The present invention therefore provides
metallo-oligonucleotide derivatives. Specifically, the present
invention provides [(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbonyl propargyl
amine) Ru(II)-2'-deoxyuridine].sup.+22PF.sub.6.sup.-, [(Bpy).sub.2
bis(2,2'-bipyridine) (4'-methyl-2,2'bipyridine-4-carbonyl propargyl
amine)
[0072] Ru(II)-2'-deoxycytidine].sup.+22PF.sub.6.sup.-, [(Bpy).sub.2
bis(2,2'-bipyridine) (4'-methyl-2,2'bipyridine-4-carbonyl propargyl
amine) Ru(II)-2'-deoxyadenesine].sup.+22PF.sub.6.sup.-,
[(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbonyl propargyl
amine)
[0073] Ru(II)-2'-deoxyguanosine].sup.+22PF.sub.6 , [Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbonyl propargyl
amine) Ru(II)-2'-deoxythymidine].sup.+22PF.sub.6.sup.-,
[0074] [(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbon- yl
propargyl amine) O.sub.5 (II)-2-deoxyuridine].sup.+22PF.sub.6,
[0075] [(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbon- yl
propargyl amine) O.sub.5 (II)-2-deoxycytidine].sup.+22PF.sub.6,
[0076] [(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbon- yl
propargyl amine) O.sub.5
(II)-2-deoxyadenosine].sup.+22PF.sub.6,
[0077] [(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbon- yl
propargyl amine) O.sub.5
(II)-2-deoxyguanosine].sup.+22PF.sub.6,
[0078] [(Bpy).sub.2
bis(2,2'-bipyridine)(4'-methyl-2,2'bipyridine-4-carbon- yl
propargyl amine) O.sub.5
(II)-2-deoxythymidine].sup.+22PF.sub.6,
[0079] Nucleotide, ribose, dideoxy, ferrocene,
dimethoxytritylchloride, .alpha.-methyl-6-nitropipronyloxy carbonyl
and 2-cyanoethyl N,N-'diisopropylcholoro phosphoramidite
derivatives, individually or combined, of the above are also
provided. Derivatives of the compounds described herein which
include a photoremovable or photolabile protecting group, such as
are taught in U.S. Pat. No. 5,753,788 are also included in the
present invention.
[0080] A preferred class of photoremovable protecting groups
include aromatic compounds that absorb near-UV and visible
radiation, such as are described, for example, in McCray et al., J.
Amer. Chem. Soc. (1970) 92:6333; and Amit et al. J. Org. Chem
(1974) 39:192. Particularly preferred photoremovable protecting
groups include those of the formula 2
[0081] where R.sub.1, R.sub.2, R.sub.3, and R.sub.4 independently
are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen,
hydroxyl, alkoxyl. thio, thioether, amino, nitro, carboxyl,
formate, foramino or phosphido group, or adjacent substituents
Ri-R4 are substituted oxygen groups that together form a cyclic
acetal or ketal; R.sub.5 is hydrogen, alkoxyl, alkyl, halo, aryl or
alkenyl group, and n=0, 1, 2 or 3. Preferred photoremovable
protecting groups include the above wherein R.sub.2 and R.sub.3
together form a methylene acetal, R.sub.1 and R.sub.4 are hydrogen,
R.sub.5 is hydrogen or methyl, and n=0 or 1; and the above where
R.sub.2 and R.sub.3 are each a methoxy group, R.sub.1 and R.sub.4
are each hydrogen, R.sub.5 is methyl or hydrogen and n=0 or 1.
[0082] The present invention provides oligonucleotide synthesis and
nucleic acid sequencing methods which incorporate and utilize the
metallo-nucleotides, -nucleosides, -purines or -pyrimidines of the
present invention. Synthesis and sequencing methods and techniques
are known and described in the art, such as in any of U.S. Pat.
Nos. 5,599,695; 5,753,788; 5,047,519; 5,691,146; 5,580,732; and
5,443,791.
[0083] Once synthesized, the oligonuceotides of the present
invention may be used, for example, as hybridization probes, PCR
amplification tools to detect the presence of a specific DNA or
RNA, such as in the diagnosis or treatment of disease, to identify
specific alleles, to perform tissue typing, to detect genomic
sequences in a DNA sample, such asfor forensic DNA analysis
(including RFLP analysis or PCR product length distribution) or for
diagnosis or treatment of diseases characterized by amplification
and/or rearrangement of a characteristic gene.
[0084] The metallo-nucleotides of the present invention may be used
as probes in oligonucleotides either as bound to a solid support,
as part of a solution method or bound to an electrode probe such
as, for example, in a method of measuring changes in the rate of
electron transfer. One of skill will appreciate that the electron
transfer rate of a probe containing a metallo-nucleotide of the
present invention will be altered by binding the probe with a
complementary nucleic acid sequence. That is, the presence of
double stranded nucleic acids, for example, in an oligonucleotide
probe assay, can be determined by comparing the rate of electron
transfer for the unhybridized probe with the rate for hybridized
probes.
[0085] The metallo-nucleotides of the present invention may be used
as an electron transfer moiety which, when used in conjunction with
a second electron transfer moiety, may be used to detect hybridized
nucleic acid sequences. Generally, the electron transfer method
involves a first nucleic acid single strand which is either
modified with two different metallo-nucleotides, or separate
modifications are provided on two complementary strands. After
hybridization, the sample is excited by a laser, for example, and
the rate of photo-induced electron transfer is measured from one
metal site to another. Winkler et al., Chem. Rev. 92, 369-379
(1992).
[0086] In one embodiment, a single strand of DNA containing two (or
more) metallo-nucleic acid bases is synthesized, as described
herein. The two metallo-nucleic acid bases on the strand can be
located at any site except next to each other. Next, a solution
containing single stranded DNA is added. One of the single strands
in solution is the target-compliment strand to the modified single
strand or metallo-probe strand. The target strand binds to the
metallo-probe strand. A photo induced electron-transfer measurement
is taken. The difference in the rate of electron transfer between
the single strand metallo-probe and the double strand
target-metallo probe strand duplex is determined. This difference
in electron-transfer signifies that the compliment is bound. As
such, this homogeneous method can be used to detect hybridization
and used to collect genetic information.
[0087] In another embodiment, two single strands of nucleic acids
each containing one metallo-nucleic acid base, or nucleotide, are
synthesized, as described herein. Next a single strand of nucleic
acid is added. The two metallo-probe strands will then bind to the
one strand that is the complementary strand to form a duplex. A
photo induced electron-transfer measurement is taken. The
observation of electron-transfer and measurement of the rate
signifies that both metallo-probe strand were bound to the
complementary strand. This method can be used to measure and target
pieces of DNA.
[0088] In yet another embodiment, a single strand of
metallo-nucleic acid DNA is synthesized, as described herein and
then is attached to a surface. This probe strand is then used to
detect a single strand of target complementary nucleic acid. The
target strand is modified with a metallo-nucleic acid by means
known in the art. The target is then incubated with the probe
strand to allow for hybridization and formation of the duplex. The
photo induced or electrochemical induced electron-transfer rate is
then measured. The detection of the compliment is determined by the
change in electron-transfer rate compared to the single probe
strand.
[0089] A preferred method of the present invention provides for one
of the electron transfer moieties being the metallo-nucleotides of
the present invention and the other electron transfer moiety being
in the form of a solid support, such as an electrode.
[0090] In this embodiment, a single stranded nucleic acid probe
containing at least one electron transfer moiety is attached via
this redox hydrogel to the surface of an electrode. Hybridization
of a target sequence can then be measured as a function of
conductivity between the electron transfer moiety covalently
attached to one end of the nucleic acid and the electrode at the
other end. This may be done using equipment and techniques well
known in the art, such as those described in the references cited
herein.
[0091] In similar embodiments, two nucleic acids are utilized as
probes as described previously. For example, one nucleic acid is
attached to a solid electrode, and the other, with a covalently
attached electron transfer moiety, is free in solution. Upon
hybridization of a target sequence, the two nucleic acids are
aligned such that electron transfer between the electron transfer
moiety of the hybridized nucleic acid and the electrode occurs. The
electron transfer is detected as outlined above, or by use of
amperometric, potentiometric or conductometric electrochemical
sensors using techniques well known in the art.
[0092] In a further embodiment, a single strand of nucleic acid DNA
is synthesized as described above containing a metallo-nucleic acid
(metallo-probe strand #1) and then it is attached to an electrode
surface (or non-electrode surface). A second single strand of DNA
is synthesized as above containing the metallo-nucleic acid
(metallo-probe strand #2). An electrode-induced or photoinduced
electron-transfer measurement is performed. Both probe strands are
then used to detect a single strand of compliment of DNA. The
electron-transfer rate is measured from the electrode to the
metallo-nucleic acid (or vice versa). The difference in
electron-transfer rate compared to the single strand probe and the
double stand (target-probe) is determined. Detection of the single
strand is confirmed.
[0093] In another embodiment, a single strand of DNA containing two
metallo-nucleic acids is synthesized as described above. In one of
the metallo-nucleic acids, the metal complex is modified to contain
a linker between itself and the surface (electrode or non-electrode
surface). The target strand is then added and it binds to the
compliment. The electron-transfer rate is then measured between the
metal complexes and detection of the single strand is
determined.
[0094] In yet another embodiment, a single strand of DNA containing
one metallo-nucleic acids is synthesized as described herein. The
metallo-nucleic acid is modified to contain a linker between itself
and the surface (electrode or non-electrode surface). The target
strand is then added which has been modified to contain a
metallo-nucleic acid, and it binds to the compliment. The electron
transfer rate is then measured between the metal complexes and
detection of the single strand is determined.
[0095] In the above detection methods, electron-transfer rates are
measured. The ability to detect the single strand is based on the
degree or relative change in electron-transfer. Since the electron
transfer rate is dependent of the ability to form a DNA duplex,
this technique can be used to detect other than perfect
compliments. In other words, point mutations, mismatches,
substitutions and deletions, where the double stranded nucleic acid
is not perfectly matched, will be detectable.
[0096] It will also be appreciated that, in the same manner as
described above, the detection of a triple helix can be
accomplished. In such a method, a single probe strand containing a
metallo-nucleic acid is linked to a surface. The target double
strand which binds to the single strand is added and triple helix
formation is detected by the standard methods described above.
Conversely, a probe double strand can be used to detect a single
strand of nucleic acid (DNA or RNA).
[0097] While this embodiment has been described by reference to a
single probe on the surface of an electrode, the present invention
also includes formation and use of a multiple array of probes which
may be packaged in, for example, a chip such as has been described
Pease et al., (PNAS 91, 5022-5026 (May 1994)) and U.S. Pat. Nos.
5,753,788 and 5,599,695 which may be interrogated after
hybridization of target or sample sequences by electron transfer
rather than fluoresence. The photolabile derivatives of the
nucleoside and nucleotides of the present invention are
particularly preferred in making these multiple probe arrays. The
chip or substrate of this embodiment may be used as a multiple
electrode array for detection of hybridization. Alternatively,
combinations of electron transfer and fluorescent probes may be
used in the method of this embodiment.
[0098] A further embodiment of the present invention provides an
improved method of labeled nucleic acid polymer synthesis. A
preferred method of this embodiment provides use of a purine or
pyrimidine, or derivatives thereof, which have been modified, for
example, in the C-8 and C-5 positions, respectively, with a group
that can be subsequently replaced with a label or reporter group or
a detectable marker, such as is described herein or generally known
in the art, after nucleic acid polymer synthesis. An alternative
method of this embodiment provides modification of nucleotides or
nucleosides in the C-2, C-3 or C-5 of the sugar with a group that
can be subsequently replaced with a label, reporter or detectable
marker. The novel method of the present invention preferably
employs standard solid phase chemistry, such as is described, for
example, in U.S. Pat. No. 5,047,519; 4,849,513 or 5,428,149;
wherein the modified nucleic acid is introduced into an
oligonucleotide during synthesis. This embodiment of the present
invention provides for coupling of the reporter or labeling group
to the nucleoside after synthesis of the nucleic acid polymer or
oligonucleotide, while the oligonucleotide is attached to the solid
phase, as opposed to conventional methods of adding a labeled
nucleotide to the automated synthesis process. While the synthesis
method is exemplified herein as being applicable to conventional
solid-phase 3' to 5' synthesis, the method may also be applicable
in 5' to 3' synthesis, such as is described in Caruthers (U.S. Pat.
No. 4,415,732) and/or in chip manufacture, such as in U.S. Pat.
Nos. 5,753,788 and 5,599,695.
[0099] This improved method of the present invention provides for
the direct incorporation of "probes" (labels, reporter groups or
detectable markers) into oligonucleotides using solid phase
technology. The advantages of this synthetic strategy include: 1)
fewer synthetic steps, 2) efficient coupling, 3) ease of
purification, and 4) ability to incorporate a wide-range of organic
and inorganic probes or reporter groups.
[0100] One of skill in the art will appreciate that DNA synthesis
may be accomplished as follows. A reactive 3' phosphorous group of
one nucleoside is coupled to the 5' hydroxyl of another nucleoside.
The former is preferably a monomer, delivered in solution; the
latter is preferably immobilized on a solid support. At least three
other chemical reactions are necessary to prepare the growing chain
of DNA for the next coupling, as described below. In his way, a
synthesis cycle is conducted, adding one nucleoside monomer at a
time. When the oligonucleotide chain is completed, the crude DNA is
cleaved from the support and protecting groups removed from the
bases. It is then ready to be desalted or purified and used.
[0101] More specifically, for example, the first step of the
synthesis cycle is detritylation of a tritylated nucleoside, where
the dimethoxytrityl (DMT) group is removed to free the 5' hydroxyl
for the coupling reaction. The next step is coupling, in which an
activated intermediate is created by simultaneously adding a
phosphoramidite nucleoside monomer and for example, tetrazole, or
other weak acid. The tetrazole protonates the nitrogen of the
phosphoramidite, making it susceptible to nucleophilic attack. The
next step, capping, terminates any chains that did not undergo
coupling. Since the unreacted chains have a free 5' OH, they can be
terminated or capped by acetylation. These unreacted chains become
"failure products." Capping is done, for example, with acetic
anhydride and 1-methylimidazole. Since the chains that reacted with
the phosphoramidite in the previous step are still blocked with the
dimethoxytrityl group, they are not affected by this step. Although
capping is not absolutely required for DNA synthesis, it minimizes
the length of impurities, making it possible to do post-synthesis
trityl-selective purification of the final product. Finally, the
intemucleotide linkage is converted from the phosphite to the more
stable phosphotriester by oxidation with an iodine solution. For
the synthesis of phosphorothioate oligonucleotides, the
intemucleotide phosphite is sulfurized between coupling and
capping. After oxidation, the DMT group is removed with
trichloroacetic acid and the cycle repeated until chain elongation
is complete. At this point, the oligonucleotide is cleaved from the
solid support with concentrated ammonium hydroxide. Ammonia
treatment also removes the cyanoethyl phosphate protecting groups.
The crude DNA in ammonium hydroxide solution is then heated to
remove the protecting groups on the exocyclic amines of the
bases.
[0102] The improved method of the present invention involves
addition of at least one halodonucleoside (where halo may be any of
fluorine, chlorine iodine or bromine, such as any of fluorouridine,
fluoroadenosine, fluoroguanosine, fluorocytidine, fluorothymidine,
chlorouridine, chloroadenosine, chloroguanosine, chlorocytidine,
chlorothymidine, iodouridine, iodoadenosine, iodoguanosine, wr:
iodocytidine, iodothymidine, bromouridine, bromoadenosine,
bromoguanosine, bromocytidine and bromothymidine) into the growing
oligonucleotide during synthesis, with subsequent attachment of a
reporter group to same through Pd(O) or other metal mediated
reaction forming a C--C bond, such as a Heck or Sonogashira
reaction either prior to or subsequent to removal of the
oligonucleotide from the solid support. In this way, the improved
method of the present invention allows for synthesis of labeled
oligonucleotides without the need for using labeled nucleosides
during the oligonucleotide synthesis process. The present invention
provides therefore an improved method of oligonucleotide synthesis
which includes combining, for example, phosphoramidite nucleoside
derivatives in combination with a solid support wherein the
improvement includes adding at least one phosphoramidite
halonucleoside during the synthesis and subsequently labeling the
halonucleoside with a marker in a metal catalyzed reaction.
[0103] A preferred means of introducing the detectable labels of
the present invention involve the use of halodonucleoside
intermediates, such as are described U.S. Pat. No. 5,047,519, which
may be incorporated in automated and manual oligonucleotide
synthesis techniques. One common method of introducing a side chain
to a nucleic acid involves the use of palladium and mercury. This
acetoxymercuration reaction was developed for introducing
covalently bound mercury atoms into the 5-position of the
pyrimidine ring, the C-8 position of the purine ring or the C-7
position of a 7-deazapurine ring, of nucleotides. Organomercurial
compounds would then react with an olifinic compound in the
presence of a palladium catalyst to form a new carbon-carbon bond.
The palladium coupling reaction, or Heck reaction, has been
previously used to construct carbon-carbon bonds as described in
JACS 1968, 90, 5518; JACS 1968, 90, 5526; JACS 1968, 90, 5531; JACS
1968, 90, 5535; and JACS 1969, 91, 6707. The use of this reaction
in modified nucleic acid synthesis is described in PNAS, 1973, 70,
2238; Biochem. 1975, 14, 2447; JACS, 1978, 100, 8106; and JACS
1980, 102, 2033. This procedure is performed in solution with the
mercurial labeled nucleic acid and a palladium catalyst. However,
the use of mercury and the purification procedures make this
methodology unfavorable, although extensively used.
[0104] A number of nucleotide analogs that contain potential
organic and inorganic probe ligands covalently attached to the C-5
pyrimidine ring that can be a used as an affinity reagent have been
synthesized to exemplify this improved method. Accordingly, this
improved method has included the synthesis, and characterization of
organic and inorganic labeled derivatives of 5'-deoxyuridine, and
their direct incorporation into an oligonucleotide on solid support
using the Heck reaction.
[0105] The C-5 position of deoxyuridine, for example, has been the
target of extensive modifications. When incorporated into duplex
DNA such modifications are located in the major groove, and as such
do not disrupt Watson-Crick base pairing. These C-5 deoxyuridine
analogs, for example, can be incorporated into a DNA oligomer using
solid phase synthesis and standard phosphoramidite chemistry, as
exemplified herein. Modification of the sugar of nucleosides and
nucleotides, as described and exemplified herein, may also provide
advantages.
[0106] One application of modified nucleic acids involves the use
of sequence specific antisense or antigene oligonucleotides to
control gene expression. This is a promising approach to treating a
range of diseases that include viral infection and cancers. In
recent years considerable efforts have been invested in the
synthesis of chemically modified oligonucleotides to improve
nuclease stability, target binding affinity and cellular uptake
properties.
[0107] The present invention provides an improved method of
oligonucleotide synthesis therefore which includes attaching a 3'
nucleoside, such as any of adenosine, guanosine, cytidine or
uridine, of the oligonucleotide to be synthesized to a solid
support through a labile linker arm, preferably an acid- or base-
labile linker arm, such as a phosphoramidite derivative. The
nucleotide preferably includes a di-p-ansylphenylmethyl (or
dimthoxytrityl) group on the 5' carbon of the sugar, such as ribose
or deoxyribose, as is commonly used in automated oligonucleotide
systems.
[0108] The improvement of the presently disclosed method includes
incorporating at least one nucleotide containing at least one
halonucleoside into the oligonucleotide during the automated or
manual synthesis method followed by attaching a detectable marker
to the at least one nucleotide through preferably, an alkynylamine
group. Preferably, the iodo- or bromo-nucleoside is a 5-iodo- or
5-bromo-pyrimidine or 7-iodo- or 7-bromo-purine. 7-deazapurines,
8-aza-7-deazapurines and 6-azapyrimidines may also be used. The
labeled alkynylamino-nucleotides of this aspect of the present
invention are preferably prepared through a palladium catalyzed
reaction.
[0109] The palladium catalyzed reaction is preferably carried out
in the presence of a Cu(I) co-catalyst. Suitable palladium
catalysts include Pd(0) complexes, for example,
tetrakis(triarylphosphine)Pd(0). The amount of Pd catalyst used is
generally 1-25 mol % (based on the iodonucleoside), preferably 5-15
mol %. The mole ratio of Cu(I) co-catalyst to Pd(0) catalyst is
more than 1 but less than 20.
[0110] The following Examples serve to more fully describe the
manner of using the above-described invention. It is understood
that these examples in no way serve to limit the scope if this
invention, but rather are presented for illustrative purposes.
Unless defined otherwise, the following abbreviations have been
used herein: MLCT is Metal to ligand charge transfer; HEPES is
4-(2-hydroxyethyl)-1-piperazinrrthanesulfonic acid; HOBt is
1-hydroxybenzotriazole; DIPEA is N,N-diisopropylethylamine; DMF is
N,N-dimethylformamide; TEA is triethylamine; DMT is
dimethoxytrityl; FPAU is Ferrocene parpagyl amide uridine; MsCl is
methanesulfonyl chloride; CDI is carbonyldiimidozole; LH-20 is
Sephadex LH20 resin; DIPEA is N,N-diisopropylethylamine; DCC is
dicyclohexylcarbodiimide; BPLC is High Pressure Liquid
Chromatography; FAB-MS is Fast atom bondarment mass
spectrometry;
[0111] TEAA is triethylamine acetate; MALDI is Matrix Assisted
Laser Desorption Ionization-time of flight mass spectoscopy; Tm is
melting temperature; 4-m-4'-ca-bpy is 2,2'-bipyridine=bpy;
4-Methyl-2, 2'-bipyridine-4'-carboxylic acid; M. W. is molecular
weight; EDTA is Ethylenediaminetetraacetic acid; B-DNA is B-form
DNA; CDI is carbonyl diimidazole; ODN is Oligonucleotide; ACN is
acetonitrile; ITO is indium tin oxide; and DCU is
dicyclohexylcarbodiimide.
EXAMPLE 1
[0112] 5-Iodo-2'-deoxyuridine and benzoyl chloride were dissolved
in dry pyridine and stirred at room temperature for overnight. The
solvent was then removed by vacuum distillation and the resulting
yellow oil was dissolved in ethyl acetate, washed with 5% sodium
bicarbonate, 0.5 M HCI, water and the solution was then dried over
sodium sulfate. The solvent was removed by rotary evaporator and
the crude product was purified on a silica gel column (in 2%
MeOH(CHCl.sub.3) in 20% hexane/chloroform. FAB-MS: molecular weight
of 562; NMR peaks are consistent with the structure.
EXAMPLE 2
[0113] Ferrocene monocarboxylic acid, propargylamine HC1, HOBt and
TEA were dissolved in DMF/DCM at 0.degree. C. DCC was dissolved in
DMF and than added dropwise to the reaction mixture. The mixture
was stirred and allowed to reach room temperature overnight.
Solvent was next removed by vacuum distillation. The solid ws then
dissolved in ethyl acetate, and DCU was removed by filtration. The
ethyl acetate solution was washed with NaHCO.sub.3(5%),0.5 N HCl,
brine and H.sub.2O and dried over sodium sulfate. Solvent removed
by rotary evaporation. Product purified FAB-MS: M. W. 269, 1; NMR
peaks are consistent with the structure.
EXAMPLE 3
[0114] FPA, 5-iodo-3',5dibenzolyloxy-2'-deoxyuridine,
(PPh.sub.3).sub.4Pd(0) and Cul were dissolved in dry DMF, and then
TEA added to the mixture. The reaction mixture was stirred for 6
hours at room temp. The solvent was next removed by vacuum
distillation, and chloroform was added. This mixture was then
washed with 10% EDTA, water and dried over sodium sulfate and
solvent removed by rot. evap. An orange oil dissolved in the
chloroform which was subsequently crashed out of hexane. The solid
compound was purified further on silica gel column (in 2%
MeOH/chloroform). FAB-MS: molecular weight of compound expected to
be 701; mass spectrum shown peak at 805. NMR: characteristic peaks
of FPA and uridine moieties.
EXAMPLE 4
[0115] The orange FPAU was dissolved in methanolic ammonia solution
and allowed to react for 48 hours. The solvent was removed by
rotary evaporation. A solid was washed with ether and dried on high
vacuum. FAB-MS: molecular weight 492,9, NMR assigned.
EXAMPLE 5
[0116] Deprotected FPAU was dissolved n dry pyridine and the
solvent was removed by vacuum distillation. This process was
repeated 2 times. Finally the FPAU was dissolved in dry pyridine
and dimethoxytrityl chloride (DMT-Cl) was added to the flask. The
mixture ws stirred at room temperature under nitrogen
overnight.
[0117] Methanol was then added to the reaction mixture to consume
and excess DMT-Cl. The solvent was removed by high vac. A solid was
then dissolved in CHCl.sub.3 and crashed out of ether. FAB-MS:
molecular ion peak of 795.3, NMR assigned.
EXAMPLE 6
[0118] The DMT-FPAU was dissolved in dry acetonitrile and
diisopropylethylamine and
2-cyanoethylchloro-N,N-diisopropylphosphramidit- e was added
dropwise. The reaction mixture was stirred under N.sub.2 for 4
hours. The reaction mixture was then added dropwise to a stirring
ether solution and a precipitate formed. The solid was dried on KOH
pellets under high vacuum. The sample was then used in a DNA
synthesizer without further purification.
EXAMPLE 7
[0119] 4'-Methyl-2,2'-bipyridine-4-carboxylic acid, propargylarnine
hydrochloride, HOBt and TEA dissolved in DMF 0.degree. C. DCC was
dissolved in DMF and added dropwise to the reaction mixture. The
mixture was stirred at room temperature overnight. Solvent was
removed by vacuum distillation. The solid compound was dissolved in
ethyl acetate and DCU was removed by filtration. The ethyl acetate
solution was washed with NaHCO.sub.3(5%), 0.5 N HCl, brine and
dried over sodium sulfate. Solvent was removed by rot. evap. and
the compound was purified by column chromatography using 2%
methanol in chloroform as eluent. FAB-MS: M. W. 251[M+]; NMR
assigned.
EXAMPLE 8
[0120] Rubpy.sub.2Cl.sub.2 and
4'-methyl-2',2'-bipyridine-4-carbonyl propargyl amine were
suspended in 70% ethanol and refluxed for 10 hours. Reaction
mixture was cooled and ethanol was removed. The water solution
after standing for 4 hours at room temperature, the mixture was
filtered and the solid compound washed with cold water. In
filtrate, a saturate aqueous solution NH.sub.4PF.sub.6 was added
until no further ppt. was observed and the mixture was kept at room
temperature for 2 hours and then filtered, washed with cold water,
ether and dried overnight to get pure orange color compound.
EXAMPLE 9
[0121]
Bis(2,2'bipyridine)(4'-methyl-2,2'-bipyridine-4-carbonylpropargyl
amine) ruthenium(II), 5-iodo-3',5'-dibenzoyloxy-2'-deoxyuridine,
(PPh.sub.3).sub.4Pd(0), and Cul were dissolved in dry DMF, and TEA
was added to mixture. The reaction mixture was stirred for 8 hours
at room temp. The solvent was removed by vacuum distillation
keeping the temperature less than 45.degree. C. The orange oil was
dissolved in dry acetonitrile and precipitated by added dry ether.
The orange compound was filtered and washed with dry ether and
dried over KOH pellets at high vacuum. FAB-MS: 1989. NMR:
assigned.
EXAMPLE 10
[0122] The
[(bpy).sub.2(bpy-N-propargyl)Ru(II)-3',5'-dibenzolyloxy-2'-deox- y
uridine]2PF.sub.6- was dissolved in methanolic ammonia solution and
left for 48 hours with occasional shaking. The solvent was removed
by rotary evaporation. A orange solid compound was dissolved in dry
CH.sub.3CN and added dropwise to an ether solution. The product was
precipitated, filtered and dried over KOH pellets under high
vacuum. FAB-MS: MW 1181 (M.sup.+-2PF.sub.6.sup.-891,
M.sup.+-PF.sub.6.sup.-1036.) NMR: assigned.
EXAMPLE 11
[0123] The [(bpy).sub.2(bpy-N-propargyl)Ru(II)-2'-deoxy
uridine]2PF.sub.6.sup.- was dissolved in dry pyridine and the
solvent was removed by vacuum distillation. This process was
repeated 2 times. Finally the compound was dissolved in dry
pyridine and dimethoxytrityl chloride (DMT-Cl) was added to the
flask. The mixture was stirred overnight at room temperature under
nitrogen. Methanol was added to the reaction mixture to consume any
excess DMT-Cl. The solvent was then removed under high vacuum. An
orange solid was then dissolved n CH.sub.3CN and precipitated with
ether. The product was dried over KOH pellets under high vacuum.
FAB-MS: MW 1402; (M.sup.+-2PF.sub.6.sup.-1192)- ; NMR assigned.
EXAMPLE 12
[0124] The [(bpy).sub.2(bpy-N-propargyl)Ru(II)-5'-DMT-2'-deoxy
uridine] 2PF.sub.6.sup.- was dissolved in dry ACN and
diisopropylethylamine was added to flask followed by slow addition
of 2-cyanoethylchloro-N,N-diisop- ropylphosphramidite under argon.
The reaction mixture was stirred for 8 hours under argon and
solvent evaporated under vacuum. The orange solid compound, thus
obtained, was dissolved in CH.sub.3CN, precipitated with dry ether
and dried over KOH pellets under high vacuum. The sample was then
used in a DNA synthesizer without further purification. FAB-MS MW
1683 (M.sup.+-2PF6-) NMR assigned
EXAMPLE 13
[0125] Bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carbonyl
propargyl amine) osmium(II),
5-iodo-3',5'-dibenzoyloxy-2'-deoxyuridine, (PPh.sub.3).sub.4Pd(O),
and Cul were dissolved in dry DMF, and TEA was added to mixture.
The reaction mixture was stirred for 8 hours at room temp. The
solvent removed by vacuum distillation keeping the temperature less
than 45.degree. C. The green oil was dissolved in dry acetonitrile
and precipitated by adding dry ether. The orange compound was
filtered and washed with dry ether and dried over KOH pellets at
high vacuum.
EXAMPLE 14
[0126] The
[(bpy).sub.2(bpy-N-propargyl)Os(II)-3',5'-dibenzoyloxy-2'-deoxy
uridine] 2PF.sub.6- was dissolved in methanolic ammonia solution
and left for 48 hours with occasional shaking. The solvent was
removed by rotary evaporation. A green solid compound was dissolved
in dry CH.sub.3CN and added dropwise to an ether solution. The
product was precipitated, filtered and dried over KOH pellets under
high vacuum.
EXAMPLE 15
[0127] [(Bpy).sub.2(bpy-N-propargyl)Os(II)-2'deoxy uridine]
2PF.sub.6- was dissolved in dry pyridine and the solvent was
removed by vacuum distillation. This process was repeated 2 times.
Finally the compound was dissolved in dry pyridine and
dimethoxytrityl chloride (DMT-Cl) was added to the flask. The
mixture was stirred overnight at room temperature under nitrogen.
Methanol was added to the reaction mixture to consume any excess
DMT-Cl. The solvent was then removed under high vacuum. An green
solid was then dissolved in CH.sub.3CN and precipitated with ether.
The product was dried over KOH pellets under high vacuum.
EXAMPLE 16
[0128] The [(bpy).sub.2(bpy-N-propargyl)Os(II)-5'-DMT-2'-deoxy
uridine] 2PF.sub.6- was dissolved in dry ACN and
diisopropylethylamine was added to flask followed by slow addition
of 2-cyanoethylchloro-N,N-diisopropylp- hosphramidite under argon.
The reaction mixture was stirred for 8 hours under argon and
solvent evaporated under vacuum. The green solid compound, thus
obtained, was dissolved in CH.sub.3CN, precipitated with dry ether
and dried over KOH pellets under high vacuum.
EXAMPLE 17
[0129] The oligonucleotides shown in Table 1 were synthesized from
the 3' to 5' end on both the 0.2 and 1.0 .mu.mol scale using
standard automated DNA synthesis on an ABI 395 DNA/RNA synthesizer
and standard reagents purchased from Glen Research. A 0.1 M
solution of ruthenium-labeled
5'-dimethoxytrityl-3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite-2'-de-
oxyuridine in dry acteonitrile was prepared with DMT--ON and end
protection protocol except that the reaction time for the modified
phosphoramidite was increased. After synthesis the oligonucleotide
was collected in a vial and incubated at 55.degree. C. in NH.sub.3
overnight to completely deprotect the oligonucleotide. Collection
and analysis of the DMT fractions during automated synthesis showed
efficient phosphoramidite coupling (>95%) for the unmodified
oligonucleotides (9-11) and until the ruthenium-labeled nucleoside
phosphorarmidite was added. The coupling of the ruthenium
nucleoside phosphoramidite occurred at .apprxeq.40%, and additional
couplings after incorporation of this modified nucleoside were high
(>95%).
EXAMPLE 18
[0130] t-Butyloxycarbonyl Propargyl Amine 3
[0131] Propargyl amine hydrocloride(0.92 g, 10 mmol) was dissolved
in water (15 ml) and NaHCO.sub.3 (0.84 g,10 mmol)was added and
stirred. Di-t-butyl dicarbonate (2.18 g, 10mmol) was dissolved in
CHCl.sub.3 (20 ml) and added dropwise. The reaction mixture was
stirred for 3 hr and the CHCl.sub.3 layer was separated, and the
water phase was further extracted with CHCl.sub.3. We subsequently
combined the CHCl.sub.3 layers and washed with water, dried over
Na.sub.2SO.sub.4, and concentrated to give 1.35 g (87%) as oil,
which crystallized on standing at -4.degree. C. Mass Spect. (FAB)
MW 155 [M+]; NMR assigned Haralambidis, J.; Chai, M.; Treager, G.
W. Nucleic Acid Res. 1987, 15, 4857-4876
EXAMPLE 19
[0132] N-Propagyl trifluoroacetainide 4
[0133] Propargyl amine (1.03 ml, 15 mmol) was dissolved in dry THF
(15ml) and cooled to 0.degree. C. Trifluoroacetic anhydride (2.8 g,
20 mmol) was added dropwise and the reaction mixture was stirred
for 3 hrs. Water(20 ml) was then added to reaction mixture and
extracted with ether. The ether layer was washed with water, dried
over Na.sub.2SO.sub.4, and concentrated to give a dark color oil
2.2 g (82%). Mass Spect. (FAB) MW 152 [M+]; NMR assigned.
Cruickshank, K. A.; Stockwell, D. L. Tetrahedron Lett. 1988, 29,
5221-5224
EXAMPLE 20
[0134] 6-((6-((Biotinoyl)aniino)hexanoyl)amino)hexanoyl propargyl
amnine 5
[0135] To a stirred solution of
6-((6-((Biotinoyl)arnino)hexanoyl)amino)he- xanoic acid,
succinimnidyl ester (biotin-NHS ester) (0.2 g, 0.35 mmol) in dry
DMF (5 ml), propargyl amine (0.04 ml, 0.6 mmol) was added. A white
precipitate formed within 15 min and the mixture was stirred
overnight. The solvent was removed in vacuo and the compound was
dissolved in CHCl.sub.3. Next the CHCl.sub.3 was slowly added to a
stirred solution of hexane, forming a white precipitate, which on
drying afforded 0.15 g (83%). Mass Spect. (FAB) MW 508 [M+]; NMR
assigned. Crisp, G. T.; Gore, J. Syn. Comm. 1997, 27, 2203-2125
EXAMPLE 21
[0136] 4'-Methyl-2,2'-bipyridine-4-carboxaldehyde
[0137] A solution of 4,4'-dimethyl-2,2'-bipyridine (5.27 g, 28.6
mmol) in dioxane (150 ml), SeO.sub.2 (3.48 g, 31.4 mmol) was added
and refluxed for 24 hr with stirring and filtered hot. The dioxane
was removed by rotary evaporator, and the residue was dissolved in
ethylacetate and filtred to remove additional solid material. The
ethylacetate layer was then extracted with 1M Na.sub.2CO.sub.3
(2*100 ml) to remove additional carboxylic acid and 0.3M
Na.sub.2S.sub.2O (3* 100 ml) to form the aldehyde bisulfite. The
combined aquous extracts were adjusted to pH 10 with
Na.sub.2CO.sub.3 and extract with DCM (4* 10ml). Evaporation of
solvent to get 1.9 g (33%) pure white solid compound. Mass Spect.
(FAB) MW 199 [M+]; NMR assigned.
EXAMPLE 22
[0138] 4'-Methyl-2,2'-bipyridine-4-carboxylic acid
[0139] A suspension of 4'-methyl-2,2'-bipyridine-4-carboxaldehyde
(3.5 g, 15 mmol) in 95% EtOH (150 ml) was added a solution of
AgNO.sub.3 (3.15 g) in water (32mnl). The suspension was stirred
rapidly and 1M NaOH (79 ml) solution was added dropwise over 20 min
to form Ag.sub.2O. The dark black solution was stirred for an
additional 15 hrs. EtOH was removed by rotary evaporator and
filtred to remove Ag.sub.2O and unreacted metallic Ag. The residue
was washed with 1.3M NaOH (2*20 ml) and H.sub.20 20 ml. The
combined basic filterate was extracted with DCM to remove unreacted
aldehyde and adjusted to pH 3.5 with 1:1 (v/v) 4N HCl/AcOH to
afford white compound. After keeping overnight at -10.degree. C.
the compound was collected and dried to afford pure compound 2.9 g
(77%). Mass Spect. (FAB) MW 215 [M+]; NMR assigned.
EXAMPLE 23
[0140] 4'-Methyl-2,2'-bipyridine-4-carbonyl propargyl amine
[0141] 4'-Methyl-2,2'-bipyridine-4-carboxylic acid (0.22 g, 1
mmol), propargylamine hydrochloride (0.092 g, Immol), HOBt (0.15 g,
1 mmol) and DIPEA (0.21 ml) were dissolved in dry DMF (15 ml) and
cooled to 0.degree. C. DCC (0.25 g, 1.2 mmol) was dissolved in DMF
(3 ml) and added dropwise to the reaction mixture. The mixture was
stirred at room temperature overnight. DCU was filtered off and
solvent was removed by vacuum distillation. The remaining solid
compound was dissolved in ethyl acetate and washed with
NaHCO.sub.3(5%), 0.5 N HCl, brine and dried over sodium sulfate.
Solvent was removed by rotary evaporator and the compound was
purified by column chromatography using 2% methanol in chloroform
as eluent afforded 0.19 g (76%). FAB-MS: M. W. 251[M+]; NMR
assigned.
EXAMPLE 24
[0142]
Bis(2,2'-bipyridine)(4'-Methyl-2,2'-bipyridine-4-carbonypropargylam-
ine)ruthenium(II) bis(hexafluorophosphate) 6
[0143] The compound 4'-methyl-2',2'-bipyridine-4-carbonyl propargyl
amine (0.08 g, 0.3 mmol) in 70% ethanol/H20 (25 ml) was added
Ru(bpY).sub.2Cl.sub.2 (0.15 g, 0.3nunol) and refluxed for 10 hr.
The reaction mixture was cooled and ethanol was removed in vacuo.
The solution after standing for 4 hours at room temperature, was
filtered and the solid compound washed with cold water. A saturated
aqueous solution of NH.sub.4PF.sub.6 was added until no further
ppt. was observed and the mixture was kept at room temperature for
2 hours and then finally filtered, washed with cold water, ether
and dried overnight to get pure orange colour compound 0.45 g(82%).
Mass Spect. (FAB) MW 809 [M++PF6]; NMR assigned.
EXAMPLE 25
[0144]
Bis(2,2'-bipyridine)(4'-Methyl-2,2'-bipyridine4-carbonypropargylami-
ne)osmium(II) bis(hexafluorophosphate)
[0145] Solid Os(bpy).sub.2Cl.sub.2 (0.12 g, 0.2 mmol) and
4'-methyl-2',2'-bipyridine4-carbonyl propargyl amine (0.08 g, 0.3
mmol) were suspended in 70% ethanol/H.sub.2O (50 ml) and refluxed
for 10 hr. The reaction mixture was cooled and ethanol was removed.
The water solution after standing for 4 hours at room temperature,
was filtered and the solid compound washed with cold water. In
filterate, a saturated aqueous solution of NH.sub.4PF.sub.6 was
added until no further ppt. was observed and the mixture was kept
at room temperature for 2 hours and then filtered, washed with cold
water, ether and dried overnight to get pure black colour compound
0.2 5 g(76%). Mass Spect. (FAB) MW 898 [M++PF 6]; NMR assigned.
EXAMPLE 26
[0146] 5'-O-(4,4'-Dimethoxytrityl)-2'-deoxy-5-iodo uridine
[0147] 2'-Deoxy-5-iodo uridine (1.1 g, 3 mmol) was dissolved in dry
pyridine (5 ml) and the solvent was removed on high vacuum. This
process was repeated two times. Finally the compound was dissolved
in dry pyridine (15 ml) and dimethoxytrityl chloride (DMT-Cl) (1.4
g, 3.5 mmol) was added to the flask in two portion. The mixture was
stirred for 4 hr at room temperature. Methanol was added to the
reaction mixture to consume any excess DMT-Cl. The solvent was then
removed and the compound was dissolved in CHCl.sub.3 and washed
with 5% NaHCO.sub.3 and finally with water and dried over
Na.sub.2SO.sub.4 and concentrated to get crude product.
Purification by flash column chromatography afforded white pure
compound 1.7 g(85%). Product was dried over KOH pellets under high
vacuum. Mass Spect. (FAB) MW 641 [M+]; NMR assigned.
EXAMPLE 27
[0148]
5'-O-(4,4'-Dimethoxytrityl)-3'-O-(2-cyanoethyl)-N,N'-diisopropylpho-
sphoramdite-2'-deoxy-5-iodo uridine
[0149] 5'-O-(4,4'-Dimethoxytrityl)-2'-deoxy-5-iodo uridine (1.28 g,
2 mmol) was dissolved in dry DCM (40 ml) and diisopropylethylamine
(0.7 ml, 4 mmol) was added to flask and cooled in ice bath. Next,
2-cyanoethylchloro-N,N-diisopropylphosphoramidite (0.7 ml, 3 mmol)
was added slowly under nitrogen. The reaction mixture was stirred
for 2 hours under argon and then DCM (10 mil) was added. The
solvent was removed and the compound was dissolved in CHC13 and
washed with 5% NaHCO.sub.3, water, dried over Na.sub.2SO.sub.4, and
concentrated to give the product 1.5 g(89%). The compound was used
as such without further purification after drying over KOH pellets
under vacuum. Mass Spect. (FAB) MW 842 [M++PF6]; NMR assigned.
EXAMPLE 28
[0150] Synthesis of Oligonucleotide
[0151] The ODN were synthesized from 5' to 3' end on a 0.2 and
1:mol scale. The 0.1M solution of dried monomer iodouridine
phosphoramidite was prepared in anhydrous acetonitrile. Synthesizer
bottle number 5 was filled with
5'-dimethoxytrityl-3'-(cyanoethyl-N-diisopropyl)phosphoramidi-
te-2'-deoxy-5-iodouridine and manually diluted with dry
acetonitrile to give a concentration 0.1M. Solid-phase synthesis
was then preformed in such a way that the sequence was stopped
after incorportion of the iodouridine, but keeping the DMT-ON and
with no end deprotection from the resin. The column was taken out
from the synthesizer and dried over N.sub.2 thus maintaining the
anhydrous condition. Next, the column cap was opened and the
coupling compound, side chain, catalyst (PdPh.sub.3P).sub.4, copper
iodide were added and dried with N.sub.2 for 30 minutes. All
addition were carried out under N.sub.2 atmosphere. 250 ml of dry
DMF: Et.sub.3N(3.5: 1.5) solution was added and cap of the column
closed. The column was placed on a shaker for 3 hrs. When the
reaction completed, the column was washed with DMF:Et.sub.3N (9:1),
acetonitrile (40 ml), dried with N.sub.2 for 30 min, and
reinstalled on the synthesizer and additional DNA bases were
added.
[0152] Collection and analysis of the trityl fractions during
automated synthesis showed efficient phosphoramidite coupling
throughout the procedure, with both the standard pyrimidine and
purine nucleosides as well as with 5-iodo-uridine (>95% ).
Finally, the modified oligonucleotide was collected and incubated
at 55.degree. C. in NH.sub.3 overnight, to completely deprotect the
oligonucleotide. Analysis of the HPLC traces of the crude
oligonucleotide products showed efficient Pd(0) cross-coupling
reactions for each functional group (yields ranged from 85 to 92%).
Enzymatic digestion of the modified oligonucleotides showed
selective coupling to 5-iodouridine with no side reactions observed
with the other bases. Of the groups synthesized, the type, size,
3-dimensional shape, or charge of the functional group did not
significantly affect the Pd(0) cross-coupling reaction nor the
ability to synthesize both short and long modified
oligonucleotides. The mass spectrum of each sequence was observed
using either FAB or MALDI.
[0153] The following sequences were synthesized (5'-3'):
3 1) DMT-TU*G CA * = CCCH.sub.2NH-Boc
(N-t-butyloxycarbonylpropargyl amine) 2) DMT-TU*C A * =
-CCCH.sub.2NHCOBP(Me)Ru(BP).sub.2 (PF.sub.6).sub.2
(bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-
carbonylpropargylamine) ruthenium(II) bis(hexafluorophosphate) 3)
GTT U*GA * =
CCCH.sub.2NHCO(CH.sub.2).sub.5NHCO(CH.sub.2).sub.5NHCO-biotin
(6-(6-biotinoylaminohexanoyl)aminohexanoyl) propargyl amine 4) CTU*
AGC A * = CCCH.sub.2NHCOCF.sub.3 (N-trifluoracetylpropargyl amine)
5) TCA ACAGTTTGU*AGCA (SEQ ID NO: 17) * =
CCCH.sub.2NHCOBP(Me)Ru(BP).sub.2 (PF6).sub.2 6) TCAACAGTTTGU*AGC A
(SEQ ID NO: 18) * = CCCH.sub.2NHCOBP(Me)Os(BP).sub.2 (PF6).sub.2
(bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carbonylpropargylamine)
osmium(II) bis(hexafluorophosphate) 7) TGCTACAAACTGU*TG A (SEQ ID
NO: 18) * = CCCH.sub.2NHCOBP(Me)Ru(BP).sub.2 (PF.sub.6).sub.2 8)
TGCTACAAACTGU*TG A (SEQ ID NO: 20) * =
CCCH.sub.2NHCOBP(Me)Os(BP).sub.2 (PF.sub.6).sub.2 9) TACATCCTAU*CT
(SEQ ID NO: 21) * = CCCH.sub.2NHCO(CH.sub.2).sub.5NHCO(CH.s-
ub.2).sub.5NHCO-biotin 10) GGTCTTATTCACCACAATAACCTCAGTU*CT (SEQ ID
NO: 22) * = CCCH.sub.2NHCO(CH.sub.2).sub.5NHCO(CH.sub.2).sub.5NHC-
O-biotin 11) DMT-TU*CA * = CCCH.sub.2NHCOBP(Me)Ru(BP).sub.- 2
(PF.sub.6).sub.2
EXAMPLE 29
[0154] 5'-O-mesyl-2'-deoxythymidine (Compound 2 of FIG. 7)
[0155] 2'-deoxythymidine 1 (2.90 g, 12.0 mmol) was dissolved in 10
mL dry pyridine and cooled to -10.degree. C. Next, mesyl chloride
(14 mmol) was added dropwise with magnetic stirring and over a
period of 20 minutes. The reaction mixture was then held at
0.degree. for 12 h. The next day, 10 mL methanol was added to
quench the reaction and the solvents were evaporated via high
vacuum. The resulting crude product was checked by TLC and purified
by column chromatagraphy. A white powdered solid 2 was obtained
(2.95 g, 77% yield). 1H NMR DMSO, d 1.78 (s, 3H, 5-methyl),
2.08-2.22 (m, 2H, C2'), 3.22 (s, 3H, mesyl), 3.98 (q, 1H, C4'),
4.28 (m, 1H, C3'), 4.40 (m, 2H, C5'), 5.50 (s, 1H, 3-OH), 6.22 t,
1H, C1'), 7,48 (s, 1H, C6), 11.25 (s, 1H, N3). MS(FAB): calculated
MW=320, found M+H 321.
EXAMPLE 30
[0156] 5'-azido-2'-deoxythymidine (Compound 3 of FIG. 7)
[0157] A solution of 2 (1.84 g, 5.75 mmol) in 15 mL of DMF
containing lithium azide (1.60 g, 32.6 mmol) was stirred at
90.degree. C. under nitrogen. After three hours, the reaction was
stopped and mixture was cooled to room temperature, and then poured
into 600 mL ice-water. The resulting white precipitate was obtained
via filtration. Column chromatagraphy yielded 3 (1.12 g, 73%
yield). 1H NMR DMSO, d 1.78 (s, 3H, 5-methyl), 2.08-2.22 (m, 2H,
C2'), 3.57 (d, 2H, C5'), 3.85 (q, 1H, C4'), 4.20 (m, 1H, C3'), 5.50
(s, 1H, 3'-OH), 6.22 (t, 1H, Cl'), 7.48 (s, 1H, C6), 11.25 (s, 1H,
N3).
EXAMPLE 31
[0158] 5'-amino-2'-deoxythymidine (Compound 4 of FIG. 7)
[0159] 5'-azido-2'-deoxythymidine, (Compound 3 of FIG. 7), (1.12 g,
4.2 mmol) and triphenylphosphine (1.74 g, 6.7 mmol) were dissolved
in 30 rnL dioxane and stirred for three hours. A concentrated
ammonia solution (15 mL) was then added to the reaction mixture.
The reaction was determined to be complete by TLC 12 hours later.
The solvents were removed, and the resulting residue was partially
dissolved in ether/petrolum ether (1:1). A solid was obtained via
filtration, and was further purified by column chromatography to
yield 4 (0.73 g, 72% yield). 1H NMR DMSO, d 1.78 (s, 3H, 5-methyl),
2.08-2.18 (m, 2H, C2'), 2.75 (s, 2H, amine), 3.37 (s, 2H, C5'),
3.75 (q, 1H, C4'), 4.20 (m, 1H, C3'), 5.20 (s, 1H, 3'-OH), 6.20 (t,
1H, C1'), 7.68 (s, 1H, C6). MS (FAB): calculated MW=241, found M+H
242; MS (FAB -HR) C.sub.10H.sub.15N.sub.3O.sub.4.
EXAMPLE 32
[0160] 4'-Methyl-2,2'-bipyridine-4-carboxylic acid was synthesized
following the procedure described by Peck, B. M.; Ross, G. T.;
Edwards, S. W.; Meyer, G. I.; Meyer, T. J.; Erickson, B. W.
Synthesis of Redox Derivatives of Lysine and Related Peptides
Containing Phenothiezine or tris(2,2'bipyridine)ruthenium(II) Int.
J. Pept. and Protein Res. 1991, 38, 114-123 with a yield of 77%;
FAB-MS calculated C.sub.12H.sub.10N.sub.2O.sub.2 214, found [M+H]
215; 1H NMR (DMSO-d6) d 2.5 (s, 3H, CH.sub.3); 7.15 -9 (m, 6H,
py).
EXAMPLE 33
[0161] bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carboxylic
acid) ruthenium(II) bis(hexafluorophosphate)
[0162] A solution of cis-dichlorobisbipyridine Ru(II) (0.54 g, 1.0
mmol) in 50 mL ethanol was stirred under N.sub.2 for 10 minutes.
Next, 4-methyl-2,2'-bipyridine-4'-carboxylic acid (0.26 g, 1/2
mmol) was added to the solution and the reaction was refluxed for
five hours. The reaction was stopped and the mixture was cooled to
25.degree. C. before adding a saturated aqueous solution of
NH.sub.4PF.sub.6.sup.-. The red precipitate was collected by
filtration and yielded 0.87 g of product (91% yield).
EXAMPLE 34
[0163]
5'-[bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine4-carboxylic
acid) ruthenium(II) bis(hexafluorophosphate) 2'-deoxythymidine
(Compound 5 of FIG. 7)
[0164] A solution of (4-methyl, 4'-carboxylic acid) bipyridine
bisbipyridine ruthenium (0.20 g, 0.21 mmol) and carbonyl
diimidazole CDI (0.06 g, 0.38 mmol) in 2 mL dry DMF was stirred
under nitrogen at 25.degree. C. for an hour. The mixture was then
diluted with 3.5 niL dry THF and then the LH-20 resin (0.09 g) was
added to quench excess CDI. An hour later, LH-20 was removed and 4
(0.05 g, 0.22 mmol) was added to the reaction mixture. After
stirring for 12 h, the reaction was stopped and the solvents
removed. Column chromatography yielded a red solid (compound 5)
(0.19 g, 80% yield). MS(FAB) calculated MW=1140, found
(MW-PF.sub.6)+H 996. MS(HR-FAB): C42 H39 05 N9 F6 P Ru
EXAMPLE 35
[0165]
5'-[bis(2,2'-bipyridine)(4'-methyl-2,2'-bipyridine-4-carboxylic
acid) ruthenium(II) bis(hexafluorophosphate)] 3'-phosphoramidite
2'-deoxythymidine (Compound 6 of FIG. 7)
[0166] 2-cyanoethylchloro-diisopropylphosphoramidite (50 :L, 0.22
mmol) was added to a solution of compound 5 of FIG. 7 (0.193 g,
0.17 mmol) in dry diisopropylethyl amine (0. 1mL) and dry
CH.sub.3CN (8.5 mL). The reaction mixture proceeded under nitrogen
for two hours. Solvents were then removed and residue was rinsed
with hexane. The red solid was then checked by .sup.31P NMR,
CDCl.sub.3: d 150 and 148 ppm.
EXAMPLE 36
[0167] Oligonucleotide Syntheses
[0168] The oligonucleotide syntheses were performed on a commercial
ABI 395 DNA/RNA synthesizer from the 3' to 5' end using standard
automated DNA synthesis protocols as shown in FIG. 8 (0.2 :mol
scale). A 0.1 M solution of 5'-DMT-3'-cyanoethyl-N,N'-diisopropyl
phosphoramidite-2'-deox- y-5-iodouridine in dry acetonitrile was
prepared and installed on the DNA synthesizer in a standard reagent
bottle. Normal solid-phase oligonucleotide synthesis was performed.
In the last step, the ruthenium-modified thymidine phosphoramidite
was introduced and allowed to react with the oligonucleotide for 15
minutes. The Ru(bpy).sub.3.sup.2+ labeled oligonucleotide was
cleaved from the column and deprotected. The ruthenium-modified
oligonucleotided exhibited one peak in an HPLC trace, with
retention times greater than the corresponding unmodified
oligonucleotide. Electrospray mass spectrometry of the metallo-awls
oligonucleotide confirmed formation [e.g., Compound 7, Electrospray
( calculated 4545; found +2 (2275.33 and +3 (1516.79) ion stated;
Compound 10 calculated (5789 found +2 (2896.77) and +(1931.34) ion
states]. Collection and analysis of the trityl fractions during
automated synthesis showed efficient phosphoramidite coupling
throughout the procedure, with both the standard pyrimidine and
purine nucleosides (>95%). The coupling of Compound 6 of FIG. 8
to the oligonucleotide in good yield ( 50%).
EXAMPLE 37
[0169] HPLC Purification of the Oligonucleotides
[0170] HPLC purification of the modified oligonucleotides was
accomplished on a Ranin HPLC instrument. Reverse phase
chromatography was performed on a C18 column (25 cm.times.4.6 mm)
with acetonitrile (ACN) and 0.1 M triethylamine acetate (TEAA) as
eluting solvents. A flow rate of 1 mL/min was used and the
concentration of ACN was increased from 5% to 50% over 35 minutes.
The retention times of the modified oligonucleotides were well
separated from the unmodified oligonucleotide products (>2
minutes in retention time).
EXAMPLE 38
[0171] Melting Curves
[0172] The stability of the duplex formed between the two
complimentary oligonucleotides was determined by analyzing the
melting curve profile as a function of temperature. Briefly stated,
2 mM stock solutions of the separate oligonucleotides were prepared
and diluted to a working solution of 0.5 absorbance units. Next the
two solutions are combined and the solution containing the
complimentary oligomers was heated to 90.degree. C. for 5 minutes.
The solution was then allowed to cool to room temperature over 3
hrs.
[0173] After cooling, the thermal denaturation experiment was
performed using the following parameters on a HP UV-VIS: a)
monitoring wavelength, 260 nm, b) temperature range, 20-75.degree.
C., c) temperature step, 0.1.degree. C., d) averaging time
constant, 15 s, e) rate of change for the temperature step,
1.degree. C./minute, and f) equilibrium time, 30 s.
EXAMPLE 39
[0174] S.sup.2FTIR
[0175] The transient data reported here were measured on a
step-scan modified Bruker IFS88 spectrometer with a standard globar
source and dry air purge. The sample was dissolved in CD.sub.3CN to
give an IR absorbance between 0.125 and 0.5 in a 250 mm pathlength
cell for the amide bond analyzed. Samples were deoxygenated by
sparging with argon for 60 minutes and were loaded into a
CaF.sub.2-window cell by syringe under argon. Data collection and
analysis was performed as previously reported. Khan, S. I.;
Beilstein, A. E.; Smith, G. D.; Sykora, M.; Grinstaff, M. W.
Synthesis and Excited-State Properties of a Novel Ruthenium
Nucleoside Inorg. Chem. 1999, 38, 2411-2415.
EXAMPLE 40
[0176] Emission Spectra
[0177] Emission spectra were recorded on a Spex Fluorolog-2
emission spectrometer equipped with a 450 W Xe lamp and cooled
Hammamatsu R928 photomultiplier. The recorded emission spectra were
corrected for spectrometer response. The calibration curve was
obtained using NIST calibrated standard lamp (Optronics
Laboratories, Inc. Model 220M), controlled with a precision current
source at 6.5 W (Optronics Laboratories, Inc. Model 65) by
following the procedure recommended by manufacturer. The spectra
were obtained in buffer at room temperature in a 1 cm quartz cell
using right angle observation of emitted light.
EXAMPLE 41
[0178] Lifetimes
[0179] A Laser Photonics LN1000 Nitrogen Laser-LN102 dye laser
(coumarin 460 dye, Exciton) was used as the irradiation source. The
emission was monitored at a right angle using a Macpherson 272
monochromator and Hammamatsu R666-10 PMT. The signal was processed
by a LeCroy 7200A transient digitizer interfaced with an IBM-PC.
The excitation wavelength was 460 nm and the monitoring wavelength
was 640 nm. Power at the sample was 41:J/pulse as measured by a
Molectron J3-09 power meter. The measured instrument lifetime
response is 10 ns (FWHM). The acquired emission decay curves were
analyzed by locally written software based on the Marquardt
algorithm.
EXAMPLE 42
[0180] Electron Transfer
[0181] The ruthenium-uridine described herein is a suitable
chromophore for reductive quenching studies since it is
photochemically stable, inert to ligand substitution reactions,
possesses an energetic excited state (0.84 eV), and a long lifetime
in fluid solution. Moreover, the excited-state electron is
localized on the bipyridine attached to the uridine. The
electron-transfer quencher, phenothiazine is known to be a very
efficient electron donor for quenching *Ru(bpy).sub.3.sup.2+. The
biomolecular electron-transfer reaction between compounds 4 and 7
of FIGS. 1A-1B was studied in solution by varying the quencher
concentration. Stem-Volmer analysis yielded a quenching rate
constant (kq) of 1.3.times.10.sup.9 M.sup.-1 s.sup.-1. Based on the
reduction potential of PTZ+/0 (0.76 eV), the driving force for this
electron-transfer reaction was estimated to be approximately 0.1
eV.
[0182] In this DNA-mediated electron-transfer system, the electron
donor and acceptor were covalently attached to different
oligonucleotide strands and separated by about 30 .ANG.. First, the
complimentary duplex containing only the ruthenium acceptor (5'-TCA
ACA GU*T TGT AGC A-3'; 5'-TGC TAC AAA CTG TTG A-3') was synthesized
(U*=Ru(diimine).sub.3.sup.2+ linked uridine). The emission maximum
for this ruthenium-labeled oligonucleotide duplex was centered at
660 nm and the emission lifetime was measured to be 540 ns at
20.degree. C. in phosphate buffer (monitoring at 640 nm after 455
nm pulse excitation). Next, phenothiazine (PTZ) was attached to the
5'-terminal of the complimentary sequence of the ruthenium labeled
oligonucleotide (5'-TCA ACA GU*T TGT AGC A-3'; 5'-PTZ-TGC TAC AAA
CTG TTG A-3'). Reductive quenching of the excited state was
observed, and the rate constant was determined to be
2.6.times.10.sup.5 s.sup.-1. The lifetime and electron-transfer
rates were measured using a Laser Photonics LN1000 Nitrogen
Laser-LN102 dye laser (coumarin 460 dye). The emission was
monitored at right angle with a Macpherson 272 monochromator and
Hammamatsu R666-10 PMT at 22.degree. C. The signal was processed by
a LeCroy 7200A transient digitizer interfaced with an IBM-PC. The
excitation wavelength was 455 nm and the monitoring wavelength was
640 nm. Power at the sample was 60 W/pulse.mm.sup.3 as measured by
a Molectron J3-09 power meter. The measured instrument response
function is 10 ns (FWHM). The acquired emission decay curves were
analyzed by a locally written software based on the Marquardt
algorithm. The data were fit to a single exponential. The residuals
between the experimental and fitted curves were less than 2%. The
electron-transfer rate constant was determined using the following
equation: k=1/t-1/t.sub.o. Over the temperature range of 5 to
30.degree. C., the rate constant increased slightly from 2.5 to
2.8.times.10.sup.5 s.sup.-1.
[0183] All references cited herein, including the following, are
incorporated herein by reference in their entirety. Palladium(0)
Catalyzed Modification of Oligonucleotides during Automated
Solid-Phase Synthesis." Shoeb I. Khan and Mark W. Grinstaff. J. Am.
Chem. Soc. 1999, 121, 47044705. "Automated Solid-Phase Synthesis of
Site Specifically Labeled Ruthenium-Oligonucleotides." Shoeb I.
Khan, Amy E. Beilstein, and Mark W. Grinstaff. Inorg. Chem. 1999,
38, 418-419. "Synthesis and Excited-State Properties of a Novel
Ruthenium Nucleoside: Ru(bpy).sub.2(5-bpy-2'-deoxyuridine).sup.2+."
Shoeb I. Khan, Amy E. Beilstein, Gregory D. Smith, Milan Sykora,
and Mark W. Grinstaff. Ilnorg. Chem. 1999, 38, 241 1-2415. "The
Alkylation of Iodouridine by a Heterogeneous Palladium Catalyst."
Shoeb I. Khan and Mark W. Grinstaff. J. Org. Chem. 1999, 64,
1077-1078.
Sequence CWU 0
0
* * * * *