(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(43) International Publication Date
`21 June 2001 (21.06.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/44220 A2
`
`(51) International Patent Classification7:
`333/04, C07H 21100
`
`C07D 307/04,
`
`(72) Inventor: KOOL, Eric, T.; Department of Chemistry,
`Stanford University, Stanford, CA 94305-5080 (US).
`
`(21) International Application Number: PCT/US00/33681
`
`(22) International Filing Date:
`13 December 2000 (13.12.2000)
`
`(74) Agent: GROLZ, Edward, W.; Scully, Scott, Murphy &
`Presser, 400 Garden City Plaza, Garden City, NY 11530
`(US).
`
`(81) Designated States (national): CA, JP.
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(84) Designated States (regional): European patent (AT, BE,
`CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, MC,
`NL, PT, SE, TR).
`
`(30) Priority Data:
`09/461,636
`
`14 December 1999 (14.12.1999) US
`
`Published:
`Without international search report and to be republished
`upon receipt of that report.
`
`(71) Applicant: RESEARCH CORPORATION TECH(cid:173)
`NOLOGIES, INC. [US/US]; 101 N. Wilmot Road, Suite
`600, Tucson, AZ 85711-335 (US).
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
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`M (54) Title: FLUORESCENT NUCLEOSIDE ANALOGS AND COMBINATORIAL FLUOROPHORE ARRAYS COMPRISING
`<SAME
`
`0
`(57) Abstract: The present invention provides fluorescent nucleoside analogs which comprise a fluorescent cyclic compound joined
`M
`to a carbon of a sugar molecule such as pentose, hexose, ribose or deoxyribose or analogs thereof in either an a or ~ configuration.
`~ The subject compounds are useful as probes in the study of the structure and dynamics of nucleid acids and their complexes with
`~ proteins. In addition, the subject compounds are useful in any technique which uses labeled oligonucleotides for detection. Non-flu(cid:173)
`...._ orescent spacer molecules in which a cyclohexane, cyclohexene, decalin, or benzene is joined to a carbon of a sugar moiety such as
`
`S pentose, hexose, ribose or deoxyribose are also provided. Also provided are the 5' dimethoxytrityl-3'-0-phosphoramidite deriva-
`0 comprising oligomers of the subjects nucleoside analogs attached to one or more solid supports are also provided as are methods of
`> selecting fluorophores from the CFA libraries. The present invention also provides oligonucleotide analogs comprising one or more
`~ of the subject nucleoside analogs in place of the DNA or RNA base.
`
`tives, suitable for incorporation into oligonucleotides by automated synthesizers. Combinatorial fluorophore array (CFA) libraries
`
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`FLUORESCENT NUCLEOSIDE ANALOGS AND
`COMBINATORIAL FLUOROl?HORE ARRAYS CO:t<Il?RISING SAME
`
`BACKGROUND OF THE INVENTION
`Fluorescence methods are extremely widespread in
`chemistry and biology. The methods give useful information on
`structure, distance, orientation, complexation, and location
`for biomolecules [1].
`In addition, time-resolved methods are
`increasingly used in measurements of dynamics and kinetics (2]
`As .a result, many strategies for fluorescence labeling of
`biomolecules, such as nucleic acids, have been developed (3]
`In the case of DNA, one of the most convenient and useful
`methods for fluorescence labeling is to add a fluorescent
`moiety during the DNA synthesis itself. Addition of the
`fluorescent moiety during DNA synthesis avoids the extra steps
`required for post-synthesis labeling and purification. The
`majority of labels commonly used d~ring DNA synthesis are
`attached to the DNA by tethers that are often 5 to 11 atoms
`long. These flexible tethers can at times be problematic,
`since they allow the dye to tumble independently of the DNA and
`make the location of the dye difficult to determine precisely
`(4]. There are very few examples of dye conjugates that hold
`the dye close to the DNA,
`thus avoiding these problems. Among
`the known dyes of this class are ethenodeoxyadenosine [5) and
`2-aminopurinedeoxyriboside [6] .. These latter two compounds
`have modified DNA bases that are themselves fluorescent, and
`have found much use as probes .of enzymatic activities such c.s
`DNA synthesis, editing, and repair [7-9].
`The present invention provides fluorescent labels for
`nucleic acids which, rather than modifying an existing nucleic
`acid base, replace one or more DNA or RNA bases with a
`fluorescent cyclic compou~d. Since the replacement fluorescent
`cyclic compound is also a fla~ cyclic structure, only small
`perturbations to the ove~all ~ucleic acid structure occur upo~
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`its use. The fluorescent label may be thought of as a
`nucleoside analog in which a known fluorescent cyclic compound
`is joined to a carbon atom of a sugar moiety. The subject
`nucleoside analogs allow for close interaction, including
`stacking, with a neighboring RNA or .. DNA helix. There are many
`known cyclic fluorophores which may be joined to a carbon atom
`of a sugar moiety to form the nucleoside analogs of the present
`invention. Many of the known cyclic fluorophores have high
`quantum yields with varied excitation and emission
`characteristics. Moreover, their lack of functional groups
`makes them relatively simple to work with in preparing
`conjugates.
`The literature has reported incorporation of 4-
`methylindole, naphthalene, phenanthrene, and pyrene
`In a
`fluorophores at the C1-position of deoxyribose [10,11]
`similar strategy, the substitution of a coumarin dye at the C1
`position of deoxyribose has also been reported[12]. The
`methylindole derivative has recently found use as a fluorescent
`
`reporter of DNA repair activities [13].
`
`In addition, the Cla
`
`pyrene derivative has been shown to be useful in DNA
`diagnostics strategies, where it efficiently forms excimers
`with neighboring pyrene labels [14]. The Cl~ pyrene derivative
`stabilizes DNA helices markedly (due to its low polarity)
`[15,16], and can be enzymatically incorporated into the DNA
`helix [ 17] . Thus, this new nucleic acid labeling strategy has
`many useful applications.
`The present invention provides nucleoside analogs
`with improved fluorescence characteristics, increasing the
`range of emission wavelengths over those previously studied.
`The subject nucleoside analogs a~e more generally useful in
`biophysical and diagnostics applications. These new compounds
`significantly broaden the ~ange of fluorescence properties
`available for automated incorporation into DNA.
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`Although not all interactions between fluorophores
`are well understood, it is clear that there is more than one
`type of interaction between light-absorbing molecules. One
`useful class of interaction is Forster energy transfer, also
`called fluorescence resonance energy transfer, or FRET.
`In
`this interaction, fluorescence emission is transferred from a
`donor to an acceptor fluorophore. The extent of transfer
`depends on distance and on overlap in emission and abs~rption
`of donor and acceptor.
`FRET can occur over relatively long
`distances (tens of Angstroms). A second form of energy
`transfer is exciplex formation, which involves bonding between
`an excited-state fluorophore and a neighboring ground-state
`fluorophore. This results in a long red shift to fluorescence.
`Exciplexes can form only between molecules in direct contact or
`very nearly so. Exciplexes between two of the same molecules
`are known as excimers. Another class of interaction involving
`a fluorophore is quenching, in which a molecule causes the
`quantum yield of nearby fluorescent molecule to be lowered.
`These forms of energy transfer are not well explored
`in systems where more than two chromophores are involved.
`FRET is now well known between pairs of well understood and
`widely used dyes, such as fluorescein, rhodamine, acridine, or
`cyanine dyes. Heretofore, FRET between more than two dyes has
`been unknown and unexplored. Similarly, while· interactions
`between a few excimer-forming dyes such as stilbene and pyrene
`are known, exciplex interactions have not been widely explored
`for combinations of dyes. Little is known about the
`interactions among more than two fluorophores. Reasons for the
`dearth of study in this area include lack of available methods
`for assembling fluorophores in a regular designed fashion. The
`study of more complex molecules could entail an inordinate
`number of combinations even where only a few dyes are used.
`Assembling even a small fraction of these possibilities for
`study heretofore has been a daunting task. Even if carried ouc,
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`many combinations of fluorophores lead to undesirable
`properties such as quenching. For example, placing two
`fluorescein labels close together results in very weak
`fluorescence emission.
`The present invention allows for the generation of
`nucleoside analogs and nucleic acids incorporating the subject
`nucleoside analogs resulting in many types of fluorescence
`properties such as emission wavelength, emission intensity, and
`Stokes shift. Combinatorial arrays of fluorophores built on a
`nucleic acid backbone may be generated and screened for
`fluorophores having useful fluorescent properties such as high
`molar absorptivities which leads to high localized fluorescense
`intensities. Fluorophores providing multiple energy transfer,
`leading to very large Stokes shifts may also be identified from
`a library of combinatorial arrays. Large Stokes shifts are
`useful in avoiding background interference in fluorescence.
`FRET and exciplex forms of energy transfer usually lead to
`large changes in emission wavelength, resulting in many
`possible colors for ease of detection.
`
`SUMMARY OF THE INVENTION
`The present invention provides nucleoside analogs
`comprising a fluorescent cyclic compound joined to a carbon of
`a pentose, hexose, ribose or deoxyribose sugar moiety in either
`an a or 0 configuration.
`In a preferred embodiment, the
`fluorescent cyclic compound is joined to the Cl position of the
`sugar moiety.
`Examples of fluorescent cyclic compounds which may be
`joined to the sugar moiety include oligomers of varying length
`selected from the group consisting of oligothiophene,
`oligobenzothiophene, oligo(phenylene vinylene), and
`oligo(phenylene acetylene). Preferably, an oligomer has a
`length of from 1 to 16. Terthiophene and sexithiophene are
`examples of oligothiophenes useful as cyclic compounds in the
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`nucleoside analogs of the present invention.
`Benzoterthiophene and terbenzothiophene are examples
`of oligobenzothiophenes useful as fluorescent cyclic compounds
`joined to a sugar moiety. Dimethylamino stilbene and
`styrylstilbene are examples of oligo(phenylene vinylenes)
`useful as fluorescent cyclic compounds joined to carbon of a
`sugar moiety.
`Diphenylacetylene and phenyl(ethynyl)
`diphenylacetylene are examples of oligo(phenylene
`acetylenes)useful as fluorescent cyclic compounds joined to a
`carbon of sugar moiety.
`Other fluorescent cyclic compounds useful as
`fluorescent cyclic compounds joined to a carbon of a sugar
`moiety include p-terphenyl, perylene, azobenzene, phenazine,
`napthalene, phenanthroline, acridine, thioxanthrene, chrysene,
`rubrene, coronene, cyanine, perylene imide, and perylene amide.
`Also provided are nucleoside analogs comprising a
`non-fluorescent cyclic compound joined to a carbon of a
`pentose, hexose, ribose or deoxyribose sugar moiety wherein the
`cyclic compound is cyclohexane, cyclohexene, decalin, benzene
`or dimethylamino benzene.
`The nucleoside analogs of the present invention may
`be derivatized at an available carbon position with a
`substituent selected from the group consisting of methoxy,
`ethoA-y, alkoxy, alkyl, dimethylamino, diethylamine, nitro,
`methyl, cyano, carboxy, fluoro, chloro, bromo,
`iodo and amino.
`The present invention also provides nucleic acid
`molecules comprising at least one subject nucleoside analog.
`Oligomers comprising the subject nucleoside analogs are also
`provided.
`
`Also in accordance with the present inve~tion, there
`are provided phosphoramidite derivatives of the subject
`nucleoside analogs wherein the phosphoramidite is joined to the
`sugar moiety at the 3' position. Examples of phosphoramidite
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`derivatives include N,N-diisopropyl-0-cyanoethyl
`phosphoramidite or 0-methyl-phosphoramidite derivatized at the
`3' alcohol of the nucleoside analog.
`The present invention also provides nucleoside 5'-3 '(cid:173)
`paratoluoyl diesters derivatized at the C-1 atom of a sugar
`moiety with a fluorescent cyclic compound.
`Methods of synthesizing the subject nucleoside
`analogs are also provided. The methods comprise the steps of
`coupling an organocadmium or organozinc derivative of a
`fluorescent cyclic compound to a carbon of Hoffer's a(cid:173)
`chlorosugar and removing the protecting groups with a
`methanolic base.
`Also provided are methods of synthesizing a
`phosphoramidite derivative of a subject nucleoside analog. The
`method comprises:coupling an organocadmium or organozinc
`derivative of a fluorescent cyclic compound to a carbon atom of
`Hoffer's a-chlorosugar, removing the protecting groups with a
`methanolic base; tritilating the 5'-0H with
`dimeoxytritylchloride in the presence of a base; and
`phosphytilating the 3'-0H with a phosphytilating agent.
`In addition, the present invention provides a method
`of preparing a fluorescently labeled nucleic acid molecule
`which comprises incorporating a subject nucleoside analog into
`an RNA or DNA molecule under conditions sufficient to
`incorporate said nucleoside.
`A method of detecting a target nucleic acid in a
`sample to be tested is also provided. The method comprises
`contacting the target nucleic acid with a nucleic acid probe
`comprising at least one subject nucleoside analog for a
`time
`and under conditions sufficient to permit hybridization becween
`the target and the probe and then detecting said hybridizaLion.
`Also provided by the present invention are
`combinatorial fluoropho:::-e array (CF!\) libraries which cor,~:::-::..se
`multiple solid supports or multiple locations on a solic
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`support, each support or location having attached thereto an
`oligomer comprising the subject fluorescent nucleoside analogs.
`A combinatorial fluorophore array (CFA) library may also
`comprise one or more unlabeled nucleosides wherein the one or
`more unlabeled nucleosides are positioned 5' or 3' to the
`oligomer of fluorescent nucleoside analogs or interspaced
`between the fluorescent nucleoside analogs.
`In addition, a
`CFA library may ~urther comprise one or more non-fluorescent
`nucleotide analogs selected from the group consisting of
`cyclohexene-2-deoxyriboside, cyclohexane-2-deoxyriboside,
`decalin-2-deoxyrib?side, and benzene-2-deoxyriboside wherein
`said one or more non-fluorescent nucleotide analogs is
`interspaced between the flourescent nucleotide analogs or
`between the fluorescent nucleoside analogs and the and non(cid:173)
`labeled nucleosides.
`The present invention also provides a method of
`selecting a fluorophore suitable for use in labeling a nucleic
`acid molecule which comprises constructing a subject
`combinatorial fluorophore array library and selecting a
`fluorophore emitting the most intense fluorescence or emitting
`a specific wavelength of light.
`Also provided is a method of identifying a
`fluorophore emitting a large Stokes shifts which comprises
`constructing a subject combinatorial fluorophore array library,
`exciting the library at short wavelength, and selecting a
`fluorophore which emits light at a much longer wavelength.
`A method of identifying a fluorbphore involved in
`energy transfer is also provided. The method comprises
`constructing a subject combinatorial fluorophore array library,
`hybridizing a nucleic acid comprising a donor or acceptor dye
`to a nucleic acid sequence in the CFA library and correlating
`any change in color exhibited by the hybridized molecules with
`energy transfer.
`fv!embers of the library which give greater
`changes in acceptor emission intensity are most efficient at
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`energy transfer.
`A method for identifying a fluorophore sequence that
`changes its emission wavelength or intensity on binding an
`analyte is also provided. The method comprises constructing a
`subject combinatorial fluorophore array library, incorporating
`an analyte affinity molecule, allowing an analyte solution to
`contact the library, and selecting library members that change
`emission intensity or wavelength on binding of the
`analyte molecule.
`The present invention also provides oligonucleotide
`analogs comprisin~ one or more of the subject nucleoside
`analogs in place of a DNA or RNA base. Further, the subject
`oligonucleotide analogs may comprise a modification to the
`sugar-phosphate backbone such as that found in phosphorothioate
`DNA, 2'-0-methyl RNA, phosphoramidite DNA, 2'fluoroDNA, peptide
`nucleic acid (PNA) or alpha DNA.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`Figure 1 shows the chemical structures of the
`nucleoside analogs terthiophene-2-deoxyriboside (3a),
`benzoterthiophene-2-deoxyriboside (3b), p-terphenyl-2-
`deoxyriboside (3c), pyrene-2-deoxyriboside (3d), stilbene-2-
`deoxyriboside (3e), cyclohexene-2-deoxyriboside (3f).
`Figure 2 shows the preparation of C-nucleosides by
`cadmium- or zinc-mediated reaction of·Grignard derivatives of
`cyclic compounds with Hoffer's chlorosugar. 2a through 2f
`represent the nucleoside 5'-3'-paratoluoyl diester derivatized
`at the C-1 atom of a sugar moiety with terthiophene (2a),
`benzoterthiophene (2b), p-terphenyl (2c), pyrene (2d), stilbene
`(2e) or cyclohexene (2f).
`Figure 3 shows the synthesis of 5-bromo-2,2' :5'
`ter-chiophene.
`Figure 4 shows the absorpcion and normalized emission
`speccra of free nucleosides 3a-3e of Figure 1 at 10 ul'-'1
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`concentration in methanol. Solid lines show absorption
`spectra; dashed lines show normalized emission spectra
`(arbitrary intensity units), with excitation at the absorption
`maxima.
`
`Figure 5 illustrates three members of a combinatorial
`fluorophore array (CFA) library. Large dark circles represent
`solid supports. Small dark circles represent phosphate groups
`on a nucleic acid molecule of five nucleosides. The different
`sized and differently shaded rectangular shapes attached to the
`sugar moieties represent varied cyclic fluorophores.
`
`DETAILED DESCRIPTION OF THE INVENTION
`The present invention provides fluorescent labels for
`nucleic acids which, rather than modifying an existing nucleic
`acid base, replace one or more DNA or RNA bases with a
`fluorescent cyclic compound.
`In accordance with the present
`invention, there are provided fluorescent nucleoside and
`nucleotide analogs comprising a fluorescent cyclic compound
`attached to a carbon of a sugar moiety. The sugar moiety may
`include, for example, pentose, hexose, ribose, or deoxyribose.
`In a preferred embodiment, the fluorescent cyclic compound is
`attached to the Cl position of the sugar moiety. The
`fluorescent cyclic compound attached to the sugar moiety may
`include such molecules as oligothiophenes of varying length,
`oligbenzothiophenes, oligo(phenylene vinylenes), p-terphenyl,
`perylene, dimethylamino benzene, oligo(phenylene acetylenes) of
`varying length, azobenzene, phenazine, napthalene,
`phe_nanthrol ine, acridine, thioxanthrene, chrysene, rubrene,
`coronene, cyanines, perylene imide and perylene amide. Due to
`the location of the fluorescent cyclic compound on the sugar
`moiety,
`the nucleoside analogs of the present invention act as
`DNA or RNA base analogs. The subject nucleoside and nucleotide
`analogs stack neatly in an RNA or DNA helix. As used herein,
`"nucleoside" also encompasses "nucleotide" which is a phospha:e
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`ester of a nucleoside. Thus, any reference herein to
`"nucleoside" or "nucleoside analog" is also meant to include
`"nucleotide" or "nucleotide analog". As used herein
`"nucleoside" is also meant to include hucleotide triphosphates.
`In addition, the present invention provides a non(cid:173)
`fluorescent nucleoside analog having a cyclic compound such as
`cyclohexane, cyclohexene, decalin, or benzene joined to a
`carbon atom of a sugar molecule.
`In a preferred embodiment,
`the cyclic compound is joined to the C-1 position of a sugar
`moiety. In another preferred embodiment, the sugar molecule is
`pentose, hexose, ribose or deoxyribose. Such a nucleoside
`analog is useful as a non-fluorescent spacer which may be
`inserted between the subject fluorescent nucleoside analogs and
`unlabeled nucleic acid bases.
`Insertion of the non-fluorescent
`nucleoside in a nucleic acid molecule limits quenching which
`may occur between the subject fluorophores.
`Examples of oligothiophenes of varying length useful
`as a fluorescent cyclic compound attached to a sugar moiety
`include terthiophene and sexithiophene. Examples of an
`oligobenzothiophene useful as a fluorescent cyclic compound
`attached to a sugar moiety include benzoterthiophene and
`terbenzothiophene. Dimethylamino stilbene and styrylstilbene
`are examples of oligo(phenylene vinylenes) useful as
`fluorescent cyclic compound attached to a sugar moiety.
`Examples of oligo(phenylene acetylenes) of varying length
`useful as a fluorescent cyclic compound attached to a sugar
`moiety include diphenylacetylene and
`phenylethynyl(diphenylacetylene).
`In those particular cases
`where the polycyclic hydrocarbon is an oligomer, e.g.,
`oligothiophene or oligo(phenylene acetylene), oligomer lengths
`from about one to about sixteen are contemplated.
`In accordance with the present invention, a subject
`nucleoside analog may be substituted at various positions on
`its ring structure with one or more alkoA~, alkylamino,
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`dialkylamino, alkyl, alkenyl, alkynyl, hydroxy, or halide
`groups. Examples include but are not limited to methoxy,
`ethoxy, dimethylamino, diethylamine, nitro, methyl, cyano,
`carboxy, fluoro, chloro, bromo,
`iodo, or amino groups.
`A fluorescent cyclic compound is attached at any
`available position on its ring structure to the sugar moiety by
`a carbon-carbon bond in a subject fluorescent nucleoside
`analog. Both alpha and beta anomers of the subject nucleosides
`are contemplated by the present invention. Similarly, a non(cid:173)
`fluorescent cyclic compound is attached at any available
`position on its ring structure to a sugar moiety by a carbon(cid:173)
`carbon bond in a subject non-fluorescent nucleoside analog.
`In one embodiment of the invention, an
`oligothiophene-derivatized nucleoside has the following general
`structure:
`
`HO
`
`In another embodiment of the invention, an
`oligothiophene-derivatized nucleoside is terthiophene-2-
`deoxyriboside, having_the following structure:
`
`In another embodiment of the invention, a~
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`oligothiophene-derivatized nucleoside is a sexithiophene-2-
`deoxyriboside, having the following structure:
`
`In another embodiment of the invention, an
`oligobenzothiophene-derivatized nucleoside is
`benzoterthiophene-2-deoxyriboside having the following
`structure:
`
`In another embodiment of the invention, an
`oligobenzothiophene-derivatized nucleoside is
`ter(benzothiophene)-2-deo~~riboside having the follo~ving
`structure:
`
`HO
`
`.
`
`~
`I\
`\'
`. '\_..--o--;.-_-1~ Df?' :
`~\;;, ·~
`.,.: ~~ !/
`:
`\\
`-~~
`.
`~ ~ n
`s -
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`In another aspect of the invention, an
`oligo(phenylene vinylene)-derivatized nucleoside such as an
`oligo(phenylene vinylene)-derivatized deoxynucleoside has the
`general structure:
`
`r:o
`
`In another aspect of the invention, an
`oligo(phenylene vinylene)-derivatized nucleoside lS
`styrylstilbene-2-deoxyriboside having the following structure:
`
`In yet another embodiment of the invention, a
`p-terphenyl-derivatized nucleoside such as a p-terphenyl-2-
`deoxyriboside has at least one of the follmv·ing structures:
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`In another embodiment of the invention, a perylene(cid:173)
`derivatized nucleoside such as a perylene-2-deoxyriboside has
`the following structure:
`
`A perylene imide-derivatized nucleoside such as a
`perylene imide-2-deoxyriboside has the following structure:
`
`A perylene amide-derivatized nucleoside such as
`perylene amide-2-deoxyriboside has the following structure:
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`In another embodiment of the invention, c
`dimethylamino benzene-derivatized nucleoside such as
`dimethylamino benzene-derivatized deoxynucleoside has the
`following structure:
`
`In accordance with the present invention, an oligo
`(phenylene acetylene) of varying length may be attached to a
`carbon atom of a sugar. Such a deoxyriboside analog may have
`the following chemical structure:
`
`~n example of such an oligo(phenyle~e acetylene)
`lS e.g., diphenylacetylene-2-deo;~riboside having one of the
`following structures:
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`In yet another embodiment of the invention. an
`oligo(phenylene acetylene) of varying length such as e.g.,
`phenyl(ethynyl)diphenylacetylene-2-deoxyriboside has one of. the
`following structures:
`
`In accordance with the present invention, an
`azobenzene-derivatized nucleoside such as an azobenzene-2-
`deoxyriboside has the following structure:
`
`In another embodiment of the invention, a phenazine(cid:173)
`derivatized nucleoside such as phenazine-2-deoxyriboside hc.s
`one of the following structures:
`
`16
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`In still another embodiment of the invention, a
`napchalene-derivatized nucleoside such as napthalene-2-
`deoxyriboside has the following structure:
`
`HO
`
`In yet another embodiment of the invention, a
`phenanthroline-derivatized nucleoside such as phenanthroline-2-
`oeoxyriboside has the following structure:
`
`In still anothe= embodiment, an acridine-derivatized
`nucleoside such as acriciir.e-2-deoxyriboside has the follov1ing
`structure:
`
`17
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`In.another embodiment of the invention, a
`thioxanthrene-derivatized nucleoside such as thioxanthrene-2-
`deoxyriboside has the following structure:
`
`HO
`
`In still another embodiment of the invention, a
`chrysene-derivatized nucleoside such as chrysene-2-
`deoxyriboside has the following structure:
`
`In still another embodiment of the invention. a
`rubrene-derivatized nucleoside such as rubrene-2-deoxyriboside
`has the following structure:
`
`18
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`SUBSTITUTE SHEET (RULE 26)
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`In still another embodiment of the invention, a
`coronene-derivatized nucleoside such as coronene-2-
`deo~yriboside has the following structure:
`
`In yet ~nother embodiment, a cyanine-derivatized
`nucleoside such as a cyanine-2-deoxyriboside has the following
`structure:
`
`(X:: S,O)
`(r.=O.l)
`
`In still another embodiment of the invention, a non(cid:173)
`fluorescent nucleoside analog is provided such as cyclohexa~e-
`2-deo:~riboside having the following structure:
`
`HO\_-O-;.._.,
`
`~~
`u
`
`-'0
`
`19
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`SUBSTITUTE SHEET (RULE 26)
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`20
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`In another embodiment, a non-fluorescent nucleoside
`analog may be cyclohexene-2-deoxyriboside having the following
`structure:
`
`HO?u
`
`In still another embodiment, a non-fluorescent
`nucleoside analog may be decalin-2-deoxyriboside having the
`following structure:
`
`r.o
`
`In yet anothe~ embodiment, a non-fluorescent
`nucleoside analog may be benzene-2-deoxyriboside having the
`following structure:
`
`20
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`useful intermediates provided by the present
`invention includes the fluorescent and non-fluorescent cyclic
`compound-derivatized nucleoside 5'-3'-paratoluoyl diesters.
`Examples are illustrated as 2a-2f of Fig. 2.
`Other particularly useful intermediates provided by
`the present invention include, for example, the fluorescent and
`non fluorescent cyclic compound-derivatized S'-dimethoxy trityl
`ethers. Examples are illustrated as 4a-4f in Fig. 2.
`Useful phosphoramidite derivatives provided by the
`present invention include N,N-diisopropyl-0-cyanoethyl
`phosphoramidite derivitized at the 3' alcohol of the subject
`nucleoside analogs. Examples are illustrated as Sa-Sf of
`Fig. 2.
`
`The subject fluorescent cyclic compound-derivatized
`nucleosides, (also termed herein "subject nucleoside analogs",
`"subject fluorescent nucleosides" and "subject fluorophores")
`when incorporated into a nucleic acid such as RNA or DNA,
`provide fluorescence at a range of from about 350 nm to about
`1100 nm emission maxima.
`The subject fluorescent nucleosides of the present
`invention can be synthesized by coupling a fluorescent cyclic
`compound to a sugar using a modification of the organocadmium
`strategy described in Schweitzer and Kool (1995) J. Am. Chern.
`Soc. 117:1863. The non-fluorescent spacer molecules may be
`made the same methodology. The disclosure of this article and
`of all other articles cited in this application are
`incorporated herein as if fully set forth.
`The C-nucleoside coupling involves the reaction of
`organocadmium or organozinc derivatives of the cyclic species
`with the well known a-chlorosugar synthon of Hoffer (Hoffer,
`(1960) Chern Ber. 23..:2777). The glycosidic coupling of a
`M.,
`cyclic compound to a sugar coupling results in a mixture of
`alpha and beta anomers in isolated yields of between about 54-
`81%. The primary product of this coupling reaction is the Cl-
`
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`SUBSTITUTE SHEET (RULE 26)
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`coupled product formed with retention of configuration.
`Alpha-
`anomeric c-nucleotides are the primary reaction products.
`Although the alpha orientation is not the same as for natural
`0-nucleotides, alpha nucleosides are also known to form DNA-
`like helices (20) and models indicate that they can still
`interact well with natural bases in neighboring positions.
`Toluoyl protecting groups may be removed in
`methanolic base. Thus, in accordance with the present
`invention, free unprotected nucleosides can be produced ln as
`little as two steps: cyclic coupling and ester deprotection
`(Fig. 2). The alpha-anomers may be converted to the beta
`configuration by a third step, acid-catalyzed equilibration. A
`preferred acid catalyzed equilibration reaction uses
`benzenesulfonic acid in refluxing xylene, in the presence of a
`small amount of water.
`The present invention also provides use of an
`oligomer of the subject nucleoside analogs which can be
`attached to generally any compound via a chemical bridge such
`as a thiol group. Methods for joining molecules can be found,
`for example, in S.L. Beaucage and R.P. Iyer (1993) Tetrahedron
`.1.2.:1925-1963.
`In addition, the present invention provides for
`oligonucleotide analogs in which fluorescent cyclic compounds
`replace some or all of the DNA or RNA bases. Natural
`oligonucleotides are strings of nucleosides bridged by
`phosphodiesters. Oligonucleotide analogs are oligonucleotides
`in which the structures of the bases, sugars, and/or
`phosphodiesters are modified to change or enhance molecular
`properties.
`A chemical structure of a fully fluorophore(cid:173)
`substituted subject oligonucleotide analog is provided below.
`In the structure depicted below, "fluorophore" is meant to
`encompass any of the subject nucleoside analogs described
`herein.
`
`22
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`SUBSTITUTE SHEET (RULE 26)
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`23
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`HO
`
`~'"'"'ho"
`
`0
`I P.-0.
`j~o
`0
`
`~