(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`2 November 2006 (02.11.2006)
`
` (10) International Publication Number
`
`WO 2006/116476 Al
`
`(51) International Patent Classification:
`C07H 21/04 (2006.01)
`CO7H 21/02 (2006.01)
`
`(21) International Application Number:
`PCT/US2006/015773
`
`(22) International Filing Date:
`
`25 April 2006 (25.04.2006)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/675,135
`
`27 April 2005 (27.04.2005)
`
`US
`
`(71) Applicant (for all designated States except US): SIGMA-
`ALDRICH CO. [US/US]; 3050 Spruce Street, St. Louis,
`MO 63103 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW,BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU,ID, IL, IN, IS, JP, KE,
`KG, KM, KN,KP, KR, KZ, LC, LK, LR, LS, LT, LU,LV,
`LY, MA, MD, MG, MK, MN, MW,MX, MZ, NA, NG,NI,
`NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG,
`SK, SL, SM, SY, ‘TJ, ‘TM, TN, TR, ‘VI, TZ, UA, UG, US,
`UZ, VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`7.W), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR,HU,IE,IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO,SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(74) Agent: DOTY, Kathryn; Polsinelli Shalton Welte
`Suelthuas, 100 South Fourth Street, Suite 1100, St. Louis,
`MO 63102-1825 (US).
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations" appearing at the begin-
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: ACTIVATORS FOR OLIGONUCLEOTIDE AND PHOSPHORAMIDITE SYNTHESIS
`
`Inventors; and
`(72)
`Published:
`(75)
`Inventors/Applicants (for US only): LEUCK, Michael
`—_with international search report
`[DE/DE]; Scheideholzweg 47G, 21149 Hamburg (DE).
`before the expiration of the time limit for amending the
`WOLTER, Andreas [DE/DE]; Julius-Leber-Str.
`28,
`claims and to be republished in the event of receipt of
`amendments
`22765 Hamburg (DE).
`
`
`
`116476AcIMITUINOTINANVUOIRIOAYAAA
`
`BS (57) Abstract: The present invention discloses methods for the synthesis of oligonuclelotidesa and nucleoside phosphoramidites.
`06
`‘The methods are based on employing aryl-substituted 5-pheny1-1H-tetrazoles with perfluroalkyl groups on the aromatic ring as acti-
`©} vators. In one aspect the activators are used in the synthesis of oligonucleoties via the phosphoramidite approach. In this aspect the
`activators are highly efficient and can be applied with very short coupling times. In a further aspect, the activators of the invention are
`used in the synthesis of phosphoramidites through the reaction of nucleosides comprising a free hydroxyl group with phosphitylating
`agents. This aspect of the activators provide very pure phosphoramidites under mild conditions. The activators of the invention are
`further characterized as being highly soluble, non-hygroscopic and non-hazardous.
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`ACTIVATORS FOR OLIGONUCLEOTIDE
`AND PHOSPHORAMIDITE SYNTHESIS
`
`FIELD OF THE INVENTION
`
`[0001]
`
`The present invention relates to the fields of nucleotide and oligonucleotide
`
`chemistry. More specifically, the invention relates to improved methods for the preparation
`of oligonucleotides and nucleoside phosphoramidites. In particular, the methods utilize
`
`activators which have advantages over the activators ofthe priorart.
`
`BACKGROUND OF THE INVENTION
`
`[0002]
`
`Life science research has stimulated an enormous increase in the demand for
`
`synthetic oligonucleotides over the last decades. A number of methods in molecular biology
`and DNA-based diagnostics to amplify, detect, analyze and quantify nucleic acids are now
`dependent on chemically synthesized oligonucleotides which are employed as primers and
`probes to amplify or to detect nucleic acid targets. Synthetic nucleic acids are also employed
`as active ingredients in a variety of novel therapeutics. They are used to block the expression
`of genes through hybridization to messenger RNA(antisense oligonucleotides), to inhibit the
`transcription of genes through their specific binding to transcription factors (decoy
`oligonucleotides), to stimulate the immune system (immunostimulatory sequences) and to
`bind to a variety of protein and other moleculartargets due their engineered three-
`dimensional shape in a highly selective manner (aptamers). A particularly important and
`promising application is the use of short, double stranded ribonucleotides to invoke RNA
`
`interference in order to down regulate individual genes based on their sequence (siRNA).
`Molecular tagging of industrial products orlifestock, the sequence-directed formation of
`nanoscale structures and molecular computing are additional important applications of
`syntheticoligonucleotides. Synthetic nucleic acidstherefore represent a highly promising
`class of molecules which are very likely to have many indusirial uses and to positively affect
`the quality oflife.
`
`Synthetic oligonucleotides are prepared through the repeated condensation of
`[0003]
`nucleosides or oligomeric nucleotide derivatives. Such condensation reactions are termed
`"coupling reactions". The most prominent chemical method to perform coupling reactionsis
`the phosphoramidite approach which is displayed in Scheme 1. In this approach, a nucleotide
`monomer or oligomer phosphoramidite (1) is reacted with a nucleoside monomeror
`oligonucleotide (2) that comprises a hydroxyl group in the presence of a catalyst, termed
`
`

`

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`"activator". The reaction productis a phosphorousacid triester (3) that is subsequently
`oxidized to a phosphoricacid triester (4). The phosphoramidite approachis largely based on
`developments reported by Beaucage and Caruthers (1981) Tetrahedron Letters 22:1859-1862,
`McBride and Caruthers (1983) Tetrahedron Letters 24:245-248, and Sinha e¢ al. (1984)
`Nucleic Acids Res. 12:4539-4557, and has been reviewed by Beaucage and lyer (1992)
`
`Tetrahedron 48:2223-2311, each of which is incorporated herein by reference inits entirety.
`
`Nul
`0
`Nul-O
`Nul-O__R'
`PN + HO-Nu2 ——>P-O-Nu2 —> O=P-O-Nu2
`RO R"
`Activator
`R-O
`
`OR
`
`(1)
`
`(2)
`
`(3)
`
`(4)
`
`Scheme 1
`
`wherein
`
`Nul, Nu2 = monomeric or oligomeric nucleoside/nucleotide groups;
`
`R = phosphate protective group, e.g. B-cyanoethyl; and
`
`R', R"= alkyl groups, e.g. diisopropyl.
`
`[0004]
`
`Coupling reactions are either performed in solution or, with the nucleoside
`
`monomeror oligonucleotide (2) being immobilized on a solid support (solid phase
`
`oligonucleotide synthesis = SPOS), whichis the preferred method for the synthesis of
`
`oligonucleotides. In SPOS oligonucleotides are assembled in a cyclical manner, each cycle
`
`consisting of a series of three chemical reactions. Thefirst reaction is a deblocking reaction,
`
`i.e. the removal of a front-end protective group from the nucleosideor oligonucleotide bound
`
`to the support, for example the removal of a dimethoxytrityl protective group. The second
`
`reaction is the coupling reaction of a nucleotide monomeror oligomer phosphoramidite to the
`partially deprotected nucleoside or oligonucleotide on the Supportinthe presence ofan
`activator. Thethird reaction is the oxidation of the phosphite triester coupling product to a
`phosphate triester. Optionally, a capping reaction is included in each cycle either directly
`
`before or after the oxidation reaction in order to block those support bound nucleosides or
`
`oligonucleotides which failed to react in the coupling reaction and to prevent them from
`
`further growth in subsequent chain elongation steps.
`
`[0005]
`
`Oneof the major methods of preparing phosphoramidites (1) is to react a
`
`nucleoside (5) with a phosphitylating agent (6) in the presence ofa catalyst, as displayed in
`
`Scheme 2. The catalyst applied in this process is termed an “activator” analogousto the
`
`catalyst applied in the synthesis of oligonucleotides mentioned above.
`
`

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`
`R'NER" _ Nu-O x
`Nu-OH + BORER"
`Activator
`RO R"
`
`R'
`
`(5)
`
`(6)
`
`Scheme 2
`
`wherein
`
`Nu = monomeric or oligomeric nucleoside/nucleotide group;
`
`R = phosphate protective group, e.g. B-cyanoethyl; and
`
`R', R" = alkyl groups, e.g. diisopropyl.
`
`[0006]
`
`Several compoundshave been described as activators for the synthesis of
`
`oligonucleotides via the phosphoramidite approach in the technical literature, most of them
`
`being either azoles or azolium salts formed from azoles with strong acids, e.g. azolium
`triflates. The common feature of the described activators is that they represent weak proton
`acids as well as good nucleophiles (the nucleophile being either the respective compound
`itself or its conjugated base). Both functions, i.c. providing a weak proton acid and a
`nucleophile, are deemed important in the mechanism of phosphoramidite coupling reactions
`as discussed by Dahlet al. (1987) Nucleic Acids Res. 15:1729-43, and Berneret al. (1989)
`Nucleic Acids Res. 17:853-864, each of whichis incorporated herein by reference in its
`entirety. Activators for the synthesis of oligonucleotides are also applied as activators for the
`synthesis ofphosphoramidites. Ammonium salts of azoles, e.g. the diisopropylammonium
`salts of 1/7-tetrazole or 4,5-dicyanoimidazole, are also used for this purpose.
`[0007]
`A numberofactivators are being marketed commercially for the synthesis of
`oligonucleotides. Examples of commercially available activators include 1H-tetrazole,
`described byBeaucage andCaruthers(1981) Tetrahedron Letters 22:1859-1862, 4,5-
`dicyanoimidazole ("DCI"), described by Vargeese et al. (1998) Nucleic Acids Res. 26:1046-
`1050, 5-ethylthio-1H-tetrazole ("ETT"), described by Wrightet al. (1993) Tetrahedron
`Letters 34:3373-3376, and 5-benzylthio-1H-tetrazole ("BTT"), described by Welz and Muller
`(2002) Tetrahedron Letters 43:795-797. Each of these referencesis specifically incorporated
`herein by reference inits entirety.
`[0008]
`Otheractivators that have been describedin the literature include 5-(4-
`nitrophenyl)-1#-tetrazole, described by Froehler and Matteucci (1983) Tetrahedron Letters
`24:3171-3174, 5-(3-nitrophenyl)-1-tetrazole, described by Raoef al. (1993) J. Chem. Soc.
`Perkin Trans. I 43-55, N-methylanilinium trifluoracetate, described by Fourrey and Varenne
`
`

`

`WO 2006/116476
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`PCT/US2006/015773
`
`(1984) Tetrahedron Letters 25:4511-4514, 2,4-dinitrophenol, described by Dabkowskietal.
`(2000) Tetrahedron Letters 41:7535-7539, 1-hydroxybenzotriazole, described by Eritja
`(1990) Tetrahedron 46:721-730, 2,4-dinitrobenzoic acid, described by Reddy and Farooqui.,
`U.S. Pat. No. 5,574,146, benzimidazolium triflate and other azolium salts, e.g. N-
`phenylimidazolium triflate, described by Hayakawaetal. (1996) J. Org. Chem. 61:7996-7997
`and (2001) J. Am. Chem. Soc. 123:8165-8176, 1,2,3-benzotriazole and 5-substituted
`derivatives thereof, described by Hudson and Cook, U.S. Pat. No. 4,474,948, pyridinium
`hydrochloride, described by Gryaznov and Letsinger (1992) Nucleic Acids Res. 20:1879-
`1882, 1-methyl-5-mercaptotetrazole, described by Efimovefal. (1996) Russ. J. Bioorg.
`Chem. 22:128-130, a combination of pyridinium trifluoracetate and 1-methylimidazole,
`
`described by Sanghvi and Manoharan, U.S. Pat. No. 6,274,725, saccharin, described by Sinha
`and Revell, International Publication No. WO 03/004512, and other compounds. Each of
`
`these references is specifically incorporated herein by reference in its entirety.
`
`[0009]
`
`The activators described to date have certain disadvantages that warrant the
`
`search for new and improved activator molecules. For instance, some of the described
`activators are hygroscopic, such as pyridinium chloride, which requires very strict exclusion
`
`of moisture for their storage and handling. The application of such activators in the synthesis
`
`of oligonucleotides results in a high risk for synthesis failure due to the great sensitivity of
`
`coupling reactions with respect to moisture. This risk is particularly pronounced when a low
`
`molar excess of phosphoramidite is applied, for instance in large scale solid phase
`
`oligonucleotide synthesis or in coupling reactions which are performed in solution.
`
`[0010]
`
`Other activators are sensitive to heat and/or mechanical impacts and may
`
`cause explosions under such conditions, e.g. 1H-tetrazole and ETT. 14-Tetrazole tests
`
`positive in a test of mechanical sensitivity with respect to shock (“Fallhammer-test’’), and is
`
`classifiedasaCategory A explosivein.Germany. ETTis classified as a-Category-C -
`
`explosive in Germany dueto its thermal sensitivity. These activators, when used as powders,
`
`require special handling and safety procedures during their purification, storage, shipping, use
`and disposal which increasesthe cost in routine applications. They also raise safety concerns
`
`as they may cause great damage to personnel, equipment and buildings under special
`
`conditions, e.g. in case of a fire, when stored in large quantities.
`
`[0011]
`
`Other activators such as 5-(4-nitrophenyl)-1H-tetrazole (7) have low solubility
`
`in acetonitrile, the preferred solvent for coupling reactions, and may crystallize in the lines
`
`and valves of automated oligonucleotide synthesis instruments upon slight variations of the
`
`environmental temperature, thus blocking the instrument and causing synthesis failures. This
`
`

`

`WO 2006/116476
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`
`undesired phenomenonalso occurs with 1H-tetrazole, which is routinely applied at
`concentrations near its maximum solubility in acetonitrile (appr. 0.45 M at 25°C).
`
`OyOlH
`
`(7)
`
`NO,
`
`[0012]
`
`Otheractivators, likc DCI, may cause precipitation and associated line
`
`clogging or valve failure in certain DNA/RNAsynthesis instruments due to the
`
`incompatibility of solutions of these activators in acetonitrile with other synthesis solutions
`
`applied in such instruments. For instance, a mixture consisting of 10 volume percent of a 0.5
`
`M DCI-solution in acetonitrile and 90 volumepercent of a 3% solutionof trichloroacetic acid
`
`in dichloromethane (w/v) becomes cloudy instantaneously after mixing of the two
`
`components and a precipitate is deposited from the resulting suspension. It appearslikely
`that such precipitation is caused by the low solubility of DCI in dichloromethane. A solution
`of 3% trichloroacetic acid in dichloromethane is widely applied in DNA/RNA synthesis
`instruments for the removal of dimethoxytrityl protective groups in the deblocking reaction of
`SPOScycles. The deblocking reaction is immediately followed by the coupling reaction in an
`SPOScycle, thus causing a risk of precipitate forming in the machine, because the employed
`activator solution may comein direct contact with the solution employed in the deblocking
`reaction.
`
`In the field of phosphoramidite-mediated RNA synthesis, the activators
`[0013]
`described so far are generally not active enough to promote coupling efficiencies comparable
`to those observed in DNA synthesis. This phenomenonespecially relates to the most widely
`used RNA amidites, i.e. 2’-O-tert-butyl-dimethylsilyl protected RNA amidites. With existing
`activators these amidites require longer couplingtimes at higher activator and/or amidite
`. concentration, butstill result in inferior product yields and purity compared to DNA
`synthesis. Consequently,it is desirable to develop alternative activators that promote the
`highly efficient synthesis of RNA oligonucleotides.
`
`Although, as discussed above, a variety of activators for the synthesis of
`[0014]
`oligonucleotides and phosphoramidites have been described, and someofthe described
`activators are commercially available, there is a need to find improved activators that
`combine the desired features of high activation efficiency and goodsolubility with easy, safe
`and economic handling, whichlead to superior synthesis results. In particular, the synthesis
`
`

`

`WO 2006/116476
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`
`of RNA oligonucleotides and other oligonucleotides prepared from sterically demanding
`
`phosphoramidites requires more efficient activators than are currently available.
`
`(0015]
`
`The present invention discloses novel methodsandactivators for the synthesis
`
`of oligonucleotides and nucleoside phosphoramidites based on aryl substituted 5-phenyl-1H-
`
`tetrazoles wherein at least one of the substituents on the aromatic ring is a perfluoroalkyl
`
`substituent. The disclosed activators are highly efficient in coupling DNA-, RNA-andother
`
`phosphoramidites, highly soluble, non-hygroscopic and non-hazardous.
`
`SUMMARY OF THE INVENTION
`
`[0016]
`
`The present invention discloses novel methodsfor the synthesis of
`
`oligonucleotides via the phosphoramidite approach. Included in the present invention are
`
`novel methodsfor the synthesis of phosphoramidites. The methods are based on the
`
`application of novel aryl-substituted 5-phenyl-14-tetrazoles as catalysts in coupling reactions
`of the phosphoramidite approach for the synthesis of oligonucleotides and in the synthesis of
`phosphoramidites with phosphitylating agents.
`
`[0017]
`
`In one embodiment, the present invention discloses novel methodsfor the
`
`synthesis of oligonucleotides via the phosphoramidite approach, wherein the coupling
`reaction is performed in the presence of certain aryl-substituted 5-phenyl-1H-tetrazoles as
`
`catalysts. The catalysts employed in the methods of the invention are characterized by
`carrying perfluoroalkyl substituents on the phenyl ring and can be represented by the
`
`following generalstructure:
`
`caH‘GS(R)n
`
`| wherein R is as defined below and wherein at least one R contains a perfluoroalkyl
`substituent and wherein n is an integer selected from 1-5. These catalysts are highly soluble
`in the solvent ofthe coupling reaction and promotethe highly efficient synthesis of DNA,
`RNAand modified oligonucleotides. In one preferred embodimentthe aryl-substituted 5-
`phenyl-1#-tetrazole is 5-(3,5-bis(trifluoromethyl)phenyl)-1A-tetrazole (8).
`
`N-oc3
`
`H
`
`CF;
`(8)
`
`

`

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`[0018]
`
`In one embodiment of the invention the synthesis of oligonucleotidesis
`
`conducted as solid phase oligonucleotide synthesis (SPOS). In a preferred embodiment, the
`
`synthesis of oligonucleotides is conducted as solid phase oligonucleotide synthesis and the
`
`aryl-substituted 5-phenyl-1H-tetrazole is 5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole (8).
`
`[0019]
`
`Using the method ofthis invention, the coupling time employed in the
`
`synthesis of DNA oligonucleotides is shorter than the coupling time employed in the
`
`conventional methodsin the art. In preferred embodiments, the maximum coupling timefor
`
`DNAphosphoramidites is about 15 seconds and the maximum coupling time for 2'-O-tert-
`
`butyldimethylsilyl RNA phosphoramidites is about 5 minutes.
`
`[0020]
`
`In other embodiments, the novel activators of the invention are applied in the
`
`presence of a nucleophilic catalyst. In a preferred embodiment, the nucleophilic catalystis
`
`N-methylimidazole.
`
`[0021]
`
`In another embodiment, the present invention discloses novel methodsfor the
`
`synthesis of nucleoside phosphoramidites through the reaction of a nucleoside comprising a
`
`free hydroxyl group with a phosphitylating agent in the presence of aryl-substituted 5-phenyl-
`
`1H-tetrazoles as catalysts wherein the substituents on the phenyl ring are perfluoroalkyl
`
`substituents.
`
`[0022]
`
`In a preferred embodiment the synthesis of phosphoramidites is conducted in
`
`the presence of 5-(3,5-bis(trifluoromethyl)phenyl)-14-tetrazole (8) as catalyst. In another
`
`preferred embodimentthe synthesis of phosphoramidites is conducted employing
`
`bis(diisopropylamino)-2-cyanoethoxyphosphaneas phosphitylating agent.
`
`In a particularly preferred embodiment, the synthesis of phosphoramidites is
`[0023]
`conducted in the presence of 5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole (8) as catalyst
`and bis(diisopropylamino)-2-cyanoethoxyphosphane is employed as phosphitylating agent.
`[0024] —_—_— Additional objectivesand advantagesof the present inyentionwillbeapparent.
`to those skilled in the art upon examination of the detailed description that follows.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figures 1A and 1B display the anion exchange chromatogramsof the 51-mer
`{0025}
`DNAoligonucleotide product (21) and the 103-mer DNAoligonucleotide product (22)
`synthesized using a 0.1 M solution of 5-(3,5-bis(trifluoromethyl)phenyl)-1-tetrazole (8) as
`activator solution, as described in Example 4.
`
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`

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`Figure 2 depicts the time course of the coupling reaction of DMT-rA(tac)-
`[0026]
`amidite (23) with dT-si (24) in the presenceofeither 5-(3,5-bis(trifluoromethyl)phenyl)-14-
`
`tetrazole (8) or ETT asactivators, as described in Example 8.
`[0027]
`Figures 3A and 3B display the reversed phase HPLC chromatogram of the
`RNAoligonucleotide products (27) and (28) synthesized using a 0.1 M solution of 5-(3,5-
`bis(trifluoromethyl)phenyl)-14-tetrazole (8) as activator solution, as described in Examples
`
`10 and 11.
`
`[0028]
`
`Figure 4 depicts the reversed phase HPLC chromatogram of 5-(3,5-
`
`bis(trifluoromethyl)phenyl)-1#-tetrazole (8) synthesized in Example 15.
`
`[0029]
`
`Figure 5 displays the reversed phase HPLC chromatogram of dG(ib)-amidite
`
`(36) synthesized in Example 17.
`
`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
`
`[0030]
`
`Various terms are used herein to refer to aspects of the present invention. To
`
`aid in the clarification of the description of the components of the invention, the following
`
`descriptions are provided.
`
`[0031]
`
`It is to be noted that the term "a" or "an" entity refers to one or more of that
`
`entity; for example, an oligonucleotide refers to one or more oligonucleotides. As such, the
`
`terms "a" or "an," "one or more" and "at least one" are used interchangeably herein and are
`
`not intendedto limit the scope of the invention.
`
`[0032]
`
`The term "oligonucleotide" as used herein refers to a single stranded chain of
`
`either deoxyribonucleotides or ribonucleotides or chemical modifications thereof, such as
`
`nucleotides with a 2’-O-4'C-methylene bridge in their sugar portion, which are the
`
`constituting nucleotides of locked nucleic acids (LNA). Modifications include, but are not
`
`_limited.to, those which provide other chemical groups that incorporate additional-charge,-
`
`polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual
`
`nucleotides or their corresponding basesorto the oligonucleotides as a whole. Such
`
`modifications include, but are not limited to, modified bases such as sugar modifications at
`
`the 2'-position of the nucleoside, pyrimidine modificationsat the 5-position of the
`
`heterocyclic base, purine modifications at the 8-position of the heterocyclic base,
`
`modifications at the exocyclic amine group of cytosine bases, methylations, bases that can be
`
`part of unusual base-pairing combinations such as the isobases isocytidine and isoguanidine
`and the like. Modifications further include attached labels and reporter molecules, such as
`
`fluorescent dyes, biotin, minor groove binders and the like that are known to those skilled in
`
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`

`WO 2006/116476
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`
`the art. In addition modifications include modified phosphate groups of the oligonucleotides,
`examples being phosphorothioate oligonucleotides, methylphosphonate oligonucleotides,
`
`phosphoramidate oligonucleotides, phosphorodithioate oligonucleotides and other
`
`modifications known to those skilled in the art and reviewed by Micklefield, (2001) Current
`
`Medicinal Chemistry 8:1157-1179, which is incorporated herein by referenceinits entirety.
`
`Oligonucleotides, as referred to in this invention can consist of any combinations of the
`
`nucleotides and their modifications described above and can haveeither a few, e.g. up to 20,
`
`or many, e.g. 20 to several hundred or more, nucleotides incorporated in its chain.
`
`[0033]
`
`An "RNA oligonucleotide" as used herein consists either entirely or to a large
`
`part, i.e. to more than 50% of the nucleotides which constitute the oligonucleotide, of
`
`ribonucleotides or 2'-modified ribonucleotides like 2'-O-methyl-ribonucleotides, 2'-O-
`
`methoxyethyl- ribonucieotides, 2'-fluoro-ribonucleotides or the like, whereas a "DNA
`
`oligonucleotide" as used herein consists either entirely or to a large part, i.e. to more than
`50% of the nucleotides which constitute the oligonucleotide, of deoxyribonucleotides.
`[0034]
`The term "phosphoramidite" as used herein refers to a phosphorous acid
`diester dialkylamide as depicted in formula (9), which is comprised ofa trivalent
`phosphorous atom bondedto one dialkylamino group (NR'R") and two alkoxy or aryloxy
`groups O-R, and O-R2.
`
` R
`RyO_
`PN
`R,-O-
`RY"
`(9)
`
`wherein R; and R2, R' and R" each taken separately are selected from anyofthe substituents
`which would be knownto those of skill in the art. By way of non-limiting example, R; and
`Ro, R' and R" each taken separately, may represent alkyl, aralkyl, cycloalkyl and-
`cycloalkylalkyl. In the context of oligonucleotide synthesis phosphoramidites may either
`contain an alkoxy group which comprisea nucleoside or oligonucleotide moiety, or may
`exclusively contain non-nucleosidic alkoxy or aryloxy groups. Thelatter are used to
`introduce modifications into oligonucleotides such as terminal phosphate groups, reporter
`groups, haptens or other modifications knownto those skilled in the art and as reviewed by
`Beaucage and Iyer (1993) Tetrahedron 49:1925-1963, whichis incorporated herein by
`reference in its entirety. In the presence ofa suitable catalyst phosphoramidites react with
`hydroxyl groups to form phosphitetriesters.
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`"Nucleoside phosphoramidites" as used herein are phosphoramidites in
`[0035]
`which phosphorousacid is esterified to a protected nucleoside or a protected oligonucleotide
`(group R, of formula (9)). The second phosphorousacid ester function is comprised of a
`
`phosphate protective group (group R2 of formula (9)). Nucleoside phosphoramidites carry a
`
`temporary protective group which is removed in the course of the synthesis of
`
`oligonucleotides and may contain one or more additional protective groups attached to the
`
`nucleoside portion of the molecule which are removedafter the synthesis of the
`
`oligonucleotides. In order to furtherillustrate the term nucleoside phosphoramidite
`
`commercially marketed examples of nucleoside phosphoramidites are depicted in formulae
`
`(10) and (11). Formula (10) depicts a DNA phosphoramidite and formula (11) an RNA
`
`phosphoramidite. Formula (12) depicts a dimer phosphoramidite as an example of a
`
`nucleoside phosphoramidite derived from a protected oligonucleotide.
`
`MeO
`
`(> 6
`
`O
`
`Base
`
`MeO
`
`O
`
`Base
`
`(I+ 5
`
`wherein Base, Basel and Base2 = protected or unprotected nucleobases.
`[0036]
`The term nucleoside phosphoramidite is, however, not limited in any way by
`the nature of the examples (10) to (12). Any other nucleosides, either DNA nucleosides or
`RNA nucleosides or any modifications thereof, including, but not limited to 2'-modified
`nucleosides such as 2'-O-methylnucleosides, 2'-O-methoxyethylnucleosides and 2'-
`fluoronucleosides, nucleosides with bicyclic sugar moieties such as LNA nucleosides,
`
`10
`
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`arabino-nucleosides, nucleosides with 6-membered sugar rings such as D-altriol nucleosides
`as described by Allart et al. (1999) Chem Eur. J. 5:2424-2431, or anhydrohexitol nucleosides
`
`as described by Van Aerschotet al. (2001) Nucleic Acids Res. 29:4187-94, or any other
`
`modified nucleosides knownto those skilled in the art could be part of nucleoside
`
`phosphoramidites as used herein. The nucleobases of such nucleosides may either be one of
`
`the main naturally occurring nucleobases,i.e. the purine bases adenine and guanine and the
`
`pyrimidine bases thymine, cytosine and uracil, or be modified nucleobases, including, but not
`
`limited to 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other
`
`alkyl derivatives of adenine and guanosine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 7-
`
`deazaguanine, 7-deazaadenine and any other modified nucleobase knownto those skilled in
`
`the art. In many instances, but not in any case the nucleobase of nucleoside
`
`phosphoramidites is protected wherein one or more functionalities of the nucleobase bears a
`
`protective group, non-limiting examples being in N6-benzoyladenine, N4-benzoylcytidine,
`
`N2-isobutyrylguanine, N2-(N,N-dimethylformamidino)guanosine, N3-anisoylthymine or 06-
`
`dichlorophenyl-N2-isobutyrylguanine. A great variety of such protective groups for
`
`nucleobases has been disclosed in the technicalliterature as described e.g. by Beaucageet al.
`
`(1992) Tetrahedron 48:2223-2311. The term nucleoside phosphoramidite as used herein
`
`includes any suitable combination of protective groups and nucleobases knownto those
`
`skilled in the art. The nucleobase of nucleoside phosphoramidites as used herein may,
`however, also be unprotected, for instance in commercially available deoxythymidine
`
`phosphoramidites, or in nucleoside phosphoramidites of other nucleobases as demonstrated
`
`by Gryaznov and Letsinger (1992) Nucleic Acids Res. 20:1879-1882, and Hayakawaet al.
`(1998) J. Am. Chem. Soc. 120:12395-12401, each of which is incorporated herein by
`referencein its entirety.
`
`[0037]===‘ Thephosphoramidites(10) to (12) carry the commonly employed_bis(4-
`methoxyphenyl)phenylmethyl (dimethoxytrityl = "DMT") group as temporary protective
`group. Nucleoside phosphoramidites as used herein are, however, not limited by the nature
`of the employed temporary protective group. Temporary protective groups for nucleoside
`phosphoramidites include, but are not limited to substituted triphenylmethyl groups other
`than the DMT-group,including butnot limited to the 9-phenylxanthen-9-yl ("pixyl") group,
`the 9-fluorenylmethoxycarbonyl ("Fmoc") group and photolabile protective groups, e.g. the
`((a-methyl-2-nitropiperonyl)-oxy)carbonyl ("MeNPOC") group as described by McGallet ai.
`(1997) J. Am. Chem. Soc. 119:5081-5090. Temporary protective groups of nucleotide
`synthons are reviewed by Seliger (2000) in Current Protocols in Nucleic Acid Chemistry,
`
`11
`
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`2.3.1-2.3.34, eds. Beaucage, S.L., Bergstrom, G.D. Glick, G.D. and Jones, R.A., J. Wiley &
`
`Sons Inc. NY, which is incorporated herein by reference in its entirety. Nucleoside
`
`phosphoramidites as used herein may carry any suitable temporary protective group known to
`
`those skilled in the art.
`
`[0038]
`
`The RNA phosphoramidite (11) carries a tert-butyldimethylsilyl protective
`
`group at the 2'-O-position. RNA nucleoside phosphoramidites as used herein are, however,
`
`not limited by the nature of the employed 2'-O-protective group. 2'-O-protective groups for
`RNAnucleoside phosphoramidites as used herein include, but are not limited to the
`
`(triisopropylsilyl)oxymethyl ("TOM") group as described by Pitsch e¢ al. (2001) Helv. Chim.
`
`Acta 84:3773-3795, the methoxytetrahydropyranyl ("MTHP") group as described by
`
`Lehmann e¢ af. (1989) Nucleic Acids Res. 17:2379-2390, the 1-(2-fluorophenyl)-4-
`methoxypiperidin-4-yl ("Fpmp") group as described by Capaldi and Reese (1994) Nucleic
`Acids Res. 22:2209-2216, and any other 2'-O-protective group for RNA phosphoramidites
`knownto those skilled in the art. Each of these literature referencesis specifically
`incorporated herein by referencein its entirety.
`
`The phosphoramidites (10) to (12) are phosphorousacid diisopropylamides.
`[0039]
`Nucleoside phosphoramidites as used herein are, however, not limited by the nature of the
`phosphorousacid amide group. In the phosphorous acid amide group of the nucleoside
`phosphoramidites as used herein -N(R',R") the substituents R' and R" are independently
`selected from an alkyl group having from about one to about ten carbons,or taken together R'
`and R"together form a cyclic alkylene group having from about twoto up to about twenty
`carbons which may or maynot have additional alkyl substituents attached to it and which
`may contain up to 3 heteroatoms selected from N, O and S includedin the cyclic alkylene
`
`group.
`
`[0040] == =—«The phosphoramidites (10) to (12) carry 2-cyanoethyl phosphate protective
`groups. Nucleoside phosphoramidites as used herein are, however, not limited by the nature
`of the phosphate protective group. Examples of other phosphate protective groups of
`nucleoside phosphoramidites as used herein include, but are not limited to, methyl-, allyl-, p-