WO2002079216A1 - Labeled oligonucleotides, methods for making same, and compounds useful therefor - Google Patents
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- LABELED OLIGONUCLEOTIDES METHODS FOR MAKING SAME, AND COMPOUNDS USEFUL THEREFOR
- the present invention is directed to labeled oligonucleotides, methods for making the same, and compounds useful therefor. More specifically, this invention relates to oligonucleotides selectively functionalized at one or more of the 3'-terminaInucleotide, 5'-terminalnucleotide, and intemucleotides with conjugate groups, methods for making the same, and compounds useful therefor.
- Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. The widespread use of such oligonucleotides has increased the demand for rapid, inexpensive and efficient procedures for their modification and synthesis. Early synthetic approaches to oligonucleotide synthesis included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72, 209, 1972; Reese, Tetrahedron Lett. 34, 3143-3179, 1978. These approaches eventually gave way to more efficient modem methods, such as the use of phosphoramidites and H-phosphonates. Beaucage and Carathers, Tetrahedron Lett., 22, 1859-1862, 1981; Agrawal and Zamecnik, U.S. Patent No. 5,149,798, issued 1992.
- cyanoethyl phosphoramidite monomers are quite expensive. Although considerable quantities of monomer go unreacted in a typical phosphoramidite coupling, unreacted monomer can be recovered, if at all, only with great difficulty.
- N-trifluoroacetyl-aminoalkanols Similar methods using N-trifluoroacetyl-aminoalkanols as phosphate protecting groups has also been reported by Wilk et al., J. Org. Chem., 62, 6712-6713, 1997. This deprotection is governed by a mechanism that involves removal of N-trifluoroacetyl group followed by cyclization of aminoalkyl phosphotri esters to azacyclanes, which is accompanied by the release of the phosphodiester group.
- Solid phase techniques continue to play a large role in oligonucleotidic synthetic approaches.
- the 3'-most nucleoside is anchored to a solid support which is functionalized with hydroxyl or amino residues.
- the additional nucleosides are subsequently added in a step-wise fashion to form the desired linkages between the 3'- functional group of the incoming nucleoside, and the 5 '-hydroxyl group of the support bound nucleoside.
- Implicit to this step-wise assembly is the judicious choice of suitable phosphorus protecting groups.
- Such protecting groups serve to shield phosphorus moieties of the nucleoside base portion of the growing oligomer until such time that it is cleaved from the solid support. Consequently, new protecting groups, which are versatile in oligonucleotidic synthesis, are needed.
- modifications to naturally occurring oligonucleotides include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
- nonisotopic labels e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
- Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2'-O- methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution.
- oligonucleotides especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence.
- mRNA target messenger RNA
- oligonucleotides are currently undergoing clinical trials for such use.
- Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
- Other mechanisms of action have also been proposed.
- oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
- Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
- Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra.
- Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses.
- modifications include those designed to increase binding to a target strand (i.e.
- Tm melting temperatures
- antisense oligonucleotides have been modified to be conjugated with lipophilic molecules.
- oligonucleotides conjugated with lipophilic molecules are able to enhance the free uptake of the oligonucleotides without the need for any transfection agents in cell culture studies.
- Conjugated oligonucleotides are also able to improve the protein binding of oligonucleotides containing phosphodiester linkages.
- oligonucleotides selectively labeled with two different reporter groups have attained a widespread interest due to their unique properties.
- FRET fluorescence resonance energy transfer
- oligonucleotide complexes with the nucleic acid
- the donor and acceptor groups are forced to move away from each other, thereby restoring the fluorescence signal.
- Oligonucleotides that exhibit such a hybridization-dependent fluorescence have been termed "molecular beacons.”
- Molecular beacons may be useful in numerous applications, such as real-time monitoring of hybridization in PCR, in molecular biosensors, on surfaces, in blood, in living cells, and in vivo. Synthesis of double labeled oligonucleotides has been performed with the aid of dye-labeled solid supports and phosphoramidites.
- oligonucleotides that bear an amino and an activated thiol group at opposite ends have been synthesized using modified phosphoramidites and solid supports, where chemoselective labeling is performed post-synthetically.
- the known methods for synthesizing double labeled oligonucleotides are restricted to the use of only certain dyes that are available as phosphoramidite, solid support, and chemoselective reagents.
- oligonucleotides In light of the foregoing, there is a continued need for selectively labeled oligonucleotides, methods for making the oligonucleotides, and compounds useful therefor.
- the labeled oligonucleotides should provide improved cellular permeation, enhanced free uptake of the oligonucleotide in cell culture studies, and improved protein binding, especially for oligonucleotides containing phosphodiester linkages.
- methods for producing oligonucleotides selectively labeled at one or more of the 3 '-terminal nucleotide, 5 '-terminal nucleotide, and intemucleotides with one or more different conjugate groups The methods should also provide for such labeled oligonucleotides without the need for post-synthetic labeling.
- the present invention allows for the selective functionalization of oligonucleotides with conjugate groups.
- the oligonucleotides can be selectively functionalized with a first conjugate group at the 3'-terminal nucleotide and optionally functionalized with a second conjugate group at the 5 '-terminal nucleotide and/or one or more intemucleotides.
- the oligonucleotides can be selectively functionalized with a first conjugate group at the 5'-terminal nucleotide and optionally functionalized with a second conjugate group at one or more intemucleotides.
- the oligonucleotides can be functionalized with a first conjugate group at one or more intemucleotides and with a second conjugate group at one or more different intemucleotides.
- Rl is hydroxyl, a protected hydroxyl or a group having the formula:
- Qo O or S
- Rt is O " , hydroxyl or a protected hydroxyl
- R is hydroxyl, a protected hydroxyl or a group having the formula:
- Li, L and each of said L 3 are, independently, a conjugate group
- FIG. 1 is an RP HPLC profile of compound 120 in its crude form prior to removal of the DMT group
- FIG. 2 is an RP HPLC profile of compound 120 in its crude form after removal of the DMT group
- FIG. 3 is an RP HPLC profile of compound 120 in its final form
- FIG. 4 is an RP HPLC profile of compound 129 in its crude form prior to removal of the DMT group;
- FIG. 5 is an RP HPLC profile of compound 129 in its crude form after removal of the DMT group
- FIG. 6 is an RP HPLC profile of compound 129 in its final form
- FIG. 7 is an RP HPLC profile of compound 126 in its crude form after removal of the DMT group
- FIG. 8 is an RP HPLC profile of compound 126 in its final form
- FIG. 9 is an RP HPLC profile of compound 121 in its crude form after removal of the DMT group.
- FIG. 10 is an RP HPLC profile of compound 121 in its final form.
- the present invention provides methods for preparing compounds that comprise a plurality of linked nucleotides wherein the linked nucleotides are selectively functionalized or labeled with conjugate groups.
- the compounds can be functionalized at the 3 '-terminal nucleotide, at the 5 '-terminal nucleotide, at an intemucleotide, at the 3'- terminal nucleotide with a first conjugate group and at the 5 '-terminal nucleotide with a second conjugate group, at the 3'-terminal nucleotide with a first conjugate group and at one or more intemucleotides with a second conjugate group, at the 5'-terminal nucleotide with a first conjugate group and at one or more intemucleotides with a second conjugate group, or at one or more intemucleotides with a first conjugate group and one or more intemucleotides with a second conjugate group.
- oligomer and “oligomeric compound” refer to compounds containing a plurality of monomeric subunits that are joined by phosphorus-containing linkages, such as phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate linkages. Oligomeric compounds therefore include oligonucleotides, their analogs, and synthetic oligonucleotides. The methods of the invention are used for the preparation of oligonucleotides and their analogs. As used herein, the term "oligonucleotide analog" means compounds that can contain both naturally occurring (i.e.
- oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides.
- oligonucleotide analogs include all such stractures which function effectively to mimic the structure and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target.
- synthetic nucleoside for the purpose of the present invention, refers to a modified nucleoside. Representative modifications include modification of a heterocyclic base portion of a nucleoside to give a non-naturally occurring nucleobase, a sugar portion of a nucleoside, or both simultaneously.
- the present invention relates to processes for preparing an oligonucleotide having the formula:
- Ri is hydroxyl, a protected hydroxyl or a group having the formula:
- Qo O or S
- R4 is O " , hydroxyl or protected hydroxyl
- R 2 is hydroxyl, a protected hydroxyl or a group having the formula:
- L ⁇ , L 2 and each of said L 3 are, independently, a conjugate group.
- K comprises a first conjugate group and R 2 optionally comprises a second conjugate group.
- R 2 optionally comprises a second conjugate group.
- . ⁇ comprises a first conjugate group and one or more R3 optionally comprise a second conjugate group.
- R 2 comprises a first conjugate group and one or more R 3 optionally comprise a second conjugate group.
- one or more R 3 comprise a first conjugate group and one or more R 3 optionally comprise a second conjugate group.
- R comprises a first conjugate group, R optionally comprises a second conjugate group, and at least one R 3 comprises a hydroxyl group.
- R comprises a first conjugate group and at least one R 3 comprises a hydroxyl group.
- Oligonucleotides, or more broadly oligomeric compounds, according to the present invention preferably comprise from about 3 to about 50 nucleosides. It is more preferred that such compounds comprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosides being particularly preferred.
- smaller oligomeric compounds are preferred.
- Libraries of dimeric, trimeric, or higher order compounds can be prepared for use as synthons in the methods of the invention. The use of small sequences synthesized via solution phase chemistries in automated synthesis of larger oligonucleotides enhances the coupling efficiency and the purity of the final oligonucleotides.
- conjugate groups can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
- Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
- Typical conjugates groups include cholesterols, phosphohpids, biotin, phenazine, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
- Groups that enhance the pharmacodynamic properties include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA.
- Groups that enhance the pharmacokinetic properties include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed October 23, 1992, United States Patent No.
- Preferred conjugate groups amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al, Bioorg. Med. Chem.
- lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g
- RNA cleavers include o-phenanthroline/Cu complexes and Ru(bipyridine) 3 + complexes. The Ru(bpy) 3 2+ complexes interact with nucleic acids and cleave nucleic acids photochemically.
- Metal chelators are include EDTA, DTP A, and o-phenanthroline.
- Alkylators include compounds such as iodoacetamide.
- Po ⁇ hyrins include po ⁇ hine, its substituted forms, and metal complexes.
- Pyrenes include pyrene and other pyrene-based carboxylic acids that could be conjugated using the similar protocols.
- Hybrid intercalator/ligands include the photonuclease/intercalator ligand 6-[[[[9-
- Photo-crosslinking agents include aryl azides such as, for example, N- hydroxysucciniimidyl-4-azidobenzoate (HSAB) and N-succinimidyl-6(-4'-azido-2'- nitrophenyl-amino)hexanoate (SANPAH).
- HSAB N- hydroxysucciniimidyl-4-azidobenzoate
- SANPAH N-succinimidyl-6(-4'-azido-2'- nitrophenyl-amino)hexanoate
- Aryl azides conjugated to oligonucleotides effect crosslinking with nucleic acids and proteins upon irradiation, They also crosslink with carrier proteins (such as KLH or BSA), raising antibody against the oligonucleotides.
- Vitamins according to the invention generally can be classified as water soluble or lipid soluble.
- Water soluble vitamins include thiamine, riboflavin, nicotinic acid or niacin, the vitamin B 6 pyridoxal group, pantothenic acid, biotin, folic acid, the B 1 cobamide coenzymes, inositol, choline and ascorbic acid.
- Lipid soluble vitamins include the vitamin A family, vitamin D, the vitamin E tocopherol family and vitamin K (and phytols).
- the vitamin A family including retinoic acid and retinol, are absorbed and transported to target tissues through their interaction with specific proteins such as cytosol retinol-binding protein type II (CRBP-II), retinol-binding protein (RBP), and cellular retinol-binding protein (CRBP).
- CRBP-II cytosol retinol-binding protein type II
- RBP retinol-binding protein
- CRBP cellular retinol-binding protein
- the conjugate groups, Li, L , and/or L 3 are optionally attached to the oligonucleotides of the present invention through a linking group.
- Suitable linking groups include, but are not limited to, dialkylglycerol linkers.
- Preferred dialkylglycerol linkers have the structure:
- L is Li, L 2 , or L 3 .
- substituent groups refers to groups that are attached to selected sugar moieties at the 2'-position.
- substituent groups can alternatively be attached to other positions of the sugar moieties (e.g., the 3'- and/or 5'- positions), selected heterocyclic base moieties, or at both the heterocyclic base and the sugar moiety.
- a representative list of substituent groups amenable to the present invention include hydrogen, hydroxyl, C ⁇ -C 0 alkyl, C -C 0 alkenyl, C -C 0 alkynyl, C 5 -C o aryl, O- alkyl, O-alkenyl, O-alkynyl, O-alkylamino, O-alkylalkoxy, O-alkylaminoalkyl, O-alkyl imidazole, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, halogen (particularly fluoro), amino, thiol, keto, carboxyl, nitro, nitroso, nitrile
- polyethers linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi et al. (Drug Design and Discovery 1992, 9, 93), Ravasio et al. (J. Org. Chem. 1991, 56, 4329) and Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249), each of which is herein inco ⁇ orated by reference in its entirety. Further sugar modifications are disclosed in Cook, P.D., Anti-Cancer Drug Design, 1991, 6, 585-607.
- Additional substituent groups amenable to the present invention include -SR and -NR 2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl.
- 2'-SR nucleosides are disclosed in United States Patent No. 5,670,633, issued September 23, 1997, hereby inco ⁇ orated by reference in its entirety. The inco ⁇ oration of 2'-SR monomer synthons are disclosed by Hamm et al, J. Org. Chem., 1997, 62, 3415-3420.
- 2'-NR2 nucleosides are disclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al, Tetrahedron Lett, 1996, 37, 3227-3230.
- substituent groups can include groups having the stracture of one of formula I or II:
- each R 8 , R , R 10 , R ⁇ and R 2 is, independently, hydrogen, C(O)R ⁇ 3 , substituted or unsubstituted -Cio alkyl, substituted or unsubstituted C 2 -C ⁇ 0 alkenyl, substituted or unsubstituted C 2 -C ⁇ o alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; or optionally, R 9 and Rio, together form a phthalimido moiety with the nitrogen atom to which they are attached; or optionally, R ⁇ and R 1 , together form a phthalimido moiety with the nitrogen atom
- R 5 is T-L
- T is a bond or a linking moiety
- Zi, Z 2 and Z 3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
- Z 5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R 5 )(R 6 ) OR 5 , halo, SR 5 or CN; each qi is, independently, an integer from 1 to 10; each q 2 is, independently, 0 or 1; q 3 is 0 or an integer from 1 to 10; q 4 is an integer from 1 to 10; provided that when q 3 is 0, q is greater than 1.
- Particularly preferred sugar substituent groups include O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , 0(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
- Some preferred oligomeric compounds of the invention contain, at least one nucleoside having one of the following substituent groups: C ⁇ to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligomeric compound, or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties.
- a preferred modification includes 2'-methoxyethoxy [2'-O- CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE] (Martin et al, Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group.
- a further preferred modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH ) 2 ON(CH ) 2 group, also known as 2'-DMAOE.
- nucleosides and oligomers include 2'-methoxy (2'-O-CH ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on nucleosides and oligomers, particularly the 3' position of the sugar on the 3' terminal nucleoside or at a 3'-position of a nucleoside that has a linkage from the 2'-position such as a 2'-5' linked oligomer and at the 5' position of a 5' terminal nucleoside. Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
- B and “Bx,” as used herein, is intended to indicate a heterocyclic base moiety.
- a heterocyclic base moiety (often referred to in the art simply as a “base” or a “nucleobase”) amenable to the present invention includes both naturally and non- naturally occurring nucleobases.
- the heterocyclic base moiety further may be protected wherein one or more functionalities of the base bears a protecting group.
- “unmodified” or “natural” nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil.
- Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substi
- nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858- 859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. Certain heterocyclic base moieties are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention to complementary targets.
- 5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
- 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (Id., pages 276-278) and are presently preferred base substitutions, even more particularly when combined with selected 2 '-sugar modifications such as 2'-methoxyethyl groups.
- Representative United States patents that teach the preparation of heterocyclic base moieties include, but are not limited to, U.S.
- the present invention provides oligomeric compounds comprising a plurality of linked nucleosides wherein the preferred intemucleoside linkage is a 3',5'-linkage.
- the preferred intemucleoside linkage is a 3',5'-linkage.
- 2', 5 '-linkages can be used (as described in U.S. Application Serial No. 09/115,043, filed July 14, 1998).
- a 2',5'-linkage is one that covalently connects the 2'-position of the sugar portion of one nucleotide subunit with the 5'- position of the sugar portion of an adjacent nucleotide subunit.
- the compounds described herein may have asymmetric centers. Unless otherwise indicated, all chiral, diastereomeric, and racemic forms are included in the present invention. Geometric isomers may also be present in the compounds described herein, and all such stable isomers are contemplated by the present invention. It will be appreciated that compounds in accordance with the present invention that contain asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms or by synthesis.
- the present invention includes all isotopes of atoms occurring in the intermediates or final compounds.
- Isotopes include those atoms having the same atomic number but different mass numbers.
- isotopes of hydrogen include tritium and deuterium.
- the present invention provides a process for preparing an oligonucleotide having the formula:
- R! is hydroxyl, a protected hydroxyl or a group having the formula:
- Qo O or S
- R4. is O " , hydroxyl, or a protected hydroxyl
- R 2 is hydroxyl, a protected hydroxyl or a group having the formula:
- the oligonucleotides have at least two different ligands attached covalently thereto. Further, it is particularly preferred that Li is different than L 2 , L 2 is different than each of Li and L 3 , and each L 3 attached at a particular position is different from a second L 3 attached at a different position.
- said two different ligands covalently attached thereto are preferably positioned to said oligonucleotide at Li and L 2 , Li and L 3 , L 2 and L 3 , or two of said L 3 groups.
- the process comprises the steps of: a) providing a derivatized solid support for oligonucleotide synthesis, said derivatized solid support being derivatized with a group having one of the structures:
- T is a bifunctional linking moiety linked to the solid support; and Qi is an acid labile hydroxyl protecting group; b) treating said solid support with an acidic reagent to deblock said acid labile hydroxyl protecting group to give a free hydroxyl group; c) reacting said free hydroxyl group with a phosphoramidite composition to form an extended compound, said phosphoramidite composition having the formula: wherein
- Q is a 5'-terminal acid labile hydroxyl protecting group
- Q is a phosphorus protecting group; and Z 6 and Z 7 are, independently, C ⁇ - 6 alkyl; or Z 6 and Z 7 are joined together to form a 4- to 7-membered heterocyclic ring system including the nitrogen atom to which Z 6 and Z are attached, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S; d) optionally treating said extended compound with a capping agent to form a capped compound; e) optionally oxidizing said capped compound to form an oxidized compound; f) repeating steps b) through e) at least three times to form a further extended compound; g) optionally treating said further extended compound with an acidic reagent effective to deblock said acid labile hydroxyl protecting group to give a free hydroxyl group and reacting said free hydroxyl group of said further extended compound with a compound of the formula:
- the methods of the present invention are useful for the preparation of all compounds containing phosphorus functionalities.
- functionality includes, but is not limited to phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate residues, and oligomeric compounds containing monomeric subunits that are joined by a variety of functionality linkages, including phosphite, phosphodiester, phosphorothioate, and/or phosphorodithioate linkages.
- the oligomeric compounds in accordance with the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier.
- oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein.
- Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention.
- each cell of multicellular eukaryotes can be treated, as they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity.
- many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms.
- single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.
- the reactions of the synthetic methods claimed herein are carried out in suitable solvents which may be readily understood by those skilled in the art of organic synthesis, the suitable solvents generally being any solvent which is substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures may range from the solvent's freezing temperature to the solvent's boiling temperature.
- a given reaction may be carried out in one solvent or a mixture of more than one solvent.
- suitable solvents for a particular reaction step may be selected.
- Methods for assembling oligomers in accordance with the present invention include both solution phase and solid phase chemistries. Representative solution phase techniques are described in United States Patent No.
- the methods of the present invention are employed for use in iterative solid phase oligonucleotide synthetic regimes.
- Representative solid phase techniques are those typically employed for DNA and RNA synthesis utilizing standard phosphoramidite chemistry, (see, e.g., Protocols For Oligonucleotides And Analogs, Agrawal, S., ed., Humana Press, Totowa, NJ, 1993, hereby inco ⁇ orated by reference in its entirety).
- Solid supports according to the invention include those generally known in the art to be suitable for use in solid phase methodologies, including, for example, controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al, Nucleic Acids Research 1991, 19, 1527, hereby inco ⁇ orated by reference in its entirety), TentaGel Support — an aminopolyethyleneglycol derivatized support (see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373, hereby inco ⁇ orated by reference in its entirety) and Poros — a copolymer of polystyrene/ divinylbenzene.
- CPG controlled pore glass
- oxalyl-controlled pore glass see, e.g., Alul, et al, Nucleic Acids Research 1991, 19, 1527, hereby inco ⁇ orated by reference in its entirety
- TentaGel Support an aminopoly
- the solid support is derivatized to provide an acid labile trialkoxytrityl group, such as a trimethoxytrityl group (TMT).
- TMT trimethoxytrityl group
- a diglycoate linker is optionally introduced onto the support.
- a preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphate compounds, h this technique, a phosphoramidite monomer is reacted with a free hydroxyl on the growing oligomer chain to produce an intermediate phosphite compound, which is subsequently oxidized to the P v state using standard methods.
- This technique is commonly used for the synthesis of several types of linkages including phosphodiester, phosphorothioate, and phosphorodithioate linkages.
- the first step in such a process is attachment of a first monomer or higher order subunit to a solid support using standard methods and procedures known in the art.
- Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Carathers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
- a linker is optionally positioned between the terminal nucleotide and the solid support.
- Linkers are known in the art as short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., RL Press, NN, 1991, Chapter 1, pages 1-23, hereby inco ⁇ orated by reference in its entirety.
- the support-bound monomer or higher order synthon is then treated to remove the protecting group from the free terminal end. Typically, this is accomplished by treatment with acid.
- the solid support bound monomer, or higher order oligomer is then reacted with individual monomeric or higher order building blocks (i.e., synthons) to form a compound which has a phosphite or thiophosphite linkage.
- synthons reacted under anhydrous conditions in the presence of an activating agent such as, for example, IH-tetrazole, 5-(4-nitrophenyl)-lH-tetrazole, or diisopropylamino tetrazolide.
- the oligonucleotides are assembled according to the method described in U.S. Patent No. 6,121,437, which is hereby inco ⁇ orated by reference in its entirety. Accordingly, the phosphate groups of intemucleosidic nucleotides that are to be functionalized are protected with derivatives of 2- benzamidoethyl groups.
- the oligonucleotides are formed by reacting a compound of Formula V:
- W is selected independently from O and S;
- X is selected independently from O and S;
- Y is selected independently from O and ⁇ R 22 ;
- Z is selected independently from a single bond, O, and ⁇ R ;
- 91 R is selected independently from C ⁇ _ 6 alkyl, C _ 6 alkenyl,
- R at each occurrence, is selected independently from H, C ⁇ - 6 alkyl, C . 6 alkenyl, C 2 _ 6 alkynyl, C 3 . 6 cycloalkyl, and phenyl;
- R 20 is selected independently from hydrogen, C ⁇ _ 6 alkyl, C 2 . 6 alkenyl, C 2 . 6 alkynyl, C 3 . 6 cycloalkyl, and phenyl;
- R 23 is selected independently from C ⁇ . 6 alkyl, C 3 - 6 cycloalkyl, and phenyl;
- R 24 and R 25 are selected independently from C ⁇ _ 6 alkyl, C 3 . 6 cycloalkyl, and phenyl;
- n is selected independently from 0, 1, 2, and 3;
- m is selected independently from 0, 1, 2, and 3;
- R 17 is selected independently from H, a hydroxyl protecting group, and a linker connected to a solid support;
- R at each occurrence, is independently H, hydroxyl, C ⁇ - 20 alkyl, C _ 0 alkenyl, C 2 . 20 alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N- phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, or one of formula
- R 26 and R 27 are independently selected from H, C MO alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support, or alternatively R 26 and R 27 , together, are joined in a nitrogen protecting group or a ring stracture that can include at least one additional heteroatom selected from N and O; q 1 is from 1 to 10; q 3 is O or 1;
- Z n is OR 28 , SR 28 , or N(R 28 ) 2 ;
- R 29 is H or C ⁇ -C 8 alkyl;
- Z 8 , Z 9 and Z ⁇ 0 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
- Zn is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R 26 )(R 27 ) OR 26 , halo, SR 26 or CN; and q 4 is, 0, 1 or 2;
- R 19 is selected independently from NR 30 R 31 , and a 5-6 membered heterocyclic system containing 1-4 heteroatoms selected independently from N, O, and S;
- R and R are selected independently from Ci-io alkyl, C 3 . 7 cycloalkyl, and isopropyl;
- X 1 is selected independently from O and S;
- B at each occurrence, is independently selected from a protected or unprotected naturally occurring nucleobase, and a protected or unprotected non-naturally occurring nucleobase; q is selected independently from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; p is an integer selected independently from 0 to about 50;
- Q at each occurrence, is selected independently from OH, SH, and
- R 32 is selected independently from a hydroxyl protecting group, and a linker connected to a solid support; and p' is an integer selected independently from 0 to about 50.
- oligonucleotides are assembled from building blocks having the formula VIII:
- DMTr is a 4,4'- dimethoxytrityl group
- iPr is an isopropyl group
- R is hydrogen or an isopropyl group
- B is a heterocyclic base.
- Treatment with an acid replaces the hydroxyl protecting group at the unbound terminus of the oligonucleotide, and thus enables the solid support bound oligomer to participate in the next synthetic iteration. This process is repeated until an oligomer of desired length is produced.
- one or more of the 2'-, 3'-, and/or 5'-positions of the oligonucleotide comprises a hydroxyl protecting group.
- a wide variety of hydroxyl protecting groups can be employed in the methods of the invention.
- the protecting group is stable under basic conditions but can be removed under acidic conditions.
- protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
- Representative hydroxyl protecting groups are disclosed by Beaucage, et al, Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which are hereby inco ⁇ orated by reference in their entirety.
- Preferred protecting groups include trimethoxytrityl, dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox).
- the protecting group can be removed from oligomeric compounds of the invention by techniques well known in the art to form the free hydroxyl.
- a wide variety of bases can be used to initiate the removal of protecting groups. These bases include aqueous ammonium hydroxide, aqueous methylamine, DBU (1,8- diazabicyclo[5.4.0]undec-7-ene) and carbonates containing counterions such as lithium, potassium, sodium, and cesium. Most preferred is potassium carbonate and ammonia.
- Removal of the protecting groups maybe performed in a variety of suitable solvents. These solvents include those known to be suitable for protecting group removal in i oligonucleotide synthesis. In the case of ammonia, water is the preferred solvent, whereas when using carbonates, alcohols are preferred.
- dimethoxytrityl protecting groups can be removed by protic acids such as formic acid, dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid or with Lewis acids such as for example zinc bromide. See for example, Greene and Wuts, supra.
- protic acids such as formic acid, dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid or with Lewis acids such as for example zinc bromide. See for example, Greene and Wuts, supra.
- treatment of the oligonucleotide with ammonium hydroxide at room temperature for about 48 hours can be used to remove isopropyl substituted benzamidoethyl and cyanoethoxy protecting groups.
- NH analogs of benzamidoethyl protecting groups can be removed by treating the oligonucleotide with ammonium hydroxide at about 55°C for about 48 hours. Further, allyl protecting groups require the use of paladium zero and silyl protecting groups can be removed by fluoro. In certain preferred embodiments, conditions for removal of the oxygen or sulfur protecting group also effect cleavage of the oligomeric compound from the solid support. The deprotected terminal nucleotide and/or intemucleosidic nucleotides can then be functionalized.
- phosphite or thiophosphite compounds are oxidized or sulfurized.
- the choice of oxidizing or sulfurizing agent will determine whether the linkage will be oxidized or sulfurized to a phosphotriester, thiophosphotriester, or a dithiophosphotriester linkage.
- Sulfurizing agents used during oxidation to form phosphorothioate and phosphorodithioate linkages include Beaucage reagent (see e.g. Iyer, R.P., et. al, J. Chem. Soc, 1990, 112, 1253-1254, and Iyer, R.P., et. al, J. Org.
- Suitable oxidizing agents for forming the phosphodiester or phosphorothioate linkages include iodine/tetrahydrofuran/ water/pyridine or hydrogen peroxide/water or tert-butyl hydroperoxide or any peracid like m-chloroperbenzoic acid.
- sulfurization the reaction is performed under anhydrous conditions with the exclusion of air, in particular oxygen whereas in the case of oxidation the reaction can be performed under aqueous conditions.
- the terms "phosphorus protecting group” and "phosphorus blocking group” refers to a group that is initially bound to the phosphoras atom of a phosphoramidite.
- the phosphorus blocking group functions to protect the phosphorus containing internucleotide linkage or linkages during, for example, solid phase oligonucleotide synthetic regimes.
- Treatment of the intemucleotide linkage or linkages that have a phosphorus blocking group thereon with a deblocking agent, such as aqueous ammonium hydroxide, will result in the removal of the phosphorus blocking group and leave a hydroxyl or thiol group in its place.
- phosphorus blocking groups known in the art which are useful in the present invention including, but not limited, to, cyanoethyl, diphenylsilylethyl, cyanobutenyl, cyano p-xylyl (CPX), methyl-N-trifluoroacetyl ethyl (META) and acetoxy phenoxy ethyl (APOE) groups.
- Phosphorus protecting groups are further described in Beaucage, S.L. and Iyer, R.P., Tetrahedron, 1993, 49, 1925-1963; Beaucage, S.L. and Iyer, R.P., Tetrahedron, 1993, 49, 10441-10488; and Beaucage, S.L.
- phosphoramidites 2 and 3 bearing pyrene or fluorescein reporter groups, respectively, were synthesized (Scheme 1, below).
- phosphoramidite 4 reference sample
- phosphoramidites 5-7 for 5 '-terminal phosphorylation were obtained.
- the phosphoramidites 2-6 were inco ⁇ orated in a standard manner at the last step of oligonucleotide synthesis (Scheme 3).
- the solid support bound oligonucleotides were deprotected with concentrated ammonium hydroxide to give crude 8-15.
- solid support 32 was synthesized (Scheme 2, below). The utility of 32 was verified in preparation of oligonucleotides 33-38 (Scheme 4 and Table 3, below). Oligonucleotide 32 demonstrated favorable properties in two respects. First, the TMT protecting group in 32 was removed with 3% Cl 2 HCCO H in CH 2 C1 2 , i.e. under milder conditions than the previously used for the removal of DMT group (3% F 2 CCO 2 H in CH 2 C1 2 ). Second, the diglycolyl linker in 32 was cleaved with aqueous bases faster than previously reported for the malonyl linker. The reactivity of the 3'-terminal phosphorothioate group was verified by labeling
- oligonucleotides labeled with two different fluorescent reporter groups at the 5'- and 3 '-termini were accomplished according to Schemes 6 and 7.
- protected oligonucleotides 41a and 41b were assembled on solid support 32.
- 5 '-O-(4,4' -dimethoxytrityl) - 3'-O-(N,N- diisopropylamino) [2-(4- methoxybenzamido) ethoxy] phosphinyl-2'-deoxythymidine was used.
- diethyl bis-(hydroxymethyl)malonate (11.5 g, 55 mmol) was treated overnight with 4,4',4"-trimethoxytrityl chloride (19.2 g, 52 mmol) in pyridine (16 mL) and dioxane (100 mL), and the solvent was evaporated. The residue was dissolved in CH 2 C1 2 (500 mL), washed with 5% aqueous NaHCO 3 (3 ' 100 mL), washed with brine (2 ' 100 mL), dried over Na 2 SO and evaporated.
- Extracts were washed with brine (3 ' 25 mL), dried over Na SO , and evaporated. The residue was dissolved in toluene (50 mL), applied on a silica gel column, and separated eluting with a gradient from 5:91:4 to 40:56:4 ethyl acetate / hexane / triethylamine. Collected fractions were evaporated, co-evaporated with dry MeCN (3 ' 50 mL), dry toluene (3 ' 50 mL), and dried on an oil pump to give 3 (1400 mg, 66.0%) as a yellow foam.
- Compound 3a was prepared analogously from diethyl 2-[[[bis (4- methoxyphenyl) phenylmethyl] oxy] methyl] -2-hydroxymethylpropanedioate la (829 mg, 1.5 mmol), chloro bis[(N,N,-diisopropyl)amino]phosphite (440 mg, 1.65 mmol), and 6-[[3',6'-bi (2,2-dimethylpropionyloxy)-3- oxospiro[isobenzofura ⁇ -l(3H),9'-[9H] xanthen]-5-yl] carboxamidojhexanol (483 mg, 0.5 mmol).
- Compound 2 was prepared from 1 (1630 mg, 2.95 mmol), chloro bis[(N,N,- diisopropyl)amino]phosphite (944 mg, 3.54 mmol), and 4-(l-pyrene)butanol (1015 mg, 3.7 mmol) as described for 3. Isolation on a silica gel column using gradient from 5:91 :4 to 40:56:4 ethyl acetate / hexane / triethylamine gave 2 (2185 mg, 77.5%) as a white foam.
- HR FAB MS found m/z 955.4421; C 57 H 67 NO ⁇ oP requires 955.4424.
- Compound 4 was prepared from 1 (553 mg, 1.0 mmol), chloro bis[(N,N,- diisopropyl)amino]phosphite (307 mg, 1.15 mmol), and ethanol (69 mg, 1.5 mmol) as described for 3. Isolation on a silica gel column using gradient from 5:93:2 to 40:58:2 ethyl acetate / hexane / triethylamine gave 4 (662 mg, 91%) as a colorless oil.
- HR FAB MS found m/z 727.3486; C 39 H 54 NO ⁇ 0 P requires 727.3485.
- Compound 5 was prepared from 1 (553 mg, 1.0 mmol), chloro bis[(N,N,- diisopropyl)amino]phosphite (307 mg, 1.15 mmol), and 2-(4-methoxybenzamido)ethanol (254 mg, 1.3 mmol) as described for 3. Isolation on a silica gel column using gradient from 0:95:5 to 40:55:5 ethyl acetate / hexane / triethylamine gave 5 (652 mg, 74.4%) as a colorless oil.
- HR FAB MS found m/z 876.3963; C 47 H 6 ⁇ N 2 O ⁇ 2 P requires 876.3962.
- oligonucleotide synthesis was carried out on an ABI 380B DNA Synthesizer on 1 to 20 ⁇ mol scale using phosphoramidite chemistry.
- 0.1 M solutions of phosphoramidite building blocks in MeCN for 2, 0.1 M solution in CH 2 Cl 2 :MeCN 50:50, v/v or in CH 2 C1 2 ) were used; 0.45 M IH-tetrazole was used as an activator.
- the coupling time for phosphoramidites 2, 3, and 5 was 600 s.
- oligonucleotides were assembled using the standard base protection strategy: N-benzoyl protected dA, dC, 2'-O-(2-methoxyethyl)-A, and 2'-O-(2-methoxyethyl)- 5-methyl-C phosphoramidites and N-isobutyryl protected dG and 2'-O-(2- methoxyethyl)-G phosphoramidites. Additionally, 9-12 were assembled using N- phenoxyacetyl dA, dC, and dG phosphoramidites.
- oligonucleotides 8-13, 33-35, 41b, and 54b For the preparation of oligonucleotides 8-13, 33-35, 41b, and 54b, phosphoramidites protected with 2- cyanoethyl group at the P(III) were used. 5 For the synthesis of oligonucleotides 41a and 54a phosphoramidites protected with N-isopropyl-(4-methoxybenzamido)ethyl group were used. 1 The 5 '-terminal phosphorylation in preparation of 8 and 9 was performed using phosphoramidites 6 and 7 to demonstrate a very similar performance of both reagents.
- a commercial iodine oxidizer or t-BuOOH (10% in MeCN) were used for sulfurization.
- 3H-l,2-benzodithiol-3-one 1,1-dioxide 4 (0.05 M in MeCN) was used as the sulfur-transfer reagent.
- oligonucleotides 8, 11, 14, 15, 33 were assembled using the iodine oxidizer.
- Oligonucleotides 9, 10, 12, 13, 35 were assembled using 3H-l,2-benzodithiol-3-one 1,1-dioxide.
- EXAMPLE 10 Releasing oligonucleotides from solid support and deprotection.
- concentrated ammonium hydroxide was used as a deprotecting agent.
- the oligonucleotides 8, 14, and 15 were treated for 2 h at room temperature.
- the oligonucleotides 9, 33-35, and 41 were deprotected for 6 h at 55 °C.
- the oligonucleotides 10-13 and 54 that possessed 5'- terminal phosphotriester moiety and phenoxyacetyl as the base protecting groups were treated at room temperature for 2 h.
- 10-13 and 54 were deprotected for 2 days at room temperature.
- the deprotected oligonucleotides were isolated by reversed phase HPLC and characterized by ESMS as described below.
- Oligonucleotides 8, 9, 33-35, and 42 were deprotected with 10% aqueous AcOH (3 mL and 100 mL for 1 and 20 ⁇ mol scale, correspondingly) for 30 min.
- Oligonucleotides 10-15, 25 were dissolved in 10% aqueous AcOH (3 mL and 100 mL for 1 and 20 ⁇ mol scale, correspondingly). When the desalted material was subjected to the removal of TMT protecting group, the deprotection was complete in 90 min. When the samples of oligonucleotides contained the HPLC buffer, NH OAc, the reaction mixture was kept for 4 to 5 h at room temperature. When the detritylation was complete, the reaction mixtures were evaporated. The oligonucleotides 36-38 and 43 were desalted and characterized (Tables 3 and 4, respectively). The oligonucleotides 16-22 (Tables 1 and 2) were coevaporated with water and the 5 '-terminal 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl-l group was removed as described below.
- Oligonucleotides 16-22 were dissolved in 0.1 M aqueous piperidine (3 mL and 100 mL for 1 and 20 mmol scale, respectively). The reaction mixture was left for 30 min at room temperature. The solvent was evaporated, and the residue was re-dissolved in water (1 to 5 mL). The target oligonucleotides, 24-31 (Tables 1 and 2) were desalted by reversed phase HPLC.
- oligonucleotides 8-31 were analyzed, and, for syntheses on 1 to 2 mmol scale, isolated by reverse phase chromatography on a Waters DeltaPak C18 column (15 ⁇ m; 300A; 3.9300 mm).
- As buffers A and B 0.1 M NH4OAc and 80% aqueous MeCN were respectively used at a flow rate 1.5 mL min-1. Linear gradients from 0 to 60% B in 40 min (Gradient 1) and 0 to 100% in 40 min (Gradient 2) were employed. Retention times for oligonucleotides 8-31 are presented in Table 1.
- oligonucleotide 37 and N-(l- pyrenylmethyl)iodoacetamide, 44, were added to DMSO.
- the reaction was kept for 4 h at 37 °C and diluted with water.
- the product was isolated and desalted by reverse phase
- oligonucleotide 43 (36 OD, 1.0 mM solution in 200 mM ethyldiisopropylammonium acetate, pH 7.0, 200 ⁇ L) and N-(l- pyrenylmethyl)iodoacetamide, 44, (2.0 mg, 25 mM solution in DMSO, 200 mL) were added to DMSO (400 ⁇ L).
- the reaction was kept for 4 h at 37 °C and diluted to 10 mL with water.
- the product was isolated and desalted by reverse phase HPLC to give 45 (25 OD, 70%) as a triethylammonium salt (Table 1).
- oligonucleotide 46 (5 OD) and compound 44 (0.5 mg) were reacted in 50 mM ethyldiisopropylammonium acetate buffer (75% aqueous DMSO; pH 7.0; 200 mL) for 8 h at 37 °C as described for 45. The solution was diluted to 10 mL with water. The product was isolated and desalted by reverse phase HPLC to give 50 (3.5 OD, 70%o) as a triethylammonium salt (Table 4).
- Oligonucleotide 46 (5 OD) and 4-chloro-7-nitrobenzofurazan, 47, (0.2 mg) were reacted in 0.1 M ethyldiisopropylammonium acetate buffer (60%> aq MeCN; pH 7.0; 400 mL) for 6 h at 37 °C and diluted to 4 mL with water.
- the product was isolated and desalted by reverse phase HPLC to give 51 (4 OD, 80%) as a triethylammonium salt (Table 4).
- Oligonucleotide 46 (5 OD) and monobromobimane, 48, (0.27 mg) were reacted in 0.1 M ethyldiisopropylammonium acetate buffer (60%> aq MeCN; pH 7.0; 400 mL) for 2 h at 37 °C and diluted to 4 mL with water.
- the product was isolated and desalted by reverse phase HPLC to give 52 (4.5 OD, 90%) as a triethylammonium salt (Table 4).
- EXAMPLE 20 Oligonucleotide 53. Oligonucleotide 46 (25 OD) and 5-iodoacetamidofluorescein, 49, (0.52 mg) were reacted in 0.1 M ethyldiisopropylammonium acetate buffer (60% aq MeCN; pH 7.0; 400 mL) for 10 h at 37 °C and diluted to 4 mL with water. The product was isolated and desalted by reverse phase HPLC to give 53 (19 OD, 75%>) as a triethylammonium salt (Table 4).
- ethyldiisopropylammonium acetate buffer 60% aq MeCN; pH 7.0; 400 mL
- the product was isolated and desalted by reverse phase HPLC to give 53 (19 OD, 75%>) as a triethylammonium salt (Table 4).
- oligonucleotide 55 (125 OD) and 5- iodoacetamidofluorescein, 49, (1.5 mg) were reacted in 0.1 M ethyldiisopropylammonium acetate buffer (60%> aq MeCN; pH 7.0; 400 mL) for 10 h at 37 °C and diluted to 4 mL with water.
- the product was isolated by reverse phase HPLC, evaporated, and dissolved in concentrated ammonium hydroxide (5 mL). The solution was heated for 48 h at 55 °C and evaporated.
- Oligonucleotide sequence p*TGCATC 5 AG 2 C 2 AC 2 ATp** ⁇ SEQ. ID. NO. 11> where p* and p** are modified phosphorothioate groups (See Schemes 8 and 9).
- EXAMPLE 22 Oligonucleotide 57.
- oligonucleotide 56 7 OD
- compound 44 7 h
- 50 mM ethyldiisopropylammonium acetate buffer 75% aqueous DMSO; pH 7.0; 250 mL
- the solution was diluted to 10 mL with water.
- the product was isolated and desalted by reverse phase HPLC to give 57 (5.2 OD, 75%) as a triethylammonium salt (Table 5).
- Oligonucleotide 56 (10 OD) and monobromobimane, 48, (0.53 mg) were reacted in 0.1 M ethyldiisopropylammonium acetate buffer (60%> aq MeCN; pH 7.0; 400 uL) for 2 h at 37 °C and diluted to 4 mL with water.
- the product was isolated and desalted by reverse phase HPLC to give 58 (8 OD, 80%>) as a triethylammonium salt (Table 5).
- EXAMPLE 24 Oligonucleotide 59. Oligonucleotide 56 (15 OD) and 5-iodoacetamido fluorescein, 49, (0.5 mg) were reacted in 0.1 M ethyldiisopropylammonium acetate buffer (60% aq MeCN; pH 7.0; 400 uL) for 10 h at 37 °C and diluted to 4 mL with water. The product was isolated and desalted by reverse phase HPLC to give 59 (10.5 OD, 70%o) as a triethylammonium salt (Table 5).
- 1,2-Di-O-hexadecyl-rac-glycerol succinimidyl carbamate (102) Referring to Scheme 10, 1,2-Di-O-hexadecyl-rac-glycerol (10.00 g, 18.5 mmol) was dissolved in anhydrous CH 2 C1 2 (150 ml). To the solution were added disuccinimidyl carbonate 7.11 g, 27.7 mmol), Et 3 N (10.0 ml), and MeCN (50 ml). The reaction mixture was stirred at room temperature under Ar for 6.5 h and then evaporated to dryness. The residue was dissolved in CH 2 CI 2 (300 ml).
- the reaction mixture was stirred at room temperature for 1 h. It was then poured into 5% NaHCO 3 aqueous solution, shaken and separated. The aqueous layer was extracted with CH 2 C1 2 (3 x 60 ml). The combined organic layer was washed with 5% NaHCO 3 aqueous solution (100 ml) and saturated NaCl aqueous solution (2 x 120 ml), dried over Na 2 SO 4 . The solid was filtered out. The filtrate was evaporated to dryness giving a gel that was further dried in high vacuum furnishing a yellow foam (4.77 g).
- DSC Disuccinimidyl carbonate
- DEC 1-(3-Dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride
- ⁇ -N-Cholesteryloxycarbonylaminocaproic acid 109)
- the ⁇ - aminocaproic acid (3.93 g, 30 mmol) was suspended in pyridine (60 ml).
- the flask was flushed with nitrogen and to the mixture was added N,O-bis(trimethylsilyl)acetamide (10 ml, 70 mmol) under stirring.
- the reaction mixture was stirred at room temperature for 35 min. and then cooled in ice bath.
- Cholesteryl chloroformate (13.5 g, 30 mmol) was added into the reaction mixture in two portions over 2 h. The reaction was continued by stirring at room temperature for another 4 h.
- Phosphoramidite derived from dihexadecylglycerol containing non-nucleosidic linker
- EXAMPLE 29 The cholesterol- and dialkylglycerol-conjugated oligonucleotides, compounds 120-129, presented in the Tables 6-9 below were prepared using the phosphoramidites and solid support described above in connection with Examples 27 and 28.
- the RP HPLC profiles of compound 120 in its crade form prior to removal of the DMT group, in its crade form after removal of the DMT group, and in its final form are shown in FIGS. 1-3, respectively.
- the RP HPLC profiles of compound 129 in its crude form prior to removal of the DMT group, in its crade form after removal of the DMT group, and in its final form are shown in FIGS. 4-6, respectively.
- the RP HPLC profiles of compound 126 in its crude form after removal of the DMT group and in its final form are shown in FIGS. 7 and 8, respectively.
- the RP HPLC profiles of compound 121 in its crude form after removal of the DMT group and in its final form are shown in FIGS. 9 and 10, respectively.
- RNA oligomers are synthesized using 2'-O-t : butyldimethylsilyl (TBDMS) protected RNA monomers available from Glen Research, McLean, NA. These monomers have ⁇ -cyanoethyl protecting group for phosphoramidite functionality and phenoxy acetyl group for A and G and acetyl group for C as base protecting groups.
- TDMS butyldimethylsilyl
- a 50:50 aqueous methylamine: ammonium hydroxide mixture is used at 65°C for 10 minutes.
- the solution is evaporated, and the residue is treated with sterile water.
- the solution is purified by reverse phase HPLC, 5 '-the trityl group is removed, and characterized by capillary gel electrophoresis and elctrospray mass spectrometry.
- EXAMPLE 31 R ⁇ A phosphorothioate synthesis
- Beaucage reagent (lg in 100ml of acetonitrile solution).
- Beaucage reagent (R.P. Iyer, W. Egan, J.B. Regan, and S.L. Beaucage, J. Am. Chem. Soc. 1990 112 1253-1254) during the oxidation step of the RNA synthesis.
- the cholesterol conjugated to RNA at the 5 '-end is synthesized using the reagent 112 at the final step of coupling procedure in repeating the procedures described in examples.
- the DMT group is removed by treatment with the acid and the material is purified by RP-HPLC.
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Abstract
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US20210171490A1 (en) * | 2019-12-09 | 2021-06-10 | Howard Hughes Medical Institute | Red-shifted fluorophores |
US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
US11981703B2 (en) | 2016-08-17 | 2024-05-14 | Sirius Therapeutics, Inc. | Polynucleotide constructs |
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US20030198966A1 (en) * | 2002-04-19 | 2003-10-23 | Stojanovic Milan N. | Displacement assay for detection of small molecules |
US20040203007A1 (en) * | 2003-04-14 | 2004-10-14 | Stojanovic Milan N. | Cross reactive arrays of three-way junction sensors for steroid determination |
US7470516B2 (en) * | 2003-04-14 | 2008-12-30 | The Trustees Of Columbia University In The City Of New York | Cross reactive arrays of three-way junction sensors for steroid determination |
US10174323B2 (en) * | 2012-05-16 | 2019-01-08 | The General Hospital Corporation | Compositions and methods for modulating ATP2A2 expression |
WO2013173601A1 (en) * | 2012-05-16 | 2013-11-21 | Rana Therapeutics, Inc. | Compositions and methods for modulating bdnf expression |
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EA201492122A1 (en) * | 2012-05-16 | 2015-10-30 | Рана Терапьютикс, Инк. | COMPOSITIONS AND METHODS FOR MODULATING UTRN EXPRESSION |
US10837014B2 (en) | 2012-05-16 | 2020-11-17 | Translate Bio Ma, Inc. | Compositions and methods for modulating SMN gene family expression |
CN113024621A (en) * | 2021-03-01 | 2021-06-25 | 通用生物系统(安徽)有限公司 | Preparation method of high-efficiency phosphorylation labeled nucleic acid for second-generation sequencing |
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2001
- 2001-03-30 US US09/823,031 patent/US6825338B2/en not_active Expired - Fee Related
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2002
- 2002-03-29 CA CA002442230A patent/CA2442230A1/en not_active Abandoned
- 2002-03-29 WO PCT/US2002/010178 patent/WO2002079216A1/en not_active Application Discontinuation
- 2002-03-29 EP EP02723725A patent/EP1379541A1/en not_active Withdrawn
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US6194598B1 (en) * | 1997-02-14 | 2001-02-27 | Isis Pharmaceuticals, Inc. | Aminooxy-modified oligonucleotide synthetic intermediates |
US6277982B1 (en) * | 1999-08-20 | 2001-08-21 | Isis Pharmaceuticals, Inc. | Alkylation of alcohols, amines, thiols and their derivatives by cyclic sulfate intermediates |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11981703B2 (en) | 2016-08-17 | 2024-05-14 | Sirius Therapeutics, Inc. | Polynucleotide constructs |
US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
US20210171490A1 (en) * | 2019-12-09 | 2021-06-10 | Howard Hughes Medical Institute | Red-shifted fluorophores |
Also Published As
Publication number | Publication date |
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EP1379541A1 (en) | 2004-01-14 |
CA2442230A1 (en) | 2002-10-10 |
US20030208061A1 (en) | 2003-11-06 |
US6825338B2 (en) | 2004-11-30 |
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