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US20050267300A1 - Processes and reagents for oligonucleotide synthesis and purification - Google Patents

Processes and reagents for oligonucleotide synthesis and purification Download PDF

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Publication number
US20050267300A1
US20050267300A1 US11/099,430 US9943005A US2005267300A1 US 20050267300 A1 US20050267300 A1 US 20050267300A1 US 9943005 A US9943005 A US 9943005A US 2005267300 A1 US2005267300 A1 US 2005267300A1
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alkyl
aryl
aralkyl
occurrence
oligonucleotide
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Muthiah Manoharan
Michael Jung
Kallanthottathil Rajeev
Rajendra Pandey
Gang Wang
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Alnylam Pharmaceuticals Inc
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Assigned to ALNYLAM PHARMACEUTICALS reassignment ALNYLAM PHARMACEUTICALS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JUNG, MICHAEL E., MANOHARAN, MUTHIAH, PANDEY, RAJENDRA K., RAJEEV, KALLANTHOTTATHIL G., WANG, GANG
Publication of US20050267300A1 publication Critical patent/US20050267300A1/en
Priority to US12/050,633 priority patent/US8063198B2/en
Priority to US12/351,605 priority patent/US8058448B2/en
Priority to US13/036,788 priority patent/US8431693B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • oligonucleotides The study of oligonucleotides is a key area of research for many academic and industrial laboratories. See S. Agrawal Trends in Biotechnology 1996, 14, 375-382; J. Marr Drug Discovery Today 1996, 1, 94-102; and W. Rush Science 1997, 276, 1192-1193.
  • the therapeutic and diagnostic potential of oligonucleotides has sparked a substantial amount of research activity.
  • One important application of oligonucleotides is the ability to modulate gene and protein function in a sequence-specific manner.
  • a method to produce large quantities of oligonucleotide compounds having high purity would greatly facilitate oligonucleotide research.
  • the synthesis of oligonucleotides and their analogs is often a tedious and costly process.
  • RNA is generally synthesized and purified by methodologies based on the following steps: phosphoramidite coupling using tetrazole as the activating agent, oxidation of the phosphorus linker to the diester, deprotection of exocyclic amino protecting groups using NH 4 OH, removal of 2′-OH alkylsilyl protecting groups using tetra-n-butylammonium fluoride (TBAF), and gel purification and analysis of the deprotected RNA. Examples of chemical synthesis, deprotection, purification and analysis procedures are provided by Usman et al. in J. Am. Chem. Soc. 1987, 109, 7845; Scaringe et al. in Nucleic Acids Res.
  • TBAF tetra-n-butylammonium fluoride
  • Odai and coworkers describe reverse-phase chromatographic purification of RNA fragments used to form a ribozyme. See Odai et al. FEBS Lett. 1990, 267, 150-152. Unfortunately, the aforementioned chemical synthesis, deprotection, purification and analysis procedures are time consuming (10-15 min.
  • oligonucleotide analogues are compounds that have a phosphorothioate in place of the phosphodiester linkage.
  • Phosphorothioate analogues are important compounds in nucleic acid research and protein research. For example, phosphorothioate-containing antisense oligonucleotides have been used in vitro and in vivo as inhibitors of gene expression. Site-specific attachment of reporter groups onto the DNA or RNA backbone is facilitated by incorporation of single phosphorothioate linkages. Phosphorothioates have also been introduced into oligonucleotides for mechanistic studies on DNA-protein and RNA-protein interactions, as well as catalytic RNAs.
  • Introduction of phosphorothioate linkages into oligonucleotides, assembled by solid-phase synthesis, can be achieved using either an H-phosphonate approach or a phosphoramidite approach.
  • the H-phosphonate approach involves a single sulfur-transfer step, carried out after the desired sequence has been assembled, to convert all of the internucleotide linkages to phosphorothioates.
  • the phosphoramidite approach features a choice at each synthetic cycle: a standard oxidation provides the normal phosphodiester internucleotide linkage, whereas a sulfurization step introduces a phosphorothioate at that specific position in the sequence.
  • RNA interference is an evolutionarily conserved gene-silencing mechanism, originally discovered in studies of the nematode Caenorhabditis elegans (Lee et al, Cell 75:843 (1993); Reinhart et al., Nature 403:901 (2000)). It is triggered by introducing dsRNA into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA.
  • the mechanism involves conversion of dsRNA into short RNAs that direct ribonucleases to homologous mRNA targets (summarized, Ruvkun, Science 2294:797 (2001)). This process is related to normal defenses against viruses and the mobilization of transposons.
  • Double-stranded ribonucleic acids are naturally rare and have been found only in certain microorganisms, such as yeasts or viruses. Recent reports indicate that dsRNAs are involved in phenomena of regulation of expression, as well as in the initiation of the synthesis of interferon by cells (Declerq et al., Meth. Enzymol. 78:291 (1981); Wu-Li, Biol. Chem. 265:5470 (1990)). In addition, dsRNA has been reported to have anti-proliferative properties, which makes it possible also to envisage therapeutic applications (Aubel et al., Proc. Natl. Acad. Sci., USA 88:906 (1991)).
  • dsRNA has been shown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci. USA, 62:357-361 (1969)), is active in the treatment of leukemic mice (Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128 (1969)); and inhibits chemically-induced tumorigenesis in mouse skin (Gelboin et al., Science 167:205-207 (1970)).
  • RNAi can be induced in adult fruit flies by injecting dsRNA into the abdomen of anesthetized Drosophila , and that this method can also target genes expressed in the central nervous system (Mol. Psychiatry 6(6):665-670 (2001)). Both transgenes and endogenous genes were successfully silenced in adult Drosophila by intra-abdominal injection of their respective dsRNA.
  • RNAi provides a rapid method to test the function of genes in the nematode Caenorhabditis elegans ; and most of the genes on C. elegans chromosome I and III have now been tested for RNAi phenotypes (Barstead, Curr. Opin. Chem. Biol. 5(1):63-66 (2001); Tavemarakis, Nat. Genet. 24(2):180-183 (2000); Zamore, Nat. Struct. Biol. 8(9):746-750 (2001).).
  • RNAi was used to analyze a random set of ovarian transcripts and have identified 81 genes with essential roles in C.
  • RNAi has also been used to disrupt the pupal hemocyte protein of Sarcophaga (Nishikawa et al., Eur. J. Biochem. 268(20):5295-5299 (2001)).
  • RNAi in invertebrate animals post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in invertebrates only post-transcriptional RNAi has been reported to date (Bernstein et al., Nature 409(6818):295-296 (2001). Indeed, both involve double-stranded RNA (dsRNA), spread within the organism from a localized initiating area, to correlate with the accumulation of small interfering RNA (siRNA) and require putative RNA-dependent RNA polymerases, RNA helicases and proteins of unknown functions containing PAZ and Piwi domains.
  • dsRNA double-stranded RNA
  • siRNA small interfering RNA
  • RNAi and PTGS were reported by Vaucheret et al., J. Cell Sci. 114(Pt 17):3083-3091 (2001).
  • PTGS in plants requires at least two genes—SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA-methyltransferase)—that are absent in C. elegans , and thus are not required for RNAi.
  • SGS3 which encodes a protein of unknown function containing a coil-coiled domain
  • MET1 which encodes a DNA-methyltransferase
  • RNAi-mediated oncogene silencing has also been reported to confer resistance to crown gall tumorigenesis (Escobar et al., Proc. Natl. Acad. Sci. USA, 98(23):13437-13442 (2001)).
  • RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger.
  • RISC RNA-induced silencing complex
  • RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity remained unknown.
  • RNAi effector nuclease from cultured Drosophila cells
  • protein microsequencing of a ribonucleoprotein complex of the active fraction showed that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora , and Arabidopsis .
  • This observation suggests links between the genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.
  • RNAi provides a suitable and robust approach to study the function of dormant maternal mRNAs in mouse oocytes.
  • Mos originally known as c-mos
  • tissue plasminogen activator mRNAs are dormant maternal mRNAs that are recruited during oocyte maturation, and translation of Mos mRNA results in the activation of MAP kinase.
  • the dsRNA directed towards Mos or TPA mRNAs in mouse oocytes specifically reduced the targeted mRNA in both a time- and concentration-dependent manner, and inhibited the appearance of MAP kinase activity. See also, Svoboda et al. Biochem. Biophys. Res. Commun. 287(5):1099-1104 (2001).
  • RNA conjugates having improved pharmacologic properties.
  • the oligonucleotide sequences have poor serum solubility, poor cellular distribution and uptake, and are rapidly excreted through the kidneys.
  • the stability of oligonucleotides has been increased by converting the P ⁇ O linkages to P ⁇ S linkages which are less susceptible to degradation by nucleases in vivo.
  • the phosphate group can be converted to a phosphoramidate or alkyl phosphonate, both of which are less prone to enzymatic degradation than the native phosphate.
  • Modifications to the sugar groups of the oligonucleotide can confer stability to enzymatic degradation.
  • oligonucleotides comprising ribonucleic acids are less prone to nucleolytic degradation if the 2′-OH group of the sugar is converted to a methoxyethoxy group. See M. Manoharan Chem Bio Chem. 2002, 3, 1257 and references therein.
  • the present invention relates to processes and reagents for oligonucleotide synthesis and purification.
  • One aspect of the present invention relates to compounds useful for activating phosphoramidites in oligonucleotide synthesis.
  • Another aspect of the present invention relates to a method of preparing oligonucleotides via the phosphoramidite method using an activator of the invention.
  • Another aspect of the present invention relates to sulfur-transfer agents.
  • the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one.
  • Another aspect of the present invention relates to a method of preparing a phosphorothioate by treating a phosphite with a sulfur-transfer reagent of the invention.
  • the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one.
  • Another aspect of the present invention relates to compounds that scavenge acrylonitrile produced during the deprotection of phosphate groups bearing ethylnitrile protecting groups.
  • the acrylonitrile scavenger is a polymer-bound thiol.
  • Another aspect of the present invention relates to agents used to oxidize a phosphite to a phosphate.
  • the oxidizing agent is sodium chlorite, chloroamine, or pyridine-N-oxide.
  • Another aspect of the present invention relates to methods of purifying an oligonucleotide by annealing a first single-stranded oligonucleotide and second single-stranded oligonucleotide to form a double-stranded oligonucleotide; and subjecting the double-stranded oligonucleotide to chromatographic purification.
  • the chromatographic purification is high-performance liquid chromatography.
  • FIG. 1 depicts activator compounds useful in phosphoramidite-mediated oligonucleotide synthesis.
  • FIG. 2 depicts activating agents useful in phosphoramidite-mediated oligonucleotide synthesis.
  • FIG. 3 depicts activating agents useful in phosphoramidite-mediated oligonucleotide synthesis.
  • FIG. 4 depicts sulfur-transfer agents useful in preparing phosphorothioate linkages in oligonucleotides.
  • FIG. 5 depicts sulfur-transfer agents useful in preparing phosphorothioate linkages in oligonucleotides.
  • FIG. 6 depicts the results of the synthesis of 25 and 26 with PADS or EDITH.
  • 25 5′-GsCsGGAUCAAACCUCACCAsAsdTsdT-3′
  • 26 5′-UsUsGGUGAGGUUUGAUCCGsCsdTsdT-3′
  • PADS fresh
  • PADS aged
  • nd indicates that the value was not determined.
  • PADS refers to the compound (benzylC(O)S) 2 .
  • EDITH refers to 3-ethoxy-1,2,4-dithiazolidine-5-one.
  • FIG. 7 depicts desilylating reagents and assorted bases used in oligonucleotide synthesis.
  • FIG. 8 depicts acrylonitrile quenching agents.
  • FIG. 9 depicts a flow chart for siRNA purification and QC. Note: LC-MS indicates liquid-chromatography mass spectrophotometric analysis; and CGE indicates capillary gel electrophoresis analysis.
  • FIG. 10 depicts the structure of AL-4112, AL-4180, AL-DP-4014, AL-2200, AL-2201, AL-DP-4127, AL-2299, AL-2300, AL-DP-4139, AL-2281, AL-2282, and AL-DP-4140.
  • FIG. 11 depicts the first part of the two-strand approach to purification of AL-DP-4014, the components of which are AL-4112 and AL-4180.
  • FIG. 12 depicts the second part of the two-strand approach to purification of AL-DP-4014, the components of which are AL-4112 and AL-4180.
  • RP HPLC indicates reverse phase high-performance liquid chromatographic analysis.
  • IEX HPLC indicates ion exchange high-performance liquid chromatographic analysis.
  • FIG. 13 depicts a reverse phase HPLC chromatogram of AL-DP-4014.
  • FIG. 14 depicts a LC-MS chromatogram of AL-DP-4014.
  • FIG. 15 depicts a mass spectrum of the peak at 9.913 minutes in the LC chromatogram of AL-DP-4014 shown in FIG. 14 .
  • FIG. 16 depicts a capillary gel electrophoresis chromatogram of AL-DP-4014.
  • FIG. 17 depicts a reverse phase HPLC chromatogram of AL-DP-4014.
  • FIG. 18 depicts an ion exchange chromatogram of AL-DP-4014.
  • FIG. 19 depicts a LC-MS chromatogram of AL-DP-4127.
  • FIG. 20 depicts a mass spectrum of the peak at 10.616 minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 19 .
  • FIG. 21 depicts a mass spectrum of the peak at 12.921 minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 19 .
  • FIG. 22 depicts a mass spectrum of the peak at 16.556 minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 19 .
  • FIG. 23 depicts a LC-MS chromatogram of AL-DP-4127.
  • FIG. 24 depicts a mass spectrum of a minor contaminant which appears as a peak at 13.397 minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 23 .
  • FIG. 25 depicts a mass spectrum of a minor contaminant which appears as a peak at 13.201 minutes in the LC chromatogram of AL-DP-4127 shown in FIG. 23 .
  • FIG. 26 depicts a capillary gel electrophoresis chromatogram of AL-DP-4127.
  • FIG. 27 depicts a reverse phase HPLC chromatogram of AL-DP-4127.
  • FIG. 28 depicts an ion exchange chromatogram of AL-DP-4127.
  • FIG. 29 depicts a LC-MS chromatogram of AL-DP-4139.
  • FIG. 30 depicts a mass spectrum of the peak at 13.005 minutes in the LC chromatogram of AL-DP-4139 shown in FIG. 29 .
  • FIG. 31 depicts a capillary gel electrophoresis chromatogram of AL-DP-4139.
  • FIG. 32 depicts a reverse phase HPLC chromatogram of AL-DP-4139.
  • FIG. 33 depicts an ion exchange chromatogram of AL-DP-4139.
  • FIG. 34 depicts a LC-MS chromatogram of AL-DP-4140.
  • FIG. 35 depicts a mass spectrum of the peak at 13.965 minutes in the LC chromatogram of AL-DP-4140 shown in FIG. 34 .
  • FIG. 36 depicts a mass spectrum of the peak at 17.696 minutes in the LC chromatogram of AL-DP-4140 shown in FIG. 34 .
  • FIG. 37 depicts a capillary gel electrophoresis chromatogram of AL-DP-4140.
  • FIG. 38 depicts a reverse phase HPLC chromatogram of AL-DP-4140.
  • FIG. 39 depicts an ion exchange chromatogram of AL-DP-4140.
  • FIG. 40 depicts alternative steps for the two-strand RNA purification procedure.
  • FIG. 41 depicts alternative steps for the two-strand RNA purification procedure.
  • FIG. 42 depicts alternative steps for the two-strand RNA purification procedure.
  • FIG. 43 depicts alternative steps for the two-strand RNA purification procedure.
  • FIG. 44 depicts nucleosides bearing various 2′-protecting groups.
  • the term “B” indicates protected C, G, A, U, or 5-Me-U.
  • the term “X” indicates CN, NO 2 , CF 3 , SO 2 R, or CO 2 R.
  • the term “X′” indicates CN, NO 2 , CF 3 , F, or OMe.
  • the term “Z” indicates H or alkyl.
  • R 1 indicates oxazole, thiazole, or azole.
  • FIG. 45 depicts nucleosides bearing various 2′-protecting groups which can be removed by enzymatic cleavage.
  • the term “B” indicates U, 5-Me-U, 5-Me-C, G, or A.
  • the term “X” indicates H, CN, NO 2 , CF 3 .
  • the term “X′” indicates H, CN, NO 2 , CF 3 , SO 2 R, or CO 2 R.
  • FIG. 46 depicts nucleosides bearing various base protecting groups amenable to the present invention. Note R is H, OMe, F, MOE, or TOM.
  • FIG. 47 depicts RNA building blocks amenable to the present invention, wherein the nucleoside has a TOM protecting group.
  • FIG. 48 depicts 5′-silyl protected RNA suitable for the silyl deprotection methods described herein.
  • Base is N-benzoyladenine, N-acetylcytosine, N-isoputyrylguanine, or uracil.
  • R is cyclooctyl for guanosine and uridine.
  • R is cyclododecyl for adenosine and cytidine. See Scaringe, S. A.; Wincott, F, E and Caruthers, M. H. J. Am. Chem. Soc. 1998, 120, 11820-21.
  • FIG. 49 depicts a general procedure for solid-phase RNA synthesis.
  • FIG. 50 depicts sulfur-transfer agents useful in preparing phosphorothioate linkages in oligonucleotides.
  • FIG. 51 depicts building blocks for conjugation of cholesteryl- and aminoalkyl-hydroxyprolinol at the 5′ and 3′-ends of oligonucleotides. I and III are for 5′-conjugation, and II and IV are for 3′-conjugation. See Example 8.
  • the present invention relates to processes and reagents for oligonucleotide synthesis and purification. Aspects of the processes and reagents are described in the paragraphs below.
  • phosphoramidite The most commonly used process in oligonucleotide synthesis using solid phase chemistry is the phosphoramidite approach.
  • a phosphoramidite is reacted with a support-bound nucleotide, or oligonucleotide, in the presence of an activator.
  • the phosphoroamidite coupling-product is oxidized to afford a protected phosphate.
  • a variety of different phosphoramidite derivatives are known to be compatible with this procedure, and the most commonly used activator is 1H-tetrazole. Similar processes have been described using a soluble support. See Bonora et al. Nucleic Acids Res., 1993, 21, 1213-1217.
  • phosphoramidite approach is also widely used in solution phase chemistries for oligonucleotide synthesis.
  • deoxyribonucleoside phosphoramidite derivatives have been used in the synthesis of oligonucleotides. See Beaucage et al. Tetrahedron Lett. 1981, 22, 1859-1862.
  • Phosphoramidites derivatives from a variety of nucleosides are commercially available. 3′-O-phosphoramidites are the most widely used amidites, but the synthesis of oligonucleotides can involve the use of 5′-O- and 2′-O-phosphoramidites. See Wagner et al. Nuclosides & Nuclotides 1997, 17, 1657-1660 and Bhan et al. Nuclosides & Nuclotides 1997, 17, 1195-1199. There are also many phosphoramidites available that are not nucleosides (Cruachem Inc., Dulles, Va.; Clontech, Palo Alto, Calif., Glen Research, Sterling, Va., ChemGenes, Wilmington, Mass.).
  • the 3′-OH group of the 5′-O-protected nucleoside has to be phosphityled. Additionally, exocyclic amino groups and other functional groups present on nucleobase moieties are normally protected prior to phosphitylation.
  • phosphitylation of nucleosides is performed by treatment of the protected nucleosides with a phosphitylating reagent such as chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine which is very reactive and does not require an activator or 2-cyanoethyl-N,N,N′,N′-tetraiso-propylphosphorodiamidite (bis amidite reagent) which requires an activator.
  • the nucleoside 3′-O-phosphoramidite is coupled to a 5′-OH group of a nucleoside, nucleotide, oligonucleoside or oligonucleotide.
  • the activator most commonly used in phosphitylation reactions is 1H-tetrazole.
  • 1H-tetrazole is known to be explosive.
  • MSDS material safety data sheet
  • 1H-tetrazole (1H-tetrazole, 98%) can be harmful if inhaled, ingested or absorbed through the skin.
  • the MSDS also states that 1H-tetrazole can explode if heated above its melting temperature of 155° C. and may form very sensitive explosive metallic compounds.
  • 1H-tetrazole requires special handling during its storage, use, and disposal.
  • 1H-tetrazole is acidic and can cause deblocking of the 5′-O-protecting group and can also cause depurination during the phosphitylation step of amidite synthesis. See Krotz et al. Tetrahedron Lett. 1997, 38, 3875-3878. Inadvertent deblocking of the 5′-O-protecting group is also a problem when chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine is used.
  • Activators with a higher pKa (i.e., less acidic) than 1H-tetrazole (pKa 4.9) such as 4,5-dicyanoimidazole (pKa 5.2) have been used in the phosphitylation of 5′-O-DMT thymidine. See C. Vargeese Nucleic Acids Res. 1998, 26, 1046-1050.
  • the solubility of 1H-tetrazole is also a factor in the large-scale synthesis of phosphoramidites, oligonucleotides and their analogs.
  • the solubility of 1H-tetrazole is about 0.5 M in acetonitrile. This low solubility is a limiting factor on the volume of solvent that is necessary to run a phosphitylation reaction. An activator having higher solubility would be preferred in order to minimize the volume of solvents used in the reactions, thereby lowering the cost and the production of waste effluents.
  • the activator compounds of the invention have superior properties for activating phosphoramidites used in oligonucleotide synthesis.
  • the activator compounds are generally less explosive and more soluble in acetonitrile than 1H-tetrazole.
  • the activator compounds of the invention required shorter reaction times in the synthesis of a decamer RNA molecule compared to 1H-tetrazole. See Example 1.
  • the activator compound of the invention has an electron-withdrawing group to decrease the pKa of the compound. More acidic activator compounds can increase the rate of the phosphoramidite coupling reaction in certain instances. Importantly, shorter reaction times minimize the opportunity for side reactions to occur, thereby providing the desired product in higher purity.
  • activator compounds of the invention can be the free heterocyclic compound or a mixture of the activator and its corresponding monoalkyl, dialkyl, or trialkyl ammonium salt with varying salt to activator molar ratio.
  • Select preferred activator compounds of the invention are presented in FIGS. 1, 2 , and 3 .
  • One aspect of the present invention relates to a compound represented by formula I: wherein
  • the present invention relates to the aforementioned compound, wherein X is C(R 6 ).
  • the present invention relates to the aforementioned compound, wherein X is N.
  • the present invention relates to the aforementioned compound, wherein X is C(R 6 ); R 1 , R 2 , R 3 , and R 6 each independently represent H, —NO 2 , or —CN; R 4 is absent; and R 5 is H.
  • the present invention relates to the aforementioned compound, wherein X is C(R 6 ); R 1 , R 2 , R 3 , and R 6 are H; R 4 is absent; and R 5 is H.
  • the present invention relates to the aforementioned compound, wherein X is N; R 1 , R 2 , and R 3 are H; R 4 is absent; and R 5 is H.
  • Another aspect of the present invention relates to a compound represented by formula II: wherein
  • the present invention relates to the aforementioned compound, wherein R 1 and R 3 each represent independently H, —NO 2 , or —CN; R 2 is absent; and R 4 is H.
  • the present invention relates to the aforementioned compound, wherein R 1 is H; R 3 is —NO 2 ; R 2 is absent; and R 4 is H.
  • Another aspect of the present invention relates to a compound represented by formula II: wherein
  • the present invention relates to the aforementioned compound, wherein R 1 and R 2 each represent independently H, —NO 2 , or —CN; R 4 is absent; and R 4 is H.
  • the present invention relates to the aforementioned compound, wherein R 1 is H; R 2 is —NO 2 ; R 3 is absent; and R 4 is H.
  • Another aspect of the present invention relates to a compound represented by formula IV: wherein
  • the present invention relates to the aforementioned compound, wherein R 1 is —SR 5 , alkyl, aryl, —N(R 4 ) 2 , or —(C(R 4 ) 2 ) m CO 2 R 5 .
  • the present invention relates to the aforementioned compound, wherein R 2 is absent, and R 3 is H.
  • the present invention relates to the aforementioned compound, wherein R 1 is —SR 5 , alkyl, aryl, —N(R 4 ) 2 , or —(C(R 4 ) 2 ) m CO 2 R 5 ; R 2 is absent; R 3 is H; R 4 is H; R 5 is alkyl or aralkyl; and m is 1.
  • Another aspect of the present invention relates to a compound represented by formula V: wherein
  • the present invention relates to the aforementioned compound, wherein R 2 is absent, and R 5 is H.
  • the present invention relates to the aforementioned compound, wherein R 1 is H, R 2 is absent, R 3 and R 4 are —CN, and R 5 is H.
  • Another aspect of the present invention relates to a method of forming a phosphite compound, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said phosphoramidite is a 3′-nucleoside phosphoramidite, 3′-nucleotide phosphoramidite, or 3′-oligonucleotide phosphoramidite.
  • the present invention relates to the aforementioned method, wherein said phosphoramidite is represented by formula A: wherein
  • the present invention relates to the aforementioned method, wherein R 1 is —CH 2 CH 2 CN.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 2 is a nucleoside or nucleotide.
  • the present invention relates to the aforementioned method, wherein R 3 and R 4 are alkyl.
  • the present invention relates to the aforementioned method, wherein said alcohol is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein said alcohol is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein alcohol is a nucleoside, nucleotide, or oligonucleotide.
  • the present invention relates to the aforementioned method, wherein said alcohol is represented by R 5 —OH, wherein R 5 is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or —(C(R 6 ) 2 ) p heterocycloalkyl; R 6 is H or alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the present invention relates to the aforementioned method, wherein R 5 is —(C(R 6 ) 2 ) p heterocycloalkyl.
  • the present invention relates to the aforementioned method, further comprising the step of admixing a proton-shuttle compound to the mixture comprising said phosphoramidite, said alcohol, and said activating agent, wherein the pKa of said proton-shuttle compound is greater than the pKa of said activating agent, and the pKa of said proton-shuttle compound is less than the pKa of said phosphoramidite.
  • the present invention relates to the aforementioned method, wherein said proton-shuttle compound is a primary, secondary, or tertiary amine.
  • the present invention relates to the aforementioned method, wherein said proton-shuttle compound is represented by N(R 7 )(R 8 )R 9 , wherein R 7 , R 8 , and R 9 each represent independently for each occurrence H, alkyl, cycloalkyl, aryl, aralkyl, alkenyl; or R 7 and R 8 taken together form a 3-8 membered ring; and R 9 is H, alkyl, cycloalkyl, aryl, or aralkyl.
  • Modified oligonucleotides are of great value in molecular biological research and in applications such as anti-viral therapy. Modified oligonucleotides which can block RNA translation, and are nuclease resistant, are useful as antisense reagents. Sulfurized oligonucleotides containing phosphorothioate (P ⁇ S) linkages are of interest in these areas. Phosphorothioate-containing oligonucleotides are also useful in determining the stereochemical pathways of certain enzymes which recognize nucleic acids.
  • P ⁇ S phosphorothioate
  • Standard techniques for sulfurization of phosphorus-containing compounds have been applied to the synthesis of sulfurized deoxyribonucleotides.
  • sulfurization reagents which have been used include elemental sulfur, dibenzoyl tetrasulfide, 3-H-1,2-benzidithiol-3-one 1,1-dioxide (also known as Beaucage reagent), tetraethylthiuram disulfide (TETD), and bis(O,O-diisopropoxy phosphinothioyl) disulfide (known as Stec reagent).
  • Most of the known sulfurization reagents however, have one or more significant disadvantages.
  • Elemental sulfur presents problems and is not suitable for automation because of its insolubility in most organic solvents.
  • carbon disulfide a preferred source of sulfur, has undesirable volatility and an undesirably low flash point. Unwanted side products are often observed with the use of dibenzoyl tetrasulfide.
  • the Beaucage reagent while a relatively efficient sulfurization reagent, is difficult to synthesize and not particularly stable. Furthermore, use of Beaucage reagent forms a secondary reaction product which is a potent oxidizing agent. See R. P. Iyer et al. J. Am. Chem. Soc. 1990, 112, 1253-1254 and R. P. Iyer et al. J. Org. Chem.
  • Tetraethylthiuram disulfide while relatively inexpensive and stable, has a sulfinurization reaction rate which can be undesirable slow.
  • a method for producing a phosphorothioate ester by reaction of a phosphite ester with an acyl disulfide is disclosed in Dutch patent application No. 8902521.
  • the disclosed method is applied to a purified phosphotriester dimer utilizing solution-phase chemistry.
  • the method is time and labor intensive in that it was only shown to work in a complex scheme which involved carrying out the first stage of synthesis (formation of a phosphite) in acetonitrile, removing the acetonitrile, purifying the intermediate phosphotriester, and proceeding with the sulfinurization in a solvent mixture of dichloroethane (DCE) and 2,4,6-collidine.
  • DCE dichloroethane
  • a thioanhydride derivative EDITH (3-ethoxy-1,2,4-dithiazolidine-5-one) is disclosed in U.S. Pat. No. 5,852,168 (the '168 application).
  • this reagent can be used in the synthesis of 2′-substituted RNA and chimeric RNA. Importantly, even though these reaction conditions are basic they do not result in elimination of the 2′-substitutent or other degredation of the RNA.
  • PADS phenylacetyl disulfide
  • U.S. Pat. Nos. 6,242,591 and 6,114,519 disclose a methof of sulfurization carried out by contacting a deoxynucleic acid with an acetyl disulfide for a time suffiient to effect formation of a phosphorothioate functional group.
  • these patents do not provide examples of such a reaction in the syntheis of RNA (including 2′-substituted RNA and chimeric RNA), as is demonstrated herein.
  • these reaction conditions are basic they do not result in elimination of the 2′-substitutent or other degredation of the RNA.
  • the present invention relates to sulfur-transfer reagents and methods for the formation of phosphorothioates.
  • the methods are amenable to the formation of phosphorothioate linkages in oligonucleotides or derivatives, without the need for complex solvent mixtures, repeated washing, or solvent changes.
  • FIGS. 4, 5 , and 50 Certain preferred sulfur-transfer reagents of the invention are presented in FIGS. 4, 5 , and 50 .
  • One aspect of the present invention relates to the compound represented by formula D: R 1 —X S n X—R 2 D wherein
  • the present invention relates to the aforementioned compound, wherein n is 2.
  • the present invention relates to the aforementioned compound, wherein R 1 and R 2 are phenyl, benzyl, cyclohexyl, pyrrole, pyridine, or —CH 2 -pyridine.
  • the present invention relates to the aforementioned compound, wherein X is C(O), R 1 is phenyl, and R 2 is phenyl.
  • the present invention relates to the aforementioned compound, wherein X is SO 2 , R 1 is phenyl, and R 2 is phenyl.
  • the present invention relates to the aforementioned compound, wherein X is C(O), R 1 is pyrrole, and R 2 is pyrrole.
  • the present invention relates to the aforementioned compound, wherein X is C(O), and R 1 and R 2 taken together form a phenyl ring.
  • Another aspect of the present invention relates to the compound represented by formula D1: X S n Y D1 wherein
  • the present invention relates to the aforementioned compound, wherein n is 2.
  • the present invention relates to the aforementioned compound, wherein Y is CN.
  • the present invention relates to the aforementioned compound, wherein Y is P(OR 2 ) 2 .
  • Another aspect of the present invention relates to the compound represented by formula E: wherein X is O or S;
  • the present invention relates to the aforementioned compound, wherein X is O.
  • the present invention relates to the aforementioned compound, wherein R 2 is H, alkyl, or cycloalkyl.
  • the present invention relates to the aforementioned compound, wherein R 2 is aryl or aralkyl.
  • the present invention relates to the aforementioned compound, wherein R 2 is —C(O)N(R 3 )R 4 , —C(S)N(R 3 )R 4 , —C(S)N(R 3 ) 2 , —C(S)OR 4 , —CO 2 R 4 , —C(O)R 4 , or —C(S)R 4 .
  • the present invention relates to the aforementioned compound, wherein R 3 is H.
  • the present invention relates to the aforementioned compound, wherein R 4 is alkyl or aryl.
  • the present invention relates to the aforementioned compound, wherein X is O, and R 2 is H.
  • Another aspect of the present invention relates to a compound formed by the process, comprising the steps of:
  • Another aspect of the present invention relates to a method of forming a phosphorothioate compound, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said phosphite is represented by formula F: wherein
  • the present invention relates to the aforementioned method, wherein R 1 is —CH 2 CH 2 CN.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 2 is a nucleoside, nucleotide, or oligonucleotide.
  • the present invention relates to the aforementioned method, wherein R 2 is wherein R′ 1 represents independently for each occurrence alkyl, aryl, aralkyl, or —Si(R 4 ) 3 ; wherein said alkyl, aryl, and aralkyl group is optionally substituted with —CN, —NO 2 , —CF 3 , or halogen; and n 1 is 1 to 50 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 25 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 15 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 10 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 5 inclusive.
  • Ethylnitrile is a common phosphate protecting group used in oligonucleotide synthesis.
  • This protecting group is that it can be easily removed by treating the protected phosphate with a base. The overall transformation is illustrated below.
  • the acrylonitrile generated from the deprotection reaction is a good electrophile which can react with nucleophilic functional groups on the desired nucleotide or oligonucleotide product.
  • This side-reaction reduces the yield of the desired product and introduces impurities which can be difficult to remove. Therefore, the need exists for a reagent that will react selectively with the acrylonitrile produced during the deprotection reaction.
  • Representative examples of compounds that would serve as acrylonitrile scavenging agents during the deprotection reaction are polymer-bound thiols, alkane thiol having at least 10 carbon a toms, heteroarylthiol, the sodium salt of an alkane thiol, and thiols that have sufficiently low volitility so that they are odorless, e.g., thiols that have a high molecular weight.
  • Odorless thiols have been described by K. Nishide and M. Node in Green Chem. 2004, 6, 142. Some examples of odorless thiols include dodecanethiol, 4-n-heptylphenylmethanethiol, 4-trimethylsilylphenylmethanethiol, and 4-trimethylsilylbenzenethiol. For additional examples see Development of Odorless Thiols and Sulfides and Their Applications to Organic Synthesis. Nishide, Kiyoharu; Ohsugi, Shin-ichi; Miyamoto, Tetsuo; Kumar, Kamal; Node, Manabu. Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto, Japan.
  • FIG. 8 Representative examples of acrylonitrile quenching agents are shown in FIG. 8 .
  • One aspect of the present invention relates to a method of removing an ethylcyanide protecting group, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said acrylonitrile scavenger is
  • the present invention relates to the aforementioned method, wherein said phosphate compound is an oligonucleotide.
  • the present invention relates to the aforementioned method, wherein said phosphate compound is an oligonucleotide containing at least one phosphorothioate group.
  • the present invention relates to the aforementioned method, wherein said phosphate compound is an oligomer of ribonucleotides.
  • the present invention relates to the aforementioned method, wherein said phosphate is represented by formula G: wherein
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 1 is a nucleoside, nucleotide, or oligonucleotide.
  • the present invention relates to the aforementioned method, wherein R 1 is wherein R′ 1 represents independently for each occurrence alkyl, aryl, aralkyl, or —Si(R 4 ) 3 ; wherein said alkyl, aryl, and aralkyl group is optionally substituted with —CN, —NO 2 , —CF 3 , or halogen; R 4 is alkyl, aryl, or aralkyl; and n 1 is 1 to 50 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 25 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 15 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 10 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 5 inclusive.
  • the P ⁇ S bond of phosphorothioate nucleotides is sensitive to oxidizing agents, resulting in conversion of the P ⁇ S bond to a P ⁇ O bond.
  • One aspect of the present invention relates to methods of preventing unwanted oxidation of the P ⁇ S bond.
  • One method of preventing unwanted oxidation of the P ⁇ S bond is to mix a compound which is more readily oxidized than the P ⁇ S bond of a phosphothioate group with the phosphorothioate-containing nucleotide. Examples of compounds that are oxidized more readily than the P ⁇ S bond of a phosphothioate group include 2-hydroxylethanethiol, EDTA, vitamin E, thiols including odorless thiols, and vitamin C.
  • oligonucleotides having a p hosphorothioate linkage are promising therapeutic agents.
  • it is advantageous to prepare an oligonucleotide having a mixture of phosphate and phosphorothioate linkages One procedure to prepare oligonucleotides having a mixture of phosphate and phosphorothioate linkages involves attaching a first oligonucleotide to a second oligonucleotide, wherein the first oligonucleotide consists of nucleosides linked via phosphorothioate groups, and the second oligonucleotide consists of nucleosides linked by phosphite groups.
  • phosphite groups are oxidized to give the phosphate linkage.
  • oligonucleotides can be added sequentially to the first oligonucleotide using the phosphoramide method.
  • the newly added nucleosides, which are linked via phosphite groups are oxidized to convert the phosphite linkage to a phosphate linkage.
  • One of the most commonly used oxidizing agents for converting a phosphite to a phosphate is I 2 /amine. Consequently, the I 2 /amine reagent is a very strong oxidant which also oxidizes phosphorothioates to phosphates.
  • milder oxidizing agents are needed which will oxidize a phosphite to a phosphate, but will not oxidize a phosphorothioate group.
  • oxidizing agents that will oxidize a phosphite to a phosphate, but will not oxidize a phosphorothioate group, are NaClO 2 , chloroamine, and pyridine-N-oxide.
  • Additional oxidizing agents amenable to the present invention are CCl 4 , CCl 4 /water/acetonitrile, CCl 4 /water/pyridine, dimethyl carbonate, mixture of KNO 3 /TMSCl in CH 2 Cl 2 , NBS, NCS, or a combination of oxidizing agent, an aprotic organic solvent, a base and water.
  • One aspect of the present invention relates to a method of oxidizing a phosphite to a phosphate, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said oxidizing agent is NaClO 2 , chloroamine, or pyridine-N-oxide.
  • the present invention relates to the aforementioned method, wherein said phosphite is an oligomer of a nucleoside linked via phosphite groups.
  • the present invention relates to the aforementioned method, wherein said nucleoside is a ribonucleoside.
  • the present invention relates to the aforementioned method, wherein said phosphite is represented by formula H: wherein
  • the present invention relates to the aforementioned method, wherein R 1 is —CH 2 CH 2 CN.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 2 is a nucleoside, nucleotide, or oligonucleotide.
  • the present invention relates to the aforementioned method, wherein R 2 is wherein R′ 1 , represents independently for each occurrence alkyl, aryl, aralkyl, or —Si(R 4 ) 3 ; wherein said alkyl, aryl, and aralkyl group is optionally substituted with —CN, —NO 2 , —CF 3 , or halogen; and n 1 is 1 to 50 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 25 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 15 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 10 inclusive.
  • the present invention relates to the aforementioned method, wherein n 1 is 1 to 5 inclusive.
  • RNA is often synthesized and purified by methodologies based on: tetrazole to activate the RNA amidite, NH 4 OH to remove the exocyclic amino protecting groups, n-tetrabutylammonium fluoride (TBAF) to remove the 2′-OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA.
  • TBAF n-tetrabutylammonium fluoride
  • the RNA compounds may be formed either chemically or using enzymatic methods.
  • oligonucleotide synthesis One important component of oligonucleotide synthesis is the installation and removal of protecting groups. Incomplete installation or removal of a protecting group lowers the overall yield of the synthesis and introduces impurities that are often very difficult to remove from the final product. In order to obtain a reasonable yield of a large RNA molecule (i.e., about 20 to 40 nucleotide bases), the protection of the amino functions of the bases requires either amide or substituted amide protecting groups. The amide or substituted amide protecting groups must be stable enough to survive the conditions of synthesis, and yet removable at the end of the synthesis.
  • amide protecting groups benzoyl for adenosine, isobutyryl or benzoyl for cytidine, and isobutyryl for guanosine.
  • the amide protecting groups are often removed at the end of the synthesis by incubating the RNA in NH 3 /EtOH or 40% aqueous MeNH 2 .
  • an incubation in ethanolic ammonia for 4 h at 65° C. is used to obtain complete removal of these protecting groups.
  • One aspect of the present invention relates to amino compounds with relatively low volatility capable of effecting the amide deprotection reaction.
  • the classes of compounds with the aforementioned desirable characteristics are listed below. In certain instances, preferred embodiments within each class of compounds are listed as well.
  • the polyamine compound used in the invention relates to polymers containing at least two amine functional groups, wherein the amine functional group has at least one hydrogen atom.
  • the polymer can have a wide range of molecular weights. In certain embodiment, the polyamine compound has a molecular weight of greater than about 5000 g/mol. In other embodiments, the polyamine compound compound has a molecular weight of greater than about 10,000; 20,000, or 30,000 g/mol. 2) PEHA 3) PEG-NH 2
  • the PEG-NH 2 compound used in the invention relates to polyethylene glycol polymers comprising amine functional groups, wherein the amine functional group has at least one hydrogen atom.
  • the polymer can have a wide range of molecular weights.
  • the PEG-NH 2 compound has a molecular weight of greater than about 5000 g/mol.
  • the PEG-NH 2 compound has a molecular weight of greater than about 10, 000; 20,000, or 30,000 g/mol.
  • the short PEG-NH 2 compounds used in the invention relate to polyethylene glycol polymers comprising amine functional groups, wherein the amine functional group has at least one hydrogen atom.
  • the polymer has a relatively low molecular weight range.
  • the cycloalkylamines used in the invention relate to cycloalkyl compounds comprising at least one amine functional group, wherein the amine functional group has at least one hydrogen atom.
  • the hydroxycycloalkyl amines used in the invention relate to cycloalkyl compounds comprising at least one amine functional group and at least one hydroxyl functional group, wherein the amine functional group has at least one hydrogen atom. Representative examples are listed below. 6) Hydroxyamines
  • the hydroxyamines used in the invention relate to alkyl, aryl, and aralkyl compounds comprising at least one amine functional group and at least one hydroxyl functional group, wherein the amine functional group has at least one hydrogen atom.
  • Representative examples are 9-aminononanol, 4-aminophenol, and 4-hydroxybenzylamine.
  • One aspect of the present invention relates to a method of removing an amide protecting group from an oligonucleotide, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said oligonucleotide is an oligomer of ribonucleotides.
  • aryl amine-HF reagents useful in this invention include compounds represented by AA: wherein
  • aryl amines of the hydrofluoride salts are selected from the group consisting of (dialkyl)arylamines, (alkyl)diarylamines, (alkyl)(aralkyl)arylamines, (diaralkyl)arylamines, (dialkyl)heteroarylamines, (alkyl)diheteroarylamines, (alkyl)(heteroaryl)arylamines, (alkyl)(heteroaralkyl)arylamines, (alkyl)(aralkyl)heteroarylamines, (diaralkyl)heteroarylamines, (diheteoroaralkyl)heteroarylamines, and (aralkyl)(heteroaralkyl)heteroarylamines.
  • the rate of the deprotection reaction can be excelerated by conducting the deprotection reaction in the presence of microwave radiation.
  • the tert-butyldimethylsilyl groups on a 10-mer or 12-mer could be removed in 2 minutes or 4 minutes, respectively, by treatment with 1 M TBAF in THF, Et 3 N—HF, or pyridine-HF/DBU in the presence of microwave radiation (300 Watts, 2450 MHz).
  • One aspect of the present invention relates to a method removing a silyl protecting group from a oligonucleotide, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said oligonucleotide is an oligomer of ribonucleotides.
  • the present invention relates to the aforementioned method, wherein the reaction is carried out in the presence of microwave radiation.
  • Solid-phase oligonucleotide synthesis is often performed on controlled pore glass. However, solid-phase oligonucleotide synthesis can be carried out on:
  • the oligonucleotide is generally attached to the solid support via a linking group.
  • Suitable linking groups are an oxalyl linker, succinyl, dicarboxylic acid linkers, glycolyl linker, or thioglycolyl linker.
  • Silyl linkers can also be used. See, e.g., DiBlasi, C. M.; Macks, D. E.; Tan, D. S. “An Acid-Stable tert-Butyldiarylsilyl (TBDAS) Linker for Solid-Phase Organic Synthesis” Org. Lett. 2005; ASAP Web Release Date: 30-Mar.-2005; (Letter) DOI: 10.1021/o1050370y. DiBlasi et al.
  • TBDAS linker is stable to aqueous HF in CH 3 CN, which allows for the use of orthogonal HF-labile protecting groups in solid-phase synthetic schemes. In one approach, they established that cleavage of the linker could be achieved with tris(dimethylamino)-sulfonium (trimethylsilyl)-difluoride (TAS-F).
  • TAS-F tris(dimethylamino)-sulfonium
  • oligonucleotides can be prepared using non-halogenated solvents.
  • oligonucleotides can be prepared using toluene, tetrahydrofuran, or 1,4-dioxane as the solvent.
  • RNA using the H-phosphonate coupling method involves reacting a nucleoside substituted with an H-phosphonate with the hydroxyl group of a second nucleoside in the presence of an activating agent.
  • activating agent is pivaloyl chloride.
  • pivaloyl chloride is not ideal for large-scale preparations because it is flammable, corrosive, volatile (bp 105-106° C.), and has a relatively low flashpoint (Fp 8° C.). Therefore, the need exists for new activating agents devoid of the aforementioned drawbacks.
  • Useful condensing reagents include acid chlorides, chlorophosphates, carbonates, carbonium type compounds and phosphonium type compounds.
  • the condensing reagent is selected from a group consisting of pivaloyl chloride, adamantyl chloride, 2,4,6-triisopropyl-benzenesulfonyl chloride, 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, diphenyl phosphorochloridate, bis(2-oxo-3-oxazolidinyl)phosphinic chloride, bis(pentafluorophenyl)carbonate, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, 6-(trifluoromethyl)benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexaflufluor
  • activating agents that can be used in the H-phosphonate coupling method.
  • Classes of compound that are better activating agents include acid chlorides of long-chain alkyl groups, acid chlorides of aromatic groups, acid chlorides of alkyl groups substituted with aromatic groups, and polymer bound acyl chlorides.
  • Representative examples of activiting agents are decanoyl chloride, dodecanoyl chloride, benzoyl chloride, 1,2-dibenzyl ethanoyl chloride, naphthoyl chloride, anthracenecarbonyl chloride, and fluorenecarbonyl chloride.
  • oxidizing agents that can be used in the H-phosphonate coupling method.
  • One of the most common oxidizing agents is iodine.
  • iodine is a very strong oxidizing agent that can lead to unwanted oxidation of sensitive functional groups on the nucleotide or oligonucleotide.
  • Representative examples of oxidizing agents that can be used in the H-phosphonate coupling method include: camphorylsulfonyloxazaridine and N,O-bis(trimethylsilyl)-acetamide in MeCN/pyridine, CCl 4 /pyridine/water/MeCN, and DMAP in pyridine/CCl 4 /water.
  • Another aspect of the present invention relates to a method of forming a phosphodiester compound, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said activating agent is decanoyl chloride, dodecanoyl chloride, benzoyl chloride, 1,2-dibenzyl ethanoyl chloride, naphthoyl chloride, anthracenecarbonyl chloride, or fluorenecarbonyl chloride.
  • the present invention relates to the aforementioned method, wherein said H-phosphonate is represented by formula I: wherein
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 1 is a nucleoside or nucleotide.
  • the present invention relates to the aforementioned method, wherein said alcohol is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein said alcohol is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein said alcohol is a nucleoside, nucleotide, or oligonucleotide.
  • the present invention relates to the aforementioned method, wherein said alcohol is represented by R 5 —OH, wherein R 5 is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, alkenyl, or —(C(R 6 ) 2 ) p heterocycloalkyl; R 6 is H or alkyl; and p is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the present invention relates to the aforementioned method, wherein said phosphodiester is represented by formula J: wherein
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 1 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 1 is a nucleoside or nucleotide.
  • the present invention relates to the aforementioned method, wherein R 2 is (C(R 6 ) 2 ) p heterocycloalkyl.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted ribose.
  • the present invention relates to the aforementioned method, wherein R 2 is an optionally substituted deoxyribose.
  • the present invention relates to the aforementioned method, wherein R 2 is a nucleoside or nucleotide.
  • RNA preparation One common problem encountered in RNA preparation is obtaining the desired oligonucleotide in high purity. In many cases, reactions used to prepare the oligonucleotide do not achieve 100% conversion, or they generate side-products. Unfortunately, the unreacted starting materials and side-products often have similar chemical properties, making it very difficult to separate the desired product from these impurities.
  • RNA sequences are often performed using a two-step chromatographic procedure in which the molecule is first purified on a reverse phase column with either the trityl group at the 5′ position on or off. This purification is carried out using an acetonitrile gradient with triethylammonium or bicarbonate salts as the aqueous phase.
  • the trityl group may be removed by the addition of an acid and drying of the partially purified RNA molecule.
  • the final purification is carried out on an anion exchange column, using alkali metal perchlorate salt gradients to elute the fully purified RNA molecule as the appropriate metal salts, e.g. Na + , Li + etc.
  • a final de-salting step on a small reverse-phase cartridge completes the purification procedure.
  • RNA purification of long RNA molecules is carried out using anion exchange chromatography, particularly in conjunction with alkali perchlorate salts.
  • This system is used to purify very long RNA molecules.
  • a Dionex NUCLEOPAK 100® or a Pharmacia MONO Q® anion exchange column for the purification of RNA by the anion exchange method.
  • This anion exchange purification may be used following a reverse-phase purification or prior to reverse-phase purification. This method results in the formation of a sodium salt of the ribozyme during the chromatography.
  • Replacement of the sodium alkali earth salt by other metal salts, e.g., lithium, magnesium or calcium perchlorate yields the corresponding salt of the RNA molecule during the purification.
  • the reverse-phase purification is usually perfomed using polymeric, e.g., polystyrene based, reverse-phase media using either a 5′-trityl-on or 5′-trityl-off method. Either molecule may be recovered using this reverse-phase method, and then, once detritylated, the two fractions may be pooled and submitted to an anion exchange purification step as described above.
  • polymeric e.g., polystyrene based
  • reverse-phase media using either a 5′-trityl-on or 5′-trityl-off method.
  • Either molecule may be recovered using this reverse-phase method, and then, once detritylated, the two fractions may be pooled and submitted to an anion exchange purification step as described above.
  • FIG. 9 A diagram illustrating the overall procedure is presented in FIG. 9 .
  • the structure of AL-4112, AL-4180, AL-DP-4014, AL-2200, AL-22-1, AL-DP-4127, AL-2299, AL-2300, AL-DP-4139, AL-2281, AL-2282, and AL-DP-4140 is presented in FIG. 10 .
  • the specific procedure for the purification of AL-DP-4014, the components of which are AL-4112 and AL-4180, is shown in FIGS. 11 and 12 .
  • AL-DP-4127, AL-DP-4139, and AL-DP-4140 were also purified using the procedures described in FIGS. 9, 11 , and 12 .
  • the results from the analyses are presented in FIGS. 19-39 .
  • FIGS. 40-43 Alternative procedures of RNA purification using the two-strand method are presented in FIGS. 40-43 .
  • One aspect of the present invention relates to a method of purifying an oligonucleotide, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said annealing a first oligonucleotide with a second oligonuclotide is done at a temperature between a first temperature and a second temperature, wherein said first temperature is about the T m of a double-stranded oligonucleotide consisting of said first oligonucleotide and a third oligonuclotide, wherein said third oligonuclotide is the antisense sequence corresponding to the first oligonuclotide, and said second temperature is about 5 degrees below said first temperature.
  • the present invention relates to the aforementioned method, wherein said chromatographic purification is liquid chromatography.
  • the present invention relates to the aforementioned method, wherein said chromatographic purification is high-performance liquid chromatography.
  • the present invention relates to the aforementioned method, wherein said first oligonucleotide is an oligomer of ribonucleotides.
  • the present invention relates to the aforementioned method, wherein said second oligonucleotide is an oligomer of ribonucleotides.
  • the present invention relates to the aforementioned method, wherein said first oligonucleotide is an oligomer of ribonucleotides, and said second oligonucleotide is an oligomer of ribonucleotides.
  • HPLC high-peformance liquid chromatography
  • protecting groups play a critical role in RNA synthesis.
  • the Applicants describe herein several new protecting groups that can be used in RNA synthesis.
  • One class of 2′-protecting groups that can be used in RNA synthesis is carbonates.
  • One preferred carbonate is propargyl carbonate shown below.
  • the propargyl carbonate can be removed using benzyltriethylammonium tetrathiomolybdate as described in Org. Lett. 2002, 4, 4731.
  • acetals Another class of 2′-protecting groups that can be used in RNA synthesis is acetals. Acetal groups can be deprotected using aqueous acid. Several representative acetal protecting groups are shown below. See FIG. 44 for additional examples.
  • RNA synthesis involves protecting both the 2′-hydroxyl group of the ribose and the phosphate attached to the 3′-position of the ribose with a silyl group.
  • a representative example is presented below in FIG. 44 .
  • DMT dimethoxytrityl
  • MMT monomethoxytrityl
  • Pixyl 9-phenylxanthen-9-yl
  • Mox 9-(p-methoxyphenyl)xanthen-9-yl
  • Aralkyl esters represented by —O 2 CCH 2 R, wherein R is phenyl, pyridinyl, aniline, quinoline, or isoquinoline can be removed from the 2′-position of a nucleoside by enzymatic cleavage using penicillin G acylase.
  • Representative examples of nucleosides bearing aralkyl ester protecting groups at the 2′-position of the ribose ring are presented in FIG. 45 .
  • certain internal amidites including those shown in FIG. 45 , can be removed by enzymatic cleavage.
  • One aspect of the present invention relates to a method of removing a protecting group, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein said protecting group is an aralkyl ester.
  • the present invention relates to the aforementioned method, wherein said protecting group is represented by the formula —O 2 CCH 2 R, wherein R is phenyl, pyridinyl, aniline, quinoline, or isoquinoline.
  • the present invention relates to the aforementioned method, wherein said enzyme is penicillin G acylase.
  • the present invention relates to the aforementioned method, wherein said ribose is a ribonucleotide oligomer.
  • an oligonucleotide comprising two adjacent thymidine nucleotides.
  • the thymidine nucleotides are located at the 3′ end of the oligonucleotide.
  • the thymidine-thymidine (TT) nucleotide unit can be prepared using solution-phase chemistry, and then the TT unit is attached to a solid support.
  • the TT unit is linked via a phosphorothioate group.
  • the different stereoisomers of the phosphorothioate TT unit may be separated prior to attachment of the TT unit to the solid support.
  • the remainder of the oligonucleotide strand can be synthesized via standard solid-phase synthesis techniques using the TT-support bound unit as a primer.
  • the thymidine-thymidine nucleotide unit is made of deoxythymidine residues.
  • One aspect of the present invention relates to a method of preparing an oligonucleotide comprising a dinucleoside unit, comprising the steps of:
  • the present invention relates to the aforementioned method, wherein each nucleoside residue of said dinucleoside group is independently a natural or unnatural nucleoside.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and said nucleobase is natural or unnatural.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is an L-ribose or L-deoxyribose, and said nucleobase is natural or unnatural.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two thymidine residues.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two deoxythymidine residues.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two 2′-modified 5-methyl uridine or uridine residues, wherein the 2′-modifications are 2′-O-TBDMS, 2′-OMe, 2′-F, 2′-O—CH2—CH2-O-Me, or 2′-O-alkylamino derivatives.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises a phosphorothioate linkage, phosphorodithioate linkage, alkyl phosphonate linkage, or boranophosphate linkage.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises a phosphorothioate linkage, alkyl phosphonate linkage, or boranophosphate linkage; and said dinucleoside group is a single stereoisomer at the phosphorus atom.
  • the present invention relates to the aforementioned method, wherein the linkage between the nucleoside residues of said dinucleoside group is a 3′-5′ linkage.
  • the present invention relates to the aforementioned method, wherein the linkage between the nucleoside residues of said dinucleoside group is a 2′-5′ linkage.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and said nucleobase is natural or unnatural; and the linkage between the nucleoside residues of said dinucleotide group is unnatural and non-phosphate.
  • the present invention relates to the aforementioned method, wherein said dinucleoside group comprises two nucleoside residues each independently comprising a sugar and a nucleobase, wherein said sugar is an L-ribose or L-deoxyribose, and said nucleobase is natural or unnatural; and the linkage between the nucleoside residues of said dinucleotide group is MMI, amide linkage, or guanidinium linkage.
  • any one of the above-mentioned improvements can be used alone with standard methods of preparing nucleosides, nuclotides, and oligonucleotides, or more than one of the above-mentioned improvements can be used together with standard methods of preparing nucleosides, nuclotides, and oligonucleotides.
  • one of ordinary skill in the art can readily determine the optimal conditions for each of the improvements described above.
  • the present invention relates to processes and reagents for oligonucleotide synthesis and purification.
  • the following description is meant to briefly describe some of the major types and structural features of oligonucleotides. Importantly, the following section is only representative and not meant to limit the scope of the present invention.
  • Oligonucleotides can be made of ribonucleotides, deoxyribonucleotides, or mixtures of ribonucleotides and deoxyribonucleotides.
  • the nucleotides can be natural or unnatural.
  • Oligonucleotides can be single stranded or double stranded.
  • Various modifications to the sugar, base, and phosphate components of oligonucleotides are described below.
  • oligonucleotides having modified backbones or internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their intersugar backbone can also be considered to be oligonucleosides.
  • oligonucleotide chemical modifications are described below. It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the following modifications may be incorporated in a single siRNA compound or even in a single nucleotide thereof.
  • Preferred modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalklyphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.
  • Various salts, mixed salts and free-acid forms are also included.
  • Preferred modified internucleoside linkages or backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleoside units are replaced with novel groups.
  • the nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid PNA
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.
  • Some preferred embodiments of the present invention employ oligonucleotides with phosphorothioate linkages and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 —[known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 —, and —O—N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above referenced U.S.
  • Oligonucleotides may additionally or alternatively comprise nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base” modifications or substitutions.
  • “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
  • 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-substit
  • nucleobases include those disclosed in U.S. Pat. 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 of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotides of the invention.
  • 5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 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 2′-O-methoxyethyl sugar modifications.
  • the oligonucleotides may additionally or alternatively comprise one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, 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 oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, 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 includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533, filed on Jan. 30, 1998, the contents of which are incorporated by reference.
  • modifications include 2′-methoxy (2′-O—CH 3 ), 2′-O-methoxyethyl, 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 the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides.
  • sugar substituent group or “2′-substituent group” includes groups attached to the 2′-position of the ribofuranosyl moiety with or without an oxygen atom.
  • Sugar substituent groups include, but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula (O-alkyl) m , wherein m is 1 to about 10.
  • 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 hereby incorporated by reference in its entirety. Further sugar modifications are disclosed by Cook ( Anti - Cancer Drug Design, 1991, 6, 585-607).
  • Additional sugar substituent groups amenable to the present invention include 2′-SR and 2′-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 U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in its entirety. The incorporation of 2′-SR monomer synthons is disclosed by Hamm et al. ( J. Org. Chem. 1997, 62, 3415-3420). 2′-NR nucleosides are disclosed by Goettingen, M. J. Org.
  • Representative 2′-O-sugar substituent groups of formula I are disclosed in U.S. Pat. No. 6,172,209, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety.
  • Representative cyclic 2′-O-sugar substituent groups of formula II are disclosed in U.S. Pat. No. 6,271,358, filed Jul. 27, 1998, entitled “RNA Targeted 2′-Modified Oligonucleotides that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.
  • Sugars having O-substitutions on the ribosyl ring are also amenable to the present invention.
  • Representative substitutions for ring 0 include, but are not limited to, NH, NR, S, CH 2 , CHF, and CF 2 . See, e.g., Secrist et al., Abstract 21 , Program & Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications , Park City, Utah, Sep. 16-20, 1992.
  • Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties, hexoses, cyclohexenyl in place of the pentofuranosyl sugar.
  • sugar mimetics such as cyclobutyl moieties, hexoses, cyclohexenyl in place of the pentofuranosyl sugar.
  • Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos.
  • oligonucleotide may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide.
  • one modification of oligonucleotides involves chemically linking to the oligonucleotide one or more additional moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to 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.
  • Oligonucleotides can be substantially chirally pure with regard to particular positions within the oligonucleotides.
  • Examples of substantially chirally pure oligonucleotides include, but are not limited to, those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those having substantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).
  • RNA molecules and derivatives thereof that catalyze highly specific endoribonuclease activities are known as ribozymes.
  • ribozymes See, generally, U.S. Pat. No. 5,543,508 to Haseloff et al., issued Aug. 6, 1996, and U.S. Pat. No. 5,545,729 to Goodchild et al., issued Aug. 13, 1996.
  • the cleavage reactions are catalyzed by the RNA molecules themselves.
  • the sites of self-catalyzed cleavage are located within highly conserved regions of RNA secondary structure (Buzayan et al., Proc. Natl. Acad. Sci.
  • RNA molecules Naturally occurring autocatalytic RNA molecules have been modified to generate ribozymes which can be targeted to a particular cellular or pathogenic RNA molecule with a high degree of specificity.
  • ribozymes serve the same general purpose as antisense oligonucleotides (i.e., modulation of expression of a specific gene) and, like oligonucleotides, are nucleic acids possessing significant portions of single-strandedness. That is, ribozymes have substantial chemical and functional identity with oligonucleotides and are thus considered to be equivalents for purposes of the present invention.
  • the oligonucleotide may be modified by a moiety.
  • moieties have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature.
  • Such moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. A cad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
  • a thioether e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl.
  • Acids Res., 1990, 18:3777 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923).
  • Typical conjugation protocols involve the synthesis of oligonucleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.
  • RNA short interfering RNA
  • the backbone of the oligonucleotide can be modified to improve the therapeutic or diagnostic properties of the siRNA compound.
  • the two strands of the siRNA compound can be complementary, partially complementary, or chimeric oligonucleotides.
  • at least one of the bases or at least one of the sugars of the oligonucleotide has been modified to improve the therapeutic or diagnostic properties of the siRNA compound.
  • the siRNA agent can include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the siRNA agent, or a fragment thereof, can mediate down regulation of the target gene.
  • the term “ribonucleotide” or “nucleotide” can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.
  • the siRNA agent is or includes a region which is at least partially complementary to the target RNA. In certain embodiments, the siRNA agent is fully complementary to the target RNA.
  • siRNA agent it is not necessary that there be perfect complementarity between the siRNA agent and the target, but the correspondence must be sufficient to enable the siRNA agent, or a cleavage product thereof, to direct sequence specific silencing, such as by RNAi cleavage of the target RNA.
  • Complementarity, or degree of homology with the target strand is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches with respect to the target RNA.
  • the mismatches are most tolerated in the terminal regions, and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus.
  • the sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double-strand character of the molecule.
  • an siRNA agent will often be modified or include nucleoside surrogates.
  • Single stranded regions of an siRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates.
  • Modification to stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored.
  • Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.
  • siRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein.
  • sRNA agent or shorter iRNA agent refers to an iRNA agent that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs.
  • the sRNA agent, or a cleavage product thereof can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.
  • Each strand of a sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length.
  • the strand is preferably at least 19 nucleotides in length.
  • each strand can be between 21 and 25 nucleotides in length.
  • Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3′ overhangs, of 2-3 nucleotides.
  • an siRNA agent will preferably have one or more of the following properties:
  • a “single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure.
  • Single strand iRNA agents are preferably antisense with regard to the target molecule.
  • a single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA.
  • a single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.
  • Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs.
  • the duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
  • the hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3′, and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.
  • Chimeric oligonucleotides are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such oligonucleotides have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.
  • the chimeric oligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, or RNA-DNA-RNA-DNA, wherein the oligonucleotide is between 5 and 60 nucleotides in length.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1 -C 30 for straight chain, C 3 -C 30 for branched chain), and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • aryl group e.g., an aromatic or heteroaromatic group.
  • a benzyl group PhCH 2 — is an aralkyl group.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • aryl as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, anthracene, naphthalene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF 3 , —CN, or the like.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively.
  • 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
  • heterocyclyl or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles.
  • Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, o
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF 3 , —CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxy
  • polycyclyl or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed “bridged” rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF 3 , —CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, si
  • nitro means —NO 2 ;
  • halogen designates —F, —Cl, —Br or —I;
  • sulfhydryl means —SH;
  • hydroxyl means —OH; and
  • sulfonyl means —SO 2 —.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: wherein R 9 , R 10 and R′ 10 each independently represent a group permitted by the rules of valence.
  • acylamino is art-recognized and refers to a moiety that can be represented by the general formula: wherein R 9 is as defined above, and R′ 11 represents a hydrogen, an alkyl, an alkenyl or —(CH 2 ) m —R 8 , where m and R 8 are as defined above.
  • amido is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: wherein R 9 , R 10 are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the “alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, —S-alkynyl, and —S—(CH 2 ) m —R 8 , wherein m and R 8 are defined above.
  • Representative alkylthio groups include methylthio, ethyl thio, and the like.
  • carbonyl is art recognized and includes such moieties as can be represented by the general formula: wherein X is a bond or represents an oxygen or a sulfur, and R 11 represents a hydrogen, an alkyl, an alkenyl, —(CH 2 ) m —R 8 or a pharmaceutically acceptable salt, R′ 11 represents a hydrogen, an alkyl, an alkenyl or —(CH 2 ) m —R 8 , where m and R 8 are as defined above. Where X is an oxygen and R 11 or R′ 11 is not hydrogen, the formula represents an “ester”.
  • alkoxyl refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH 2 ) m —R 8 , where m and R 8 are described above.
  • sulfonate is art recognized and includes a moiety that can be represented by the general formula: in which R 41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
  • triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
  • triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
  • Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively.
  • a more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry ; this list is typically presented in a table entitled Standard List of Abbreviations .
  • the abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
  • sulfonyl refers to a moiety that can be represented by the general formula: in which R 44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.
  • sulfoxido refers to a moiety that can be represented by the general formula: in which R 44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.
  • a “selenoalkyl” refers to an alkyl group having a substituted seleno group attached thereto.
  • Exemplary “selenoethers” which may be substituted on the alkyl are selected from one of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH 2 ) m —R 7 , m and R 7 being defined above.
  • Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
  • each expression e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • the substituent can be halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF 3 , —CN, and the like.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2 nd ed.; Wiley: New York, 1991).
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., functioning as analgesics), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in binding to sigma receptors.
  • the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
  • the strength of the activator is increased by forming the activated salt resulting in decreased coupling time for RNA Synthesis.
  • RNA molecules 49, 5′-CAUCGCTGAdT-3′ was synthesized on a 394 ABI machine (ALN 0208) using the standard 98 step cycle written by the manufacturer with modifications to a few wait steps as described below.
  • the solid support was controlled pore glass (CPG, prepacked, 1 ⁇ mole, 500, Proligo Biochemie GmbH) and the monomers were RNA phosphoramidites with fast deprotecting groups obtained from Pierce Nucleic Acid Technologies used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated.
  • RNA phosphoramidites were 5′-O-Dimethoxytrityl-N 6 -phenoxyacetyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-( ⁇ -cyanoethyl-N,N′-diisopropyl)phosphoramidite, 5′-O-Dimethoxytrityl-N 2 -p-isopropylphenoxyacetyl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-( ⁇ -cyanoethyl-N,N′-diisopropyl)phosphoramidite, 5′-O-Dimethoxytrityl-N 4 -acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O—( ⁇ -cyanoethyl-N,N′-diisopropyl)phosphoramidite, and 5′-O-O-
  • the coupling times were either 1, 3 or 5 minutes for the different salt concentrations which themselves were 10, 20 and 40 mol % relative to the 5-(ethylthio)-1H-tetrazole (ETT, 0.25 M, Glen Research).
  • Diisopropylammonium salt of ETT with required mol % was obtained by adding calculated amount of anhydrous diisopropylamine to 0.25 M ETT solution and stored over molecular sieves for 4-6 h.
  • the CPG was transferred to a screw cap RNase free microfuge tube.
  • the oligonucleotide was cleaved from the CPG with simultaneous deprotection of base and phosphate groups with 1.0 mL of a mixture of 40% methylamine: ammonia (1:1)] for 30 minutes at 65° C.
  • the solution was then lyophilized to dryness.
  • Activator was 0.6 M 5-Ethylthiotetrazole (American International Chemical; Natick, Mass.) in MeCN and was used at three-fold excess relative to RNA CEPs and at 4.5-fold excess to DNA CEPs.
  • Oxidation was via 50 mM I 2 in 90% pyridine 10% H 2 O or with 0.05 M 3-ethoxy-1,2,4-dithiazolidine-5-one (EDITH) in MeCN (Q. Xu, et al. Nucleic Acids Research , Vol. 24, No. 18, pp. 3643-3644).
  • Capping was with 10% acetic anhydride (Ac 2 O) 10% 1-methylimidazole (1-MeIm) 15% 2,6-lutidine in MeCN.
  • Buffer A was 1 mM EDTA, 25 mM Tris pH 8, 20 mM NaClO 4 .
  • Buffer B was 1 mM EDTA, 25 mM Tris pH 8, 0.4 M NaClO 4 . Separation was performed on a 0-40% B gradient with buffers and column heated to 65° C.
  • the solutions containing the crude material were diluted 4-6 fold, loaded onto the column in 1-3kAU amounts at 10 mL/min and eluted with a segmented gradient from 0-60% B. Appropriate fractions were pooled and this pooled material desalted in 30 mL amounts over Sephadex G-25 on a BioPilot column (6 cm dia. ⁇ 7.5 cm) against H 2 O. The eluate was vacuum evaporated to less than 25 mL, shell frozen and lyophilized.
  • RNA CEPs Pulierce Nucleic Acids; Milwaukee, Wis.
  • MeCN acetonitrile
  • Activator was 0.6 M 5-Ethylthiotetrazole (American International Chemical; Natick, Mass.) in MeCN and was used at threefold excess relative to RNA CEPs and at 4.5-fold excess to DNA CEPs.
  • Oxidation was via 50 mM I 2 in 90% pyridine 10% H 2 O.
  • Thiolation was with 0.2 M phenyl acetyl disulfide (PADS) in 1:1 3-picoline:MeCN or with 0.05 M 3-ethoxy-1,2,4-dithiazolidine-5-one (EDITH) in MeCN (Q.
  • PADS phenyl acetyl disulfide
  • Buffer A was 1 mM EDTA, 25 mM Tris pH 9, 50 mM NaClO 4 , 20% MeCN.
  • Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO 4 , 20% MeCN. Separation was performed on a 0-65% B segmented gradient with buffers and column heated to 65° C.
  • P O backbone 33:5′ UU GGUGAGGUUUGAUCC GC d T dT.
  • P S backbone Method 1
  • a ⁇ 1 umole sample of 27 was deprotected by MeNH 2 at 65° C. for 20 mins and dried. Then it was treated with a mixture of 0.1 mL TEA.3HF, 0.075 mL TEA and 0.15 mL DMSO at 65° C. for 1.5 hours. The yield on HPLC was 47/54% (260 nm and 280 nm) on anion exchange HPLC.
  • the yield was 55/53% after 10 mins, 57/57% after 20 mins, 57/58% after 30 mins and 57/57% after 1 hour.
  • the pH of this 1:5 mixture was found out to be about 10 by adding in water. Therefore, ⁇ 0.5 ⁇ mole of the MeNH 2 deprotected and dried 27 was deprotected by premixed 6.5 ⁇ L Py.HF, 27.4 ⁇ L DBU and 26 ⁇ L DMSO at 65° C. for 15 mins and 70 mins. The yield was 57/57% after 15 mins and 70 mins. A ⁇ 4 ⁇ mole sample of 27 was deprotected by concentrated ammonia at 65° C. for 1 hour and dried.
  • Compound 29 was synthesized at 1 ⁇ mole scale. It was deprotected by ethanolic ammonia at 65° C. for 1 hour, then divided to half (71 OD and 77 OD) and dried. 27 ⁇ L Py.HF, 108 ⁇ L DBU and 135 ⁇ L DMSO were mixed. Half of this mixture was used to treat the 77 OD sample for 20 mins at 65° C., the other half was used to treat the 71 OD sample for 30 mins. The yield was 64/63% after 20 mins and 62/63% after 30 mins. The fully thioated 31 was deprotected by ethanolic ammonia at 65° C. for 45 mins. The crude mixture was divided into half and dried, 76 OD in each sample.
  • Part of 28 was deprotected with MeNH 2 at 65° C. for 20 mins. The crude mixture was divided into ⁇ 40 OD samples and dried. The other part was deprotected with ethanolic ammonia at 65° C. for 40 mins, and also divided into ⁇ 40 OD samples and dried.
  • MeNH 2 deprotected sample was desilylated with standard procedures (16 ⁇ L TEA.3HF, 12 ⁇ L TEA and 24 ⁇ L DMSO at 65° C.), the yield was 37/36% after 30 mins, 41/49% after 1 hour, 38/43% after 1.5 hours and 42/42% after 2.5 hours.
  • MeNH 2 deprotected sample was desilylated with premixed 9 ⁇ L Py.HF, 36 ⁇ L DBU and 36 ⁇ L DMSO at 65° C., and the yield was 44/45% after 15 mins, 46/45% after 30 mins, 45/44% after 1 hour, 45/44% after 1.5 hr and 44/48% after 2.5 hrs.
  • Another portion of MeNH 2 deprotected sample was desilylated with premixed 9 ⁇ L Py.HF, 31.5 ⁇ L DBU and 31.5 ⁇ L DMSO at 65° C., and the yield was 42/45% after 15 mins, 45/47% after 30 mins, 45/44% after 1 hour, 45/48% after 1.5 hr and 39/47% after 2.5 hrs.
  • ethanolic ammonia deprotected sample was desilylated with standard procedures (16 ⁇ L TEA.3HF, 12 ⁇ L TEA and 24 ⁇ L DMSO at 65° C.), the yield was 40/39% after 30 mins, 49/51% after 1 hour, 49/51% after 1.5 hour and 47/49% after 2.5 hour.
  • Second portion of ethanolic ammonia deprotected sample was desilylated with premixed 9 ⁇ L Py.HF, 36 ⁇ L DBU and 36 ⁇ L DMSO at 65° C., and the yield was 50/50% after 15 mins, 49/49% after 30 mins, 53/54% after 1 hour, 55/58% after 1.5 hour and 54/54% after 2.5 hrs.
  • Silyl deprotection reagent 4 volume desilylation mixture (1 mL Py.HF, 3.5 mL DBU, 4 mL DMSO) per 1 volume of ethanolic ammonia at 60° C. for 20 mins.
  • TAS-F Tris(dimethylamino)sulfur diflurotrimethylsilane
  • a ⁇ 40 OD sample of MeNH 2 deprotected (65° C. 20 mins) and dried 28 sample was treated with 41 mg TASF and 90 ⁇ L DMF at 65° C. Injections were done after 30 mins, 1 hr, 2 hr, and then at RT overnight. The reaction did not yield noticeable amount of product.
  • Another ⁇ 40OD sample was treated with 41 mg TASF, 90 ⁇ L DMF and 40 ⁇ L water at 65° C. Injections were done after 30 min, 1 hr, 2 hr, and then at RT overnight. No major peak was detected in the HPLC for the product. Same deprotection conditions were applied on ⁇ 40OD samples of 28 deprotected by ethanolic ammonia (65° C., 40 min.) and same results were observed: no major peak.
  • the oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 2.0 mL of a mixture of ammonia and 8 M ethanolic methylamine [1:1] for 30 min at 65° C.
  • the vial was cooled briefly on ice and then the ethanolic ammonia mixture was transferred to a new microfuge tube.
  • the CPG was washed with 2 ⁇ 0.1 mL portions of deionized water, put in dry ice for 10 min, and then dried in speed vac.
  • Condition B The reaction was then quenched with 400 ⁇ L of isopropoxytrimethylsilane (iPrOSiMe 3 , Aldrich) and further incubated on the heating block leaving the caps open for 10 min. (This causes the volatile isopropxytrimethylsilylfluoride adduct to vaporize). The residual quenching reagent was removed by drying in a speed vac. Added 1.5 mL of 3% triethylamine in diethyl ether and pelleted by centrifuging. The supernatant was pipetted out without disturbing the pellet. Dry the pellet in speed vac. The crude RNA was obtained as a white fluffy material in the microfuge tube.
  • isopropoxytrimethylsilane iPrOSiMe 3 , Aldrich
  • FIG. 9 A diagram illustrating the overall purification procedure is presented in FIG. 9 .
  • the specific procedure used for the purification of AL-DP-4014 is presented in FIGS. 11 and 12 .
  • FIGS. 11 and 12 The specific procedure used for the purification of AL-DP-4014 is presented in FIGS. 11 and 12 .
  • the chromatographic data presented in FIGS. 14-18 indicate that the purification procedure produced AL-DP-4014 in substantially pure form.
  • the purification procedure was performed as described above for AL-DP-4127, AL-DP-4139, AND AL-DP-414.
  • the results from analytical analyses are presented in FIGS. 19-39 .
  • the alkylamine would generate the acrylonitlile which would be scavenged by the thiol. This is an improvement over the process described by Capaldi et al. Org. Process Res. Dev. 2003, 7, 832-838.
  • oligonucleotides were synthesized on an AKTAoligopilot synthesizer.
  • Commercially available controlled pore glass solid supports dT-CPG, rC-CPG, rU-CPG, from Prime Synthesis
  • the in-house synthesized solid supports phthalimido-hydroxy-prolinol-CPG, hydroxyprolinol-cholesterol-CPG described in patent applications: provisional 60/600,703 Filed Aug. 10, 2004 and PCT/US04/11829 Filed Apr. 16, 2004
  • RNA phosphoramidites and 2′-O-methyl modified RNA phosphoramidites with standard protecting groups (5′-O-dimethoxytrityl-N-6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N-4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N-2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-2′
  • the 2′-F phosphoramidites (5′-O-dimethoxytrityl-N-4-acetyl-2′-fluro-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramidite) were obtained from Promega.
  • the cholesterol and amino-linker phosphoramidites were synthesized in house, and used at a concentration of 0.1 M in dichloromethane for cholesterol and 0.2 M in CH 3 CN for the amino-linker. Coupling/recycling time for both the cholesterol and the amino-linker phosphoramidites was 16 minutes.
  • RNAs without the 2′-fluoro modification After completion of synthesis, the support was transferred to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 40 mL of a 40% aq. methyl amine (Aldrich) 90 mins at 45° C. The bottle was cooled briefly on ice and then the methylamine was filtered into a new 500 mL bottle. The CPG was washed three times with 40 mL portions of DMSO. The mixture was then cooled on dry ice.
  • the dried residue was resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (Et3N.3HF), and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position.
  • the reaction was then quenched with 50 mL of 20 mM sodium acetate and the pH was adjusted to 6.5, and the solution was stored in freezer until purification.
  • Unconjugated oligonucleotides The unconjugated crude oligonucleotides were first analyzed by HPLC (Dionex PA 100). The buffers were 20 mM phosphate, pH 11 (buffer A); and 20 mM phosphate, 1.8 M NaBr, pH 11 (buffer B). The flow rate 1.0 m L/min and monitored wavelength was 260-280 nm. Injections of 5-15 ⁇ L were done for each sample.
  • the unconjugated samples were purified by HPLC on an TSK-Gel SuperQ-5PW (20) column packed in house (17.3 ⁇ 5 cm).
  • the buffers were 20 mM phosphate in 10% CH 3 CN, pH 8.5 (buffer A) and 20 mM phosphate, 1.0 M NaBr in 10% CH 3 CN, pH 8.5 (buffer B).
  • the flow rate was 50.0 mL/min and wavelengths of 260 and 294 nm were monitored.
  • the fractions containing the full-length oligonucleotides were pooled together, evaporated, and reconstituted to about 100 mL with deionised water.
  • (b) Cholesterol-conjugated oligonucleotides The cholesterol-conjugated crude oligonucleotides were first analyzed by LC/MS to determine purity. The 5′-cholesterol conjugated sequences were HPLC purified on an RPC-Source15 reverse-phase column packed in house. The buffers were 20 mM TEAA in 10% CH 3 CN (buffer A) and 20 mM TEAA in 70% CH 3 CN (buffer B). The fractions containing the full-length oligonucleotides were then pooled together, evaporated, and reconstituted to 100 mL with deionised water.
  • the 3′-cholesterol conjugated sequences were HPLC purified on an RPC-Source15 reverse-phase column packed in house.
  • the buffers were 20 mM NaOAc in 10% CH 3 CN (buffer A) and 20 mM NaOAc in 70% CH 3 CN (buffer B).
  • the fractions containing the full-length oligonucleotides were pooled, evaporated, and reconstituted to 100 mL with deionised water.
  • the purified oligonucleotides were desalted on an AKTA Explorer system (Amersham Biosciences) using a Sephadex G-25 column. First, the column was washed with water at a flow rate of 25 mL/min for 20-30 min. The sample was then applied in 25 mL fractions. The eluted salt-free fractions were combined, dried, and reconstituted in 50 mL of RNase free water.
  • Step 6 Purity Analysis by Capillary Gel Electrophoresis (CGE), Ion-Exchange HPLC, and Electrospray LC/Ms
  • Lower case “s” indicates a phosphorothioate linkage.
  • the lower case “d” indicates a deoxy residue.
  • “HP-NH2” or “NH2-HP” indicates a hydroxyprolinol amine conjugate.
  • “Chol-” indicates a hydroxyprolinol cholesterol conjugate.
  • Subscript “OMe” indicates a 2′-O-methyl sugar and subscript “F” indicates a 2′-fluoro modified sugar. Purity was determined by CGE except where indicated by an asterisk (in these two cases, purity was determined by ion-exchange chromatography).
  • RNA with 2′-OMe, PS, or cholesterol modifications
  • PVPHF polyvinylpyridine polyHF
  • oligonucleotides were synthesized on an AKTA oligopilot synthesizer.
  • Commercially available controlled pore glass solid support dT-CPG, U-CPG 500′
  • the hydroxy-prolinol-cholesterol solid support described in patent applications: provisional 60/600,703 Filed Aug. 10, 2004 and PCT/US04/11829 Filed Apr. 16, 2004 was used.
  • RNA phosphoramidites with standard protecting groups 5′-O-dimethoxytrityl-N-6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethyloxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N-2-isobutryl-2′-t-butyldimethylsilyl- guanosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine
  • oligonucleotides synthesized, scale, support type, amount and loading are listed below: Alnylam Support, Mass Mass of Loading Scale Synthesis SQ No. (gram) support (g) ( ⁇ mol/g) (umol) column 5718 dT 4.15 84 349 12 mL 5719 dT 4.15 84 349 12 mL 3216 dT 4.01 87 349 12 mL 3218 dT 4.08 87 355 12 mL 5474 Hydroxy prolinol 4.1 68.6 281 12 mL cholesterol 5475 rU 3.9 83 324 12 mL Step 2, Deprotection
  • Step 1) cleavage of oligonucleotide from support with simultaneous removal of base and phosphate protecting groups from the oligonucleotide
  • Step 2) deprotection of 2′-O-TBDMS groups.
  • the purified oligonucleotides were desalted on a Waters 5 cm column with size exclusion resin Sephadex G-25. The flow rate was 25 mL/min. The eluted salt-free fractions were combined together, dried down and reconstituted in RNase-free water.
  • Step 5 Capillary Gel Electrophoresis (CGE) and Electrospray LC/Ms
  • oligonucleotide was diluted in water to 150 ⁇ L. Mass of the product and purity (as shown below) were determined by LC/MS analysis and anion exchange HPLC or CGE. AL-SQ Cal. Obs. Purity Deprotect.
  • Lower case “s” indicates a phosphorothioate linkage.
  • the lower case “d” indicates a deoxy residue.
  • Subscript “OMe” indicates a 2′-O-methyl sugar.
  • “Chol-” indicates a hydroxyprolinol cholesterol conjugate.
  • Step 1) cleavage of oligonucleotide from support with simultaneous removal of base protecting groups from the oligonucleotide and Step 2) deprotection of 2′-O-TBDMS groups
  • the following procedure was used. Since the pyridine present in the crude oligonucleotide solution absorbs at 254 nm, the absorbance was measured at 280 nm. A small amount of the crude support was subjected to deprotection using TEA.3HF instead of HF in pyridine. Absorbance was measured for this sample at 254 nm and 280 nm. Based on the ratio of A 254 to A 280 of this sample, the absorbance at 254 nm for the sample containing pyridine was estimated.
  • the amount of full-length product was determined by anion exchange HPLC.
  • the full-length product was 73% of the total strand concentration and for AL-SQ-5549 full-length product was 67%.
  • the crude yield was 143 OD/ ⁇ mole.
  • RNA phosphoramidites and 2′-O-methyl modified RNA phosphoramidites with standard protecting groups (5′-O-dimethoxytrityl-N-6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N-4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N-2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3
  • the 2′-F phosphoramidites (5′-O-dimethoxytrityl-N-4-acetyl-2′-fluro-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramidite) were obtained from Promega.
  • RNA amidite coupling/recycling time was 23 minutes and 2 equivalents of amidite were used.
  • DNA coupling cycle used 60% activator, 7 min recycling, and 2.0 equivalents of phosphoramidite.
  • a UV watch was introduced in the “push” step before the “recycle” step to assure consistency in each coupling step.
  • the activator was 0.6 M ethylthiotetrazole.
  • 50 mM iodine in water/pyridine (10:90 v/v) was used; 4.5 equivalents were added in 2.5 min.
  • the CPG was mixed with 180 mL of aqueous methylamine (Aldrich) in a 250 mL Schott bottle. The mixture was placed in a shaker oven at 45° C. for 75 min. The mixture was cooled, filtered into a 1 L Schott bottle and the CPG was washed three times with 160 mL of DMSO. The filtrates were combined and cooled for 10 min in dry ice. TEA.3HF (Alfa Aesar, 270 mL) was added to the mixture. The bottle was placed in a shaker oven at 40° C. for 65 min. The mixture was cooled to room temperature and the reaction was quenched with 1 L of 50 mM sodium acetate.
  • aqueous methylamine Aldrich
  • the oligonucleotides were purified by reverse phase HPLC using a matrix of TSK-GEL, SuperQ-5PW (20) in a 5 cm ⁇ 17-18 cm column. The temperature was maintained at 55° C. to 65° C.
  • the buffers were 20 mM sodium phosphate, 10% ACN v/v, pH 8.5 (buffer A) and 20 mM sodium phosphate, 1 M NaBr, 10% ACN, pH 8.5 (buffer B).
  • the flow rates was 60 mL/min.
  • the gradient was from 20% B to 40% B in 160 min.
  • the solution of crude oligonucleotide was diluted 5-fold with buffer A and loaded directly onto the purification column using a flow rate that loaded about 20 mg crude material (based on A 260 readings) per mL of column volume. Fractions of 50 mL were collected.

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CA2561741A1 (en) 2005-10-20
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US20110196145A1 (en) 2011-08-11
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