Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/22—Amides of acids of phosphorus
  • C07F9/24—Esteramides
  • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
  • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds

Definitions

• R 3 has the formula -LA
• A is Ci-C 3 operfluoroalkyl
• a or “an” refers to one or more; for example, a polymer refers to one or more polymers. As such, the terms “a” or “an” are used interchangeably herein.
• heterooligo refers to an oligomer that comprises two or more different monomer residues.
• oligomers and “polymers” refer generally to molecules that are made by linking together repeating units of one or more small molecules called monomers. Generally, oligomers include fewer monomer units than polymers, although the precise border between an oligomer and a polymer in not well defined and for the purposes of this invention the terms are used interchangeably to encompass the full scope of both terms.
• the oligomers may have differing numbers of repeat units.
• the oligomers may be attached to tags or labels.
• a “sequence” of a nucleic acid refers to the order and identity of nucleotides in the nucleic acid. A sequence is typically read in the 5' to 3' direction.
• oligonucleotide is typically single-stranded or double-stranded and will generally contain phosphodiester bonds, although in some cases, as outlined herein, nucleic acid analogs are included that may have alternate backbones, including, for example and without limitation, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; SRocl et al. (1977) Eur. J. Biochem. 81 :579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al. (1988) J Am. Chem. Soc. 110:4470; and
• nucleoside refers to a nucleic acid component that comprises a base or basic group (e.g., comprising at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, and/or the like) covalently linked to a sugar moiety (e.g., a ribose sugar, etc.), a derivative of a sugar moiety, or a functional equivalent of a sugar moiety (e.g., an analog, such as carbocyclic ring).
• a base is typically linked to a l'-position of that sugar moiety.
• the oligomers are attached to a solid support for steps (a) and (b). In other embodiments, the oligomers are cleaved from the solid support before step (c) wherein the non-capped oligomers of the desired target sequence are separated from capped truncated oligomers of by fluorous affinity methodology.
• solid supports suitable for the present invention include, but are not limited to, glass, typically a derivatized controlled pore glass (CPG); silica, alumina, zeolite, synthetic polymers or copolymers such as polystyrene; combinations thereof and the like.
• Figure 1 depicts the synthesis cycle of an oligonucleotide that produces a mixture of an oligonucleotide with a correct sequence and capped oligonucleotides with incorrect or failure sequences in various ratios according to certain embodiments of the invention.
• Nucleotides can be extended via the hydroxyl group at either the 3'- or 5'-position of an intact sugar ring (e.g., pentose sugar ring) or a sugar analog ring (e.g., carbocyclic ring, etc.).
• Figure 1 shows the synthesis is carried out in the 3' to 5' direction by adding nucleotides to the 5' end of the growing chain.
• Figure 1 only shows the extension and capping of a pair of monomers
• the present invention is not limited by the number or size of the nucleic acids being synthesized or purified. Synthesis in this direction is carried out using nucleotide phosphoramidites in which the phosphoramidite group is attached to the 3 '-oxygen and a protecting or blocking group (e.g., a negatively charged blocking group, a bulky blocking group, and/or the like).
• a protecting or blocking group e.g., a negatively charged blocking group, a bulky blocking group, and/or the like.
• oligonucleotide synthesis can be carried out in the 5' to 3' direction by adding nucleotides to the 3' end of the growing chain. Synthesis in this direction is carried out using nucleotide phosphoramidites in which the phosphoramidite group is attached to the 5 '-oxygen and a protecting group, again typically a dimethoxytrityl group, is attached to the 3 '-oxygen.
• a protecting group again typically a dimethoxytrityl group
• the 5'-hydroxyl protecting group is removed to form a compound with a free 5'-hydroxyl 2a and 2b.
• the conditions to remove a particular protecting group depend on the protecting group used. In the case of DMT, this can be done by adding an acid, such as dichloroacetic acid (DCA) or trichloracetic acid (TCA) in dichloromethane.
• DCA dichloroacetic acid
• TCA trichloracetic acid
• L is Cj-Cjoalkyleneoxy-, which is optionally substituted with from 1-3 substituents selected from the group consisting of Ci-C ⁇ alkyl-, Ci-C ⁇ haloalkyl-, Ci- C 6 alkoxy-, arylC ! -C 6 alkoxy-, oxo- and Ci-C ⁇ alkoxycarbonyl-; and
• A is Ci-C 30 perfluoroalkyl. .
• the undesired materials having a fluorous-capped oligonucleotide 4a interact with the adsorbent, so that washing the adsorbent with at least a first suitable solvent allows the desired non-capped oligonucleotide 5b to elute, leaving only the complex 5a. Dissociation of the undesired fluorous-capped oligonucleotides 5b from the adsorbent may then be accomplished by washing with a second, more fluorophilic solvent. In cases where the fluorous-capped failure sequence oligonucletides are retained, the non-capped oligonucleotide 5b is the final purified target compound.
• Nu is a nucleoside
• R 3 has the formula -LA
• kits for example for the synthesis and purification of oligonucleotides
• the kits include as a component at least one capping reagent as described herein.
• the kit further includes one or more of: (a) at least one extendible monomer, e.g. a nucleotides or a modified nucleotide or a phosphoramidite; (b) at least one solid support; (c) at least one catalyst for use in extending the oligonucleotides; (d) at least one buffer; (e) at least one set of instructions for extending the oligonucleotides, e.g. nucleic acid, using with the components of the kit; and (f) at least one a container for packing the components of the kit.
• Figure 7 shows an HPLC analysis of the impurities released from the column after filtration through a fluorous cartridge by washing the column with 40% acetonitrile in 0.1 M TEAA.
• the eluant shows the removal of the failure sequences.
• Figure 8 shows the HPLC analysis of the filtrate containing the fluorous-purified 15-mer oligonucleotide after a NAP-10 desalting step. These examples show that the present method allows for an easy separation of the full-length oligonucleotide away from failure sequences. Because of the efficiency of the purification the full length oligonucleotide may be obtained in high yield and purity.

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• 2007
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