Classifications

• • 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

Definitions

• the present invention is directed to oligonucleo ides containing a 3 1 - and/or 5'-capped terminal and which are thereby rendered resistant to degradation by exonucleases.
• the exonuclease-resistant oligonucleotides have two or more phosphoramidate internucleotide linkages at one or both termini which render the oligonucleotides resistant to degradation.
• DNA molecules contain internucleotide phosphodiester linkages which are degraded by exonucleases present in cells, culture media and human serum. For example, degradation by exonucleases in tissue culture media of DNA may be observed within about 30 minutes to about six hours.
• Synthetic oligodeoxy- nucleotides with phosphodiester linkages are routinely used in genetic engineering, for example, to locate specific RNA or DNA fragments from a library.
• the long- term stability of an oligonucleotide for this utility is not a major concern, since the oligonucleotide is usually not exposed to the relatively stringent environment of the culture medium, therefore exonuclease degradation is not a substantial problem.
• a oligodeoxynucleotide with phosphodiester linkages can be used to block protein synthesis by hydrogen bonding to complementary messenger RNA thereby providing a tool for use in an antisense fashion.
• Exonuclease-stable oligodeoxynucleotides could also be utilized to form triple-helix DNA which would interfere with the transcription process or with DNA replication, by competing with naturally occurring binding factors or by gene destruction.
• synthetic oligonucleotides in this manner, they must be stable to exonucleases, the major activity of which in cells and serum appears to be 3 1 to 5 , i.e., digestion of oligonucleotides begins starting at the 3' end.
• the present invention is accordingly directed to such exonuclease-stable oligonucleotides.
• It a further object of the invention to provide methods of making such exonuclease-resistant oligo ⁇ nucleotides. It is still a further object of the invention to provide a method for end-capping oligonucleotides with moieties which can perform multiple functions, such as aiding in transport, serving as chromophoric tags, or enabling cross-linking.
• the present invention provides oligonucleotides having two or more phos ⁇ phoramidate linkages at the 3 1 terminus and/or 5' terminus, which oligonucleotides are resistant to exonuclease degradation.
• the number of phosphoramidate linkages is at least 1 and less than a number which would interfere with hybridization to a complementary oligo ⁇ nucleotide strand, and/or less than a number which would interfere with RNAse activity when said oligonucleotide is hybridized to RNA.
• at least 2, and more preferably on the order of about 2 to 10 phosphorami ⁇ date linkages are incorporated at either or both the 3 ' terminus and the 5' terminus.
• the phosphoramidate linkages may be substituted with any one of a number of different types of moieties as will be described in detail hereinbelow.
• exonuclease-resistant which have the following formulas I, II or III, i.e., containing phosphoramidate linkages as just described as well as phosphoromonothioate and/or phosphorodithioate linkages:
• each n, m, i, j and s is independently an integer and each s is in the range of about 2 to 10; each n and m is independently from 1 to about 50; s + n in formulas I and II is less than 100; and s + s + in formula III is less than about 100; each i varies from 1 to n; each j varies from 1 to m; T is hydrogen or a hydroxyl- protecting group; R and R are moities independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and oxyhydrocarbyl of 20 carbon atoms or less and 1-3 oxy groups, wherein said hydrocarbyl and oxyhydrocarbyl substituents are linear or branched alkyl of 1 to 20 carbon atoms, linear or branched alkenyl of 2 to 20 carbon atoms, cycloalkyl or cycloalkenyl of 3 to 20 carbon atoms, linear or branched alk
• the present invention also provides methods for preparing such end-capped oligonucleotides.
• polynucleotide and oligonucleotide shall be generic to polydeoxyribo- nucleotides (containing 2'-deoxy-D-ribose or modified forms thereof) , to polyribonucleotides (containing D- ribose or modified forms thereof) , and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine bases, or modified purine or pyrimidine bases.
• nucleoside will similarly be generic to ribonucleosides, deoxyribonucleosides, or to any other nucleoside which is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
• polynucleotide and oligonucleotide
• nucleoside and “nucleotides” will include those moieties which contain not only the known purine and pyrimidine bases, i.e., adenine, thy ine, cytosine, 5 P
• guanine and uracil but also other heterocyclic bases which contain protecting groups or have been otherwise modified or derivatized.
• modified nucleosides or “modified nucleotides” as used herein are intended to include those compounds containing one or more protecting groups such as acyl, isobutyryl, benzoyl, or the like, as well as any of the wide range of modified and derivatized bases as known in the art.
• protecting groups such as acyl, isobutyryl, benzoyl, or the like
• examples of such modified or deriva- tized bases include 5-fluorouracil, 5-bromouracil,
• Modified nucleosides or nucleotides can also include modifications on the sugar moiety, for example, wherein one or more of the hydroxyl groups are replaced with halogen or aliphatic groups, or functionalized as ethers, amines, etc.
• the polynucleotides according to the present invention may be of any length, but lengths of about three to about fifty nucleotides are particularly useful for most genetic engineering applications.
• the 3' end and/or the 5' end of the polynucleotide will contain at least two phosphoramidate internucleotide linkages.
• the remaining internucleotide linkages may be phosphodiester linkages, phosphorothioate linkages or phosphorodithioate linkages, or any other internucleotide linkage, other than a phosphoramidate, or combinations of these other linkages.
• Internucleotide phosphodiester linkages are prepared from hydrogen phosphonate linkages preferably by oxidation with, e.g., aqueous iodine.
• a typical procedure involves treatment of the hydrogen phosphonate in 0.1 M iodine in Pyr/NMI/H 2 0/THF (5:1:5:90) for about 2-3 minutes, followed by treatment with 0.1 M iodine in Et 3 /H 2 0/THF (5:5:90) for another approximately 2-3 minutes.
• Phosphoromonothioate linkages are formed from the initially present hydrogen phosphonate linkages by treatment with sulfur.
• the reaction is carried out at approximately room temperature for on the order of 20 minutes in a solvent system which typically includes a sulfur solvent such as CS 2 along with a basic solvent such as pyridine.
• a solvent system typically includes a sulfur solvent such as CS 2 along with a basic solvent such as pyridine.
• CS 2 is preferred as the sulfur solvent because it acts to dissolve elemental sulfur.
• the oligonucleotides of the invention are resistant to degradation under both physio ⁇ logical and tissue culture conditions, and in particular are resistant to degradation by exonucleases.
• the oligonucleotide In order that the oligonucleotide be resistant to such enzymatic degradation, it is modified so that phosphodiester linkages initially present at the 3' terminus are replaced with a selected number of phos ⁇ phoramidate linkages, that number being at least one and less than a number which would cause interference with hybridization to a complementary oligonucleotide strand, and/or less than a number which would interfere with RNAseH activity when said the oligonucleotide is hybridized to RNA. Such a modification may additionally or alternatively be made at the 5' terminus.
• the number of phosphorami ⁇ date linkages be selected such that the melting temperature of any duplex formed with complement is lowered by less than about 10°C relative to that obtained with an oligonucleotide containing only the initial phosphodiester linkages.
• the number of phosphoramidate linkages is such that the melting temperature of a duplex formed is lowered by less than about 5°C.
• the number of phosphoramidate linkages present is typically and preferably between about 2 and 10, more preferably between about 2 and 8, and most preferably between about 2 and 6.
• the phosphoramidate linkage has the formula
• R 1 and R2 moieti.es are substituents which must be selected so as not to interfere with hybridization with complement.
• the groups 1 and R2 are independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and oxyhydrocarbyl substituents of 20 carbon atoms or less containing 1-3 oxy groups, with the proviso that R and R are not both hydrogen, i.e., the phosphoramidate linkages herein are always N-substituted. In this case, it is preferred that one of the two substituents be hydrogen.
• Suitable hydrocarbyl and oxyhydrocarbyl substituents include, for example, linear or branched alkyl of 1-20 carbon atoms, linear or branched alkenyl of 2-20 carbon atoms, cycloalkyl or cycloalkenyl of 3-20 carbon atoms, linear or branched alkoxy of 1-20 carbon atoms, or aryl of 6-18 carbon atoms.
• the hydrocarbyl substituent may be, for example, an alkoxy substituent having the formula CH 3 0-(CH 2 ) ⁇ - or a straight chain alkyl group having the formula CH 3 (CH 2 ) - where x is an integer in the range of 1-20, inclusive, preferably in the range of 1-10, inclusive, and y is an integer in the range of 0-15, inclusive.
• Examples of preferred oligonucleotide linkages within the aforementioned groups are wherein one of R 1 and R 2 is H and the other is either 2-methoxyethyl, dodecyl, or n- propyl.
• the 2-methoxyethyl and dodecyl linkages are sometimes referred to herein as "MEA” and "C12", respectively.
• the R 1 and R2 groups may also be, in addition to the foregoing, macromolecular species such as sugars, polypeptides, chromophoric groups, lipophilic groups, polymers, steroid hormones, or the like.
• Lipophilic groups refer to moieties which are chemically compatible with the outer cell surface, i.e., so as to enable the oligonucleotide to attach to, merge with and cross the cell membrane. Examples of such lipophilic groups are fatty acids and fatty alcohols (in addition to the long chain hydrocarbyl groups described above) .
• Examples of preferred polypeptides that can be used for 1 and/or 2 include transferrin and epidermal
• growth factor growth factor
• suitable non-polypeptide polymers include ionic, nonionic and zwitterionic polymers.
• examples of a particularly preferred polymer is polyethylene glycol.
• Steroid substituents include any of the general fat ⁇ ily of lipid compounds which comprise sterols, bioacids, cardiac glycosides, seponans, and sex hormones, which include the following basic structure:
• steroids examples include natural corticosteroid hormones (produced by the adrenal glands) , sex hormones (progesterone, androgens, and estrogens) .
• R1 or 2 is a polymer such as polyethylene glycol, a polypeptide or a lipophilic group such as a long-chain hydrocarbyl moiety, such a group may facilitate transport or permeation of the oligonucleotide through cell membranes, thus increasing the cellular uptake of the oligonucleotide.
• the R 1 or R group may also be a group which affects target DNA or RNA to which the oligonucleotide will bind, such as providing covalent linkages to the target strand to facilitate cleavage or intercalation of the oligonucleotide to the target strand.
• the R 1 and R2 groups may addi.ti.onally serve a cutting function (e.g., a site for cutting the complementary strand), or a receptor function (e.g., a receptor ligand) .
• oligonucleotides of the present invention can include other phosphoramidate N-substituents not explicitly disclosed herein so long as those substituents confer exonuclease resistance and do not interfere with hybridization to a complementary oligonucleotide strand.
• the invention also encompasses oligonucleotide compositions containing oligonucleotides of the following formula I, II or III, i.e., wherein phosphoromonothioate and/or phosphorodithioate linkages are incorporated in addition to the phosphoramidate linkages:
• the 3 '-capped oligonucleotides may be prepared by first preparing a polymer-bound polynucleoside with the formula IV
• P is a solid state polymeric support, or other type of solid support
• B the base portion of a nucleoside, i.e., a purine or pyrimidine base, or any modified purine or pyrimidine base.
• the functional groups on the base i.e., the amine groups, will be appropriately protected during the course of the synthesis and removed after the completed polynucleotide is removed from the polymer support.
• the linkage to the polymer support is through the 3' hydroxy group, the free hydroxy group is the 5' group of the nucleoside.
• the group T is a conventional hydroxy-protecting group used in oligonucleotide synthesis, preferably the DMT group (dimethoxytrityl) or MMT group (monomethoxytrityl) .
• the polymer-bound polynucleoside hydrogen phosphonate (IV) is preferably prepared by treating the DBU (1.8-diazabicyclo[5.4.0]undec-7-ene ammonium salt) of a 5*-protected (preferably, 5 DMT) nucleoside hydrogen phosphonate with a polymer-bound nucleoside, linked to support through its 3'-hydroxyl group in the presence of an activating agent, as is known in the art.
• nucleoside hydrogen phosphonates may be added (to make the two or more internucleotide linkages at the 3' end of the polynucleotide) by sequentially deprotecting the
• oligonucleotide chain elongation will proceed in conformance with a predetermined sequence in a series of condensations, each one of which results in the addition of another nucleoside to the oligomer.
• the condensation is typically accomplished with dehydrating agents, which are suitably phosphorylating agents or acylating * agents such as isobutylchloroformate, diphenylchlorophosphate, organic acid anhydrides (such as acetic anhydride, isobutyric anhydride or trimethyl acetic anhydride) and organic acid halides such as pivaloyl chloride, pivaloyl bromide, 1-adamantyl- carboxylic chloride or benzoyl chloride.
• the preferred condensing agent is pivaloyl chloride in pyridine acetonitrile. Prior to the addition of each successive nucleoside hydrogen phosphonate, the 5*-protecting group or the carrier bound nucleotide is removed.
• the carrier is preferably washed with anhydrous pyridine/acetonitrile (l/l,v/v) and the condensation reaction is completed in as many cycles as are required to form the desired number of 3'-end internucleotide bonds which will be converted to phosphoramidates.
• the carrier- bound polynucleotide hydrogen phosphonate is oxidized to convert the hydrogen phosphonate internucleotide linkages to phosphoramidate linkages, preferably by treatment with the desired amine NHR X R 2 with R 1 and R 2 as defined earlier and CC1 4 as described in Froehler, et al.. Nucleic Acids Research 16:4831-4839 (1988).
• the oligonucleotide is then completed by methods which form nonphosphoramidate linkages, such as phosphodiester linkages, phosphorothioate linkages or phosphorodithioate linkages, by methods known in the art referenced above and incorporated by reference herein.
• the preferred method for completing the oligonucleotide is to continue the sequence using 5'-protected nucleoside hydrogen- phosphonates.
• all of the hydrogen phosphonate linkages are oxidized to produce diester linkages, preferably by aqueous iodine oxidation or oxidation using other oxidizing agents, such as N-chlorosuccinimide, N-bromosuccinimide or salts or periodic acid. This will result in all of the internucleotide linkages, except for the 3'-end capped linkages which are phosphoramidate linkages, being phosphodiester linkages.
• the oligonucleotide may be separated from the carrier, using conventional methods, which in the preferred instance is incubation with concentrated ammonium hydroxide. Any protecting groups may be removed as described above using about 2% dichloroacetic acid/CH 2 Cl 2 , or about 80% acetic acid, or by other conventional methods, depending on the nature of the protecting groups.
• the desired oligonucleotide is then purified by HPLC, polyacrylamide gel electro- phoresis or using other conventional techniques.
• ⁇ protecting group or solid state carrier i: varies from 1 to 5
• Q hydrogen or -NR X R 2 (with the proviso that at least one Q j is hydrogen)
• B a purine or pyrimidine base Scheme 2a
• the two or more phosphoramidate linkages need not each contain the same 1 and R2 groups. This may be accomplished by generating the first internucleotide hydrogen phosphonate linkage, and then oxidizing it with a first amine, generating the second hydrogen phosphonate internucleotide linkage, and then oxidizing it in the presence of a second (different) amine. This would result in a capped oligonucleotide having mixed phosphoramidate internucleotide linkages.
• a 5'-capped oligonucleotide may be made.
• the above method may be modified by first forming a polymer-bound oligonucleotide having only hydrogen phosphonate internucleotide linkages which may then be oxidized to form phosphodiesters (or phosphorothioate or phosphorodithioate linkages) . Then for the last two (or more) cycles, the 5'-end cap is formed when the last two or more nucleosides are added, followed by reaction with the amine NHR X R 2 .
• the 5' end may be added by adding a polynucleotide, such as a tri- or tetranucleotide containing the desired phosphoramidate internucleotide linkages.
• a combination of both of the above methods for making a 5' and a 3' end- capped oligonucleotide may be utilized.
• the first two (or more) internucleotide linkages on the 3 '-bound oligonucleotide may be oxidized to form the phosphoramidate linkages, then the non-terminal portion of the oligonucleotide may be made (having phosphodiesters, phosphorothioate or phosphorodithioate internucleotide linkages) , with the final two (or more) linkages being phosphoramidates, formed as described above.
• 5•- or 3'-phosphoramidate-capped oligonucleotides as made in accordance with the present invention may be as therapeutic agents against viral diseases (such as HIV, hepatitis B, cytomegalovirus) , cancers (such as leukemias, lung cancer, breast cancer, colon cancer) or metabolic disorders, immune modulation agents, or the like, since the present end-capped oligonucleotides are stable within the environment of a cell as well as in extracellular fluids such as serum, and can be used to selectively block protein synthesis, transcription, replication of RNA and/or DNA which is uniquely associated with the disease or disorder.
• viral diseases such as HIV, hepatitis B, cytomegalovirus
• cancers such as leukemias, lung cancer, breast cancer, colon cancer
• immune modulation agents or the like
• the end-capped oligonucleotides of the invention may also be used as therapeutics in animal health care, plant gene regulation (such as plant growth promoters) or in human diagnostics, such as to stabilize DNA probes to detect microorganisms, oncogenes, genetic defects, and the like, and as research reagents to study gene functions in animal cells, plant cells, microorganisms,- and viruses.
• plant gene regulation such as plant growth promoters
• human diagnostics such as to stabilize DNA probes to detect microorganisms, oncogenes, genetic defects, and the like
• research reagents to study gene functions in animal cells, plant cells, microorganisms,- and viruses.
• dermatologic applications for treatment of diseases or for cosmetic purposes.
• There are many other potential uses which derive from the stability of the oligonucleotide to exonuclease degradation, thus prolonging oligonucleotide integrity within the relatively stringent environment of the cells.
• Example 1 Polymer-bound polynucleoside H-phosphonates were prepared as described by Froehler et al., supra, on control pore glass using the DBU salt of the protected nucleoside H-phosphonate.
• the diester linkages were generated by aqueous I 2 oxidation and the amidate linkages by amine/CC1 4 oxidation.
• the polynucleoside H-phosphonate was oxidized with a solution of 2-methoxyethylamine in Pyr/CCl. (1:5:5) (20 min.) followed by twelve more couplings and oxidation with aq.
• Example 2 Polymer-bound polynucleoside H-phosphonates were prepared as in the preceding example on control pore glass using the DBU salt of the protected nucleoside H-phosphonate. After twelve couplings the polynucleoside H-phosphonate was oxidized with aq. I 2 (0.1 M in N-methyl morpholine/water/THF, 5:5:90) followed by two more couplings and oxidation with a solution of 2-methoxy- ethylamine in Pyr/CCl 4 (1:5:5) (20 min.) to generate a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end.
• aq. I 2 0.1 M in N-methyl morpholine/water/THF, 5:5:90
• 2-methoxy- ethylamine in Pyr/CCl 4 (1:5:5) 20 min.
• the oligomer was removed from the solid support, deprotected with cone. NH 4 OH (45°C/18 hr.) and purified by HPLC (PRP) using an acetonitrile (CH 3 CN) gradient in 50 M aqueous TEAP. The DMT was removed from the product fraction (80% acetic acid/R.T./2 hrs.), evaporated, desalted and evaporated.
• Example 3 Polymer-bound polynucleoside H-phosphonates were prepared as described as in the preceding examples on control pore glass using the DBU salt of the protected nucleoside H-phosphonate.
• the diester linkages were generated by aqueous I 2 oxidation and the amidate linkages by amine/CCl 4 .
• the polynucleoside H-phosphonate was oxidized with a solution of 2-methoxyethylamine in Pyr/CCl 4 (1:5:5) (20 min.) followed by ten more couplings and oxidation with aq.
• Example 4 The procedure of Example 1 was repeated using dodecylamine to generate a 15-mer containing two phos ⁇ phoramidate linkages at the 3* end and twelve diester linkages, wherein the phosphoramidate linkages are such 5
• R 1 and R 2 as defined earlier herein is hydrogen and the other is dodecyl.
• Example 5 The procedure of Example 2 was repeated using dodecylamine in place of 2-methoxyethylamine, so as to yield a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end, wherein the phosphoramidate linkages are substituted as
• Example 6 The procedure of Example 3 was repeated using dodecylamine in place of 2-methoxyethylamine, to give . rise to a 15-mer containing two phosphoramidate linkages at the 3' end, ten diester linkages, and two phosphor ⁇ amidate linkages at the 5' end, wherein the phosphor ⁇ amidate is N-substituted as in the preceding two examples.
• Example 7 The procedure of Example 1 was repeated using propylamine to generate a 15-mer containing two phos- phoramidate linkages at the 3• end and twelve diester linkages, wherein the phosphoramidate linkages are such that one of R 1 and R2 as defi.ned earli.er herei.n is hydrogen and the other is ji-propyl.
• Example 2 The procedure of Example 2 was repeated using propylamine in place of 2-methoxyethylamine, so as to yield a 15-mer containing twelve diester linkages at the 3' end and two phosphoramidate linkages at the 5' end, wherein the phosphoramidate linkages are substituted as in the preceding example, i.e., one of R 1 and R 2 is hydrogen and the other is n-propyl.
• Example 9 The procedure of Example 3 was repeated using propylamine in place of 2-methoxyethylamine, to give rise to a 15-mer containing two phosphoramidate linkages at the 3' end, ten diester linkages, and two phosphor ⁇ amidate linkages at the 5' end, wherein the phosphor- amidate is N-substituted as in the preceding two examples.
• Example 10 The following Example describes hybridization stability studies performed using end-capped oligo ⁇ nucleotides as described and claimed herein.
• Oligonucleotides containing end-caps were tested for their ability to form stable duplexes with complementary single-stranded DNA sequences; the various oligonucleotides tested were outlined below in Table 1. Duplex stability was measured by determining the melting temperature T m in solution over a range of temperatures. The experiment was conducted in a solution containing 150 mM NaCl, 5 mM Na 2 HP0 4 and 3 ⁇ M DNA at a pH of 7.1. The results obtained and set forth in Table 1 show that binding to complementary sequences is not materially affected by 3'-end-cap modification.
• Example 11 Several additional oligonucleotides also end-capped at the 3' terminal two internucleotide linkages were tested for their ability to form stable duplexes with complementary single stranded DNA sequences, as described in the preceding example. Results are set forth in Table 2. Table 2
• Example 12 The following example was used to determine the efficacy of end-capped oligodeoxynucleotides virus inhibition and cellular toxieity using oligonucleotides capped at two terminal 3'-end internucleotide linkages with 2-methoxyethylamine and dodecylamine.
• the acute infection assay used the MOLT-4 cell line which is susceptible to HIV infection.
• Measurement of HIV p24 was used to assay for inhibition of virus replication 7 days after infection with virus at a multiplicity of infection of approximately 0.1. Approximately 1 x 106 cells were preincubated with oligonucleotide, washed, infected with virus stock and then incubated for 7 days in oligonucleotide.
• HIV p24 levels in the supernatant were measured by radioimmunoassay and compared with control infections lacking oligonucleotide. Results are expressed as the percent of control p24 found in cultures containing oligonucleotide. Sequences of antisense oligonucleotides were complementary to HIV targets listed in Table 3. Toxieity data was obtained by incubation of 3'-end-capped oligonucleotides with uninfected cells, followed by a comparison with cell numbers with control cultures incubated in the absence of oligonucleotide. Toxieity results are expressed as the percent reduction of cell numbers obtained by incubation in oligonucleotide for 7 days compared to controls.

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