WO1996039414A1 - Groupes nouveaux de protection des bases pendant la synthese d'oligonucleotides - Google Patents

Groupes nouveaux de protection des bases pendant la synthese d'oligonucleotides Download PDF

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WO1996039414A1
WO1996039414A1 PCT/US1996/008136 US9608136W WO9639414A1 WO 1996039414 A1 WO1996039414 A1 WO 1996039414A1 US 9608136 W US9608136 W US 9608136W WO 9639414 A1 WO9639414 A1 WO 9639414A1
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nucleoside
oligonucleotide
group
hydrogen
nitrogen
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PCT/US1996/008136
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English (en)
Inventor
Radhakrishnan P. Iyer
Theresa Devlin
Ivan Habus
Dong Yu
Sudhir Agrawal
Nan-Hui Ho
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Hybridon, Inc.
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Priority claimed from US08/518,921 external-priority patent/US5614622A/en
Priority claimed from US08/519,318 external-priority patent/US6140482A/en
Priority claimed from US08/570,390 external-priority patent/US5955599A/en
Priority claimed from US08/598,320 external-priority patent/US6531589B1/en
Priority claimed from US08/606,915 external-priority patent/US5962674A/en
Application filed by Hybridon, Inc. filed Critical Hybridon, Inc.
Priority to AU59581/96A priority Critical patent/AU5958196A/en
Publication of WO1996039414A1 publication Critical patent/WO1996039414A1/fr
Priority to US08/846,303 priority patent/US6509459B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric 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
    • 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

  • the invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis.
  • Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides. The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e . g. , Methods in Molecular Biology, Vol 20 : Protocols for Oligonucleotides and Analogs pp. 165-189 (S.
  • this approach comprises anchoring the 3 '-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the 5 additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3 ' functional group of the incoming nucleoside and the 5' hydroxyl group of the 5 '-most nucleoside of the nascent, support-bound oligonucleotide. 0 Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (lOumol to 1 mmol and higher) . See Padmapriya et al., Antisense Res . Dev. : 185 (1994) . Several modifications of the standard phosphoramidite processes have 5 already been reported to facilitate the synthesis
  • oligonucleotides The routine synthesis of oligonucleotides is presently carried out using various ⁇ -acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine) , and benzoyl for adenine and cytosine.
  • the protecting groups are removed by treatment with ammonia at 55-60"C for 5-10 hours. Using these protecting groups, PO oligonucleotides and other modified oligonucleotides can be synthesized.
  • modified oligonucleotides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out.
  • base- sensitive modified oligonucleotides include, ribonucleoside- containing oligonucleotides, methylphosphotriester oligonucleotides, phosphoramidates, etc.
  • R ⁇ A is the large-scale synthesis of R ⁇ A which is required for the ribozyme-based therapeutic strategies .
  • Such synthesis presents special challenges due to two factors. These are, first, 3'-5' to 2'-5' internucleotide chain migration during preparation of nucleoside monomer precursors, during synthesis, and during removal of protecting groups from the R ⁇ A, and second, degradation of R ⁇ A.
  • Use of classical protecting groups compounds these factors.
  • oligonucleotide phosphoramidates Another example is that current synthesis procedures allow the synthesis of some, but not all possible oligonucleotide phosphoramidates, because some of these compounds are labile under the highly alkaline conditions required for deprotection of the nucleoside base. Oligonucleotides containing primary phosphoramidate internucleoside linkages, for example, have not previously been possible to synthesize for this reason. In the case of the oligonucleotide phosphoramidates, this inability to synthesize oligonucleotides containing primary phosphoramidate internucleoside linkages has probably slowed their development as optimally useful compounds for molecular biology applications and the antisense therapeutic approach.
  • oligonucleotide phosphoramidates that have been developed all have relatively large chemical substituents in place of one of the nonbridging oxygen atoms on the phosphate backbone, which may lead to steric hindrance in the ability of the oligonucleotide to bind to its target. It would be valuable to internucleotidic primary phosphoramidate linkages, since incorporation of such non-ionic linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides. For example, Galbraith et al . , Antisense Research and Development 4 .
  • oligonucleotides may potentially interfere with blood clotting.
  • oligonucleotides may potentially interfere with blood clotting.
  • Yet another example is the synthesis of oligonucleotides containing methylphosphonate internucleoside linkages.
  • Various methodologies have been used to synthesize such oligonucleotides. Miller et al . , Biochemistry 25: 5092-5095 (1986), discloses an early methodology using a polymer support. Agrawal and Goodchild, Tetrahedron Lett.
  • bz nucleoside phosphonamidite monomers in conjunction with dA iBu and dG monomers, followed by deprotection using initial exposure of the oligomer to 10% ammonium hydroxide in acetonitrile/ethanol at room temperature, then prolonged exposure to ethylenediamine.
  • Also used has been pretreatment of the protected oligonucleotide with hydrazine hydrate in pyridine/acetic acid, followed by prolonged exposure to ethylene diamine/ethano1.
  • Still another example is synthesis of oligonucleotides containing methyphosphotriester internucleoside linkages.
  • Such oligonucleotides could have many beneficial properties, because the methyl phosphotriester group is nonionic, but is similar in size and molecular shape to the phosphodiester linkage.
  • Such nonionic methyl phosphotriester linkages could result in a reduction in oligonucleotide side effects that are attributable to the polyanionic character of the oligonucleotides .
  • the difficulty in synthesizing oligonucleotides having methyl phosphotriester internucleotide linkages is due to the lability of the methyl ester bond under the oligonucleotide synthesis conditions used in the steps of deprotecting the nucleoside bases and cleaving the oligonucleotides from the solid support.
  • Alul efc al . , Nucl. Acids Res. JL9_: 1527-1532 (1991) addressed the problem of cleaving the oligonucleotide from the solid support by introducing an oxalyl-type linker that can be cleaved under conditions that preserve the methyl ester bond.
  • oligonucleotides it is desirable to have oligonucleotides still bound to the solid support.
  • Such completely deprotected oligonucleotides still bound to the solid support can be useful in a variety of applications such as those involving isolation of transcription factors and other factors or elements that interact with oligonucleotides. They are also useful for solid-phase PCR, investigation into nucleic acid protein interactions by, for example, NMR, creation and use of combinatorial libraries, screening of nucleic acid libraries, and solid support based hybridization probes (analogous to Southern and Northern blotting protocols) .
  • oligonucleotide Creating such a support bound, deprotected oligonucleotide would be greatly aided by having a protecting group that could be removed by mild conditions that would not cleave the oligonucleotide from the support.
  • These numerous examples clearly demonstrate a need for processes for oligonucleotide synthesis that allow for deprotection of the oligonucleotide under more mild conditions than existing processes.
  • nucleoside synthons having new base protecting groups that are stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups.
  • oligonucleotides which contain any of a variety of base labile functionalities.
  • the invention provides new processes for synthesizing oligonucleotides that allow for deprotection of the oligonucleotide under more mild conditions than existing processes .
  • the invention further provides a nucleoside base protecting group that is stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups, as well as nucleoside synthons having such base protecting groups .
  • the invention also provides oligonucleotides containing any of a variety of base labile functionalities and methods for using such oligonucleotides.
  • the invention provides a novel nucleoside base protecting group having the general structure I:
  • n , n and n are independently 0-10, wherein a, b, c, d and e are each independently hydrogen, carbon or nitrogen, and wherein the ring structure bearing substituent R shown may be aromatic or heterocyclic, wherein the nitrogen displayed is the protected amino moiety of the nucleoside base, wherein R R and R are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, ester, carboxyl, or aldehyde group, and wherein dotted lines represent alternative exocyclic or endocyclic double bonds.
  • a is hydrogen when n is 0 and is carbon or nitrogen when n is 1-10
  • b is hydrogen when n and n are both 0 and is carbon or nitrogen when either or both n and n are 1-10
  • c is
  • Compounds I and II protect the nucleoside base amino moieties by forming amide linkages, as in:
  • Base protecting group I and the preferred embodiment II are particularly advantageously used because such protecting group can be removed chemoselectively by treatment with a chemoselective removing agent.
  • the invention provides a process for synthesizing oligonucleotides that allows for removal of base protecting groups under more mild conditions than existing processes. This new process comprises sequentially coupling nucleoside synthons having base protecting groups according to the invention to produce a base-protected oligonucleotide, followed by deprotection using a chemoselective removing agent.
  • the process according to the invention can utilize any known or otherwise suitable oligonucleotide synthesis chemistry, including the well known H-phosphonate, phosphoramidite and phosphotriester chemistries.
  • the use of this new process provides numerous advantages. For example the process's mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as ribonucleoside-containing oligonucleotides, alkylphosphotriesters, certain base- sensitive phosphoramidate and other base-sensitive oligonucleotides .
  • the process according to this aspect of the invention can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups.
  • this new process allows for synthesis of oligonucleotides still bound to any type of solid support.
  • the processes according to this aspect of the invention are compatible with and can be used in conjunction with any of the well known oligonucleotide synthetic chemistries, including the H-phosphonate, phosphoramidate and phosphotriester chemistries.
  • the processes according to this aspect of the invention can be used to synthesize oligonucleotides having ribonucleosides and/ or primary phosphoramidate, alkylphosphonate, or methylphosphonate linkages at some internucleoside positions and other linkages at other internucleoside positions.
  • the invention provides novel synthons for use in synthesis of oligonucleotides having base-sensitive functionalities .
  • One such novel synthon can be used to prepare portions of the oligonucleotide containing deoxyribonucleosides linked by any known internucleoside linkage.
  • This monomer synthon according to the invention has the general structure III:
  • D is a 5 ' -OH blocking group (see e.g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S.
  • the protecting group (G) is the previously described structure I, or its preferred embodiment II
  • Z is hydrogen, -OG, -NG2, halogen (preferably Cl, Br or F) , an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e. g.
  • L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al . , J. Org. Chem. .60:5388-5389 (1995)) .
  • This synthon can be used alone, with any of the known synthons, or in conjunction with any of the following synthons.
  • This ribonucleoside synthon has the general structure IV:
  • B is a nucleoside base
  • D is a 5 ' -OH blocking group
  • the protecting group (G) is the previously described structure I, or its preferred embodiment II
  • L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al., J. Org. Chem. 6_0:5388-5389 (1995)) .
  • This monomer synthon according to the invention is useful for synthesizing oligonucleotides containing primary phosphoramidate internucleoside linkages.
  • This monomer synthon according to the invention has the general structure V:
  • the protecting group (G) is the previously described structure I, or its preferred embodiment II
  • Z is hydrogen, -0G, -NG2, halogen (preferably Cl, Br or F) , an -0-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an -0-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and L is an H- phosphonate or H-phosphonothioate leaving group.
  • This monomer synthon according to the invention is useful for synthesizing oligonucleotides containing alkylphosphonate internucleoside linkages.
  • This monomer synthon according to the invention has the general structure VI:
  • R is an alkylphosphonamidite (preferably a methylphosphonamidite) group
  • Z is hydrogen, - OG, -NG2, halogen (preferably Cl, Br or F) , an -0-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e . g.
  • the invention provides novel oligonucleotides containing from one to about all of a variety of base-sensitive functionalities .
  • such oligonucleotides may contain from one to about all ribonucleotides and/ or may contain from one to about all internucleoside linkages selected from the group consisting of primary phosphoramidate and methylphosphotriester linkages.
  • the other internucleoside linkages may be any of the known internucleoside linkages, or may be any internucleoside linkage not yet known that can be incorporated into an oligonucleotide according to a synthetic chemistry with which the process according to the invention is compatible. Oligonucleotides containing such a mixture of internucleoside linkages are referred to herein as mixed backbone oligonucleotides.
  • the internucleoside linkages that are not primary phosphoramidate linkages are selected from the group consisting of phosphodiester and phosphorothioate internucleoside linkages .
  • the internucleoside linkages that are not primary phosphoramidate linkages are selected from the group consisting of phosphodiester and phosphorothioate internucleoside linkages .
  • several adjacent nucleosides comprising one region of the oligonucleotide are connected by primary phosphoramidate linkages, and several other adjacent nucleosides comprising another region of the oligonucleotide are connected by a different type of internucleoside linkage.
  • Oligonucleotides according to the invention are useful for a variety of purposes. For example, they can be labelled with a reporter group and used as probes in conventional nucleic acid hybridization assays. They can also be used as antisense "probes" of specific gene function by being used to block the expression of a specific gene in an experimental cell culture or animal system and to evaluate the effect of blocking such specific gene expression. In this use, oligonucleotides according to the invention are preferable to traditional "gene knockout" approaches because they are easier to use and can be used to block specific gene expression at selected stages of development or differentiation. Finally, oligonucleotides according to the invention are useful in the antisense therapeutic approach. In this use, oligonucleotides according to the invention should have reduced polyanion- mediated side effects and improved cellular uptake.
  • the invention provides methods for using oligonucleotides containing any of a variety of base- sensitive functionalities to control the expression of specific genes. Such methods comprise administering oligonucleotides according to the invention to cells or to animals, including humans. These methods may be used to assess gene function, or as a therapeutic approach to the treatment of diseases resulting from aberrant gene expression.
  • Figure 1 shows a scheme for synthesizing ribonucleoside monomer synthons.
  • Figure 2 shows a sceme for synthesizing nucleoside methylphosphonamidite monomer synthons.
  • Figure 3 shows typical NMR results for trinucleotides having one phosphodiester or phosphorothioate internucleoside linkage and one O-methyl phosphotriester or phosphorothioate internucleoside linkage.
  • the trimers are a
  • FIG. 3 31 Figure 4 shows P-NMR analysis proving that methylphospho-triester and phosphorothioate segments were present in the correct relative proportion in chimeric oligonucleotides synthesized as described in Example 14.
  • Figure 5 shows the expected slower mobility on polyacrylamide gel electrophoresis of a methylphosphotriester-phosphorothioate chimeric oligonucleotide, relative to a phosphodiester- phosphororthioate chimera of identical sequence.
  • the invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis.
  • the patents and publications identified in this specification are within the knowledge of those skilled in this field and are hereby incorporated by reference in their entirety.
  • the invention provides new processes for synthesizing oligonucleotides that allow for deprotection of the oligonucleotide under more mild conditions than existing processes .
  • the invention further provides a nucleoside base protecting group that is stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups, as well as nucleoside synthons having such base protecting groups.
  • the invention provides oligonucleotides containing any of various base-sensitive functionalities, and methods for using such oligonucleotides to modulate specific gene expression.
  • the invention provides a novel nucleoside base protecting group having the general structure I:
  • n , n and n are independently 0-10, wherein a, b, c, d and e are each independently hydrogen, carbon or nitrogen, and wherein the ring structure bearing substituent R may be aromatic or heterocyclic, wherein the nitrogen displayed is the protected amino moiety of the nucleoside base, wherein R R and R are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, ester, carboxyl, or aldehyde group, and wherein dotted lines represent alternative exocyclic or endocyclic double bonds (i.e., any one of the dotted double bonds is present) .
  • a is hydrogen when n is 0 and is carbon or nitrogen when n is 1-10
  • b is 1 1 hydrogen when n and n are both 0 and is carbon or nitrogen when either or both n and n are 1-10
  • c is hydrogen when n is 0 and is carbon or nitrogen when n is 1-10
  • e is 2 2 hydrogen when n is 0 and is carbon or nitrogen when n is 1-10.
  • Compounds I and II protect the nucleoside base amino moieties by forming amide linkages, as in:
  • Base protecting group I and the preferred embodiment II are particularly advantageously used because such protecting group can be removed chemoselectively by treatment with a chemoselective removing agent.
  • the invention provides processes for synthesizing oligonucleotides which allow for removal of base protecting groups under more mild conditions than existing processes.
  • nucleoside synthons having base protecting groups according to the invention are sequentially coupled according to standard procedures to yield a base-protected oligonucleotide.
  • the base protecting groups are then removed by a chemoselective removing agent.
  • a nucleoside synthon means a monomeric or multimeric nucleoside derivative appropriate for synthesis of an oligonucleotide.
  • Preferred nucleoside ' synthons include monomeric nucleoside phosphoramidites, phosphotriesters, or H-phosphonates having a blocked 5' -OH, preferably blocked with a trityl or dimethoxytrityl group.
  • a chemoselective removing agent means an agent that is capable of removing a base protecting group according to the invention.
  • the chemoselective removing agent is selected from the group consisting of halogens, especially Br , Cl and I , any of which are preferably taken up in water, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or as an N-halosuccinimide.
  • non-chemoselective reagents may be used, such as aqueous ammonium hydroxide, alcoholic ammonia, alkali carbonates in organic solvents, primary or secondary amines, alkali hydroxides, or any amidolytic reagent, i.e., an agent capable of hydrolyzing an amide linkage.
  • the processes according to this aspect of the invention can utilize any suitable oligonucleotide synthesis chemistry in solid or solution phase, including the well known H- phosphonate and phosphoramidite chemistries.
  • synthesis is carried out on a suitable solid support using either H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H-phosphonate chemistry for some cycles and phosphoramidite chemistry for other cycles) .
  • Suitable solid supports include any of the standard solid supports used for solid phase oligonucleotide synthesis, such as controlled-pore glass (CPG) or polymer supports.
  • CPG controlled-pore glass
  • Synthesis on such a solid support begins with coupling a nucleoside synthon according to the invention to a nucleoside that is covalently linked the solid support (i.e., linked to a functionality on the solid support, preferably an amino or hydroxyl functionality) . More generally, the processes according to this aspect of the invention can be used with any of the chemistries commonly used for oligonucleotide synthesis, whether in solution phase or in solid phase.
  • the invention provides a process for synthesizing an oligonucleotide, such process comprising coupling a suitable nucleoside synthon, such as a nucleoside H-phosphonate, a nucleoside phosphoramidite, or a nucleoside phosphotriester to a nucleoside and deprotecting a nucleoside base with a reagent comprising (1) a halogen in water, in an ethereal solvent such as ether or THF, or in pyridine/ROH, wherein R is an alkyl, aralkyl or aryl group having 1-10 carbon atoms, or (2) a suitable halide releasing reagent, such as N- halosuccinimide, sodium hypochlorite, or N-iodosuccinimide in para-toluenesulfonic acid.
  • a suitable nucleoside synthon such as a nucleoside H-phosphonate, a nucleoside phosphoramidite
  • the nucleoside to which the nucleoside synthon is coupled may be a monomer, a multimer, or it may be the terminal nucleoside of a growing oligonucleotide chain. In either case, the nucleoside or growing oligonucleotide chain may be support-bound or free in solution.
  • this new process provides numerous advantages. For example the process's mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as ribonucleoside-containing oligonucleotides, alkylphosphotriesters, certain base- sensitive phosphoramidate and other base-sensitive oligonucleotides.
  • One preferred use of this aspect of the invention is in the synthesis of oligonucleotides containing from one to about all ribonucleosides.
  • such synthesis employs a phosphoramidite, H-phosphonate or phosphotriester nucleoside monomer synthon having novel protecting groups according to the invention on the nucleoside base, as well as on the 2' hydroxyl of the nucleoside sugar.
  • Another preferred use of this aspect of the invention is in the synthesis of an oligonucleotide containing from one to about all primary phosphoramidate internucleoside linkages.
  • the primary phosphoramidate internucleoside linkage has the structure:
  • This process comprises condensing a nucleoside H-phosphonate with another nucleoside, wherein at least one of the nucleosides has a nucleoside base-protecting group according to the invention, to produce adjacent nucleosides coupled by an H-phosphonate internucleoside linkage, wherein at least one of the nucleosides has a nucleoside base-protecting group according to the invention, aminating the H-phosphonate internucleoside linkage to produce a primary phosphoramidate linkage, and chemoselectively removing the nucleoside base- protecting group without cleaving the primary phosphoramidate linkage.
  • This process allows for synthesis, for the first time, of oligonucleotides containing primary phosphoramidate internucleoside linkages.
  • Another preferred use of this aspect of the invention is in the synthesis of oligonucleotides containing from one to about all alkylphosphonate internucleoside linkages, most preferably methylphosphonate internucleoside linkages.
  • This new process comprises sequentially coupling nucleoside alkylphosphonamidite (preferably methylphosphonamidite) synthons having base protecting groups according to the invention to produce a base-protected oligonucleotide having an alkylphosphonate internucleoside linkage, followed by deprotection using a chemoselective removing agent.
  • this aspect of the invention comprises coupling together an alkylphosphonamidite nucleoside synthon, most preferably a methylphosphonamidite nucleoside synthon, with a nucleoside or oligonucleoside having a free 5 ' hydroxyl group to produce a base-protected oligonucleotide having an alkylphosphonite (III) internucleoside linkage having as a bridging oxygen the oxygen from the free 5' hydroxyl group from the nucleoside or nucleotide, oxidizing the internucleoside linkage to an alkylphosphonate linkage, and deprotecting the oligonucleotide using a chemoselective removing agent.
  • an alkylphosphonamidite nucleoside synthon most preferably a methylphosphonamidite nucleoside synthon
  • a nucleoside or oligonucleoside having a free 5 ' hydroxyl group to produce a base-protected oligon
  • nucleoside or “oligonucleoside” include those having appropriately protected reactive functionalities, either in accordance with the present invention or with conventional protecting groups known in the art (see e. g. Sonveaux in Methods in Molecular Biology, Vol 26 : Protocols for Oligonucleotide Conjugates pp. 1-36 (S. Agrawal, Ed., Humana Press, 1994) .
  • the oxidation of the internucleoside linkage to an alkylphosphonate linkage utilizes a phosphite oxidizing agent such as tert-butyl hydroperoxide or other well known agents (see Beaucage and Iyer, Tetrahedron 4_8: 2223-2311 (1992) ) .
  • a phosphite oxidizing agent such as tert-butyl hydroperoxide or other well known agents (see Beaucage and Iyer, Tetrahedron 4_8: 2223-2311 (1992) ) .
  • Another preferred use of this aspect of the invention is in the synthesis of oligonucleotides containing from one to about all methylphosphotriester internucleoside linkages.
  • the methylphosphotriester internucleoside linkage has the structure
  • One embodiment of this new process comprises condensing in the presence of lH-tetrazole a methoxy-3 ' -0-
  • nucleoside base-protecting group to produce adjacent nucleosides coupled by a phosphite linkage, wherein at least one of the nucleosides has a nucleoside base-protecting group, oxidizing the internucleotidic phosphite linkage to yield an O- methylphosphotriester or 0- methylphosphorothioate linkage, and chemoselectively removing the nucleoside base-protecting group without demethylating the O-methylphosphotriester or 0- methylphosphorothioate linkage (s) .
  • Another embodiment comprises condensing in the presence of a suitable activator, such as pivaloyl chloride, a nucleoside H- phosphonate or thio-H-phosphonate with another nucleoside, wherein at least at least one of the nucleosides has a nucleoside base protecting group, to produce adjacent nucleosides coupled by an H-phosphonate or thio-H- phosphonate linkage, wherein at least one of the nucleosides has a nucleoside base protecting group, oxidizing the H- phosphonate linkage in carbon tetrachloride/pyridine/ methanol to produce an O-methylphosphotriester or 0- methylphosphorothioate linkage, then chemoselectively removing the nucleoside base protecting group without memethylating the O-methylphosphotriester or 0- methylphosphorothioate linkage, as described previously, and most preferably in I /pyridine/methanol.
  • a suitable activator such as
  • oligonucleotide includes polymers of two or more deoxyribonucleotide, or any modified nucleoside, including 2 '-halo-nucleosides, 2 '-O-substituted ribonucleosides, deazanucleosides or any combination thereof. Such monomers may be coupled to each other by any of the numerous known internucleoside linkages.
  • these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof.
  • oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/ or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane.
  • the term "2 '-O-substituted" means substitution of the 2 ' position of the pentose moiety with a halogen (preferably Cl, Br, or F) , or an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e . g.
  • a halogen preferably Cl, Br, or F
  • this new process provides numerous advantages. For example the process's chemoselective capacity for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as oligoribonucleotides, alkylphosphotriesters, certain base sensitive phosphoramidates and other base-sensitive oligonucleotides . Besides being able to synthesize oligonucleotides bearing "sensitive" functionalities, it can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups. In addition, this new process allows for synthesis of oligonucleotides still bound to any type of solid support.
  • support-bound oligonucleotide where an unprotected, support-bound oligonucleotide is desired, the full length support-bound oligonucleotide will have its internucleoside linkages oxidized, followed by contacting the oligonucleotide with a chemoselective removing agent to cleave the base protecting group. In the phosphoramidite approach, this is followed by treatment with anhydrous triethylamine to cleave the beta-cyanoethyl moiety.
  • support-bound branched oligonucleotides can be synthesized using, for example glycol residues in which one hydroxyl group is protected by e . g.
  • the invention provides novel synthons for use in synthesis of oligonucleotides having base-sensitive functionalities.
  • One such novel synthon can be used to prepare portions of the oligonucleotide containing deoxyribonucleosides linked by any known internucleoside linkage.
  • This monomer synthon according to the invention has the general structure III:
  • the protecting group (G) is the previously described structure I, or its preferred embodiment II
  • Z is hydrogen, -OG, -NG2, halogen (preferably Cl, Br or F) , an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyG, alkoxyG, carboxyG, carbalkoxyG;and L is a phosphoramidite, H-phosphonate, or phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al .
  • This synthon can be used alone, with any of the known synthons, or in conjunction with any of the following synthons.
  • Another such novel synthon is useful in the synthesis of oligonucleotides containing ribonucleosides.
  • This monomer synthon according to the invention has the general structure IV:
  • B is a nucleoside base
  • D is a 5 ' -OH blocking group
  • the protecting group (G) is the previously described structure I, or its preferred embodiment II
  • L is a phosphoramidite, H-phosphonate, or 0 phosphotriester leaving group, including cyclic phosphoramidite leaving groups (see Iyer et al . , J. Org. Chem. £0:5388-5389 (1995)) .
  • FIG. 1 A scheme for synthesis of such a monomer having a particularly preferred embodiment of the protecting group 5 according to the invention is shown in Figure 1.
  • the monomer synthon is synthesized from the ribonucleoside by first protecting the 3' and 5' hydroxyl groups as the cyclic silyl ether derivative using the Markiewicz reagent. Then the N-pent-4-enoyl (PNT) group is o installed at the nucleobase and the 2' hydroxyl of the ribose unit using PNT anhydride or using pent-4-enoic acid in the presence of dicyclohexylcarbodiimide (DCC) .
  • PNT N-pent-4-enoyl
  • the 3' and 5' protecting groups are removed using tetrabutylammonium fluoride, followed by conversion of the 5 diol to the 5'-0-4, 4-dimethoxytrityl 3 '-O-phosphoramidite monomer synthon by adaptation of standard phosphoramidite synthesis protocols using the appropriate chlorophosphitylation reagent.
  • a minor iso erization product resulting from 2 '-3' migration of groups is also formed, but is readily removed by chromatography. The formation of this minor product can also be substantially reduced by using bis-N, -diisopropylphosphoramidite as the phosphitylating reagent.
  • the protecting group (G) is the previously described structure I, or its preferred embodiment II
  • Z is hydrogen, -OG, -NG2, halogen (preferably Cl, Br or F) , an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e . g.
  • L is an H- phosphonate or H-phosphonocrowdedthioate (see Kers et al . , Nucleosides and Nucleotides . 15 . : 361-378 (1996)) leaving group.
  • This monomer synthon according to the invention is useful for synthesizing oligonucleotides containing alkylphosphonate internucleoside linkages.
  • This monomer synthon according to the invention has the general structure VI:
  • B is a nucleoside base
  • D is a 5'-OH blocking group (see e . g. Sonveaux in Methods in Molecular Biology, Vol 26: Protocols for Oligonucleotide Conjugates pp. 28-36 (S. Agrawal, Ed., Humana Press, 1994), preferably dimethoxytrityl or trityl
  • R is an alkylphosphonamidite
  • Z is hydrogen, - OG, -NG2, halogen (preferably Cl, Br, or F) , an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e . g. , with halogen, trifluoromethyl, cyano, nitroG, acylG, acyloxyG, alkoxyG, carboxyG, carbalkoxyG; and the protecting group (G) is the previously described structure I, or its preferred embodiment II.
  • halogen preferably Cl, Br, or F
  • alkyl group means a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an allyl group having 2-6 carbon atoms, wherein such alkyl or allyl group may be unsubstituted or may be substituted, e . g. , with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups.
  • the invention provides novel oligonucleotides containing from one to about all of a variety of base-sensitive functionalities.
  • such oligonucleotides may contain from one to about all ribonucleotides and/ or may contain from one to about all internucleoside linkages selected from the group consisting of primary phosphoramidate and methylphosphotriester linkages.
  • oligonucleosides according to this aspect of the invention may have alkylphosphonate or alkylphosphonite (III) internucleoside linkages .
  • the other internucleoside linkages may be any of the known internucleoside linkages, or may be any internucleoside linkage not yet known that can be incorporated into an oligonucleotide according to a synthetic chemistry with which the process according to the invention is compatible.
  • the other internucleoside linkages are phosphodiester or phosphorothioate linkages.
  • the linkages may be phosphorothioate mixed enantiomers or stereoregular phosphorothioates (see Iyer et al. , Tetrahedron Asymmetry 6.: 1051-1054 (1995) .
  • Oligonucleotides containing such a mixture of internucleoside linkages are referred to herein as mixed backbone oligonucleotides .
  • the internucleoside linkages that are not primary phosphoramidate or methylphosphotriester linkages are selected from the group consisting of phosphodiester and phosphorothioate internucleoside linkages .
  • mixed backbone oligonucleotides In some preferred embodiments of mixed backbone oligonucleotides according to the invention, several adjacent nucleosides comprising one region of the oligonucleotide are connected by primary phosphoramidate or methylphosphotriester linkages, and several other adjacent nucleosides comprising another region of the oligonucleotide are connected by a different type of internucleoside linkage. These preferred oligonucleotides are referred to herein as "chimeric" oligonucleotides .
  • the oligonucleotide comprises a primary phosphoramidate or methylphosphotriester region and a phosphorothioate and/or phosphodiester and/or alkylphosphonate region.
  • a "primary phosphoramidate region” or a "methylphosphotriester region” is a region within an oligonucleotide of from about 2 to about 15 contiguous nucleosides linked to each other through primary phosphoramidate or methylphosphotriester linkages according to the invention, respectively.
  • a “phosphorothioate region” is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through phosphorothioate linkages.
  • a “phosphodiester region” is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through phosphodiester linkages.
  • An “alkylphosphonate region” is a region within an oligonucleotide of from about 4 to about 20 contiguous nucleosides linked to each other through alkylphosphonate, preferably methylphosphonate linkages.
  • the oligonucleotide comprises a phosphorothioate or phosphodiester region flanked on either side by a primary phosphoramidate or methylphosphotriester region, or alternatively, a primary phosphoramidate or methylphosphotries er region flanked on either side by a phosphorothioate or phosphodiester region.
  • the nucleosides of the primary phosphoramidate or methylphosphotriester region are ribonucleosides or 2 ' -O- substituted ribonucleotides, as defined above herein.
  • Preferred chimeric oligonucleotides according to the invention are further characterized by having the ability to activate RNaseH.
  • such oligonucleotides will have from about 12 to about 50 nucleotides, most preferably from about 17 to about 35 nucleotides.
  • such oligonucleotides will have a nucleotide sequence that is complementary to a genomic region, a gene, or an RNA transcript thereof.
  • complementary means having the ability to hybridize to a genomic region, a gene, or an RNA transcript thereof under physiological conditions.
  • Such hybridization is ordinarily the result of base-specific hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can also lead to hybridization.
  • the gene sequence or RNA transcript sequence to which the modified oligonucleotide sequence is complementary will depend upon the biological effect that is sought to be modified.
  • the genomic region, gene, or RNA transcript thereof may be from a virus.
  • viruses include, without limitation, human immunodeficiency virus (type 1 or 2), influenza virus, herpes simplex virus (type 1 or 2), Epstein-Barr virus, cytomegalovirus, respiratory syncytial virus, influenza virus, hepatitis B virus, hepatitis C virus and papilloma virus.
  • the genomic region, gene, or RNA transcript thereof may be from endogenous mammalian cells
  • genomic regions, genes or RNA transcripts thereof include, without limitation, sequences encoding vascular endothelial growth factor (VEGF) , beta amyloid, DNA methyltransferase, protein kinase A, ApoE4 protein, p- glycoprotein, c-MYC protein, BCL-2 protein, protein kinase A and CAPL.
  • VEGF vascular endothelial growth factor
  • the genomic region, gene, or RNA transcript thereof may be from a eukaryotic or prokaryotic pathogen including, without limitation, Plasmodium falciparum, Plasmodium malar ie , Plasmodium ovale, Schistosoma spp . , and Mycobacterium tuberculosis .
  • the invention provides methods for using oligonucleotides containing any of a variety of base- sensitive functionalities to control the expression of specific genes .
  • Such methods comprise administering oligonucleotides according to the invention to cells or to animals, including humans. These methods may be used to assess gene function, or as a therapeutic approach to the treatment of diseases resulting from aberrant gene expression.
  • the invention further provides a method for therapeutically treating, with reduced side effects, a disease caused by aberrant gene expression, the method comprising administering to an individual having the disease a composition of matter comprising an oligonucleotide according to the invention, wherein the oligonucleotide is complementary to a gene that is aberrantly expressed, wherein such aberrant expression causes the disease.
  • aberrant gene expression means expression in a host organism of a gene required for the propagation of a virus or a prokaryotic or eukaryotic pathogen, or inappropriate expression of a host cellular gene.
  • Inappropriate host cellular gene expression includes expression of a mutant allele of a cellular gene, or underexpression or overexpression of a normal allele of a cellular gene, such that disease results from such inappropriate host cellular gene expression.
  • such administation should be parenteral, oral, sublingual, transdermal, topical, intranasal or intrarectal.
  • Administration of the therapeutic compositions can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the disease.
  • the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of oligonucleotide from about 0.01 micromolar to about 10 micromolar.
  • a total dosage of oligonucleotide will range from about 0.1 mg oligonucleotide per patient per day to about 200 mg oligonucleotide per kg body weight per day. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. In a preferred embodiment, after the composition of matter is administered, one or more measurement is taken of biological effects selected from the group consisting of complement activation, mitogenesis and inhibition of thrombin clot formation.
  • H-phosphonate nucleosides are similarly prepared in overall yields ranging from 70-90%.
  • Example 3 Solid phase coupling of nucleoside synthons and removal of base protecting groups Nucleoside synthons prepared according to Example 2 were coupled using solid phase H-phosphonate methodology
  • Example 4 Solid phase coupling of nucleoside synthons, introduction of the primary phosphoramidate linkage and removal of base protecting groups Nucleoside synthons prepared according to Example 2 were coupled using solid phase H-phosphonate methodology (Froehler ref. above) . The support bound oligonucleotide H- phosphonate was then treated with a solution of NH (0.5 M in dioxane/CCl , 1:1) at ambient temperature for 30 minutes to give the corresponding support-bound primary phosphoramidate dinucleotide.
  • NH 0.5 M in dioxane/CCl , 1:1
  • Oligonucleotides containing either all primary phosphoramidate internucleoside linkages or a mixture of primary phosphoramidate internucleoside linkages and phosphorothioate or phosphodiester internucleoside linkages in various chimeric configurations are synthesized according to Example 4, or by incorporating the protocol of Example 4 into a conventional H-phosphonate or phosphoramidite synthetic approach. Oligonucleotide phosphodiesters and phosphorothioates are synthesized according to standard procedures. The oligonucleotides have a previously described sequence that is complementary to a portion of the gag gene of HIV-l (see Agrawal and Tang, Antisense Research and Development 2 : 261-266(1992)) .
  • oligonucleotides are treated with snake venom phosphodiesterase (SVPD) .
  • SVPD snake venom phosphodiesterase
  • 260 oligonucleotide is dissolved in 500 microliters buffer (40 mM NH CO , pH 7.0, 20 mM MgCI ) and mixed with 0.1 units SVPD. The mixture is incubated at 37'C for 420 minutes. After 0, 200 and 420 minutes, 165 microliter aliquots are removed and analyzed using ion exchange HPLC.
  • 500 microliters buffer 40 mM NH CO , pH 7.0, 20 mM MgCI
  • Oligonucleotides containing primary phosphoramidate internucleoside linkages are expected to have greater nuclease resistance than oligonucleotides containing exclusively phosphodiester or phosphorothioate internucleoside linkages .
  • Oligonucleotides containing either all primary phosphoramidate internucleoside linkages or a mixture of primary phosphoramidate internucleoside linkages and phosphorothioate or phosphodiester internucleoside linkages in various chimeric configurations are synthesized according to Example 4, or by incorporating the protocol of Example 4 into a conventional H-phosphonate or phosphoramidite synthetic approach. Oligonucleotide phosphodiesters and phosphorothioates are synthesized according to standard procedures. The oligonucleotides have a previously described sequence that is complementary to a portion of the gag gene of HIV-l (see Agrawal and Tang, Antisense Research and Development 2 . : 261-266(1992)) .
  • each oligonucleotide is mixed with an equivalent quantity (0.2 A units) of its complementary oligonucleotide in 150 mM NaCl, lOmM Na PO , ImM EDTA (pH
  • Oligonucleotides according to the invention are expected to form duplexes with complementary oligodeoxyribonucleotides or oligoribonucleotides at temperatures well above physiological temperatures.
  • Oligonucleotides containing either all primary phosphoramidate internucleoside linkages or a mixture of primary phosphoramidate internucleoside linkages and phosphorothioate or phosphodiester internucleoside linkages in various chimeric configurations are synthesized according to Example 4, or by incorporating the protocol of Example 4 io into a conventional H-phosphonate or phosphoramidite synthetic approach.
  • Oligonucleotide phosphodiesters and phosphorothioates are synthesized according to standard procedures. The oligonucleotides have a previously described sequence that is complementary to a portion of the
  • Oligonucleotides are tested for their abilty to inhibit HIV-
  • H9 lymphocytes are infected with HIV-l virions (0.01-0.1 TCID /cell) for one hour at
  • the cells are then cultured for four days. At the end of four days, inhibition of HIV-l is assessed by observing or measuring reductions in syncytium formation, p24 expression and reverse transcriptase activity. All of the tested oligonucleotides according to the invention are 0 expected to show significant reductions in these parameters without significant cytotoxicity.
  • the reaction mixture was rapidly stirred and a solution of the appropriate protected nucleoside monomers (lmmol in 5 ml anhydrous methylene chloride containing 1.5 mmol N,N- diisopropylethylamine) , prepared according to Examples 1 and 8 above, was added.
  • the reaction was allowed to continue for 20 minutes, then 0.5 ml anhydrous methanol was added to destroy any residual chlorophosphonite.
  • the reaction mixture was poured into 5% aqueous sodium bicarbonate and the product was extracted with methylene chloride (3 X 20 ml) . The combined extracts were dried over anhydrous Na SO
  • P P was about 60/40.
  • Each of the nucleoside monomer synthons was readily soluble in anhydrous acetonitrile.
  • Example 10 Synthesis of Methylphosphonate Dinucleosides Thymidine nucleoside coupled to a CPG support by its 3 ' hydroxyl functionality was prepared according to standard procedures. In separate reactions, each of the nucleoside monomer synthons prepared according to Example 9 were coupled to the support-bound thymidine using conventional phosphoramidite chemistry. The coupling reaction yielded a support bound dinucleoside coupled by an internucleosidic methylphosphonite (III) linkage. This linkage was then oxidized using tert-butyl hydroperoxide (1 M in toluene) to yield a methylphosphonate internucleosidic linkage.
  • tert-butyl hydroperoxide (1 M in toluene
  • the support-bound methylphosphonate dinucleosides were then treated with aqueous ammonium hydroxide (28%, 1 hour, room temperature) to remove the PNT protecting group and to cleave the dimers from the solid support.
  • the dimers were obtained in yields of 94-96% as a mixture of R , S
  • Example 11 Synthesis of Chimeric Oligonucleotides Containing Methylphosphonate Internucleosidic Linkages Nucleoside monomer synthons prepared according to Example 9 were used under standard phosphoramidite coupling conditions to prepare chimeric oligonucleotides having methylphosphonate internucleosidic linkages in different numbers and at different positions. All syntheses were carried out on a 1-10 micromole scale.
  • a first oligonucleotide had its 5 most 5' internucleosidic linkages as methylphosphonates, with the remaining 9 internucleosidic linkages as phosphodiesters.
  • a second oligonucleotide had its 10 most 5' internucleosidic linkages as methylphosphonates, with the remaining 9 internucleosidic linkages as phosphodiesters.
  • a third oligonucleotide had 10 phosphodiester internucleosidic linkages, followed by 4 methylphosphonate internucleosidic linkages, followed by 4 phosphodiester internucleosidic linkages .
  • a fourth oligonucleotide had 5 phosphodiester internucleosidic linkages, followed by 4 methylphosphonate internucleosidic linkages, followed by 9 phosphodiester internucleosidic linkages.
  • the support-bound oligonucleotides were treated with aqueous ammonium hydroxide (28% for 1 hour at room temperature) to remove the phosphate and nucleoside base protecting groups and cleave the oligonucleotides from the support.
  • Polyacrylamide gel electrophoresis revealed that these oligonucleotides had identical mobility to oligonucleotide standards of the same structure prepared using commercially available monomer synthons under the conditions recommended by the manufacturer.
  • the PNT nucleosides prepared according to Example 1 were employed in the synthesis of beta-cyanoethyl- (CEPNT)
  • Methoxy- (MEPNT) 3 '-O- (phosphoramidite)-5 ' -O- (4,4- dimethoxytriphenyl) methyl) [DMT] monomers were coupled in a standard lH-tetrazole-mediated phosphoramidite coupling reaction to form the dinucleoside phosphites.
  • the dinucleoside phosphites were then oxidized using t-butyl hydroperoxide (IM in toluene) to yield the protected O- ethyl phosphotriester, or 3H-benzodithiol-3-one 1,1-dioxide to yield the protected O-methyl phosphorothioate.
  • Example 14 Synthesis of Triester-Containing Chimeric Oligonucleotides
  • the CEPNT and MEPNT monomers were used to prepare chimeric trinucleotides having one phosphodiester or phosphorothioate internucleoside linkage and one O-methyl phosphotriester or phosphorothioate internucleoside linkage under conditions as descibed in Example 13. Synthesis was carried out on a solid support using conventional succinyl- linked nucleoside loading.
  • the phosphodiester or phosphorothioate internucleoside linkage was assembled using the CEPNT monomer and the O-methyl phosphotriester or phosphorothioate internucleoside linkage was assembled using the MEPNT monomer.
  • the trimers thus obtained, a mixture of
  • MALDI-TOF mass spectrum revealed the expected molecular ion + at 911.7 (Na form) for the species containing the phosphorothioate and O-methylphosphorothioate linkages .
  • Oligonucleotides containing either all methyl phosphotriester internucleoside linkages or a mixture of methyl phosphotriester internucleoside linkages and phosphorothioate or phosphodiester internucleoside linkages in various chimeric configurations were synthesized according to Example 13 or 14. Oligonucleotide phos- phodiesters and phosphorothioates were synthesized according to standard procedures.
  • oligonucleotides were treated with snake venom phosphodiesterase (SVPD) . About 0.2 A units
  • oligonucleotide 260 of oligonucleotide was dissolved in 500 microliters buffer (40 mM NH CO , pH 7.0, 20 mM MgCI ) and mixed with 0.1 units SVPD. The mixture was incubated at 37"C for 420 minutes. After 0, 200 and 420 minutes, 165 microliter aliquots were removed and analyzed using ion exchange HPLC. Oligonucleotides containing methyl phosphotriester internucleoside linkages exhibited greater nuclease resistance than oligonucleotides containing exclusively phosphodiester or phosphorothioate internucleoside linkages.
  • Example 16 Duplex stability of oligonucleotides containing methyl phosphotriester internucleoside linkages
  • Oligonucleotides containing either all methyl phosphotriester internucleoside linkages or a mixture of methyl phosphotriester internucleoside linkages and phosphorothioate or phosphodiester internucleoside linkages in various chimeric configurations were synthesized using the process described in Example 13 or 14.
  • Oligonucleotide phosphodiesters and phosphorothioates were synthesized according to standard procedures. The oligonucleotides are tested for their ability to form duplexes with complementary oligodeoxyribonucleotides and oligoribonucleotides. In separate reactions, each oligonucleotide is mixed with an equivalent quantity (0.2 A units) of its complementary oligonucleotide in 150 mM NaCl, lOmM Na PO leave, ImM EDTA (pH
  • Oligonucleotides according to the invention were found to form duplexes with complementary oligodeoxyribonucleotides or oligoribonucleotides at temperatures well above physiological temperatures.
  • Oligonucleotides containing methyl phosphotriester internucleoside linkages Oligonucleotides containing either all methyl phosphotriester internucleoside linkages or a mixture of methyl phosphotriester internucleoside linkages and phosphorothioate or phosphodiester internucleoside linkages in various chimeric configurations are synthesized according to the process described in Examples 13 or 14: Oligonucleotide phosphodiesters and phosphorothioates are synthesized according to standard procedures.
  • oligonucleotides have a previously described sequence that is complementary to a portion of the gag gene of HIV-l (see Agrawal and Tang, Antisense Research and Development 2 : 261- 266(1992) ) . Oligonucleotides are tested for their ability to inhibit HIV-l in a tissue culture system. H9 lymphocytes are infected with HIV-l virions (0.01-0.1 TCID /cell) for
  • oligonucleotide 50 one hour at 37"C. After one hour, unadsorbed virions are washed away and the infected cells are divided among wells of 24 well plates. To the infected cells, an appropriate concentration (from stock solution) of oligonucleotide is added to obtain the required concentration (0.1 -10 mi ⁇ cromolar) in 2 ml media. The cells are then cultured for four days. At the end of four days, inhibition of HIV-l is assessed by observing or measuring reductions in syncytium formation, p24 expression and reverse transcriptase activity. All of the tested oligonucleotides according to the invention are expected to show significant reductions in these parameters without significant cytotoxicity.

Abstract

L'invention concerne des procédés nouveaux de synthèse d'oligonucléotides qui permettent de déprotéger les oligonucléotides dans des conditions plus douces qu'avec les procédés existants. L'invention concerne en outre un groupe protecteur des bases de nucléosides qui reste stable dans des conditions de synthèse d'oligonucléotides mais qui peut être éliminé dans des conditions plus douces que les groupes protecteurs existants. L'invention concerne également des synthons de nucléosides qui contiennent ces groupes protecteurs de base, des oligonucléotides qui contiennent des fonctionnalités labiles diverses de bases et des procédés d'utilisation de ces oligonucléotides.
PCT/US1996/008136 1995-06-01 1996-05-31 Groupes nouveaux de protection des bases pendant la synthese d'oligonucleotides WO1996039414A1 (fr)

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AU59581/96A AU5958196A (en) 1995-06-01 1996-05-31 Novel base protecting groups for oligonucleotide synthesis
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US45719895A 1995-06-01 1995-06-01
US08/457,198 1995-06-01
US08/518,921 US5614622A (en) 1995-06-01 1995-08-24 5-pentenoyl moiety as a nucleoside-amino protecting group, 4-pentenoyl-protected nucleotide synthons, and related oligonucleotide syntheses
US08/518,921 1995-08-24
US08/519,318 US6140482A (en) 1995-06-01 1995-08-25 Primary phosphoramidate internucleoside linkages and oligonucleotides containing the same
US08/519,318 1995-08-25
US08/570,390 1995-12-11
US08/570,390 US5955599A (en) 1995-06-01 1995-12-11 Process for making oligonucleotides containing o- and s- methylphosphotriester internucleoside linkages
US08/598,320 1996-02-08
US08/598,320 US6531589B1 (en) 1995-06-01 1996-02-08 Base protecting groups and synthons for oligonucleotide synthesis
US08/606,915 1996-02-26
US08/606,915 US5962674A (en) 1995-06-01 1996-02-26 Synthesis of oligonucleotides containing alkylphosphonate internucleoside linkages

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JP2002534434A (ja) * 1998-12-30 2002-10-15 オリゴス・イーティーシー・インコーポレイテッド 酸安定性骨格で修飾された末端がブロックされた核酸及びその治療的使用
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US9045522B2 (en) 2006-07-31 2015-06-02 Wanli Bi Nucleic acid amplification using a reversibly modified oligonucleotide

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WO1998049183A1 (fr) * 1997-04-30 1998-11-05 Hybridon, Inc. Groupes protecteurs de bases et procede de synthese d'oligonucleotides
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JP4688294B2 (ja) * 1998-12-30 2011-05-25 オリゴス・イーティーシー・インコーポレイテッド 酸安定性骨格で修飾された末端がブロックされた核酸及びその治療的使用
WO2008016562A3 (fr) * 2006-07-31 2008-07-17 Wanli Bi Amplification d'acide nucléique à l'aide d'un oligonucléotide modifié de façon réversible
WO2008016562A2 (fr) 2006-07-31 2008-02-07 Wanli Bi Amplification d'acide nucléique à l'aide d'un oligonucléotide modifié de façon réversible
US8334099B2 (en) 2006-07-31 2012-12-18 Wanli Bi Nucleic acid amplification using a reversibly modified oligonucleotide
US9045522B2 (en) 2006-07-31 2015-06-02 Wanli Bi Nucleic acid amplification using a reversibly modified oligonucleotide
CN106566855A (zh) * 2006-07-31 2017-04-19 毕万里 使用可逆修饰寡核苷酸扩增核酸
CN106566855B (zh) * 2006-07-31 2021-11-09 苏州新海生物科技股份有限公司 使用可逆修饰寡核苷酸扩增核酸
US8785409B2 (en) 2007-01-30 2014-07-22 Geron Corporation Compounds having anti-adhesive effects on cancer cells
US9732114B2 (en) 2007-01-30 2017-08-15 Geron Corporation Compounds having anti-adhesive effects on cancer cells

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