WO1996039413A1 - Procedes et composes pour la synthese des oligonucleotides et oligonucleotides ainsi produits - Google Patents

Procedes et composes pour la synthese des oligonucleotides et oligonucleotides ainsi produits Download PDF

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WO1996039413A1
WO1996039413A1 PCT/US1996/007430 US9607430W WO9639413A1 WO 1996039413 A1 WO1996039413 A1 WO 1996039413A1 US 9607430 W US9607430 W US 9607430W WO 9639413 A1 WO9639413 A1 WO 9639413A1
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Prior art keywords
oligonucleotide
oligonucleotides
contacting
compound
synthesizing
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PCT/US1996/007430
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English (en)
Inventor
Radhakrishnan P. Iyer
Dong Yu
Sudhir Agrawal
Weitian Tan
Theresa Devlin
Ivan Habus
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Hybridon, Inc.
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Priority claimed from US08/448,131 external-priority patent/US5750674A/en
Priority to AU58711/96A priority Critical patent/AU5871196A/en
Application filed by Hybridon, Inc. filed Critical Hybridon, Inc.
Publication of WO1996039413A1 publication Critical patent/WO1996039413A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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

Definitions

  • This invention relates to methods for the chemical synthesis of oligonucleotides, compounds use ul in the methods, and oligonucleotides thereby produced.
  • Oligonucleotides and their chemical synthesis have become indespensible tools in modern molecular biology, being used in a wide variety of techniques ranging from PCR to antisense inhibition of nucleic acid expression. Understandably, therefore, there has been an every increasing demand for oligonucleotides having desirable properties such as resistance to nucleolytic attack and increased binding affinities. Furthermore, with the widespread use of oligonucleotides for these varying purposes, there has been an ever increasing demand for fast, inexpensive, and efficient methods of synthesizing oligonucleotides having these desirable properties.
  • Antisense oligonucleotides are constructed to be sufficiently complementary to a target nucleic acid to hybridize with the target under the conditions of interest and inhibit expression of the target. Antisense oligonucleotides may be designed to bind directly to DNA (the so-called "anti-gene” approach) or to viral RNA or mRNA. Id. Expression inhibition is believed to occur by interfering with transcription processing or translation, or inducement of target mRNA cleavage by RNase H.
  • Antisense oligonucleotides can be used as research tools in vitro to determine the biological function of genes and proteins. They provide an easily used alternative to the laborious method of gene mutation (e.g. , deletion mutation) to selectively inhibit gene expression. The importance of this method is readily appreciated when one realizes that the elucidation of most known biological processes has been determined by deletion mutation.
  • Antisense oligonucleotides also may be used to treat a variety of pathogenic diseases by inhibiting gene expression of the pathogen in vivo.
  • Oligonucleotide phosphorothioates PS-oligos
  • PS-oligos Oligonucleotide phosphorothioates
  • Agrawal and Tang Antisense Res. and Dev. 2, 261 (1992) and references therein, and Bayever et al., Antisense Res. Dev. 3, 383 (1993).
  • the synthesis of oligonucleotides for antisense and diagnostic applications is now be routinely accomplished.
  • Desired internucleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the '-most nucleoside of the nascent, support- bound oligonucleotide.
  • Oligonucleotide synthesis generally begins with coupling, or "loading," of the 3'- most nucleoside of the desired oligonucleotide to a functionalized solid phase support.
  • a variety of solid supports and methods for their preparation are known in the art. E.g. , Pon, "Solid-Phase Supports for Oligonucleotide Synthesis," in Methods in Molec. Biol. , Vol.
  • the functionalized support has a plurality of long chain alkyl amines (LCAA) on the surface that serve as sites for nucleoside coupling.
  • LCAA long chain alkyl amines
  • CPG Controlled pore glass
  • oligonucleotides The routine synthesis of oligonucleotides is presently carried out using various N- acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine), and benzoyl for adenine and cytosine.
  • N- 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.
  • PO oligonucleotides and other modified oligonucleotides can be synthesized. But in certain instances where modified oligonucletides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out.
  • base-sensitive modified oligonucleotides examples include, methyl phosphotriester oligonucletides, phosohsphoramides, etc. in other applications of oligonucletides, it is desirable to have oligonucleotides still bound to a solid-support.
  • oligonucleotides still bound to the solid support will be useful in a variety of applications such as those involving isolation of transcription factors and other factors or elements that interact with oligonucleotides, solid-phase PCR, investigations 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).
  • PS-oligos synthesized by prior art methods are mixtures of 2" diastereomers, where n is the number of internucleotide phosphorothioates.
  • n is the number of internucleotide phosphorothioates.
  • a novel nucleoside base protecting group is provided.
  • This protecting group has the general structure XXI:
  • n,, n 2 , and n 3 are independently 0-10 and the nitrogen displayed is the amino moiety of the base.
  • Compounds XXI and XXII protect the nucleoside base amino moieties by forming amide linkages, as in:
  • oligonucleotides having 3 or more phosphotriester internucleotide linkages are provided. These oligonucleotides are synthesized according to the method outlined in Figure 2.
  • phosphorothioate oligonucleotides bound or unbound to a solid support are provided.
  • This aspect of the invention is is schematically displayed in Figure 3. If free (unbound) oligonucleotide is desired, the oligonucleotide can be synthesized according to the phosphoramidite method using the base protecting groups of the invention. When the full length support bound oligonucleotide is complete, it can be contacted with ammonia for 1 to 2 hours to yield the free, unprotected oligonucleotide.
  • support-bound oligonucleotide is contacted with I 2 in water to cleave the base protecting group and then with anhydrous triethylamine to cleave the ⁇ -cyano moiety.
  • base-protecting group can also be used to protect hydroxyl moieties
  • support-bound branched oligonucleotides can be synthesized using, for example, glycol residues in which one hydroxyl group is protected by DMT and the other by a protecting group according to the invention. Then the DMT group may be selectively removed and an oligonucleotide synthesized from the resulting unprotected hydroxyl.
  • the hydroxyl moiety protected by the protecting group according to the invention can be deprotected with I 2 and water and another, different oligonucleotide synthesized from it. Such an approach is useful in producing combinatorial libraries.
  • the unprotected support bound oligonucleotides of the invention can have phosphodiester, or phosphotriester internucleotide linkages of the form -O-PO(XR)-O- where X is O, NH, or S and R is a C, - C 20 alkyl or an aryl moiety.
  • the resulting unprotected support-bound oligonucleotide is useful for a variety of purposes such as applications involving isolation of transcription factors and other factors or elements that interact with oligonucleotides, solid-phase PCR, investigations 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).
  • the present invention also provides additional methods for the synthesis of oligonucleotides. These methods use phosphoramidite chemistry with novel phosphoramidite reagents, which form another aspect of the invention.
  • novel synthons and synthetic methods of the invention can be used for the synthesis of diastereomeric mixtures of oligonucleotides and for the synthesis of oligonucleotides enriched in a particular diastereomer.
  • the invention also provides oligonucleotides produced from these compounds by these methods. Oligonucleotides produced using the synthons and methods of the invention are useful for any purpose for which oligonucleotides produced using prior art techniques are used, such as PCR and as inhibitors of nucleic acid expression.
  • compounds of structure XIII can be made to be diastereomerically enriched or a mixture of diastereomers.
  • Diastereomerically enriched synthons as well as derivatives and analogs thereof are useful in the synthesis of diastereomerically pure oligonucleotides by the phosphoramidite method. They can be used as a substitute for the well-known ⁇ -cyanoethyl-protected phosphoramidate.
  • synthon XIII has the form:
  • XIV where chiral centers 4 and 5 can be, respectively, R and S, S and R, R and R, or S and S.
  • synthon XIII has the form:
  • the present invention provides a phosphoramidite monomer synthon Via:
  • a method of synthesizing the diastereomerically enriched monomer synthons XIII, XIV, and VI is provided.
  • the method comprises
  • XVII takes the form:
  • the R p isomer predominates ( > 95%).
  • Contacting IV with a 5 ' -DMT-protected mononucleoside having a free 3 ' hydroxyl group yields the monomer synthon VI in high yield (84%).
  • the monomer synthon Via is synthesized by contacting:
  • thiophosphoramidate monomer synthons enriched in a particular stereoisomer are provided. These compounds have the general structures:
  • thiophosphoramidate monomer synthons having ry ⁇ -(VIIIb) and anti- (Villa) conformations are provided:
  • XV, XVI, Villa and VHIb are all made by oxidatively thiolating the monomer synthon precursors XIII, XTV, and VI, respectively, with a sulfurizing agent such as the 3H-1,2- benzodithiole-3-one- 1,1 -dioxide reagent.
  • a sulfurizing agent such as the 3H-1,2- benzodithiole-3-one- 1,1 -dioxide reagent.
  • the result is about 90% retention of configuration.
  • the stereoisomers can be separated by flash chromatography.
  • the phosphorothioate monomer synthon VIIIc is provided: vmc
  • VIIIc is made by oxidatively thiolating the monomer synthon precursor Via with a sulfurizing agent such as the 3 H-l ,2-benzodithiole-3 -one- 1,1 -dioxide reagent.
  • oligonucleotides having one or more P-chiral centers predominantly in the S configuration and methods for their synthesis have been developed.
  • these oligonucleotides can be synthesized via the well-known phosphoramidate approach using XIII, XrV, or VI instead of the well known ⁇ -cyanoethyl phosphoramidite synthon.
  • the intermediate phosphite linkage may be oxidized with, for example, I 2 and H 2 O in THF to yield a phosphodiester linkage, or oxidatively thiolated with a sulfurizing agent, such as the Beaucage reagent, to yield a phosphorothioate linkage.
  • Oligonucleotides synthesized according to this embodiment of the invention will have predominantly S p configuration ( - 60%) at each internucleotide linkage in which compound XIII, XTV, or VI was employed during synthesis.
  • one of XV, XVI, Villa or VHIb is contacted with a nascent oligonucleotide having a free 5 ' hydroxyl group.
  • a nascent oligonucleotide having a free 5 ' hydroxyl group When either Villa or VIHb is used, the result is an oligonucleotide having a 5 ' phosphorothioate internucleotide linkage with an R p :S p ratio of about 70:30 when Villa is used and 10:90 when VHIb is used.
  • Similar results are obtained from compound XVI when R 1 and R 2 are both anti- or both syn- with respect to the nucleoside and compound XV when all of the R j are anti- or syn- with respect to the nucleoside.
  • each of the foregoing monomer synthons and oligonucleotides can be synthesized using the methods of the present invention to be in enantiomeric excess.
  • One advantage of the methods of the present invention is that the stereochemistry of the precursors is maintained in the products, and, if the reactants are in enantiomeric excess, the products are predominantly in one stereoconfiguration.
  • the present method provides a convenient method of oligonucleotide synthesis using the phosphoramidite method wherein the well-known ⁇ -cyanomethol phosphoramidite synthon is replaced by compound Via.
  • the intermediate phosphite linkage may be oxidized with, for example, l 2 and H in THF to yield a phosphodiester linkage, or oxidatively thiolated with a sulfurizing agent, such as the Beaucage reagent, to yield a phosphorothioate linkage.
  • a sulfurizing agent such as the Beaucage reagent
  • compound VIIIc is used to synthesize oligonucleotides having phosphorothioate internucleotide linkages by contacting VIIIc with a nascent oligonucleotide having a free 5 '-hydroxyl group.
  • Oligonucleotides according to the invention are useful for both in vitro and in vivo applications.
  • the present oligonucleotides are useful as research tools in determining gene function by effecting gene modulation and as hybridization probes, for example.
  • Oligonucleotides according to the invention are also useful for in vivo applications, such as the treatment of pathogen-caused diseases. Oligonucleotides according to the invention can be synthesized to have a sequence sufficiently complementary to a region of a nucleic acid essential for the growth, reproduction, and/or metabolism of the pathogen to inhibit expression of the nucleic acid under physiological conditions.
  • Figure 1 displays the synthetic pathway for making the compounds of the present invention.
  • Figure 2 displays synthesis of phosphodiester and phosphotriester oligonucleotides using H-phosphonate chemistry and the base protecting group of the invention.
  • Figure 3 displays synthesis of free and support-bound unprotected oligonucleotides according to the method and reagents of the invention.
  • a novel nucleoside base protecting group is provided.
  • This protecting group has the general structure XXI:
  • n l t n 2 , and n 3 are independently 0-10 and the nitrogen displayed is the amino moiety of the base.
  • Compounds XXI and XXII protect the nucleoside base amino moieties by forming amide linkages, as in:
  • oligonucleotides having 3 or more phosphotriester internucleotide linkages are provided. These oligonucleotides are synthesized according to the method outlined in Figure 2. After the complete support- bound oligonucleotide is synthesized according to the H-phosphonate method using the base amino-protecting group of the present invention, the oligonucleotide is contacted carbon tetrachloride, N-methyl imidazole and RXH (where X is O, NH, or S and R is a C C 20 alkyl or aryl) and then with iodine in water to remove the protecting group.
  • RXH where X is O, NH, or S and R is a C C 20 alkyl or aryl
  • phosphorothioate oligonucleotides bound or unbound to a solid support are provided.
  • This aspect of the invention is is schematically displayed in Figure 3. If free (unbound) oligonucleotide is desired, the oligonucleotide can be synthesized according to the phosphoramidite method using the base protecting groups of the invention. When the full length support bound oligonucleotide is complete, it can be contacted with ammonia for 1 to 2 hours to yield the free, unprotected oligonucleotide.
  • the full length support-bound oligonucleotide is contacted with I 2 in water to cleave the base protecting group and then with anhydrous triethylamine to cleave the ⁇ -cyano moiety.
  • support-bound branched oligonucleotides can be synthesized using, for example glycol residues in which one hydroxyl group is protected by DMT and the other by a protecting group according to the invention. Then the DMT group may be selectively removed and an oligonucleotide synthesized from the resulting unprotected hydroxyl. Upon completion of that oligonucleotide, the hydroxyl moiety protected by the protecting group according to the invention can be deprotected with I 2 and water and another, different oligonucleotide synthesized from it.
  • the unprotected support bound oligonucleotides of the invention can have phosphodiester, or phosphotriester internucleotide linkages of the fo ⁇ n -O-PO(XR)-O- where X is O, NH, or S and R is a C, - C 20 alkyl or an aryl moiety.
  • X is O, NH, or S and R is a C, - C 20 alkyl or an aryl moiety.
  • the resulting unprotected support-bound oligonucleotide is useful for a variety of purposes such as applications.
  • the present invention also provides a sructurally novel class of antisense oligonucleotides useful for modulation of nucleic acid expression in vitro and in vivo.
  • the present invention also provides novel methods for synthesizing this class of oligonucleotides using new synthons.
  • the present invention also provides additional methods for the synthesis of oligonucleotides.
  • novel synthons and synthetic methods of the invention can be used for the synthesis of diastereomeric mixtures of oligonucleotides and for the synthesis of oligonucleotides enriched in a particular diastereomer.
  • the invention also provides oligonucleotides produced from these compounds by these methods. Oligonucleotides produced using the synthons and methods of the invention are useful for any purpose for which oligonucleotides produced using prior art techniques are used, such as PCR and as inhibitors of nucleic acid expression.
  • monomer synthons having the structure:
  • R* and R b , and each R ' are independently H, C, - C 20 alkyl, aryl, heterocyclic, C r
  • R is a suitable protecting group, such as DMT
  • n is 1-3
  • i is 1-n
  • X* is C, O, S, or N, such that if n > 1 the identity of each X* (i.e. , each of X ' . . . X 5 is independent of the identity of every other X s and the identity of each substituent R ' (i.e., R 1 . . . R n ) is independent of every other R', each R' is covalently bound to the corresponding X ' (e.g. , X'-R l . . .
  • X n -R n the X' are arranged consecutively such that X l is bound to the N and X" is bound to the O, and B is any suitably protected, modified or unmodified, purine or pyrimidine base.
  • aryl means a polyaromatic-ring structure having from 1 to 5 linearly or angularly fused aromatic rings, such as phenyl and naphthyl.
  • heterocyclic means a 5 or 6 membered ring having from 1 to 5 heteroatoms (i.e. , N, S, or O) that may be located at any position within the ring.
  • Furan and thiophene are examples of heterocyclic moieties encompassed by this definition.
  • Compound XIII is synthesized according to the methods of the present invention (infra) to be predominantly in one stereoconfiguration.
  • the stereochemistry of the product XIII depends on the stereochemistry of the starting material. Synthesis of XIII from its precursor is accomplished in a stereorentive manner, infra.
  • R* is H
  • n is 2
  • X ' and X 2 are each C, which has the structure XIV:
  • the configurations at carbons 4 and 5 can be, respectively, R and S, S and R, R and R, or S and S, each of which can be obtained in pure form.
  • n is 2, X 1 and X 2 are each C, R ' is methyl, R is phenyl, R ' is H, R b is methyl, and the compound has the R,, configuration as shown in structure VI:
  • the present invention provides a phosphoramidite monomer synthon Via:
  • the present method provides a method of synthesizing oligonucleotides according to the phosphoramidite method using the phosphoramidite Via.
  • the synthetic protocol for incorporating XIII, XIV, and VI in a nascent oligonucleotide is the same as that for the ⁇ -cyanoethyl-protected phosphoramidates.
  • cent oligonucleotide means a solid support-bound nucleotide chain having at least one nucleotide.
  • XVIII is contacted with a 5 '-protected mononucleoside having an unprotected 3 '-hydroxyl to yield XIII.
  • Compounds XIII and XVIII are obtained from their precursors (XVIII and XVII, respectively) a stereoretentive manner, i.e. , the stereoconfiguration of the precursor is maintained in the reaction.
  • a method of synthesizing the diastereomerically enriched monomer synthon VI comprises contacting (IR, 2S)-(-)-ephedrine (V) with PC1 3 at between -100 and 40 °C for between one and 40 hours.
  • the two compounds are allowed to react in N-methyl morpholine and toluene at -78 °C for 3 hours and then at 22 °C for 12 hours.
  • Other suitable solvents are benzene, tetrahydrofuran, ether, and dioxane. The result is about a 75 % yield of the chlorophosphoramidite product:
  • the R p isomer predominates ( > 95%).
  • Contacting IV with a 5 '-DMT-protected mononucleoside having a free 3 '-hydroxyl group yields the monomer synthon VI in high yield (84%).
  • the mononucleoside and IV are allowed to react in ethyl ether and triethylamine as a scavenger of HCl liberated during the reaction.
  • Other scavengers such as pyridine and 2,6-lutidine can also be used.
  • the reaction can be conducted at temperatures ranging from between -100 and 40 °C for between 1 and 40 hours.
  • the mixture is allowed to react at -78 °C for 3 hours and then at 22 °C for 12 hours.
  • Other suitable solvents such as benzene, tetrahydrofuran,
  • Compound IV is fairly stable, undergoing no apparent decomposition (as evaluated by 31 P-NMR) after being stored at -5 °C for several days.
  • the monomer synthon Via is synthesized by contacting:
  • thiophosphoramidate monomer synthons enriched in a particular stereoisomer are provided. These compounds have the general structures:
  • the phosphorothioate monomer synthon VIIIc is provided:
  • VIIIc is made by oxidatively thiolating the monomer synthon precursor Via Any suitable method of oxidative thiolation may be used, such as elemental sulfur. E.g. , Stec et al., J Am. Chem. Soc. 106, 6077 (1984).
  • the thiophosphoramidate monomer synthons are synthesized by contacting the phosphoramidite precursors with the Beaucage reagent, 3H-l ,2-benzodithiol-3-one-l, l-dioxide:
  • reagent I is used as a 2% solution in acetonitrile and the mixture is allowed to react for 30 seconds at about room temperature. All of the various diastereomers (e.g. , Villa and Vlllb) are easily separated by conventional chromatography or crystallization.
  • oligonucleotides having from one to all nucleotide P-chiral centers independently predominantly in the S configuration and methods for synthesizing them are provided. As used herein, the term "predominantly" means more than half.
  • these oligonucleotides can be synthesized via the well-known phosphoramidate approach (e.g. , Beaucage in Methods in Molecular Biology, Vol 20, Protocols for Oligonculeotides and Analogs, supra, pp. 33-61 and references cited therein) using XIII in place of XII.
  • phosphoramidate approach e.g. , Beaucage in Methods in Molecular Biology, Vol 20, Protocols for Oligonculeotides and Analogs, supra, pp. 33-61 and references cited therein
  • XIV is used in place of XII.
  • VI is used.
  • a nascent oligonucleotide having a free 5 ' hydroxyl is contacted with XIV, XV, or VI in the presence of tetrazole.
  • a phosphate linkage is thereby formed.
  • the phosphite linkage may then be oxidized with, for example, I 2 and H 2 O in THF to yield a phosphodiester linkage or oxidatively thiolated with I to yield a phosphorothioate linkage.
  • Phosphorothioate oligonucleotides synthesized according to this embodiment of the invention have predominantly S p configuration (-60%) at each internucleotide linkage in which compound XIV, XV, or VI was employed during synthesis.
  • oligonucleotide synthesis uses the foregoing synthetic methods with compound Via.
  • compound VIIIc is used to synthesize oligonucleotides having phosphorothioate internucleotide linkages by contacting VIIIc with a nascent oligonucleotide having a freee 5 '-hydroxyl group.
  • oligonucleotides having one or more phosphorothioate internucleotide linkages that are independently predominantly in the R or S configuration are provided.
  • one of the stereoisomers of XV or XVI is contacted with a nascent oligonucleotide having an unprotected 5 ' hydroxyl group.
  • Villa or VIHb is used, resulting in an oligonucleotide having a 5 ' phosphorothioate internucleotide linkage with an R,:S P ratio of about 70:30
  • the antisense oligonucleotides of the present invention may be designed to incorporate a number of additional features that have been demonstrated to increase efficacy. For example, they may be designed to be "self-stabilized," i.e. , having a first region sufficiently complementary to a second region to allow for intramolecular hybridization, thereby rendering the oligonucleotide less susceptible to nucleolytic attack.
  • self-stabilized i.e. , having a first region sufficiently complementary to a second region to allow for intramolecular hybridization, thereby rendering the oligonucleotide less susceptible to nucleolytic attack.
  • Such oligonucleotides are described in PCT International Application Publication No. WO 94/01550. All of the foregoing methods can be used with RNA and DNA and with any solid support. See, e.g., Pon in Methods in Molecular Biology, Vol. 20, pp. 465-496.
  • oligonucleotides may be designed to be
  • fold-back triplex forming i.e., having a first region complementary to a target nucleic acid and a second region having a sequence that allows for triplex formation by Hoogsteen base pairing between it and the duplex formed by the first region and the target nucleic acid, as described in PCT International Application Publication No. WO 94/17091.
  • Oligonucleotides according to the invention are useful for both in vitro and in vivo applications.
  • the present oligonucleotides are useful as research tools in determining gene function. Because they can be prepared to be complementary to a particular sequence, the present oligonucleotides can be used to selectively inhibit expression of a target gene.
  • the present oligonucleotides thus provide an attractive and easily used alternative to the laborious method of gene inhibition by mutation (e.g. , deletion mutation). The significance of this will be appreciated when one realizes that the elucidation of most biological pathways now known has been determined by deletion mutations.
  • Oligonucleotides according to the invention are also useful in standard hybridization assays.
  • oligonucleotides of the present invention are also useful as therapeutic agents for diseases or physiological conditions involving expression of specific genes.
  • Oligonucleotides useful for treating a disease or condition will have a nucleotide sequence sufficiently complementary to the target nucleic acid to bind to the target nucleic acid under physiological conditions.
  • complementary and “sufficiently complementary” are used interchangeably and, when used to describe the sequence of an antisense oligonucleotide, mean that the oligonucleotide sequence is such that the oligonucleotide inhibits expression of the target nucleic acid under the conditions of interest (e.g. , in vitro experimental conditions or physiological conditions).
  • oligonucleotides according to the invention will have a sequence complementary to a nucleic acid (e.g., a gene or mRNA) that is essential to a biological process.
  • a nucleic acid e.g., a gene or mRNA
  • processes include reproduction and metabolic processes of pathogens and other disease-causing infectious agents.
  • the biological process can be a naturally occurring one whose inhibition is desirable, e.g. , spermatogenesis in men and ovulation in women desiring contraception.
  • the oligonucleotides of the invention can also be complementary to a gene or other nucleic acid whose expression causes or is involved in a diseased or otherwise abnormal state of the organism.
  • the presently claimed oligonucleotides are also useful for treating diseases arising from genetic abnormalities that cause under- or over-expression of a gene.
  • diseases in which an abnormal gene is expressed or a normal gene is over-expressed for example, the presently claimed oligonucleotides may be designed to target the abnormal or normal gene directly, or, in the alternative, to target the gene encoding the protein that promotes expression of the abnormal or normal gene.
  • a normal gene is under-expressed, one may design an oligonucleotide that suppresses expression of a gene encoding a protein that suppresses expression of the normal gene.
  • the target nucleic acid sequence will be a viral nucleic acid sequence.
  • the use of antisense oligonucleotides to inhibit various viruses is well known and has been reviewed in Agrawal, Trends in Biotechnology 10, 152 (1992).
  • Viral nucleic acid sequences that hybridize to effective antisense oligonucleotides have been described for many viruses, including human immunodeficiency virus type I (U.S. Patent No. 4,806,463), Herpes simplex virus (U.S. patent No. 4,689,320), Influenzavirus (U.S. Patent No. 5,194,428), and Human papilloma virus (Storey et al., Nucleic Acids Res. 12, 4109 (1991)).
  • nucleotide sequences complementary to nucleic acid sequences from any other virus can be used, as can nucleotide sequences complementary to nucleic acid sequences from any other virus.
  • Additional viruses that have known nucleic acid sequences against which an antisense oligonucleotide according to the invention can be prepared include, but are not limited to, Foot and Mouth Disease Virus (See Robertson et al. , /. Virology 54, 651 (1985); Harris et al., J. Virology 36, 659 (1980)), Yellow Fever Virus (see Rice et al. , Science 229, 726 (1985)), Varicella-Zoster Virus (see Davison and Scott, J. Gen.
  • the oligonucleotides of the invention can have a nucleotide sequence complementary to a nucleic acid sequence of a pathogenic organism.
  • the nucleic acid sequences of many pathogenic organisms have been described, including the malaria organism, Plasmodiumfalciparum and many pathogemc bacteria.
  • pathogenic eukaryotes having known nucleic acid sequences against which oligonucleotides of the present can be prepared include, but are not limited to Trypanosoma brucei gambiense and Leishmania (see Campbell et al., Nature 311, 350 (1984)), and Fasciola hepatica (see Zurita et al., Proc. Natl. Acad Sci.
  • Antifungal oligonucleotides can be prepared having a nucleotide sequence that is complementary to a nucleic acid sequence from, e.g. , the chitin synthetase gene, and antibacterial oligonucleotides according to the invention can be prepared using, e.g. , the alanine racemase gene.
  • the oligonucleotides can have a nucleotide sequence complementary to a cellular gene or gene transcript, the abnormal expression or product of which results in a disease state.
  • the nucleic acid sequences of several such cellular genes have been described, including prion protein (Stahl and Prusiner, FASEB J. 5, 2799 (1991)), the amyloid-like protein associated with Alzheimer's disease (PCT International Application Publication No. WO 95/09236), and various well-known oncogenes and proto-oncogenes, such as c-myb, c-myc, c-abl, and n-ras..
  • oligonucleotides that inhibit the synthesis of structural proteins or enzymes involved largely or exclusively in spe ⁇ natogenesis, sperm motility, the binding of the sperm to the egg or any other step affecting sperm viability may be used as contraceptives for men.
  • contraceptives for women may be oligonucleotides that inhibit production of proteins or enzymes involved in ovulation, fertilization, implantation or in the biosynthesis of hormones involved in those processes.
  • Hypertension can be controlled by oligonucleotides that suppress the synthesis of angiotensin converting enzyme or related enzymes in the renin angiotensin system; platelet aggregation can be controlled by suppression of the synthesis of enzymes necessary for the synthesis of thromboxane A2 for use in myocardial and cerebral circulatory disorders, infarcts, arteriosclerosis, embolism and thrombosis; deposition of cholesterol in arterial wall can be inhibited by suppression of the synthesis of fatty acyl co-enzyme A: cholesterol acyl transferase in arteriosclerosis; inhibition of the synthesis of cholinephosphotransferase may be useful in hypoiipidemia.
  • oligonucleotides of the present invention can be used to reduce or eliminate adverse effects of the disorder. For example, suppression of the synthesis of monoamine oxidase can be used in Parkinson's disease; suppression of catechol O-methyl transferase can be used to treat depression; and suppression of indole N-methyl transferase can be used in treating schizophrenia.
  • Suppression of selected enzymes in the arachidonic acid cascade may be useful in the control of platelet aggregation, allergy, inflammation, pain and asthma.
  • Suppression of the protein expressed by the multi-drug resistance (mdr) gene which is responsible for development of resistance to a variety of anti-cancer drugs and is a major impediment in chemotherapy may prove to be beneficial in the treatment of cancer.
  • Nucleotide sequences complementary to nucleic acid sequences from any of these genes can be used for the oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to any other cellular gene or gene transcript, the abnormal expression or product of which results in a disease state.
  • oligonucleotides of the invention should be very broad. Still, certain diseases are of
  • a variety of viral diseases may be treated by oligonucleotides having one or more S-triesterphosphorothioates internucleotide linkages, including AIDS, ARC, oral or genital herpes, papilloma warts, flu, foot and mouth disease, yellow fever, chicken pox, shingles, HTLV-leukemia, and hepatitis.
  • oligonucleotides Among fungal diseases treatable by oligonucleotides according to the invention are candidiasis, histoplasmosis, cryptococcocis, blastomycosis, aspergillosis, sporotrichosis, chromomycosis, dematophytosis and coccidioidomycosis.
  • the method can also be used to treat rickettsial diseases (e.g. , typhus, Rocky Mountain spotted fever), as well as sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum.
  • a variety of parasitic diseases can be treated by oligonucleotides of the present invention, including amebiasis, Chagas' disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, and Pneumocystis carini pneumonia; also worm (helminthic diseases) such as ascariasis, filariasis, trichinosis, schistosomiasis and nematode or cestode infections. Malaria can be treated by oligonucleotides of the present invention, regardless of whether it is caused by P. falciparum, P.vivax, P. orale, or P. malaria.
  • infectious diseases identified above can all be treated with oligonucleotides according to the invention because the infectious agents and their gene sequences for these diseases are known, and, thus, oligonucleotides according to the invention can be prepared having a nucleotide sequence that hybridizes to a nucleic acid sequence that is an essential
  • nucleic acid sequence for the propagation of the infectious agent such as an essential gene.
  • an essential gene or nucleic acid is one that is required for a biological process and without which the biological process does not occur.
  • oxazaphospholidine (TV) was obtained by mixing 8.14 g of lR,2S-ephedrine (V) and 10.4
  • structure IV as being the R isomer in which the chlorine atom is disposed trans relative
  • IV could be stored as a solid in a desiccator at -5 °C for several days with no apparent decomposition (as evaluated by
  • the 31 P-NMR spectrum of VI has a signal at ⁇ 140 ppm, corresponding to a single
  • VI can be formulated as having the
  • phosphoramidite VI is a white solid and is stable when stored dry at 0 - 5 ⁇ C.
  • the stage was set for its use in solid-phase coupling with CPG-bound nucleoside.
  • Oligonucleotides are synthesized on a 1 mmoi scale following the standard protocol
  • an automated synthesizer e.g. , Millipore 8700 DNA Synthesizer, Bedford, MA.
  • the phosphoramidite VI is used by dissolving it in dry acetonitrile at a concentration of 50 mg/ml.
  • the iodine oxidation step is replaced by sulftirization with
  • Oligonucleotides are purified by reverse-phase HPLC and/or PAGE, and desalted by using C-1 SEP-PAK cartridges.
  • Diazabicyclononane (DBU) (296 mg, 1.95 mmoi) is dissolved in anhydrous THF
  • Human T cell and leukemia cell line H9 are used in this study. They are cultured in RPMI media supplemented with 10% fetal bovine serum (heat inactivated to 56 °C for
  • Fiuorescein is conjugated to the 5 ' end of the oligonucleotides by either an automated DNA synthesizer or by a manual procedure using a "FLUORESCEIN-ON" phosphoramidite.
  • the efficiency of fiuorescein labeling is determined by using a spectrofluorometer (excitation 488 nm, emission 520 nm).
  • the concentrations of the fiuorescein labeled and unlabelled oligonucleotides in the samples are measured by a spectrofluorometer and UV spectroscopy and adjusted to be the same by adding the corresponding unlabelled oligonucleotides.
  • HBSS Hank's balanced salt solution
  • Propidium iodide (final concentration 10 ⁇ l/ml) is used to distinguish viable cells
  • Flow cytometric data on 5,000 viable cell is acquired in list mode on Epics XL (Coulter, Hialeah, FL), and data are analyzed by Epics XL (version 1.5 software) after gating on living cells by forward scatter versus side scatter and propidium iodide staining.
  • oligonucleotides of the invention to inhibit HIV-1 replication, and thus syncytia formation, in tissue culture is tested in T cell cultures according to the method of Agrawal and Sarin, Advanced Drug Delivery Rev. 6, 251 (1991). Briefly, CEM cells are infected with HIV-1 virions (0.01 - 0.1 TCID 50 /cell) for one hour at 37°C. After one hour unadsorbed virions are washed and the infected cells are divided among walls of 24 well plates. To the infected cells, an appropriate concentration (from stock solution) of oligonucleotide is added to obtain the required concentration in 2 ml medium. The cells
  • HIV expression can be determined by measuring the level of viral protein p24
  • PBS buffered saline
  • oligonucleotide-treated and untreated cells are incubated in a humid chamber at 37°C.
  • the slides are labelled with goat anti-mouse IgG for 30 min and then washed in PBS overnight. The percentage of cells fluorescing in oligonucleotide-treated and untreated cells is compared.
  • This assay is performed essentially as described in Agrawal and Sarin, supra.
  • virus particles precipitated with poly(ethyleneglycol).
  • the virus pellet is suspended in 300 ⁇ l of buffer containing 50 mM Tris-HCl (pH 6.8), 5 mM dithiothreitol
  • solubilized pellet is assayed in a 50 ⁇ l reaction mixture containing 50 mM Tris-HCl (pH
  • Triethyl amine 35 ml, 250 mmoi
  • reaction mixture is allowed to warm to room
  • 2'-Deoxy adenosine (Mallinkckrodt) (2.5 g, 10 mmoi) is dried by repeated evaporation from anhydrous pyridine and is suspended in 50 ml of anhydrous pyridine. Trichloromethylsilane (64. ml, 50 mmoi) is added and the reaction is stirred for about 1 h. Then, 4-pentenoic anhydride (4g, 20 mmoi) is added and the contents stirred. After 15 min triethyl amine (3 ml) was added and the contents stirred for 2-3 h. The reaction
  • the aqueous layer was separated and concentrated to a small volume. Upon leaving at
  • H-phosphonate nucleosides were similarly prepared in overall yields ranging from 70-90% .
  • the support-bound H-phosphonate oligonucleotide is treated with a 10% solution
  • the CPG-bound oligonucleotide is next treated with
  • the solution is evaporated to dryness and the oligonucleotide can be purified by reverse
  • backbone modified oligonucleotides can be prepared by using ROH (for alkyl
  • the support-bound oligo is treated with the iodine solution, as above, to remove the base-protecting group and then with anhydrous triethylamine to remove the phosphate protecting group. Finally cleavage

Abstract

L'invention concerne de nouveaux synthons de mononucléotides présentant un rôle utile dans la synthèse des oligonucléotides dont un à tous les centres chiraux-P se situent de manière prédominante et indépendante dans la configuration en R ou S. L'invention traite également de procédés de synthèse de ces synthons, de procédés de synthèse des oligonucléotides dont un à tous les centres chiraux-P se situent de manière prédominante et indépendante dans la configuration en R ou en S, et ces oligonucléotides. Les oligonucléotides synthétisés avec les nouveaux synthons sont utilisés pour moduler l'expression de l'acide nucléique, tant in vitro que in vivo, ainsi que dans les essais d'hybridation classiques.
PCT/US1996/007430 1995-05-23 1996-05-23 Procedes et composes pour la synthese des oligonucleotides et oligonucleotides ainsi produits WO1996039413A1 (fr)

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US08/448,131 1995-05-23
US08/447,494 1995-05-23
US08/448,131 US5750674A (en) 1995-05-23 1995-05-23 Methods and compounds for the stereoselective enrichment of oligonucleotide diastereomers and oligonucleotides thereby produced
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US5734041A (en) * 1995-10-20 1998-03-31 Mcgill University Preparation of chiral phosphorothioate oligomers
WO1999038878A1 (fr) * 1998-01-29 1999-08-05 Hybridon, Inc. Synthons d'un type nouveau permettant la synthese d'oligonucleotides
US6121437A (en) * 1999-03-16 2000-09-19 Isis Pharmaceuticals, Inc. Phosphate and thiophosphate protecting groups
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EP1111068A1 (fr) * 1999-12-21 2001-06-27 LION Bioscience AG Composé ramifié pour l'utilisation dans des réactions d'analyse et de détection des acides nucléiques
WO2001046464A1 (fr) * 1999-12-21 2001-06-28 Vbc-Genomics Bioscience Research Gmbh Compose ramifie s'utilisant dans des reactions de detection et d'analyse d'acide nucleique
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US9605019B2 (en) 2011-07-19 2017-03-28 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9617547B2 (en) 2012-07-13 2017-04-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant
US9744183B2 (en) 2009-07-06 2017-08-29 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US9982257B2 (en) 2012-07-13 2018-05-29 Wave Life Sciences Ltd. Chiral control
US10144933B2 (en) 2014-01-15 2018-12-04 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
US10149905B2 (en) 2014-01-15 2018-12-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10160969B2 (en) 2014-01-16 2018-12-25 Wave Life Sciences Ltd. Chiral design
US10322173B2 (en) 2014-01-15 2019-06-18 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries

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US5945521A (en) * 1995-10-20 1999-08-31 Mcgill University Preparation of phosphorothioate oligomers
US6031092A (en) * 1995-10-20 2000-02-29 Mc Gill University Preparation of phosphorothioate oligomers
US6160109A (en) * 1995-10-20 2000-12-12 Isis Pharmaceuticals, Inc. Preparation of phosphorothioate and boranophosphate oligomers
US6861518B2 (en) 1995-10-20 2005-03-01 Mcgill University Preparation of phosphorothioate oligomers
US5734041A (en) * 1995-10-20 1998-03-31 Mcgill University Preparation of chiral phosphorothioate oligomers
US6476216B1 (en) 1995-10-20 2002-11-05 Mcgill University Preparation of phosphorothioate oligomers
WO1999038878A1 (fr) * 1998-01-29 1999-08-05 Hybridon, Inc. Synthons d'un type nouveau permettant la synthese d'oligonucleotides
US6121437A (en) * 1999-03-16 2000-09-19 Isis Pharmaceuticals, Inc. Phosphate and thiophosphate protecting groups
US6610837B1 (en) 1999-03-16 2003-08-26 Isis Pharmaceuticals, Inc. Phosphate and thiophosphate protecting groups
US6620796B1 (en) 1999-11-08 2003-09-16 Micrologix Biotech Inc. Combinatorial library synthesis and pharmaceutically active compounds produced thereby
WO2001034622A1 (fr) * 1999-11-08 2001-05-17 Origenix Technologies, Inc. Synthese de bibliotheque combinatoire et composes pharmaceutiquement actifs ainsi produits
WO2001046464A1 (fr) * 1999-12-21 2001-06-28 Vbc-Genomics Bioscience Research Gmbh Compose ramifie s'utilisant dans des reactions de detection et d'analyse d'acide nucleique
EP1111068A1 (fr) * 1999-12-21 2001-06-27 LION Bioscience AG Composé ramifié pour l'utilisation dans des réactions d'analyse et de détection des acides nucléiques
US6825338B2 (en) 2001-03-30 2004-11-30 Isis Pharmaceuticals, Inc. Labeled oligonucleotides, methods for making same, and compounds useful therefor
US7256179B2 (en) 2001-05-16 2007-08-14 Migenix, Inc. Nucleic acid-based compounds and methods of use thereof
US7709449B2 (en) 2001-05-16 2010-05-04 Migenix, Inc. Nucleic acid-based compounds and methods of use thereof
JP2015044842A (ja) * 2008-12-02 2015-03-12 株式会社Ｗａｖｅ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ Ｊａｐａｎ リン原子修飾核酸の合成方法
US9394333B2 (en) 2008-12-02 2016-07-19 Wave Life Sciences Japan Method for the synthesis of phosphorus atom modified nucleic acids
JP2012510460A (ja) * 2008-12-02 2012-05-10 株式会社キラルジェン リン原子修飾核酸の合成方法
US9695211B2 (en) 2008-12-02 2017-07-04 Wave Life Sciences Japan, Inc. Method for the synthesis of phosphorus atom modified nucleic acids
US10329318B2 (en) 2008-12-02 2019-06-25 Wave Life Sciences Ltd. Method for the synthesis of phosphorus atom modified nucleic acids
US9744183B2 (en) 2009-07-06 2017-08-29 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US10307434B2 (en) 2009-07-06 2019-06-04 Wave Life Sciences Ltd. Nucleic acid prodrugs and methods of use thereof
US10428019B2 (en) 2010-09-24 2019-10-01 Wave Life Sciences Ltd. Chiral auxiliaries
US10280192B2 (en) 2011-07-19 2019-05-07 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9605019B2 (en) 2011-07-19 2017-03-28 Wave Life Sciences Ltd. Methods for the synthesis of functionalized nucleic acids
US9617547B2 (en) 2012-07-13 2017-04-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant
US10167309B2 (en) 2012-07-13 2019-01-01 Wave Life Sciences Ltd. Asymmetric auxiliary group
US9982257B2 (en) 2012-07-13 2018-05-29 Wave Life Sciences Ltd. Chiral control
US9598458B2 (en) 2012-07-13 2017-03-21 Wave Life Sciences Japan, Inc. Asymmetric auxiliary group
US10590413B2 (en) 2012-07-13 2020-03-17 Wave Life Sciences Ltd. Chiral control
US10149905B2 (en) 2014-01-15 2018-12-11 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having antitumor effect and antitumor agent
US10144933B2 (en) 2014-01-15 2018-12-04 Shin Nippon Biomedical Laboratories, Ltd. Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
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