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WO1986007362A1 - Methode de synthese de desoxyoligonucleotides - Google Patents

Methode de synthese de desoxyoligonucleotides Download PDF

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Publication number
WO1986007362A1
WO1986007362A1 PCT/US1985/001148 US8501148W WO8607362A1 WO 1986007362 A1 WO1986007362 A1 WO 1986007362A1 US 8501148 W US8501148 W US 8501148W WO 8607362 A1 WO8607362 A1 WO 8607362A1
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WO
WIPO (PCT)
Prior art keywords
compound
formula
phosphoramidites
morpholino
bis
Prior art date
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Ceased
Application number
PCT/US1985/001148
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English (en)
Inventor
Marvin H. Caruthers
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University Patents Inc
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University Patents Inc
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Publication date
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Priority to PCT/US1985/001148 priority Critical patent/WO1986007362A1/fr
Priority to EP19850903503 priority patent/EP0229053A1/fr
Publication of WO1986007362A1 publication Critical patent/WO1986007362A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/6533—Six-membered rings
    • 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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • the present invention relates to a new and novel method for the preparation of deoxynucleoside phos ⁇ phoramidites in situ from deoxynucleosides, bis- dial ylaminophosphines, and the corresponding dialkyl ⁇ amine hydrotetrazolide or tetrazole as catalyst for the reaction.
  • These phosphoramidites lead to the direct synthesis of deoxyoligonucleotides on various polymer supports.
  • Modified inorganic polymers are known in the prior art, primarily for use as absorption materials, for example, in liquid chromatography.
  • the attach ⁇ ment of nucleosidephosphates to silica gel using a trityl linking group is described in the prior art but the method is apparently applicable only to pyri idine nucleosides.
  • the cleavage of the nucleo- side from the silica support can only be accomplished with acid to which the purine nucleosides are sensi ⁇ tive.
  • phosphotriester derivatives of oligothymidylates is described in the prior art by reaction of a phosphorodichloridite with a 5'-0 blocked thymidine and subsequent reaction of the product with a 3'-0 blocked ' thymidine. This is followed by oxidation of the resulting phosphite to a phosphate and removal of blocking groups to obtain the phosphotriesters.
  • the process requires separation and purification of products at each stage to ensure proper sequencing of the added nucleosides. Separation techniques including precipi ⁇ tation and washing of precipitates are necessary to implement each successive stage reaction.
  • U.S. Patent No. 4,415,732 describes a new class of nucleoside phosphoramidites derived from saturated secondary amines, which are relatively stable. This permits isolation and storage of the compounds, at room temperature. These phosphoramidite compounds have been found to have excellent use in the forma ⁇ tion of the internucleotide bonds described in the earlier mentioned U.S. Patent. The disclosure of this patent is incorporated in toto herein.
  • B may be 1-thyminyl; l-(N-4 benzoyl cyto- sinyl) ; 9-(N-2-isobutyrylquaninyl) ; or 9-(N-6-benzoyl- adeninyl) , wherein Z may be morpholino or N(lower alkyl)-, preferably (i-propyl)_; and wherein DMT is di-p-anisylphenyl methyl.
  • phosphoramidites are activated by tetra ⁇ zole, with the 5'-hydroxyl group of a deoxynucleoside or deoxyoligonucleotide attached covalently to a polymer support (see, for example, Caruthers, M. H. , Beaucage, S. L., Becker, C. , Efcavitch, J. . , Fisher, E. F. , Galluppi, G. , Goldman, R. A., de- Haseth, P. L., Martin, F. , Matteucci, M. D. and Stabinsky, Y. ,(1982) in Genetic Engineering, Setlow, J. and Hollaender, A. Eds., Vol.
  • phosphoramidites can be prepared by existing methods (see, for example, Beaucage, S. L. and Caruthers, M. H. (1981) Tetrahedron Lett. 22, 1859-1862; and McBride, L. J. and Caruthers, M. H. (1983) ibid. 24, 245-248), from the appropriately protected deoxynucleosides of the following struc ⁇ tural formula:
  • DMT and B are as defined for the compounds of Formula I above, and chlorphosphines of the following structural formula:
  • chlorophosphines used in the preparation of the compounds of Formula I, are difficult to prepare and easily react with trace amounts of water.
  • the high reactivity of the chlorophosphines and the concomitant production- of insoluble amine hydrochloride salts preclude their use for any strategy involving the in situ generation of deoxynu ⁇ cleoside phosphoramidites for deoxyoligonucleotide synthesis on solid supports, (see, for example Fourrey, J. L. and Shire, D. (1981) Tetrahedron Lett. 22, 729-732) .
  • the reaction is furthermore found to be catalytic in either tetrazole or the salt, as well as a greater selectivity of activation.
  • This preparation has been successfully applied to the synthesis of deoxyoligo- nucleotides directly on a solid support via an in situ approach.
  • Example IV shows a typical experi ⁇ mental procedure for the preparation of phosphor ⁇ amidites according to the present invention. Al ⁇ though the description is specific for the formation of the preparation of the compound of general Formula I wherein B is 1-thyminyl and Z is diisopropyl amino, the remaining compounds of general Formula I may be prepared along similar protocols by those skilled in the synthesis art.
  • the 31P NMR spectral data for phosphoramidites prepared following the typical procedure given in Example IV are contained in Table
  • the bis-morpholinomethoxyphosphine was prepared in a similar manner (80% yield) and had stability comparable to that obtained for the bis-(diisopropylamino)methoxy ⁇ phosphine.
  • the mixed (diisopropylaminomorpholino) methoxyphosphine could not be obtained cleanly in the same one pot procedure as above; however, treatment of the mixed (diisopropylaminochloro)methoxyphosphine with one equivalent of morpholine and triethylamine gave a 77% yield of methoxyphosphine contaminated with about 2% of the thermodynamically more stable bis-(diisopropylamino) methoxyphosphine.
  • the typical preparation of phosphoramidites depicted in Example IV required the addition of 1.1 equivalents of the diisopropyl phosphine to a mixture of the nucleoside and 0.5 equivalents of the catalyst (general Formula V) in dichloromethane.
  • the TLC showed the reaction to be complete in about 20 min. After an aqueous work-up and precipitation of the material in cold hexanes, the product was obtained in 87% yield as an amorphous solid.
  • the 31P NMR spectrum showed two signals corresponding to a 1:1 diastereo eric mixture of phosphoramidites.
  • Phosphoramidites prepared using the procedure according to the present invention were tested as syn- thons by constructing d(GGGAATTCCC) , a self-complementary segment containing the EcoRI recognition sequence.
  • the deoxyoligonucleotide was synthesized and deprotected using standard procedures according to Caruthers in Chemical and Enzymatic Synthesis of Gene Fragments, A Laboratory Manual, and isolated by gel electrophoresis in a 57% yield.
  • the average coupling yield (measured spectrophotometrically from the dimethoxytrityl cation,
  • Bis-(diisopropylamino) ethoxyphosphine was also tested as part of an _in situ synthesis protocol.
  • Phos ⁇ phoramidites according to general Formula I were each prepared as 0.1M solutions in dry acetonitrile containing bis-(diisopropylamino)methoxyphosphine (1.0 eq) and the diisopropylamine tetrazolides of general Formula V (0.5 eq) .
  • the segment d(GGGAATTCCC) was then prepared in 50% isolated yield (average coupling yield was 94%) using the following procedure.
  • the present invention demonstrates that very stable dialkylamino phosphines according to general Formula IV can be used as phosphitylating reagents to form deoxynucleoside phosphoramidites cleanly and in good yields.
  • the reactions are catalytic in either tetrazole or the corresponding amine hydrotetrazolides. These reactions have furthermore been found to be selective to the formation of only 3'-deoxynucleoside phosphoramidites without concurrent hydrolysis to phosphonus acid or synthesis of the 3'-3 * dinucleoside phosphit-3. This selectivity renders these reagents attractive for the in situ generation of phosphoramidites useful ' for DNA synthesis on solid supports.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)

Abstract

Des phosphoramidites de désoxynucléosides sont préparés avec un rendement élevé à partir de désoxynucléosides, de bis-dialkylamino-phosphines, avec l'hydrotétrazolure de dialkylamine ou le tétrazole correspondant comme catalyseur pour la réaction. Ces phosphoramidites produits in situ conduisent à la synthèse directe de désoxyoligonucléotides sur des supports polymères.
PCT/US1985/001148 1985-06-14 1985-06-14 Methode de synthese de desoxyoligonucleotides Ceased WO1986007362A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1985/001148 WO1986007362A1 (fr) 1985-06-14 1985-06-14 Methode de synthese de desoxyoligonucleotides
EP19850903503 EP0229053A1 (fr) 1985-06-14 1985-06-14 Methode de synthese de desoxyoligonucleotides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1985/001148 WO1986007362A1 (fr) 1985-06-14 1985-06-14 Methode de synthese de desoxyoligonucleotides

Publications (1)

Publication Number Publication Date
WO1986007362A1 true WO1986007362A1 (fr) 1986-12-18

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EP (1) EP0229053A1 (fr)
WO (1) WO1986007362A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0266168A3 (fr) * 1986-10-31 1990-10-03 Amoco Corporation Compositions et méthodes de synthèse d'oligonucléotides dont l'extrémité 5' est phosphorylée
EP0476071A4 (en) * 1989-06-05 1992-11-04 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides and methods for preparing the same
US5256775A (en) * 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
EP0595839A4 (fr) * 1991-07-12 1994-08-03 Applied Biosystems, Inc.
US5623068A (en) * 1994-03-07 1997-04-22 Beckman Instruments, Inc. Synthesis of DNA using substituted phenylacetyl-protected nucleotides
WO1997019092A1 (fr) * 1995-11-17 1997-05-29 Isis Pharmaceuticals, Inc. Procede ameliore de synthese de composes oligomeres
WO1997042208A1 (fr) * 1996-05-03 1997-11-13 Hybridon, Inc. Preparation in situ de phosphoramidites de nucleosides et leur utilisation pour la synthese d'oligonucleotides
WO1999051547A3 (fr) * 1998-04-07 2000-07-06 Chemicus Inc Synthese en phase solide de composes organiques au moyen de reactifs de phosphitylation
US6451998B1 (en) 1999-10-18 2002-09-17 Agilent Technologies, Inc. Capping and de-capping during oligonucleotide synthesis

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4415732A (en) * 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
EP0097805A2 (fr) * 1980-02-29 1984-01-11 University Patents, Inc. Nulcléoside phosphites
WO1984001778A1 (fr) * 1982-10-28 1984-05-10 Hubert Koester Procede de production de phosphonates d'oligonucleosides
DE3247923A1 (de) * 1982-12-24 1984-06-28 Dr. Karl Thomae Gmbh, 7950 Biberach Neue oligonucleotide und verfahren zu ihrer herstellung
US4458066A (en) * 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) * 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0097805A2 (fr) * 1980-02-29 1984-01-11 University Patents, Inc. Nulcléoside phosphites
US4458066A (en) * 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) * 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US4415732A (en) * 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
WO1984001778A1 (fr) * 1982-10-28 1984-05-10 Hubert Koester Procede de production de phosphonates d'oligonucleosides
DE3247923A1 (de) * 1982-12-24 1984-06-28 Dr. Karl Thomae Gmbh, 7950 Biberach Neue oligonucleotide und verfahren zu ihrer herstellung

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 101 No. 19, issued 1984, LEE et al., "Bis-(N, N-Dialkylamino)Alkoxy Phosphines as a New Class of Phosphite Coupling Agent for the Synthesis of Oligonucleotides" No. 101: 171651w *
CHEMICAL ABSTRACTS, Volume 101 No. 23, issued 1984, BARONE et al., "In Situ Activation of Bisdialkyl-Aminophosphines-A New Method for Synthesizing Deoxyligonucleotides on Polymer Supports", No. 101:211639g *
CHEMICAL ABSTRACTS, Volume 102 No. 15, issued 1985, BEAUCAGE, "Nucleoside Phosphoramidite Inter-Mediates", No. 102: 132425b *
CHEMICAL ABSTRACTS, Volume 75 No. 5, issued 1971, NIFANT'EV et al. "Reaction of Tetraethylphosphorodiamidous Acid Esters with Hydroquinone" No. 75: 35063y *
CHEMICAL ABSTRACTS, Volume 86 No. 22, issued 19778 DOL'NIK, N. et al. "Reactions of Amidophosphites with Phenols and Bisphenols" No. 86: 156030w *
CHEMICAL ABSTRACTS, Volume 87 No. 5, issued 1977, BATYEVA et al "Reaction of Isothiocyanates with Amides of Phosphorous (III) Acids" No. 87: 39598X *
CHEMICAL ABSTRACTS, Volume 96 No. 17, issued 1982, NURETDINOVA et al., "Synthesis and Reactivity of some Sulfer-Containing Three-and Four-Membered Rings" No. 96: 142586b *
Tetrahedron, Volume 23, issued 1967, ZWIERAK, A. et al, "Phosphorous Acid Amides-II, pages 2243-2252 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0266168A3 (fr) * 1986-10-31 1990-10-03 Amoco Corporation Compositions et méthodes de synthèse d'oligonucléotides dont l'extrémité 5' est phosphorylée
EP0476071A4 (en) * 1989-06-05 1992-11-04 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides and methods for preparing the same
US5256775A (en) * 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
EP0595839A4 (fr) * 1991-07-12 1994-08-03 Applied Biosystems, Inc.
US5623068A (en) * 1994-03-07 1997-04-22 Beckman Instruments, Inc. Synthesis of DNA using substituted phenylacetyl-protected nucleotides
US6051699A (en) * 1995-11-17 2000-04-18 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US5705621A (en) * 1995-11-17 1998-01-06 Isis Pharmaceuticals, Inc. Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same
US5859232A (en) * 1995-11-17 1999-01-12 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric phosphite, phosphodiester, phosphorothioate and phosphorodithioate compounds
WO1997019092A1 (fr) * 1995-11-17 1997-05-29 Isis Pharmaceuticals, Inc. Procede ameliore de synthese de composes oligomeres
WO1997042208A1 (fr) * 1996-05-03 1997-11-13 Hybridon, Inc. Preparation in situ de phosphoramidites de nucleosides et leur utilisation pour la synthese d'oligonucleotides
WO1999051547A3 (fr) * 1998-04-07 2000-07-06 Chemicus Inc Synthese en phase solide de composes organiques au moyen de reactifs de phosphitylation
US6248877B1 (en) 1998-04-07 2001-06-19 Biolink Partners Solid phase synthesis of organic compounds via phosphitylating reagents
US6451998B1 (en) 1999-10-18 2002-09-17 Agilent Technologies, Inc. Capping and de-capping during oligonucleotide synthesis

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Publication number Publication date
EP0229053A1 (fr) 1987-07-22

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