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WO1998017675A1 - Procedes ameliores de synthese de mono et d'oligonucleotides recourant au h-phosphonate - Google Patents

Procedes ameliores de synthese de mono et d'oligonucleotides recourant au h-phosphonate Download PDF

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Publication number
WO1998017675A1
WO1998017675A1 PCT/US1996/016636 US9616636W WO9817675A1 WO 1998017675 A1 WO1998017675 A1 WO 1998017675A1 US 9616636 W US9616636 W US 9616636W WO 9817675 A1 WO9817675 A1 WO 9817675A1
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WO
WIPO (PCT)
Prior art keywords
phosphonate
nucleotide
hydroxyl
oligonucleotide
moiety
Prior art date
Application number
PCT/US1996/016636
Other languages
English (en)
Inventor
Nandkumar Bhongle
Original Assignee
Hybridon, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hybridon, Inc. filed Critical Hybridon, Inc.
Priority to PCT/US1996/016636 priority Critical patent/WO1998017675A1/fr
Priority to CA002205218A priority patent/CA2205218C/fr
Priority to AU74499/96A priority patent/AU7449996A/en
Publication of WO1998017675A1 publication Critical patent/WO1998017675A1/fr

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Classifications

    • 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
    • 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
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • This invention relates to new methods of synthesizing mononucleoside H- phosphonates and oiigonucleotides using the H-phosphonate method.
  • Antisense oiigonucleotides 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 oiigonucleotides may be designed to bind directly to DNA (the so-called "anti-gene” approach) or to mRNA. Id. Expression inhibition is believed to occur by prevention of transcription or translation * or inducement of target mRNA cleavage by RNase H.
  • Antisense oiigonucleotides can be used as a research tool in vitro to determine the biological function of genes and proteins. They provide an easily used alternative
  • Antisense oiigonucleotides also may be used to treat a variety of pathogenic diseases by inhibiting nucleic acid expression of the pathogen in vivo.
  • the first nucleotide (monomer 1) is bound by its 3' hydroxyl to a solid matrix while its 5' hydroxyl remains available for binding.
  • the synthesis of the first internucleotide link is carried out by mixing bound monomer 1 with a second nucleotide that has a reactive
  • the H-phosphonate method involves condensing the 5' hydroxyl group of the nascent oligonucleotide with a nucleoside having a 3'
  • a new method of synthesizing mononucleoside H-phosphonates comprises contacting a 5'- and base- protected mononucleoside with phosphonic acid and benzoic anhydride at room temperature. The resulting product is the desired mononucleoside H-
  • a catalytic amount of triphosgene is added to the reaction mixture.
  • the resulting product is the desired mononucleoside H-phosphonate.
  • triphosgene accelerates the reaction
  • a new method of coupling nucleosides comprises contacting a 5 '-protected nucleoside or oligonucleotide having a 3' phosphonate moiety with a 3 '-protected mononucleoside having a free 5 ' hydroxyl in the presence of benzoic anhydride.
  • the coupling reaction is accelerated by the addition of a catalytic amount of triphosgene.
  • a new method of synthesizing oligonucleotide phosphodiesters and phosphorothioates comprises repeated nucleoside coupling according to the first and/or second aspect of the invention followed by oxidation to produce the phosphodiester or oxidative sulfurization to produce the phosphorothioate.
  • Benzoic anhydride is a stable, inexpensive, commercially available crystalline solid. Like triphosgene, it offers many of the same advantages
  • the present invention generally comprises new methods for synthesizing nucleotide monomers useful for constructing oiigonucleotides by the H-phosphonate method, as well as methods for constructing oiigonucleotides.
  • a new method is provided for the synthesis of nucleoside H-phosphonate monomers.
  • the method comprises contacting a mononucleoside having a 3' hydroxyl moiety with benzoic anhydride and an excess of phosphonic acid (H 3 PO 3 ).
  • H 3 PO 3 phosphonic acid
  • the reaction product is the desired mononucleoside H-phosphonate.
  • the mononucleoside is suitably protected, for example, at the 5' position (e.g. , with DMT) and, if necessary, at the
  • suitable and “suitably,” when used to describe a general class of compounds, methods, or techniques (as the case may be) that serves a desired function means any compound, method, or technique of that class that does not cause or induce undesirable side effects that would either defeat the purpose for which the compound, method, or technique is used, or, on balance, outweigh the benefits of using the particular compound, method, or technique.
  • a “suitable solvent” is any solvent that is capable of dissolving the starting materials, permits the reaction to proceed, and does not itself chemically react with the starting or ending materials.
  • a suitable protecting group is one that prevents reaction
  • protecting group encompasses not only moieties traditionally used to prevent side reactions at the site to which it is bound (e.g. , DMT or an acetyl moiety), but also any other chemical moiety (such as a mono- or oligonucleotide) that effects a protecting group type function.
  • the 3' most N-l nucleotides of a N-mer oligonucleotide will serve as a 3 ' protecting group for the 5 ' nucleotide of the N-mer.
  • oligonucleotide refers to any nucleic acid chain comprising at least two nucleotides and that can be chemically synthesized.
  • a catalytic amount of triphosgene is added to accelerate the coupling reaction.
  • a "catalytic amount" of triphosgene means an amount that is about 10-15 times less than the amount of benzoic anhydride used. In this embodiment the reaction is slightly exothermic on a small scale. Care should be taken, therefore, in large scale preparations.
  • a new method for the synthesis of dinucleotides.
  • This method comprises contacting, in the presence of benzoic anhydride, a first mononucleoside having a 3' H-phosphonate with a second mononucleoside having a free 5' hydroxyl.
  • the result is a dinucleoside H- phosphonate.
  • the first mononucleoside is suitably protected, for example, at the 5'-0 position (e.g., by DMT) and, if necessary, at the base, and the second
  • a mononucleoside is suitably protected at the '-O (e.g. , by an acetyl moiety) and, if necessary, the base. Any suitable solvent may be used. In a preferred embodiment, a catalytic amount of triphosgene is added to accelerate the reaction. In a third aspect of the present invention, a new method is provided for the synthesis of oiigonucleotides.
  • This method comprises contacting, in the presence of benzoic anhydride, a nascent oligonucleotide having a free 5' hydroxyl moiety with a mono- or oligo- nucleotide having a 3 ' H-phosphonate moiety to yield an oligonucleotide H-phosphonate that is one or more nucleotide(s) greater in length
  • oligonucleotide can then be treated with additional nucleotides (mono- or oligo-) in the presence of benzoic anhydride to further increase its length. This procedure is repeated until the desired oligonucleotide sequence has been synthesized.
  • the nascent oligonucleotide can be of any conveniently synthesized length and is preferably anchored to a solid support. Preferably, the oligonucleotide is suitably protected, for example, at its 3' end (e.g.
  • the solid support and, if necessary, at the bases, and the mono- or oligo- nucleotide is suitably protected at the 5 '-O end (e.g. , by DMT) and, if necessary the base or bases.
  • a catalytic amount of triphosgene is added to accelerate the reaction.
  • the method of synthesizing an oligonucleotide comprises sequentially:
  • the reaction mixture generally comprises one or more suitable solvent
  • the desired oligonucleotide is incrementally synthesized
  • the desired oligonucleotide is synthesized in increments
  • Oiigonucleotides synthesized according to either the second or third aspects of the invention may be subjected to oxidation with, for example, iodine to yield an oligonucleotide in which the H-phosphonate internucleoside linkages are converted to phosphodiesters. Froehler, id. at 63-80.
  • the oligonucleotide H- phosphonate may be subjected to oxidative sulfurization (e.g. , Iyer et al. , J. Org. Chem. 55, 4693 (1990)) to yield an oligonucleotide in which the H-phosphonate internucleoside linkages have been converted to phosphorothioates.
  • the methods according to the invention are advantageously used with either ribo- or deoxyribo- mononucleosides and oiigonucleotides. Ribonucleotides will have to be protected at the 2'-0 position.
  • the benzoic anhydride activator is preferably added to a mixture of the reactants and, if it is to be used, the triphosgene catalyst added subsequent to the benzoic anhydride.
  • Table 1 displays the data from synthesizing four different mononucleotide H- phosphonates by the foregoing protocol. All yields correspond to isolated pure compounds. The reaction conditions were not optimized. 31 P NMR of the crude products confirmed the H-phosphonate product structures. TLC of the pure products was identical to the commercially available materials.
  • the resin is washed with acetonitrile several times.
  • the resin is washed with pyridine/acetonitrile several times.
  • Oxidation An I 2 solution or sulfur solution is used to oxidize the H-phosphonate linkages to phosphate (PO or PS) linkages. This reaction can be performed in a round bottom flask and, therefore, need not be conducted in an automated synthesizer.

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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)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention concerne de nouveaux procédés de synthèse H-phosphonates de mono et d'oligonucléotides. Ces procédés comprennent la mise en contact d'un mononucléoside avec de l'acide phosphonique et de l'anhydride de benzoyle pour obtenir le H-phosphanate de mononucléoside correspondant. De préférence, on utilise aussi une quantité catalytique de triphosgène. On peut recourir à une procédure similaire pour coupler un premier ou à un deuxième mononucléoside ou à un oligonucléotide, le procédé comprenant la mise en contact, en présence d'anhydride benzoïque et, de préférence, d'une quantité catalytique de triphosgène, d'un mononucléotide ou d'un oligonucléotide ayant un radical hydroxyle 5' libre avec un mononucléoside ayant un fragment phosphoreux (de préférence du H-phosphonate) portant un radical hydroxyle 3'.
PCT/US1996/016636 1995-10-20 1996-10-18 Procedes ameliores de synthese de mono et d'oligonucleotides recourant au h-phosphonate WO1998017675A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/US1996/016636 WO1998017675A1 (fr) 1996-10-18 1996-10-18 Procedes ameliores de synthese de mono et d'oligonucleotides recourant au h-phosphonate
CA002205218A CA2205218C (fr) 1995-10-20 1996-10-18 Methodes ameliorees pour la synthese de mono- et d'oligonucleotides
AU74499/96A AU7449996A (en) 1996-10-18 1996-10-18 Improved methods for h-phosphonate synthesis of mono- and oligonucleotides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1996/016636 WO1998017675A1 (fr) 1996-10-18 1996-10-18 Procedes ameliores de synthese de mono et d'oligonucleotides recourant au h-phosphonate

Publications (1)

Publication Number Publication Date
WO1998017675A1 true WO1998017675A1 (fr) 1998-04-30

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AU (1) AU7449996A (fr)
WO (1) WO1998017675A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0219342A2 (fr) * 1985-10-15 1987-04-22 Genentech, Inc. Méthode et réactifs pour la synthèse in vitro d'oligonucléotides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0219342A2 (fr) * 1985-10-15 1987-04-22 Genentech, Inc. Méthode et réactifs pour la synthèse in vitro d'oligonucléotides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BHONGLE, NANDKUMAR N. ET AL: "A convenient synthesis of nucleoside 3'-H- phosphonate monoesters using triphosgene", TETRAHEDRON LETTERS, vol. 36, no. 38, 1995, OXFORD GB, pages 6803 - 6806, XP000570437 *
SEKINE M. ET AL: "A convenient method for the synthesis of deoxyribonucleoside 3'-hydrogenphosphonates", TETRAHEDRON LETTERS, vol. 29, no. 9, 1988, OXFORD GB, pages 1037 - 1040, XP002026387 *
STAWINSKI J ET AL: "NUCLEOSIDE H-PHOSPHONATES. XI A CONVENIENT METHOD FOR THE PREPARATION OF NUCLEOSIDE H-PHOSPHONATES", NUCLEOSIDES & NUCLEOTIDES, vol. 9, no. 1, 1 January 1990 (1990-01-01), pages 129 - 135, XP000571749 *
STAWINSKI J. AND THELIN M.: "Studies on the Activation Pathway of Phophonic Acid using Acyl Chlorides as Activators", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 2, 1990, LETCHWORTH GB, pages 849 - 853, XP002005191 *

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Publication number Publication date
AU7449996A (en) 1998-05-15

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