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WO1993013118A1 - Synthese des analogues phosphorothioate des oligonucleotides et polynucleotides - Google Patents

Synthese des analogues phosphorothioate des oligonucleotides et polynucleotides Download PDF

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Publication number
WO1993013118A1
WO1993013118A1 PCT/GB1992/002395 GB9202395W WO9313118A1 WO 1993013118 A1 WO1993013118 A1 WO 1993013118A1 GB 9202395 W GB9202395 W GB 9202395W WO 9313118 A1 WO9313118 A1 WO 9313118A1
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WIPO (PCT)
Prior art keywords
sulphur
oligo
defined above
transfer agent
dibenzoyl
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
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PCT/GB1992/002395
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English (en)
Inventor
Colin Bernard Reese
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Kings College London
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Kings College London
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Filing date
Publication date
Application filed by Kings College London filed Critical Kings College London
Publication of WO1993013118A1 publication Critical patent/WO1993013118A1/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
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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 the synthesis of phosphorothioate analogues of oligo- and poly- nucleotides; more particularly, it relates to the solid phase synthesis of phosphorothioate analogues of, preferably, oligo- and poly- deoxyribonucleotides from phosphora idite building blocks using certain diacyl tri- and tetra-sulphides as sulphur-transfer agents.
  • Antisense oligodeoxyribonucleotides (see, for example, "Oligodeoxynucleotides - Antisense inhibitors of gene expression", Cohen, J.S., Ed., Macmillan, London, 1989), which are complementary to particular RNA targets, may inhibit translation and thereby provide a highly specific method for the inhibition of gene expression.
  • Phosphorothioate analogues of oligonucleotides in which one of the non-bridging oxygen atoms of the internucleotide linkages is replaced by a sulphur atom are much more resistant to nuclease-promoted degradation than corresponding unmodified oligomers, (see, for example, Eckstein, F., Ann. Rev. Biochem. 54 . , 367-402, 1985) , and are therefore potentially valuable antisense therapeutic agents.
  • oligonucleotide phosphorothioates have been prepared by the phosphotriester approach in solution, (see, for example, Burgers, P.M.J. , and Eckstein, F. , Biochemistry, 18., 592-596, 1979; and Kemal, 0., et al, J. Chem. Soc, Chem. Co mun., 591-593, 1983) , they are at present much more conveniently prepared by solid phase synthesis using suitably-protected nucleoside H-phosphonate, (see, for example, Froehler, B.C., Tet.
  • the H-phosphonate approach does not permit the synthesis of oligonucleotides containing both natural phosphodiester and modified (e.g. phosphorothioate diester) internucleotide linkages, and is considered by some workers to be less efficient than the phosphoramidite approach, (see, for example, Iyer, R.P., et a_l, J. Org. Chem., 55., 4693-4699, 1990; and Vu, H. , and Hirschbein, B.L., Tet. Lett., .32 . , 3005-3008, 1991).
  • n 3 or ;
  • X represents RC(O)- or RC(S)- wherein R represents alkyl, cycloalkyl or aryl, (R'O)C(O)- wherein R represents alkyl, (R 2 N)C(0)- or (R 2 N)C(S)- wherein R is as defined above and R 2 NH may be a cyclic 10 secondary amine,
  • an easily prepared crystalline solid may 5 be used as an efficient sulphur-transfer agent which appears both to have good solubility properties and to transfer sulphur very rapidly.
  • the presently-preferred reagent may be readily prepared in 81% yield in a one-step reaction from thiobenzoic acid and sulphur monochloride, 0 which are both relatively cheap, commercially-available starting materials; it may be isolated as a stable, pale yellow crystalline solid that is extremely soluble in chloroform, dichloro ethane and tetrahydrofuran, but not in acetonitrile. 5
  • the presently-preferred dibenzoyl tetrasulphide was prepared by an advantageous modification of the original literature procedure.
  • a solution of triethylamine (16.2 ml, 0.12 mol) in dry tetrahydrofuran (100 ml) was added dropwise over a period of 20 minutes to a stirred solution of redistilled thiobenzoic acid (16.5 g, 0.12 mol) and sulphur monochloride (6.0 ml, 0.075 mol) in dry tetrahydrofuran (150 ml) at 0°C (ice-water bath) .
  • the products were stirred for a further period of 1 hour at room temperature and were then filtered.
  • one preferred embodiment of the present invention concerns the use of, in particular, dibenzoyl tetrasulphide as a sulphur-transfer agent, it is not so- limited.
  • dibenzoyl tetrasulphide as a sulphur-transfer agent
  • the main requirements for a sulphur-transfer agent in the context of oligonucleotide phosphorothioate synthesis are as follows:
  • X-S 4 -X and X-S 3 -X may be considered for use as effective sulphur-transfer agents.
  • R represents an aryl group, which may be substituted, for example, with para- or meta- methyl.
  • the preparation of such reagents is within the competence of those skilled in the art.
  • dibenzoyl tetrasulphide As dibenzoyl tetrasulphide was found to be the most reactive of the three reagents here examined in most detail, it was decided to investigate its use in solid phase oligonucleotide synthesis.
  • the efficacy of dibenzoyl tetrasulphide as a sulphur-transfer agent was compared with that of the recently-recommended tetraethylthiura disulphide in the solid phase synthesis of the nonadecadeoxyribonucleoside octadecaphosphorothioate , d[A(S)T(S)T(S)C(S)C(S)G(S)G(S)A(S)C(S)T(S)C(S)G(S)T(S)C(S)- C(S)A(S) C(S) C(S)C(S)A] .
  • Solid phase synthesis was carried out on a 0.2 ⁇ molar scale in an Applied Biosystems 381A DNA Synthesizer. 5'-0-(4,4'-dimethoxytrityl) -2'-deoxy- ribonucleoside 3 '-(2-cyanoethyl) -N,N-diisopropylphosphor- a idites with standard base protecting groups (i.e. benzoyl on adenine and cytosine, and isobutyryl on guanine) were used as building blocks, and lH-tetrazole was used as activating agent.
  • standard base protecting groups i.e. benzoyl on adenine and cytosine, and isobutyryl on guanine
  • Sulphur-transfer was effected in each of the 18 synthetic cycles either with (a) 0.4M dibenzoyl tetrasulphide in commercially-supplied, base-free 0.025% butylated-hydr ⁇ xytoluene(anti-peroxidation stabilizer) - containing tetrahydrofuran for 60 seconds or (b) 0.5M tetraethylthiuram disulphide in acetonitrile for 900 seconds as recommended by Vu and Hirschbein (loc c_it) .

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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)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention décrit un procédé pour la production en phase solide d'un analogue phosphorothioate d'un oligonucléotide ou polynucléotide à partir des constituants de la phosphoramidite en présence d'un agent de transfert sulfurique. Le procédé se caractérise par le fait que l'agent de transfert sulfurique correspond à la formule générale suivante X - Sn - X dans laquelle n vaut 3 ou 4; et X représente RC(O)- ou RC(S)- dans laquelle R représente un alkyle, un cycloalkyle ou un aryle, (R'O)C(O)- dans laquelle R' représente un alkyle, (R2N)C(O)- ou (R2N)C(S)- dans laquelle R est défini tel que ci-dessus et R2NH peut être une amine secondaire cyclique, RSO2- dans laquelle R est défini tel qui ci-dessus, (RO)2P(O)- ou (RO)2P(S)- dans laquelle R est defini tel que ci-dessus.
PCT/GB1992/002395 1991-12-23 1992-12-23 Synthese des analogues phosphorothioate des oligonucleotides et polynucleotides Ceased WO1993013118A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9127243.5 1991-12-23
GB919127243A GB9127243D0 (en) 1991-12-23 1991-12-23 Synthesis of phosphorothioate analogues of oligonucleotides

Publications (1)

Publication Number Publication Date
WO1993013118A1 true WO1993013118A1 (fr) 1993-07-08

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1992/002395 Ceased WO1993013118A1 (fr) 1991-12-23 1992-12-23 Synthese des analogues phosphorothioate des oligonucleotides et polynucleotides

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GB (1) GB9127243D0 (fr)
WO (1) WO1993013118A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0723973A1 (fr) * 1995-01-25 1996-07-31 King's College London Dérivés de nucléosides phosphorothioatés, leur synthèse et leur utilisation
US6172217B1 (en) 1996-12-27 2001-01-09 Isis Pharmaceuticals Inc. Method of synthesizing phosphorothioate oligonucleotides
EP1023310A4 (fr) * 1997-10-15 2001-05-16 Isis Pharmaceuticals Inc Synthese amelioree d'oligonucleotides soufres
US7378516B2 (en) 1997-10-15 2008-05-27 Isis Pharmaceuticals, Inc. Synthesis of sulfurized oligonucleotides
JP2008266331A (ja) * 2004-04-05 2008-11-06 Alnylam Pharmaceuticals Inc オリゴヌクレオチドの合成および精製に使用する方法および反応試薬

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BERICHTE DER DEUTSCHEN CHEMISCHEN GESELLSCHAFT vol. 53, 1920, WEINHEIM DE pages 961 - 977 I.BLOCH ET AL. 'Uber Trisulfide und Tetrasulfide einiger Carbonsauren. VI. Mitteilung uber Wasserstoffpersulfide.' cited in the application *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. vol. 106, no. 20, 3 October 1984, GASTON, PA US pages 6077 - 9 W.J.STEC ET AL. 'Automated Solid-Phase Synthesis, Separation and Stereochemistry of Phosphorothioate Analogues of Oligodeoxyribonucleotides.' cited in the application *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. vol. 112, no. 3, 31 January 1990, GASTON, PA US pages 1253 - 4 R.P.IYER ET AL. '3H-1,2-Benzodithiole-3-one 1,1'-Dioxide as an Improved Sulfurizing Reagent in the Solid-Phase Synthesis of Oligodeoxyribonucleoside Phosphorothioates.' cited in the application *
TETRAHEDRON LETTERS. vol. 30, no. 48, 1989, OXFORD GB pages 6757 - 6760 P.C.J.KAMER ET AL. 'An efficient Approach Toward the Synthesis of Phosphorothioate Diesters via the Schonberg reaction.' cited in the application *
TETRAHEDRON LETTERS. vol. 32, no. 26, June 1991, OXFORD GB pages 3005 - 8 H.VU ET AL. 'Internucleotide Phosphite Sulfurization with Tetraethylthiuram Disulfide. Phosphorothioate Oligonucleotide Synthesis via Phosphoramidite Chemistry.' cited in the application *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0723973A1 (fr) * 1995-01-25 1996-07-31 King's College London Dérivés de nucléosides phosphorothioatés, leur synthèse et leur utilisation
US5708161A (en) * 1995-01-25 1998-01-13 King's College London Nucleoside phosphorothioate derivatives, synthesis and use thereof
US6172217B1 (en) 1996-12-27 2001-01-09 Isis Pharmaceuticals Inc. Method of synthesizing phosphorothioate oligonucleotides
US6403781B2 (en) 1996-12-27 2002-06-11 Isis Pharmaceuticals, Inc. Method of synthesizing phosphorothioate oligonucleotides
US6780989B2 (en) 1996-12-27 2004-08-24 Isis Pharmaceuticals, Inc. Diribonucleoside Phosphoramidites
EP1023310A4 (fr) * 1997-10-15 2001-05-16 Isis Pharmaceuticals Inc Synthese amelioree d'oligonucleotides soufres
US7378516B2 (en) 1997-10-15 2008-05-27 Isis Pharmaceuticals, Inc. Synthesis of sulfurized oligonucleotides
US7723511B2 (en) 2000-01-11 2010-05-25 Isis Pharmaceuticals, Inc. Synthesis of sulfurized oligonucleotides
JP2008266331A (ja) * 2004-04-05 2008-11-06 Alnylam Pharmaceuticals Inc オリゴヌクレオチドの合成および精製に使用する方法および反応試薬
US8058448B2 (en) 2004-04-05 2011-11-15 Alnylam Pharmaceuticals, Inc. Processes and reagents for sulfurization of oligonucleotides
US8063198B2 (en) 2004-04-05 2011-11-22 Alnylam Pharmaceuticals, Inc. Processes and reagents for desilylation of oligonucleotides
US8431693B2 (en) 2004-04-05 2013-04-30 Alnylam Pharmaceuticals, Inc. Process for desilylation of oligonucleotides

Also Published As

Publication number Publication date
GB9127243D0 (en) 1992-02-19

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