[go: up one dir, main page]

WO1996014277A1 - Synthese d'oligonucleotides marques au 35s a l'aide de 3h-1,2-benzodithiol-3-one-1,1-dioxyde - Google Patents

Synthese d'oligonucleotides marques au 35s a l'aide de 3h-1,2-benzodithiol-3-one-1,1-dioxyde Download PDF

Info

Publication number
WO1996014277A1
WO1996014277A1 PCT/US1995/014259 US9514259W WO9614277A1 WO 1996014277 A1 WO1996014277 A1 WO 1996014277A1 US 9514259 W US9514259 W US 9514259W WO 9614277 A1 WO9614277 A1 WO 9614277A1
Authority
WO
WIPO (PCT)
Prior art keywords
benzodithiol
acid
dioxide
compound
oligonucleotides
Prior art date
Application number
PCT/US1995/014259
Other languages
English (en)
Inventor
Radhakrishnan P. Iyer
Sudhir Agrawal
Weitian Tan
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
Priority claimed from US08/335,100 external-priority patent/US5833944A/en
Application filed by Hybridon, Inc. filed Critical Hybridon, Inc.
Priority to EP95939047A priority Critical patent/EP0790965A1/fr
Priority to JP8515428A priority patent/JPH10509447A/ja
Priority to AU41013/96A priority patent/AU4101396A/en
Publication of WO1996014277A1 publication Critical patent/WO1996014277A1/fr

Links

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
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds

Definitions

  • the invention relates to synthesis of 35 S-labeled 3H-1,2 benzodithiol-3-one-l,l dioxide (1) and its use in the preparation of site-specifically 35 S-labeled oligonucleotides.
  • Zamecnik U.S. Patent No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
  • H-phosphonate chemistry Garegg et al., Chem. Scr. 25, 280-282 (1985). It is difficult to achieve site-specific labeling of phosphorothioates using H-phosphonate chemistry, however, and it is inconvenient to carry out preparation of 35 S-labeled oligonucleoside phosphorothioate constructs, such as those with (a) mixed ribonucleotide- deoxyribonucleotide population ("hybrid oligos"), (b) heterogeneous backbones, e.g., deoxyribonucleotide-methyl phosphonate ("chimeric oligos”) and (c) mixed phosphodiester-phosphorothioate (PO-PS) backbones. In order to ensure stereochemically
  • the present invention provides new compounds and improved methods for synthesizing 35 S-labeled oligonucleoside phosphorothioates.
  • This invention comprises several aspects.
  • the present invention provides a novel compound useful for synthesizing oligonucleotide phosphorodiioates labelled with 33 S.
  • This compound 35 S-3H-l,2-benzodithiol-3-one-l,l dioxide (1), has the structure
  • a new method of synthesizing 35 S-3H-1,2- benzodithiol-3-one-l,l dioxide (1) is provided.
  • An important consequence of this method is that it allows for the preparation of a variety of 35 S-labeled oligonucleotide phosphorothioates and thereby facilitates pharmacokinetic studies of these compounds.
  • the method of synthesizing 3$ S-3H-l,2-benzodithiol-3-one-l,l dioxide (1) is depicted in Fig. 2 and comprises first contacting 35 S-thiobenzoic acid (4) with thiosalicylic acid (5) to yield the condensation product, 35 S-3 H 1 ,2-benzodithiol-3-one (2).
  • 35 S-3 H 1 ,2- benzodithiol-3-one (2) is then oxidized to yield the desired product, 35 S-3H-1,2- benzodithiol-3-one-l,l dioxide (1).
  • a new method of synthesizing 35 S-labelled oligonucleotides comprises contacting 35 S-3H-l,2-benzodithiol- 3-one-l,l dioxide (1) with an oligonucleotide susceptable to oxidative sulfurization.
  • the method of 35 S labelling an oligonucleotide synthesized via the phosphoramidite method is depicted in Fig. 3.
  • Other methods are contemplated, however, such as oxidative sulfurization of alkyl- and/or aryl-phosphites to yield the corresponding 35 S-labelled alkyl- and/or aryl-phosphonothioate.
  • 35 S-3H-l,2-benzodithiol-3-one-l,l dioxide (1) can be used for any purpose and in any way that its unlabelled analog, 3H-1,2- benzodithiol-3-one-l,l dioxide, can be used.
  • Figure 1 depicts the synthesis of 35 S-thiobenzoic acid (4) from 35 S elemental sulfur and thiobenzoic acid.
  • Figure 2 depicts the synthesis of 35 S-3H-l,2-benzodithiol-3-one-l,l dioxide (1) from 35 S-thiobenzoic acid (4) and thiosalicylic acid (5) via the intermediate 35 S-3H-1,2- benzodithiol-3-one (2).
  • Figure 3 depicts the 3 S labelling of an oligonucleotide synthesized by the phosphoramidate method.
  • Figure 5 displays 35 S-labelled oligonucleotides synthesized according to the methods of the present invention.
  • Figure 6 displays and autoradiogram of oligonucleotides SEQ. ID NOs. 5-7 (purified) and SEQ. ID. NO. 8 (crude) subjected to PAGE.
  • oligonucleotides Because of the ever-increasing interest in antisense oligonucleotides as therapeutic agents, there is a need to provide methods whereby the pharmacokinetic properties of these compounds can be tested. It is necessary to determine biodistribution, as well as to determine the half-lives and degradation products.
  • One method of accomplishing these tasks is to label the oligonucleotides with 35 S, a common isotopic label used for tracing and detecting biological compounds.
  • the present invention provides a new compound useful for synthesizing 35 S- labelled antisense oligonucleotides, a new method of synthesizing the compound and new methods for 3S S-labelling oligonucleotides.
  • the first aspect of the invention comprises a new compound, 35 S-3H-1,2- benzodithiol-3-one-l,l dioxide (1), having the following structure
  • a second aspect of the invention comprises a new method of synthesizing 35 S -3H-l,2-benzodithiol-3-one- 1,1 dioxide.
  • This method is a modification of the method of Beaucage et al. '097, for example.
  • An important benefit of this method is that it enables production of 35 S-3H-l,2-benzodithiol-3-one-l,l dioxide (1).
  • the method of this invention uses a reactant in which the 35 S label is easily incorporated. The prior art teaches that the precursor to 3H-l,2-benzodithiol-3-one-l,l dioxide,
  • 3H-l,2-benzodithiol-3-one can be produced by mixing 2-thiolbenzoic acid and thiolacetic acid in sulfuric acid.
  • 2-thiolbenzoic acid and thiolacetic acid in sulfuric acid E.g., Beaucage et al. '097.
  • 35 S-thiolacetic acid was prepared by a high temperature (125°C) exchange reaction between thiolacetic acid and elemental 35 S using a reported procedure.
  • 125°C 125°C
  • the desired product 35 S-1 as a white crystalline solid in 30% chemical yield (based on the amount of 5 used) and having a specific activity of 90 ⁇ Ci/ ⁇ mol.
  • the reaction mixture should be worked up immediately after its completion to avoid decomposition of 35 S-1.
  • the synthetic method according to this aspect of the invention comprises first contacting 35 S-thiobenzoic acid (4) with thiosalicylic acid (5) to yield the condensation product, 35 S-3 H l,2-benzodithiol-3-one (2).
  • This reaction is acid catalyzed.
  • sulfuric acid is used, although any suitably strong acid may be used.
  • 35 S-3H 1 ,2-benzodithiol-3-one (2) is then oxidized to yield the desired product, 5 S-3H- 1 ,2- benzodithiol-3-one-l,l dioxide (1).
  • oxidizing agent e.g., hydrogen peroxide and trifluoroacetic acid, trifluoro peroxyacetic acid or other oxidizing agents such as oxone, sodium periodate NaOCl, RuCl 3 and reagents used in the oxidation of sulfide to sulfone. See, e.g., M. ⁇ udlicky, Oxidation in Organic Chemistry, ACS Monograph 186, 1990.
  • oxidation is accomplished with hydrogen peroxide and trifluoroacetic acid. This scheme is depicted in Fig. 2.
  • the third aspect of the present invention comprises a new method for 35 S-labelling oligonucleotides.
  • the method can be used to selectively place the 3$ S at any desired internucleoside linkage. Anywhere from one to all internucleoside linkages may be labelled with 35 S.
  • the method comprises contacting 35 S-3H-l,2-benzodithiol-3-one-l,l dioxide (1) with an oligonucleotide susceptible to oxidative sulfurization.
  • the oligonucleotide is synthesized by the phosphoramidite method, and 35 S- 3H- 1 ,2-benzodithiol-3-one- 1 , 1 dioxide (1) is contacted with the oligonucleotide having one or more ⁇ -cyanoethyl phosphotriester internucleoside linkages under standard conditions known in the art.
  • an oligonucleotide having one or more alkyl- and/or aryl-phosphite internucleotide linkages is contacted with 35 S-3H-1,2- benzodithiol-3-one-l,l dioxide (1) to yield the corresponding 35 S-labelled alkyl- and/or aryl-phosphonothioate.
  • This reaction using the non-radiolabeled oxidative sulfurization agent, is taught by Padmapriya et al., supra.
  • 35 S-3H-l,2-benzodithiol-3-one-l,l dioxide (1) can be used to 35 S-label any compound that is capable of being sulfurized by the unlabeled analog 3H-l,2-benzodithiol-3-one-l,l dioxide.
  • the present method is capable of 35 S labelling carbohydrates, proteins, and any macromolecule into which one can incorporate an 35 S label by oxidative thiolation.
  • RNA can be labelled with 3S S in a site-specific manner, as can phosphopeptides.
  • Phosphorothioate and sulfur analogs of phospholipids, glycerophospholipids, and phosphocar bohydtrates can also be labeled with 35 S.
  • 35 S can be inserted into thiophosphates and thiotriphosphates (e.g., ATP) and then incorporated into any molecule using chemical or enzymatic phosphorylation reactions.
  • thiophosphates and thiotriphosphates e.g., ATP
  • Ion-exchange ⁇ PLC was done using a GEN-PAK FAX column (4.6 X 100 mm) at 65°C using a gradient (80% A to 100 % B over 50 min.) of Buffer A (25 raM Tris ⁇ CL. p ⁇ 8.5, 10% C ⁇ 3 CN) to Buffer B (25 mM Tris HC1, 2 M LiCl, pH 8.5, % CH 3 CN) and a flow rate of 0.5 ml/min.
  • Buffer A 25 raM Tris ⁇ CL. p ⁇ 8.5, 10% C ⁇ 3 CN
  • Buffer B 25 mM Tris HC1, 2 M LiCl, pH 8.5, % CH 3 CN

Landscapes

  • 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)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Heterocyclic Compounds Containing Sulfur Atoms (AREA)

Abstract

L'invention concerne un nouveau composé, qui permet de marquer des oligonucléotides au ?35S, le 35¿S-3H-1,2-benzodithiol-3-one-1,1-dioxyde (1) où l'astérisque indique la position du 35S. On décrit aussi un procédé de synthèse de ce composé qui consiste d'abord à mettre en contact de l'acide 35S-thiobenzoïque (4) avec de l'acide thiosalicylique (5) en milieu acide pour obtenir le produit de condensation, 35S-3 H 1,2-benzodithiol-3-one (2), qui est alors oxydé avec un agent oxydant approprié tel que de l'acide trifluoroacétique et du peroxyde d'hydrogène pour donner le produit souhaité, le 35S-3 H 1,2-benzodithiol-3-one-1,1-dioxyde (1). Tout oligonucléotide pouvant subir une sulfuration oxydative due au 3H-1,2-benzodithiol-3-one-1,1-dioxyde peut être marqué au 35S-3H-1,2-benzodithiol-3-one-1,1-dioxyde (1). L'invention concerne donc aussi de nouveaux procédés permettant de marquer des oligonucléotides au 35S. Ce composé et ces procédés permettent de suivre la biorépartition et la dégradation d'oligonucléotides antisens dans le cadre d'études pharmacocinétiques.
PCT/US1995/014259 1994-11-07 1995-11-06 Synthese d'oligonucleotides marques au 35s a l'aide de 3h-1,2-benzodithiol-3-one-1,1-dioxyde WO1996014277A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP95939047A EP0790965A1 (fr) 1994-11-07 1995-11-06 Synthese d'oligonucleotides marques au ?35 s a l'aide de 3h-1,2-benzodithiol-3-one-1,1-dioxyde
JP8515428A JPH10509447A (ja) 1994-11-07 1995-11-06 ３ｈ−１，２−ベンゾジチオール−３−１，１−ジオキシドを用いた▲上３５▼ｓ−標識オリゴヌクレオチドの合成
AU41013/96A AU4101396A (en) 1994-11-07 1995-11-06 Synthesis of 35s-labeled oligonucleotides with 3h-1,2-benzodithiol-3-1,1-dioxide

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US08/335,100 US5833944A (en) 1994-11-07 1994-11-07 Procedure for the solid phase synthesis of 35 S-labeled oligonucleotides with 3H-1,2-benzodithiol-3-one-1,1-dioxide
US08/335,100 1994-11-07
US49333995A 1995-06-21 1995-06-21
US49325795A 1995-06-21 1995-06-21
US08/493,257 1995-06-21
US08/493,339 1995-06-21

Publications (1)

Publication Number Publication Date
WO1996014277A1 true WO1996014277A1 (fr) 1996-05-17

Family

ID=27407027

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/014259 WO1996014277A1 (fr) 1994-11-07 1995-11-06 Synthese d'oligonucleotides marques au 35s a l'aide de 3h-1,2-benzodithiol-3-one-1,1-dioxyde

Country Status (5)

Country Link
EP (1) EP0790965A1 (fr)
JP (1) JPH10509447A (fr)
CN (1) CN1165508A (fr)
AU (1) AU4101396A (fr)
WO (1) WO1996014277A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9388415B2 (en) 2003-09-09 2016-07-12 Geron Corporation Modified oligonucleotides for telomerase inhibition
US9796747B2 (en) 2014-05-01 2017-10-24 Geron Corporation Oligonucleotide compositions and methods of making the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5003097A (en) * 1989-10-02 1991-03-26 The United States Of America As Represented By The Department Of Health And Human Services Method for the sulfurization of phosphorous groups in compounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5003097A (en) * 1989-10-02 1991-03-26 The United States Of America As Represented By The Department Of Health And Human Services Method for the sulfurization of phosphorous groups in compounds

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
C. A. STEIN ET AL: "Preparation of 35S-labeled polyphosphorothioate oligodeoxyribonucleotides by use of hydrogen phosphonate chemistry", ANALYTICAL BIOCHEMISTRY, vol. 188, no. 1, 1 July 1990 (1990-07-01), DULUTH, pages 11 - 16 *
S. AGRAWAL ET AL: "Pharmacokinetics, biodistribution, and stability of oligonucleotide phosphorothioates in mice", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, no. 17, 1 September 1991 (1991-09-01), WASHINGTON US, pages 7595 - 7599 *
S. KAWAMURA ET AL: "Reaction of acetyl aralkyl disulfides with triphenylphosphine", CHEMISTRY LETTERS, no. 12, pages 1231 - 1234 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9388415B2 (en) 2003-09-09 2016-07-12 Geron Corporation Modified oligonucleotides for telomerase inhibition
US9388416B2 (en) 2003-09-09 2016-07-12 Geron Corporation Modified oligonucleotides for telomerase inhibition
US9404112B2 (en) 2003-09-09 2016-08-02 Geron Corporation Modified oligonucleotides for telomerase inhibition
US9657296B2 (en) 2003-09-09 2017-05-23 Geron Corporation Modified oligonucleotides for telomerase inhibition
US10196641B2 (en) 2003-09-09 2019-02-05 Geron Corporation Modified oligonucleotides for telomerase inhibition
US9796747B2 (en) 2014-05-01 2017-10-24 Geron Corporation Oligonucleotide compositions and methods of making the same
US10392418B2 (en) 2014-05-01 2019-08-27 Geron Corporation Oligonucleotide compositions and methods of making the same
US11299511B2 (en) 2014-05-01 2022-04-12 Geron Corporation Oligonucleotide compositions and methods of making the same
US11739114B2 (en) 2014-05-01 2023-08-29 Geron Corporation Oligonucleotide compositions and methods of making the same

Also Published As

Publication number Publication date
AU4101396A (en) 1996-05-31
EP0790965A1 (fr) 1997-08-27
JPH10509447A (ja) 1998-09-14
CN1165508A (zh) 1997-11-19

Similar Documents

Publication Publication Date Title
EP1028124B1 (fr) Procédé amélioré de déprotection pour la synthèse de composés oligomériques
CA2386221C (fr) Composes de protection des hydroxyles et procedes d'utilisation
US6531590B1 (en) Processes for the synthesis of oligonucleotide compounds
Mag et al. Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged intemucleotide 5′-phosphorothioate linkage
US6291669B1 (en) Solid phase synthesis
CA2627208C (fr) Polynucleotide contenant un mimetique de phosphate
US6846922B1 (en) Activators for oligonucleotide synthesis
WO2000027859A1 (fr) Procedes ameliores de synthese de composes oligomeres
KR0151153B1 (ko) 알파 tnf와 반대방향인 대항 메신저 rna의 올리고클레오티드 서열, 그의 제조방법, 약제로서의 용도 및 제약 조성물
Kuijpers et al. Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent
US6310198B1 (en) Extremely high purity oligonucleotides and methods of synthesizing them using dimer blocks
Sun et al. Synthesis of 3'-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry.
EP1244682A1 (fr) Procede de preparation de composes oligomeres
US5833944A (en) Procedure for the solid phase synthesis of 35 S-labeled oligonucleotides with 3H-1,2-benzodithiol-3-one-1,1-dioxide
WO1997029116A1 (fr) Phosphoramidites de dinucleotides contenant du soufre
WO1996014277A1 (fr) Synthese d'oligonucleotides marques au 35s a l'aide de 3h-1,2-benzodithiol-3-one-1,1-dioxyde
Krotz et al. Phosphorothioate oligonucleotides: largely reduced (N-1)-mer and phosphodiester content through the use of dimeric phosphoramidite synthons
NZ261480A (en) Preparation of dimeric nucleotide derivatives containing phosphonothionate internucleotide linkages
Karamychev, Michael W. Reed, Ronald D. Neumann, Igor G. Panyutin Distribution of DNA strand breaks produced by iodine-123 and indium-111 in synthetic oligodeoxynucleotides
US5847104A (en) Method of tritium labeling oligonucleotide
US5631361A (en) Method of synthesizing radioisotopically labeled oligonucleotides by direct solid-phase 5' phosphitylation
US5668262A (en) Method of tritium labeling oligonucleotides
Iyer et al. Synthesis of [35S] 3H-1, 2-benzodithiole-3-one-1, 1-dioxide: Application in the Preparation of site-specifically 35S-labeled oligonucleotides
US5639875A (en) Methods for H-phosphonate syntheis of oligonucleotides using triphosgene
Metelev et al. Oligodeoxyribonucleotides with Internucleotidic or Terminal Phosphorothioate Groups: Different Pathways in the Reaction with Water-Soluble Carbodhmide

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 95196093.8

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG US US US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 296413

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 1995939047

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1995939047

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995939047

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA