WO1998049181A1 - Nouveaux reactifs de transfert de soufre pour la synthese d'oligonucleotides - Google Patents

Nouveaux reactifs de transfert de soufre pour la synthese d'oligonucleotides Download PDF

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Publication number
WO1998049181A1
WO1998049181A1 PCT/US1998/008763 US9808763W WO9849181A1 WO 1998049181 A1 WO1998049181 A1 WO 1998049181A1 US 9808763 W US9808763 W US 9808763W WO 9849181 A1 WO9849181 A1 WO 9849181A1
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WIPO (PCT)
Prior art keywords
sulfur transfer
transfer reagent
sulfur
reagent according
lower alkyl
Prior art date
Application number
PCT/US1998/008763
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English (en)
Inventor
Jin Yan Tang
Zhaoda Zhang
Alexandra Nichols
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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.)
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Publication date
Application filed by Hybridon, Inc. filed Critical Hybridon, Inc.
Priority to AU72710/98A priority Critical patent/AU7271098A/en
Publication of WO1998049181A1 publication Critical patent/WO1998049181A1/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
    • 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 System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/16—Esters of thiophosphoric acids or thiophosphorous acids
    • C07F9/165—Esters of thiophosphoric acids
    • C07F9/1654—Compounds containing the structure P(=X)n-X-acyl, P(=X)n-X-heteroatom, P(=X)n-X-CN (X = O, S, Se; n = 0, 1)
    • C07F9/1655—Compounds containing the structure P(=X)n-S-(S)x- (X = O, S, Se; n=0,1; x>=1)

Definitions

  • Patent No. 5,149,798 (1992) discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
  • Solid phase synthesis of oligonucleotides by each of the foregoing processes involves the same generalized protocol. Briefly, this approach comprises anchoring the 3'-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Internucleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the 5'-most nucleoside of the nascent, support-bound oligonucleotide.
  • the internucleoside linkage is a phosphite linkage
  • the H-phosphonate approach it is an H-phosphonate internucleoside linkage.
  • the phosphite or H-phosphonate linkage must be oxidized by an appropriate sulfur transfer reagent.
  • this sulfurization is carried out on all of the H-phosphonate linkages in a single step following the completion of oligonucleotide chain assembly, typically using elemental sulfur in a mixed solvent, such as CS 2 /p ridine.
  • the phosphoramidite approach allows stepwise sulfurization to take place after each coupling, thereby providing the capability to control the state of each linkage in a site-specific manner.
  • Tetrahedron Lett. 3g: 6741-6744 (1994) discloses benzyltriethyl-ammonium tetrathiomolybate (BTTM) as a sulfur transfer reagent. Effimov et al., Nucl.
  • Beaucage reagent has sub-optimal synthesis and stability characteristics and during sulfurization forms a by-product, 3H-2,1- benzoxathiolan-3-one-l-oxide, which is a powerful oxidizing agent capable of producing unwanted phosphodiester internucleoside linkages.
  • 3H-2,1- benzoxathiolan-3-one-l-oxide is a powerful oxidizing agent capable of producing unwanted phosphodiester internucleoside linkages.
  • the invention provides new sulfur transfer reagents and processes for their use in sulfurizing oligonucleotides.
  • the sulfur transfer reagents according to the invention are inexpensive to make, stable in storage, and highly efficient in sulfurization.
  • the invention provides novel sulfur transfer reagents having the general structure according to Formula 1:
  • R 2 , R 3 , and R 4 are selected from the group consisting of Cl, Br, I, - CN, NO 2 , CH 3 , and CH 2 CH 3 , and wherein R l , R 2 , R 3 , and R ⁇ taken separately, are hydrogen, lower alkyl, electron-withdrawing substituted lower alkyl, lower alkyl- or halo-substituted aryl, or a heterocycle containing nitrogen, oxygen, or sulfur and from 5-8 carbon atoms.
  • R l , R 2 , R 3 , and R 4 taken separately, are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n- pentyl, cyclopentylmethyl, isopentyl, neopentyl, n-hexyl, neohexyl, isohexyl, cyclohexylmethyl, beta-cyclopentylethyl, nitro, lower alkyl-nitro-, or halo- substituted phenyl, lower alkyl - or halo-substituted benzyl, or lower alkyl- nitro-,or halo-substituted phenylethyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, beta-electron-with-drawing-substituted
  • the electron-withdrawing substituent of beta- electron-withdrawing-substituted ethyl is cyano, nitro, phenylsulphonyl, or phenylester.
  • the beta-electron-withdrawing-substituted ethyl is beta-cyanoethyl.
  • the lower alkyl-nitro-, or halo- substituents of the lower alkyl-nitro-, or halo-substituted phenyl and benzyl are methyl, chloro, or bromo.
  • morpholinyl, thiomorpholinyl, and piperidinyl are morpholino, thiomorpholino, and piperidinio, respectively.
  • R 1 , R 2 , R 3 , and R* taken separately, are methyl, ethyl, or isopropyl.
  • R 1 and R 2 when taken together are cycloalkyl having from 4 to 7 carbon atoms or a heterocycle containing nitrogen, oxygen, or sulfur and from 3 to 6 carbon atoms; more preferably, when taken together, R 1 and R 2 are cycloalkyl having 4 carbon atoms.
  • R 3 and R 4 when taken together are cycloalkyl having from 4 to 7 carbon atoms or a heterocycle containing nitrogen, oxygen, or sulfur and from 3 to 6 carbon atoms; more preferably, when taken together, R 3 and R 4 are cycloalkyl having 4 carbon atoms.
  • R 2 and R 3 when taken together are a bond so that the compound of Formula 1 is a substituted 1,2 dithio-4,5-diaza heterocycle.
  • the invention provides novel processes for adding a sulfur group to an internucleoside linkage of an oligonucleotide using the novel sulfur transfer reagents according to the invention.
  • the novel processes according to the invention comprise contacting an oligonucleotide having at least one sulfurizable internucleoside linkage with a novel sulfur transfer reagent according to the invention for a time sufficient for sulfurization of the sulfurizable internucleoside linkage(s) to occur.
  • Figure 1 shows a scheme for the synthesis of novel sulfur transfer reagents according to the invention.
  • Figure 1A shows the synthesis of 4- Chlorobenzenesulfonyl(O,O-Diethoxyphosphinothioyl)sulfide.
  • Figure IB shows the synthesis of O,0-Diethoxyphosphinothioyl(p-toluenesulfonyl)disulfide.
  • the invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to sulfurization of the internucleoside linkages of oligonucleotides.
  • the invention provides new sulfur transfer reagents and processes for their use in sulfurizing oligonucleotides.
  • the sulfur transfer reagents according to the invention are inexpensive to make, stable in storage, and highly efficient in sulfurization.
  • the invention provides novel sulfur transfer reagents having the general structure:
  • R 2 , R 3 , and R 4 are selected from the group consisting of Cl, Br, I, - CN, NO 2 , CH 3 , and CH 2 CH 3 , and wherein R l , R 2 , R 3 , and R 4 , taken separately, are hydrogen, lower alkyl, electron-withdrawing substituted lower alkyl, lower alkyl- or halo-substituted aryl, or a heterocycle containing nitrogen, oxygen, or sulfur and from 5-8 carbon atoms.
  • R 1 , R 2 , R 3 , and R 4 taken separately, are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n- pentyl, cyclopentylmethyl, isopentyl, neopentyl, n-hexyl, neohexyl, isohexyl, cyclohexylmethyl, beta-cyclopentylethyl, nitro, lower alkyl-nitro-, or halo- substituted phenyl, lower alkyl - or halo-substituted benzyl, or lower alkyl- nitro-,or halo-substituted phenylethyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, beta-electron-withdrawing-substituted ethyl, morph
  • the electron-withdrawing substituent of beta- electron-withdrawing-substituted ethyl is cyano, nitro, phenylsulphonyl, or phenylester.
  • the beta-electron-withdrawing-substituted ethyl is beta-cyanoethyl.
  • the lower alkyl-nitro-, or halo- substituents of the lower alkyl-nitro-, or halo-substituted phenyl and benzyl are methyl, chloro, or bromo.
  • morpholinyl, thiomorpholinyl, and piperidinyl are morpholino, thiomorpholino, and piperidinio, respectively.
  • R 1 , R 2 , R 3 , and R 4 taken separately, are methyl, ethyl, or isopropyl.
  • R 1 and R 2 when taken together are cycloalkyl having from 4 to 7 carbon atoms or a heterocycle containing nitrogen, oxygen, or sulfur and from 3 to 6 carbon atoms; more preferably, when taken together, R 1 and R 2 are cycloalkyl having 4 carbon atoms.
  • R 3 and R 4 when taken together are cycloalkyl having from 4 to 7 carbon atoms or a heterocycle containing nitrogen, oxygen, or sulfur and from 3 to 6 carbon atoms; more preferably, when taken together, R 3 and R 4 are cycloalkyl having 4 carbon atoms.
  • R 2 and R 3 when taken together are a bond so that the compound of Formula 1 is a substituted 1,2 dithio-4,5-diaza heterocycle.
  • novel sulfur transfer reagents according to the invention are conveniently prepared according to the scheme shown in Figure 1.
  • novel sulfur transfer reagents can easily be prepared from the starting material, reagents, and solvents listed in Table 1. Table 1
  • the invention provides novel processes for adding a sulfur group to an internucleoside linkage of an oligonucleotide using the novel sulfur transfer reagents according to the invention.
  • the novel processes according to the invention comprise contacting an oligonucleotide having at least one sulfurizable internucleoside linkage with a novel sulfur transfer reagent according to the invention for a time sufficient for sulfurization of the sulfurizable internucleoside linkage(s) to occur.
  • Each sulfurizable internucleoside linkage preferably contains a phosphorous (III) atom.
  • the sulfurizable internucleoside linkage is a phosphite, thiophosphite, H- phosphonate, thio-H-phosphonate, or alkylphosphite (especially methylphosphite) internucleoside linkage.
  • the sulfurization reaction would be allowed to proceed to a sulfur transfer efficiency greater than that expected for the prior art compounds, as measured by 31 P-NMR. In typical synthesis conditions such efficiency is achieved within from about 1 to about 5 minutes reaction time with the novel transfer reagents. Typically, the reaction takes place in pyridine, THF, or mixtures thereof.
  • oligonucleotide includes linear polymers of two or more natural deoxyribonucieotide, ribonucleotide, or 2'- O-substituted ribonucleotide monomers, or any combination thereof.
  • the term oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/ or non-nucleosidic analogs linked by phosphodiester bonds or analogs thereof ranging in size from a few monomeric units, e.g., 2-3, to several hundred monomeric units and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane.
  • oligonucleotides may also include non-natural oligomers having phosphorous-containing internucleoside linkages whose phosphorous (HI) precursors are amenable to sulfurization, (See, e.g., Takeshita et al., /. Biol.
  • the term "2*-O- substituted" means substitution of the 2' position of the pentose moiety with an - -lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxy, or amino groups; or with a hydroxy, an amino or a halogen group, but not with a 2'-H group.
  • Such oligonucleotides may include any of the internucleoside linkages which are known in the art, including without limitation phosphorothioate, phosphorodithioate, alkylphosphonate (especially methylphosphonate), phosphoramidate, amide (PNA), carbamate, and alkylphosphonothioate linkages.
  • the oligonucleotide is bound to a solid support, but such oligonucleotides may be sulfurized in solution phase as well.
  • Reverse phase HPLC was performed on a Waters 600E with a Waters 440 absorbance detector, Waters 746 integrator and a Waters Nova-Pak Radial-Pak C18 (8 X 100 mm) column at flow rate of 2 mL/min.
  • Eluents were (A) 100 mM ammonium acetate and (B) 80% acetonitrile/20% 100 mM ammonium acetate. Starting at 90% A, the gradient was: 10 to 60% B in 40 min; to 100% B in 5 min; to 0% B in 5 min.
  • Ion-exchange HPLC was performed on a Beckman System Gold 126 with a Beckman 166 absorbance detector, Beckman 507 autosampler and NUCLEO PACTM PA-100 GUARD column (Dionex Corporation, Sunnyvale, CA) at flow rate of 2 mL/min.
  • Eluents were (A) 25mM Tris, 1 mM EDTA, 10% CH 3 CN,pH8 and (B) 2.5 M ammonium chloride, 25 mM Tris, 1 mM EDTA, 10% CH 3 CN, ⁇ H8. Starting at 0% B, the gradient was: 0-100% B in 5 min, maintained 100% B for 2 min, 100%-0% B in 0.5 min.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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Abstract

Synthèse chimique d'oligonucléotides et entités chimiques utiles pour ladite synthèse. Plus particulièrement, la présente invention concerne la sulfurisation des liaisons internucléosidiques d'oligonucléotides. Elle concerne encore de nouveaux réactifs de transfert de soufre de formule (1) et des procédés d'utilisation desdits réactifs pour la sulfurisation des oligonucléotides. Dans la formule (1), n est 1 à 4, R peut être identique à R1 ou différent de lui, R et R1 sont choisis dans le groupe constitué de CH¿3?, CH2CH3, tert-butyle et de substances de formule (2), (3), et (4) dans lesquelles R?2, R3 et R4¿ sont choisis dans le groupe constitué de Cl, Br, I, CN, NO¿2?, CH3 et CH2CH3. R?1, R2, R3 et R4¿ pris séparément sont hydrogène, alkyle inférieur, alkyle inférieur substitué à attraction d'électrons, aryle à substitution alkyle inférieur ou halo, ou un hétérocycle contenant azote, oxygène ou soufre et de 5 à 8 atomes de carbone. Les réactifs selon la présente invention sont peu onéreux pour ce qui est de leur fabrication, stables au stockage et provoquent une sulfurisation très efficace.
PCT/US1998/008763 1997-04-30 1998-04-30 Nouveaux reactifs de transfert de soufre pour la synthese d'oligonucleotides WO1998049181A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010072831A1 (fr) * 2008-12-23 2010-07-01 Girindus America, Inc Réactifs de sulfurisation et leur utilisation pour la synthèse d'oligonucléotides
WO2012001126A1 (fr) * 2010-06-30 2012-01-05 Girindus America, Inc. Nouveau procédé d'utilisation des composés n-thio pour la synthèse d'oligonucléotides

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Publication number Priority date Publication date Assignee Title
US3923763A (en) * 1972-09-15 1975-12-02 Petro Tex Chem Corp Novel sulfur compound modifiers for chloroprene polymerization
JPS6215649A (ja) * 1985-07-12 1987-01-24 ウオング・ラボラトリ−ズ・インコ−ポレ−テツド 複数のポ−トと複数のチヤンネルとの間を選択的に接続する手段

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XU Q ET AL: "USE OF 1,2,4-DITHIAZOLIDINE-3,5-DIONE (DTSNH) AND 3-ETHOXY-1,2,4-DITHIAZOLINE-5-ONE (EDITH) FOR SYNTHESIS OF PHOSPHOROTHIOATE-CONTAINING OLIGODEOXYRIBONUCLEOTIDES", NUCLEIC ACIDS RESEARCH, vol. 24, no. 9, 1 May 1996 (1996-05-01), pages 1602 - 1607, XP002037973 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010072831A1 (fr) * 2008-12-23 2010-07-01 Girindus America, Inc Réactifs de sulfurisation et leur utilisation pour la synthèse d'oligonucléotides
JP2012513450A (ja) * 2008-12-23 2012-06-14 ギリンダス・アメリカ・インコーポレイテッド 硫化剤およびオリゴヌクレオチドを合成するためのその使用
WO2012001126A1 (fr) * 2010-06-30 2012-01-05 Girindus America, Inc. Nouveau procédé d'utilisation des composés n-thio pour la synthèse d'oligonucléotides
CN103298823A (zh) * 2010-06-30 2013-09-11 集润德斯股份公司 一种使用n-硫代化合物用于寡核苷酸合成的新方法
US8710210B2 (en) 2010-06-30 2014-04-29 Girindus America, Inc. Method of using N-thio compounds for oligonucleotide synthesis

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