EP1181301A1 - Synthese d'oligonucleotides utilisant des acides de lewis comme activateurs - Google Patents

Synthese d'oligonucleotides utilisant des acides de lewis comme activateurs

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Publication number
EP1181301A1
EP1181301A1 EP00926516A EP00926516A EP1181301A1 EP 1181301 A1 EP1181301 A1 EP 1181301A1 EP 00926516 A EP00926516 A EP 00926516A EP 00926516 A EP00926516 A EP 00926516A EP 1181301 A1 EP1181301 A1 EP 1181301A1
Authority
EP
European Patent Office
Prior art keywords
group
chloride
composition according
alkoxy
magnesium
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.)
Withdrawn
Application number
EP00926516A
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German (de)
English (en)
Inventor
Xiu C. Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
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Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP1181301A1 publication Critical patent/EP1181301A1/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
    • 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
    • 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

  • the present invention relates to a process utilizing Lewis acids as activators in the preparation of oligonucleotides by phosphoramidite chemistry.
  • the present invention relates generally to the fields of organic chemistry and biology.
  • the present invention is directed to compositions and methods for use in oligonucleotide synthesis.
  • Phosphoramidite chemistry (Beaucage, S. L., and Lyer, R. P. Tetrahedron (1992), 48, 2223-2311) has become by far the most widely used coupling chemistry for the synthesis of oligonucleotides.
  • phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activating agent to form activated intermediates, followed by sequential addition of the activated intermediates to the growing oligonucleotide chain (generally anchored at one end to a suitable solid support) to form the oligonucleotide product.
  • Tetrazole is commonly used for the activation of the nucleoside phosphoramidite monomers; the activation occurs by the mechanism depicted in Scheme I.
  • Tetrazole has an acidic proton which presumably protonates the basic nitrogen of the diisopropylamino phosphine group, thus making the diisopropylamino group a leaving group.
  • the negatively charged tetrazolium ion then makes an attack on the trivalent phosphorous, forming a transient phosphorous tetrazolide species.
  • the 5'-OH group of the solid support bound nucleoside then attacks the active trivalent phosphorous species, resulting in the formation of the internucleotide linkage.
  • tetrazole Principal drawbacks of tetrazole are its cost and instability which includes its potential to explode (Material Safety Data Sheets or MSDS lists IH-tetrazole as a severe explosion hazard). Because of its inherent instability, sublimed tetrazole is generally required to ensure desired coupling yields. Further, tetrazole (which is typically used near its saturated solubility of 0.5M) tends to precipitate out of acetonitrile solution at cold temperatures; this can lead to valve blockage on some automated DNA synthesizers. Other activators which work almost as efficiently as tetrazole have similar drawbacks to those of tetrazole as discussed above.
  • activators which are all proton donors, include the following members of the tetrazole class of activators: 5-(p- nitrophenyl) tetrazole (Froehler, B. C. and Mattcucci, M. D., Tetrahedron Letters (1983), 24, 3171-3174); 5-(p-nitrophenyl) tetrazole and DMAP (Pon, R.T., Tetrahedron Letters (1987), 28, 3643-3646); and 5-(ethylthio)- IH-tetrazole (Wright, P. et al., Tetrahedron
  • a 1 :1 mixture of benzimidazole and BF 3 etherate is disclosed wherein the BF 3 component acts to increase the acidity of the benzimidazole proton necessary for activation of the phosphoramidite (intermediates).
  • the benzimidazole BF 3 complex acts in a manner similar to tetrazole described in Scheme 1.
  • the present invention does not activate phosphoramidite intermediates with a proton donor but instead utilizes Lewis acids for activation.
  • BF 3 etherate is used in the present invention.
  • the advantages of BF 3 etherate over the benzimidazole BF 3 complex include commercial availability and ease of removal of diethyl ether versus removal of benzimidazole.
  • the activated phosphoramidite intermediates are highly sensitive to moisture. An excess of 50% to 100% of the highly valuable phosphoramidites are required for sequencing even with anhydrous solvents ( ⁇ 20 ppm moisture content). The presence of trace amounts of moisture results in considerable loss of yield and an increase in deleted sequencing impurities.
  • Lewis acids can act as moisture scavengers minimizing decomposition of the phosphoramidite. Therefore, the use of Lewis acids for activation of phosphoramidite intermediates leads to improved coupling efficiency, lower cost, and convenient material handling and operation.
  • B 1 is selected from the group consisting of a purine base and a pyrimidine base;
  • R 1 is a secondary amine, a preferred amine is diisopropylamine;
  • R 2 is selected from the group consisting of alkoxy, alkyl, alkylsulfonylalkoxy arylsulfonylalkoxy, cyanoalkoxy, and haloalkoxy;
  • R 3 is a hydroxy-protecting group, a preferred group is 4-4'-dimethoxytrityl
  • R 4 is selected from the group consisting of hydrogen and -OR 7 wherein, R 7 is a hydroxy- protecting group; comprising treating the phosphoramidite monomers of formula I with an optional amount of pyridine and a Lewis acid, preferred Lewis acids are selected from aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride and zirconium(IN) chloride.
  • preferred Lewis acids are selected from aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride and zirconium(IN) chloride.
  • alkoxy refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein.
  • alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and the like.
  • alkyl refers to a straight or branched chain hydrocarbon containing from 1 to 5 carbon atoms.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, and the like.
  • alkylcarbonyl refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a carbonyl group, as defined herein.
  • Representative examples of alkylcarbonyl include, but are not limited to, acetyl, ethylcarbonyl, and the like.
  • alkylcarbonyloxy refers to an alkylcarbonyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein.
  • Representative examples of alkylcarbonyloxy include, but are not limited to, acetyloxy, ethylcarbonyloxy, t-butylcarbonyloxy, and the like.
  • alkylsulfonyl refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.
  • Representative examples of alkylsulfonyl include, but are not limited to, methylsulfonyl, ethylsulfonyl, and the like.
  • alkylsulfonylalkoxy refers to an alkylsulfonyl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.
  • Representative examples of alkylsulfonylalkoxy include, but are not limited to, 2-methylsulfonylethoxy, 2-ethylsulfonylethoxy, and the like.
  • amino refers to a -NH 2 group.
  • amino-protecting group or “N-protecting group,” refer to groups intended to protect an amino group against undersirable reactions during synthetic procedures. Commonly used nitrogen-protecting groups are disclosed in Greene, T. W., &
  • nitrogen-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).
  • aryl refers to an aromatic monocyclic ring system, or a bicyclic-fused ring system wherein one or both of the fused rings are aromatic.
  • aryl include, but are not limited to, azulene, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • the aryl groups of this invention can be substituted with 1, 2, or 3 substituents independently selected from alkyl, cyano, halogen, haloalkyl, and nitro.
  • arylalkoxy refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.
  • arylalkoxy include, but are not limited to, 2-phenylethoxy, 2-naphthylethoxy, 2-(4-nitrophenyl)ethoxy, and the like.
  • arylsulfonyl refers to an aryl group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.
  • arylsulfonyl include, but are not limited to, phenylsulfonyl, naphthylsulfonyl, and the like.
  • arylsulfonylalkoxy refers to an arylsulfonyl group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.
  • Representative examples of arylsulfonylalkoxy include, but are not limited to, 2-phenylsulfonylethoxy, 3-phenylsulfonylpropoxy, and the like.
  • carbonyl refers to a -C(O)- group.
  • catechol refers to a C 6 H 4 - 1 ,2-(O-) 2 group, wherein both oxygen atoms are attached to M, as defined herein.
  • cyano refers to a -CN group.
  • cyanoalkoxy refers to a cyano group, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein.
  • Representative examples of cyanoalkoxy include, but are not limited to, 2-cyanoethoxy, 3-cyanopropoxy, 1 -methyl-2-cyanoethoxy, l,l-dimethyl-2-cyanoethoxy, and the like.
  • halo refers to -Cl, -Br, -I or -F.
  • haloalkoxy refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of haloalkoxy include, but are not limited to, 2,2,2- trichloroethoxy, l,l-dimethyl-2,2,2-trichloroethoxy, trifluoromethoxy, and the like.
  • haloalkyl refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2- fluoroethyl, trifiuoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.
  • hydroxy-protecting group or "O-protecting group” refers to groups intended to protect a hydroxy group against undesirable reactions during synthetic procedures. Commonly used hydroxy-protecting groups are disclosed in T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis. 2nd edition, John Wiley & Sons, New York (1991), which is hereby incorporated by reference.
  • hydroxy- protecting groups include, but are not limited to, substituted methyl ethers, for example, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)- ethoxymethyl, benzyl, and triphenylmethyl; tetrahydropyranyl ethers; substituted ethyl ethers, for example, 2,2,2-trichloroethyl and t-butyl; silyl ethers, for example, trimethylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl; cyclic acetals and ketals, for example, methylene acetal, acetonide and benzylidene acetal; cyclic ortho esters, for example, methoxymethylene; cyclic carbonates; cyclic boronates; carbonyl derivatives, for example, acetyl, p-phenylazopheny
  • Lewis acid refers to a chemical species, other than a proton, that has a vacant orbital or accepts an electron pair. It is to be understood that Lewis acids can be purchased or prepared as complexes including but not limited to, etherates, hydrates, and thioetherates. It is to be further understood that complexes purchased or prepared for the present invention do not contain an acidic proton.
  • Lewis acid examples include, but are not limited to, aluminum chloride, bismuth(III) chloride, boron trifluoride, iron(II) chloride, iron(III) chloride, magnesium bromide, magnesium chloride, magnesium trifluoromethanesulfonate, manganese(II) chloride, zinc bromide, zinc chloride, zirconium(IV) chloride, and the like.
  • methylenedioxy refers to a -OC(R 80 )(R 81 )O- group wherein R 80 and R 81 are independently selected from hydrogen and alkyl. The oxygen atoms of the methylenedioxy group are attached to the parent molecular moiety through two adjacent carbon atoms.
  • methylenedioxy group examples include, but are not limited to, 1,3-dioxolanyl, 2,2-dimethyl-l,3-dioxolanyl, 2-methyl-l,3- dioxolanyl, and the like.
  • purine base refers to an organic base selected from 9H-purin-6-ylamine (adenine) and 2-amino-l,9-dihydro-6H-purin-6-one (guanine).
  • adenine 9H-purin-6-ylamine
  • guanine 2-amino-l,9-dihydro-6H-purin-6-one
  • the amino group attached to adenine can be protected with a nitrogen-protecting group.
  • proto refers to H + .
  • pyrimidine base refers to an organic base selected from 2,4(lH,3H)-pyrimidinedione (uracil), 5-methyl-2,4(lH,3H)-pyrimidinedione (thymine), and 4-amino-2(lH)-pyrimidinone (cytosine).
  • uracil 2,4(lH,3H)-pyrimidinedione
  • thymine 5-methyl-2,4(lH,3H)-pyrimidinedione
  • cytosine 4-amino-2(lH)-pyrimidinone
  • the amino group attached to cytosine can be protected with a nitrogen-protecting group.
  • sulfonyl refers to a -SO 2 - group.
  • trifluoromethane refers to a -CF 3 group.
  • trifluoromethanesulfonyl refers to a trifluoromethane group, as defined herein, appended to the parent molecular moiety through a sulfonyl group, as defined herein.
  • trifluoromethanesulfonyloxy refers to a trifluoromethanesulfonyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein.
  • dimer (iii) can be oxidizied using standard conditions know to those of ordinary skill in the art to give the phosphate, (J. Am. Chem. Soc, (1976), 98, 3655-3661).
  • the 5'-OH of the oxidized dimer can be deprotected and treated with a Lewis acid activated phophoramidite monomer to form a trimer. This sequence of steps can be repeated until an oligonucleotide of desired length has been synthesized such that the process of the present invention can be used for preparing oligonucleotides, including solid phase synthesis thereof.

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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
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  • Engineering & Computer Science (AREA)
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Abstract

L'invention concerne un procédé permettant d'effectuer la synthèse d'oligonucléotides par chimie des phosphoramidites, l'amélioration consistant à utiliser des acides de Lewis comme activateurs, afin de créer la liaison phosphore-oxygène.
EP00926516A 1999-06-03 2000-05-08 Synthese d'oligonucleotides utilisant des acides de lewis comme activateurs Withdrawn EP1181301A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US32505599A 1999-06-03 1999-06-03
US325055 1999-06-03
PCT/US2000/012530 WO2000075157A1 (fr) 1999-06-03 2000-05-08 Synthese d'oligonucleotides utilisant des acides de lewis comme activateurs

Publications (1)

Publication Number Publication Date
EP1181301A1 true EP1181301A1 (fr) 2002-02-27

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EP (1) EP1181301A1 (fr)
JP (1) JP2003514766A (fr)
CA (1) CA2376016A1 (fr)
MX (1) MXPA01012444A (fr)
WO (1) WO2000075157A1 (fr)

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JP5283178B2 (ja) * 2009-03-05 2013-09-04 株式会社島津製作所 マトリックス支援レーザ脱離イオン化質量分析用マトリックス
CA2773773C (fr) 2009-09-21 2019-04-23 Gilead Sciences, Inc. Procedes et intermediaires pour preparer des analogues de carba-nucleoside 1'-substitues
SG186830A1 (en) 2010-07-22 2013-02-28 Gilead Sciences Inc Methods and compounds for treating paramyxoviridae virus infections
TWI687432B (zh) * 2014-10-29 2020-03-11 美商基利科學股份有限公司 絲狀病毒科病毒感染之治療
LT3785717T (lt) 2015-09-16 2022-04-11 Gilead Sciences, Inc. Coronaviridae infekcijų gydymo būdai
US10682368B2 (en) 2017-03-14 2020-06-16 Gilead Sciences, Inc. Methods of treating feline coronavirus infections
CN111093627B (zh) 2017-07-11 2024-03-08 吉利德科学公司 用于治疗病毒感染的包含rna聚合酶抑制剂和环糊精的组合物
TWI789695B (zh) 2020-01-27 2023-01-11 美商基利科學股份有限公司 治療sars cov-2感染之方法
AU2021234308C1 (en) 2020-03-12 2024-02-22 Gilead Sciences, Inc. Methods of preparing 1'-cyano nucleosides
WO2021207049A1 (fr) 2020-04-06 2021-10-14 Gilead Sciences, Inc. Formulations d'inhalation d'analogues de carbanucléosides à substitution 1'-cyano
TW202203941A (zh) 2020-05-29 2022-02-01 美商基利科學股份有限公司 瑞德西韋之治療方法
CN115996928A (zh) 2020-06-24 2023-04-21 吉利德科学公司 1’-氰基核苷类似物及其用途
TW202233204A (zh) 2020-08-27 2022-09-01 美商基利科學股份有限公司 用於治療病毒感染之化合物及方法
WO2023167944A1 (fr) 2022-03-02 2023-09-07 Gilead Sciences, Inc. Composés et méthodes pour traiter des infections virales

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JPH08301878A (ja) * 1995-04-28 1996-11-19 Toagosei Co Ltd 三フッ化ホウ素配位化合物およびオリゴヌクレオチドの合成方法

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See references of WO0075157A1 *

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Publication number Publication date
MXPA01012444A (es) 2002-07-30
JP2003514766A (ja) 2003-04-22
WO2000075157A1 (fr) 2000-12-14
CA2376016A1 (fr) 2000-12-14

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