[go: up one dir, main page]

WO2006095740A1 - Procede de synthese d’acide nucleique utilisant un groupe carbamoyle substitue en tant que groupe protecteur - Google Patents

Procede de synthese d’acide nucleique utilisant un groupe carbamoyle substitue en tant que groupe protecteur Download PDF

Info

Publication number
WO2006095740A1
WO2006095740A1 PCT/JP2006/304400 JP2006304400W WO2006095740A1 WO 2006095740 A1 WO2006095740 A1 WO 2006095740A1 JP 2006304400 W JP2006304400 W JP 2006304400W WO 2006095740 A1 WO2006095740 A1 WO 2006095740A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
general formula
nucleic acid
compound represented
trophenyl
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
Application number
PCT/JP2006/304400
Other languages
English (en)
Japanese (ja)
Inventor
Mitsuo Sekine
Kenichi Miyata
Ryuji Tamamushi
Kohji Seio
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.)
Tokyo Institute of Technology NUC
Original Assignee
Tokyo Institute of Technology NUC
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 Tokyo Institute of Technology NUC filed Critical Tokyo Institute of Technology NUC
Publication of WO2006095740A1 publication Critical patent/WO2006095740A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • 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
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a method for synthesizing a nucleic acid using a substitutional power ruberamoyl group as a protecting group, and a method for deprotecting the protecting group.
  • the amino group of the nucleobase portion requires a protecting group when chemically modifying other nucleic acid sites such as a highly reactive sugar moiety or when synthesizing DNA or RNA oligomers.
  • protecting groups include the trityl skeleton, the acyl skeleton, the force rubamate skeleton, the amidine skeleton (Strength Protocols in Nucleic Acid Chemistry, Chapter 2, Unit 2.1 John Wiley and Sons). And a protecting group.
  • both protecting groups require acidic or basic conditions for deprotection, and the development of new protecting groups that can be deprotected under neutral conditions is required.
  • Nucleobase amino protecting groups for the synthesis of DNA and RNA oligomers are commonly used having an acyl skeleton and an amidine skeleton. All protecting groups must be deprotected using ammonia or methylamine, and oligomers containing molecules that are unstable under basic conditions cannot be synthesized.
  • a method using a carboxy group that can be deprotected under neutral conditions as a protecting group for the nucleobase amino group (The Journal of Organic Chemistry 51 ⁇ P2400—2402 1986) Although it has been developed, there is a problem because it is necessary to use a transition metal for deprotection.
  • the base-part unprotected phosphoramidite method using no protecting group in the base part Journal of the American Chemi- nox Society 12684 P10884-10896 2004 has been developed by the present inventors.
  • Non-Patent Document 1 Tetrahedron Letters 45 ⁇ P9365— 9368 2004
  • the present invention has been made under the above technical background, and an object of the present invention is to provide a novel means for protecting an amino group of a nucleobase.
  • Non-patent Document 1 N allylcarbamoyl deoxycytidine
  • the present invention provides the following (1) to (7).
  • X represents a moiety other than the amino group of a nucleoside or a similar compound containing a nucleobase having an amino group.
  • Y in the general formulas (II) and (III) may have a substituent! /, May have an aryl group or a substituent, or may have an aryl group.
  • X represents a moiety other than the amino group of a nucleoside containing a nucleobase having an amino group or an analogous compound, and Y represents an electron-withdrawing group.
  • a method for deprotecting an amino group in a nucleobase which comprises converting to a compound represented by the formula:
  • Y in the general formula (III) may have a substituent! /, May have an aryl group or a substituent, and may have an aryl group.
  • Y force in general formula (III) is characterized by being 4-trophenyl group, 3-trophenyl group, 2-trophenyl group, or phenylsulfol group ( 4) A method for deprotecting an amino group in the nucleobase.
  • X represents a moiety other than an amino group of a nucleoside containing a nucleobase having an amino group or an analogous compound
  • Y represents a 4-trophenyl group, a 3-trophenyl group, or a 2-trophenyl group. Or a phenylsulfol group.
  • the present invention provides a novel method for synthesizing a nucleic acid using a substitutional ruberamoyl group as a protecting group for an amino group of a nucleobase.
  • the substitution force rubamoyl group can be removed by heating or the like under neutral conditions, and can be easily introduced by an isocyanate derivative, and has very excellent properties as a protecting group. Yes.
  • the nucleic acid synthesis method of the present invention is characterized by including at least the following protecting group introduction step and deprotection step.
  • the protective group introduction step is represented by the general formula (I)
  • X in the general formulas (I) and (III) represents a nucleoside containing a nucleobase having an amino group, or a moiety other than the amino group of an analogous compound.
  • nucleoside containing a nucleobase having an amino group refers to all substances in which ribose 1-yl or 2-deoxyribose 1-yl group and any heteroaromatic ring having an amino group are bonded. Examples thereof include, but are not limited to, 2′-deoxyadenosine, 2′-deoxyguanosine, 2 ′ deoxycytidine, adenosine, guanosine, cytidine, and the like.
  • Similar nucleoside compound refers to a naturally-occurring or artificially synthesized compound containing a nucleobase in its molecule as in the case of a nucleoside. At least a nucleoside derivative, a nucleoside phosphoramidite Monomers of compounds, oligo DNAs, oligo RNAs, PNAs, and PNAs are included in “analogues of nucleosides”.
  • Nucleoside derivative refers to a compound in which a part of a nucleoside molecule is replaced with another atom or group, for example, a part of a hydroxyl group present at a sugar site in a nucleoside.
  • all compounds protected by appropriate protecting groups include compounds in which the hydrogen atom at the 2-position of deoxyribose is substituted with a functional group other than a hydroxyl group.
  • Nucleoside phosphoramidite complex refers to a compound in which the hydroxyl group at the 5-position of ribose or deoxyribose is protected with an appropriate protecting group and the compound at the 3-position is a free hydroxyl group. Is a compound having a group represented by the formula (IV) bonded thereto.
  • R 3 wherein R 2 is the same or different alkyl group, or R 1 and R 2 may be bonded to each other to contain a hetero atom!
  • R 3 represents a phosphate group, and R 3 represents a phosphate group Represents a protecting group.
  • R 2 include an isopropyl group, and examples of R 3 include a 2-cyanoethyl group, but are not limited thereto.
  • Oligo DNA and oligo RNA include those that have undergone chemical modification.
  • Y in the general formulas (II) and (III) represents an electron-withdrawing group.
  • the electron-withdrawing group may include a substituent, may be an aryl group, may have a substituent! /, May have a arylsulfonyl group, and the like, more specifically.
  • Examples thereof include 4--trophenyl group, 3--trophenyl group, 2--phenol group, and phenylsulfol group.
  • a 2-trophenyl group and a phenolsulfol group can be mentioned as particularly suitable groups.
  • the compound represented by the general formula (III) is novel. It is a compound.
  • the reaction of the compound represented by the general formula (I) with the isocyanate derivative represented by the general formula (II) is carried out by a known method (for example, Tetrahedron Letters 45 ⁇ P9365-9368 2004). Described method).
  • the compound represented by the general formula (I) and the isocyanate derivative represented by the general formula ( ⁇ ) should not inhibit the reaction! ⁇
  • Organic solvent a plurality of organic solvents can be mixed at any volume ratio.
  • the compound represented by the general formula (III) can be obtained by mixing in a mixed organic solvent in step 2) and stirring for a certain period of time.
  • the deprotection step involves heating or microwave irradiation of the compound represented by the general formula (III). And a step of obtaining a compound represented by the general formula (I).
  • the heating temperature is not particularly limited as long as it can be deprotected, but it is preferably 40 to 100 ° C, more preferably 50 to 90 ° C.
  • Heating time is a force that varies depending on the type of substituent of the force rubamoyl group.For example, if the substituent is 2--phenol group, the caloric heat time is preferably 1 to 30 hours. 3 to 20 hours Is more preferable.
  • the frequency of the microwave to be irradiated is not limited as long as it can be deprotected, but it is preferably 1000 to 10,000 MHz, and more preferably 1000 to 5000 MHz.
  • the irradiation time of the microwave required for deprotection varies depending on the type of substituent of the force rubamoyl group. For example, if the substituent is a 2-trophenyl group, the irradiation time is preferably 1 to 30 hours. More preferably, it is 20 hours.
  • the above-described heat treatment or the like is usually performed in a solvent.
  • a solvent a solvent obtained by mixing an organic solvent that does not inhibit the reaction and an aqueous solvent at an arbitrary volume ratio of 0: 100-100: 0 can be used.
  • the organic solvent the deprotection reaction is not inhibited.
  • a mixed organic solvent obtained by mixing a plurality of organic solvents at an arbitrary volume ratio may be used.
  • the aqueous solvent not only water but also any buffer solution can be used, and a mixed buffer solution obtained by mixing a plurality of buffer solutions at an arbitrary concentration may be used.
  • Additives that do not inhibit the reaction include, for example, ammonia, primary amine, secondary amine, tertiary amine, amidine compound, guanidine compound, tetra (n-butyl) ammonium fluoride, carbonate , Carboxylates, metal alkoxides, metal hydroxides, metal hydrides and the like can be used in combination.
  • the nucleic acid synthesis method of the present invention can be carried out in the same manner as a general nucleic acid synthesis method (for example, phosphoramidite method) except for the protecting group introduction step and the deprotection step described above.
  • An example of a synthesis method using the phosphoramidite method is shown below.
  • a nucleoside phosphoramidite complex containing a nucleobase having an amino group is synthesized.
  • a protecting group is introduced into the amino group of this nucleoside phosphoroamidaitoi compound according to the protecting group introduction step described above.
  • a nucleoside phosphoramidite complex having a protecting group introduced is sequentially ligated to synthesize an oligonucleic acid containing a protecting group.
  • the oligonucleic acid containing this protecting group is removed according to the deprotection step described above to obtain an oligonucleic acid.
  • nucleic acid to be synthesized in the synthesis method of the present invention includes a non-natural PNA or the like. Types of nucleic acids are also included.
  • Deoxycytidine hydrochloride (264 mg, 1.0 mmol) was suspended in dimethylformamide (10 ml), and triethylamine (141 n 1.0 mmol) was stirred for 5 minutes. Subsequently, 4-trophenyl isocyanate (164 mg, 1.0 mmol) was added and stirred for 30 minutes, and the solvent was distilled off under reduced pressure. The precipitate formed by adding isopropyl alcohol was dried to obtain the title compound (334 mg, yield 88%).
  • Deoxyadenosine (502 mg, 2.0 mmol) was suspended in pyridine (20 mL), and trimethinoresilinorechloride (758 L, 6.0 mmol) was calored and stirred for 30 minutes. Then, 2-nitrophenol isocyanate (361 mg, 2.2 mmol) was stirred for 90 minutes. After diluting with pyridine (20 mL), concentrated aqueous ammonia (20 mL) was added and stirred for 2 hours. Chloroform (50 mL) and aqueous sodium chloride solution (50 mL) were added, and the mixture was extracted 3 times with black mouth form-pyridine (1: 1, v / v, 30 mL). The organic layer was recovered, the solvent was distilled off under reduced pressure, and water was added. The resulting precipitate was collected and dried to obtain the title compound (740 mg, yield 89%).
  • Deoxycytidine protected with various substitution force rubamoyl groups was heat-treated, and the time until deprotection was measured.
  • Table 1 shows the time (T) until deoxycytidine is deprotected.
  • the amount of protected nucleoside was 1 OmM, and the volume ratio of D 2 0 to DMSO d 6 was 1: 3.
  • the amount of c-protected nucleoside was 7 mM, and the volume ratio of D 20 to DMS O—d 6 was 1: 5.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Saccharide Compounds (AREA)

Abstract

L’invention décrit un procédé de synthèse d’un acide nucléique caractérisé en ce qu’il comprend une étape au cours de laquelle on obtient un composé de formule générale (III) ci-dessous en faisant réagir un composé de formule générale (I) ci-dessous avec un dérivé isocyanate de formule générale (II) ci-dessous, et une autre étape au cours de laquelle on obtient un composé de formule générale (I) ci-dessous en chauffant le composé de formule générale (III) ou en l’irradiant par des micro-ondes. NH2-X (I) (Dans la formule (I), X représente un fragment autre qu’un groupe amino dans une base d’acide nucléique ou autre espèce similaire ayant un groupe amino). O=C=N-Y (II) (Dans la formule (II), Y représente un groupe accepteur d’électrons.) [Formule chimique 3] (III) (Dans la formule (III), X et Y sont tels que précédemment définis).
PCT/JP2006/304400 2005-03-09 2006-03-07 Procede de synthese d’acide nucleique utilisant un groupe carbamoyle substitue en tant que groupe protecteur Ceased WO2006095740A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005-064892 2005-03-09
JP2005064892A JP2006248931A (ja) 2005-03-09 2005-03-09 置換カルバモイル基を保護基とした核酸の合成方法

Publications (1)

Publication Number Publication Date
WO2006095740A1 true WO2006095740A1 (fr) 2006-09-14

Family

ID=36953334

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/304400 Ceased WO2006095740A1 (fr) 2005-03-09 2006-03-07 Procede de synthese d’acide nucleique utilisant un groupe carbamoyle substitue en tant que groupe protecteur

Country Status (2)

Country Link
JP (1) JP2006248931A (fr)
WO (1) WO2006095740A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5397960B2 (ja) 2008-03-11 2014-01-22 国立大学法人 東京大学 粘着末端を有するｄｎａ断片の調製方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030749A1 (fr) * 1999-10-28 2001-05-03 New Pharma Research Sweden Ab Nouveaux composes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030749A1 (fr) * 1999-10-28 2001-05-03 New Pharma Research Sweden Ab Nouveaux composes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BEAUCAGE S.L. ET AL.: "Advances in the Synthesis of Oligonucleosides by the Phosphoramidite Approach", TETRAHEDRON, vol. 48, no. 12, 1992, pages 2223 - 2311, XP000915225 *
GREENE T.W. ET AL.: "protective Groups in Orgaic Synthesis", vol. SEC ED., 1991, JOHN WILEY & SONS, INC., pages: 346, XP003005808 *
SIGMUND H. ET AL.: "Nucleosides. Part LVI. Aminolysis of carbamates of adenosine and cytidine", HELVETICA CHIMICA ACTA, vol. 77, no. 5, 1994, pages 1267 - 1280, XP003005807 *

Also Published As

Publication number Publication date
JP2006248931A (ja) 2006-09-21

Similar Documents

Publication Publication Date Title
US11512106B2 (en) Nucleoside analogue, preparation method and application
US5760209A (en) Protecting group for synthesizing oligonucleotide analogs
JP4580870B2 (ja) リボヌクレオチド又はリボヌクレオチド誘導体の製造方法
CN107474091B (zh) 光敏保护基保护的5-醛基胞嘧啶亚磷酰胺单体及其制备方法和寡聚核苷酸的合成和应用
CN108424433B (zh) 一种α核苷合成方法
WO2006095740A1 (fr) Procede de synthese d’acide nucleique utilisant un groupe carbamoyle substitue en tant que groupe protecteur
Srivastava et al. Achiral, acyclic nucleic acids: synthesis and biophysical studies of a possible prebiotic polymer
JPH06135988A (ja) ヌクレオシド誘導体
Apostle et al. Synthesis of Sensitive RNAs Using Fluoride‐Cleavable Groups as Linkers and Amino‐Group Protection
Obika et al. Recognition of T· A interruption by 2′, 4′-BNAs bearing heteroaromatic nucleobases through parallel motif triplex formation
EP1397377B1 (fr) Hybrides de calix 4 arene-nucleoside et de calix 4 arene-oligonucleotide
Sekine et al. Synthesis of N-(Trimethylsilylethoxycarbonyl)-deoxycytidine and Deoxyadenosine Derivatives as Key Intermediates for the DNA Synthesis using Fluoride Ion-Promoted Deprotection Strategy
EP1812457A1 (fr) SYNTHÈSE STÉRÉOSÉLECTIVE DE ß-NUCLÉOSIDES
EP1186612B1 (fr) Procede de preparation de derives de cytidine
JP6429264B2 (ja) ボラノホスフェート化合物、及び核酸オリゴマー
EP2841401B1 (fr) Procédé pour la protection de groupes fonctionnels hydroxyle, amine ou thiol à l'aide du tétraisopropyldisilane et composés protégés correspondants
WO2006095739A1 (fr) Procede de deblocage des groupes 2'-hydroxyle des ribonucleosides
JP2003002895A (ja) 核酸の合成法
Nakahara et al. A 2-Amino-6-methylpyridin-5-yl Nucleobase for GC Base Pair Recognition in the Parallel Triplex DNA
JP2005514462A6 (ja) 新規カリックス［４］アレーン−ヌクレオシドおよびカリックス［４］アレーン−オリゴヌクレオチドハイブリッド
WO2004048376A1 (fr) Nucleosides de napthylidine bicycliques
JPS59152399A (ja) 核酸誘導体の製造方法
JP2010509938A (ja) Ｒｎａ脱シリル化方法
JPH06135989A (ja) ヌクレオシド誘導体
JPH01224392A (ja) 新規核酸誘導体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06728719

Country of ref document: EP

Kind code of ref document: A1