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Aromatic amino acid labeled with stable isotope, method for incorporating the same into target protein and method for analyzing protein structure using nmr 実績あり

外国特許コード F110003613
整理番号 A202-01WO
掲載日 2011年6月30日
出願国 欧州特許庁(EPO)
出願番号 04799421
公報番号 1679302
公報番号 1679302
出願日 平成16年11月1日(2004.11.1)
公報発行日 平成18年7月12日(2006.7.12)
公報発行日 平成26年4月30日(2014.4.30)
国際出願番号 JP2004016215
国際公開番号 WO2005042469
国際出願日 平成16年11月1日(2004.11.1)
国際公開日 平成17年5月12日(2005.5.12)
優先権データ
  • 2004JP016215 (2004.11.1) WO
  • 特願2003-373304 (2003.10.31) JP
発明の名称 (英語) Aromatic amino acid labeled with stable isotope, method for incorporating the same into target protein and method for analyzing protein structure using nmr 実績あり
発明の概要(英語) The present invention herein provides, for instance, a stable isotope-labeled phenylalanine wherein a carbon atom of the phenyl group linked to an amino acid residue is **13C, 2 to 4 carbon atoms of the remaining 5, carbon atoms constituting the phenyl group are **12C atoms to which deuterium atoms are bonded, and the remaining carbon atoms are **13C atoms to which hydrogen atoms are linked, and a stable isotope-labeled tyrosine wherein a carbon atom of the phenyl group linked to an amino acid residue is **13C, the carbon atom bonded to the hydroxyl group (OH group) of the phenyl group is **12C or **13C, 2 to 4 carbon atoms of the remaining 4 carbon atoms constituting the phenyl group are **12C atoms to which deuterium atoms are bonded, and the remaining carbon atoms are **13C atoms to which hydrogen atoms are linked.
The stable isotope-labeled amino acid permits the elimination of such a conventional problem concerning the compl exity of the NMR signals ascribed to aromatic rings, the complexity being a principal cause of making the NMR analysis difficult, encountered when using the conventional uniformly labeled amino acid residue.
Moreover, the isotope-labeled amino acid likewise permits the substantial improvement of the sensitivity thereof to the NMR spectroscopic analysis.
特許請求の範囲(英語) [claim1]
1. A stable isotope-labeled aromatic amino acid selected from those listed below: A stable isotope-labeled phenylalanine wherein a carbon atom of the phenyl group linked to an amino acid residue represented by the following general formula A is **13C, 2 to 4 carbon atoms of the remaining 5 carbon atoms constituting the phenyl group are **12C atoms to which deuterium atoms are bonded, and the remaining carbon atoms are **13C atoms to which hydrogen atoms are linked; A stable isotope-labeled tyrosine wherein a carbon atom of the phenyl group linked to an amino acid residue represented by the following general formula A is **13C, the carbon atom bonded to the hydroxyl group (OH group) of the phenyl group is **12C or **13C, 2 to 4 carbon atoms of the remaining 4 carbon atoms constituting the phenyl group are **12C atoms to which deuterium atoms are bonded, and the remaining carbon atoms are **13C atoms to which hydrogen atoms are linked; A stable isotope-labeled tryptophan selected from the group consisting of the following formulae (8) to (13): (see diagramm)(see diagramm) wherein C represents **12C or **13C, N represents **14N or **15N, Z represents a hydrogen atom or a deuterium atom and R represents a group represented by the following formula: (see diagramm) wherein each of X, Y and Z represents a hydrogen atom or a deuterium atom;
and A stable isotope-labeled histidine wherein a carbon atom of the imidazolyl group linked to an amino acid residue represented by the following general formula A is **13C, the both of the remaining two carbon atoms constituting the imidazolyl group are **13C atoms to which hydrogen atoms are linked or one of these two carbon atoms is **12C to which a deuterium atom is bonded, while the other carbon atom is **13C to which a hydrogen atom is linked, one of the two nitrogen atoms constituting the imidazolyl group is **15N, while the other nitrogen atom is **14N, and the hydrogen atom constituting the NH group is not a deuterium atom:

- * **1C(X) (Y) - * **2C(Z) ( **15NH 2) (* **3COOH) (A)

wherein each of * **1C, * **2C, and * **3C represents **12C or **13C atom, each of X, Y and Z represents a hydrogen or deuterium atom.
[claim2]
2. The stable isotope-labeled aromatic amino acid as set forth in claim 1, wherein each of * **1C, * **2C, and * **3C appearing in the general formula A is **13C atom.
[claim3]
3. The stable isotope-labeled aromatic amino acid as set forth in claim 1, wherein it is represented by one of the following general formula (1) to (7), (14) and (15): (see diagramm)(see diagramm)(see diagramm) wherein C represents **12C or **13C, Z represents a hydrogen atom or a deuterium atom and R represents a group represented by the following formula: (see diagramm) wherein each of X, Y and Z represents a hydrogen atom or a deuterium atom.
[claim4]
4. The stable isotope-labeled aromatic amino acid as set forth in claim 1 or claim 3, wherein it is an amino acid represented by the general formula (1), (2), (3), (4), (7) or (8).
[claim5]
5. A combination of stable isotope-labeled amino acids constituting a target protein wherein the aromatic amino acids constituting the target protein are stable isotope-labeled aromatic amino acids as set forth in any one of claims 1 to 4 and the aliphatic amino acids constituting the target protein are stable isotope-labeled aliphatic amino acids which satisfy the following labeled pattern: (a) In case where a methylene group carrying two hydrogen atoms is present, one of the methylene hydrogen atoms is deuterated; (b) In case where a prochiral gem-methyl group is present, all of the hydrogen atoms on one of the methyl groups are completely deuterated, while the hydrogen atoms on the other methyl group are partially deuterated; (d) In case where a methyl group other than the foregoing ones is present, all of the hydrogen atoms on the methyl group except for one hydrogen atom are deuterated or all of the hydrogen atoms on the methyl group are deuterated; (e) After the deuteration in the foregoing items (a), (b) and (d), not less than 15 atom% of the carbon atoms of hydrogen atom-carrying methylene and/or methyl groups are replaced with **13C atoms; and (f) All of the nitrogen atoms present are completely replaced with **15N atoms.
[claim6]
6. The combination of stable isotope-labeled amino acids constituting a target protein as set forth in claim 5 wherein the aliphatic amino acids constituting the target protein are stable isotope-labeled aliphatic amino acids wherein the requirement (e) for the label pattern is: (e) After the deuteration in the foregoing requirements (a), (b) and (d), all of the carbon atoms of hydrogen atom-carrying methylene and/or methyl groups are replaced with **13C atoms.
[claim7]
7. The combination of stable isotope-labeled amino acids constituting a target protein as set forth in claim 6, wherein the stable isotope-labeled aliphatic amino acid satisfies the requirement (e) after the deuteration in the foregoing requirements (a), (b) and (d), all of the carbon atoms of hydrogen atom- carrying methylene and methyl groups are replaced with **13C atoms.
[claim8]
8. The combination of stable isotope-labeled amino acids as set forth in claim 7, wherein the carbon atoms constituting the carbonyl and guanidyl groups of the stable isotope-labeled aliphatic amino acids are replaced with **13C atoms.
[claim9]
9. A combination of stable isotope-labeled amino acids constituting a target protein wherein the aromatic amino acids constituting the target protein are stable isotope-labeled aromatic amino acids as set forth in any one of claims 1 to 4 and the aliphatic amino acids constituting the target protein are stable isotope-labeled aliphatic amino acids which satisfy the following labeled pattern: (a) In case where a methylene group carrying two hydrogen atoms is present, one of the methylene hydrogen atoms is deuterated; (b) In case where a prochiral gem-methyl group is present, all of the hydrogen atoms on one of the methyl groups are completely deuterated, while the hydrogen atoms on the other methyl group are partially deuterated; (d) In case where a methyl group other than the foregoing ones is present, all of the hydrogen atoms on the methyl group except for one hydrogen atom are deuterated or all of the hydrogen atoms on the methyl group are deuterated; (e) After the deuteration in the foregoing items (a), (b) and (d), all of the carbon atoms of hydrogen atom-carrying methylene and/or methyl groups are **12C atoms; and (f) All of the nitrogen atoms present are completely replaced with **15N atoms.
[claim10]
10. A method for incorporating, into a target protein, stable isotope-labeled aromatic amino acids as set forth in any one of claims 1 to 4, wherein the isotope-labeled aromatic amino acids are incorporated into the protein through the cell-free protein synthesis technique.
[claim11]
11. A method for incorporating, into a target protein, a combination of stable isotope-labeled aromatic amino acids as set forth in any one of claims 5 to 9, wherein the isotope-labeled aromatic amino acids are incorporated into the protein through the cell-free protein synthesis technique.
[claim12]
12. A method for the preparation of a target protein constituted by stable isotope-labeled amino acids comprising the step of synthesizing the target protein according to the cell-free protein synthesis technique while making use of combinations of stable isotope-labeled amino acids as set forth in any one of claims 5 to 9, as the whole amino acids components constituting the target protein.
[claim13]
13. A method for the NMR spectroscopic structural analysis of a target protein comprising the steps of incorporating stable isotope-labeled amino acids as set forth in any one of claims 1 to 4 into the target protein and then carrying out the NMR spectrometric measurement on the target protein to thus structurally analyze the same.
[claim14]
14. A method for the NMR spectroscopic structural analysis of a target protein comprising the step of structurally analyzing a target protein whose constituent amino acids are completely replaced with a combination of stable isotope-labeled amino acids as set forth in any one of claims 5 to 9 according to the NMR spectrometry.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • KAINOSHO MASATSUNE
  • TERAUCHI TSUTOMU
国際特許分類(IPC)
欧州特許分類/主・副
  • C07C047/54
  • C07C047/575
  • C07C229/08
  • C07C229/36
  • C07D209/20
  • C07D233/64
  • C07K001/00
  • C12P021/02
  • G01N024/08
  • G01R033/465
  • M07B200/05
指定国 Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LU MC NL PL PT RO SE SI SK TR
参考情報 (研究プロジェクト等) CREST Understanding the Brain (Mechanisms of Brain) AREA
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