TOP > 外国特許検索 > Stable isotope-labeled aromatic amino acids, method for incorporating the same in target protein and method for NMR-structural analysis of proteins

Stable isotope-labeled aromatic amino acids, method for incorporating the same in target protein and method for NMR-structural analysis of proteins 実績あり

外国特許コード F110003335
整理番号 A202-01US2
掲載日 2011年6月22日
出願国 アメリカ合衆国
出願番号 55908909
公報番号 20100056799
公報番号 8440169
出願日 平成21年9月14日(2009.9.14)
公報発行日 平成22年3月4日(2010.3.4)
公報発行日 平成25年5月14日(2013.5.14)
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
  • 特願2003-373304 (2003.10.31) JP
  • 11/414,756 (2006.4.28) US
  • 2004JP016215 (2004.11.1) WO
発明の名称 (英語) Stable isotope-labeled aromatic amino acids, method for incorporating the same in target protein and method for NMR-structural analysis of proteins 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 complexity 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.
従来技術、競合技術の概要(英語) BACKGROUND ART
The structural analysis of a protein by the NMR spectrometry should always be carried out while taking into consideration such problems as any possible overlapping between NMR signals and the reduction of signal intensities due to the relaxation phenomenon.
In this respect, it would be essential to the solution of this problem to develop an advanced NMR measurement and analysis technique.
However, proteins each having a molecular weight on the order of about 20,000 can presently be analyzed without being accompanied by any significant error because of the application of the multi-nuclear and multi-dimensional NMR spectroscopic technique developed in the early 1990s to the protein and the development of a technique for the mass-production of stable isotope-labeled proteins, which has been advanced along with the progress of the NMR spectroscopic technique.
However, all of these methods are ones for obtaining information on the three-dimensional structure of a high molecular weight protein at the sacrifice of the precision of the determination of the higher-order structure thereof.
Therefore, these techniques are limited in the subject to be analyzed and the effectiveness thereof.
In this respect, Patent Document 1 specified below discloses an invention which can solve these conventional problems, which permits the deuterium-exchange of a protein without adversely affecting the sensitivity of the remaining hydrogen nuclei to the NMR spectroscopic measurement and which simultaneously permits the rapid and highly reliable analysis of the NMR spectra observed for a protein having a molecular weight higher than the conventional limit and the determination of the higher-order structure thereof with a high accuracy.
However, this invention never specifies the isotope-labeling pattern on the aromatic ring portion present in an aromatic amino acid.
On the other hand, aromatic amino acids such as Phe, Tyr and Trp play important roles along with the amino acids each carrying a long chain alkyl group such as Leu, Val and Ile in the formation of the three-dimensional structure of the hydrophobic core portion contained in a globular protein.
In addition, these aromatic amino acids likewise play important roles in the manifestation of protein functions typical of the substrate-recognizing function, while making the most use of functional groups such as the hydroxyl group of Tyr and the nitrogen derived from the indole ring of Trp, or the pi -electrons common to the aromatic rings, in addition to the roles in the formation of the three-dimensional structure.
In this respect, however, if using a sample uniformly labeled with stable isotope (13C, 15N, 2H) disclosed in Patent Document 1 or a sample (non-labeled) having a natural abundance ratio of isotopes, the proton NMR signals, ascribed to the ring portions of the aromatic amino acids, in particular, Phe and Trp, show chemical shifts quite close to one another and the chemical shifts of the carbon atoms (13C) to which they are bonded likewise come close to one another.
Accordingly, quite complicated signals are obtained for such a uniformly labeled derivative, this accordingly results in the deterioration of the sensitivity thereof to the NMR spectroscopic measurement and this makes, quite difficult, the individual observation of signals and the assignment thereof to each corresponding sequence.
Under such circumstances, there have been proposed a variety of methods for overcoming these difficulties, for collecting the information of nuclear Overhauser effects (NOE) concerning aromatic ring protons serving as the distance-limiting information quite important for the determination of the three-dimensional structure and for accurately measuring information on the local structure of aromatic rings.
However, all of the conventional methods are ones developed while aiming at the sample uniformly labeled with stable isotope (13C, 15N, 2H), whose preparation is quite easy and therefore, there has not yet been developed any method quite excellent from the viewpoint of the practicability.
Patent Document 1: International Publication WO 03/053910A1

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 a group 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; and
A stable isotope-labeled tyrosine wherein a carbon atom of the phenyl group linked to a group 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;
-*1C(X)(Y) -- *2C(Z)(15NH)(*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 the following general formula (1) to (8):

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:

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 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 claim 1 and the aliphatic amino acids constituting the target protein are stable isotope-labeled aliphatic amino acids which satisfy the following requirements of labeled patterns: (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 requirements (a), (b) and (d), all 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 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/or methyl groups are replaced with 13C atoms.
[claim7]
7. The combination of stable isotope-labeled amino acids as set forth in claim 6, wherein the carbon atoms constituting the carbonyl and guanidyl groups of the stable isotope-labeled aliphatic amino acids are replaced with 13C atoms.
[claim8]
8. 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 claim 1 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.
[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 claim 1 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.
  • 発明者/出願人(英語)
  • KAINOSHO MASATSUNE
  • TERAUCHI TSUTOMU
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 424/9.34
  • 435/106
  • 435/131
  • 530/350
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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