Process for the specific isotopic labeling of methyl groups of Val, Leu and Ile

Patent metadata

Field	Value
Publication number	US-10053396-B2
Application number	US-201715620850-A
Country	US
Kind code	B2
Filing date	Jun 13, 2017
Priority date	Jan 6, 2010
Publication date	Aug 21, 2018
Grant date	Aug 21, 2018

Abstract

A process for the specific isotopic labeling of an amino acid selected from Valine (Val), Leucine (Leu), and Isoleucine (Ile), in proteins and biomolecular assemblies by introducing, in a medium containing bacteria overexpressing a protein, an acetolactate derivative of Formula I of the application.

First claim

The invention claimed is:

1. A process for the specific isotopic labeling of an amino acid selected from Valine (Val), Leucine (Leu), and Isoleucine (Ile), in proteins and biomolecular assemblies comprising:
a) introducing, in a medium containing bacteria overexpressing a protein, a composition consisting essentially of an acetolactate derivative of Formula I:

wherein:
X is 1 H or 2 H (D),
each Y is independently from the others 12 C or 13 C,
R 1 is a methyl group in which the carbon atom is 13 C or 12 C and the hydrogen atoms are independently from each other 1 H or 2 H (D),
R 2 is either a methyl group in which the carbon atom is 13 C or 12 C and the hydrogen atoms are independently from each other 1 H or 2 H (D), or an ethyl group in which the carbon atoms are independently from each other 13 C or 12 C and the hydrogen atoms are independently from each other 1 H or 2 H (D),

with the proviso that:
the hydrogen atoms of R 1 and the hydrogen atoms of R 2 are not all, at the same time, either 1 H or 2 H (D).

2. The process of claim 1 further comprising:
b) overexpression of the protein by the bacteria contained in the medium, and
c) purification of the protein.

3. The process of claim 1 , wherein in the acetolactate derivative of Formula I:
R 1 is chosen from the following groups:
12 CH 3 , 12 CD 3 , 13 CH 3 , 13 CD 3 , 13 CHD 2 , 13 CH 2 D, and
R 2 is chosen from the following groups:
12 CH 3 , 12 CD 3 , 13 CH 3 , 13 CD 3 , 13 CHD 2 , 13 CH 2 D, 12 CH 3 12 CD 2 , 12 CD 3 12 CD 2 , 13 CH 3 12 CD 2 , 13 CH 3 13 CD 2 , 13 CD 3 13 CD 2 , 13 CHD 2 13 CD 2 , 13 CH 2 D 13 CD 2 , 13 CHD 2 12 CD 2 , 13 CH 2 D 12 CD 2 .

4. The process of claim 1 , wherein the acetolactate derivative of Formula I is selected from the following compounds:
2-hydroxy-2-( 13 C)methyl-3-oxo-4( 2 H 3 )butanoic acid,
2-hydroxy-2-( 2 H 3 )methyl-3-oxo-4( 13 C)butanoic acid,
2-( 2 H 5 )ethyl-2-hydroxy-3-oxo-4-( 13 C)methylbutanoic acid,
1,2,3,4-( 13 C)-2-( 2 H 5 )ethyl-2-hydroxy-3-oxobutanoic acid,
1,2,3-( 13 C)-2-( 13 C)methyl-2-hydroxy-3-oxo-4-( 2 H 3 )butanoic acid,
1,2,3,4-( 13 C)-2-( 2 H 3 )methyl-2-hydroxy-3-oxobutanoic acid,
1,2,3-( 13 C)-2-(1′-( 2 H 2 ), 13 C 2 )ethyl)-2-hydroxy-3-oxo-4-( 2 H 3 )butanoic acid,
3,4-( 13 C)-2-( 13 C)methyl-2-hydroxy-3-oxo-4-( 2 H 3 )butanoic acid,
3,4-( 13 C)-2-( 2 H 3 , 13 C)methyl-2-hydroxy-3-oxobutanoic acid,
3,4-( 13 C)-2-(1′-( 2 H 2 ), 13 C 2 )ethyl-2-hydroxy-3-oxo-4-( 2 H 3 )butanoic acid,
3,4-( 13 C)-2-( 2 H 5 , 13 C 2 )ethyl-2-hydroxy-3-oxobutanoic acid, and
3,4-( 13 C)-2-(1′-( 2 H 2 ), 13 C 2 )ethyl-2-hydroxy-3-oxobutanoic acid.

5. The process of claim 1 , wherein the acetolactate derivative of Formula I is selected from the following compounds:
2-hydroxy-2-( 2 H 2 , 13 C)methyl-3-oxo-4-( 2 H 3 )butanoic acid,
2-hydroxy-2-( 2 H 3 )methyl-3-oxo-4-( 2 H 2 , 13 C)butanoic acid,
2-( 2 H 5 )ethyl-2-hydroxy-3-oxo-4-( 2 H 2 , 13 C)butanoic acid, and
2-(1′-( 2 H 2 ),2′-( 2 H),2′-( 13 C))ethyl-2-hydroxy-3-oxo-4-( 2 H 3 )butanoic acid.

6. The process of claim 1 , wherein the acetolactate derivative of Formula I is selected from the following compounds:
2-hydroxy-2-( 2 H, 13 C)methyl-3-oxo-4-( 2 H 3 )butanoic acid,
2-hydroxy-2-( 2 H 3 )methyl-3-oxo-4-( 2 H, 13 C)butanoic acid, and
2-( 2 H 5 )ethyl-2-hydroxy-3-oxo-4-( 2 H, 13 C)butanoic acid.

7. The process of claim 1 , wherein said process specifically isotopically labels Valine in proteins and biomolecular assemblies.

8. The process of claim 1 , wherein said process specifically isotopically labels Leucine in proteins and biomolecular assemblies.

9. The process of claim 1 , wherein said process specifically isotopically labels Isoleucine in proteins and biomolecular assemblies.

10. The process of claim 1 , wherein said process specifically isotopically labels methyl groups of Leucine and Valine in proteins and biomolecular assemblies.

11. The process of claim 1 , wherein said process specifically isotopically labels γ2-methyl groups of Isoleucine in proteins and biomolecular assemblies.

12. A process for analyzing a protein by NMR, comprising labeling a protein to be analyzed by the process of claim 1 .

Assignees

• Commissariat Energie Atomique
• Centre Nat Rech Scient
• Univ Joseph Fourier
• Centre Nat Rech Scient

Inventors

• Gans Pierre
• Boisbouvier Jerome
• Ayala Isabel
• Hamelin Olivier

Classifications

• C07B59/008 Primary

  Peptides; Proteins · CPC title

• C07C59/215

  containing singly bound oxygen containing groups · CPC title

• G01N24/087

  Structure determination of a chemical compound, e.g. of a biomolecule such as a protein · CPC title

• C07B59/001

  Acyclic or carbocyclic compounds · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title