Norbornene modified peptides and their labelling with tetrazine compounds

Patent metadata

Field	Value
Publication number	US-9968690-B2
Application number	US-201314373292-A
Country	US
Kind code	B2
Filing date	Jan 21, 2013
Priority date	Jan 20, 2012
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

The invention relates to a polypeptide comprising an amino acid having a norbornene group. Suitably said norbornene group is present as an amino acid residue of a norbornene lysine. The invention also relates to a method of producing a polypeptide comprising a norbornene group, said method comprising genetically incorporating an amino acid comprising a norbornene group into a polypeptide.

First claim

The invention claimed is:

1. A method of producing a polypeptide comprising a Nε-5-norbornene-2-yloxycarbonyl-L-lysine, said method comprising (i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal amber codon (TAG) encoding said Nε-5-norbornene-2-yloxycarbonyl-L-lysine; (ii) translating said nucleic acid in the presence of said Nε-5-norbornene-2-yloxycarbonyl-L-lysine, a MbtRNA CUA and a MbPylRS tRNA synthetase, wherein said MbPylRS tRNA synthetase contains the following amino acid substitutions in SEQ ID NO: 1: L274A, C313S, and M315I, recognizes said MbtRNA CUA and said Nε-5-norbornene-2-yloxycarbonyl-L-lysine and attaches said Nε-5-norbornene-2-yloxycarbonyl-L-lysine acid to said MbtRNA CUA and said MbtRNA CUA recognizes said orthogonal amber codon and incorporates said Nε-5-norbornene-2-yloxycarbonyl-L-lysine into the polypeptide chain at said orthogonal amber codon.

2. A method according to claim 1 , wherein said Nε-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated at a position corresponding to a lysine residue in the polypeptide.

3. A method according to claim 1 , wherein said Nε-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated at a position corresponding to a serine residue in the polypeptide.

4. A method according to claim 1 , wherein said Nε-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated at a position corresponding to an asparagine residue in the polypeptide.

5. A method according to claim 1 , wherein said polypeptide contains a single Nε-5-norbornene-2-yloxycarbonyl-L-lysine.

6. A method according to claim 1 , wherein said Nε-5-norbornene-2-yloxycarbonyl-L-lysine is joined to a tetrazine group after said Nε-5-norbornene-2-yloxycarbonyl-L-lysine is incorporated into said polypeptide via a reaction between the tetrazine group and norbornene.

7. The method of claim 6 , wherein said tetrazine group has a structure selected from the groups consisting of:

wherein X is CH or N and R is tert-butyloxycarbonyl (Boc).

8. A method according to claim 6 , wherein said tetrazine group is further joined to a fluorophore or to a PEG group.

9. A method according to claim 8 , wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).

Assignees

• Medical Res Council
• Univ North Carolina State

Inventors

• Chin Jason
• Deiters Alexander
• Lang Kathrin

Classifications

• C12Y601/01026

  Pyrrolysine-tRNAPyl ligase (6.1.1.26) · CPC title

• C12P21/02

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

• C07C2601/16

  the ring being unsaturated · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C07C2602/42

  the bicyclo ring system containing seven carbon atoms · CPC title