Methods of incorporating an amino acid comprising a BCN group into a polypeptide using an orthogonal codon encoding it and an orthogonal pylrs synthase

1. A method of producing a polypeptide comprising a bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) group in a cell, said method comprising genetically incorporating an amino acid comprising a BCN group into a polypeptide in a cell, wherein said amino acid comprising a BCN group is a BCN lysine, and wherein producing the polypeptide comprises

(i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the amino acid having a BCN group; and

(ii) translating said nucleic acid in the cell in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognizing said orthogonal codon and incorporating said amino acid having a BCN group into the polypeptide chain,

wherein the tRNA synthetase consists of SEQ ID NO: 1 with the three following mutations: Y271M, 274G, and C313A; and

wherein said BCN group is in the exo form,

wherein said BCN lysine has the structure:

2. The method according to claim 1 , wherein said orthogonal codon comprises an amber codon (TAG) and said tRNA is mbtRNA CUA .

3. The method according to claim 1 , wherein said amino acid having a BCN group is incorporated at a position corresponding to a lysine residue in the wild type polypeptide.

4. The method according to claim 1 , wherein the method produces a polypeptide comprising a single BCN group.

5. The method according to claim 1 , further comprising:

(iii) contacting the translated polypeptide with a tetrazine compound, and incubating to allow joining of the tetrazine compound to the BCN group by an inverse electron demand Diels-Alder cycloaddition reaction.

6. The method according to claim 5 , wherein the tetrazine compound has the chemical formula of

wherein:

(i) X═CH, R═BOC (Formula VI-1);

(ii) X═N, R═BOC (Formula VI-2);

(iii) X═CH, R═TAMRA-X (Formula VI-3);

(iv) X═N, R═TAMRA-X (Formula VI-4);

(v) X═CH, R═Bodipy TMR-X (Formula VI-5); or

(vi) X═CH, R═TAMRA (Formula VI-6),

wherein:

(i) R═BOC (Formula VII-1):

(ii) R═TAMRA-X (Formula VII-2); or

(iii) R═Bodipy-FL (Formula VII-3),

or

wherein:

(i) R═BOC (Formula IX-I); or

(ii) R═CFDA (Formula IX-2).

7. The method according to claim 6 , wherein the tetrazine compound has the chemical formula selected from the group consisting of Formula VI-1, Formula VI-2, Formula VII-1 and Formula VIII-1, and wherein the pseudo first order rate constant for the reaction is at least 80 M −1 S −1 .

8. The method according to claim 5 , wherein said reaction of step (iii) is allowed to proceed for 10 minutes or less.

9. The method according to claim 5 , wherein said reaction of step (iii) is allowed to proceed for 1 minute or less.

10. The method according to claim 5 , wherein said reaction of step (iii) is allowed to proceed for 30 seconds or less.

11. The method according to claim 6 , wherein said tetrazine compound has the chemical formula selected from the group consisting of Formula VI-3, Formula VI-4, Formula VI-5, Formula VI-6, Formula VII-2, Formula VII-3, and Formula IX-2.

12. The method according to claim 5 , wherein said tetrazine compound is further joined to a fluorophore.

13. The method according to claim 12 , wherein said fluorophore comprises fluorescein, tetramethyl rhodamine (TAMRA) or boron-dipyrromethene (BODIPY).