Granted Patent B2

US 12,286,657 · App. 17/288,798 · Granted Apr 29, 2025

Inventors: Jason Chin (Cambridgeshire, GB); Nicolas Huguenin-Dezot (Basel, CH); Mohan Mahesh (Cambridgeshire, GB); Shan Tang (Cambridgeshire, GB); Duy P. Nguyen (Cambridgeshire, GB)

Assignee: United Kingdom Research and Innovation

C12N9/93C07D317/62C12N9/506C12Y304/22069C12Y601/01026C07K2319/60

Abstract

Claims (52)

1. A method of producing a polypeptide comprising 2,3-diamino propionic acid (DAP), said method comprising genetically incorporating an unnatural amino acid into a polypeptide, wherein the unnatural amino acid is of formula (I) or (II):

or salts, solvates, tautomers, isomers, or mixtures thereof;

wherein:

R 1 is H, an amino acid residue, or a peptide;

R 2 is H, C 1-6 alkyl, C 1-6 haloalkyl, or C 5-20 aryl;

q is 1, 2, or 3;

each R 3 or R 4 is independently selected from H, halo, C 1-6 alkyl, C 1-6 haloalkyl, C 5-20 aryl, C 3-20 heteroaryl, OC 1-6 alkyl, SC 1-6 alkyl, NH(C 1-6 alkyl), and N(C 1-6 alkyl) 2 ;

X is X 1 —Y, S—S—R 5 , Se—Se—R 5 , O—NH—R 5 , S—NH—R 5 , Se—NH—R 5 , X 2 —Y 1 , X 3 —Y 2 , N 3 , or NH—S(O) 2 —Y 3 ;

X 1 is S, Se, O, NH, or N(C 1-6 alkyl);

X 2 is S, Se, or O;

X 3 is NH—C(O)—O;

X 4 is NH—C(O)—O, O, S, or NH;

R 5 is selected from H, halo, C 1-6 alkyl, C 1-6 haloalkyl, C 5-20 aryl, C 3-20 heteroaryl, OC 1-6 alkyl, NH(C 1-6 alkyl), N(C 1-6 alkyl) 2 , peptides, sugars, C 3-20 heterocyclyl, and nucleic acids;

Y is a protecting group selected from:

R 6 is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, CO 2 H, CO 2 R′, SO 2 H, SO 2 R′, C 5-20 aryl, C 3-20 heteroaryl, NHC(O)R′, and NHR′;

R 7 and R 8 are independently selected from H, OH, O(C 1-6 alkyl), O(C 5-20 aryl), and O(C 3-20 heteroaryl); or R 7 and R 8 are linked together to form an O—CH 2 —O group;

each R′ is independently selected from C 1-6 alkyl, C 1-6 haloalkyl, and C 5-20 aryl;

R 9 is selected from H, C 1-6 alkyl, C 1-6 haloalkyl, CO 2 H, CO 2 R′, SO 2 H, SO 2 R′, and C 5-20 aryl;

R 10 is selected from H, C 1-6 alkyl, and C 1-6 haloalkyl;

R 11 is selected from H, C 1-6 alkyl, and C 1-6 haloalkyl;

X 5 is S, O, NH, N—C(O)—O—R′, N—S(O) 2 H, N—S(O) 2 R′, or NR′;

Y 1 is a protecting group selected from:

Y 2 is a protecting group selected from:

 t-Bu, and CH 2 Ph;

M + is Li + , Na + , K + , or N(R 13 ) 4 + ;

Z is Si or Ge;

R 12 is C 1-6 alkyl or C(O)—(C 5-20 aryl);

R 13 is H, C 1-6 alkyl, allyl, or C 5-20 aryl; and

Y 3 is a protecting group

wherein the method comprises:

(i) providing a nucleic acid encoding the polypeptide, wherein said nucleic acid comprises an orthogonal codon encoding the unnatural amino acid; and

(ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said unnatural amino acid into the polypeptide chain,

wherein said orthogonal codon comprises an amber codon (TAG), said tRNA comprises Methanosarcina barkeri tRNA CUA (MbtRNA CUA ), and said tRNA synthetase comprises the Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS synthetase) of SEQ ID NO: 3.

2. The method of claim 1 , wherein said unnatural amino acid comprises

or salts, solvates, tautomers, isomers, or mixtures thereof.

3. The method of claim 1 , wherein said method is carried out inside a live cell.

4. The method of claim 3 , wherein said live cell is an E. coli cell.

5. The method of claim 3 , wherein said live cell is a mammalian cell.

6. The method of claim 1 , wherein said unnatural amino acid is of the formula:

or salts, solvates, tautomers, isomers, or mixtures thereof.

7. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, and wherein said unnatural amino acid is of the formula:

or salts, solvates, tautomers, isomers, or mixtures thereof.

8. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, and wherein Y is:

9. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, and wherein said unnatural amino acid is of the formula:

or salts, solvates, tautomers, isomers, or mixtures thereof.

10. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, and wherein Y is selected from the group consisting of:

11. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, and wherein R 6 is selected from the group consisting of H, CH 3 , CH 2 CH 3 , CF 3 , CO 2 H, CO 2 CH 3 , CO 2 CH 2 CH 3 , and Ph.

12. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, and wherein R 6 is CH 3 .

13. The method of claim 1 , wherein said unnatural amino acid is of formula (I), or salts, solvates, tautomers, isomers, or mixtures thereof, wherein Y is:

14. The method of claim 4 , wherein the E. coli cell is a BL21 DE3 cell.

15. The method of claim 5 , wherein the mammalian cell is a HEK293T cell.

16. The method of claim 1 , further comprising deprotecting said unnatural amino acid to 2,3-diamino propionic acid (DAP).

Priority Claims (1)

Continuity (1)

References Cited (196)