US 12,286,657 B2
Methods and compositions
Jason Chin, Cambridgeshire (GB); Nicolas Huguenin-Dezot, Basel (CH); Mohan Mahesh, Cambridgeshire (GB); Shan Tang, Cambridgeshire (GB); and Duy P. Nguyen, Cambridgeshire (GB)
Assigned to United Kingdom Research and Innovation, Swindon (GB)
Appl. No. 17/288,798
Filed by United Kingdom Research and Innovation, Swindon (GB)
PCT Filed Oct. 24, 2019, PCT No. PCT/GB2019/053023
§ 371(c)(1), (2) Date Apr. 26, 2021,
PCT Pub. No. WO2020/084307, PCT Pub. Date Apr. 30, 2020.
Claims priority of application No. 1817444 (GB), filed on Oct. 26, 2018.
Prior Publication US 2022/0010296 A1, Jan. 13, 2022
Int. Cl. C12N 9/00 (2006.01); C07D 317/62 (2006.01); C12N 9/50 (2006.01)
CPC C12N 9/93 (2013.01) [C07D 317/62 (2013.01); C12N 9/506 (2013.01); C12Y 304/22069 (2013.01); C12Y 601/01026 (2013.01); C07K 2319/60 (2013.01)] 16 Claims
 
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):

OG Complex Work Unit Chemistry
or salts, solvates, tautomers, isomers, or mixtures thereof;
wherein:
R1 is H, an amino acid residue, or a peptide;
R2 is H, C1-6 alkyl, C1-6 haloalkyl, or C5-20 aryl;
q is 1, 2, or 3;
each R3 or R4 is independently selected from H, halo, C1-6 alkyl, C1-6 haloalkyl, C5-20 aryl, C3-20 heteroaryl, OC1-6 alkyl, SC1-6 alkyl, NH(C1-6 alkyl), and N(C1-6 alkyl)2;
X is X1—Y, S—S—R5, Se—Se—R5, O—NH—R5, S—NH—R5, Se—NH—R5, X2—Y1, X3—Y2, N3, or NH—S(O)2—Y3;
X1 is S, Se, O, NH, or N(C1-6 alkyl);
X2 is S, Se, or O;
X3 is NH—C(O)—O;
X4 is NH—C(O)—O, O, S, or NH;
R5 is selected from H, halo, C1-6 alkyl, C1-6 haloalkyl, C5-20 aryl, C3-20 heteroaryl, OC1-6 alkyl, NH(C1-6 alkyl), N(C1-6 alkyl)2, peptides, sugars, C3-20 heterocyclyl, and nucleic acids;
Y is a protecting group selected from:

OG Complex Work Unit Chemistry
R6 is selected from H, C1-6 alkyl, C1-6 haloalkyl, CO2H, CO2R′, SO2H, SO2R′, C5-20 aryl, C3-20 heteroaryl, NHC(O)R′, and NHR′;
R7 and R8 are independently selected from H, OH, O(C1-6 alkyl), O(C5-20 aryl), and O(C3-20 heteroaryl); or R7 and R8 are linked together to form an O—CH2—O group;
each R′ is independently selected from C1-6 alkyl, C1-6 haloalkyl, and C5-20 aryl;
R9 is selected from H, C1-6 alkyl, C1-6 haloalkyl, CO2H, CO2R′, SO2H, SO2R′, and C5-20 aryl;
R10 is selected from H, C1-6 alkyl, and C1-6 haloalkyl;
R11 is selected from H, C1-6 alkyl, and C1-6 haloalkyl;
X5 is S, O, NH, N—C(O)—O—R′, N—S(O)2H, N—S(O)2R′, or NR′;
Y1 is a protecting group selected from:

OG Complex Work Unit Chemistry
Y2 is a protecting group selected from:

OG Complex Work Unit Chemistry
 t-Bu, and CH2Ph;
M+ is Li+, Na+, K+, or N(R13)4+;
Z is Si or Ge;
R12 is C1-6 alkyl or C(O)—(C5-20 aryl);
R13 is H, C1-6 alkyl, allyl, or C5-20 aryl; and
Y3 is a protecting group

OG Complex Work Unit Chemistry
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 tRNACUA (MbtRNACUA), and said tRNA synthetase comprises the Methanosarcina barkeri pyrrolysyl-tRNA synthetase (MbPylRS synthetase) of SEQ ID NO: 3.