Granted Patent B2

US 12,359,237 · App. 17/279,634 · Granted Jul 15, 2025

Inventors: Hiroaki Suga (Tokyo, JP); Yuki Goto (Tokyo, JP); Hiroyasu Onaka (Tokyo, JP); Alexander Vinogradov (Tokyo, JP)

Assignee: The University of Tokyo

C12P21/02C40B40/10

Abstract

Claims (134)

1. An in vitro method for producing a compound library comprising two or more cyclic compounds represented by the formula (I):

wherein

m number of X a , and X b and X c each independently represent an amino acid residue selected from the group consisting of any amino acids and analogs thereof,

Z is present or absent and, when present, represents a peptide consisting of 1 to 100 amino acids and/or analogs thereof,

m is an integer selected from 2 to 40,

ring A is a nitrogen-containing 6-membered ring optionally having a substituent,

B 1 is an oxygen atom, a sulfur atom, or a NH group, and

R 1 is a hydrogen atom or a hydrocarbon group,

the method comprising allowing a macrocyclase in vitro to act on two or more peptides represented by the formula (II):

L

⁢

P

-

X

-

(

X

⁢

a

)

m

-

Y

-

Z

(

II

)

wherein

X represents a group represented by the formula (1):

wherein

R 2 is a hydrogen atom or a hydrocarbon group,

Y is a peptide residue consisting of four amino acids and/or analogs thereof and contains a group represented by the formula (2):

wherein

R 1 and B 1 are as defined above, and R 3 represents a hydrogen or a hydrocarbon group,

m number of X a , m and Z are as defined above, and

LP is present or absent and, when present, represents a peptide residue consisting of 1 to 100 amino acids and/or analogs thereof,

and forming the nitrogen-containing 6-membered ring A while eliminating LP, if present, to form the two or more cyclic compounds represented by the formula (I).

2. The production method according to claim 1 , wherein LP is a peptide residue consisting of 11 or more and 100 or less amino acids and/or analogs thereof.

3. The production method according to claim 1 , wherein m is an integer selected from 2 to 24.

4. The production method according to claim 1 , wherein Y is a group represented by the following formula (3):

wherein R 1 , R 3 , R 4 and R 5 each independently represent a hydrogen atom or a hydrocarbon group, and B 1 and B 2 each independently represent an oxygen atom, a sulfur atom or a NH group.

5. The production method according to claim 1 , wherein Y is a group represented by the following formula (3′):

wherein R 1 , R 3 , R 4 and R 5 each independently represent a hydrogen atom or a methyl group, and B 1 and B 2 each independently represent an oxygen atom or a sulfur atom, with the proviso that when B 1 and/or B 2 is a sulfur atom, each of R 1 and R 5 is a hydrogen.

6. The production method according to claim 1 , wherein Y is a group represented by the following formula (3″):

wherein R 1 , R 4 and R 5 each independently represent a hydrogen atom or a methyl group, and B 1 and B 2 each independently represent an oxygen atom or a sulfur atom, with the proviso that when B 1 and/or B 2 is a sulfur atom, each of R 1 and R 5 is a hydrogen.

7. The production method according to claim 1 , wherein Y is a group represented by the following formula (3-1):

8. The production method according to claim 1 , wherein the macrocyclase comprises LazC and/or an enzyme having homology with LazC.

9. The method for producing a compound library according to claim 1 ,

the method further comprising:

producing a mRNA library encoding precursor peptides represented by the formula (III):

LP

′

-

X

′

-

(

Xa

′

)

m

′

-

Y

′

-

Z

′

(

III

)

wherein

X′ is serine or threonine, or an analog thereof,

Y′ is a peptide consisting of four amino acids represented by -Y′(10)-Y′(11)-Y′(12)-Y′(13)-and/or analogs thereof, wherein Y′(10) is an amino acid residue selected from the group consisting of any amino acids and analogs thereof, Y′(11) is serine, cysteine, threonine or diaminopropionic acid, or an analog thereof, Y′(12) is serine or threonine, or an analog thereof, and Y′(13) is an amino acid residue selected from the group consisting of any amino acids and analogs thereof,

m′ number of X a ′, m′ and Z′ are the same as defined as m number of X a , m and Z, respectively, in the formula (I), and

LP′ is present or absent and, when present, represents a peptide consisting of 1 to 100 amino acids and/or analogs thereof;

expressing the precursor peptides by a cell-free translation system with the mRNA library to produce a first peptide library;

forming an azole ring on at least Y′(11) in the precursor peptides with an azole ring-forming enzyme;

converting at least X′ and Y′(12) in the precursor peptides to α,β-unsaturated amino acid residues with an «, β-unsaturated amino acid-forming enzyme in the presence of co-substrate tRNA Glu for glutamylation and aminoacylation enzyme GluRS; and

forming a nitrogen-containing 6-membered ring with a macrocyclase while eliminating LP′, if present.

10. The production method according to claim 9 , wherein

Y′(10) is serine or threonine, or an analog thereof, and

Y′(13) is serine, cysteine, threonine or diaminopropionic acid, or an analog thereof.

11. The production method according to claim 9 , wherein

Y′(11) is serine, cysteine or threonine, or an analog thereof, and

Y′(12) is serine or threonine, or an analog thereof.

12. The production method according to claim 9 , wherein

Y′(10) is serine or threonine, or an analog thereof,

Y′(11) is serine, cysteine or threonine, or an analog thereof,

Y′(12) is serine, and

Y′(13) is serine, cysteine or threonine, or an analog thereof.

13. The production method according to claim 9 , wherein in (X a ′) m′ in the formula (III), amino acid residue X a ′(1) adjacent to X′ is an amino acid other than an acidic amino acid or an analog thereof.

14. The production method according to claim 9 , wherein in (X a ′) m′ in the formula (III), amino acid residue X a ′(m′) at m′-th position is an amino acid other than an acidic amino acid or an analog thereof.

15. The production method according to claim 9 , wherein in (X a ′) m′ in the formula (III), amino acid residue X a ′(m′-1) at (m′-1)-th position is an amino acid other than an acidic amino acid and a basic amino acid or an analog thereof.

16. The production method according to claim 9 , wherein the azole ring-forming enzyme comprises LazD, LazE and LazF, and/or an enzyme having homology with any of them.

17. The production method according to claim 9 , wherein the α,β-unsaturated amino acid-forming enzyme comprises LazB and LazF, and/or an enzyme having homology with any of them.

18. The production method according to claim 9 , wherein the macrocyclase comprises LazC and/or an enzyme having homology with LazC.

19. The production method according to claim 9 , wherein

the cosubstrate tRNA Glu for glutamylation reaction is actinomycete-derived tRNA Glu , and the aminoacylation enzyme GluRS is actinomycete-derived GluRS.

20. The method for producing a compound library according to claim 1 ,

the method further comprising:

producing a mRNA library encoding precursor peptides represented by the formula (III):

LP

′

-

X

′

-

(

Xa

′

)

m

′

-

Y

′

-

Z

′

(

III

)

wherein

X′ is serine or threonine, or an analog thereof,

Y′ is a peptide consisting of four amino acids represented by -Y′(10)-Y′(11)-Y′(12)-Y′(13)- and/or analogs thereof, wherein Y′(10) is an amino acid residue selected from the group consisting of any amino acids and analogs thereof, Y′(11) is serine, cysteine, threonine or diaminopropionic acid, or an analog thereof, Y′(12) is serine or threonine, or an analog thereof, and Y′(13) is an amino acid residue selected from the group consisting of any amino acids and analogs thereof,

m′ number of X a ′, m′ and Z′ are the same as defined as m number of X a , m and Z, respectively, in the formula (I), and

LP′ is present or absent and, when present, represents a peptide consisting of 1 to 100 amino acids and/or analogs thereof;

binding puromycin to the 3′ end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;

expressing the precursor peptides by a cell-free translation system with the puromycin-bound mRNA library to produce a first peptide-mRNA complex library;

forming an azole ring on at least Y′(11) in the precursor peptides with an azole ring-forming enzyme;

converting at least X′ and Y′(12) in the precursor peptides to α,β-unsaturated amino acid residues with an α,β-unsaturated amino acid-forming enzyme in the presence of cosubstrate tRNA Glu for glutamylation and aminoacylation enzyme GluRS; and

forming a nitrogen-containing 6-membered ring with a macrocyclase while eliminating LP′, if present.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (16)