Triazole macrocycle systems
The present invention provides novel peptidomimetic macrocycles and methods for their preparation and use, as well as amino acid analogs and macrocycle-forming linkers, and kits useful in their production. In various embodiments, the peptidomimetic macrocycles are of Formula I: The linker L includes a triazole moiety. Peptidomimetic macrocycles according to the invention may exhibit increased α-helical or beta sheet structure in aqueous solution compared to a corresponding non-macrocyclic polypeptide.
1. A peptidomimetic macrocycle of Formula (I):
wherein:
each A, C, D, and E is independently a natural or non-natural amino acid;
B is a natural or non-natural amino acid, amino acid analog,
[—NH-L 3 -CO—], [—NH-L 3 -SO 2 —], or [—NH-L 3 -];
R 1 and R 2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each non-H group being optionally substituted with halo-;
R 3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each non-H group being optionally substituted with R 5 ;
L is a macrocycle-forming linker of the formula
L 1 , L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R 4 —K—R 4 —] n , each being optionally substituted with R 5 ;
each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, SO 2 , CO, C(═O)—O—, or CONR 3 ;
each R 5 is independently halogen, alkyl, —OR 6 , —N(R 6 ) 2 , —SR 6 , —SOR 6 , —SO 2 R 6 , —CO 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;
each R 6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
R 7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each non-H group being optionally substituted with R 5 , or part of a cyclic structure with a D residue;
R 8 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each non-H group being optionally substituted with R 5 , or part of a cyclic structure with an E residue;
v is an integer from 1-1000;
w is an integer from 1-1000;
x is an integer from 0-10;
y is an integer from 0-10;
z is an integer from 0-10; and
n is an integer from 1-5;
wherein the peptidomimetic macrocycle comprises an α-helix or beta sheet.
2. The peptidomimetic macrocycle of claim 1 , wherein L is
3. The peptidomimetic macrocycle of claim 1 , wherein L is
4. The peptidomimetic macrocycle of claim 1 , wherein at least one of R 1 and R 2 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group being optionally substituted with halo-.
5. The peptidomimetic macrocycle of claim 1 , wherein R 1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group being optionally substituted with halo-.
6. The peptidomimetic macrocycle of claim 1 , wherein at least one of R 1 and R 2 is alkyl, optionally substituted with halo-.
7. The peptidomimetic macrocycle of claim 1 , wherein R 1 and R 2 are independently alkyl, optionally substituted with halo-.
8. The peptidomimetic macrocycle of claim 1 , wherein at least one of R 1 and R 2 is methyl.
9. The peptidomimetic macrocycle of claim 1 , wherein R 1 and R 2 are methyl.
10. The peptidomimetic macrocycle of claim 1 , wherein at least one of D and E is a natural or unnatural amino acid substituted with a high molecular weight lipid or hydrocarbon.
11. The peptidomimetic macrocycle of claim 1 , wherein at least one of D and E is attached to an additional macrocycle-forming linker.
12. The peptidomimetic macrocycle of claim 1 wherein a secondary structure of the peptidomimetic macrocycle is more stable than a corresponding secondary structure of a corresponding non-macrocyclic polypeptide.
13. The peptidomimetic macrocycle of claim 1 wherein the peptidomimetic macrocycle comprises an α-helix.
14. The peptidomimetic macrocycle of claim 13 wherein the α-helix comprises from 1 turn to 5 turns.
15. The peptidomimetic macrocycle of claim 13 wherein the α-helix is more stable than an α-helix of a corresponding non-macrocyclic polypeptide.
16. The peptidomimetic macrocycle of claim 13 wherein the macrocycle-forming linker spans from 1 turn to 5 turns of the α-helix.
17. The peptidomimetic macrocycle of claim 13 wherein the macrocycle-forming linker spans approximately 1, 2, 3, 4 or 5 turns of the α-helix.
18. The peptidomimetic macrocycle of claim 13 wherein the length of the macrocycle-forming linker is about 5 Å to about 9 Å per turn of the α-helix.
19. The peptidomimetic macrocycle of claim 13 wherein the macrocycle-forming linker spans approximately 1 turn of the α-helix.
20. The peptidomimetic macrocycle of claim 19 wherein the length of the macrocycle-forming linker is approximately equal to the length of from about 6 carbon-carbon bonds to about 14 carbon-carbon bonds.
21. The peptidomimetic macrocycle of claim 19 wherein the length of the macrocycle-forming linker is approximately equal to the length of from about 8 carbon-carbon bonds to about 12 carbon-carbon bonds.
22. The peptidomimetic macrocycle of claim 19 wherein the macrocycle comprises a ring of about 18 atoms to 26 atoms.
23. The peptidomimetic macrocycle of claim 13 wherein the α-helix comprises about 2 turns.
24. The peptidomimetic macrocycle of claim 23 wherein the length of the macrocycle-forming linker is approximately equal to the length of from about 8 carbon-carbon bonds to about 16 carbon-carbon bonds.
25. The peptidomimetic macrocycle of claim 23 wherein the length of the macrocycle-forming linker is approximately equal to the length of from about 10 carbon-carbon bonds to about 13 carbon-carbon bonds.
26. The peptidomimetic macrocycle of claim 23 wherein the macrocycle comprises a ring of about 29 atoms to about 37 atoms.
27. A method for synthesizing a peptidomimetic macrocycle comprising an α-helix or beta sheet, the method comprising the steps of contacting a peptidomimetic precursor of Formula III or Formula IV:
with a macrocyclization reagent;
wherein v, w, x, y, z, A, B, C, D, E, R 1 , R 2 , R 7 , R 8 , L 1 and L 2 are as defined in claim 1 ;
R 12 is —H or alkyl;
further wherein R 12 is —H when the macrocyclization reagent is a Cu reagent and R 12 is —H or alkyl when the macrocyclization reagent is a Ru reagent; and
said contacting step results in a covalent linkage being formed between the alkyne and azide moiety in Formula III or Formula IV.
28. The method of claim 27 , wherein at least one of R 1 and R 2 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group being optionally substituted with halo-.
29. The method of claim 27 , wherein R 1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each group being optionally substituted with halo-.
30. The method of claim 27 , wherein at least one of R 1 and R 2 is alkyl, optionally substituted with halo-.
31. The method of claim 27 , wherein R 1 and R 2 are independently alkyl, optionally substituted with halo-.
32. The method of claim 27 , wherein at least one of R 1 and R 2 is methyl.
33. The method of claim 27 , wherein R 1 and R 2 are methyl.
34. The method of claim 27 , wherein the macrocyclization reagent is a Cu reagent.
35. The method of claim 27 , wherein the macrocyclization reagent is a Ru reagent.
36. The method of claim 27 , wherein the peptidomimetic precursor is purified prior to the contacting step.
37. The method of claim 27 , wherein the peptidomimetic macrocycle is purified after the contacting step.
38. The method of claim 27 , wherein the peptidomimetic macrocycle is refolded after the contacting step.
39. The method of claim 27 , wherein the method is performed in solution.
40. The method of claim 27 , wherein the method is performed on a solid support.
41. The method of claim 27 , wherein the method is performed in the presence of a target macromolecule that binds to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding.
42. The method of claim 27 , wherein the method is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor or peptidomimetic macrocycle under conditions that favor said binding.
43. The method of claim 27 , wherein the method is applied to synthesize a library of peptidomimetic macrocycles.
44. The method of claim 27 , wherein the peptidomimetic macrocycle comprises an α-helix in aqueous solution.
45. The method of claim 27 , wherein the peptidomimetic macrocycle exhibits increased α-helical structure in aqueous solution compared to a corresponding non-macrocyclic polypeptide.
46. The method of claim 27 , wherein the peptidomimetic macrocycle exhibits increased thermal stability compared to a corresponding non-macrocyclic polypeptide.
47. The method of claim 27 , wherein the peptidomimetic macrocycle exhibits increased biological activity compared to a corresponding non-macrocyclic polypeptide.
48. The method of claim 27 , wherein the peptidomimetic macrocycle exhibits increased resistance to proteolytic degradation compared to a corresponding non-macrocyclic polypeptide.
49. The method of claim 27 , wherein the peptidomimetic macrocycle exhibits increased ability to penetrate living cells compared to a corresponding non-macrocyclic polypeptide.
50. The method of claim 27 , wherein the alkyne moiety of the peptidomimetic precursor of Formula III or Formula IV is a sidechain of an amino acid selected from the group consisting of L-propargylglycine, D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, and (R)-2-amino-2-methyl-8-nonynoic acid, and the azide moiety of the peptidomimetic precursor of Formula III or Formula IV is a sidechain of an amino acid selected from the group consisting of ε-azido-L-lysine, ε-azido-D-lysine, ε-azido-alpha-methyl-L-lysine, ε-azido-alpha-methyl-D-lysine, δ-azido-alpha-methyl-L-ornithine, and δ-azido-alpha-methyl-D-ornithine.
51. The method of claim 27 , wherein x+y+z is 3, and A, B and C are independently natural or non-natural amino acids.
52. The method of claim 27 , wherein x+y+z is 6, and A, B and C are independently natural or non-natural amino acids.
53. The method of claim 27 , wherein the macrocyclization reagent is a Cu reagent and the contacting step is performed in a solvent selected from the group consisting of protic solvent, aqueous solvent, organic solvent, and mixtures thereof.
54. The method of claim 53 , wherein the solvent is H 2 O, THF/H 2 O, tBuOH/H 2 O, DMF, DIPEA, CH 3 CN, CH 2 Cl 2 or ClCH 2 CH 2 Cl.
55. The method of claim 27 , wherein the macrocyclization reagent is a Ru reagent and the contacting step is performed in an organic solvent.
56. The method of claim 55 , wherein the solvent is DMF, THF, CH 3 CN, CH 2 Cl 2 or ClCH 2 CH 2 Cl.
57. The method of claim 53 , wherein the solvent is a solvent that favors helix formation.
58. A method for synthesizing a peptidomimetic macrocycle, the method comprising the steps of contacting a peptidomimetic precursor of Formula III or Formula IV:
with a ruthenium-based macrocyclization reagent;
wherein v, w, x, y, z, A, B, C, D, E, R 1 , R 2 , R 7 , R 8 , L 1 and L 2 are as defined in claim 1 ;
R 12 is —H or alkyl; and
said contacting step results in a covalent linkage being formed between the alkyne and azide moiety in Formula III or Formula IV.
59. The method of claim 27 or 58 , wherein the peptidomimetic macrocycle comprises an α-helix.