Bis-sulfhydryl macrocyclization systems
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, are provided.
1. A method for synthesizing a peptidomimetic macrocycle, the method comprising the step of contacting a peptidomimetic precursor of the Formula III:
with a compound formula X-L 2 -Y,
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, an amino acid comprising one or more additional methylene groups between the amino and carboxyl group, an amino acid comprising an amino group which is a secondary or tertiary amine, an amino acid comprising a carboxy group replaced by an ester,
[—NH-L 4 -CO—], [—NH-L 4 -SO 2 —], or [—NH-L 4 -];
R 1 and R 2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
R 3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, unsubstituted or substituted with R 5 ;
L 1 , L 2 , L 3 and L 4 are independently alkylene, alkenylene, alkynylene, heteroalkylene, heterocycloalkylene, or [—R 4 —K—R 4 —]n, each being unsubstituted or substituted with R 5 ;
K is O, S, SO, SO 2 , CO, CO 2 , or CONR 3 ;
each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;
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 label, a radioisotope or a therapeutic agent;
each R 6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent label, a radioisotope or a therapeutic agent;
R 7 is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, unsubstituted or 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, unsubstituted or 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;
n is an integer from 1-5;
X and Y are each independently a reactive group capable of reacting with a thiol group;
x+y+z is at least 3;
wherein said contacting step results in a covalent linkage being formed between the two thiol groups in Formula III, wherein a secondary structure of the peptidomimetic macrocycle is more stable than a corresponding secondary structure of a corresponding non-macrocyclic polypeptide, and wherein the peptidomimetic macrocycle comprises an alpha-helix.
2. The method of claim 1 , wherein at least one of R 1 and R 2 is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
3. The method of claim 1 , wherein R 1 and R 2 are independently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
4. The method of claim 1 , wherein at least one of R 1 and R 2 is alkyl, unsubstituted or substituted with halo-.
5. The method of claim 1 , wherein R 1 and R 2 are independently alkyl, unsubstituted or substituted with halo-.
6. The method of claim 1 , wherein at least one of R 1 and R 2 is methyl.
7. The method of claim 1 , wherein R 1 and R 2 are methyl.
8. The method of claim 1 , wherein the peptidomimetic precursor is expressed in cells.
9. The method of claim 1 , wherein the peptidomimetic precursor is purified prior to the contacting step.
10. The method of claim 1 , wherein the peptidomimetic macrocycle is purified after the contacting step.
11. The method of claim 1 , wherein the peptidomimetic macrocycle is refolded after the contacting step.
12. The method of claim 1 , wherein the method is performed in solution.
13. The method of claim 1 , wherein the method is performed on a solid support.
14. The method of claim 1 , wherein the contacting step is performed in the presence of a target macromolecule that binds to the peptidomimetic precursor under conditions that favor said binding.
15. The method of claim 1 , wherein the contacting step is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor under conditions that favor said binding.
16. The method of claim 1 , wherein the method is applied to synthesize a library of peptidomimetic macrocycles.
17. The method of claim 1 , wherein the peptidomimetic macrocycle comprises an α-helix in aqueous solution.
18. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased α-helical structure in aqueous solution compared to a corresponding non-macrocyclic polypeptide.
19. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased thermal stability compared to a corresponding non-macrocyclic polypeptide.
20. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased biological activity compared to a corresponding non-macrocyclic polypeptide.
21. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased resistance to proteolytic degradation compared to a corresponding non-macrocyclic polypeptide.
22. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased ability to penetrate living cells compared to a corresponding non-macrocyclic polypeptide.
23. The method of claim 1 , wherein the two thiol moieties of the compound of Formula III are sidechains of an amino acid selected from the group consisting of L-cysteine, D-cysteine, α-methyl L-cysteine, and α-methyl D-cysteine.
24. The method of claim 1 , wherein x+y+z is 3, and A, B and C are independently natural or non-natural amino acids.
25. The method of claim 1 , wherein the contacting step is performed in a solvent selected from the group consisting of protic solvent, aqueous solvent, organic solvent, and mixtures thereof.
26. The method of claim 1 , wherein the solvent is DMF, dichloroethane, NH 3 , NH 3 /MeOH, NH 3 /DMF, or aqueous guanidinium-HCL.
27. The method of claim 1 , wherein the solvent is water.
28. The method of claim 1 , wherein the peptidomimetic macrocycle has Formula (I):