Bis-sulfhydryl macrocyclization systems
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 are provided.
1. A method for synthesizing a peptidomimetic macrocycle, comprising contacting a peptidomimetic precursor of Formula III:
with a compound of 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, amino acid analog,
[—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, each of which except —H is unsubstituted or substituted with halo-;
R 3 is —H, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except —H is unsubstituted or substituted with R 5 ;
L 1 , L 3 , and L 4 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R 4 —K—R 4 —] n , each being unsubstituted or substituted with R 5 ;
L 2 is cycloalkylene, cycloarylene or heterocycloarylene, each of which is 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 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 of which except —H is 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, each of which except —H is 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; and
x+y+z is at least 3,
wherein the contacting results in a covalent linkage being formed between the two thiol groups in Formula III, and wherein the peptidomimetic macrocycle exhibits increased resistance to proteolytic degradation compared to a corresponding non-macrocyclic polypeptide; further wherein the peptidomimetic macrocycle comprises an α-helix when in aqueous solution.
2. The method of claim 1 , wherein L 2 is cycloarylene unsubstituted or substituted with R 5 .
3. The method of claim 1 , wherein L 1 and L 3 are each unsubstituted alkylene.
4. The method of claim 1 , comprising expressing the peptidomimetic precursor in cells prior to the contacting.
5. The method of claim 1 , comprising purifying the peptidomimetic precursor prior to the contacting.
6. The method of claim 1 , comprising purifying the peptidomimetic macrocycle after the contacting.
7. The method of claim 1 , comprising refolding the peptidomimetic macrocycle after the contacting.
8. The method of claim 1 , wherein the method is performed in solution.
9. The method of claim 1 , wherein the method is performed on a solid support.
10. The method of claim 1 , wherein the contacting is performed in the presence of a target macromolecule that binds to the peptidomimetic precursor.
11. The method of claim 1 , wherein the contacting is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor.
12. The method of claim 1 , comprising synthesizing a library of peptidomimetic macrocycles.
13. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased thermal stability when compared to a corresponding non-macrocyclic polypeptide.
14. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased biological activity when compared to a corresponding non-macrocyclic polypeptide.
15. The method of claim 1 , wherein the peptidomimetic macrocycle exhibits increased ability to penetrate living cells when compared to a corresponding non-macrocyclic polypeptide.
16. The method of claim 1 , wherein the two thiol moieties of the compound of Formula III are sidechains of an amino acid that is selected from the group consisting of L-cysteine, D-cysteine, α-methyl L-cysteine, and α-methyl D-cysteine.
17. The method of claim 1 , wherein the sum of x+y+z is 6, and A, B and C are independently natural or non-natural amino acids.
18. The method of claim 1 , wherein the contacting is performed in a solvent selected from the group consisting of a protic solvent, an aqueous solvent, an organic solvent, and mixtures thereof.
19. The method of claim 1 , wherein the peptidomimetic macrocycle has Formula I:
20. A method for synthesizing a peptidomimetic macrocycle, comprising contacting a peptidomimetic precursor of Formula III:
with a compound of formula X-L 2 -Y,
wherein v, w, x, y, z, A, B, C, D, E, R 1 , R 2 , R 7 , R 8 , L 1 , L 2 , and L 3 are as defined in claim 1 ; X and Y are each independently a reactive group capable of reacting with a thiol group; and
x+y+z is at least 3,
wherein the contacting results in a covalent linkage being formed between the two thiol groups in Formula III, and wherein the peptidomimetic precursor is expressed in cells prior to the contacting; further wherein the peptidomimetic macrocycle comprises an α-helix when in aqueous solution.
21. The method of claim 20 , comprising refolding the peptidomimetic macrocycle after the contacting.
22. The method of claim 20 , wherein the method is performed in solution.
23. The method of claim 20 , wherein the method is performed on a solid support.
24. The method of claim 20 , wherein the contacting is performed in the presence of a target macromolecule that binds to the peptidomimetic precursor.
25. The method of claim 20 , wherein the contacting is performed in the presence of a target macromolecule that binds preferentially to the peptidomimetic precursor.
26. The method of claim 20 , comprising synthesizing a library of peptidomimetic macrocycles.