Ligation of stapled polypeptides

1. A method of preparing a stabilized, non-immunogenic, and folded protein comprising a stitched α-helical peptide segment, the method comprising steps of:

providing a stitched α-helical peptide segment, wherein the stitched α-helical peptide segment is of formula:

wherein

each instance of K, L 1 , L 2 , and M, is, independently, a bond, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene;

each instance of R a is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; or R a is a suitable amino protecting group;

each instance of R b is, independently, a suitable amino acid side chain; hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; cyano; isocyano; halo; or nitro;

each instance of R e is, independently, —R E , —OR E , —N(R E ) 2 , or —SR E , wherein each instance of R E is, independently, hydrogen, cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; or two R E groups together form a substituted or unsubstituted 5-to 6-membered heterocyclic or heteroaromatic ring;

each instance of R f is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable amino protecting group; a label optionally joined by a linker, wherein the linker is selected from cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene; or R f and R a together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring;

each instance of R KL , R LL , and R LM , is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; azido; cyano; isocyano; halo; nitro;

or two adjacent R KL groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; two adjacent R LL groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; or two adjacent R LM groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring;

each instance of X AA is, independently, a natural or unnatural amino acid;

each instance of y and z is, independently, an integer between 2 to 6;

each instance of j is, independently, an integer between 1 to 10;

each instance of p is, independently, an integer between 0 to 10;

each instance of s and t is, independently, an integer between 0 and 100;

each instance of u, v, and q, is, independently, an integer between 0 to 2; and wherein

corresponds to a single or double bond;

providing a second protein to which the stitched α-helical peptide segment is to be ligated, wherein the second protein is produced recombinantly or purified from a natural source; and

ligating the stitched α-helical peptide segment to the second protein.

2. The method of claim 1 , wherein the stitched α-helical peptide segment is less than 30 amino acids in length.

3. The method of claim 1 , wherein the stitched peptide is of formula:

4. The method of claim 1 , wherein the stitched peptide is of formula:

5. The method of claim 1 , wherein the step of providing a stitched α-helical peptide segment comprises steps of:

synthetically producing an unstitched peptide segment, wherein the peptide segment comprises unnatural amino acid residues suitable for stitching; and

stitching the unstitched peptide.

6. The method of claim 1 , wherein the step of providing a stitched α-helical peptide segment comprises steps of:

providing a peptide with at least two α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residues and at least one dialkenylglycine residue; and

stitching the peptide using a Grubbs catalyst to form at least two macrocyclic hydrocarbon staples.

7. The method of claim 6 , wherein the two α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residues are at positions i and i+3, at positions i and i+4, and/or at positions i and i+7.

8. The method of claim 6 , wherein the α-methyl,α-alkenylglycine or α-hydro, α-alkenylglycine residue is of the formula:

wherein n is an integer between 1 and 10, inclusive; and R b is H or methyl.

9. The method of claim 1 , wherein the second protein is prepared recombinantly.

10. The method of claim 1 , wherein the second protein is treated with a protease to yield an N-terminal cysteine.

11. The method of claim 10 , wherein the protease is Factor Xa, Tobacco Etch Virus, enterokinase, or ubiquitin C-terminal hydrolase.

12. The method of claim 1 , wherein the second protein is produced by having cysteine as the second residue in an expression construct following an N-terminal methionine; wherein the methionine is processed during protein expression to yield an N-terminal cysteine.

13. The method of claim 1 , wherein the step of ligating comprises ligating using Expressed Protein Ligation (EPL).

14. The method of claim 1 , wherein the step of ligating produces a scarless protein.

15. The method of claim 1 , wherein stitched protein comprises a stitched version of IL-13.

16. The method of claim 15 , wherein Helix A of IL-13 is stitched.

17. The method of claim 15 , wherein Helix D of IL-13 is stitched.

18. The method of claim 1 , wherein the stitched protein comprises a stitched version of c-myc, a stitched version of insulin, a stitched version of a transcription factor, or a stitched version of a cytokine.

19. The method of claim 1 , wherein the stitched α-helical peptide segment is less than 25 amino acids in length.

20. The method of claim 1 , wherein the stitched α-helical peptide segment is less than 20 amino acids in length.

21. The method of claim 1 , wherein the stitched α-helical peptide is a bifunctional peptide.

22. A stitched protein made by the method of claim 1 .