Methods of stapling and unstapling peptides and proteins

Patent metadata

Field	Value
Publication number	US-9850469-B2
Application number	US-201514659754-A
Country	US
Kind code	B2
Filing date	Mar 17, 2015
Priority date	Mar 17, 2014
Publication date	Dec 26, 2017
Grant date	Dec 26, 2017

Abstract

The present disclosure pertains to the field peptide stapling and/or macrocyclization, where a structural motif is used to improve the properties of amino acid sequences (e.g. protease resistance, cellular penetration, biological activity). Also within the scope of the disclosure are methods for unstapling the S,S-tetrazine-containing amino acid sequence. The disclosure is also directed to methods for the reductive removal of thiocyanates from an amino acid sequence with cysteine to recycle back to the native amino acid sequence.

First claim

What is claimed:

1. A process comprising:
preparing a phase-transfer reaction medium comprising
an aqueous solution of an unprotected amino acid sequence comprising two proximal cysteine residues or two homocysteine residues; and
an organic solution of a di-halo-tetrazine;

contacting the aqueous solution with the organic solution for a time sufficient to form an S,S-tetrazine moiety;
irradiating the S,S-tetrazine moiety with light to form an amino acid sequence comprising two proximal thiocyanate moieties; and
contacting the amino acid sequence comprising two proximal thiocyanate moieties with cysteine for a time sufficient to produce the amino acid sequence comprising two proximal cysteine residues or the amino acid sequence comprising two proximal homocysteine residues;
wherein the proximal cysteine residues, the proximal homocysteine residues, and the proximal thiocyanate moieties are separated by 0 to 35 amino acid residues.

2. The process of claim 1 , wherein each amino acid sequence is a peptide, a protein, or a protein fragment.

3. The process of claim 1 , wherein the aqueous solution has a pH of from about 5 to about 9.

4. The process of claim 1 , wherein the aqueous solution further comprises a buffer.

5. The process of claim 4 , wherein the buffer is monosodium phosphate or guanidine hydrochloride.

6. The process of claim 1 , wherein the organic solution comprises chloroform, ethyl acetate, diethyl ether, toluene, dichloromethane, cyclohexane, or a combination thereof.

7. The process of claim 6 , wherein the organic solution comprises chloroform.

8. The process of claim 1 , wherein the organic solution has a solubility in water of less than 10 g/mL at 20° C.

9. The process of claim 1 , wherein the proximal cysteine residues, the proximal homocysteine residues, and the proximal thiocyanate moieties are separated by 1, 2, 3, 4, 5, 10, or 27 amino acid residues.

10. The process of claim 1 , wherein the di-halo-tetrazine is dichlorotetrazine.

11. The process of claim 1 , wherein the irradiation is with UV-A light or UV-B light.

12. The process of claim 1 , wherein the step of contacting the amino acid sequence comprising two proximal thiocyanate moieties with cysteine occurs in aqueous solution.

13. The process of claim 1 , wherein the pH of the aqueous solution is a basic pH.

14. The process of claim 1 , wherein the amino acid sequence includes at least 4 amino acid residues.

15. The process of claim 1 , wherein the amino acid sequence includes at least 6 amino acid residues.

Assignees

• Univ Pennsylvania

Inventors

• Smith Iii Amos B
• Brown Stephen

Classifications

• C07K7/54

  with at least one abnormal peptide link in the ring · CPC title

• C12N9/0051 Primary

  acting on a sulfur group of donors (1.8) · CPC title

• C07K1/1133

  by redox-reactions involving cystein/cystin side chains · CPC title

• C07K1/1136

  by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents · CPC title