Conjugation reagents and methods using 1,2-cyclohexanediones

Patent metadata

Field	Value
Publication number	US-11639366-B2
Application number	US-202117535516-A
Country	US
Kind code	B2
Filing date	Nov 24, 2021
Priority date	Oct 30, 2020
Publication date	May 2, 2023
Grant date	May 2, 2023

Abstract

The present invention relates to methods to use cyclohexan-1,2-dione (CHD) groups to attach labels, linkers, and other molecules to a target compound comprising a CHD-reactive group such as a guanidine, amidine, urea, thiourea and the like. Methods of the invention include milder conditions than those previously known for promoting reaction of CHD with CHD-reactive groups, which makes the methods suitable for use with base-sensitive compounds and complex biomolecules. Methods of the invention are especially useful for attaching linking and labeling groups to a peptide that comprises at least one arginine residue, and can also be used to link such peptides to other target molecules such as nucleic acids. The invention also provides CHD-containing conjugation reagents and compositions comprising CHD-containing intermediates, and precursors useful for making CHD-containing compounds that can be used in the methods of the invention.

First claim

The invention claimed is:

1. A method of attaching a conjugation reagent to a target molecule, wherein:
the conjugation reagent comprises a 1,2-cyclohexanedione (CHD) group; and
the target molecule comprises a CHD-reactive group;
wherein the method comprises contacting the conjugation reagent with the target molecule under reaction conditions that cause the CHD-reactive group to react irreversibly with the CHD group to form a modified target molecule having a covalent linkage connecting the conjugation reagent and the target molecule,
wherein the reaction conditions comprise an aqueous buffered medium having a pH between 7 and 11.2 and a temperature between 37° C. and 90° C. and the medium comprises a buffer having at least 0.1 M ionic strength.

2. The method of claim 1 , wherein the CHD-reactive group is a guanidine group of an arginine amino acid residue.

3. The method of claim 1 , wherein the conjugation reagent comprises at least one additional reactive handle that is stable under the reaction conditions that cause the CHD-reactive group to react irreversibly with the CHD group.

4. The method of claim 1 , wherein the covalent linkage connecting the conjugation reagent and the target molecule comprises the following substructure (A):

or a tautomer thereof, wherein:
the dashed bond to L connects the substructure (A) to the conjugation reagent;
the dashed bond to T connects the substructure (A) to the target molecule;
R 4 is an optional substituent on the cyclopentyl ring, and each R 4 is independently selected from C 1-2 alkyl, C 1-2 alkoxy, C 1-2 haloalkyl, —COOR, SO 3 R, halo, hydroxy, and CONR 2 ;

each R is independently H or C 1-4 alkyl optionally substituted with 1-3 groups selected from halo, OH, and C 1-2 alkoxy; and
n is 0, 1, 2 or 3.

5. The method of claim 1 , wherein the reaction conditions comprise an aqueous medium that includes up to 25% of an organic co-solvent.

6. The method of claim 1 , wherein at least 25% of the target molecules present react to form the modified target molecule.

7. The method of claim 3 , wherein the method further comprises contacting the additional reactive handle on the conjugation reagent of the modified target molecule with a second target molecule having a reactive handle complementary to the additional reactive handle to form a covalent linkage between the additional reactive handle and the second target molecule, thereby forming a target molecule—conjugation reagent—second target molecule conjugate.

8. A method of attaching a peptide comprising at least one arginine residue to a polynucleotide, the method comprising the steps of:
(a) contacting the peptide with a conjugation reagent comprising a first reactive handle and a 1,2-cyclohexanedione (CHD) group under reaction conditions that allows the least one arginine residue to react irreversibly with the CHD group, wherein the reaction conditions comprise an aqueous buffered medium having a pH between 7 and 11.2 and a temperature between 37° C. and 90° C. and the medium comprises a buffer having at least 0.1 M ionic strength, and the first reactive handle is attached or is configured to be attached to the polynucleotide or to a second reactive handle attached to the polynucleotide;
(b) optionally, attaching the first reactive handle to the polynucleotide or to the second reactive handle attached to the polynucleotide.

9. The method of claim 8 , further comprising the following steps: (i) before contacting the peptide with the conjugation reagent, contacting the peptide with a site-specific protease that is configured to cleave the peptide at arginine residue(s), thereby producing at least one fragmented peptide having a single arginine residue at its carboxyl terminus; and (ii) immobilizing the at least one fragmented peptide to a solid support via a linker, wherein the at least one fragmented peptide is contacted with the conjugation reagent.

10. The method of claim 9 , wherein immobilizing comprises covalently attaching the peptide to the solid support via a cleavable linker.

11. The method of claim 9 , wherein the step (ii) is performed before the step (a).

12. A method of analyzing a peptide comprising at least one arginine residue, the method comprising the steps of:
(a) providing a conjugate of the peptide and a recording tag, the conjugate attached to a solid support, wherein the recording tag comprises a polynucleotide that is conjugated to the peptide according to the following steps:
(i) contacting the peptide with a conjugation reagent comprising a first reactive handle and a 1,2-cyclohexanedione (CHD) group under reaction conditions that allows the least one arginine residue to react irreversibly with the CHD group, wherein the reaction conditions comprise an aqueous buffered medium having a pH between 7 and 11.2 and a temperature between 37° C. and 90° C. and the medium comprises a buffer having at least 0.1 M ionic strength, and the first reactive handle is attached or is configured to be attached to the polynucleotide or to a second reactive handle attached to the polynucleotide; and
(ii) optionally, attaching the first reactive handle to the polynucleotide or to the second reactive handle attached to the polynucleotide;
(b) contacting the peptide of the conjugate with a binding agent capable of binding to the peptide, wherein the binding agent comprises a coding tag that comprises identifying information regarding the binding agent;
(c) transferring the identifying information from the coding tag to the recording tag to generate an extended recording tag; and
(d) analyzing the extended recording tag, thereby analyzing the peptide.

13. The method of claim 12 , wherein analyzing the peptide comprises identifying at least one component of the peptide.

14. The method of claim 1 , wherein the aqueous medium has a pH between 7 and 10.

15. The method of claim 1 , wherein the buffer has an ionic strength of at least 0.5 M.

16. The method of claim 1 , wherein the reaction conditions comprise temperature between 60° C. and 90° C.

17. The method of claim 8 , wherein the aqueous medium has a pH between 7 and 10.

18. The method of claim 9 wherein the buffer has an ionic strength of at least 0.5 M.

19. The method of claim 12 , wherein the aqueous medium has a pH between 7 and 10.

20. The method of claim 19 , wherein the reaction conditions comprise temperature between 60° C. and 90° C., and the aqueous medium comprises a buffer having at least 0.5 M ionic strength.

Assignees

• Encodia Inc

Inventors

• James Robert C
• Verespy Iii Stephen
• Zhou Eric Cunyu
• Okerberg Eric
• Schramma Kelsey Rose
• Huang Fei

Classifications

• C07K1/13 Primary

  Labelling of peptides · CPC title

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C07K1/1072

  by covalent attachment of residues or functional groups · CPC title

• G01N33/532

  Production of labelled immunochemicals · CPC title