Conjugation Reagents and Methods Using 1,2-Cyclohexanediones

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.

Description

Claims

1-32. (canceled)

33. A composition comprising a peptide-polynucleotide conjugate, wherein a covalent linkage connecting a peptide and a polynucleotide of the peptide-polynucleotide conjugate comprises the following substructure (D): or a tautomer thereof, wherein: the dashed bond to Nuc represents where substructure (D) is linked to the polynucleotide;the dashed bond to Pep represents where substructure (D) is linked to the peptide;R4 is an optional substituent on the cyclopentyl ring, andeach R4 is independently selected from the group consisting of C1-2 alkyl, C1-2 alkoxy, C1-2 haloalkyl, -COOR, SO3R, halo, hydroxy, and C(O)NR2;each R is independently H or C1-4 alkyl optionally substituted with up to three groups selected from the group consisting of halo, OH, C1-2 alkyl, C1-2 haloalkyl, C1-2 alkoxy, and carbonyl (oxo), or two R groups on one N are forming a 4-8 membered ring optionally containing an additional one or two heteroatoms selected from N, O and S as ring members and optionally substituted with one or two groups selected from halo, C1-2 alkyl, C1-2 haloalkyl, C1-2 alkoxy, hydroxy, and carbonyl (oxo); and n is 0, 1, 2 or 3.

34. The composition of claim 33, further comprising a solid support, wherein the peptide is attached to the solid support via a linker.

35. (canceled)

36. The composition of claim 33, wherein the covalent linkage is attached to an arginine residue of the peptide.

37. (canceled)

38. The composition of claim 34, wherein the peptide is covalently attached to the solid support and the linker is a cleavable linker.

39. (canceled)

40. The composition of claim 33, wherein the covalent linkage comprises substructure (D′): .

41-53. (canceled)

54. The composition of claim 34, wherein an N-terminal amino acid (NTAA) of the peptide forms a covalent bond with the linker.

55. The composition of claim 54, wherein the covalent bond between the NTAA and the linker is an amide bond.

56. The composition of claim 33, wherein the polynucleotide comprises a barcode.

57. The composition of claim 40, wherein the covalent linkage is attached to an arginine residue of the peptide.