Conjugation reagents and methods using 1,2-cyclohexanediones
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 CD-containing compounds that can be used in the methods of the invention.
1. 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 covalently linked to the polynucleotide;
the dashed bond to Pep represents where substructure (D) is covalently linked to the peptide;
R 4 is an optional substituent on the cyclopentyl ring, and
each R 4 is independently selected from the group consisting of C 1-2 alkyl, C 1-2 alkoxy, C 1-2 haloalkyl, —COOR, SO 3 R, halo, hydroxy, and C(O)NR 2 ;
each R is independently H or C 1-4 alkyl optionally substituted with up to three groups selected from the group consisting of halo, OH, C 1-2 alkyl, C 1-2 haloalkyl, C 1-2 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, C 1-2 alkyl, C 1-2 haloalkyl, C 1-2 alkoxy, hydroxy, and carbonyl (oxo); and n is 0, 1, 2 or 3.
2. The composition of claim 1 , further comprising a solid support, wherein the peptide is attached to the solid support via a linker.
3. The composition of claim 1 , wherein the covalent linkage is attached to an arginine residue of the peptide.
4. The composition of claim 2 , wherein the peptide is covalently attached to the solid support and the linker is a cleavable linker.
5. The composition of claim 1 , wherein the covalent linkage connecting the peptide and the polynucleotide comprises substructure (D′):
6. The composition of claim 2 , wherein an N-terminal amino acid (NTAA) of the peptide forms a covalent bond with the linker.
7. The composition of claim 6 , wherein the covalent bond between the NTAA and the linker is an amide bond.
8. The composition of claim 1 , wherein the polynucleotide comprises a barcode.
9. The composition of claim 5 , wherein the covalent linkage is attached to an arginine residue of the peptide.