Benzophenone-based chromophoric crosslinkers and reagents for incorporation of biotin or other haptens into macromolecules
Compounds for UV-Vis detectable incorporation of a crosslinker a hapten into a protein or macromolecules are disclosed. The compounds comprise bis heterofunctional crosslinkers or haptens containing a chromophoric group that is incorporated into the linker that is positioned between a reactive linking moiety and a biotin molecule. The incorporation of the crosslinker or hapten into a protein or other macromolecules may be detected by UV-Vis spectroscopy.
1. A spectrophotometrically quantifiable linker of the following formula:
wherein,
R 1 and R 2 are the same or different and are each independently selected from the group consisting of —O—, —OH, —SH, —NH, —NH 2 , —F, —Cl, —Br, —I, O—Su (N-hydroxysuccinimidyl, sulfo-N-hydroxysuccinimidyl), —O-STP (4-sulfo-2,3,5,6,-tetrafluorophenyl), —O-TFP (2,3,5,6-pentafluorophenyl), —O-benzotriazole, benzotriazole, —CO—CH 2 —I, —NR-biomolecule wherein the biomolecule is a peptide, protein, antibody, nucleotide, or oligonucleotide, —COO—, —COOH, —CO—NH—NH 2 , —O-phosphoramidite, —CHO, -maleimide, pyridylsulfide, —O—NH 2 (aminoxy), —N 3 , terminal alkyne, norbornene, tran-cyclooctene, cyclooalkyne, dibenzocyclooctyne, tetrazine, biotin, desthiobiotin, digoxigenin, and hapten;
L 1 and L 2 are the same or different and are independently selected from the group consisting of a divalent linear —(CH2) x — group, or —(CH2CH2O) x — group, wherein x is 1 to 25, a branched or cyclic alkane group, which is optionally substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and sulfur; and
one of Y 1 and Y 2 is O—, and the other of Y 1 and Y 2 is NH—, or NR,
wherein R is selected from the group consisting of a linear or cyclic —(CH2) x —CH 3 group, or -(CH2CH2O) x —CH 3 group, wherein x is 1 to 25, a branched or cyclic alkane group, which is optionally substituted by at least one atom selected from the group consisting of oxygen, substituted nitrogen, and sulfur.
2. The spectrophotometrically quantifiable linker according to claim 1 , wherein
R is a lower alkyl (C 1 -C 10 ) or PEG.
3. The spectrophotometrically quantifiable linker according to claim 1 of the following formula
4. The spectrophotometrically quantifiable linker according to claim 1 , 2 , or 3 , wherein at least one of R 1 or R 2 is hound to a biomolecule.
5. The spectrophotometrically quantifiable linker according to claims 1 , 2 , or 3 , wherein at least one of R 1 or R 2 is bound to a biomolecule and wherein the biomolecule is part of binding pair selected from the group consisting of receptor ligand binding; biotin avidin binding, peptide S ribonuclease binding, digoxigenin anti-digoxigenin antibody binding, complimentary oligonucleotide pair binding or antibody ligand pairs binding.
6. A process of jointing two molecule comprising of the following steps:
Reacting the spectrophotometrically quantifiable linker according to claims 1 , 2 , or 3 with a first the biomolecule
Reacting the spectrophotometrically quantifiable linker-biomolecule complex with a second biomolecule.
7. A kit comprising at least one spectrophotometrically quantifiable linker according to claim 1 , 2 , or 3 .
8. The kit according to claim 7 wherein at least one of R 1 or R 2 of the spectrophotometrically quantifiable linker is biotin, desthiobiotin, digoxigenin.
9. The kit according to claim 7 , wherein at least one of R 1 or R 2 of the spectrophotometrically quantifiable linker is —NR-biomolecule.
10. The kit according to claim 7 , wherein at least one of R 1 or R 2 of the spectrophotometrically quantifiable linker is biotin, desthiobiotin.
11. The spectrophotometrically quantifiable linker which is one of the following compounds,