Kits for Using Bispyridines to Improve Labeling of Nucleophiles

Abstract

Bispyridines improve the labeling of nucleophiles, including amines and thiols and are particularly useful for improving labeling with acidic and basic labels. Use of bispyridines with such labels dramatically increases labeling compared to protocols without a bispyridine. The labeled nucleophile can then be subjected to standard analytical methods.

1 - 25 . (canceled) 
     
     
         26 . A kit for labeling a nucleophile of interest, wherein said nucleophile is an amine, a glycosylamine, a thiol, a guanidine or an imidazole group on a protein or peptide, or an exocyclic amine on a nucleic acid, said kit comprising,
 (a) a bispyridine,   (b) a label reactive with said nucleophile of interest, and   (c) instructions on labeling said nucleophile with said label.   
     
     
         27 . The kit of  claim 26 , wherein said nucleophile is a glycosylamine. 
     
     
         28 . The kit of  claim 26 , wherein said label is an amine-reactive dye. 
     
     
         29 . The kit of  claim 26 , wherein said bispyridine is pyridine, 4,4′-(1,3-propanediyl)bis- or pyridine, 4,4′-(1,2-ethanediyl)bis-. 
     
     
         30 . The kit of  claim 26 , wherein said label is dry 2-(diethylamino)ethyl 4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate and said bispyridine is pyridine, 4,4′-(1,3-propanediyl)bis- or pyridine, 4,4′-(1,2-ethanediyl)bis-. 
     
     
         31 . The kit of  claim 26 , further wherein said bispyridine is in a solvent. 
     
     
         32 . The kit of  claim 31 , wherein said solvent is dimethylformamide. 
     
     
         33 . The kit of  claim 26 , wherein said nucleophile is an amine. 
     
     
         34 . The kit of  claim 28 , wherein said amine-reactive dye is 2-(diethylamino)ethyl 4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate, 8-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,5-trisulfonic acid, or 4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoic acid. 
     
     
         35 . The kit of  claim 28 , wherein said amine-reactive dye is 2-(diethylamino)ethyl 4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate, 2,5-Pyrrolidinedione, 1-[[(2′,7′-difluoro-3′,6′-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthen]-5-yl)carbonyl]oxy], 5(6)-CR 6G, SE (5-(and-6)-Carboxyrhodamine 6G, Succinimidyl Ester), 7-diethylaminocoumarin-3-carboxylic acid, 8-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,5-trisulfonic acid, or 4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoic acid. 
     
     
         36 . The kit of  claim 28 , wherein said amine-reactive dye is 2-(diethylamino)ethyl 4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate. 
     
     
         37 . The kit of  claim 26 , wherein said bispyridine is pyridine, 4,4′,4″,4′″-(2,3,4,5-thiophenetetrayl)tetrakis; 1,2-Ethanediol, 1,2-di-4-pyridinyl; methanone, di-4-pyridinyl; cyclohexanone, 2,6-bis(4-pyridinylmethylene-,(2E6E)-; 2H-Indol-2-one, 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-; 2,3-Butanediol, 2,3-di-4-pyridinyl-; 1,2-Ethanediol, 1,2-di-4-pyridinyl-, (1R,2S)-rel-; 9(10H)-Anthracenone, 10,10-bis(4-pyridinylmethyl)-; 2,4,6(1H,3H,5H)-Pyrimidinetrione, 1-(3-methoxyphenyl)-5,5-bis[2-(4-pyridinyl)ethyl]-; 2,4,6(1H,3H,5H)-Pyrimidinetrione, 1,3-dimethyl-5,5-bis[2-(4-pyridinyl)ethyl]-; Ethanone, 1,2-di-4-pyridinyl-; Pyridine, 4.4′-(1,4-butanediyl)bis-; 2,4,6(1H,3H,5H)-Pyrimidinetrione, 1-(phenylmethyl)-5,5-bis[2-(4-pyridinyl)ethyl]-; 2,4,6(1H,3H,5H)-Pyrimidinetrione, 1-(4-ethoxyphenyl)-5,5-bis[2-(4-pyridinyl)ethyl]-; 4-Pyridinepropanenitrile, α-4-pyridinyl-; 1,2-Ethanediarnine, N,N′-dimethyl-1,2-di-4-pyridinyl-(9Cl); pyridine, 4,4′,4″-(1H-imidazole-2,4,5-triyl)tris-; 4,6(1H,5H)Pyrimidinedione, dihydro-5,5-bis[2-(4-pyridinyl)ethyl]-2-thioxo-; or Pyridine, 4,4′-methylenebis-.