Preparation and photoactivation of caged fluorophores and caged proteins using a new class of heterobifunctional, photocleavable cross-linking reagents.
The design, synthesis, and spectroscopic and chemical properties of four members of a new class of heterobifunctional photocleavable (caged) cross-linking reagents were described. One of the two reactive groups of the cross-linker reacted with amino groups to form the corresponding photolabile carbamates. Amino group containing compounds or proteins caged with these reagents can be coupled through the thiol reactive oxirane group of the cross-linker to a different biomolecule or to a thiol-derivatized surface. The 3,4-dimethoxy-6-nitrophenyl photoisomerization group of the reagent was physically and chemically isolated from the cross-linking functionality, and the high extinction coefficient and red-shifted action spectrum of this chromophore make it suitable for photoactivation applications of caged compounds on surfaces or in living cells. The bifunctional, photocleavable cross-linking reagents were used to prepare a thiol reactive caged rhodamine 110. The new reagents and conjugation procedures described may be used as part of a general procedure to cage the activity of proteins by physically masking binding sites.
Ottl
J
J
Biomolecular and Cellular Dynamics Research Group, Max Planck Institute for Biochemistry, Martinsried, Germany.
Gabriel
D
D
Marriott
G
G
D000199:Actins / Q000737:chemistry; D000818:Animals; D000840:Anisoles / Q000138:chemical synthesis; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry; D015195:Drug Design; D005027:Ethylene Oxide / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007202:Indicators and Reagents; D007460:Iodoacetamide; D010777:Photochemistry; D011506:Proteins / Q000737:chemistry*; D011726:Pyridinium Compounds / Q000138:chemical synthesis; D011817:Rabbits; D012235:Rhodamines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012995:Solubility; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013450:Sulfones / Q000138:chemical synthesis; D014867:Water
C112047:4-(1-(3,4-dimethoxy-6-nitrophenyl)-2,3-epoxypropyl-1-oxocarbonyloxy)-3-oxo-2,5-diphenyl-2,3-dihydrothiophen-1,1-dioxide; D000199:Actins; D000840:Anisoles; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C112048:N-1-((3,4-dimethoxy-6-nitrophenyl)-2,3-epoxypropyloxycarbonyl)-4-(N,N-dimethylamino)pyridinium 3-oxo-2,5-diphenyl-2,3-dihydrothiophen-1,1-dioxide-4-hydroxide; D011506:Proteins; D011726:Pyridinium Compounds; D012235:Rhodamines; D013438:Sulfhydryl Compounds; D013450:Sulfones; D014867:Water; D012710:Serum Albumin, Bovine; C005358:tetramethylrhodamine; D005027:Ethylene Oxide; D007460:Iodoacetamide