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Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

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Aminopropylated glass-fiber discs were derivatized with the photosensitive reagents 3-(trifluoromethyl)-3-[m-(isothiocyano)phenyl]diazirine and p-azidophenyl isothiocyanate, respectively. Photolabel-derivatized solid supports were photo activated in the presence of a hexapeptide. Gas-phase sequence analysis of washed fiber discs yielded end-to-end sequences with photoactivated disks only, indicating efficient photo-immobilization of the applied peptide. The quantity of amino acid released with each Edman-de-gradation cycle conferred with the established philicity of amino-acid side chains for photogenerated carbenes. The procedure provides a hitherto unexplored way to immobilize biomolecules (polypeptides) through statistically occurring carbene or nitrene insertions.