Oriented and covalent immobilization of target molecules to solid supports: Synthesis and application of a light-activatable and thiol-reactive cross-linking reagent

Abstract

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.

Surface modification of titanium, titanium alloys, and related materials for biomedical applications

“…The benzophenone derivatives could be linked covalently onto the solid surface by irradiation. Clemence et al [186] and Collioud et al [187] produced a photosensitive hetero-bifunctional crosslinker, which could couple petides containing the cell-adhesive motifs of RGD and YIGSR to maleimidyl groups using thiol/maleimide chemistry. The photosensitive diazirine group generates an intermediate carbene after irradiation with non-destructive UV light (!320 nm).…”

Biomaterials in orthopaedics

“…Methods ranging from dip-coating techniques to formation of SAMs and tethering polymer chains to the surface were developed to affect the adhesion of cells, to influence proliferation and differentiation rates and to achieve more stable long-term material/tissue integration (Blawas & Reichert 1998;Scotchford et al 1998). In this context, covalent chemical binding of polymers and biomolecules has been achieved through silanized titania surfaces, using amino-and carboxyldirected immobilization mainly through glutaraldehyde chemistry, and photochemistry by 'grafting to' biomolecules with a photoactive group (Colloiud et al 1993;Xiao et al 1998Xiao et al , 2001). …”

UV Light Effects on Proteins: From Photochemistry to Nanomedicine

“…The orientation of the enzyme molecules is not controlled. A method for oriented, light-dependent, covalent immobilization of proteins on a solid support, using the heterobifunctional wetting-agent N-(m-(3-(trifluoromethyl)diazirin-3-yl)phenyl)-4-maleimidobutyramine, is described by Collioud et al (Collioud et al, 1993). The aryldiazirine function of this cross-linking reagent facilitates light-dependent, carbenemediated, covalent binding to either inert supports or to biomolecule s s u c h a s p r o t e i n s , carbohydrates and nucleic acids.…”