Synthesis and surface grafting of a ß-cyclodextrin dimer facilitating cooperative inclusion of 2,6-ANS.

A novel ß-cyclodextrin (ß-CD) dimer was synthesized and surface-grafted by click chemistry onto azide-functionalized quartz surfaces in order to introduce the cooperative features of the ß-CD dimer to solid surfaces. Using NMR and fluorescence spectroscopy, it is shown that the free ß-CD dimer forms a 1:1 complex with the fluorescent guest molecule, 2-anilinonaphthalene-6-sulfonic acid (otherwise known not to form 1:2 complexes with parent ß-CD), with an apparent association constant of 7300 M(-1). Further, it is shown using total internal reflection fluorescence spectroscopy that the inclusion of the fluorescent guest into both cavities of the ß-CD dimer is maintained when grafted onto a solid surface.

Direct site-directed photocoupling of proteins onto surfaces coated with beta-cyclodextrins.

A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A molecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K(d) of 1.65 mM was determined using (1)H NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment.

Nonfouling tunable ßCD dextran polymer films for protein applications.

Polymeric ß-cyclodextrin (ßCD) films tunable with respect to thickness and ßCD content were prepared in order to develop a suitable platform, allowing for inclusion of nonpolar guest molecules in the ßCD cavity, while suppressing nonspecific protein adsorption. The ßCD films were synthesized from linear ßCD dextran polymers, and grafted onto silicon oxide surfaces by "click" chemistry. Topographic and morphological characteristics are controllable by reaction conditions and polymer type, with average film heights from 2.5 to 12.5 nm. Reversible introduction of electrostatic charges in the ßCD dextran by complex formation with 1-adamantanecarboxylic acid prior to surface grafting resulted in a thinner and denser film, presumably by decompaction of the polymers. Total internal reflection fluorescence spectroscopy (TIRF) was employed to evaluate the accessibility of ßCD cavities to the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid. Only a minor fraction of the ßCD cavities was accessible in the thicker and less dense films; however, accessibility was largely improved with increased ionic strength using NaCl up to 1 M. Antifouling properties of the ßCD dextran polymer films were assessed by TIRF real-time monitoring, using bovine serum albumin as a model protein, and showed a 5- to 10-fold reduction in nonspecific adsorption as compared to a bare quartz surface with the degree of reduction reflecting film thickness and interfacial polymer density.