Despite that thiol-maleimide ‘click’ chemistry progresses under mild reaction conditions and is generally considered to be both highly-reactive and specific, applications for surface immobilization of thiol-containing biomolecules remains a challenge due to the spontaneous surface-dissociation as a consequence of the retro-Michael addition degradation pathway. To retain the attractive aspects of thiol-maleimide ‘click’ chemistry and address issues of poor stability, we describe an immobilization protocol that exploits the degradation qualities of an aryl-maleimide ‘click’ precursor in place of the more commonly used aliphatic-maleimide. The enhanced stability described is a consequence of the aryl-thiosuccinimide’s susceptibility to a ring-opening hydrolysis which spontaneously converts the thiosuccinimide to a surface-stable succinamic acid thioether. To monitor the interfacial reactions in-situ, we perform the immobilization chemistry at mesoporous silica and characterize the internal composition of these materials with confocal-Raman microscopy. To immobilized DNA at thiol-terminated silica, we perform a sequential two-step thiol cross-linking, which involves first reacting a slight molar excess of thiol-terminated DNA with 1,4-phenylenedimaleimdie in free-solution. The primary product of the first ‘click’ reaction is a DNA-monomer presenting an aryl-maleimide group that, without requiring a purification step, can be reacted with a thiol-terminated substrate. Using the confocal-Raman microscopy methodology outlined, we monitor the second ‘click’ reaction in-situ and detect a loss in the thiol stretch (2580 cm^-1) accompanied by the addition of DNA (1094 cm^-1 PO₂^-1 stretch) and thiosuccinimide (1770 cm^-1) vibrational modes. Stabilization of the surface conjugated DNA under hydrolyzing conditions was observed by a decrease in the thiosuccinimide stretch (1770 cm^-1) and the addition of bands characteristic of an amide (1257, 1550, 1656 cm^-1), revealing the formation of the succinamic acid thioether degradation product. Following the hydrolysis step, we show ~90% of the surface-bound DNA is preserved at the surface. No detectable dissociation of the stabilized surface population was observed after 2-weeks of storage in a standard PBS buffer.

Joel Harris

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