Electrochemically Active Crosslinking Reaction for Fluorescent Labeling

Crosslinking reaction is a valuable method for the integration of two or more functionalities or properties to facilitate the effective design and fabrication of bioconjugates and nanomaterials. In particular, crosslinking reactions using photoactive linkers or click-chemistry for fluorescent labeling offer versatile strategies to manufacture biomolecular probes, which allow surface modification through stable covalent bonds. Although electroorganic synthetic methods have produced a wide variety of useful intermediate species under mild conditions, their application to practical crosslinking is restricted due to the difficulty of mass transfer, i.e., the unstable reactive intermediate generated from the electrode must be transported to another location. To date, attempts in solid-phase electroorganic synthesis have been limited mostly to immobilization of substrates on an electrode or indirect electroorganic synthesis. Furthermore, solid-phase syntheses often require excess amounts of reagents to complete target reactions due to low reactivity, and typical electroorganic approaches therefore suffer from in situgeneration of unstable intermediates trapped within the solid-supports. Therefore, the development of electrochemically active crosslinkers for the modification of insoluble targets, nonconductive samples, and biomolecules remains a significant challenge.

 We have recently accomplished intermolecular carbon-carbon bond formation reactions with aliphatic alkenes viaanodically generated cation intermediates to afford a variety of polycyclic systems. Furthermore, less reactive alkenes could be coupled with electrochemically activated substrates, which can be trapped in a thermomorphic solution-phase, spatially separated from the electrode, by using cyclohexane-nitromethane as a medium. These solution-phase reaction systems effectively accelerated intermolecular interactions between the less reactive olefins and unstable cation intermediates. We hypothesized that this approach would allow selective, direct and stable modification of alkene-attached insoluble targets with electrochemically active species. The electrochemically active crosslinking system could also enable the construction of a fluorescent probe, triggered by the formation of a covalent linkage. This strategy addresses the time-consuming, multistep post-processing problems associated with fluorescent labeling, such as washing and purification, and reduces false signals arising from nonspecific adsorption of a fluorophore or its undesired cleavage in the detection environment.

To study the selectivity of the functional groups in this reaction system, electrochemical coupling reactions were conducted using phenol 1 in the presence of terminal mono-substituted olefins and trialkyl-substituted olefin 2 (Table1). These results indicate that the phenoxonium cations anodically generated in the LiClO₄-MeNO₂ reaction system can be selectively trapped with trialkyl-substituted olefins to afford the cycloadduct as a fluorescent unit in high yields.

Based on these results, we also attempted to synthesize a fluorescent probe composed of 2,7-dimethyl-6-octenoic acid on amino-modified silica and unactivated alkenes to confirm the lifetime of the phenoxonium cation. The size of the amino-silica particles was ca.0.6-0.7 μm and the amino group content was 0.4 mmol/g. After the reaction, the mixture was washed in water and collected by filtration. Fluorescence on the silica was observed by fluorescent microscopy. The reactive site of the alkenes cannot approach the electrode because the silica molecule is too large, and thus, it was assumed that the phenoxonium cation was stable until reacting on the silica surface.

In conclusion, an electrochemically active crosslinking reaction was developed to attach fluorescent probes to aliphatic alkenes. Several dihydrobenzofuran derivatives formed via[3+2] cyclization reactions exhibited fluorescence properties. Furthermore, this approach allows the effective and stable modification of alkene-attached silica gel as an insoluble target with electrochemically active species, which enables the construction of fluorescent probes, triggered by the formation of a covalent linkage.

References

Shokaku Kim, Kumi Hirose, Jumpei Uematsu, Yuzuru Mikami, Kazuhiro Chiba, Chem. Eur. J., 2012, 18(20), 6284-6288.