UC San Diego

This dissertation will first explore the use of methylcyclopropene as an activated dienophile in the [4 + 2] inverse Diels–Alder cycloaddition with a tetrazine coupling partner for applications in bioimaging. Recently, bioorthogonal chemistries to label and track biomolecules in their native environment has received considerable interest from biochemists and chemical biologists. The tetrazine ligation is one such example, exhibiting robust kinetics and is mutually exclusive to other bioorthogonal reactions

making it feasible to carryout multiple-color labeling experiments. However, the classical coupling partners of tetrazine are bulky dienophiles, like norbornene and trans-cyclooctene. This potentially limits live-cell applications requiring sterically small labeling probes. In contrast, methylcyclopropene is the smallest cyclic alkene and as a tetrazine coupling partner it provides minimal steric impact that is often desired in intracellular investigations. In addition to the fast kinetics (k 13 M−1s−1), fluorophore conjugated tetrazines can also exhibit a fluorogenic “turn-on” upon cycloaddition with methylcyclopropene, making them well suited for live-cell imaging probes.

In the second investigation, this dissertation will explore two fundamental features of a phospholipid bilayer; their ability to encapsulate macromolecules and reconstitute transmembrane proteins. Phospholipid liposomes are akin to micron-sized flasks that can function as a delivery system and/or a bioreactor. In both of these applications high encapsulation efficiency is greatly desired or even necessary, but there are few liposomal methodologies that can achieve this. In order to integrate genetic circuits in liposomes we employed the inverted emulsion technique to make giant unilamellar vesicles that can be visualized by light microscopy. In addition, this method achieves greater than 90% encapsulation efficiency for polar macromolecules. Building off this technique we show it is possible to encapsulate live bacteria and yeast at high densities, which was previously only possible via microfluidics.

An alternative methodology of encapsulation can be accomplished with synthetic lipids that are composed of two clickable precursors, comprising of an alkyl chain and a lysophospholipid. Initial we demonstrated how this could work between an oleoyl azide and an alkyne lysophospholipid to form a triazole phospholipid, but due to the low solubility of the azide oil in aqueous solutions we pioneered vesicle formation by native chemical ligation (NCL). In this system both precursors are water soluble allowing for higher encapsulation efficiency and similarly to the Cu(I)-catalyzed azide–alkyne cycloaddition, the NCL system can also spontaneously reconstitute active transmembrane proteins during membrane growth.