Ladderane lipids produced by anammox bacteria constitute some of the most structurally fascinating yet poorly studied molecules among biological membrane lipids. Slow growth of the producing organism and the inherent difficulty of purifying complex lipid mixtures have prohibited isolation of useful amounts of natural ladderane lipids. We have devised a highly selective total synthesis of ladderane lipid tails and a full phosphatidylcholine to enable biophysical studies on chemically homogeneous samples of these molecules. Additionally, we report the first proof of absolute configuration of a natural ladderane.

Ladderane lipids are unique to anaerobic ammonium-oxidizing (anammox) bacteria and are enriched in the membrane of the anammoxosome, an organelle thought to compartmentalize the anammox process, which involves the toxic intermediate hydrazine (NH). Due to the slow growth rate of anammox bacteria and difficulty of isolating pure ladderane lipids, experimental evidence of the biological function of ladderanes is lacking. We have synthesized two natural and one unnatural ladderane phosphatidylcholine lipids and compared their thermotropic properties in self-assembled bilayers to distinguish between [3]- and [5]-ladderane function. We developed a hydrazine transmembrane diffusion assay using a water-soluble derivative of a hydrazine sensor and determined that ladderane membranes are as permeable to hydrazine as straight-chain lipid bilayers. However, pH equilibration across ladderane membranes occurs 5-10 times more slowly than across straight-chain lipid membranes. Langmuir monolayer analysis and the rates of fluorescence recovery after photobleaching suggest that dense ladderane packing may preclude formation of proton/hydroxide-conducting water wires. These data support the hypothesis that ladderanes prevent the breakdown of the proton motive force rather than blocking hydrazine transmembrane diffusion in anammox bacteria.

Highly functionalized cyclopropanecarboxylates were readily prepared by rhodium-catalyzed cyclopropanation of alkenes with aryldiazoacetates and styryldiazoaceates, in which the ester functionality is either trimethylsilylethyl (TMSE) or trichlorethyl (TCE). By having labile protecting groups on the ester, chiral triarylcyclopropane carboxylate ligands were conveniently prepared. The asymmetric induction during cyclopropanation is dependent on the nature of the ester group and the chiral dirhodium tetracarboxylate catalyst. The prolinate catalyst Rh2(S-DOSP)4 was the optimum catalyst for asymmetric intermolecular cyclopropanation of TMSE diazoesters with styrene, while Rh2(R-BPCP)4 was the optimum catalyst for TCE diazoesters.

The rhodium-catalyzed decomposition of 2-(triisopropylsilyl)ethyl aryl- and vinyldiazoacetates results in the stereoselective formation of Z-allylsilanes. The transformation is considered to proceed by silyl-directed intramolecular C-H functionalization to form a β-lactone intermediate followed by a silyl-activated extrusion of carbon dioxide.

Anaerobic ammonium oxidizing (anammox) bacteria form a critical part of the nitrogen cycle by converting ammonium and nitrite to nitrogen gas. Anammox bacteria carry out their metabolism in the anammoxosome, a membrane-bound organelle that is comprised of the unique ladderane lipids. Ladderane lipids, named for the ladder-like structure of fused cyclobutane rings in their fatty acid tails, have not been found anywhere else in nature, suggesting that they play a critical role in the anammox metabolism. It has been hypothesized that ladderane lipids prevent the diffusion of hydrazine (an anammox intermediate), protons, or other species across the anammoxosome membrane. As anammox bacteria have not been grown in axenic culture and grow extremely slowly in enrichment cultures, researchers have not been able to isolate sufficient quantities of pure ladderane lipids to determine the biophysical properties of ladderane membranes. Without knowledge of the physical properties of ladderane lipids or genetic tools for studying the lipids in vivo, their biological function remains unknown. We have developed efficient synthetic routes to naturally occurring ladderane phospholipids and unnatural analogs. We show that ladderane lipids have physical properties that are distinct from conventional straight-chain lipids. Ladderane lipids form dense bilayers with slow lateral diffusion and dense monolayers with low compressibility. By varying the identities of the fatty acid tails, we establish structure-function relationships for the different ladderane structures. These physical properties result in membranes with much slower rates of transbilayer diffusion of protons, which are pumped across the anammoxosome membrane and used to produce ATP. These results suggest that ladderane lipids in the anammoxosome may prevent the dissipation of the proton gradient during the slow anammox metabolism. This role for ladderane lipids in the anammoxosome may partially explain why anammox bacteria evolved such unique lipids.