Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea‐Inspired Tetraether Lipids

Abstract

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies.

Effect of Headgroups on Small‐Ion Permeability across Archaea‐Inspired Tetraether Lipid Membranes

“…We recently reported a systematic study of the dependence of membrane leakage on the presence of small rings incorporated into the tethered, membrane‐spanning segments of synthetic glycerolmonoalkyl glycerol tetraether (GMGT) lipids . We found that incorporating a single transmembrane tether (with or without rings), phytanyl hydrophobic side chains, and ether linkages between the glycerol backbone made it possible to generate membranes with approximately two orders of magnitude slower rate of leakage of small ions (H + , OH − , Na + , Cl − , buffer ions) at room temperature compared to membranes generated from commercial EggPC lipids.…”

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum‐Stable Membranes with Low Leakage

“…[18] We also demonstrated that pure GcGTPC-CH liposomes exhibited a % 30-foldr eduction in leakage of an eutrally charged drug, gemcitabine, compared to POPC liposomes with 40 mol %a dded cholesterol,and a % 10-fold reduction in leakage of the drug compared to GMGTPC-CH/chol liposomes. We demonstrated that membranes formed from pure GcGTPC-CHl ipids exhibit a5 0-fold reduction in membrane permeability to small ions when comparedw ith membranes comprised of at ypicald iacyl lipid, POPC, and af ive-fold reduction in membrane permeability compared to ap reviously reporteda rchaea-inspired tetraether lipid, GMGTPC-CH, that lacks acovalently attached cholesterolg roup.…”

“…[17] We previously reported an ew class of archaea-inspired singlyt ethered lipids, namely,g lycerol monoalkyl glycerolt etraether with phosphocholine (PC) headgroups (GMGTPC-CH, Figure 1. [18] In contrast, the addition of free cholesterol to lipid formulations has been commonly used in research laboratories to reduce membrane leakage by improving membrane integrity ( Figure 1A). Figure 2).…”

“…[23] The free terminala lcohol in 1 was then converted to the corresponding tosylate, which successively reacted with 2-phenyl-1,3-dioxan-5-ol followed by as elective ring-opening of the dioxolane moiety using diisobutylaluminium hydride (DIBAL-H) to form the benzyl protected glycerolb ackbone 2.D ibromo alkane 3 [18] was then connected to the glycerolb ackbone in 2 to generate protected lipid 4 under basic conditions. We designed the GcGTPC-CHl ipid to comprise ac holesterol group attachedt ot he lipid headgroup via an ethylene glycol spacertoimprove flexibility and increase access for synthetic modification of the steroid.…”

“…This result was in agreement with previousr eports, which showed that adding4 0-50 mol %o ff ree cholesterol to diacyl lipids [25] maintains af luid phase and diminishes its phase transition. Furthermore, liposomes remained structurally stable throughout the time frame of leakage experiments andw edid not observe Using ap reviously described pH equilibrationm ethodt oe stimate initial rates of leakageo fs mall ions (e.g.,H + ,O H À ,N a + , Cl À ,a nd other buffer ions) from liposomes, [18,26] we first evaluated the observed initial rate of small ion membrane leakage to study whether the addition of free cholesterol (40 mol %) to 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), GMGTPC-CH, or GcGTPC-CH liposomal formulations affectedm embrane permeationo fs malli ons (see Figure S4 in the Supporting Information). Furthermore, liposomes remained structurally stable throughout the time frame of leakage experiments andw edid not observe Using ap reviously described pH equilibrationm ethodt oe stimate initial rates of leakageo fs mall ions (e.g.,H + ,O H À ,N a + , Cl À ,a nd other buffer ions) from liposomes, [18,26] we first evaluated the observed initial rate of small ion membrane leakage to study whether the addition of free cholesterol (40 mol %) to 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), GMGTPC-CH, or GcGTPC-CH liposomal formulations affectedm embrane permeationo fs malli ons (see Figure S4 in the Supporting Information).…”