Azide-Modified Membrane Lipids: Synthesis, Properties, and Reactivity

Abstract

In the present work, we describe the synthesis and the temperature-dependent behavior of photoreactive membrane lipids as well as their capability to study peptide/lipid interactions. The modified phospholipids contain an azide group either in the middle part or at the end of an alkyl chain and also differ in the linkage (ester vs ether) of the second alkyl chain. The temperature-dependent aggregation behavior of the azidolipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small-angle X-ray scattering (SAXS). Aggregate structures were visualized by stain and cryo transmission electron microscopy (TEM) and were further characterized by dynamic light scattering (DLS). We show that the position of the azide group and the type of linkage of the alkyl chain at the sn-2 position of the glycerol influences the type of aggregates formed as well as their long-term stability: P10AzSPC and r12AzSHPC show the formation of extrudable liposomes, which are stable in size during storage. In contrast, azidolipids that carry a terminal azido moiety either form extrudable liposomes, which show time-dependent vesicle fusion (P15AzPdPC), or self-assemble in large sheet-like, nonextrudable aggregates (r15AzPdHPC) where the lipid molecules are arranged in an interdigitated orientation at temperatures below T (LI phase). Finally, a P10AzSPC:DMPC mixture was used for photochemically induced cross-linking experiments with a transmembrane peptide (WAL-peptide) to demonstrate the applicability of the azidolipids for the analysis of peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and mass spectrometry (MS).

15N-Azides as practical and effective tags for developing long-lived hyperpolarized agents

“…7 d ,8 As the most commonly used chemical reporter, the azides have been used in the studies of nearly every class of biomolecules, including carbohydrates, 9 amino acids, 10 cholesterol, 11 nucleosides, 12 and lipids. 13 Thus, the ( 15 N) 3 -azide group, in addition to being a potential long-lived molecular tag for hyperpolarized NMR and MRI, can be used simultaneously in a bioorthogonal reaction to introduce other imaging readouts, 7 d ,8,14 allowing for development of multimodal imaging agents.…”

A Diazirine‐Modified Membrane Lipid to Study Peptide/Lipid Interactions – Chances and Challenges

“…[10] In all previously reported studies, it has been assumed that the physicochemical behaviour of modified and unmodified membrane lipids are entirely comparable, thus ensuring complete miscibility, which is not necessarily the case. We have recently shown that the position of an azide moiety in the alkyl chain of a membrane lipid will indeed influence the type of aggregates formed in aqueous dispersion, [11] and affects the miscibility of azidemodified lipids with saturated membrane lipids. [12] This highlights the need for more detailed investigations on both aspects of modified lipid behaviour.…”

“…Importantly, T m of DiazPC is higher thanother lipids with symmetry-breaking chain modifications, for example the insertion of one double bond as in 1-palmitoyl-1-oleoyl-snglycero-3-phosphocholine (POPC; T m = À 2.5 °C) [18] or one azide moiety at position 10 of the stearic acid as in P10AzSPC (T m < 5 °C). [11] This could be due to the restricted rotatability at the carbon atom carrying the diazirine group. The thermotropic transition of DiazPC coincides with a fluidization of the alkyl chains, i. e. a gel-to-liquid crystalline phase transition as evidenced by infrared (IR) spectroscopic analysis of the temperature-dependent positions of both methylene stretching vibrational bands (Figure 2b and Figure S7a).…”

“… [12] This highlights the need for more detailed investigations on both aspects of modified lipid behaviour. Although we have demonstrated the general feasibility of using azide‐modified lipids for XL‐MS studies,[ 11 , 13 ] the cross‐linking yield of azides was found to be rather low. [ 11 , 14 ] We therefore specifically designed a diazirine‐modified lipid (DiazPC, Figure 1 a) for use in XL‐MS studies.…”

“…Although we have demonstrated the general feasibility of using azide‐modified lipids for XL‐MS studies,[ 11 , 13 ] the cross‐linking yield of azides was found to be rather low. [ 11 , 14 ] We therefore specifically designed a diazirine‐modified lipid (DiazPC, Figure 1 a) for use in XL‐MS studies. The main question to be addressed in peptide/lipid and protein/lipid XL‐MS studies is: Is it possible to determine the exact cross‐link position between a membrane‐embedded peptide (or protein) and a photo‐reactive membrane lipid?…”