Archaeal Lipids: Extraction, Separation, and Identification via Natural Product Chemistry Perspective

Abstract

Archaeal lipids, defining a primordial life domain alongside Bacteria and Eukarya, are distinguished by their unique glycerol-1-phosphate backbone and ether-linked isoprenoid chains. Serving as critical geochemical biomarkers, archaeal lipids like glycerol dialkyl glycerol tetraethers (GDGTs) underpin paleoclimate proxies, while their phylum-specific distributions illuminate phylogenetic divergence. Despite the maturity of Mass Spectrometry-based quantitative biomarkers-predominantly those with established structures-becoming well-established in geochemical research, systematic investigation of archaeal lipids as natural products has notably lagged. This deficit manifests across three key dimensions: (1) Extraction methodology lacks universal protocols adapted to diverse archaeal taxa and sample matrices. While comparative studies exist, theoretical frameworks guiding method selection remain underexplored. (2) Purification challenges persist due to the unique structures and complex isomerization profiles of archaeal lipids, hindering standardized separation protocols. (3) Most critically, structural characterization predominantly depends on decades-old foundational studies. However, the existing reviews prioritize chemical structural, biosynthetic, and applied aspects of archaeal lipids over analytical workflows. This review addresses this gap by adopting a natural product chemistry perspective, integrating three key aspects: (1) the clarification of applicable objects, scopes, and methodological mechanisms of various extraction technologies for archaeal lipids, encompassing both cultured and environmental samples; (2) the elucidation of separation principles underlying polar-gradient lipid fractionation processes, leveraging advanced chromatographic technologies; (3) the detailed exploration of applications for NMR in resolving complex lipid structures, with specialized emphasis on determining the stereochemical configuration. By synthesizing six decades of methodological evolution, we establish a comprehensive analytical framework, from lipids extraction to structural identification. This integrated approach constructs a systematic methodological paradigm for archaeal lipid analysis, bridging theoretical principles with practical implementation.

Keywords: archaea; extraction; identification; lipids; natural product chemistry; separation.

Figure 1

The basic skeletal structure of phospholipids in Archaea (a) and Bacteria and Eukarya (b); three core dimensions are marked with different colors.

Figure 2

The chemical structures of representative archaeal lipids, where the R group in all compounds is either a polar head group or H; R = H, compounds 1–5 are diether lipids: 1: sn-2,3-di-O-geranylgeranylglyceryl alcohol (DGGGOH); 2: 2,3-di-O-phytanyl-sn-glycerol; 3: 2-O-sesterpanyl-3-O-phytanyl-sn-glycerol; 4: 2,3-di-O-sesterpanyl-sn-glycerol; and 5: macrocyclic diether. Compounds 6–9 are GDGTs: R₁ = H; 6: GDGT-0; 7: GMGT-0; 8: GDGT-1; 10: backbone of glycerol dialkyl nonitol tetraether (GDNT); and 9: GDGT-4; R₁ = OH; 6: hydroxy-GDGT-0.

Figure 3

Illustration of the Bligh and Dyer method. Solvent 1 is a mixture of chloroform (or DCM) and methanol (1:2, v/v) or a triphasic solvent system with methanol, chloroform (or DCM), and water (e.g., 2:1:0.8, v/v/v), and solvent 2 is a chloroform (or DCM) and water mixture (1:1, v/v).

Figure 4

Schematic diagram of the hydrolysis of the intact polar archaeal lipids: ester bonds are hydrolyzed under alkaline conditions, and ether bonds are hydrolyzed under acidic conditions.

Figure 5

Schematic diagram of archaeal lipids using column chromatography.

Figure 6

The structural elucidation of crenarchaeol, key HMBC (arrows pointing from H to C), and NOESY correlations; HMBC experiments confirmed the connectivity of its six-membered ring system (Va and Vb), and NOESY correlations established the stereochemical configuration of its cyclopentane moieties (Vc and Vd).

Figure 7

Schematic diagram illustrates conceptual TLC analysis of halophilic archaeal lipid extractions; (A) lipid extract from Natronorubrum aibiense stained with 0.5% α-naphthol reagent, showing dark yellow spots corresponding to phosphatidylglycerol derivatives (PGs), and purple spots representing triglycosyl diethers (TGDs) and diglycosyl diethers (DGDs) [128]; (B) lipid extract from Haloterrigena longa stained with Zinzadze reagent, where blue spots indicate phosphatidylglycerol species (PGs) [129]. Both analyses employed chloroform/methanol/acetic acid/water (85:22.5:10:4, v/v) as the developing solvent. Black spots denote the TLC origin. Green and red dashed lines demarcate the sample application origin and solvent front, respectively. Abbreviations: PGP-Me (phosphatidylglycerol phosphate methyl ester); PGs (phosphatidylglycerols); TGDs (triglycosyl diethers); DGDs (diglycosyl diethers). Note: Rf values shown do not represent actual experimental data.