Phospholipase D-Catalyzed Transphosphatidylation for the Synthesis of Rare Complex Phospholipid Species─Hemi-bis(monoacylglycero)phosphate and Bis(diacylglycero)phosphate

Abstract

Progress in developing synthetic pathways for novel and complex phospholipid species, such as Hemi-bis-(monoacylglycero)phosphates (Hemi-BMPs) and bis-(diacylglycero)phosphates (BDPs), is essential for expanding the knowledge and availability of rare and uncommon phospholipid species. These structurally complex phospholipid species have recently gained more attention with promising applications, as active pharmaceutical ingredient carriers in multiple COVID-19 vaccines, or biomarkers for numerous lysosomal storage disorders and certain types of cancers. The presented work facilitates the production of a range of structurally diverse Hemi-BMP and BDP products intending to increase the availability and thereby the understanding of the underlying chemistry for these high-valuable compounds. The transphosphatidylation of phosphatidylcholine with a variety of structurally diverse monoacylglycerols and diacylglycerols is proceeded by phospholipase D (PLD) catalysis in a biphasic system. Optimization in regard to enzyme loading (5 U), substrate mole ratio (1:5 mol/mol), temperature (30 °C), and aqueous concentration of (18% v/v) afforded the highest conversion for the model transphosphatidylation of phosphatidylcholine with monoolein, yielding 87% in 2 h. The study additionally proposes a reaction mechanism based on molecular simulation, elegantly elaborating the structural constraints (substrate configuration and character of the fatty acid residues) for access to the active site of PLD accordingly for lower yield of BDPs. The successful system designed for the production of high-valuable Hemi-BMP and BDP-analogues demonstrated in this work promises to enhance the understanding of these complex phospholipids, leading to new scientific breakthroughs.

A sustainable and regioselective synthesis of Hemi-bis(monoacylglycero)phosphates and bis(diacylglycero)phosphates

“…Based on the findings from the previously published work is it possible to ensure that both the samples collected were representative and that the analytical methods were accurate, the samples were re-dissolved and analyzed on both NMR and HPLC-ELSD and can be seen in the adopted figures ( Figures 2 B and 2C). 33 A comparison of the analysis results indicates a small variance between the systems with the isolation of individual phospholipid species, presenting an average error margin of 5%, which verifies and accredits the accuracy and reliability of the two analytical methods ( Figure 2 ). Previously reported studies have also utilized High-Performance-TLC (HPTLC) as an analytical tool for BMP detection, which should be of consideration as an alternative.…”

“…The further possible entrapment of unreacted lipid substrates in the formed lipid aggregates has not been shown to have any significant effect on the diffusion and access to the active site of the enzyme, because previous results show no substantial variation in homogeneous and heterogeneous produced systems. 33 The fact that the experimental setup was carried out on a smaller scale in comparison to the previously developed system by Bogojevic et al., 2022., 33 and still illustrates the same tendencies in regard to lipid aggregation and formation of heterogeneous systems, further verifies the preference for creating homogeneous systems. 1

Figure 2 Experimental setup overview (A) Illustrates the A: experimental setup in the PLD-catalyzed transphosphatidylation/hydrolysis of phosphatidylcholine, highlighting the possible entrapment of starting lipid species.

…”

“…The transphosphatidylation were developed and carried out with slight modifications from a previously established method. 33 0.031 mmol of PC (egg yolk, soybean, and marine oil) and 0.163 mmol of respective alcohol donors (monoacylglycerols and diacylglycerols) were dissolved in 1.125 ml of ethyl acetate at room temperature. The enzyme loading was defined in terms of Unit (U), in which one U will liberate 1.0 μmol of choline from L-α-phosphatidylcholine per hour at pH 5.6 and at 30°C.…”

Synthesis of diacylglycerol‐terminated lipid–polymer conjugates: Application to cell surface modification

“…Natural amphiphilic phospholipid compounds are vital components of biological systems and are essential in preserving cellular equilibrium. [1][2][3] These compounds comprise one or more hydrophobic fatty acid tail groups and a hydrophilic head group incorporating a phosphorus component. 4,5 Although these compounds show remarkable biocompatibility, [6][7][8] their biomedical application is limited due to their simple structure and single molecular weight.…”

“…Natural amphiphilic phospholipid compounds are vital components of biological systems and are essential in preserving cellular equilibrium 1–3 . These compounds comprise one or more hydrophobic fatty acid tail groups and a hydrophilic head group incorporating a phosphorus component 4,5 .…”