All living organisms synthesize phospholipids as the primary constituent of their cell membranes. While phospholipids can spontaneously self-assemble in water to form membranebound vesicles, their aqueous synthesis requires pre-existing membrane-embedded enzymes. This limitation has led to models in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate that a combination of ion pairing and self-assembly of reactants allows highyielding synthesis of cellular phospholipids in water. Acylation of 2-lysophospholipids using cationic thioesters occurs in mildly alkaline solutions resulting in the formation of cell-like membranes. A variety of membrane-forming natural phospholipids can be synthesized. Membrane formation takes place in water from natural alkaline sources, such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained.Main Text: Cellular membranes composed of glycerophospholipids are found in all living organisms (1). Bacterial and eukaryotic membranes consist of diacylphospholipids, in which two ester linkages connect a polar head group to two hydrophobic tails. Cells synthesize diacylphospholipids through enzymatic acylation of lysophospholipids (2). As several enzymes involved in phospholipid biosynthesis must themselves be membrane-bound for enzyme activity, it is unclear if phospholipid membranes could have formed in the absence of advanced enzymatic machinery (3-6). Enzyme-free synthesis of glycerophospholipids can occur using wet-dry cycling (7, 8) and acylation of glycerophosphates can take place in the presence of a large excess of activated long-chain acyl imidazole derivatives, but the reactions require organic co-solvent due to the hydrophobicity of the acylating precursors and result in unnatural phospholipids (9, 10). A high-yielding synthesis of natural phospholipids in water would shed light on the origin and evolution of cellular membranes and open up new routes for lipid synthesis in artificial cells (11,12).
We report the genome-guided discovery of sungeidines, a class of microbial secondary metabolites with unique structural features. Despite evolutionary relationships with dynemicin-type enediynes, the sungeidines are produced by a biosynthetic gene cluster (BGC) that exhibits distinct differences from known enediyne BGCs. Our studies suggest that the sungeidines are assembled from two octaketide chains that are processed differently than those of the dynemicin-type enediynes. The biosynthesis also involves a unique activating sulfotransferase that promotes a dehydration reaction. The loss of genes, including a putative epoxidase gene, is likely to be the main cause of the divergence of the sungeidine pathway from other canonical enediyne pathways. The findings disclose the surprising evolvability of enediyne pathways and set the stage for characterizing the intriguing enzymatic steps in sungeidine biosynthesis.
New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a nonnatural, functionally naïve zinc-binding protein into a highly active catalyst for an abiological hetero-Diels-Alder reaction. The artificial metalloenzyme achieves >10 4 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a chemical proficiency (1/K TS = 2.9 x 10 10 M -1 ) that exceeds all previously characterised Diels-Alderases. These properties capitalise on effective Lewis acid catalysis, a chemical strategy for accelerating Diels-Alder reactions common in the lab but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.
We report here the orchestration of molecular ion networking and a set of computationally assisted structural elucidation approaches in the discovery of a new class of pyrroloiminoquinone alkaloids that possess selective bioactivity against pancreatic cancer cell lines. Aleutianamine represents the first in a new class of pyrroloiminoquinone alkaloids possessing a highly strained multibridged ring system, discovered from Latrunculia (Latrunculia) austini Samaai, Kelly & Gibbons, 2006 (class Demospongiae, order Poecilosclerida, family Latrunculiidae) recovered during a NOAA deep-water exploration of the Aleutian Islands. The molecule was identified with the guidance of mass spectrometry, nuclear magnetic resonance, and molecular ion networking (MoIN) analysis. The structure of aleutianamine was determined using extensive spectroscopic analysis in conjunction with computationally assisted quantifiable structure elucidation tools. Aleutianamine exhibited potent and selective cytotoxicity toward solid tumor cell lines including pancreatic cancer (PANC-1) with an IC 50 of 25 nM and colon cancer (HCT-116) with an IC 50 of 1 μM, and represents a potent and selective candidate for advanced preclinical studies.
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