In Situ Vesicle Formation by Native Chemical Ligation

Abstract

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non-enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water-soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self-assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction enables compatibility of in situ membrane formation with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes. Keywordsnative chemical ligation; phospholipid; self-assembly; artificial cell; synthetic biology Phospholipid membranes are routinely employed in several practical applications such as the study of protein-membrane interactions, [1] drug-delivery, [2] origin-of-life studies, [3] bottomup synthetic biology, [4] and synthetic reactors. [5] While the capability of phospholipids to self-assemble into membranes is well studied, [6] the de novo synthesis and assembly of membranes from simple non-membrane forming reactive precursors is poorly understood. Living organisms are capable of in situ synthesis of lipid membranes by utilizing membrane bound acyltransferases and reactive thioester precursors. [7] Given the well-documented challenges associated with reconstituting lipid synthesizing membrane proteins into vesicles, [8] several groups have explored methods to generate lipid membranes de novo from reactive amphiphilic precursors. [9], [10] However, these methods generally suffer from a range of issues, including the necessity of catalysts, lack of soluble starting materials, use of biologically incompatible reactive precursors and the formation of lipids that have limited structural similarity to natural phospholipids. [9] Phospholipid membranes are often Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript advantageous in complex environments due to their stability and biocompatibility. [11] Simpler and more robust methods for phospholipid synthesis could also find use as drivers for the growth and division of primitive protocells. [11], [12] Therefore, it would be exciting to develop a catalyst-free method to generate and grow phospholipid membrane vesicles from reactive soluble precursors. Furthermore, it would be interesting to utilize water-soluble thioester precursors, analogous to living cells. [7], [13] Here we demonstrate that the chemoselective native chemical ligation (NCL) [14] is capable of in situ synthesis of phospholipid vesicles from long-chain thioesters. The lipids are capable o...

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes

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Genetically controlled membrane synthesis in liposomes

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Traceless synthesis of ceramides in living cells reveals saturation-dependent apoptotic effects

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