title: Design and synthesis of nucleolipids as possible activated precursors for oligomer formation via intramolecular catalysis: stability study and supramolecular organization.
abstract: BACKGROUND
  Fatty acid vesicles are an important part of protocell models currently studied. As protocells can be considered as pre-biological precursors of cells, the models try to contribute to a better understanding of the (cellular) origin of life and emphasize on 2 major aspects: compartmentalization and replication. It has been demonstrated that lipid-based membranes are amenable to growth and division (shell replication). Furthermore compartmentalization creates a unique micro-environment in which biomolecules can accumulate and reactions can occur. Pioneering research by Sugawara, Deamer, Luisi, Szostak and Rasmussen gave more insight in obtaining autocatalytic, self-replicating vesicles capable of containing and reproducing nucleic acid sequences (core replication). Linking both core and shell replication is a challenging feat requiring thorough understanding of membrane dynamics and (auto)catalytic systems. A possible solution may lie in a class of compounds called nucleolipids, who combine a nucleoside, nucleotide or nucleobase with a lipophilic moiety. Early contributions by the group of Yanagawa mentions the prebiotic significance (as a primitive helical template) arising from the supramolecular organization of these compounds. Further contributions, exploring the supramolecular scope regarding phospoliponucleosides (e.g. 5'-dioleylphosphatidyl derivatives of adenosine, uridine and cytidine) can be accounted to Baglioni, Luisi and Berti. This emerging field of amphiphiles is being investigated for surface behavior, supramolecular assembly and even drug ability.
  
  
  RESULTS
  A series of α/β-hydroxy fatty acids and α-amino fatty acids, covalently bound to nucleoside-5'-monophosphates via a hydroxyl or amino group on the fatty acid was examined for spontaneous self-assembly in spherical aggregates and their stability towards intramolecular cleavage. Staining the resulting hydrophobic aggregates with BODIPY-dyes followed by fluorescent microscopy gave several distinct images of vesicles varying from small, isolated spheres to higher order aggregates and large, multimicrometer sized particles. Other observations include rod-like vesicle precursors. NMR was used to assess the stability of a representative sample of nucleolipids. 1D31P NMR revealed that β-hydroxy fatty acids containing nucleotides were pH-stable while the α-analogs are acid labile. Degradation products identified by [1H-31P] heteroTOCSY revealed that phosphoesters are cleaved between sugar and phosphate, while phosphoramidates are also cleaved at the lipid-phosphate bond. For the latter compounds, the ratio between both degradation pathways is influenced by the nucleobase moiety. However no oligomerization of nucleotides was observed; nor the formation of 3'-5'-cyclic nucleotides, possible intermediates for oligonucleotide synthesis.
  
  
  CONCLUSIONS
  The nucleolipids with a deoxyribose sugar moiety form small or large vesicles, rod-like structures, vesicle aggregates or large vesicles. Some of these aggregates can be considered as intermediate forms in vesicle formation or division. However, we could not observe nucleotide polymerization or cyclic nucleotide function of these nucleolipids, regardless of the sugar moiety that is investigated (deoxyribose, ribose, xylose). To unravel this observation, the chemical stability of the constructs was studied. While the nucleolipids containing β-hydroxy fatty acids are stable as well in base as in acid circumstances, others degraded in acidic conditions. Phosphoramidate nucleolipids hydrolyzed by P-N as well as P-O bond cleavage where the ratio between both pathways depends on the nucleobase. Diester constructs with an α-hydroxy stearic acid degraded exclusively by hydrolysis of the 5'-O-nucleoside ester bond. As the compounds are too stable and harsh conditions would destruct the material itself, more reactive species such as lipid imidazolates of nucleotides need to be synthesized to further analyze the potential polymerization process. Graphical AbstractVesicle information of a nucleolipid consisting of a nucleoside 5'-monophosphate and a α-hydroxy fatty acid.
authors.affiliation: Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, 3000 Leuven, Belgium.
authors.forename: Kishore Lingam
authors.initials: KL
authors.lastname: Gangadhara
authors.affiliation: Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, 3000 Leuven, Belgium.
authors.forename: Puneet
authors.initials: P
authors.lastname: Srivastava
authors.affiliation: Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, 3000 Leuven, Belgium.
authors.forename: Jef
authors.initials: J
authors.lastname: Rozenski
authors.affiliation: Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, 3000 Leuven, Belgium ; Department of Chemistry, Molecular Design and Synthesis, KU Leuven, Leuven, Belgium.
authors.forename: Henri-Philippe
authors.initials: HP
authors.lastname: Mattelaer
authors.affiliation: Department of Chemistry, Molecular Design and Synthesis, KU Leuven, Leuven, Belgium.
authors.forename: Volker
authors.initials: V
authors.lastname: Leen
authors.affiliation: Department of Chemistry, Molecular Design and Synthesis, KU Leuven, Leuven, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Dehaen
authors.affiliation: Department of Chemistry, Molecular Imaging and Photonics, KU Leuven, Leuven, Belgium.
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
authors.affiliation: Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, 3000 Leuven, Belgium.
authors.forename: Eveline
authors.initials: E
authors.lastname: Lescrinier
authors.affiliation: Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Leuven, Minderbroederstraat-10, 3000 Leuven, Belgium.
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
keywords: BODIPY; Chemical stability; Fluorescence microscopy; Hydroxy fatty acids; Intramolecular catalysis; NMR stability study; Nucleolipid; Protocell; Supramolecular assembly; Vesicles