Comprehensive Characterization of an "Off/On" Rhodol-Based Lysosomal Tracker for Orthogonal Cellular Analysis by Confocal Imaging.

Two florescent xanthene-cyanamide lysosomal trackers emitting strongly at ∼525 nm were prepared from fluorescein and rhodol methyl esters in microwave-assisted reactions. Both forms named "off" (nonfluorescent lactam) and "on" (strongly fluorescent ring-opened amide) have been comprehensively characterized out by using a combination of NMR spectroscopy, X-ray analysis, fluorimetry and confocal microscopy. Known rhodamines bearing electron-withdrawing groups (EWGs) exhibit an equilibrium between non-fluorescent (off) and fluorescent (on) depending on the dielectric constant of the medium. Here, cyanamide was introduced as EWG amine into the fluorescein and rhodol framework. Unlike rhodamine-type dyes, the ring-opened forms of fluorescein- and rhodol-cyanamides are stable in protic solvents under circumneutral and basic pH conditions. The osteoblastic cell line MC3T3-E1 from C57BL/6 mouse calvaria was used for confocal imaging where the different organelles and nuclei were distinguished by using an orthogonal combination of fluorescent dyes.

Towards the preparation of synthetic outer membrane vesicle models with micromolar affinity to wheat germ agglutinin using a dialkyl thioglycoside.

A series of alkyl thioglycosides and alkyl thiodiglycosides bearing glucose and N-acetylglucosamine residues were prepared by thiol-ene coupling in moderate to good yields (40-85%). Their binding ability towards wheat germ agglutinin was measured by competitive enzyme-linked lectin assays. One of the synthetic compounds presenting two GlcNAc units showed the highest inhibitory effect of this study with an IC50 of 11 µM corresponding to a 3182-fold improvement compared to GlcNAc. These synthetic molecules were used to produce giant vesicles, alone or in mixture with phospholipids, mimicking bacterial outer membrane vesicles (OMV) with potential antiadhesive properties.

Glass Microsphere-Supported Giant Vesicles for the Observation of Self-Reproduction of Lipid Boundaries.

Growth and division experiments on phospholipid boundaries were carried out using glass microsphere-supported phospholipid (DOPC) giant vesicles (GVs) fed with a fatty acid solution (oleic acid) at two distinct feeding rates. Both fast and slow feeding methods produced daughter GVs. Under slow feeding conditions the membrane growth process (evagination, buds, filaments) was observed in detail by fluorescence microscopy. The density difference between supported mother vesicles and newly formed daughter vesicles allowed their easy separation. Mass spectrometric analysis of the resulting mother and daughter GVs showed that the composition of both vesicle types was a mixture of original supported phospholipids and added fatty acids reflecting the total composition of amphiphiles after the feeding process. Thus, self-reproduction of phospholipid vesicles can take place under preservation of the lipid composition but different aggregate size.

Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions.

Giant lipid vesicles resemble compartments of biological cells, mimicking them in their dimension, membrane structure and partly in their membrane composition. The spontanenous appearance of closed membranes composed of bilayers of self-assembling amphiphiles was likely a prerequisite for Darwinian competitive behavior to set in at the molecular level. Such compartments should be dynamic in their membrane composition (evolvable), and sufficiently stable to harbor macromolecules (leak-free), yet semi-permeable for reactive small molecules to get across the membrane (stay away from chemical equilibrium). Here we describe bottom-up experiments simulating prebiotic environments that support the formation of simple amphiphilic molecules capable of self-assembling into vesicular objects on the micrometer scale. Long-chain alkyl phosphates, together with related amphiphilic compounds, were formed under simulated prebiotic phosphorylation conditions by using cyanamide, a recognized prebiotic chemical activator and a precursor for several compound classes. Crude dry material of the thus obtained prebiotic mixtures formed multilamellar giant vesicles once rehydrated at the appropriate pH and in the presence of plausibly prebiotic co-surfactants, as observed by optical microscopy. The size and the shape of lipid aggregates tentatively suggest that prebiotic lipid assemblies could encapsulate peptides or nucleic acids that could be formed under similar chemical prebiotic conditions. The formation of prebiotic amphiphiles was monitored by using TLC, IR, NMR and ESI-MS and UPLC-HRMS. In addition we provide a spectroscopic analysis of cyanamide under simulated prebiotic conditions in the presence of phosphate sources and spectroscopic analysis of O-phosphorylethanolamine as a plausible precursor for phosphoethanolamine lipids.

Correction: Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions.

Correction for 'Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions' by Michele Fiore et al., Org. Biomol. Chem., 2017, 15, 4231-4240.

Bringing Prebiotic Nucleosides and Nucleotides Down to Earth.

There may be more than one way leading to RNA: Recent discoveries in the synthesis of nucleoside and nucleotide precursors are described and put into the wider context of prebiotic systems chemistry. Mixing Butlerow's carbohydrate precursors with Traube's 5-formylaminopyrimidines has led to the formation of prebiotic purine nucleosides whereas the mixing of 5-phosphoribose with barbituric acid and melamine gave supramolecular fibers from stacks of Whitesides' rosettas.

A hydrophobic disordered peptide spontaneously anchors a covalently bound RNA hairpin to giant lipidic vesicles.

The attraction of nucleic acids to lipidic compartments is the first step for carriers of potentially inheritable information to self-organise in functionalised synthetic cells. Confocal fluorescence imaging shows that a synthetic amphiphilic peptidyl RNA molecule spontaneously accumulates at the outer bilayer membranes of phospho- and glycolipidic giant vesicles. Cooperatively attractive interactions of -3.4 to -4.0 kcal mol(-1) between a random coil hydrophobic peptide and lipid membranes can thus pilot lipophobic RNA to its compartmentation. The separation of mixed lipid phases in the membranes further enhances the local concentration of anchored RNA.

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA.

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

From the RNA-Peptide World: Prebiotic Reaction Conditions Compatible with Lipid Membranes for the Formation of Lipophilic Random Peptides in the Presence of Short Oligonucleotides, and More.

Deciphering the origins of life on a molecular level includes unravelling the numerous interactions that could occur between the most important biomolecules being the lipids, peptides and nucleotides. They were likely all present on the early Earth and all necessary for the emergence of cellular life. In this study, we intended to explore conditions that were at the same time conducive to chemical reactions critical for the origins of life (peptide-oligonucleotide couplings and templated ligation of oligonucleotides) and compatible with the presence of prebiotic lipid vesicles. For that, random peptides were generated from activated amino acids and analysed using NMR and MS, whereas short oligonucleotides were produced through solid-support synthesis, manually deprotected and purified using HPLC. After chemical activation in prebiotic conditions, the resulting mixtures were analysed using LC-MS. Vesicles could be produced through gentle hydration in similar conditions and observed using epifluorescence microscopy. Despite the absence of coupling or ligation, our results help to pave the way for future investigations on the origins of life that may gather all three types of biomolecules rather than studying them separately, as it is still too often the case.

Microwave-Assisted Syntheses of Rhodamine, Rhodol, and Fluorescein Derivatives.

A series of rhodol-; fluorescein- and rhodamines-based spirolactam compounds, bearing electron donor amines have been prepared. For this purpose we have redesigned the synthesis of the rhodol scaffold using 2-(2,4-dihydroxybenzoyl)benzoic acid obtaining one example rhodol methyl ester in good yields (25-30 %) Thus, one set of non-cytotoxic rhodamine-based compounds has been prepared using thermal and microwave assisted synthesis (40-78 %) and tested as high affinity ATP chemo-sensors.

The shortest synthetic route to puromycin analogues using a modified Robins approach.

We are reporting on the utility of commercial vinyl isocyanate for a practical synthetic route from adenosine to N(6)-bis-demethylpuromycin in seven steps and 65% overall yield. A clean one-pot conversion of 3'-bromo-2'-carbamoyl derivative 8 to 3'-amino-3'-deoxyadenosine derivative 10 is the main feature of this synthetic pathway. This synthesis is the shortest synthetic route toward 3'-(aminoacylamido)deoxyadenosines to date.

Chemical Analysis of Lipid Boundaries after Consecutive Growth and Division of Supported Giant Vesicles.

The reproduction of the shape of giant vesicles usually results in the increase of their "population" size. This may be achieved on giant vesicles by appropriately supplying "mother" vesicles with membranogenic amphiphiles. The next "generation" of "daughter" vesicles obtained from this "feeding" is inherently difficult to distinguish from the original mothers. Here we report on a method for the consecutive feeding with different fatty acids that each provoke membrane growth and detachment of daughter vesicles from glass microsphere-supported phospholipidic mother vesicles. We discovered that a saturated fatty acid was carried over to the next generation of mothers better than two unsaturated congeners. This has an important bearing on the growth and replication of primitive compartments at the early stages of life. Microsphere-supported vesicles are also a precise analytical tool.

Total syntheses of a conformationally locked North-type methanocarba puromycin analogue and a dinucleotide derivative.

An original synthetic approach for the first synthesis of an enantiopure methanocarba puromycin (3'-alpha-aminoacylamino-3'-deoxyadenosine) analogue and its cytidine dinucleotide derivative is described. Each compound is conformationally locked in a North-type pucker and exhibits both a pseudoaxial hydroxy group and a pseudoequatorial aminoacyl group. The syntheses were accomplished from D-ribose in 18 and 19 steps, respectively, with key steps being a ring-closing metathesis, a Luche reduction, a Simmons-Smith cyclopropanation, a Mitsunobu coupling, a Mattocks bromoacetylation, a regioselective and a stereoselective nucleophilic substitution, a chemoselective phosphoramidite coupling and a Staudinger-Vilarrasa coupling. Both molecules are being tested for peptidyl transfer efficiency in ribosomes for comparison with the peptidyl transfer kinetics of natural puromycin and other natural and synthetic ribosomal A site substrates.

The Essence of Systems Chemistry.

Systems Chemistry investigates the upkeep of specific interactions of an exceptionally broad choice of objects over longer periods of time than the average time of existence of the objects themselves. This maintenance of a dynamic state focuses on conditions where the objects are thermodynamically not very stable and should be rare or virtually inexistent. It does not matter whether they are homochirally enriched populations of chiral molecules, a specific composition of some sort of aggregate, supramolecules, or even a set of chemically relatively unstable molecules that constantly transform one into another. What does matter is that these specific interactions prevail in complex mixtures and eventually grow in numbers and frequency through the enhancing action of autocatalysis, which makes such systems ultimately resemble living cells and interacting living populations. Such chemical systems need to be correctly understood, but also intuitively described. They may be so complex that metaphors become practically more important, as a means of communication, than the precise and correct technical description of chemical models and complex molecular or supramolecular relations. This puts systems chemists on a tightrope walk of science communication, between the complex reality and an imaginative model world. This essay addresses, both, scientists who would like to read "A Brief History of Systems Chemistry", that is, about its "essence", and systems chemists who work with and communicate complex life-like chemical systems. I illustrate for the external reader a light mantra, that I call "to make more of it", and I charily draw systems chemists to reflect upon the fact that chemists are not always good at drawing a clear line between a model and "the reality": The real thing. We are in a constant danger of taking metaphors for real. Yet in real life, we do know very well that we cannot smoke with Magritte's pipe, don't we?

Low-Digit and High-Digit Polymers in the Origin of Life.

Extant life uses two kinds of linear biopolymers that mutually control their own production, as well as the cellular metabolism and the production and homeostatic maintenance of other biopolymers. Nucleic acids are linear polymers composed of a relatively low structural variety of monomeric residues, and thus a low diversity per accessed volume. Proteins are more compact linear polymers that dispose of a huge compositional diversity even at the monomeric level, and thus bear a much higher catalytic potential. The fine-grained diversity of proteins makes an unambiguous information transfer from protein templates too error-prone, so they need to be resynthesized in every generation. But proteins can catalyse both their own reproduction as well as the efficient and faithful replication of nucleic acids, which resolves in a most straightforward way an issue termed "Eigen's paradox". Here the importance of the existence of both kinds of linear biopolymers is discussed in the context of the emergence of cellular life, be it for the historic orgin of life on Earth, on some other habitable planet, or in the test tube. An immediate consequence of this analysis is the necessity for translation to appear early during the evolution of life.

The Beginning of Systems Chemistry.

Systems Chemistry has its roots in the research on the autocatalytic self-replication of biological macromolecules, first of all of synthetic deoxyribonucleic acids. A personal tour through the early works of the founder of Systems Chemistry, and of his first followers, recalls what's most important in this new era of chemistry: the growth and evolution of compartmented macromolecular populations, when provided with "food" and "fuel" and disposed of "waste".

Synthesis of conformationally locked versions of puromycin analogues.

Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North (2 and 3) and South (6 and 7) 3'-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine. North azides 2 and 3 were reported earlier. The 3'-azido intermediates 6 and 7 that are necessary for the synthesis of the South puromycin analogues are described herein for the first time.

Rapid purification of giant lipid vesicles by microfiltration.

Giant lipid vesicles (GVs) are emerging models for investigating the properties and reactivity of cell-like microcompartments, providing useful information about plausible protocellular structures in primitive times, as well as for the modern synthetic biology goal of constructing the first artificial cell from its reconstituted and partly modified components. Here we explore a novel methodology of GV purification by microfiltration under reduced pressure, operated by a simple apparatus. The method has been characterized in terms of flow rate, amount of lipid loss, quality of recovered GVs, and size distribution. A case study is reported to show the practicability of GV microfiltration. A clickable fluorescent probe was encapsulated inside GVs; more than 99.9% of the non-entrapped probe was easily and rapidly removed by multiple microfiltrations. This novel methodology is briefly discussed as a future tool for selection experiments on GV populations.

Facile and rapid access to inosine puromycin analogues through the use of adenylate deaminase.

To study the ribosomal peptidyl transfer, puromycin analogues are of interest in which adenine has been replaced by hypoxanthine. We synthesized inosine puromycin analogues from 3'-azidodeoxyadenosine derivatives using adenylate deaminase for the quantitative transformation of the N-heterocycle. The amino acid coupling was carried out under Staudinger-Vilarrasa conditions in 94% yield starting from the protected and in 82% using the unprotected azide, thus, in the presence of two hydroxyls and a lactam function.

Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments.

It is an open question how the chemical structure of prebiotic vesicle-forming amphiphiles complexified to produce robust primitive compartments that could safely host foreign molecules. Previous work suggests that comparingly labile vesicles composed of plausibly prebiotic fatty acids were eventually chemically transformed with glycerol and a suitable phosphate source into phospholipids that would form robust vesicles. Here we show that phosphatidic acid (PA) and phosphatidylethanolamine (PE) lipids can be obtained from racemic dioleoyl glycerol under plausibly prebiotic phosphorylation conditions. Upon in situ hydration of the crude phosphorylation mixtures only those that contained rac-DOPA (not rac-DOPE) generated stable giant vesicles that were capable of encapsulating water-soluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction side-products (identified by IR and MS and quantified by 1H NMR) acted as co-surfactants and facilitated vesicle formation. To mimic the compositional variation of such primitive lipid mixtures, self-assembly of a combinatorial set of the above amphiphiles was tested, revealing that too high dioleoyl glycerol contents inhibited vesicle formation. We conclude that a decisive driving force for the gradual transition from unstable fatty acid vesicles to robust diacylglyceryl phosphate vesicles was to avoid the accumulation of unphosphorylated diacylglycerols in primitive vesicle membranes.