Membraneless polyester microdroplets as primordial compartments at the origins of life.

Compartmentalization was likely essential for primitive chemical systems during the emergence of life, both for preventing leakage of important components, i.e., genetic materials, and for enhancing chemical reactions. Although life as we know it uses lipid bilayer-based compartments, the diversity of prebiotic chemistry may have enabled primitive living systems to start from other types of boundary systems. Here, we demonstrate membraneless compartmentalization based on prebiotically available organic compounds, α-hydroxy acids (αHAs), which are generally coproduced along with α-amino acids in prebiotic settings. Facile polymerization of αHAs provides a model pathway for the assembly of combinatorially diverse primitive compartments on early Earth. We characterized membraneless microdroplets generated from homo- and heteropolyesters synthesized from drying solutions of αHAs endowed with various side chains. These compartments can preferentially and differentially segregate and compartmentalize fluorescent dyes and fluorescently tagged RNA, providing readily available compartments that could have facilitated chemical evolution by protecting, exchanging, and encapsulating primitive components. Protein function within and RNA function in the presence of certain droplets is also preserved, suggesting the potential relevance of such droplets to various origins of life models. As a lipid amphiphile can also assemble around certain droplets, this further shows the droplets' potential compatibility with and scaffolding ability for nascent biomolecular systems that could have coexisted in complex chemical systems. These model compartments could have been more accessible in a "messy" prebiotic environment, enabling the localization of a variety of protometabolic and replication processes that could be subjected to further chemical evolution before the advent of the Last Universal Common Ancestor.

Common and Potentially Prebiotic Origin for Precursors of Nucleotide Synthesis and Activation.

We have recently shown that 2-aminoimidazole is a superior nucleotide activating group for nonenzymatic RNA copying. Here we describe a prebiotic synthesis of 2-aminoimidazole that shares a common mechanistic pathway with that of 2-aminooxazole, a previously described key intermediate in prebiotic nucleotide synthesis. In the presence of glycolaldehyde, cyanamide, phosphate and ammonium ion, both 2-aminoimidazole and 2-aminooxazole are produced, with higher concentrations of ammonium ion and acidic pH favoring the former. Given a 1:1 mixture of 2-aminoimidazole and 2-aminooxazole, glyceraldehyde preferentially reacts and cyclizes with the latter, forming a mixture of pentose aminooxazolines, and leaving free 2-aminoimidazole available for nucleotide activation. The common synthetic origin of 2-aminoimidazole and 2-aminooxazole and their distinct reactivities are suggestive of a reaction network that could lead to both the synthesis of RNA monomers and to their subsequent chemical activation.

Structural revision of kynapcin-12 by total synthesis, and inhibitory activities against prolyl oligopeptidase and cancer cells.

Kynapcin-12 is a prolyl oligopeptidase (POP) inhibitor isolated from Polyozellus multiplex, and its structure was assigned as 1 having a p-hydroquinone moiety by spectroscopic analyses and chemical means. This Letter describes the total syntheses of the proposed structure 1 for kynapcin-12 and 2',3'-diacetoxy-1,5',6',4″-tetrahydroxy-p-terphenyl 2 isolated from Boletopsis grisea, revising the structure of kynapcin-12 to the latter. These syntheses involved double Suzuki-Miyaura coupling, CAN oxidation, and LTA oxidation as key steps. The inhibitory activities of synthetic compounds against POP and cancer cells were also evaluated.

Structural elucidation and synthesis of vialinin C, a new inhibitor of TNF-α production.

A new inhibitor of TNF-α production (IC50=0.89 µM) named vialinin C (1) was isolated from dry fruiting bodies of an edible Chinese mushroom, Thelephora vialis. The structure of 1 was determined by high-resolution MS, NMR spectroscopic analysis, and confirmed by synthesis. Synthesis of ganbajunin B (5) obtained from the same origin was also described.

Mobilize a Proton to Transform the Collision-Induced Dissociation Spectral Pattern of a Cyclic Peptide.

The collision-induced dissociation (CID) behaviors of protonated molecules of anabaenopeptins, a group of cyanobacterial cyclic peptides, were investigated in detail using liquid chromatography-tandem mass spectrometry. Although anabaenopeptin A and B share a macrocyclic peptide structure, they give strikingly different fragmentation patterns; the former gives a variety of product ions including cleavages in the cyclic peptide structure, which is useful for structural analysis; whereas the latter gives far fewer product ions and no fragmentation in the cyclic moiety. Energy-resolved CID experiments clarified the mechanism behind the striking difference attributable to the difference in exocyclic amino acid residues, Tyr or Arg. The guanidino group in Arg-containing analogue, anabaenopeptin B, should be by far the most preferred protonation site; the proton would be sequestered at the guanidino group in the protonated molecule, with the lack of proton mobility prohibiting opening of the charge-directed fragmentation channels in the cyclic moiety. Enzymatic hydrolysis of the guanidino group to give citrullinated-anabaenopeptin B restored proton mobility. The fragmentation pattern of the citrullinated peptide became almost identical to that of anabaenopeptin A. The observed fragmentation behaviors of these cyclic peptides were consistent with those of linear peptides, which have been well understood based on the mobile proton model.

Prebiotic Environments with Mg2+ and Thiophilic Metal Ions Increase the Thermal Stability of Cysteine and Non-cysteine Peptides.

Wet-dry cycles driven by heating to high temperatures are frequently invoked for the prebiotic synthesis of peptides. Similarly, iron-sulfur clusters are often cited as an example of an ancient catalyst that helped prune early chemical systems into metabolic-like pathways. Because extant iron-sulfur clusters are metallocofactors of protein enzymes and nearly ubiquitous across biology, a reasonable hypothesis is that prebiotic iron-sulfur peptides formed on the early Earth. However, iron-sulfur clusters are coordinated by multiple cysteine residues, and the stability of cysteines to the heat steps of wet-dry cycles has not been determined. It, therefore, has remained unclear if the peptides needed to stabilize the formation of iron-sulfur clusters could have formed. If not, then iron-sulfur-dependent activity may have emerged later, when milder, more biological-like peptide synthesis machinery took hold. Here, we report the thermal stability of cysteine-containing peptides. We show that temperatures of 150 °C lead to the rapid degradation of cysteinyl peptides. However, the presence of Mg2+ at environmentally reasonable concentrations leads to significant protection. Thiophilic metal ions also protect against degradation at 150 °C but require concentrations not frequently observed in the environment. Nevertheless, cysteine-containing peptides are stable at lower, prebiotically plausible temperatures in seawater, carbonate lake, and ferrous lake conditions. The data are consistent with the persistence of cysteine-containing peptides on the early Earth in environments rich in metal ions. High concentrations of Mg2+ are common intra- and extra-cellularly, suggesting that the protection afforded by Mg2+ may reflect conditions that were present on the prebiotic Earth.

Synthesis of all possible isomers corresponding to the proposed structure of montanacin E, and their antitumor activity.

Total synthesis of 4 and its three diastereomers is described. The key steps involve stereoselective formation of the tetrahydrofuran ring by a cascade cyclization of hydroxy tosylate 7 and an intermolecular cross metathesis between a tetrahydrofuran 5 and a gamma-lactone 6. Spectroscopic data of 4 and biosynthetic hypothesis strongly suggest it to be montanacin E. Inhibitory activities of 4 and its isomers against six human solid tumor cell lines were also evaluated.

Formation of Abasic Oligomers in Nonenzymatic Polymerization of Canonical Nucleotides.

Polymerization of nucleotides under prebiotically plausible conditions has been a focus of several origins of life studies. Non-activated nucleotides have been shown to undergo polymerization under geothermal conditions when subjected to dry-wet cycles. They do so by a mechanism similar to acid-catalyzed ester-bond formation. However, one study showed that the low pH of these reactions resulted in predominantly depurination, thereby resulting in the formation of abasic sites in the oligomers. In this study, we aimed to systematically characterize the nature of the oligomers that resulted in reactions that involved one or more of the canonical ribonucleotides. All the reactions analyzed showed the presence of abasic oligomers, with purine nucleotides being affected the most due to deglycosylation. Even in the reactions that contained nucleotide mixtures, the presence of abasic oligomers was detected, which suggested that information transfer would be severely hampered due to losing the capacity to base pair via H-bonds. Importantly, the stability of the N-glycosidic linkage, under conditions used for dry-wet cycling, was also determined. Results from this study further strengthen the hypothesis that chemical evolution in a pre-RNA World would have been vital for the evolution of informational molecules of an RNA World. This is evident in the high degree of instability displayed by N-glycosidic bonds of canonical purine ribonucleotides under the same geothermal conditions that otherwise readily favors polymerization. Significantly, the resultant product characterization in the reactions concerned underscores the difficulty associated with analyzing complex prebiotically relevant reactions due to inherent limitation of current analytical methods.

Synthesis of imidazole-activated ribonucleotides using cyanogen chloride.

We report the syntheses of ribonucleoside 5'-monophosphates activated with imidazole, using a mechanism which relies on the in situ generation of cyanogen chloride from the reaction of cyanide anion with hypochlorous acid. Cyanogen chloride reacts rapidly with imidazole to form diimidazole imine as the major product, a species which affords the activation of ribonucleoside 5'-monophosphates to their 5'-phosphorimidazolides.

Estimating the capacity for production of formamide by radioactive minerals on the prebiotic Earth.

Water creates special problems for prebiotic chemistry, as it is thermodynamically favorable for amide and phosphodiester bonds to hydrolyze. The availability of alternative solvents with more favorable properties for the formation of prebiotic molecules on the early Earth may have helped bypass this so-called "water paradox". Formamide (FA) is one such solvent, and can serve as a nucleobase precursor, but it is difficult to envision how FA could have been generated in large quantities or accumulated in terrestrial surface environments. We report here the conversion of aqueous acetonitrile (ACN) via hydrogen cyanide (HCN) as an intermediate into FA by γ-irradiation under conditions mimicking exposure to radioactive minerals. We estimate that a radioactive placer deposit could produce 0.1‒0.8 mol FA km-2 year-1. A uraninite fission zone comparable to the Oklo reactors in Gabon can produce 0.1‒1 mol m-2 year-1, orders of magnitude greater than other scenarios of FA production or delivery for which reaching sizeable concentrations of FA are problematic. Radioactive mineral deposits may be favorable settings for prebiotic compound formation through emergent geologic processes and FA-mediated organic chemistry.

Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

Cytoprotective Effects of Lysophospholipids from Sea Cucumber Holothuria atra.

Lysophospholipids are important signaling molecules in animals and metazoan cells. They are widely distributed among marine invertebrates, where their physiological roles are unknown. Sea cucumbers produce unique lysophospholipids. In this study, two lysophospholipids were detected in Holothuria atra for the first time, lyso-platelet activating factor and lysophosphatidylcholine, with nuclear magnetic resonance and liquid chromatography-time-of-flight mass spectrometric analyses. The lipid fraction of H. atra contained lyso-platelet activating factor and lysophosphatidylcholine, and inhibited H2O2-induced apoptosis in the macrophage cell line J774A.1. The antioxidant activity of the lysophospholipid-containing lipid fraction of H. atra was confirmed with the oxygen radical absorbance capacity method. Our results suggest that the lysophospholipids from H. atra are potential therapeutic agents for the inflammation induced by oxidative stress.

Detection of oxygen addition peaks for terpendole E and related indole-diterpene alkaloids in a positive-mode ESI-MS.

This report describes that a regular positive electrospray ionization mass spectrometry (MS) analysis of terpendoles often causes unexpected oxygen additions to form [M + H + O](+) and [M + H + 2O](+), which might be a troublesome in the characterization of new natural analogues. The intensities of [M + H + O](+) and [M + H + 2O](+) among terpendoles were unpredictable and fluctuated largely. Simple electrochemical oxidation in electrospray ionization was insufficient to explain the phenomenon. So we studied factors to form [M + H + O](+) and [M + H + 2O](+) using terpendole E and natural terpendoles together with some model indole alkaloids. Similar oxygen addition was observed for 1,2,3,4-tetrahydrocyclopent[b]indole, which is corresponding to the substructure of terpendole E. In tandem MS experiments, a major fragment ion at m/z 130 from protonated terpendole E was assigned to the substructure containing indole. When the [M + H + O](+) was selected as a precursor ion, the ion shifted to m/z 146. The same 16 Da shift of fragments was also observed for 1,2,3,4-tetrahydrocyclopent[b]indole, indicating that the oxygen addition of terpendole E took place at the indole portion. However, the oxygen addition was absent for some terpendoles, even whose structure resembles terpendole E. The breakdown curves characterized the tandem MS features of terpendoles. Preferential dissociation into m/z 130 suggested the protonation tendency at the indole site. Terpendoles that are preferentially protonated at indole tend to form oxygen addition peaks, suggesting that the protonation feature contributes to the oxygen additions in some degrees.

RK-1355A and B, novel quinomycin derivatives isolated from a microbial metabolites fraction library based on NPPlot screening.

Two novel quinomycin derivatives, RK-1355A (1) and B (2), and one known quinomycin derivative, UK-63,598 (3), were isolated from a microbial metabolites fraction library of Streptomyces sp. RK88-1355 based on Natural Products Plot screening. The structural elucidation of 1 and 2 was established through two-dimensional NMR and mass spectrometric measurements. They belong to a class of quinomycin antibiotics family having 3-hydroxyquinaldic acid and a sulfoxide moiety. They are the first examples for natural products as a quinoline type quinomycin having a sulfoxide on the intramolecular cross-linkage. They showed potent antiproliferative activities against various cancer cell lines and they were also found to exhibit moderate antibacterial activity.

Synthesis and structural revision of phomopsin B, a novel polyketide carrying a 10-membered cyclic-ether ring.

Total synthesis of the proposed structure 2 for phomopsin B was achieved by using an intramolecular olefin metathesis as a key step. The spectral data, however, did not match with those of the natural product reported. Re-examination of the reported NMR data led to the structural revision of phomopsin B to known dothiorelone A 18. The R configuration of dothiorelone A was determined by total synthesis through a cross-metathesis with a chiral olefin 19.

Structural determination of montanacin D by total synthesis.

The first total syntheses of acetogenin 3 and its 4 S,8 R-isomer are described. The key step involves intermolecular metathesis of an alpha,beta-unsaturated ketone carrying a tetrahydropyranyl lactone with a tetrahydrofuran derivative. Compound 3 has spectroscopic and physical data consistent with those of natural montanacin D, suggesting that the absolute configuration of the natural product is as shown in 3.

Structural revision of terpenoids with a (3Z)-2-methyl-3-penten-2-ol moiety by the synthesis of (23E)- and (23Z)-cycloart-23-ene-3beta,25-diols.

Synthesis of (23E)-cycloart-23-ene-3beta,25-diol (1) and its 23Z-isomer 2 was achieved by using cycloartenol as a starting material, thus revising the proposed structure of natural 2 to 1 unequivocally. These synthetic studies revealed that the structural revision (Z-form --> E-form) should also be applied to terpenoids such as (23Z)-3beta-acetoxyeupha-7,23-diene-25-ol, (23Z)-tirucalla-7,23-diene-3beta,25-diol, quadrangularol A, quadrangularic acid K, and daurichromene C.

Convergent synthesis of pyragonicin.

This paper describes a second-generation synthesis of an antitumor tetrahydropyran (THP) acetogenin, pyragonicin. The key step involved an olefin cross-metathesis between the THP segment and the terminal gamma-lactone residue. The coupling reaction in the presence of Grubbs' second-generation catalyst resulted in an unseparable mixture of a desired coupling product and its one-carbon eliminated product while the use of Grubbs' first-generation catalyst afforded the former exclusively. A novel MOM-migrating reaction found in a cyclization reaction is also discussed.