Removal of Paramagnetic Ions Prior to Analysis of Organic Reactions in Aqueous Solutions by NMR Spectroscopy.

This article describes a method for improving 1H NMR spectra of aqueous samples containing paramagnetic metals by precipitation of metal cations with a variety of counteranions. The addition of hydroxide, phosphate, carbonate, and arsenate to solutions of transition metals such as Fe2+ and Mn2+ can reduce line broadening and improve the ability of a spectrometer to lock on the signal of deuterium. The method is most effective under strongly alkaline conditions, and care must be taken to observe whether the organic substrates undergo side reactions or are themselves removed from solution upon addition of the precipitating salts. As a demonstration of the practical value of the method, we show that NMR spectroscopy can be used to monitor the transition-metal-mediated hydrolysis of glycylglycine (Gly2).

Design of Hydrolytically Degradable Polyethylene Glycol Crosslinkers for Facile Control of Hydrogel Degradation.

Hydrogels, whose degradability can be controlled while also preserving cell viability or biomolecule stability, are in demand. Degradable polyethylene glycol crosslinkers are hydrolytically designed for use in hydrogels. Degradation is controlled by crosslinker chemical structure, such as introducing local hydrophobicity, steric hindrance, or electron-withdrawing moieties near a degradable ester moiety. Hydrogels made using these crosslinkers have gelation times from 1 to 22 min, storage moduli from 3 to 10 kPa, mesh sizes from 10 to 13 nm, and degradation times from 18 h to 16 d. However, when reaction conditions are modified to achieve similar gelation time, hydrogels have similar initial properties but preserve the wide range of degradation times. All crosslinkers support high cell viability upon hydrogel encapsulation or exposure to leachables and degradation products. This innovation in controlling degradation can help realize the hydrogels' potential for drug delivery or as matrices for cell encapsulation and transplantation.

The Prebiotic Provenance of Semi-Aqueous Solvents.

The numerous and varied roles of phosphorylated organic molecules in biochemistry suggest they may have been important to the origin of life. The prominence of phosphorylated molecules presents a conundrum given that phosphorylation is a thermodynamically unfavorable, endergonic process in water, and most natural sources of phosphate are poorly soluble. We recently demonstrated that a semi-aqueous solvent consisting of urea, ammonium formate, and water (UAFW) supports the dissolution of phosphate and the phosphorylation of nucleosides. However, the prebiotic feasibility and robustness of the UAFW system are unclear. Here, we study the UAFW system as a medium in which phosphate minerals are potentially solubilized. Specifically, we conduct a series of chemical experiments alongside thermodynamic models that simulate the formation of ammonium formate from the hydrolysis of hydrogen cyanide, and demonstrate the stability of formamide in such solvents (as an aqueous mixture). The dissolution of hydroxylapatite requires a liquid medium, and we investigate whether a UAFW system is solid or liquid over varied conditions, finding that this characteristic is controlled by the molar ratios of the three components. For liquid UAFW mixtures, we also find the solubility of phosphate is higher when the quantity of ammonium formate is greater than urea. We suggest the urea within the system can lower the activity of water, help create a stable and persistent solution, and may act as a condensing agent/catalyst to improve nucleoside phosphorylation yields.

Prebiotic condensation through wet-dry cycling regulated by deliquescence.

Wet-dry cycling is widely regarded as a means of driving condensation reactions under prebiotic conditions to generate mixtures of prospective biopolymers. A criticism of this model is its reliance on unpredictable rehydration events, like rainstorms. Here, we report the ability of deliquescent minerals to mediate the oligomerization of glycine during iterative wet-dry cycles. The reaction mixtures evaporate to dryness at high temperatures and spontaneously reacquire water vapor to form aqueous solutions at low temperatures. Deliquescent mixtures can foster yields of oligomerization over ten-fold higher than non-deliquescent controls. The deliquescent mixtures tightly regulate their moisture content, which is crucial, as too little water precludes dissolution of the reactants while too much water favors hydrolysis over condensation. The model also suggests a potential reason why life evolved to favor the enrichment of potassium: so living systems could acquire and retain sufficient water to serve as a solvent for biochemical reactions.

Quantitative Analysis of Glycine Oligomerization by Ion-Pair Chromatography.

This paper describes a method for the quantitative analysis of mixtures of glycine and its oligomers by ion-pair high-performance liquid chromatography (IP-HPLC), with a particular focus on applications in origins-of-life research. We demonstrate the identification of glycine oligomers (Gly n ) up to 14 residues long-the approximate detectable limit of their solubility in water-and measurement of the concentration of these species in the product mixture of an oligomerization reaction. The molar response factors for higher oligomers of glycine-which are impractical to obtain as pure samples-are extrapolated from direct analysis of pure standards of n = 3-6, which established a clear linear trend. We compare and contrast our method to those in previous reports with respect to accuracy and practicality. While the data reported here are specific to the analysis of oligomers of glycine, the approach should be applicable to the design of methods for the analysis of oligomerization of other amino acids.

The drive to life on wet and icy worlds.

This paper presents a reformulation of the submarine alkaline hydrothermal theory for the emergence of life in response to recent experimental findings. The theory views life, like other self-organizing systems in the Universe, as an inevitable outcome of particular disequilibria. In this case, the disequilibria were two: (1) in redox potential, between hydrogen plus methane with the circuit-completing electron acceptors such as nitrite, nitrate, ferric iron, and carbon dioxide, and (2) in pH gradient between an acidulous external ocean and an alkaline hydrothermal fluid. Both CO2 and CH4 were equally the ultimate sources of organic carbon, and the metal sulfides and oxyhydroxides acted as protoenzymatic catalysts. The realization, now 50 years old, that membrane-spanning gradients, rather than organic intermediates, play a vital role in life's operations calls into question the idea of "prebiotic chemistry." It informs our own suggestion that experimentation should look to the kind of nanoengines that must have been the precursors to molecular motors-such as pyrophosphate synthetase and the like driven by these gradients-that make life work. It is these putative free energy or disequilibria converters, presumably constructed from minerals comprising the earliest inorganic membranes, that, as obstacles to vectorial ionic flows, present themselves as the candidates for future experiments. Key Words: Methanotrophy-Origin of life. Astrobiology 14, 308-343. The fixation of inorganic carbon into organic material (autotrophy) is a prerequisite for life and sets the starting point of biological evolution. (Fuchs, 2011 ) Further significant progress with the tightly membrane-bound H(+)-PPase family should lead to an increased insight into basic requirements for the biological transport of protons through membranes and its coupling to phosphorylation. (Baltscheffsky et al., 1999 ).

Thermodynamic analysis to assist in the design of recombinant antibodies.

This critical review examines the thermodynamics of binding of bivalent antibodies (IgG and IgE) to soluble ligands with two or more binding moieties joined covalently (multivalent ligands) and to surfaces functionalized with multiple identical ligands. Given the prevalence of antibodies in nature, the goal of this paper is to begin to understand what aspects of bivalent antibodies are important relative to their monovalent counterparts (Fab). We provide a brief introduction to the thermodynamic parameters of importance to bivalent binding, guidance as to which of these parameters are most useful for the comparison of disparate systems, and a re-examination of binding studies of bivalent antibodies from the literature. For all of the cases we examined, the intramolecular free energy of binding (ΔG°intra) was less favorable than the intermolecular free energy (ΔG°inter). The effective molarity (EM) and the ratio of the free energies of intramolecular and intermolecular binding (ΔG°intra/ΔG°inter) are tools to assess the particular contribution of intramolecular binding to the thermodynamics of bivalent association. The paper concludes with guidance to the reader on what to consider when designing experiments to study bivalent systems.

The relative rates of thiol-thioester exchange and hydrolysis for alkyl and aryl thioalkanoates in water.

This article reports rate constants for thiol-thioester exchange (k (ex)), and for acid-mediated (k (a)), base-mediated (k (b)), and pH-independent (k (w)) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate-model alkyl and aryl thioalkanoates, respectively-in water. Reactions such as thiol-thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k (a) = 1.5 × 10(-5) M(-1) s(-1), k (b) = 1.6 × 10(-1) M(-1) s(-1), and k (w) = 3.6 × 10(-8) s(-1). At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol-thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k (ex) = 1.7 M(-1) s(-1). At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK (a) of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol-thioester exchange exceed those of hydrolysis by several orders of magnitude.

Patterned paper as a template for the delivery of reactants in the fabrication of planar materials.

This account reviews the use of templates, fabricated by patterning paper, for the delivery of aqueous solutions of reactants (predominantly, ions) in the preparation of structured, thin materials (e.g., films of ionotropic hydrogels). In these methods, a patterned sheet of paper transfers an aqueous solution of reagent to a second phase-either solid or liquid-brought into contact with the template; this process can form solid structures with thicknesses that are typically ≤1.5 mm. The shape of the template and the pattern of a hydrophobic barrier on the paper control the shape of the product, in its plane, by restricting the delivery of the reagent in two dimensions. The concentration of the reagents, and the duration that the template remains in contact with the second phase, control growth in the third dimension (i.e., thickness). The method is especially useful in fabricating shaped films of ionotropic hydrogels (e.g., calcium alginate) by controlling the delivery of solutions of multivalent cations to solutions of anionic polymers. The templates can also be used to direct reactions that generate patterns of solid precipitates within sheets of paper. This review examines applications of the method for: (i) patterning bacteria in two dimensions within a hydrogel film, (ii) manipulating hydrogel films and sheets of paper magnetically, and (iii) generating dynamic 3-D structures (e.g., a cylinder of rising bubbles of O(2)) from sheets of paper with 2-D patterns of a catalyst (e.g., Pd(0)) immersed in appropriate reagents (e.g., 1% H(2)O(2) in water).

Heterogeneous films of ionotropic hydrogels fabricated from delivery templates of patterned paper.

The use of delivery templates makes it possible to fabricate shaped, millimeter-thick heterogeneously patterned films of ionotropic hydrogels. These structures include two-dimensional (2-D) patterns of a polymer cross-linked by different ions (e.g., alginic acid cross-linked with Ca2+ and Fe3+) and patterns of step gradients in the concentration of a single cross-linking ion. The delivery templates consist of stacked sheets of chromatography paper patterned with hydrophobic barriers (waterproof tape, transparency film, or toner deposited by a color laser printer). Each layer of paper serves as a reservoir for a different solution of cross-linking ions, while the hydrophobic barriers prevent solutions on adjacent sheets from mixing. Holes cut through the sheets expose different solutions of cross-linking ions to the surface of the templates. Films with shaped regions of hydrogels cross-linked by paramagnetic ions can be oriented with a bar magnet. Variations in the concentrations of cations used to cross-link the gel can control the mechanical properties of the film: for single alginate films composed of areas cross-linked with different concentrations of Fe3+, the regions cross-linked with high concentrations of Fe3+ are more rigid than regions cross-linked with low concentrations of Fe3+. The heterogeneous hydrogel films can be used to culture bacteria in various 2-D designs. The pattern of toxic and nontoxic ions used to cross-link the polymer determines the pattern of viable colonies of Escherichia coli within the film.

Electrically addressable parallel nanowires with 30 nm spacing from micromolding and nanoskiving.

This paper describes the fabrication of arrays of parallel, electrically addressable metallic nanowires by depositing alternating layers of thin films of metal and polymer-both planar and topographically patterned-and sectioning the laminated structures with an ultramicrotome (nanoskiving). The structures that resulted from this process had two distinct regions: one in which parallel Au nanowires were separated by a minimum distance of 30 nm, and one in which the nanowires diverged such that the distal ends were individually addressable by low-resolution (> or =10 microm) photolithography. Conductive polyaniline (PANI) was electrochemically deposited across the nanowire electrodes to demonstrate their electrical addressability, continuity, and physical separation. Before deposition, the wires were electrically isolated; with the PANI, they were electrically connected. After dry etching to remove the polymer, the gap between the nanowire electrodes returned to an insulating state. This procedure provides a method for making wires with dimensions and separations of <50 nm without the use of e-beam or focused-ion-beam "writing" and opens applications in organic and molecular electronics, chemical and biological sensing, and other fields where nanoscale distances between parallel conductive electrodes are desirable.

Dependence of effective molarity on linker length for an intramolecular protein-ligand system.

This paper reports dissociation constants and "effective molarities" (M(eff)) for the intramolecular binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EG(n)) linkers of different lengths (n = 0, 2, 5, 10, and 20) and compares these experimental values with theoretical estimates from polymer theory. As expected, the value of M(eff) is lowest when the linker is too short (n = 0) to allow the ligand to bind noncovalently at the active site of the protein without strain, is highest when the linker is the optimal length (n = 2) to allow such binding to occur, and decreases monotonically as the length increases past this optimal value (but only by a factor of approximately 8 from n = 2 to n = 20). These experimental results are not compatible with a model in which the single bonds of the linker are completely restricted when the ligand has bound noncovalently to the active site of the protein, but they are quantitatively compatible with a model that treats the linker as a random-coil polymer. Calorimetry revealed that enthalpic interactions between the linker and the protein are not important in determining the thermodynamics of the system. Taken together, these results suggest that the manifestation of the linker in the thermodynamics of binding is exclusively entropic. The values of M(eff) are, theoretically, intrinsic properties of the EG(n) linkers and can be used to predict the avidities of multivalent ligands with these linkers for multivalent proteins. The weak dependence of M(eff) on linker length suggests that multivalent ligands containing flexible linkers that are longer than the spacing between the binding sites of a multivalent protein will be effective in binding, and that the use of flexible linkers with lengths somewhat greater than the optimal distance between binding sites is a justifiable strategy for the design of multivalent ligands.

Fabrication and Manipulation of Ionotropic Hydrogels Crosslinked by Paramagnetic Ions.

This manuscript describes the fabrication and manipulation of millimeter-scale spheres fabricated from ionotropic hydrogels that are crosslinked with paramagnetic metal ions (e.g., Ho(3+)). These ionotropic hydrogels experience a force in a magnetic field gradient that correlates with the concentration of the paramagnetic cations crosslinking the polymer. In an externally applied magnetic field, the paramagnetic hydrogel spheres assemble into ordered arrays or confined geometrical structures in the regions of highest magnetic field. These spheres can be separated from heterogeneous mixtures of diamagnetic materials using a simple bar magnet. Two applications using these recoverable hydrogel spheres were demonstrated: i) When prepared with embedded indicator dyes bound to paper, the spheres were used as colorimetric sensors for pH. ii) When prepared with embedded activated carbon powder, they were used to remove organic materials from aqueous solutions.

[60]fullerene-stoppered porphyrinorotaxanes: pronounced elongation of charge-separated-state lifetimes.

A series of Sauvage-type porphyrinorotaxanes containing [60]fullerene stoppers have been synthesized by a convergent route. Photoinduced energy transfer and electron-transfer reactions in these rotaxanes yield long-distance charge-separated radical-pair states, whose lifetimes in solution at ambient temperatures are as high as 32 mus, depending on the distance between the fullerene and zinc porphyrin chromophores.