A Synthetic Model for the Possible FeIV2(µ-O)2 Core of Methane Monooxygenase Intermediate Q Derived from a Structurally Characterized FeIIIFeIV(µ-O)2 Complex.

A bis(µ-oxo)diiron(IV,IV) complex as a model for intermediate Q in the methane monooxygenase reaction cycle has been prepared. The precursor complex with a [FeIIIFeIV(µ-O)2] core was fully characterized by X-ray crystallography and other spectroscopic analyses and was converted to the [FeIV2(µ-O)2] complex via electrochemical oxidation at 1000 mV (vs Ag/Ag+) in acetone at 193 K. The UV-vis spectral features, Mössbauer parameters (ΔEQ = 2.079 mm/s and δ = -0.027 mm/s), and EXAFS analysis (Fe-O/N = 1.73/1.96 Å and Fe···Fe = 2.76 Å) support the structure of the low-spin (S = 1, for each Fe) [FeIV2(µ-O)2] core. The rate constants of the hydrogen abstraction reaction from 9,10-dihydroanthracene at 243 K suggest the high reactivity of these synthetic bis(µ-oxo)diiron complexes supported by simple N4 tripodal ligand.

Antimicrobial Activity of ε-Poly-l-lysine after Forming a Water-Insoluble Complex with an Anionic Surfactant.

ε-Poly-l-lysine (ε-PL) is one of the few homopoly(amino-acid)s occurring in nature. ε-PL, which possesses multiple amino groups, is highly soluble in water, where it forms the antimicrobial polycationic chain (PLn+). Although the high water-solubility is advantageous for the use of ε-PL as a food preservative, it has limited the applicability of ε-PL as a biopolymer plastic. Here, we report on the preparation and availability of a water-insoluble complex formed with PLn+ and an anionic surfactant, bis(2-ethylhexyl) sulfosuccinate (BEHS-, is also commercialized as AOT) anion. The PLn+/BEHS--complex, which is soluble in organic solvents, was successfully used as a coating material for a cellulose acetate membrane to create a water-resistant antimicrobial membrane. In addition, the thermoplastic PLn+/BEHS--complex was able to be uniformly mixed with polypropylene by heating, resulting in materials exhibiting antimicrobial activities.

Nanoparticle-assisted laser desorption/ionization using sinapic acid-modified iron oxide nanoparticles for mass spectrometry analysis.

Iron oxide-based nanoparticles (NP) were covalently modified with sinapic acid (SA) through a condensation reaction to assist the ionization of both large and small molecules. The morphology of SA-modified NPs (SA-NP) was characterized by transmission electron microscopy (TEM), and the modification of the NP surface with SA was confirmed using ultraviolet (UV) and infrared (IR) spectroscopy. The number of SA molecules was estimated to be 6 per NP. SA-NP-assisted laser desorption/ionization was carried out on small molecules, such as pesticides and plant hormones, and large molecules, such as peptides and proteins. A peptide fragment from degraded proteins was detected more efficiently compared with conventional methods.

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase.

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na2SiO3, 600 mM Na2MoO4, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.

ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis.

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-ß-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-ß-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-ß-lysine molecules adenylated by ORF 19 are used to elongate an L-ß-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-ß-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.

Effect of the alkyl chain length of α,ω-dichloroalkane on the Gibbs energy of transfer for functional groups.

Ion-transfer reactions of alkyl and perfluoroalkyl carboxylate ions (CH3(CH2)n-2COO- with n = 8-12 and CF3(CF2)n-2COO- with n = 3-9) were investigated at the polarized Cl-(CH2)m-Cl with m = 2, 4, 6, and 8 (O) | water (W) interface to evaluate the effects of n and m on the solvation energy of the ions, as well as on their methylene and terminal groups. These ions exhibited reversible or quasi-reversible voltammetric waves due to their transfer across the O | W interfaces, enabling the determination of formal potentials and formal Gibbs transfer energies from O to W, Δ G tr,o → w 0 ' The Δ G tr,o → w 0 ' values for CH3(CH2)n-2COO- and CF3(CF2)n-2COO- increased linearly with n, allowing the estimation of Δ G tr,o → w 0 ' for methylene and difluoromethylene groups, Δ G tr,o → w 0 ' (CH2) and Δ G tr,o → w 0 ' (CF2), and for their terminal groups, Δ G tr,o → w 0 ' (COO- + CH3) and Δ G tr,o → w 0 ' (COO- + CF3). Whereas the Δ G tr,o → w 0 ' (CH2) and Δ G tr,o → w 0 ' (CF2) hardly changed with the variation in m, the Δ G tr,o → w 0 ' (COO- + CH3) and Δ G tr,o → w 0 ' (COO- + CF3) decreased noticeably. These results suggest that the solvation energy for ions in Cl-(CH2)m-Cl increases with m, regardless of hydrophilic or lipophilic nature of the ions. Based on these findings, the advantage of using Cl-(CH2)m-Cl with a large m as a non-aqueous solvent for ion-transfer voltammetry was discussed.

Separation of an ε-poly-L-lysine derivative by solvent extraction under a controlled interfacial potential difference.

This paper describes the availability of a 1,2-dichloroethane (DCE)-water (W) interfacial system under a controlled interfacial potential difference for the separation of polycationic species. The system was applied to the production of polyethylene glycol-modified ε-poly-L-lysine (PEG-εPL). PEG-εPL is produced by a fermentation process, and the crude product contains a significant amount of non-modified εPL, which is hardly separated by conventional chromatographic techniques. Both εPL species exist in fully protonated forms under certain acidic conditions, and an extractant, dibenzo-18-crown-6 (DB18C6), associates with their ammonium groups to stabilize the polycations in DCE. Despite the polydispersity of the samples, the εPL and crude PEG-εPL give well-defined cyclic voltammetric waves due to the DB18C6-assisted transfer of the polycations at the polarizable DB18C6 (DCE) | (W, pH ~ 3) interface with midpoint potentials useful for a rough prediction of ion partition equilibria. Thus, the partition experiment was performed using the DB18C6, Bu4N[(CF3SO2)2N] (DCE) | crude PEG-εPL, Li[(CF3SO2)2N] (W, pH ~ 3) interfacial system, of which the potential difference was controlled to enable selective extraction of polycationic PEG-εPL by partition of the [(CF3SO2)2N]- ion. The extract could be collected from the DCE phase and was found to consist of highly purified PEG-εPL.

Determination of protamine and heparin based on their effects on a glucose oxidase enzymatic reaction.

This paper describes a sensitive method for determining protamine and heparin by utilizing a glucose oxidase enzymatic reaction. Polycationic protamine significantly promoted the enzymatic reaction rate with [Fe(CN)6]3-, so that the increase could be used to determine protamine. The promotion effect was stoichiometrically decreased by the addition of polyanionic heparin through the polyion complex formation with protamine, so that the enzymatic reaction also allowed for the determination of heparin. We thus applied the proposed method to blood plasma containing heparin and found that heparin did not stoichiometrically form a polyion complex with protamine, likely due to strong interactions between heparin and some components of the plasma. The proposed method allowed for the detection of free protamine (and/or weakly binding protamine with heparin) existing in the condition that protamine did not neutralize all of the heparin in the plasma. The method also permitted for the estimation of heparin concentrations using calibration curves. Thus, the proposed method would help reduce the risks of protamine overdose in heparin neutralization and would be a helpful tool in clinical practices that use heparin and protamine.

Assay of enzymes forming AMP+PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate.

The formation of 18-molybdopyrophosphate anion has been studied to develop a simple and rapid assay of the enzymatic reaction involving ATP→AMP+PPi(P(2)O(7)(4-)). By the addition of P(2)O(7)(4-) anion to an acidic acetonitrile-water solution containing MoO(4)(2-) anion, the colorless Mo(VI) solution immediately became yellow due to the formation of 18-molybdopyrophosphate anion. The absorbance of the P(2)O(7)(4-)-Mo(VI) mixture at, for example, 450nm was proportional to the analytical concentration of P(2)O(7)(4-) anion. Although the test Mo(VI) solution remained colorless by the addition of AMP, it gradually turned to yellow by ATP. The undesired color development is attributed to the formation of a yellow molybdophosphate species accompanied by the dissociation of PO(4)(3-) from the unstable ATP molecule. However, the color development became much slower when ethylenediaminetetraacetic acid was added into an assay mixture, where ATP may form a kinetically stable species. Thus, P(2)O(7)(4-) anion can be determined spectrophotometrically in the enzymatic reaction mixture containing ATP. By the addition of ascorbic acid, the yellow P(2)O(7)(4-)-Mo(VI) mixture turned to blue due to the reduction of the molybdopyrophosphate anion. Thus, P(2)O(7)(4-) anion can be detected colorimetrically by the blueness. The spectrophotometric and colorimetric methods could be applied advantageously to the assay of acetyl-CoA synthetase.

Determination of polyanion utilizing a promoted glucose oxidase enzymatic reaction by ε-poly-L-lysine.

In this paper, a sensitive determination method for polyanion using a glucose oxidase (GOx) enzymatic reaction with ferricyanide ion is described. We previously reported that the GOx enzymatic reaction was significantly promoted by a cationic polymer of ε-poly-L-lysine (εPL), and the enzymatic reaction could be utilized for the determination of εPL. Generally, polycation stoichiometrically forms polyion complex with polyanion. Thus, it is expected that the promotion effect of εPL on the enzymatic reaction is interfered by polyanion, and the enzymatic reaction is also applicable to the determination of polyanion. Predictably, the promotion effect of εPL was stoichiometrically interfered by polyanions, such as polyvinyl sulfate and polyacrylate, and the interference effect allowed for the determination of the polyanions. The detection limit of polyanion was estimated to be ~ 0.3 µeq L-1. As a preliminary application, the proposed method was applied to the determination of anionic polymer of heparin in a human plasma.

Tetraphenylantimony(V)-assisted transfer of hydroxide and fluoride anions across the 1,6-dichlorohexane | water interface.

The ion-transfer reaction at the 1,6-dichlorohexane (DCH) | water (W) interface in the presence of an organometallic cation, tetraphenylantimony (TPhSb+), in DCH was studied voltammetrically. When TPhSb+ salt with [(C4F9SO2)2N]- ion was added to the DCH-phase and the W-phase was buffered at pH < 6, a reversible cyclic voltammogram due to the simple transfer of TPhSb+ ion across the DCH | W interface was observed within the polarizable potential window. When the W-phase was buffered at pH > 7, the midpoint potential shifted to more positive potentials with increasing pH. The voltammogram could be attributed to the transfer of the OH- ion assisted by the formation of TPhSbOH, which is stable in DCH. Also, a 7reversible voltammogram due to the TPhSb+-assisted transfer of F- ion was observed at the TPhSb+ (DCH) | F- (W, unbuffered) interfacial system. The same results were achieved when TPhSb[(C4F9SO2)2N] in DCH was replaced by TPhSbOH or TPhSbF, indicating the applicability of the TPhSb+ and TPhSbOH (DCH) | OH- (W) interfacial system to a pH sensor for alkaline solution and that of the TPhSb+ and TPhSbF (DCH) | F- (W) interface to a F- ion sensor.

First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-L-α-lysine (ε-PαL) and ε-oligo-L-ß-lysine (ε-OßL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OßL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OßL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.

Ion-Transfer Voltammetry at Fluorous Ether | Water Interfaces.

This paper describes that fluorous ethers, 1,1,2,2-tetrafluoroethyl-2,2,3,3-tetrafluoropropyl ether and 1H,1H,5H-octafluoropentyl-1,1,2,2-tetrafluoroethyl ether, can be used for a non-aqueous medium in electrochemistry at a liquid | liquid interface. These solvents dissolved a high concentration of tetraalkylammonium salts with highly-fluorinated anions, such as bis(nonafluorobutanesulfonyl)imide and tetrakis[3,5-bis(trifluoromethyl)phenyl]borate ions, and the solution had a high conductivity. The fluorous ether | water interfaces exhibited a substantial polarizable potential window, and various ions, including tetraphenylarsonium and tetraphenylborate ions, gave a reversible voltammetric wave due to their ion transfer across the interfaces. Using the tetraphenylarsonium-tetraphenylborate assumption, the formal potentials for the ion transfer and the formal Gibbs energies of ion transfer from the ethers to water were estimated. The Gibbs energies were close to those from a previously reported fluorous solvent, 1,1,1,2,3,4,4,5,5,5-decafluoropentane; the fluorous ethers also exhibited a higher affinity for fluorinated ions. Because of a lower volatility, the fluorous ethers would be more advantageously used in two-phase electrochemistry, particularly concerning analytical purposes.

Fluorination Effect on the Gibbs Transfer Energy for Methylene Group from 1,2-Dichloroethane or 1,1,1,2,3,4,4,5,5,5-​Decafluoropentane to Water.

The ion-transfer reactions of alkyl and perfluoroalkyl carboxylate ions (CH3(CH2)n-2COO- and CF3(CF2)n-2COO-) at 1,2-dichloroethane (DCE) | water (W) and 1,1,1,2,3,4,4,5,5,5-decafluoropentane (DFP) | W interfaces were investigated. These ions gave reversible or quasi-reversible voltammetric waves due to their ion transfer across the interfaces, and the formal potentials and the formal Gibbs transfer energies from a non-aqueous solvent to water, ΔG°'tr,α→W (α = DCE and DFP), were determined. The ΔG°'tr,α→W for CH3(CH2)n-2COO- and CF3(CF2)n-2COO- linearly increased with n, allowing for estimating ΔG°'tr,α→W for methylene groups. The estimated value of ΔG°'tr,DCE→W for the -CH2- group was higher than that of ΔG°'tr,DCE→W for the -CH2- group, whereas the ΔG°'tr,DCE→W for the -CF2- group was lower than that of ΔG°'tr,DCE→W for the -CF2- group, indicating that the -CH2- (or -CF2-) group is more favorably (or unfavorably) solvated in the DCE phase compared to the DFP phase. From the estimated values, the fluorination effect of alkyl chains on partitioning the alkyl group between the biphase media has also been discussed.

A Theoretical Approach to the Fluorophilicity of Ions via the Gibbs Energy of Ion Transfer at the Fluorous Solvent/Water Interface.

The non-Bornian solvation model has been applied to a theoretical consideration of the Gibbs free energy for the transfer of fluorinated anions, non-fluorinated cations, and non-fluorinated anions at the 2H,3H-decafluoropentane (DFP)/water (W) and 1,2-dichloroethane (DCE)/W interfaces. According to our previous experimental results, the fluorinated anions are more stable in DFP than DCE, while the non-fluorinated cations and anions are less stable in DFP. To understand this characteristic feature of DFP, energy decomposition analyses have been performed for the hypothetical transfer of ions at the DFP/DCE interface. In conclusion, the characteristics of DFP as a fluorous solvent should be explained in terms of the higher repulsive interaction of the solvent molecule with ions, particularly with non-fluorinated ions.

Structure and electrochemical properties of (µ-O)2Mn2(iii,iii) and (µ-O)2Mn2(iii,iv) complexes supported by pyridine-, quinoline-, isoquinoline- and quinoxaline-based tetranitrogen ligands.

Seven new bis(µ-oxo)dimanganese complexes with Mn2(iii,iii) or Mn2(iii,iv) oxidation states were prepared using quinoline- and isoquinoline-based tetraamine ligands. The structures of the ligands include ethylenediamine, trans-1,2-cyclohexanediamine and tripodal amine, bearing two or three nitrogen-containing heteroaromatics. Regardless of the skeleton and number of aliphatic nitrogen atoms in the ligands, quinoline complexes stabilize the Mn2(iii,iii) oxidation state, whereas, isoquinoline ligands afford Mn2(iii,iv) complexes. A systematic comparison of the differences in structural parameters and redox potentials of a total of 14 complexes with a (µ-O)2Mn2 diamond core, which includes corresponding pyridine and quinoxaline derivatives as supporting ligands, highlights the distinct deviation of quinoline and tripodal amine motifs in this ligand series.

Direct label-free electrochemical detection of proteins using the polarized oil/water interface.

Voltammetric behaviors of various globular proteins, including cytochrome c, ribonuclease A, lysozyme, albumin, myoglobin, and alpha-lactalbumin, were studied at the polarized 1,2-dichloroethane/water (DCE/W) interface in the presence of four different anionic surfactants, that is, dinonylnaphthalenesulfonate (DNNS), bis(2-ethylhexyl)sulfosuccinate (Aerosol-OT; AOT), bis(2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl)sulfosuccinate (BDFHS), and bis(2-ethylhexyl)phosphate (BEHP). When the W phase was acidic (pH = approximately 3.4), the surfactants (except for BEHP) added to DCE facilitated the adsorption of the above proteins to the DCE/W interface and gave a well-developed voltammetric wave due to the adsorption/desorption of the proteins. This voltammetric wave, which we here call "protein wave", is promising for direct label-free electrochemical detection of proteins. The current for the adsorption of a protein to the interface showed a linear dependence on the protein concentration in the presence of excess surfactant. The foot potential at which the protein wave appeared in cyclic voltammetry showed different values depending on the natures of the protein and surfactant. Multivariate analysis for the foot potentials determined for different proteins with different surfactants revealed that the protein selectivity should depend on the charged, polar, and nonpolar surface areas of a protein molecule. On the basis of these voltammetric studies, it was shown in principle that online electrochemical separation/determination of proteins could be performed using a two-step oil/water-type flow-cell system.

Determination of Polyhexamethylene Biguanide Utilizing a Glucose Oxidase Enzymatic Reaction.

Polyhexamethylene biguanide (PHMB) is a cationic disinfectant widely used for personal-care products and for sanitizers in swimming pools. This paper describes a promotion effect of PHMB on a glucose oxidase (GOx) enzymatic reaction with ferricyanide ion and its analytical application. The promotion effect arose from a polyion complex formation between polycationic PHMB and polyanionic GOx. The promoted GOx reaction was analyzed by Michaelis-Menten equation, and the Michalis constant and catalytic constant were estimated to be 240 µM and 31 s-1, respectively. Utilizing the promotion effect, PHMB was successfully determined in the range of 0.05 to 0.40 ppm, and the detection limit was calculated to be 0.027 ppm. The visual detection and semi-determination of PHMB with the same concentration level were also possible. As an application, the method was applied to the determination of PHMB in a contact lens detergent and its model solution.

Gibbs Transfer Energies of Ions from a Mixed Solvent of 2H,3H-Decafluoropentane and 1,2-Dichloroethane to Water.

The transfer of hydrophilic, lipophilic, and fluorophilic ions at the interface between water (W) and a mixed solvent (MIX) of 2H,3H-decafluoropentane (DFP) and 1,2-dichloroethane (DCE) was studied voltammetrically and potentiometrically, and the formal Gibbs transfer energies of the ions from MIX to W, ΔG0'tr,MIX→W, were determined. The ΔG0'tr,MIX→W values of all the ions tested were higher than those from DFP to W. Namely, the ions would exist more stably in MIX than DFP, even for fluorophilic ions. This is due to the addition of DCE, which has a higher dielectric constant. A comparison of ΔG0'tr,MIX→W with that from DCE to W showed a superior affinity of fluorophilic ions to the fluorous solvent in spite of equivolume addition of DCE. Therefore, the mixed solvent would be a practically superior extraction medium for fluorophilic ions. In practice, the MIX | methylene blue+ (W) system showed higher extractability of a fluorophilic ion C8F17SO3- than the DFP | W and DCE | W systems.