High-throughput quantification of lincomycin traces in fermentation broth of genetically modified Streptomyces spp. Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography with UV detection

A new separation and quantification method using ultra-performance liquid chromatography (UPLC) with UV detection was developed for detection of lincomycin traces in fermentation broth of different Streptomyces spp. A similar high-performance liquid chromatography (HPLC) protocol was simultaneously developed for comparison purposes. Both methods were validated and showed a linear range of detector response for quantification of lincomycin in concentration from 3.125 to 1000.0 microgml(-1) with correlation coefficient 0.999 and recoveries ranging from 81.5 to 89.85% with precision < or =5%. Compared with the HPLC, the UPLC method offered high sample throughput and about 10 times lower consumption of solvents. The developed assays were used for determination of lincomycin production in genetically manipulated production strain Streptomyces lincolnensis and for determination of lincomycin production after heterologous expression of lincomycin biosynthetic gene cluster in non-producing strain Streptomyces coelicolor.     

Heterobi- to heterotetrametallic transition metal complexes constructed from ferrocenecarboxylate and [{[Ti](μ-σ,π-CCSiMe3)2}M] units

A series of copper(I) and silver(I) carboxylates received from various ferrocenecarboxylic acids was synthesized and used in the preparation of heterooligometallic Ti-Cu(Ag)-Fe complexes. The silver(I) salts [FcCO₂Ag] (2a) and [FcCHCHCO₂Ag] (2b) (Fc = ferrocenyl, (η⁵-C₅H₄)Fe(η⁵-C₅H₅)) were obtained through deprotonation of the respective acids FcCO₂H (1a) and FcCHCHCO₂H (1b) with NEt₃, followed by a reaction with [AgNO₃]. The heterotrimetallic complexes {[Ti](μ-σ,π-CCSiMe₃)₂}AgO₂CFc (4a) and {[Ti](μ-σ,π-CCSiMe₃)₂}AgO₂CCHCHFc (4b), where [Ti] denotes the (η⁵-C₅H₄SiMe₃)₂Ti unit, were obtained from the reaction of 2a and 2b with the organometallic π-tweezer compound [Ti](CCSiMe₃)₂ (3). The related heterotrimetallic copper(I) complex {[Ti](μ-σ,π-CCSiMe₃)₂}CuO₂CFc (9a) was prepared via two synthetic routes. First, salt 2a was reacted with [(η²-Me₃SiCCSiMe₃)CuBr]₂ (10) to give the alkyne-stabilized copper(I) carboxylate [(η²-Me₃SiCCSiMe₃)(CuO₂CFc)₂]₂ (11). Subsequent reaction of 11 with four equivalents of 3 afforded 9a. Alternatively, 9a and its analogues {[Ti](μ-σ,π-CCSiMe₃)₂}CuO₂C-E-Fc (E = trans-CHCH (9b), CH₂CH₂ (9c)), were prepared from acidolysis of the Cu-C_Me bond in {[Ti](μ-σ,π-CCSiMe₃)₂}CuMe (8) with acids 1a-1c. An analogous reaction between HO₂CfcPPh₂M(CO)₅ (M = Cr (14a), Mo (14b), W (14c); fc = ferrocene-1,1′-diyl) and 8 at−30 °C gave the alkyne/ferrocene-bridged heterotetrametallic compounds {[Ti](μ-σ,π-CCSiMe₃)₂}CuO₂CfcPPh₂M(CO)₅ (M = Cr (15a), Mo (15b), W (15c)). Reversing the reaction steps so that {[Ti](μ-σ,π-CCSiMe₃)₂}CuO₂CfcPPh₂ (12) was prepared first and then reacted with M(CO)₅(thf) (M = Cr (13a), Mo (13b), W (13a)) gave complicated reaction mixtures from which pure 15a-15c could not be isolated. The solid-state structures of 5, 7, 9a, and 11 have been corroborated by single-crystal X-ray structural studies and the electrochemical behavior of acids 1a-1c and of complexes 4a, 4b and 9a-9c was studied by cyclic voltammetry.     

Structure of N,C,N-chelated organotin(IV) fluorides

The set of starting tri-, di- and monoorganotin(IV) halides containing N,C,N-chelating ligand (L^NCN = {1,3-[(CH₃)₂NCH₂]₂C₆H₃}⁻) has been prepared (1-5) and two compounds structurally characterized ([L^NCNPh₂Sn]⁺I₃⁻ (1c), L^NCNSnBr₃ (5)) in the solid state. These compounds were reacted with KF with 18-crown-6, NH₄F or L^CNnBu₂SnF to give derivatives containing fluorine atom(s). Triorganotin(IV) fluorides L^NCNMe₂SnF (2a) and L^NCNnBu₂SnF (3a) revealed monomeric structural arrangement with covalent Sn-F bond both in the coordinating and non-coordinating solvents, except the behaviour of 3a that was ionized in the methanol solution at low temperature. The products of fluorination of L^NCNSnPhCl₂ (4) and 5 were described by NMR in solution as the ionic hypervalent fluorostannates or the oligomeric species reacting with chloroform, methanol or moisture to zwitterionic monomeric stannate L^NCN(H)⁺SnF₄⁻ (5c), which was confirmed by XRD analysis in the solid state.     

Existence and reactivity of three forms of orthophthalaldehyde in aqueous solutions. Polarographic, voltammetric, and spectrophotometric study

Orthophthalaldehyde (1,2-dicarboxaldehyde) (OPA) forms in the presence of a strong nucleophile with amino acids isoindole derivatives. The reaction is used in fluorometric determination of amino acids. The mechanism of these processes is not understood. OPA is present in aqueous solutions in three forms: unhydrated (I(a)), monohydrated acyclic (I(b)), and cyclic hemiacetal (I(c)). The absence of data for the molar absorptivities of these forms, together with overlap of their absorption bands, limits the application of spectrophotometry. Measurement of polarographic limiting currents of forms I(a) and I(b) enables determination of equilibrium constants K1 (formation of I(b)) and K2 (for the ring formation). The presence of these forms was supported by 1H NMR and 13C NMR. The rate of hydration of OPA is general-acid-base-catalyzed, but that of dehydration shows only specific-acid-base catalysis. The rate of hydration is controlled by general-acid-base-catalyzed addition of water to I(a). The rate of dehydration depends on the opening of the ring in I(c), which is specific-acid-base-catalyzed. At pH > 10 OPA undergoes a complex set of acid-base reactions (Scheme 3). The presence of polarographic anodic waves and oxidation on the gold electrode indicates the importance of the presence of a geminal diol form (II(a)). Establishment of equilibria among the three forms of OPA together with reactions at pH > 10 has to be considered in elucidating the reaction scheme of procedures using OPA as a reagent in the determination of amino acids.     

Fragmentation dynamics of size-selected pyrrole clusters prepared by electron impact ionization: forming a solvated dimer ion core

Structure and dynamics of size-selected charged pyrrole clusters have been studied by means of molecular beam scattering experiments and ab initio calculations. Small neutral Pyn clusters were produced in Py/He mixture expansions, and the scattering experiment with a secondary beam of He-atoms was exploited to select the neutral clusters of different sizes. The complete size-selected fragmentation patterns for the neutral dimer to the tetramer after an electron impact ionization at 70 eV were obtained from the measurements of the angular and velocity distributions at different fragment masses. All the investigated cluster sizes decay mainly to the monomer ions Py+1 (from 60 to 80% of the corresponding neutral size) and to the dimer ion Py+2 (20-30%). The trimer ions Py+3 are generated to less than 10% from the neutral trimer and tetramer. To explain the observed results, we have calculated the structures and energetics of pyrrole clusters up to the trimer for the neutral and the ionic state using DFT and PMP2 methods. The ab initio calculations show that ionized pyrrole clusters are formed with a dimeric core that is solvated by neutral pyrrole molecules. In addition, the ground and ionic state of Py-Ar complexes were calculated at CCSD(T) level with extended basis in relevance to the mixed clusters produced in supersonic expansions of Py seeded in Ar. The calculated dissociation energies of the Py-Ar and (Py-Ar)+ complexes indicate that Ar atoms are able to rapidly evaporate after ionization. The combined analysis of the fragmentation probabilities, and calculations allowed us to estimate the distribution of energy deposited in the clusters after the electron impact, which peaks above 1 eV and has a tail up to 5 eV.     

Separation of curium from americium using composite sorbents and complexing agent solutions: part 2

TODGA–PAN composite sorbent and (PhSO₃H)₂–BTPhen in nitric acid solution were employed as a system for separation of curium from americium. The influence of aqueous phase composition (complexing agent and nitric acid concentrations) on weight distribution coefficients and Cm/Am separation factor was studied in batch experiments with trace amounts of ²⁴¹Am and ²⁴⁴Cm. Based on the results obtained, column experiment was designed and conducted. The Cm/Am separation factor of 3.8 ± 0.1 found in batch experiments with TODGA–PAN could be reproduced also in column experiment resulting in good separation of Cm from Am. The efficiency of Cm separation from Am in the TODGA–PAN system was compared with the analogous system with DGA resin (Triskem International). After separation on a 0.5 mL column (φ4.7 × 29 mm) the Cm fraction containing 93% of Cm(III) contained only 3% of Am(III) in optimum conditions.

     

Advanced Analysis of Isobaric Heat Capacities by Mathematical Gnostics

Two novel imidazolium based ether-functionalized room temperature ionic liquids were synthesized to study the temperature dependence of the isobaric heat capacity in the temperature range 298.15–348.15 K. In-house synthesized ionic liquids are often available in small amounts only, owing to the high cost of the precursors and difficulties during the synthesis itself. It is therefore impossible to repeat measurement many times so that a statistically significant data sample can be obtained. In addition, to obtain at least several values of the measured property, the ionic liquid must be recycled and reused. In this work two recycling techniques were used and their influence on the values of the experimental isobaric heat capacity was analyzed. Advantages of a modern nonstatistical data analysis technique, namely mathematical gnostics, were demonstrated.     

Cytotoxicity of poly(<Emphasis Type="Italic">p</Emphasis>-phenylenediamine)

Although poly(p-phenylenediamine) is an electric non-conductor, it exhibits, analogously to conducting polymers, redox activity and could, therefore, find applications in biomedicine. In the current work, the cytotoxicity of poly(p-phenylenediamine) polymer powder produced by the chemical oxidation of p-phenylenediamine with ammonium peroxydisulfate in acidic aqueous media has been studied. Primary mouse embryonic fibroblasts were used for this purpose. Interestingly, the standard methods for the determination of polymer cytotoxicity based on international standard EN ISO 10993-5 could not be applied. The reason was the interaction of polymer extracts with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. On the basis of the evaluation of flow cytometry and micrographs taken by fluorescence microscopy on cells treated with extracts of poly(p-phenylenediamine), it can be concluded that the powder polymer possesses severe cytotoxicity. The results suggest that practical application of the polymer within biomedicine is, at the current state of knowledge, difficult, and modification of the preparation techniques and/or subsequent purification of poly(p-phenylenediamine) is needed.     

Cellulose nanocrystals prepared in H<Subscript>3</Subscript>PW<Subscript>12</Subscript>O<Subscript>40</Subscript>-acetic acid system

Cellulose nanocrystals (CNC) were prepared by destruction of cotton cellulose using phosphotungstic acid in acetic acid medium. The study shows the influence of the heteropolyacid concentration, pre-activation, and hydrogen peroxide addition on the size of CNC particles. CNC were characterized using the methods of dynamic light scattering, FTIR-spectroscopy, thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction, scanning and transmission electron microscopy, and potentiometric titration. CNC particles have highly-crystalline structure and rod-like morphology. Freeze-dried CNC hydrosols form lamellar or fibrous agglomerates, depending on the initial concentration of the CNC sol. Potentiometric titration results show increase in surface activity index and offset of pK_a values for CNC active centers in comparison to initial cellulose material.     

Interior regularity of space derivatives to an evolutionary,symmetric $$\varphi $$-Laplacian

We consider the Orlicz-growth generalization to the evolutionary p-Laplacian and to the evolutionary symmetric p-Laplacian. We derive the spatial second-order Caccioppoli-type estimate for a local weak solution to these systems. Our result is new even for the p-case.