Total synthesis of rutamycin B and oligomycin C

The asymmetric synthesis of the macrolide antibiotics (+)-rutamycin B (1) and (+)-oligomycin C (2) is described. The approach relied on the synthesis and coupling of the individual spiroketal fragments 3a and 3b with the C1-C17 polyproprionate fragment 4. The preparation of the spiroketal fragments was achieved using chiral (E)-crotylsilane bond construction methodology, which allowed the introduction of the stereogenic centers prior to spiroketalization. The present work details the synthesis of the C19-C28 and C29-C34 subunits as well as their convergent assembly through an alkylation reaction of the lithiated N,N-dimethylhydrazones 6 and 8 to afford the individual linear spiroketal intermediates 5a and 5b, respectively. After functional group adjustment, these advanced intermediates were cyclized to their respective spiroketal-coupling partners 40 and 41. The requisite polypropionate fragment was assembled in a convergent manner using asymmetric crotylation methodology for the introduction of six of the nine-stereogenic centers. The use of three consecutive crotylation reactions was used for the construction of the C3-C12 subunit 32. A Mukaiyama-type aldol reaction of 35 with the chiral alpha-methyl aldehyde 39 was used for the introduction of the C12-C13 stereocenters. This anti aldol finished the construction of the C3-C17 advanced intermediate 36. A two-carbon homologation completed the construction of the polypropionate fragment 38. The completion of the synthesis of the two macrolide antibiotics was accomplished by the union of two principal fragments that was achieved with an intermolecular palladium-(0) catalyzed cross-coupling reaction between the terminal vinylstannanes of the individual spiroketals 3a and 3b and the polypropionate fragment 4. The individual carboxylic acids 46 and 47 were cyclized to their respective macrocyclic lactones 48 and 49 under Yamaguchi reaction conditions. Deprotection of these macrolides completed the synthesis of the rutamycin B and oligomycin C.     

A comparative study of the single crystal X-ray determination and molecular modelling of the binding of oligomycin to ATP Synthase

Recently published X-ray structures of three common forms, A, B and C, of oligomycin, including absolute configurations, are investigated to examine their binding to ATP Synthase. The X-ray studies reveal regions with differences in three-dimensional structure and hydrogen bonding propensity between the oligomycins, which may be associated with their potential to bind to sites on ATP Synthase. Computational docking studies carried out using MOE with the X-ray structures and an homology model of the F_O domain of ATP Synthase from Escherichia coli, are used to derive an induced fit pocket. Docking of all oligomycins to this pocket indicate that the B and C forms bind more tightly than the A form. Consideration of the single crystal X-ray data alone indicate the B form may be the best inhibitor and that O(24) is the most important ligating group for binding, this is supported by the docking data. The latter reveals Asn214 and other key proton translocating residues to be the main residues contacted by the inhibitor. These data allow the binding modes of different forms of oligomycin to be deduced from X-ray single crystal data supported by molecular modelling and computational docking studies.     

Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8

A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2–356 μg ml⁻¹ with recovery of ¹²⁵I-labeled C2B8 at the target sample concentration of 125 μg ml⁻¹ equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9–4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.     

Investigation of the use of B3LYP zero-point energies and geometries in the calculation of enthalpies of formation

The use of B3LYP/6–31G^* zero-point energies and geometries in the calculation of enthalpies of formation has been investigated for the enlarged G2 test set of 148 molecules [J. Chem. Phys. 106 (1997) 1063]. A scale factor of 0.96 for the B3LYP zero-point energies gives an average absolute deviation nearly the same as scaled HF/6–31G^* zero-point energies for G2, G2(MP2), and B3LYP/6–311 + G(3df,2p) enthalpies. A scale factor of 0.98, which has been recommended in some studies, increases the average absolute deviation by about 0.2 kcal/mol. Geometries from B3LYP/6–31G^* are found to do as well as MP2/6–31G^* geometries in the calculation of the enthalpies of formation.     

Liquid chromatography of polymyxin B sulphate

A reversed-phase liquid chromatography method for analysis of polymyxin B sulphate is described. The method uses a YMC-Pack Pro, C₁₈, 5 μm, 250×4.6 mm I.D. column maintained at 30°C. The mobile phase comprises acetonitrile–sodium sulphate (0.7%, m/v)–phosphoric acid (6.8%, v/v dilution of 85%, m/m phosphoric acid)–water (22.25:50:5:22.75) at a flow-rate of 1.0 ml/min. Detection was by UV at 215 nm. The method is able to resolve polymyxin B₁, the major component, from more than thirty other components present in the complex. Robustness was evaluated by performing a full-factorial design experiment. The method showed good selectivity, repeatability, linearity and sensitivity.     

DFT studies on tautomerism of C5-substituted 1,2,4-triazoles

DFT (B3LYP/6-311++G**, B3PW91/6-311++G**) Gibbs free energy and single point CCSD(T)/6-311++G**//DFT total energy calculations were performed to investigate stability and tautomerism of C5-substituted 1,2,4-triazoles. Three different tautomers are possible for the substituted 1,2,4-triazoles: N1–H, N2–H, and N4–H. Unlike for the 1,2,3-triazoles, where the most stable is the N2–H tautomer regardless of substituent applied, for the 1,2,4-triazoles, the electron donating substituents (–OH, –F, –CN, –NH₂, and –Cl) and the C5-cation stablize the N2–H tautomer, whereas the electron withdrawing substituents (–CONH₂, –COOH, –CHO, –BH₂, and –CFO) and the C5-anion stablize the N1–H tautomer. Except for the C5-anion and C5-cation, the N4–H form is the least stable tautomer. The relative stability of the C5-substituted 1,2,4-triazole tautomers is strongly influenced by attractive and/or repulsive intramolecular interactions between substituent and electron donor or electron acceptor centres of the triazole ring.     

Vapour-liquid equilibrium of the acetone---methanol system in the presence of KSCN and comparison with other salts

The effect of KSCN at salt mole fractions of 0.01–0.05 and at saturation on the VLE of the binary acetone-methanol system has been experimentally investigated at 101.32 kPa using a modified Othmer equilibrium still. The experimental data were correlated by the electrolytic NRTL model [B. Mock, L.B. Evans and C.C. Chen, AIChE J., 32 (1986) 1655–1664], the extended UNIQUAC equation [B. Sander, A. Fredensland and P. Rasmussen, Chem. Eng. Sci., 41 (1986) 1171–1183] and the modified Wilson and NRTL models proposed by Tan [T.C. Tan, AIChE J., 31 (1985) 2083–2085; Chem. Eng. Res. Des., 65 (1987) 355–366; Trans. Inst. Chem. Eng., 68 (1990) 93–102]. The results were compared with those obtained by Tan's predictive models. A crossover between the salting-out and salting-in effects on acetone and a non-azeotropic minimum in the temperature-composition diagram were observed at low and high salt concentrations. Based on the solubility data in the mixture, comparisons with the effect of other salts on the same mixed solvent were made.     

Comparison of calculated DFT/B3LYP and experimental C and O NMR chemical shifts, ab initio HF/6-31G* optimised structures, and single crystal X-ray structures of some substituted methyl 5β-cholan-24-oates

Single crystal X-ray structures (monoclinic space group P2₁) for methyl 3-oxo-5β-cholan-24-oate and methyl 3,12-dioxo-5β-cholan-24-oate have been solved and compared with HF/6-31G* optimised structures. In the crystalline packings the side chains are connected with weak OC(sp³)HO-type of interactions between C25–H and C24–O–C25 and the keto ends with weak C(sp³)HO=C-type of interactions between C4–H and O=C3. The orientations of the side chains, which steric configurations are of great importance to the biological activity of the molecules, are compared with the experimental structure of methyl 3-hydroxy-5β-cholan-24-oate. Probable reasons for the observed differences are discussed. In addition, ¹³C and ¹⁷O NMR chemical shifts of methyl 3-oxo-5β-cholan-24-oate and methyl 3,12-dioxo-5β-cholan-24-oate as well as the epimeric methyl 3-hydroxy-5β-cholan-24-oate and methyl 3β-hydroxy-5β-cholan-24-oate have been calculated (DFT/B3LYP/6-311G*) and compared with the experimental values by linear regression analyses. In general, the correspondence between the theoretical and experimental parameters is good or excellent.     

DFT B3-LYP/3-21G geometry optimisation and effective charge values calculations for azodiazaphenanthrenes and acylaminodiazaphenanthrenes

For four azodiazaphenanthrenes 1–4 and three acylaminodiazaphenanthrenes 5–7 the geometry was optimised and their effective charge and dipole moment values were calculated using DFT B3-LYP/3-21G method. For 5–7 the results have been compared with those obtained by AM1 method. The UV experimental values of 1–4 are presented. With the use of DFT B3-LYP/6-31G^** optimised geometry the simulation of UV spectra of 5–7 by AM1 and ZINDO/S methods was made and correlations with experimental UV values have been performed.     

Comparative study of the separation of methanol–methyl acetate mixtures by pervaporation and vapor permeation using a commercial membrane

In the present study, the permeation behavior of methanol and methyl acetate in the pervaporation (PV) experiments are compared with those in vapor permeation (VP) experiments using a PVA-based composite membrane. Experiments have been carried out to study the selectivity and mass transport flux of the systems under varying operations conditions of feed temperature (40–60 °C) and feed methanol concentrations (2–34 wt%). The selected membrane was found to be methanol selective. Results show higher permeation flux but a similar separation factor for methanol in PV than in VP. For PV operation, the resulting separation factor at 60 °C shows a monotonous decrease (6.4–4.1) as the alcohol concentration in the feed mixture increases (2.3–34 wt%), whereas the total flux increases from 0.97 to 7.9 kg m⁻² h⁻¹. Based on the solution-diffusion theory, a mathematical model that describes satisfactorily the permeation fluxes of methanol and methyl acetate in both the PV and VP processes has been applied. The fluxes of both permeants can be explained by the solution-diffusion model with variable diffusion coefficients dependent on MeOH concentration in the membrane. Both PV and VP processes can be described with the same model but using different fitting parameters.     

Spectral studies on the interaction of Amido black 10B with DNA in the presence of cetyltrimethylammonium bromide

The interaction of Amido black 10B (AB) with DNA in basic medium was studied in the presence of cetyltrimethylammonium bromide (CTMAB) based on the measurements of resonance light scattering (RLS), UV–vis, CD spectra, and RLS imaging. The interaction has been proved to give a ternary complex of CTMAB–DNA–AB in Britton–Robinson buffer of pH 11.55, which exhibits strong negative Cotton effect at 233.3 nm and 642.8 nm, and strong RLS signals characterized at 469 nm. Experiments showed that the enhanced RLS intensities (ΔI_RLS) against the mixture of AB and CTMAB are proportional to the concentration of fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA), respectively over the range of 0.03–1.0 and 0.05–1.5 μg ml⁻¹, with the limits of determination (3σ) of 7.3 ng ml⁻¹ for fsDNA and 7.0 ng ml⁻¹ for ctDNA.     

Total syntheses of makaluvamines A, B, C, D and E, cytotoxic pyrroloiminoquinone alkaloids isolated from marine sponge bearing inhibitory activities against topoisomerase II

Syntheses of makaluvamines A, B, C, D and E (1 – 5), new members of tetrahydropyrroloiminoquinone alkaloids, have been successfully carried out. Particularly, olefin introduction for makaluvamines B and E could be achieved by Pd - mediated and E₂ type methodologies.     

Tri‐n‐butyltin binding to a low‐affinity site decreases the F1FO‐ATPase sensitivity to oligomycin in mussel mitochondria

In mussel digestive gland mitochondria the environmental pollutant tri‐n‐butyltin (TBT), other than strongly inhibiting ATPase activity at <1.0 μ m , at ≥1.0 μ m concentration was previously found to desensitize F₁F_O‐ATPase to the antibiotic oligomycin. While F₁F_O‐ATPase inhibition is widely known as one of the main mitochondrial damages caused by TBT, the enzyme's desensitization to oligomycin was quite unexpected. The possible mechanisms involved are here stepwise approached, aiming at enlightening the molecular mechanism(s) of TBT toxicity and the still poorly investigated oligomycin interaction with F_O. The findings strongly suggest that the oligomycin desensitization directly stems from the covalent binding of TBT to monothiols of the F₁F_O‐ATPase. This binding implies sulfur oxidation, irrespective of the possible formation of radical species in mitochondria, a mechanism which does not seem to be involved here. It is hypothesized that TBT interacts with the enzyme complex in at least two sites distinguished by different affinities: TBT binding to the high‐affinity site would lead to ATPase inhibition, while TBT binding to monothiols in the low‐affinity site could mirror the decrease in F₁F_O‐ATPase oligomycin sensitivity at ≥1.0 μ m TBT. Experiments carried out on inside‐out submitochondrial particles hint that TBT binding destabilizes the oligomycin‐blocked F_O conformation, allowing proton flux recovery within F_O, without uncoupling the catalytic function from proton channeling. Copyright © 2012 John Wiley & Sons, Ltd.     

Towards the understanding of molecular mechanisms in the early stages of heat-induced aggregation of beta-lactoglobulin AB

Heat-induced aggregation of bovine β-lactoglobulin AB (10 mg/ml) was studied at 68.5 °C at two different pH values (6.7, 4.9) using gel electrophoresis techniques and matrix-assisted laser desorption ionization mass spectrometry (MALDI–TOF MS). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) analysis under non-reducing and reducing conditions showed that in the early stages of the aggregation of β-lactoglobulin disulfide linked aggregates were formed on heating at pH 6.7, but not at pH 4.9. We related this result to the pH-dependent activity of the free thiol group at C121. Mass spectrometric analyses were conducted in two steps. The first involved the analysis of intact non-native monomers and dimers following their ultrasonic passive elution into a suitable solvent mixture in order to confirm the identity of the different gel bands. The second step comprises the analysis of in-gel digests for the determination of disulfide patterns in non-native monomers, covalent dimers and trimers. The results of in-gel digestions analyzed by mass spectrometry suggest that non-native dimers could result from the formation of inter-molecular disulfide bonds C121–C66, C160–C160, or C121–C160. Moreover, two inter-molecular bonds C121–C66 and C160–C160 between two and the same monomer units have been detected, which may play an important role in limiting the process of covalent β-lactoglobulin network formation. The combination of SDS–PAGE and MALDI–TOF MS enables us to understand the mechanism of β-lactoglobulin aggregation at the macromolecular level.     

Separation of β-lactoglobulin A, B and C variants of bovine whey using capillary zone electrophoresis

β-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the β-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pK_a range (pH 5.3–7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified β-lactoglobulin A and B proteins and a β-lactoglobulin AC whey. The method was then used to phenotype β-lactoglobulin in a sample population of New Zealand Jersey cows.     

Total synthesis and stereochemistry of pinnatoxins B and C

[Structure: see text] Pinnatoxins B and C were synthesized from diols (34R)-3b and (34S)-3a, respectively, in a stereochemically controlled manner. Through extensive analysis of the 1H NMR spectra of synthetic PnTXs B and C, the diagnostic NMR signals were first identified to differentiate (34S)- and (34R)-diastereomers and then used to establish the C34 configuration of natural PnTXs B and C as 34S and 34R, respectively.     

Competition between π–π stacking and hydrogen bonding in (1:2) picrates of 1,5-diamino-3-oxapentane, 1,8-diamino-3,6-dioxaoctane and 1,5-diamino-3-azapentane—solid state studies

Crystallographic studies of (2:1) salts of picric acid with 1,5-diamino-3-oxapentane (1OPICR), 1,8-diamino-3,6-dioxaoctane (2OPICR) and 1,5-diamino-3-azapentane (1NPICR) showed significant conformational change of the picrate ion due to numerous electrostatic, H-bonding and π–π stacking interactions present in the crystal lattice. In particular, intermolecular N–HO H-bonds were found to cause significant twisting of the o-NO₂ groups from the plane of the benzene ring, whereas overlapping of the picrate ions due to electrostatic interactions and π–π stacking caused flattening of the molecule. Analysis of the geometry of 74 picrate ions found in the Cambridge Crystallographic Database, in their various crystallochemical environments, showed that competition between essentially weak but numerous intermolecular interactions of different types led to systematic changes in geometric parameters within the picrate ion. In particular, relations found between the C1–C2–N–O (C1–C6–N–O) torsion angle and the endocyclic C1–C2–C3 (C1–C6–C5) valence angle can be explained on the basis of competition between resonance effects of the o-NO₂ group and π–π stacking.     

Molecular and crystal structure of the sesquiterpene lactone ketopelenolide B

The spatial structure of the sesquiterpene lactone ketopelenolide B has been established.Institute of Phytochemistry, Ministry of Sciences and High Education, Republic of Kazakhstan, Karaganda, Ul. Erzhanova, a/ya 19, fax 433773, arglabin@phyto.karaganda.su. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 481–483, July–August, 1999.     

Rare gas–iodine complexes in the ion-pair states

Luminescence and luminescence excitation spectra in the vicinity of the optical–optical double resonance transitions to the I₂(, v_f = 8 and 9, J_f ≈ 55) levels have been measured at the bulb conditions for the I₂ + Rg mixtures (Rg = He, Ar, Xe) at the rare gas pressures 2–20 Torr and room temperature. Luminescence attributed to the RgI₂ complexes in the ion-pair states has been observed for the first time. It is argued that the complexes can be formed by direct optical excitation from the complexes or colliding pairs. Besides, the RgI₂ complexes in the ion-pair states can be formed in nonadiabatic internal conversion processes from the one. The complexes have rather long lifetime, especially in the case of Xe, and decay radiatively and nonradiatively forming I₂ molecules in different ion-pair states.     

Pyrolysis and film growth studies of phosphinoborane compounds

Chemical vapor deposition experiments were conducted using phosphinoborane compounds of the type [R₂BPR′₂]_n, where R = CH₂CH₃ (1) or CH₃ (2), R′= C(CH₃)₃, and R = CH₂CH₃ (3) or CH₃ (4), R′ = Si(CH₃)₃. Thin films were deposited on Si substrates at 300–850°C under vacuum using 1–3, while no film formed using 4. All films contained considerable carbon (C/B = 0.67–7) and were deficient in phosphorus (P/B = 0-0.5) according to Auger electron spectroscopy. For 3, silicon was incorporated in the films (Si/B = 0.9–2). The preferential loss of phosphorus was nearly complete at higher pyrolysis temperatures that favor formation of carbon-rich films. Volatile products of the decompositions were observed by in situ mass spectroscopy and by ¹H nuclear magnetic resonance analysis of the condensate in a liquid nitrogen cooled trap. The products indicate that β-hydrogen elimination of alkene from boron is a preferred reaction pathway, while concerted elimination reactions appear to be inefficient. Although these phosphinoborane compounds do not appear to be suitable precursors for pure boron phosphide, the B-P-C films obtained are chemically inert and may be of interest as protective coatings.