Unusual CD couplet pattern observed for the pi*<--n transition of enantiopure (Z)-8-methoxy-4-cyclooctenone: an experimental and theoretical study by electronic and vibrational circular dichroism spectroscopy and density functional theory calculation

Ultraviolet absorption (UV) and electronic circular dichroism (ECD) spectra of enantiopure (Z)-8-methoxy-4-cyclooctenone (MCO) were measured in hexane to give a normal single UV absorption band at 298 nm, which is assigned to the carbonyl's pi*<--n transition. Unexpectedly, the ECD spectrum exhibited an apparent couplet pattern with vibrational fine structures. Obviously, the conventional CD exciton coupling mechanism cannot be applied to this bisignate CD signal observed for single-chromophoric MCO. Variable temperature-ECD and vibrational circular dichroism (VCD) spectral measurements, simultaneous UV and ECD spectral band resolution, and density functional theory (DFT) calculations of energy and structure revealed that this apparent CD couplet originates from a rather complicated spectral overlap of more than three conformers of MCO, two of which exhibit mirror-imaged ECD spectra at appreciably deviated wavelengths. In the simultaneous band-resolution analysis, the observed UV and ECD spectra were best fitted to four overlapping bands. Two major conformers were identified by comparing the experimental IR and VCD spectra with the simulated ones, and the other two by comparing the observed UV and ECD spectra with the theoretical ones obtained by time-dependent DFT calculations. It was shown that the combined use of experimental ECD and VCD spectra and theoretical DFT calculations can give a reasonable interpretation for the Cotton effects of the conformationally flexible molecule MCO.     

Eight new biflavonoids with lavandulyl units from the roots of Sophora flavescens and their inhibitory effect on PTP1B

In the course of studying the components from the roots of Sophora flavescens, eight new unusual biflavonoids consisting of a flavanone fused with a dihydrochalcone skeleton were isolated. These new chemical structures were elucidated by means of UV, IR, HRESIMS, NMR and ECD spectroscopic data and a comparison of experimental ECD spectra with calculated ECD spectra. Some compounds were subjected to an antidiabetic bioassay on human recombinant PTP1B inhibition, and showed strong inhibitory activity.     

Synthesis,Structural Characterization,and Chiroptical Studies of Bidentate Salen‐Type Lanthanide (III) Complexes

The salen‐type ligand prepared with (R,R) diphenylethan‐1,2‐diamine and salicylaldehyde provides stable and inert complexes KLnL₂ upon simple reaction with lanthanide halides or pseudohalides LnX₃ (Ln = Tb³⁺‐Lu³⁺; X = Cl^? or TfO^?) of its potassium salt. All the complexes were completely characterized through nuclear magnetic resonance (NMR), electronic circular dichroism (ECD) in the UV and some (Er³⁺, Tm³⁺, Yb³⁺) also with Near‐IR ECD (NIR‐ECD) and luminescence (Tb³⁺, Tm³⁺). Careful analysis of the NMR shifts demonstrated that the complexes are isostructural in solution and afforded an accurate geometry. This was further confirmed by means of Density Functional Theory (DFT) optimization of the Lu³⁺ complex, and by comparing the ligand‐centered experimental and time‐dependent TD‐DFT computed UV‐ECD spectra. As final validation, we used the NIR‐ECD spectrum of the Yb³⁺ derivative calculated by means of Richardson's equations. The excellent match between calculated and experimental ECD spectra confirm the quality of the NMR structure.  Chirality 27:857–863, 2015. © 2015 Wiley Periodicals, Inc.     

Spectroscopic Studies on Nicotine and Nornicotine in the UV Region

The UV absorption and electronic circular dichroism (ECD) spectra of (R)‐ and (S)‐nicotine and (S)‐nornicotine in aqueous solution were measured to a significantly lower wavelength range than previously reported, allowing the identification of four previously unobserved electronic transitions. The ECD spectra of the two enantiomers of nicotine were equal in magnitude and opposite in sign, while the UV absorption spectra were coincidental. In line with previous observations, (S)‐nicotine exhibited a negative cotton effect centered on 263 nm with vibronic structure (π–π₁* transition) and a broad, positive ECD signal at around 240 nm associated with the n–π₁* transition. As expected this band disappeared when the pyridyl aromatic moiety was protonated. Four further electronic transitions are reported between 215 and 180 nm; it is proposed the negative maxima around 206 nm is either an n–σ* transition or a charge transfer band resulting from the movement of charge from the pyrrolidyl N lone pair to the pyridyl π* orbital. The pyridyl π–π₂* transition may be contained within the negative ECD signal envelope at around 200 nm. Another negative maximum at 188 nm is thought to be the pyridyl π–π₃* transition, while the lowest wavelength end‐absorption and positive ECD may be associated with the π–π₄* transition. The UV absorption spectra of (S)‐nornicotine was similar to that of (S)‐nicotine in the range 280–220 nm and acidification of the aqueous solution enhanced the absorption. The ECD signals of (S)‐nornicotine were considerably less intense compared to (S)‐nicotine and declined further on acidification; in the far UV region the ECD spectra diverge considerably. Chirality 25:288–293, 2013. © 2013 Wiley Periodicals, Inc.     

Antifungal γ-Pyrone and Isoprenylated Cyclohexanoids from the Fungus Beenakia informis

Chemical investigation of the extract of the macroscopic fungus Beenakia informis led to the isolation of a previously unreported γ-pyrone and two new isoprenylated cyclohexanoids, together with speciocin N. Their structures were elucidated spectroscopically and the absolute configuration was determined by comparison of the experimental vs. calculated ECD curves. Three of the compounds showed very good to moderate activity against phytopathogenic fungi.     

Photopic spectral sensitivity of a teleost fish,the roach (Rutilus rutilus), with special reference to its ultraviolet sensitivity

Summary This study reports photopic spectral sensitivity curves (351–709 nm) for four individual roach,Rutilus rutilus, determined by two choice appetitive training. All four curves show four sensitivity maxima at 361–398 nm, 421–448 nm, 501–544 nm and 634–666 nm which are related to the four known roach photopic visual pigments (Avery et al. 1982). The overall shape of the curves at long wavelengths indicates inhibitory interactions between the red and green cone mechanisms. That the high behavioural sensitivity in the UV is caused by a specific ultraviolet visual pigment and is not due to aberrant stimulation of the other cone types is shown by the redetermination of spectral sensitivity at short wavelengths (351–501 nm) following the selective bleaching of the three longer wavelength visual pigments. This depresses the blue sensitivity to a greater degree than the relatively unaffected UV sensitivity maximum. Spectral transmission data from two corneas and four lenses show that they transmit considerable amounts of light in the near UV.     

Thermodynamics of unfolding of beta-trypsin at pH 2.8

The unfolding equilibrium of beta-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the van't Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of beta-trypsin estimated from spectroscopic data (deltaGu = 6.0 +/- 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (deltaGUH2O = 5.5 +/- 0.3 kcal/mol) at pH 2.8 and 300 degrees K.     

Sensitive electrochemical detection method for alpha-acids, beta-acids and xanthohumol in hops (Humulus lupulus L.)

A new HPLC method with coulometric detection for the quantification of xanthohumol, alpha-acids and beta-acids in hops was developed. The separation of compounds was accomplished with a C18 column and isocratic elution with methanol: 50 mM potassium phosphate: ortho-phosphoric acid=80:20:0.25 (v/v/v). The method was validated and UV and electrochemical detectors (ECD) were compared. The HPLC method with ECD was precise, accurate and very sensitive for detection of xanthohumol and alpha- and beta-acids. The detection limits of analytes were at least 8.8 to 24 times lower with ECD than those of the UV detector. The ECD method was successfully applied for quantification of studied compounds in hop pellets. The concentrations of all compounds obtained with ECD and UV were found to be equivalent. This is the first study demonstrating a very sensitive and validated method for the quantification of xanthohumol, alpha- or beta-acids in hop samples with the use of the electrochemical detector.     

Action spectrum for polarotropism of the germ tube of the liverwort Sphaerocarpos Donnellii aust.

Summary A series of dose response curves was worked out in the polarotropically active blue and UV spectral region. These dose response curves show changes in slope as a function of wavelength. In blue the slope values are higher than in UV. As a consequence, both the relative height of the peaks in blue and UV and the fine characteristics in blue of the action spectrum calculated on the basis of these dose response curves change decisively with different response levels taken for calculation. Therefore no decision can be made as to what photoreceptor(s) might be involved. Though at medium response levels the action spectra show similarity with action spectra of other blue-UV-mediated photoresponses, which generally are believed as being indicative of some flavin.     

NMR structural characterization of a minimal peptide antagonist bound to the extracellular domain of the corticotropin-releasing factor1 receptor

Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.     

Improving protein identification using complementary fragmentation techniques in fourier transform mass spectrometry

Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.     

Serum levels of the extracellular domain of the epidermal growth factor receptor in individuals exposed to arsenic in drinking water in Bangladesh

Epidermal growth factor receptor-dependent mechanisms have been implicated in growth signal transduction pathways that contribute to cancer development, including dermal carcinogenesis. Detection of the extracellular domain of the epidermal growth factor receptor (EGFR ECD) in serum has been suggested as a potential biomarker for monitoring this effect in vivo. Arsenic is a known human carcinogen, producing skin and other malignancies in populations exposed through their drinking water. One such exposed population, which we have been studying for a number of years, is in Bangladesh. The purpose of this study was to examine the EGFR ECD as a potential biomarker of arsenic exposure and/or effect in this population. Levels of the EGFR ECD were determined by enzyme-linked immunosorbent assay in the serum samples from 574 individuals with a range of arsenic exposures from drinking water in the Araihazar area of Bangladesh. In multiple regression analysis, serum EGFR ECD was found to be positively associated with three different measures of arsenic exposure (well water arsenic, urinary arsenic and a cumulative arsenic index) at statistically significant levels (p≤0.034), and this association was strongest among the individuals with arsenic-induced skin lesions (p ≤ 0.002). When the study subjects were stratified in tertiles of serum EGFR ECD levels, the risk of skin lesions increased progressively for each increase in all three arsenic measures (also stratified in tertiles) and this increasing risk became more pronounced among subjects within the highest tertile of EGFR ECD levels. These results suggest that serum EGFR ECD levels may be a potential biomarker of effect of arsenic exposure and may indicate those exposed individuals at greatest risk for the development of arsenic-induced skin lesions.     

Molecular Morphology of Eukaryotic Class-1 Translation Termination Factor eRF1 in Solution

The integral structural parameters and the shape of the molecule of human translation termination factor eRF1 were determined from the small-angle X-ray scattering in solution. The molecular shapes were found by bead modeling with nonlinear minimization of the root-mean-square deviation of the calculated from the experimental scattering curve. Comparisons of the small-angle scattering curves computed for atomic-resolution structures of eRF1 with the experimental data on scattering from solution testified that the crystal and the solution conformations are close. In the ribosome, the distance between the eRF1 motifs GGQ and NIKS must be shorter than in crystal or solution (75 versus 100–107 Å). Therefore, like its bacterial counterpart RF2, the eukaryotic eRF1 must change its conformation as it binds to the ribosome. The conformational mobility of eukaryotic and prokaryotic class-1 release factors is another feature making them functionally akin to tRNA.     

Serum levels of the extracellular domain of the epidermal growth factor receptor in individuals exposed to arsenic in drinking water in Bangladesh

Epidermal growth factor receptor-dependent mechanisms have been implicated in growth signal transduction pathways that contribute to cancer development, including dermal carcinogenesis. Detection of the extracellular domain of the epidermal growth factor receptor (EGFR ECD) in serum has been suggested as a potential biomarker for monitoring this effect in vivo. Arsenic is a known human carcinogen, producing skin and other malignancies in populations exposed through their drinking water. One such exposed population, which we have been studying for a number of years, is in Bangladesh. The purpose of this study was to examine the EGFR ECD as a potential biomarker of arsenic exposure and/or effect in this population. Levels of the EGFR ECD were determined by enzyme-linked immunosorbent assay in the serum samples from 574 individuals with a range of arsenic exposures from drinking water in the Araihazar area of Bangladesh. In multiple regression analysis, serum EGFR ECD was found to be positively associated with three different measures of arsenic exposure (well water arsenic, urinary arsenic and a cumulative arsenic index) at statistically significant levels (p≤0.034), and this association was strongest among the individuals with arsenic-induced skin lesions (p ≤ 0.002). When the study subjects were stratified in tertiles of serum EGFR ECD levels, the risk of skin lesions increased progressively for each increase in all three arsenic measures (also stratified in tertiles) and this increasing risk became more pronounced among subjects within the highest tertile of EGFR ECD levels. These results suggest that serum EGFR ECD levels may be a potential biomarker of effect of arsenic exposure and may indicate those exposed individuals at greatest risk for the development of arsenic-induced skin lesions.     

Kramers-Kronig transformation of experimental electronic circular dichroism: application to the analysis of optical rotatory dispersion in dimethyl-L-tartrate

When a limited region of the experimental electronic circular dichroism (ECD) spectrum is subjected to Kramers-Kronig (KK) transformation, the resulting optical rotatory dispersion (ORD) may or may not reproduce the experimentally measured ORD in the long-wavelength nonresonant region. If the KK transform of experimentally measured ECD in a limited wavelength region reproduces the experimentally measured ORD in the long-wavelength nonresonant region, then that observation indicates that the ORD in the long-wavelength nonresonant region should be satisfactorily predicted from the correspondingly limited number of electronic transitions in a reliable quantum mechanical calculation. On the other hand, if the KK transform of experimentally measured ECD in a limited region does not reproduce the experimentally measured ORD in the long-wavelength nonresonant region, then it should be possible to identify the ECD bands in the shorter wavelength region that are responsible for the differences between experimentally observed ORD and KK-transformed ECD. This approach helps to identify the role of ECD associated with higher energy-excited states in the nature of ORD in the long-wavelength nonresonant region. These concepts are demonstrated here by measuring the experimental ECD and ORD for dimethyl-L-tartrate in different solvents. While ECD spectra of dimethyl-L-tartrate in different solvents show little variation, ORD spectra in the long-wavelength nonresonant region show marked solvent dependence. These observations are explained using the difference between experimental ORD and KK-transformed ECD. Quantum mechanical predictions of ECD and ORD are also presented for isolated (R, R)-dimethyl tartrate at the B3LYP/aug-cc-pVDZ level.     

X-ray inactivation, Weigle reactivation, and Weigle mutagenesis of the lysogenic Vibrio kappa phage

Vibrio cholerae lysogenic kappa phage was inactivated by X-ray (60 kV) in a dose-dependent manner, the inactivation dose leading to 37% survival (D37) in phosphate-buffered saline (PBS), pH 7.4, being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. Maximum protection was offered when both histidine (10.0 mM) and cysteine (10.0 mM) were present in PBS (dose enhancement factor being 4.17). The X-irradiated kappa phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Smr. The Weigle factor or the frequency of clear-plaque mutants increased with increasing UV dose, attained a maximum at a UV dose of 2.4 J m-2, and thereafter decreased gradually with a further increase of the UV dose. The X-ray dose (D)--survival (S) curves could be empirically described by the equation S = exp[-(aD + bD2)], where a and b are constants depending on the irradiation conditions, and a good agreement between the theoretical curves and experimental data was obtained.     

Central-to-axial chirality transfer revealed by liquid crystals: a combined experimental and computational approach for the determination of absolute configuration of carboxylic acids with an α chirality centre

The conversion into 6,7-dihydro-5H-dibenz[c,e]azepine (DAZ) N-protected amides is a viable route for the determination of the absolute configuration of chiral 2-substituted carboxylic acids. The biphenyl moiety of DAZ, besides being a probe of chirality for the electronic circular dichroism (ECD) spectroscopy, makes these systems suitable for configuration assignment by exploiting the chirality amplification which occurs in nematic liquid crystals. To assess the reliability of the liquid crystal method in detecting the absolute stereochemistry of chiral amides bound to a biphenyl group, we measured the helical twisting power of a series of DAZ-N-protected amides and compared these data with the results obtained from ECD measurements. We will show that the liquid crystal method, corroborated by HTP predictions, is trustworthy with our biphenyl derivatives, even when ECD spectra are ambiguous for the presence of aryl moieties displaying strong UV absorptions in the same range of the biphenyl chromophore.     

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone‐binding domain: Possible allosteric regulation and a conserved structural motif for the chloride‐binding site

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP‐binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full‐length ANPR expressed in CHO cells. ECD without chloride (ECD(?)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X‐ray structure of the bromide‐bound ECD is essentially identical to that of the chloride‐bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP‐bound structures, indicating exchangeable and reversible halide binding. Far‐UV CD and thermal unfolding data show that ECD(?) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(?) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride‐binding site in ANPR are highly conserved among receptor‐guanylate cyclases and metabotropic glutamate receptors. The chloride‐dependent ANP binding, reversible chloride binding, and the highly conserved chloride‐binding site motif suggest a regulatory role for the receptor bound chloride. Chloride‐dependent regulation of ANPR may operate in the kidney, modulating ANP‐induced natriuresis.     

Effect of Ecdysterone on Morphological and Physiological Processes in Plants

In order to elucidate the physiological role of phytoecdysteroids in plants, we investigated the effects of exogenous ecdysterone (ECD) and phytohormones (IAA, GA₃, and 24-epibrassinolide (EBL)) on the growth of wheat coleoptiles and Arabidopsis thaliana seedlings (wild-type ecotype Columbia (Col) and its det2 mutant), on -amylase activity in the barley aleurone layer, and on the pigment content in the kidney bean senescent leaves. The range of effective ECD concentrations depended on the type of a reaction to be regulated. The regulation of growth processes was affected by a wide range of ECD concentrations (10^–13–10^–5 M), whereas some metabolic processes, such as the activation of -amylase and the retardation of leaf yellowing, by a narrow range, that is, 10^–9–10^–7 M and 10^–9–10^–8 M, respectively. We noted the synergetic effect of ECD and IAA on coleoptile elongation, the antagonistic effect of ECD and EBL on coleoptile elongation, as well as the antagonistic action of ECD and GA₃ on coleoptile elongation and -amylase activity. The data obtained demonstrate that ECD is a physiologically active compound. ECD might be supposed to act as a source of sterols or a regulator of IAA and protein synthesis. The effects of this regulator seems to be brought about by its interaction with the EBL and GA₃ receptors.     

Configurational assignments of conformationally restricted bis-monoterpene hydroquinones: Utility in exploration of endangered plants

Background

Endangered plant species are an important resource for new chemistry. Lindera melissifolia is native to the Southeastern U.S. and scarcely populates the edges of lakes and ponds. Quantum mechanics (QM) used in combination with NMR/ECD is a powerful tool for the assignment of absolute configuration in lieu of X-ray crystallography.

Methods

The EtOAc extract of L. melissifolia was subject to chromatographic analysis by VLC and HPLC. Spin–spin coupling constant (SSCC) were calculated using DFT at the MPW1PW91/6-31G(d,p) level for all staggered rotamers. ECD calculations employed Amber* force fields followed by PM6 semi-empirical optimizations. Hetero- and homo-nuclear coupling constants were extracted from 1D ¹H, E.COSY and HETLOC experiments.

Results

Two meroterpenoids, melissifolianes A (1) and B (2) were purified and their 2-D structures elucidated using NMR and HRESIMS. The relative configuration of 1 was established using the combination of NOE-based distance restraints and the comparisons of experimental and calculated SSCCs. The comparison of calculated and experimental ECD assigned the absolute configuration of 1. The relative configuration of a racemic mixture, melissifoliane B (2) was established utilizing J-based analysis combined with QM and NMR techniques.Conclusion Our study of the Lindera melissifolia metabolome exemplifies how new chemistry remains undiscovered among the numerous endangered plant species and demonstrates how analysis by ECD and NMR combined with various QM calculations is a sensible approach to support the stereochemical assignment of molecules with conformationally restricted conformations.

General significance

QM–NMR/ECD combined approaches are of utility for unambiguous assignment of 3-D structures, especially with limited plant material and when a molecule is conformationally restricted. Conservation of an endangered plant species can be supported through identification of its new chemistry and utilization of that chemistry for commercial purposes.