Improved pulse sequences for pure exchange solid-state NMR spectroscopy

Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly (15)N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated (15)N-(15)N spin-exchange experiments for a (15)N-labeled N-acetyl-L-valyl-L-leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly (15)N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin.     

Vibrational spectra of benzene chromium tricarbonyl and its mesityl analogue: a study by neutron spectroscopy

We have used high resolution neutron vibrational spectroscopy to study the vibrations of the model compound for arene-to-metal bonding, benzene chromium tricarbonyl, and its mesityl analogue. It is shown that all previously published assignment schemes, based on optical spectroscopy alone, are wrong, and new assignments are given in agreement with ab initio calculations.     

Limonene: electronic state spectroscopy by high-resolution vacuum ultraviolet photoabsorption, electron scattering, He(I) photoelectron spectroscopy and ab initio calculations

Electronic state spectroscopy of limonene has been investigated using vacuum ultraviolet photoabsorption spectroscopy in the energy range 5.0-10.8 eV. The availability of a high resolution photon beam (~0.075 nm) enabled detailed analysis of the vibrational progressions and allowed us to propose, for the first time, new assignments for several Rydberg series. Excited states located in the 7.5-8.4 eV region have been studied for the first time. A He(I) photoelectron spectrum has also been recorded from 8.2 to 9.5 eV and compared to previous low resolution works. A new value of 8.521 ± 0.002 eV for the ground ionic state adiabatic ionisation energy is proposed. Absolute photoabsorption cross sections were derived in the 10-26 eV range from electron scattering data. All spectra presented in this paper represent the highest resolution data yet reported for limonene. These experiments are complemented by new ab initio calculations performed for the three most abundant conformational isomers of limonene, which we then used in the assignment of the spectral bands.     

Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids

A novel solid-state NMR concept is introduced that allows for the detection of through-space proton-proton contacts in high spectral resolution. The proposed method not only can be utilized to infer sequential assignments and backbone or side chain conformation in solid-phase polypeptides, but it also provides information about the three-dimensional arrangement of the molecule of interest. As a result, the molecular structure can be studied without additional restrictions regarding sample labeling or magic angle spinning rates.     

High resolution 13C-NMR spectroscopy of sodium carboxy methyl cellulose

Results of NMR studies on intact sodium carboxy methyl cellulose (SCMC) are presented. Similar studies in the literature are all on partially depolymerized SCMC. The degree of substitution and the relative distribution of substituents at OH-2, OH-3, and OH-6 of anhydro D -glucose residue in intact SCMC were determined by high resolution ¹³C-NMR spectroscopy (125 MHz). It is observed that the degree of substitution at OH-6 is almost equal to that at OH-3. In two SCMC samples, which are widely different in molecular weight and degree of substitution (ds), the relative reactivity order of the hydroxyl groups was found to be OH-2 > OH-6 ? OH-3. The NMR assignments were based on calculated shifts of carbons of anhydroglucose moiety in an oligosaccharide due to substitution.     

Dynamic two-dimensional fluorescence correlation spectroscopy. Generalized correlation and experimental factors

Dynamic two-dimensional fluorescence correlation spectroscopy (2D FCS) is presented in the general form. Dynamic 2D FCS evaluates the time correlation function between two wavelength axes when an external perturbation is applied to the sample. It displays the vibronic features with similar time response functions in the synchronous correlation spectrum and the features with different time responses in the asynchronous correlation spectrum. The correlation analysis allows detailed assignments of the vibronic spectra of multicomponent samples. The emission-emission 2D FCS has proven to be able to resolve spectra with substantial overlaps, of species in equilibrium with each other, and of reacting species whose kinetic constants are linked and multiexponential. Similarly, the correlation analysis between excitation wavelengths allows the assignment of the excitation bands to fluorescent components. When a sinusoidal light source is used to excite the sample, the excitation-emission correlation requires the collection of only four spectra, two in-phase and two quadrature. The two-dimensional excitation-emission correlation analysis uncovers the association between the excitation and the emission vibronic features, enabling the complete assignment of the component spectra. The band associations and spectral assignments are facilitated by the two-dimensional phase map that is constructed from the synchronous and asynchronous correlation spectra. Spectral resolution can be optimized by varying the frequency of excitation and is not influenced by the detector phase angle used to collect the spectra. The resolution power of the 2D FCS is demonstrated with the retrieval of the anthracene emission spectrum from a pyrene-anthracene mixture when it contributes only 4% to the total fluorescence intensity.     

Conformational analysis by UV spectroscopy: the decisive contribution of environment-induced electronic Stark effects

UV chromophores are frequently used as probes of the molecular structure. In particular, they are sensitive to the electric field generated by the molecular environment, resulting in the observation of Stark effects on UV spectra. While these environment-induced electronic Stark effects (EI-ESE) are already used for conformational analysis in the condensed phase, this work explores the potential of such an approach when performed at much higher conformational resolution in the gas phase. By investigating model alkali benzylacetate and 4-phenylbutyrate ion pairs, where the electric field applied to the phenyl ring is chemically tuned by changing the nature of the alkali cation, this work demonstrates that precise conformational assignments can be proposed based on the correlation between the conformation-dependent calculated electric fields and the frequency of the electronic transitions observed in the experimental UV spectra. Remarkably, the sole analysis of Stark effects and fragmentation patterns in mass-selected UV spectra provided an accurate and complete conformational analysis, where spectral differences as small as a few cm⁻¹ between electronic transitions were rationalized. This case study illustrates that the identification of EI-ESE together with their interpretation at the modest cost of a ground state electric field calculation qualify UV spectroscopy as a powerful tool for conformational analysis.

The correlation between experimental electronic transitions and calculated electric fields leads to precise conformational assignments and opens up the possibility of interpreting electronic spectra in a quantitative manner at the wavenumber scale.     

VUV electronic state spectroscopy of 1,1-difluoroethene and difluorochloromethane by high resolution synchrotron radiation

High resolution VUV photoabsorption spectra of 1,1-difluoroethene (C(2)H(2)F(2)) and difluorochloromethane (CF(2)HCl) are reported in the wavelength range 115-200 nm (10.8-6.2 eV). New photoabsorption features are observed in C(2)H(2)F(2) and some Rydberg and vibrational assignments are proposed for the first time. Evidence for a weak vibrational pattern in CF(2)HCl is discussed here for the first time. Absolute cross section values have been obtained allowing photolysis lifetimes to be derived in the Earth's troposphere and stratosphere.     

Observation of the A-x electronic transition of the isomers and conformers of pentyl peroxy radical using cavity ringdown spectroscopy

Cavity ringdown spectra of the A-X electronic transition of all eight isomers of the pentyl peroxy radical are reported. Using the corresponding assignments from previously studied smaller alkyl peroxy radicals, assignments of origin bands are made for the pentyl peroxy isomers including some conformer-specific assignments for bands of a given isomer. Ab initio calculations also were performed to aid in the spectral assignments for neopentyl, t-butyl, and t-pentyl peroxies. In addition to the origins, vibrational bands have also been assigned for some species. Using the analyzed spectra, the relative reactivity of the primary, secondary, and tertiary hydrogen atoms in isopentane could be determined semiquantitatively.     

Two-dimensional correlation spectroscopy and multivariate curve resolution for the study of lipid oxidation in edible oils monitored by FTIR and FT-Raman spectroscopy

Accelerate oxidative degradation of six vegetable oils was monitored using FTIR and FT-Raman spectroscopy. Two-dimensional correlation spectroscopy and multivariate curve resolution alternating least squares (MCR-ALS) were applied to the analysis of the data. The use of hetero-spectral two-dimensional correlation of FTIR and FT-Raman data allowed the use of well established band assignments to interpret less clearly assigned spectral features. With a moving window approach it was possible to obtain simplified two-dimensional correlation maps and to detect compounds evolving with different kinetic. Simultaneous analysis of the oxidation experiments of the six different oils monitored by both spectroscopic techniques was performed using MCR-ALS. Although a complete resolution of the data was not possible, the spectral changes occurring during the oxidative degradation of the oils were described with a five-component model. The two fundamentally different chemometric approaches lead to coincident results.     

Assignment of 1H signals in complex overlapping spectral regions using proton-detected 2D heteronuclear shift correlation spectroscopy

The application of a heteronuclear 2D pulse sequence is demonstrated which correlates chemical shifts of carbons with directly bound protons and with remote protons belonging to the same coupled spin system by using the effect of Hartmann—Hahn cross polarization among protons. The enhanced spectral resolution obtained by spreading individual proton subsystems into the carbon domain and the option for multiplicity discrimination makes these kind of experiments most suitable for proton and carbon signal assignments of natural products as alkaloids and steroids with strongly overlapping proton resonances. With the application to a steroid we want to demonstrate that such unambiguous signal assignments are the prerequisite and the basis for detailed structural investigations using additional, more conventional NMR experiments.     

Assignments in photoelectron spectroscopy using the CNDO/2 method

In the photoelectron spectra of molecules vibrational fine structure often accompanies the observed ionization potentials (IPs). For non-diatomic molecules it is difficult to make vibrational assignments because the frequencies of the ion often differ greatly from those of the neutral molecule. In order to make both the vibrational assignments and the assignments of the IPs to molecular orbitals (MOs) using Koopmans' theorem CNDO/2 MO calculations have been carried out. Within the framework of this method a quantity E _AB has been calculated which is the change in potential energy between an atom-pair A-B for a particular MO on ionization. Application to some diatomic molecules has shown that this quantity reflects the changes in vibrational frequencies on ionization. The IP-MO assignments are made such that removal of an electron from a particular MO is in accord with the observed vibrational fine-structure; attempting to make these assignments on the basis of the calculated MO ordering alone has been shown to be very unsatisfactory.

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Zusammenfassung Photoelektronenspektren von Molekülen zeigen neben den Ionisierungspotentialen oft noch eine Schwingungsfeinstruktur, deren Zuordnung bei nicht-zweiatomigen Molekülen wegen der starken Frequenzverschiebungen vom Molekül zum Ion Schwierigkeiten macht. Um sowohl Schwingungsais auch Ionisierungs-Zuordnungen vornehmen zu können, wurden Rechnungen mit dem CNDO/2-Verfahren durchgeführt. Dabei stützt man sich auf die Änderung der potentiellen Energie zwischen dem Atompaar AB in bezug auf ein bestimmtes MO bei Ionisierung (E _AB). Die Anwendung auf zweiatomige Moleküle zeigt, daß diese Größe tatsächlich die Änderung der Schwingungsfrequenzen wiedergibt, und die Zuweisungen des IP werden so vorgenommen, daß die Entfernung eines Elektrons aus einem MO im Einklang mit der Schwingungsfeinstruktur ist. Dagegen erweist sich die einfache Zuweisung auf Grund der MO-Reihenfolge als schlechter.

     

Determining the absolute configuration of two marine compounds using vibrational chiroptical spectroscopy

Chiroptical techniques are increasingly employed for assigning the absolute configuration of chiral molecules through comparison of experimental spectra with theoretical predictions. For assignment of natural products, electronic chiroptical spectroscopies such as electronic circular dichroism (ECD) are routinely applied. However, the sensitivity of electronic spectral parameters to experimental conditions and the theoretical methods employed can lead to incorrect assignments. Vibrational chiroptical methods (vibrational circular dichroism, VCD, and Raman optical activity, ROA) provide more reliable assignments, although they, in particular ROA, have been little explored for assignments of natural products. In this study, the ECD, VCD, and ROA chiroptical spectroscopies are evaluated for the assignment of the absolute configuration of a highly flexible natural compound with two stereocenters and an asymmetrically substituted double bond, the marine antibiotic Synoxazolidinone A (SynOxA), recently isolated from the sub-Arctic ascidian Synoicum pulmonaria. Conformationally averaged nuclear magnetic resonance (NMR), ECD, Raman, ROA, infrared (IR) and VCD spectral parameters are computed for the eight possible stereoisomers of SynOxA and compared to experimental results. In contrast to previously reported results, the stereochemical assignment of SynOxA based on ECD spectral bands is found to be unreliable. On the other hand, ROA spectra allow for a reliable determination of the configuration at the double bond and the ring stereocenter. However, ROA is not able to resolve the chlorine-substituted stereogenic center on the guanidinium side chain of SynOxA. Application of the third chiroptical method, VCD, indicates unique spectral features for all eight SynOxA isomers in the theoretical spectra. Although the experimental VCD is weak and restricted by the limited amount of sample, it allows for a tentative assignment of the elusive chlorine-substituted stereocenter. VCD chiroptical analysis of a SynOxA derivative with three stereocenters, SynOxC, results in the same absolute configuration as for SynOxA. Despite the experimental challenges, the results convincingly prove that the assignment of absolute configuration based on vibrational chiroptical methods is more reliable than for ECD.     

Strategies for measurements of pseudocontact shifts in protein NMR spectroscopy.

Paramagnetic metal ions bound to proteins generate a dipolar field that can be accurately probed by pseudocontact shifts (PCS) displayed by the protein's nuclear spins. PCS are highly useful for determining the coordinates of individual spins in the molecule and for rapid structure determinations of entire protein-protein and protein-ligand complexes. However, PCS measurements require reliable resonance assignments for the molecule in its paramagnetic state and in a diamagnetic reference state. This article discusses different approaches for pairwise resonance assignments, with emphasis on a strategy which exploits chemical exchange between the two states.     

Carbon-13 n.m.r. spectroscopy of some Strychnos alkaloids

¹³C N.m.r. spectra have been determined for strychnine and a series of fourteen derivatives. The assignments are based on previously established data and on off-resonance decoupled spectra. The shifts resulting from the alterations in molecular structure are discussed and explained, in part, as a consequence of conformational changes. This detailed study indicates that changes are required in some previously published assignments.     

15N NMR spectroscopy 31—preliminary study on the insulin-a chain (bunte salt form)

The Bunte salt derivative was prepared from bovine insulin-A isolated from natural sources, and was purified by chromatography. Natural abundance 40.55 MHz ¹⁵N NMR spectra were measured in water and dimethyl sulphoxide. The latter solvent gave the best resolution, and 20 of the 22 possible amide nitrogen signals were detected. Several model oligopeptides were also measured and their usefulness for assignments is discussed.     

Carbon-13 NMR spectroscopy of hop bitter substances

The ¹³C NMR spectra of the most important hop bitter substances are analysed. All individual carbon atoms are assigned, except the oxygen bonded sp² carbon atoms. Most assignments are made partly by comparison with analogous compounds and partly by reference to literature data. Cross-correlations with the ¹H NMR shifts are made for two main products and confirm the correctness of the assignments. All data are in agreement with the known structures.     

Subtraction of atmospheric water contribution in Fourier transform infrared spectroscopy of biological membranes and proteins

The contribution of the absorption of H₂O vapor in the IR spectrum of proteins Interferes with the analysis of the shape of amide I and II bands and prevents correct assignments to be performed, in particular after Fourier self-deconvolution of the spectra Mathematical treatments of the spectra have previously been proposed to subtract the water vapor contribution from the sample spectrum. Here we propose to take advantage of the intrinsic bandwidth difference existing between the absorption bands of the water vapor and these of the liquid or solid sample. When a nominal resolution of 8 or 4 cm⁻¹ is chosen, atmospheric water bands are broad and rather featureless. The subtraction coefficient applied may vary by about 50% according to the operator. Conversely, when the spectrum of the same sample in the same conditions is recorded with a nominal resolution of 0.5 cm⁻¹, subtraction coefficients are exactly evaluated by integration and the visual evaluation can not be mistaken by more than 5%. The very sharp features arising from imperfect matching between the atmospheric water band shapes in the reference and in the sample spectrum completely disappear if the difference spectrum is now convoluted to a final resolution of 4 cm⁻¹. Incidence of the subtractions obtained at different resolutions on the evaluation of protein secondary structure is evaluated.     

Intrinsic redox states of polyaniline studied by high-resolution X-ray photoelectron spectroscopy

High-resolution X-ray photoelectron spectroscopic (XPS) measurements of the various intrinsic redox states of polyaniline (PANI), using a monochromatized Al—Kα source, were carried out. The presence of the imine, amine and positively charged nitrogen species corresponding to a particular intrinsic redox state and protonation level of the polymer was resolved quantitatively and unambiguously. The result confirmed the peak assignments of former XPS core-level studies using the lower resolution non-monochromatized Mg—Kα X-ray source. Thus, the high-resolution XPS using a monochromatized Al—Kα X-ray source is a truly unique tool for the convenient and quantitative analysis of the various intrinsic redox states of PANI. Received: 16 May 2000/Accepted: 29 August 2000     

High-resolution electron momentum spectroscopy of valence satellites of carbon disulfide

The binding energy spectrum of carbon disulphide (CS(2)) in the energy range of 9-23 eV has been measured by a high-resolution (e,2e) spectrometer employing asymmetric noncoplanar kinematics at an impact energy of 2500 eV plus the binding energy. Taking the advantage of the high energy resolution of 0.54 eV, four main peaks and five satellites in the outer-valence region are resolved. The assignments and pole strengths for these satellite states are achieved by comparing the experimental electron momentum profiles with the corresponding theoretical ones calculated using Hartree-Fock and density functional theory methods. The results are also compared in detail with the recent SAC-CI general-R calculations. General agreement is satisfactory, while the present experiment suggests cooperative contributions from (2)Π(u), (2)Σ(g)(+) states to satellite 2 and (2)Σ(g)(+), (2)Π(g) states to satellite 3. Besides, relatively low pole strength for X (2)Π(g) state is obtained which contradicts all the theoretical calculations [2ph-TDA, ADC(3), SAC-CI general-R, ADC(4)] so far.