Improving real-time measurement of H/D exchange using a FTIR biospectroscopic probe

We describe the improvement of a novel approach to investigating hydrogen/deuterium (H/D) exchange kinetics in biomolecules using transmission infrared spectroscopy. The method makes use of a Fourier transform infrared spectrometer coupled with a microdialysis flow cell to determine exchange rates of labile hydrogens. With this cell system, the monitoring of exchange reactions has been studied here as a function of some cell characteristics such as: (a) dialysis membrane surface contacting both the H₂O and D₂O compartments; (b) molecular cutoff of dialysis membrane; and (c) distance between the cell-filling holes. The best improvement has been obtained by increasing the dialysis membrane surface followed by increase of molecular cutoff. However, not significant differences were found using various distances between filling holes. The fastest exchange rate which can be measured with the cell system used here is found to be k = 0.41 ± 0.02 min⁻¹, that is, about threefold greater than the one got in a previous work. This microdialysis flow cell has been used here for the study of H/D exchange in nucleic acids with subsequent structural analysis by 2D correlation spectroscopy.     

Protein–protein interaction dynamics by amide H/H exchange mass spectrometry

Amide H/²H exchange detected by mass spectrometry provides a powerful tool for observing changes that occur upon protein–protein interactions. In general, it is possible to observe protection of surface amides, they become less solvent exposed when they are buried at the interface. The information thus obtained about the location of the protein–protein interface is useful for building a correct docked structure of the protein–protein complex. Examples of protein–protein interfaces that were correctly identified by such methods include the thrombin–thrombomodulin interaction and the interaction between the regulatory and catalytic subunit of protein kinase A. Amide exchange also affords a view into the subtle changes in the ensemble of states that occur upon protein modification or protein–protein binding. Examples of proteins in which amide exchange has been used to observe phosphorylation-induced changes include ERK2 and CheB. Amide exchange showed the pathway of communication between the cAMP-binding site and the catalytic subunit site within the regulatory subunit of protein kinase A. Clues as to how thrombomodulin regulates the catalytic activity of thrombin were also obtained.     

Quantitative 2H NMR spectroscopy. 1. Thermodynamic H/D isotope effects in N,N-dimethylformamide and cyclopentadiene molecules

A new method for quantification of the relative distribution of deuterium in molecules is proposed. The technique is based on the lineshape analysis in the ²H NMR spectra obtained at the natural abundance level of deuterium with allowance for inhomogeneity of the magnetic field. The equilibrium thermodynamic H/D isotope effects for hindered rotation about the C—N bond in the N,N-dimethylformamide molecule and for prototropic exchange in the cyclopentadiene molecule were determined. The results obtained agree with those of DFT calculations of the vibrational energies.     

Binding/antibinding analyses for diatomic interactions H 2 + system

The region-functional concept of electron density has been quantitatively examined for 1s_g, 2p_u, 2p_u, and 3d_g states of H ₂ ⁺ system on the basis of Berlin diagram which divides the three-dimensional molecular space into binding and antibinding regions. The electronic charge, Hellmann-Feynman (H-F) force, and stabilization energy of the system are partitioned into the binding and antibinding contributions by the regional integrations.Dynamic behaviors of the electron density (i.e. electron-cloud preceding and following) during the interaction processes are also clarified using the centers of electron density and force density.Differences in attractive and repulsive, and - and -type interactions are discussed from the force and density point of view.     

Specific intramolecular C-H...O interactions in 1-vinyl-2-formylimidazole and 1-vinyl-2-formylbenzimidazole studied by1H and13C NMR spectral data

According to the¹H and¹³C NMR spectral data, the vinyl group in 1-vinyl-2-formylimidazole and 1-vinyl-2-formylbenzimidazole istrans-oriented with respect to the formyl fragment, while the carbonyl group occupies theanti-position with respect to the N atom of pyridine cycle. A specific intramolecular C—H...O interaction of a weak hydrogen bond type is realized between the -H atom of the vinyl group and O atom of the carbonyl group.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1197–1201, May, 1996.     

1-Methyl-2(3,4)-(1-vinylpyrrol-2-yl)pyridinium iodides: some structural features revealed by 1H and 13C NMR spectroscopy

The reactions of 2-, 3-, and 4-(1-vinylpyrrol-2-yl)pyridines with methyl iodide afford the corresponding quaternary salts. Analysis of their ¹H and ¹³C NMR spectra showed that the quaternization of the nitrogen atom considerably enhances the -acceptor effect of the pyridine ring on the pyrrole ring and on the vinyl group. 1-Methyl-2-(1-vinylpyrrol-2-yl)pyridinium iodide contains no weak intramolecular C--H...N hydrogen bond present in the starting compound.     

13C and1H NMR spectra of substituted dihydropyrroles prepared by the reaction of 2-aminooxazoles and maleimide

Substituted dihydropyrroles were characterized by¹³C and¹H NMR spectra. The spectral patterns of these compounds and reversible hydrogen—deuterium exchange are discussed. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 697–700, April, 2000.     

Stereochemicai study of 7-aryl-1,7,8,8a-tetrahydro-3(2H)indolizinones by1H and13C NMR

Stereochemistry of 7-aryl-1,7,8,8a-tetrahydro-3(2H)-indolizinones was studied by¹H and¹³C NMR. Complete assignment of¹H NMR signals and analysis of¹H-¹H coupling constants were performed using the iterative PANIC program. Values of³ J _6,7,³ J _7,8endo, and⁴ J _5,7 allow one to unambiguously identify the correspondingexo- andendo-stereoisomers. For stereoisomers with exo-orientation of H(7), complete assignment of¹³C NMR signals was performed on the basis of analysis of the¹³C-¹H coupling constants using two dimensional heteronuclear shift-correlating spectroscopy.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 591–593, March, 1996.     

Diatomic interactions in momentum space. The 1sσg and 2pσ u states of H 2 + system

The method of momentum electron density for interatomic interactions has been applied to the two lowest σ states of the H ₂ ⁺ system. For attractive (1sσ_g) and repulsive (2pσ _u ) interactions, the behaviour of momentum density and its effect on the stabilization energy of the system are examined quantitatively. The concept of contraction and expansion of the momentum density is shown to form an important guiding principle in this approach. The origin of covalent bonding is discussed based on the energy partitioning proposed previously.     

2H Chemical-shift resolution and dipolar2H-1H,2H-15N correlations in solid-state MAS NMR spectroscopy for structure determination and distance measurements in hydrogen-bonded systems

In solid-state NMR, deuteron (²H) spectroscopy can be performed in full analogy to¹H spectroscopy, including²H chemical-shift resolution and²H-X dipolar correlation schemes, when the NMR experiments are conducted in a “rotor-synchronized” fashion under fast magic-angle spinning. Here, 2H-X NMR experiments of this type, including²H-¹⁵N and²H-¹H chemical-shift correlations and distance measurements, are introduced and demonstrated on cytosine monohydrate, whose acidic protons can readily be replaced by deuterons by recrystallization from D₂O. In this way,²H NMR spectroscopy provides information complementary to¹H NMR data, which is particularly useful for studying hydrogen bonds in supra- or biomolecular systems. Electronic supplementary material Supplementary material is available in the online versionof this article atand is accessible for authorized users.     

Fourier transform-IR and 1H NMR studies on the structure of water solubilized by reverse aggregates of calcium bis(2-ethylhexyl) sulfosuccinate in organic solvents

The structure of water solubilized by reverse aggregates of calcium bis(2-ethylhexyl) sulfosuccinate in deuterobenzene and toluene has been probed by Fourier transform-IR and ¹H NMR spectroscopies. The ν_OD band of solubilized HOD (4% D₂O in H₂O) has been recorded as a function of the [water]/[surfactant] molar ratio, W/S. Curve fitting of this band showed the presence of a main peak at 2550 ± 13 cm⁻¹ and a small one at 2405 ± 15 cm⁻¹. As a function of increasing W/S, the frequency of the main peak decreases, its full width at half-height increases, and its area increases linearly. The ¹H NMR chemical shift of solubilized H₂O–D₂O mixtures at W/S = 18.1 has been measured as a function of the deuterium content of the aqueous nanodroplet. These data were used to calculate the so-called “fractionation factor” of the aggregate-solubilized water, the value of which was found to be unity. The results of both techniques show that reverse aggregate-solubilized water, although different from bulk water, does not seem to coexist in “layers” of different degrees of structure, as suggested, for example by the two-state water-solubilization model. Received: 12 July 1999/Accepted: 30 August 1999     

Study of ion exchange selectivity of organic ions by using (31)P NMR spectroscopy

Ion exchange distribution of the phenylphosphoric acid has been studied on various types of chloride form strong base resins as a function of pH at 25 °C, at 0.1 M ionic strength. Equilibrium measurements were supplemented by the study of pH dependence of the ³¹P NMR spectra of the resin-phase phenylphosphate species. Equations were derived to describe the pH dependence of the overall distribution coefficient and the chemical shift of the resin-phase solute species. Experimental data were evaluated by using these model equations and the values of the individual distribution coefficients, ion exchange selectivity coefficients and the resin-phase ³¹P chemical shifts of the mono- and divalent ions have been calculated. Comparison of distribution data of the individual species corroborated the significance of the role of hydrophobic interaction in the selectivity of organic ion exchange processes. A well-defined correlation between the ion exchange selectivity and the resin-phase ³¹P NMR chemical shift data has been pointed out.     

Synthesis and spectroscopic studies of some new metal carbonyl derivatives of 1-(2-pyridylazo)-2-naphthol

Interaction of 1-(2-pyridylazo)-2-naphthol (PAN) with [Mo(CO)₆] in air resulted in formation of the tricarbonyl oxo-complex [Mo(O)(CO)₃(PAN)], 1. The dicarbonyl complex [Ru(CO)₂(PAN)], 3, was obtained from the reaction of [Ru₃(CO)₁₂] with PAN. In presence of triphenyl phosphine (PPh₃), the reaction of PAN with either Mo(CO)₆ or Ru₃(CO)₁₂ gave [Mo(CO)₃(PAN)(PPh₃)], 2, and [Ru(CO)₂(PAN)(PPh₃)], 4. All the complexes were characterized by elemental analysis, mass spectrometry, IR, and NMR spectroscopy. The thermal properties of the complexes were also investigated by thermogravimetry.     

Correlation NMR spectroscopy of 1,1-dichloro-2,3,4,5-tetraphenyl-1-germacyclopenta-2,4-diene

Assignment of all of the signals in the¹H and¹³C NMR spectra of 1,1-dichloro-2,3,4,5-tetraphenyl-l-germacyclopenta-2,4-diene has been carried out using two-dimensional NMR spectroscopy. Translated fromIzvestiya Akademii Nauk Seriya Khimicheskaya, No. 3, pp. 623–625, March, 1997.     

Fruit juice authentication by <Superscript>1</Superscript>H NMR spectroscopy in combination with different chemometrics tools

To discriminate orange juice from grapefruit juice in a context of fraud prevention, ¹H NMR data were submitted to different treatments to extract informative variables which were then analysed using multivariate techniques. Averaging contiguous data points of the spectrum followed by logarithmic transformation improved the results of the data analysis. Moreover, supervised variable selection methods gave better rates of classification of the juices into the correct groups. Last, independent-component analysis gave better classification results than principal-component analysis. Hence, ICA may be an efficient chemometric tool to detect differences in the ¹H NMR spectra of similar samples, and so may be useful for authentication of foods.     

First experimental determination of two-bond C isotopic effects on H NMR chemical shifts

For the first time, a simple NMR methodology is proposed for the accurate determination of the effect of the substitution of ¹²C by ¹³C on the chemical shifts of protons separated by two-bonds in small molecules in their natural abundance.     

Detection and quantification of phenolic compounds in olive oil by high resolution H nuclear magnetic resonance spectroscopy

High resolution ¹H NMR spectroscopy has been employed as a versatile and rapid method to analyze the polar fraction of extra virgin olive oils containing various classes of phenolic compounds. The strategy for identification of phenolic compounds is based on the NMR chemical shifts of a large number of model compounds assigned by using two-dimensional (2D) NMR spectroscopy. Furthermore, 2D NMR was applied to phenolic extracts in an attempt to discover additional phenolic compounds. The ¹H NMR methodology was successful in detecting simple phenols, such as p-coumaric acid, vanillic acid, homovanillyl alcohol, vanillin, free tyrosol, and free hydroxytyrosol, the flavonols apigenin and luteolin, the lignans (+) pinoresinol, (+) 1-acetoxypinoresinol and syringaresinol, two isomers of the aldehydic form of oleuropein and ligstroside, the dialdehydic form of oleuropein and ligstroside lacking a carboxymethyl group, and finally total hydroxytyrosol and total tyrosol reflecting the total amounts of free and esterified hydroxytyrol and tyrosol, respectively. The absolute amount of each phenolic constituent was determined in the polar fraction by using anhydrous 1,3,5-triazine as an internal standard.     

Cyclodextrin inclusion of four phenylurea herbicides: determination of complex stoichiometries and stability constants using solution 1H NMR spectroscopy

An inclusion complex formation between α- and β-cyclodextrin and four phenylurea analogues, namely metobromuron, monolinuron, monuron and fenuron, is reported. Complex formation was established using solution ¹H NMR spectroscopy. Complex stoichiometries were determined by the method of continuous variation using the chemically induced shifts of both the host and guest protons. An analysis of the spectroscopic data revealed the stoichiometry as 1:1 in all cases while a further analysis of the same data yielded values for the association constant K ranging from 208 to 2749 M^? 1. From the observed chemical shifts it was deduced that in all cases, only the guest aromatic ring enters the host cavities, the substituted urea moiety protruding from the secondary rim in the case of α-cyclodextrin, but from the primary rim in the case of β-cyclodextrin.     

H and C NMR spectroscopy of 9-acridinones

The ¹H and ¹³C NMR spectra of 9-acridinone and its five derivatives dissolved in CDCl₃, CD₃CN and DMSO-d₆ were measured in order to reveal the influence of the constitution of the compounds and features of the solvents on chemical shifts and ¹H-¹H coupling constants. Experimental data were compared with theoretically predicted chemical shifts, on the GIAO/DFT level of theory, for DFT (B3LYP)/6-31G^∗∗ optimized geometries of molecules—also for four other 9-acridinones. This comparison helped to ascribe resonance signals in the spectra to relevant atoms and enabled revelation of relations between chemical shifts and physicochemical features of the compounds. It was found that experimentally or theoretically determined ¹H and ¹³C chemical shifts of selected atoms correlate with theoretically predicted values of dipole moments of the molecules, as well as bond lengths, atomic partial charges and energies of HOMO.