Multiplet-Assisted Peak Alignment for 1H NMR-Based Metabolomics.

NMR-based metabolomics aims at recovering biological information by comparing spectral data from samples of biological interest and appropriate controls. Any statistical analysis performed on the data matrix relies on the proper peak alignment to produce meaningful results. Through the last decades, several peak alignment algorithms have been proposed, as well as alternatives like spectral binning or strategies for annotation and quantification, the latter depending on reference databases. Most of the alignment algorithms, mainly based on segmentation of the spectra, present limitations for regions with peak overlap or cases of frequency order exchange. Here, we present our multiplet-assisted peak alignment algorithm, a new methodology that consists of aligning peaks by matching multiplet profiles of f1 traces from J-resolved spectra. A correspondence matrix with the linked f1 traces is built, and multivariate data analysis can be performed on it to obtain useful information from the data, overcoming the issues of peak overlap and frequency crossovers. Statistical total correlation spectroscopy can be applied on the matrix as well, toward a better identification of molecules of interest. The results can be queried on one-dimensional (1D) 1H databases or can be directly coupled to our previously published Chemical Shift Multiplet Database.

Consecutive Queries to Assess Biological Correlation in NMR Metabolomics: Performance of Comprehensive Search of Multiplets over Typical 1D 1H NMR Database Search.

NMR-based metabolomics requires proper identification of metabolites to draw conclusions from the system under study. Normally, multivariate data analysis is performed using 1D 1H NMR spectra, and identification of peaks (and then compounds) relevant to the classification is accomplished using database queries as a first step. 1D 1H NMR spectra of complex mixtures often suffer from peak overlap. To overcome this issue, several studies employed the projections of the (tilted and symmetrized) 2D 1H J-resolved (JRES) spectra, p-JRES, which are similar to 1D 1H decoupled spectra. Nonetheless, there are no public databases available that allow searching for chemical shift spectral data for multiplets. We present the Chemical Shift Multiplet Database (CSMDB), built utilizing JRES spectra obtained from the Birmingham Metabolite Library. The CSMDB provides scoring accounting for both matched and unmatched peaks from a query list and the database hits. This input list is generated from a projection of a 2D statistical correlation analysis on the JRES spectra, p-(JRES-STOCSY), being able to compare the multiplets for the matched peaks, in essence, the f1 traces from the JRES-STOCSY spectrum and from the database hit. The inspection of the unmatched peaks for the database hit allows the retrieval of peaks in the query list that have a decreased correlation coefficient due to low intensities. The CSMDB is coupled to "ConQuer ABC", which permits the assessment of biological correlation by means of consecutive queries with the unmatched peaks in the first and subsequent queries.

Tackling the Peak Overlap Issue in NMR Metabolomics Studies: 1D Projected Correlation Traces from Statistical Correlation Analysis on Nontilted 2D 1H NMR J-Resolved Spectra.

The identification of metabolites in complex biological matrices is a challenging task in 1D 1H-NMR-based metabolomics studies. Statistical total correlation spectroscopy (STOCSY) has emerged for aiding the structural elucidation by revealing the peaks that present a high correlation to a driver peak of interest (which would likely belong to the same molecule). However, in these studies, the signals from metabolites are normally present as a mixture of overlapping resonances, limiting the performance of STOCSY. As an alternative to avoid the overlap issue, 2D 1H homonuclear J-resolved (JRES) spectra were projected, in their usual tilted and symmetrized processed form, and STOCSY was applied on these 1D projections (p-JRES-STOCSY). Nonetheless, this approach suffers in cases where the signals are very close. In addition, STOCSY was applied to the whole JRES spectra (also tilted) to identify correlated multiplets, although the overlap issue in itself was not addressed directly and the subsequent search in databases is complicated in cases of higher order coupling. With these limitations in mind, in the present work, we propose a new methodology based on the application of STOCSY on a set of nontilted JRES spectra, detecting peaks that would overlap in 1D spectra of the same sample set. Correlation comparison analysis for peak overlap detection (COCOA-POD) is able to reconstruct projected 1D STOCSY traces that result in more suitable database queries, as all peaks are summed at their f2 resonances instead of the resonance corresponding to the multiplet center in the tilted JRES spectra. (The peak dispersion and resolution enhancement gained are not sacrificed by the projection.) Besides improving database queries with better peak lists obtained from the projections of the 2D STOCSY analysis, the overlap region is examined, and the multiplet itself is analyzed from the correlation trace at 45° to obtain a cleaner multiplet profile, free from contributions from uncorrelated neighboring peaks.

Fast Metabolite Identification in Nuclear Magnetic Resonance Metabolomic Studies: Statistical Peak Sorting and Peak Overlap Detection for More Reliable Database Queries.

A lot of time is spent by researchers in the identification of metabolites in NMR-based metabolomic studies. The usual metabolite identification starts employing public or commercial databases to match chemical shifts thought to belong to a given compound. Statistical total correlation spectroscopy (STOCSY), in use for more than a decade, speeds the process by finding statistical correlations among peaks, being able to create a better peak list as input for the database query. However, the (normally not automated) analysis becomes challenging due to the intrinsic issue of peak overlap, where correlations of more than one compound appear in the STOCSY trace. Here we present a fully automated methodology that analyzes all STOCSY traces at once (every peak is chosen as driver peak) and overcomes the peak overlap obstacle. Peak overlap detection by clustering analysis and sorting of traces (POD-CAST) first creates an overlap matrix from the STOCSY traces, then clusters the overlap traces based on their similarity and finally calculates a cumulative overlap index (COI) to account for both strong and intermediate correlations. This information is gathered in one plot to help the user identify the groups of peaks that would belong to a single molecule and perform a more reliable database query. The simultaneous examination of all traces reduces the time of analysis, compared to viewing STOCSY traces by pairs or small groups, and condenses the redundant information in the 2D STOCSY matrix into bands containing similar traces. The COI helps in the detection of overlapping peaks, which can be added to the peak list from another cross-correlated band. POD-CAST overcomes the generally overlooked and underestimated presence of overlapping peaks and it detects them to include them in the search of all compounds contributing to the peak overlap, enabling the user to accelerate the metabolite identification process with more successful database queries and searching all tentative compounds in the sample set.

O-ylide and π-complex formation in reactions of a carbene with dibenzo and monobenzo crown ethers.

Reactions of p-nitrophenylchlorocarbene (PNPCC) with various dibenzo crown ethers produce O-ylides and π-complexes; the reactions can be followed via the spectral signatures of the carbene and the products. The O-ylides form most rapidly, but over time they decay in favor of the more stable π-complexes. Extensive computational studies support and refine appropriate structural and mechanistic conjectures. Reactions of PNPCC with monobenzo crown ethers afford only the spectral signatures of O-ylides; monobenzo π-complexes are either not formed in significant concentrations or are spectroscopically silent.

Visualizing carbene equilibria.

Reviewed herein are equilibria between halocarbenes and halocarbanions, carbenes and carbene complexes and carbenes and O-ylides. The transient species were visualized by laser flash photolysis coupled with UV-Visible spectroscopy. This methodology enabled the determination of equilibrium constants and the extraction of associated thermodynamic parameters. Parallel computational studies provided anticipated structures and energies for the transient species, as well as electronic absorption wavelengths and oscillator strengths.

Solvent polarity effects on carbene/ether-O-ylide equilibria.

p-Nitrophenylchlorocarbene (PNPCC) reacted reversibly with tetrahydrofuran (THF), tetrahydropyran (THP), 1,3-dioxane (1,3-D), and 1,4-dioxane (1,4-D) to form O-ylides 8, 9, 10, and 11, respectively. The O-ylides were visualized by their characteristic UV-vis spectroscopic signatures. Equilibrium constants (K) were determined spectroscopically, and studies of K as a function of pentane/1,2-dichloroethane (DCE) solvent blends illustrated the dependence of K on solvent polarity. Electronic structure calculations based on density functional theory provided carbene/ether O-ylide structures and energetics, as well as electronic spectroscopic parameters for use in the determination of K. Comparisons of the computed and experimental data were generally satisfactory.

Reversible O-ylide formation in carbene/ether reactions.

p-Nitrophenylchlorocarbene reacted reversibly with diethyl ether, di-n-propyl ether, or tetrahydrofuran (THF) to form O-ylides, which were visualized by their UV-visible spectroscopic signatures. Equilibrium constants (K(eq)) were determined spectroscopically and ranged from 0.10 M(-1) (di-n-propyl ether) to 7.5 M(-1) (THF) at 295 K. Studies of K(eq) as a function of temperature afforded ΔH(o), ΔS(o), and ΔG(o) for the di-n-propyl ether and THF/O-ylide equilibria. ΔH(o) was favorable for ylide formation, but ΔS(o) was quite negative, so that ΔG(o)s for the equilibria were small. Electronic structure calculations based on density functional theory provided structures, spectroscopic signatures, and energetics for the carbene/ether O-ylides.

Proton transfer from 2-naphthol to aliphatic amines in supercritical CO2.

The proton transfer from 2-naphthol to aliphatic amines was studied in supercritical CO(2) (scCO(2)) and in cyclohexane as reference solvent, by absorption and fluorescence spectroscopy and by time-resolved emission. Irradiation of 2-naphthol in scCO(2) in the presence of ethyldiisopropylamine shows dynamic fluorescence quenching of the acidic form of 2-naphthol and emission from the basic form. Fluorescence excitation spectra show that the emission of the basic form is originated upon excitation of the acidic form. The interaction between 2-naphthol and the amines is described by the formation of a complex with proton donor-acceptor character in the ground and excited states of 2-naphthol. The acidity increase of 2-naphthol upon electronic excitation to the first excited singlet in scCO(2) is as high as in water. Proton transfer quantum yields of 0.6 can be easily achieved in scCO(2). The results have implications for carrying out acid-base catalyzed reactions in scCO(2).

Photophysics and photochemistry of an asymmetrically substituted diazene: a suitable cage effect probe.

The photophysics and photochemistry of (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene were thoroughly studied by laser flash photolysis from the picosecond to the microsecond time domain. The compound has favorable features as a radical photoinitiator and as a probe for cage effect studies in liquids, supercritical fluids, and compressed gases. The biphenyl moiety acts as an antenna efficiently transferring electronic energy to the dissociative (1)n,pi* state centered on the azo moiety. By picosecond experiments irradiating at the biphenyl- and at the azo-centered transitions, we were able to demonstrate this fact as well as determine a lifetime of 0.7 ps for the buildup of 1-biphenyl-4-yl-1-methyl-ethyl radicals (BME*). The sum of in-cage reaction rate constants of BME* radicals by combination and disproportionation is 5 x 10(10) s(-1). The free radical quantum yield in solution is 0.21 (phi(BME*)) in n-hexane at room temperature, whereas the dissociation quantum yield approaches 50%. The symmetric ketone, 2,4-bis-biphenyl-4-yl-2,4-dimethyl-pentan-2-one, was used as a reference compound for the production and reaction of BME* radicals. Transient IR measurements show CO stretching bands of the excited (3)pi,pi* and (1)n,pi* states but no dissociation up to 0.5 ns. A fluorescence lifetime of 1 ns for this ketone is consistent with this observation. By transient actinometry and kinetic decays in the microsecond time range, we measured epsilon(BME*) = (2.3 +/- 0.2) x 10(4) M(-1) cm(-1) at 325 nm and a second-order rate constant of 5.8 x 10(9) M(-1) s(-1) for the consumption of BME* radicals.

Cage effect in supercritical fluids and compressed gases in the photolysis of an asymmetrically substituted diazene.

We studied the photolysis of (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene in supercritical CO(2) and Xe, as well as in compressed Kr. The compound has good solubility in the mentioned fluids, allowing the photolysis measurements to be performed in CO(2) at 1.4 K above T(c) and at pressures as low as 70 bar. We monitored relative cage effect after nanosecond laser pulses by measuring the absorbance at 320 nm (DeltaA(t-->0)) corresponding to the total amount of out-of-cage 1-biphenyl-4-yl-1-methyl-ethyl radical (BME.) produced after nitrogen loss of the diazene. In supercritical CO(2) and Xe, isothermal values of DeltaA(t-->0) showed an increase-decrease behavior with increasing pressure at constant temperature, a typical feature of the transition from the solvent energy transfer to the friction controlled regimes. The comparison of the behavior of DeltaA(t-->0) in CO(2) at reduced temperatures between 1.004 and 1.027, in Xe, and in Kr points to an absence of enhanced cage effect near the critical point. Compatibility with spectroscopic data is analyzed.