The identification of metabolites in complex biological matrices is a challenging task in 1D 1 H NMR based metabolomic studies. Statistical TOtal Correlation Spectroscopy (STOCSY) has emerged for aiding the structural elucidation by revealing the peaks that present 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. 2D 1 H homonuclear J-resolved spectra (JRES), in its usual tilted and symmetrized processed form, were projected and STOCSY was applied on these 1D projections (p-JRES-STOCSY) as an alternative to avoid the overlap issue, but this approach suffers in cases where the signals are very close. In addition, STOCSY was applied to 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 (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.
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.
A novel series of fully substituted pyrazolo[3,4-b]pyridines 4 has been prepared in a regioselective manner by the microwave-assisted reaction between N-substituted 5-aminopyrazoles 1 and 3-(3-oxo-2-benzofuran-1(3H)-ylidene)pentane-2,4-dione (2). This is the second reported example of a cyclocondensation reaction using substrate 2 as a 1,3-bis-electrophilic reagent. Remarkably, this synthesis offers functionalized products with acetyl and carboxyl groups in one step, in good yields, and with short reaction times. Additionally, the cyclization intermediate 3 was isolated, allowing us to postulate a mechanism for this reaction, which is initiated via isobenzofuranone ring opening of 2 in a Michael-type reaction. The structures of the products and regioselectivity of the reactions were determined on the basis of NMR measurements and X-ray diffraction. For this new reaction using substrate 2, the optimal reaction conditions and its scope were investigated.
A quick access toward a 1,2‐dialkyl‐5‐trifluoromethylbenzimidazoles library by a three‐step synthesis sequence starting from 1‐chloro‐2‐nitro‐4‐(trifluoromethyl)benzene is described. The synthesis proceed via o‐phenylendiamines eficiently isolated, which also are key synthetic intermediates of another valuable heterocyclic compounds. Likewise, the trifluoromethyl group is part of the obtained benzimidazoles, affording an important structural feature for their possible applications. The advantages of this methodology are the modular lipophilicity of products, easy work‐up, up to 83% overall yield, the convenient use of microwave‐assisted reactions, and the production of compounds (intermediates and products) of high‐added value using cheap reagents and simple protocols.
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