These are short reports about the research activities carried out at the SeRMN.
In them we describe the work done in collaboration with research groups, to summarize communications presented at scientific meetings, to report visits and stages at other laboratories or facilities, and to comment the meetings and workshops we have attended.
A method based on d-DNP NMR spectroscopy to study chiral recognition is described for the first time. The enantiodifferentiation of a racemic metabolite in a millimolar aqueous solution using a chiral solvating agent was performed. Hyperpolarized 13C-labeled DL-methionine enantiomers were differently observed with a single-scan 13C NMR experiment, while the chiral auxiliary at thermal equilibrium remained unobserved. The method developed entails a step forward in the chiral recognition of small molecules by NMR spectroscopy, opening new possibilities in situations where the sensitivity is limited, for example, when a low concentration of analyte is available or when the measurement of an insensitive nucleus, like 13C, is required. The advantages and current limitations of the method, as well as future perspectives, are discussed.
Selective Refocusing (GSERF or the recent PSYCHEDELIC) experiments were originally designed to determine all proton-proton coupling constants (JHH) for a selected proton resonance. They work for isolated signals on which selective excitation can be successfully applied but, as happens in other selective experiments, fail for overlapped signals. To circumvent this limitation, a doubly-selective TOCSY-GSERF scheme is presented for the measurement of JHH in protons resonating in crowded regions. This new experiment takes advantage of the editing features of an initial TOCSY transfer to uncover hidden resonances that become accessible to perform the subsequent frequency-selective refocusing.
A novel approach for the fast and efficient structural discrimination of molecules containing multiple stereochemical centers is described. A robust Jresolved HSQC experiment affording highly resolved 1JCH/1TCHsplittings along the indirect dimension and homodecoupled 1H signals in the detected dimension is proposed. The experiment enables in-situ distinction of both isotropic and anisotropic components of molecules dissolved incompressed PMMA gels, allowing a rapid and direct one-shot determination of accurate residual dipolar coupling constants from a single NMR spectrum
“Mycobacteria clumping increase their capacity to damage macrophages” by C. Brambilla, M. Llorens-Fons, E. Julián, E. Noguera-Ortega, C. Tomàs-Martínez, M. Pérez-Trujillo, T. F. Byrd, F. Alcaide and M. Luquin.
The rough morphotypes of non-tuberculous mycobacteria have been associated with the most severe illnesses in humans. This idea is consistent with the fact that Mycobacterium tuberculosis presents a stable rough morphotype. Unlike smooth morphotypes, the bacilli of rough morphotypes grow close together, leaving no spaces among them and forming large aggregates (clumps). Currently, the initial interaction of macrophages with clumps remains unclear. Thus, we infected J774 macrophages with bacterial suspensions of rough morphotypes of Mycobacterium abscessus containing clumps and suspensions of smooth morphotypes, primarily containing isolated bacilli. Using confocal laser scanning microscopy and electron microscopy, we observed clumps of at least 5 rough-morphotype bacilli inside the phagocytic vesicles of macrophages at 3 hours post-infection. These clumps grew within the phagocytic vesicles, killing 100% of the macrophages at 72 hours post-infection, whereas the proliferation of macrophages infected with smooth morphotypes remained unaltered at 96 hours post-infection. Thus, macrophages phagocytose large clumps, exceeding the bactericidal capacities of these cells. Furthermore, proinflammatory cytokines and granuloma-like structures were only produced by macrophages infected with rough morphotypes. Thus, the present study provides a foundation for further studies that consider mycobacterial clumps as virulence factors.
Figure. Content of GPL and structure of mycolic acids. (A) 1-D TLC analysis of the crude lipid extracts of M. abscessus strains. (B) 1H-NMR spectra of purified mycolic acid methyl esters from M. abscessus. (C) Relative molar ratios of molecular moieties cis-db, trans-db, cis-cp and trans-cp of mycolic acid methyl esters from M. abscessus.
Abstract: Oocytes recovered from prepubertal goats are very heterogeneous in growth and grade of atresia which make them unpredictable for IVEP programs. We have observed that oocytes from prepubertal goats obtained from >3 mm follicles develop up to blastocyst stage at a similar percentage than oocytes from adult goats (18% vs 21%), suggesting that the follicle development and the follicular fluid (FF) content are more relevant to oocyte competence than the age of the donor. The aim of this study is to characterize the FF metabolomic profile from different follicular environments through a high-resolution 1H NMR-based metabolomic study. Samples of adult (n=40) and prepubertal (n=16) FF where collected by laparoscopic ovum pick-up (LOPU) and by aspiration of slaughterhouse ovaries, respectively. FF from small (< 3 mm) and large (> 3 mm) diameter follicles where pooled for each female. Multivariate ordination principal component analysis (PCA) was performed to detect patterns of sample ordination in the metabolomes. The unsupervised method clearly differed between the FF metabolomes of large and small follicles of prepubers and between the FF of preadolescent and adult individuals.
Figure. a) PCA scores plot (PC1-PC2) from 1H NMR spectral data of follicular fluid samples of preadolescent (n=16; blue dots) and adult (n=40; black dots) goats. b) PCA heat map loadings plot (PC1-PC2) with some discriminant variables assigned.
Abstract: A fast RDC-assisted strategy involving the simultaneous determination of isotropic (scalar) and anisotropic (total) interactions is reported. The concerted use of individual 1DCH for all CHn multiplicities and 2DHH obtained from a single 1JCH/2JHH-resolved NMR spectrum offers an unambiguous assignment of diastereotopic protons and an efficient discrimination between all eight possible diastereoisomeric structures of strychnine which contains six stereocenters.
Abstract: The development of novel experimental strategies to significantly enhance signal resolution by broadband homodecoupling is a current topic of high interest in 1H NMR spectroscopy . A number of different building blocks have been implemented into 1D and 2D homo- and heteronuclear experiments in order to provide resolution-enhanced pure chemical shift 1H NMR spectra, where signals appear collapsed to singlets. On the other hand, Covariance processing methods have been used to generate challenging NMR spectral representations . We present here the first attempts towards a general solution to generate Pure Shift NMR spectra by using Generalized Indirect Covariance (psGIC) co-processing3,4 . The current strategy is based on the calculation of a new 2D psGIC spectrum from the combination of a parent homo- or heteronuclear spectrum and a reference 2D F1-homodecoupled 1H- 1H correlation spectrum only showing diagonal cross-peaks (DIAG), which share a common 1H frequency dimension. Using psGIC, the F1 dimension in the DIAG spectrum is transferred to the F2 dimension of the parent spectrum, thus generating a new pure shift 2D spectrum
André has been working as a Ph.D. candidate at the Department of Chemistry and SeRMN under the direction of Dr. Teodor Parella and Dr. Pau Nolis since November 2014, when he enrolled in the Department of Chemistry doctoral program at Universitat Autònoma de Barcelona with a fellowship from CNPq-Brazil. He is currently in his second year and expects to defend the doctoral thesis on 2017/2018.
“Chloroperoxidase-catalyzed amino alcohol oxidation: Substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal” by G. Masdeu, M. Pérez-Trujillo, J. López-Santín and Gregorio Álvaro. Process Biochemistry 2016; DOI:10.1016/j.procbio.2016.05.022