Study of Mixtures by NMR

At the present, mixtures of different complexities can be analyzed in great detail directly by NMR spectroscopy, without prior sample simplification or molecule isolation. In the past, chromatographic fractionation followed by characterization of pure, isolated compounds constituted the traditional approach of structure determination. This approach obviously excluded any molecules not robust enough to survive chromatography and it necessarily risks losing important biological information encoded in the original mixture. The study of mixtures by NMR is a transversal area of work used in many diverse fields of research. Currently, high throughput analysis are also carried out by NMR spectroscopy, for example in NMR-based metabolomics.

Published works

Electrochemical dehalogenation of dibromomethane and 1, 2‐dibromoethane to non‐toxic products using a carbon fiber brush electrode

D. Fernández‐Verdejo, M. LK Sulonen, M. Pérez‐Trujillo, E. Marco‐Urrea, A. Guisasola,* P. Blánquez

Journal of Chemical Technology & Biotechnology, 2021, 96, 335-340. DOI

Dibromomethane (DBM) and 1,2-dibromoethane (DBA) are two brominated volatile contaminants used in several industrial applications which are often detected in groundwater. The electrochemical degradation of DBM and DBA was studied at different cathode potentials in aqueous solution using an inexpensive graphite fiber brush electrode. The degradation followed first-order kinetics with respect to the nominal concentration of the brominated compounds, and the kinetic constant increased concomitantly with the decrease of the cathode potential. During the electrochemical dehalogenation 96.8% and 99.8% of the bromide in DBM and DBA was released as bromine ions, respectively. The main non-brominated compounds detected during the degradation of DBM and DBA were methane and ethene, respectively. In addition, traces of formic acid for DBM and acetic acid for DBA degradation were detected. The non-toxicity of the effluent was confirmed by a Microtox test. The efficient electrochemical degradation of DBM and DBA and the lack of toxic products open the door for a simple and non-toxic electrochemical approach for removing aliphatic brominated compounds from aquifers and other water sources.


Recyclable Mesoporous Organosilica Nanoparticles Derived from Proline-Valinol Amides for Asymmetric Organocatalysis

H. Li, M. Pérez-Trujillo, X. Cattoën, R. Pleixats*

ACS Sustainable Chem. Eng., 2019. DOI


Going a step further from bulk organosilicas to nanosized materials, we describe herein the preparation of mesoporous organosilica nanoparticles derived from mono- and bis-silylated proline-valinol amides, both by grafting on preformed mesoporous silica nanoparticles (MSN) and by a co-condensation method in neutral medium using Brij-56/CTAB as templates. This is the first report on the obtention of functionalized MSN by a co-condensation procedure with a structurally complex chiral precursor. The functionalized MSN have been characterized by elemental analysis, 29Si and 13C CP MAS NMR, transmission electron microscopy, scanning electron microscopy, N2-sorption measurements, dynamic light scattering, ζ-potential, and powder X-ray diffraction. We have evaluated the activity of these materials as recyclable catalysts in the asymmetric aldol reaction. The best organocatalysts are those derived from the monosilylated precursor, observing good diastereo- and enantiomeric ratios with a simple and environmentally friendly optimized protocol (water, 0 °C, absence of cocatalyst). The nanocatalyst was easily recovered by centrifugation and recycled up to five runs without loss of activity and selectivity. The use of organosilica nanoparticles reduces the problems of diffusion and low reaction rates encountered with bulk organosilicas.


Molecule confirmation and structure characterization of pentatriacontatrienyl mycolate in Mycobacterium smegmatis

M. Llorens-Fons, E. Julián, M. Luquin,* M. Pérez-Trujillo*

Chem Phys Lip, 2018, 212, 138-143. DOI


Mycobacterium smegmatis is often used to study the different components of mycobacterial cell wall. Mycolic acids are important components of mycobacterial cell wall that have been associated with virulence. Recently, a novel lipid containing mycolic acids has been described in M. smegmatis. However, some uncertainties regarding the structure of this molecule named mycolate ester wax have been reported. The objective of this work was to perform an in depth structural study of this molecule for its precise characterization. Using 1H and 13C NMR spectroscopy, the molecular structure of mycolate ester wax found in M. smegmatis has been elucidated. The characterization was complemented with MS analyses. This molecule is formed by a carbon chain with three methyl substituted olefinic units and a mycolate structure with trans double bonds and cis cyclopropane rings. The present molecular study will facilitate the detection and identification of pentatriacontatrienyl mycolate in future studies by the performance of a simple 1D 1H NMR experiment.



Exopolysaccharides from olive brines could reduce the adhesion of ETEC K88 to intestinal epithelial cells

Y. Zhu, G. González-Ortiz, R. Jiménez-Díaz, M. Pérez-Trujillo, T. Parella, P.López-Colom, S.María Martín-Orúe*

Food Funct., 2018, 9, 3884-3894. DOI

This study aims to explore the biological functions of the isolated exopolysaccharides (EPSs) produced during the industrial fermentation of olives against enterotoxigenic E. coli (ETEC) K88. Exopolysaccharides were isolated from five industrial fermenters. Analysis of their monosaccharide composition by GLC revealed that the main components were glucose (27%–50%) and galactose (23%–33%) followed by rhamnose (4–23%) and arabinose (6–17%). The 1H NMR spectrum showed a very similar profile between samples, and a more in-depth analysis revealed the presence of an α-pyranose in the form of α-D-Glcp-(1→) and two different α-furanoses, with chemicals shift values, suggesting the presence of α-D-Glcf and α-D-Galf. These results suggest that the EPSs produced during the fermentation of table green olives could interfere with the attachment of opportunistic pathogens onto the intestinal epithelial cells. This would open the possibility of novel functional properties for this traditional Mediterranean fermented food and for the isolated EPSs as candidates for nutraceutics to be used in human and/or animal diets in the prevention and treatment of ETEC diarrhoea.


Trehalose polyphleates, external cell wall lipids in Mycobacterium abscessus, are associated with the formation of clumps with cording morphology, which have been associated with virulence

M. Llorens-Fons, M. Pérez-Trujillo, E. Julián, E. C. Brambilla, F. Alcaide, T. F. Byrd, M. Luquin*

Front. Microbiol., 2017. DOI

Mycobacterium abscessus is a reemerging pathogen that causes pulmonary diseases similar to tuberculosis, which is caused by Mycobacterium tuberculosis. When grown in agar medium, M. abscessus strains generate rough (R) or smooth colonies (S). R morphotypes are more virulent than S morphotypes. Cording has also been related to the virulence of M. tuberculosis. In this species, the presence of mycolic acids and surface-exposed cell wall lipids has been correlated with the formation of cords. The objective of this work was to study the roles of the surface-exposed cell wall lipids and mycolic acids in the formation of cords in M. abscessus. A comparative study of the pattern and structure of mycolic acids was performed on R (cording) and S (non-cording) morphotypes derived from the same parent strains with the aid of NMR spectroscopy.



Preliminary evaluation of Pleurotus ostreatus for the removal of selected pharmaceuticals from hospital wastewater

L. Palli*, F. Castellet-Rovira, M. Pérez-Trujillo, D. Caniani, M. Sarrà-Adroguer, R. Gori

Biotechnology Progress, 2017. DOI

The fungus Pleurotus ostreatus was investigated to assess its ability to remove diclofenac, ketoprofen and atenolol spiked at 10 mg/L each one in hospital wastewater. The degradation test was carried out in a fluidized bed bioreactor testing both the batch and the continuous mode. In batch mode, diclofenac disappeared in less than 24 h, ketoprofen was degraded up to almost 50% in 5 d while atenolol was not removed. In continuous mode, diclofenac and ketoprofen removals were about 100% and 70% respectively; atenolol degradation was negligible during the first 20 d but it increased up to 60% after a peak of laccase production and notable biomass growth. Two intermediates of diclofenac and ketoprofen were detected by NMR spectroscopy. Moreover P. ostreatus was able to reduce chemical oxygen demand of the hospital wastewater which is an important advantage comparing to other fungi in order to develop a wastewater treatment process.


Chloroperoxidase-catalyzed amino alcohol oxidation: Substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal

G. Masdeu, M. Pérez-Trujillo, J. López-Santín*, G. Álvaro

Process Biochemistry, 2016. DOI

The ability of chloroperoxidase (CPO) to catalyze amino alcohol oxidations was investigated. The oxidations of compounds with different configurations with respect to the amine position towards hydroxyl – using H2O2 and tert-butyl hydroperoxide (t-BuOOH) – were analyzed in terms of the initial reaction rate, substrate conversion, and CPO operational stability. It was observed that the further the amino group from the hydroxyl, the lower the initial reaction rate. The effect of the amino-protecting group and other substituents (i.e., methyl and hydroxyl) was also examined, revealing an increase in steric hindrance due to the effect of bulky substituents. The observed reaction rates were higher with t-BuOOH, whereas CPO was more stable with H2O2. Final products and intermediates were analyzed directly from the crude by NMR spectroscopy.



Data on the identification and characterization of by-products from N-Cbz-3-aminopropanal and t-BuOOH/H2O2 chemical reaction in chloroperoxidase-catalyzed oxidations

G. Masdeu, M. Pérez-Trujillo, J. López-Santín*, G. Álvaro

Data in Brief, 2016, 8, 659-665. DOI

This data article is related to the subject of a publication in Process Biochemistry, entitled “Chloroperoxidase-catalyzed amino alcohol oxidation: Substrate specificity and novel strategy for the synthesis of N-Cbz-3-aminopropanal” (Masdeu et al., 2016) [1]. Here, the products of the chemical reaction involving the amino aldehyde (N-Cbz-3-aminopropanal) and peroxides (tert-butyl hydroperoxide and H2O2) are characterized by NMR. 1H and 13C NMR full characterization of the products was obtained based on 2D NMR, 1D selective NOESY and diffusion spectroscopy (DOSY) experiments.


Mycobacteria clumpling increase their capacity to damage macrophages

C. Brambilla, M. Llorens-Fons, E. Julián, E. Noguera-Ortega, C. Tomàs-Martínez, M. Pérez-Trujillo, T. F. Byrd, F. Alcaide, M. Luquin*

Front. Microbiol., 2016, 7:1562. DOI

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.


Diversity of pyrrolizidine alkaloids in native and invasive Senecio pterophorus (Asteraceae): Implications for toxicity

E. Castells*, P. J. Mulder and M. Pérez-Trujillo

Phytochemistry, 2014, 108:137-146. DOI

Changes in plant chemical defenses after invasion could have consequences on the invaded ecosystems by modifying the interactions between plants and herbivores and facilitating invasion success. However, no comprehensive biogeographical studies have yet determined the phenotypic levels of plant chemical defenses, as consumed by local herbivores, covering large distributional areas of a species. Senecio pterophorus is a perennial shrub native to Eastern South Africa, expanded into Western South Africa and introduced into Australia and Europe. As other Asteraceae, S. pterophorus contains pyrrolizidine alkaloids (PAs) toxic to vertebrate and invertebrate herbivores. Here we analyzed S. pterophorus PAs by LC–MS/MS and NMR spectroscopy. Plants from different origins diverged in their PA absolute and relative concentrations. Rosmarinine was the most abundant compound in Australia and South Africa, but it was nearly absent in Europe. We characterized three plant chemotypes: retrorsine– senkirkine chemotype in Eastern South Africa, rosmarinine chemotype in Australia and Western South Africa, and acetylseneciphylline chemotype in Europe. PA absolute concentrations were highest in Australia. The increased absolute and relative concentrations of retronecine PAs from Australia and Europe, respectively, indicate that S. pterophorus is potentially more toxic in the invasive range than in the native range.


Cyclopropanation of α-mycolic acids is not required for cording in non-tuberculous mycobacteria

C.Brambilla, A. Sánchez-Chardi, M. Pérez-Trujillo, E. Julián, M. Luquin*

Microbiology, 2012, 158, 1615-1621. DOI

Mycobacterium tuberculosis and Mycobacterium bovis are the principal agents responsible for human tuberculosis and tuberculosis in domestic and wild animals. Both form microscopic cords, a phenotypic characteristic that has been related to their virulence. The compounds responsible for cording are unknown, but a recent work has shown that cording could be related to the fine structure of α-mycolic acids. This investigation attributes the need of a proximal cyclopropane in α-mycolic acids for cording in Mycobacterium tuberculosis and Mycobacterium bovis BCG and proposes cyclopropanases as good targets for new chemotherapeutic agents. As in addition to M. tuberculosis and M. bovis, other Mycobacterium species form microscopic cords, it would be of great interest to know if the relationship between proximal cyclopropanation of α-mycolic acids and cording could be extended to non-tuberculous mycobacteria. In this work we have studied the correlation between cording and cyclopropanation of α-mycolic acids by Nuclear Magnetic resonance (NMR) spectroscopy and Scanning Electron Microscopy (SEM) images.



Revisited mycolic acid pattern of Mycobacterium confluentis using thin-layer chromatography

S. Secanella-Fandos, M. Luquin, M. Pérez-Trujillo, E. Julián*

Journal of Chromatography B, 2011, 879, 2821–2826. DOI

The profile of mycolic acids from Mycobacterium confluentis has not been adequately published. However, the definition of the composition of mycolic acids is a critical element for describing new mycobacterial species. Thus, an erroneously published profile can lead to confusing citations. The aim of this article is to make the protocols clear, by using thin layer chromatography as a tool, for defining the discrete pattern of mycolic acids of any newly reported mycobacterial species. By using this method, and corroborated using nuclear magnetic resonance analysis, we demonstrated that M. confluentis contains α-mycolates (type I) and epoxymycolates (type V mycolic acids).


Separation and Identification of Phenolic Compounds of Extra-Virgin Olive Oil from Olea europaea L. by HPLC-DAD-SPE-NMR/MS. Identification of a New Diastereoisomer of the Aldehydic Form of Oleuropein Aglycone

M. Pérez-Trujillo*, A. M. Gómez-Caravaca, A. Segura-Carretero, A. Fernández-Gutiérrez, T. Parella

Journal of Agricultural and Food Chemistry, 2010, 58, 9129-9136. DOI

The phenolic fraction of a monovarietal extra virgin olive oil (EVOO) from Olea europaea L. var. Cornezuelo was studied by the hyphenated HPLC-DAD-SPE-NMR/MS techniques. This survey led to the identification of 25 main compounds. One was identified as a new diastereoisomer of the aldehydic form of oleuropein aglycone (AOA) and characterized by 1D and 2D NMR techniques. The relative configuration of this new AOA was determined as 5R*,8S*,9S* on the basis of the results obtained from the combination of NOE experiments and Monte Carlo conformational search calculations. Assuming, as for the described diastereoisomers, that the new AOA comes from the natural oleuropein aglycone (OA), the absolute configuration was proposed as 5S,8R,9R.


White-rot fungus-mediated degradation of the analgesic ketoprofen Biodegradation of the analgesic naproxen by Trametes versicolor and identification of intermediates using HPLC-DAD-MS and NMR

E. Marco-Urrea, M. Pérez-Trujillo, P. Blánquez, T. Vicent, G. Caminal*

Bioresource technology, 2010, 101, 2159-2166. DOI

The white-rot fungus Trametes vesicolor degraded naproxen (10 mg L−1) in a liquid medium to non-detectable levels after 6 h. When naproxen was added in the range of concentrations typically found in the environment (55 μg L−1), it was almost completely degraded (95%) after 5 h. In vitro degradation experiments with purified laccase and purified laccase plus mediator 1-hydroxybenzotriazol showed slight and almost complete naproxen degradation, respectively. A noticeable inhibition on naproxen degradation was also observed when the cytochrome P450 inhibitor 1-aminobenzotriazole was added to the fungal cultures. These data suggest that both enzymatic systems could play a role in naproxen degradation. 2-(6-hydroxynaphthalen-2-yl)propanoic acid and 1-(6-methoxynaphthalen-2-yl)ethanone were structurally elucidated by HPLC-DAD-MS and NMR as degradation intermediates of naproxen. After 6 h of incubation, both parent compound and intermediates disappeared from the medium. The non-toxicity of the treated medium was confirmed by Microtox test.



Degradation of the drug sodium diclofenac by Trametes versicolor pellets and identification of some intermediates by NMR

E. Marco-Urrea, M. Pérez-Trujillo, C. Cruz-Morató, G. Caminal*, T. Vicent

Journal of Hazardous Materials, 2010, 176, 836-842. DOI

Degradation of diclofenac sodium, a nonsteroidal anti-inflammatory drug widely found in the aquatic environment, was assessed using the white-rot fungus Trametes versicolor. Almost complete diclofenac removal (≥94%) occurred the first hour with T. versicolor pellets when the drug was added at relatively high (10 mg L−1) and environmentally relevant low (45 μg L−1) concentrations in a defined liquid medium. In vivo and in vitro experiments using the cytochrome P450 inhibitor 1-aminobenzotriazole and purified laccase, respectively, suggested at least two different mechanisms employed by T. versicolor to initiate diclofenac degradation. Two hydroxylated metabolites, 4′-hydroxydiclofenac and 5-hydroxydiclofenac, were structurally elucidated by nuclear magnetic resonance as degradation intermediates in fungal cultures spiked with diclofenac. Both parent compound and intermediates disappeared after 24 h leading to a decrease in ecotoxicity calculated by the Microtox test. Laccase-catalyzed transformation of diclofenac led to the formation of 4-(2,6-dichlorophenylamino)-1,3-benzenedimethanol, which was not detected in in vivo experiments probably due to the low laccase activity levels observed through the first hours of incubation.


White-rot fungus-mediated degradation of the analgesic ketoprofen and identification of intermediates by HPLC–DAD–MS and NMR

E. Marco-Urrea, M. Pérez-Trujillo, C. Cruz-Morató, G. Caminal, T. Vicent*

Chemosphere, 2010, 78, 474-481. DOI

Ketoprofen is a nonsteroidal anti-inflammatory drug that has been detected in the environment in the range of ng L−1–μg L−1 due to its low degradability in some wastewater treatment plants. In this study, the use of the white-rot fungus Trametes versicolor to effectively degrade ketoprofen in a defined liquid medium was assessed. The fungus eliminated ketoprofen to nondetectable levels in 24 h when it was added at 10 mg L−1 whereas at low concentration of 40 μg L−1 it was almost completely removed (95%) after 5 h. Low extracellular laccase activity was detected in the T. versicolor cultures but the addition of the laccase-mediator system did not lead to ketoprofen oxidation. The cytochrome P-450 inhibitor 1-aminobenzotriazole reduced ketoprofen oxidation. These data suggest that the first oxidation step is cytochrome P450 mediated. During time-course degradation experiments, three intermediates were structurally elucidated and quantified by HPLC–DAD–MS and NMR: 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid, 2-[(3-hydroxy(phenyl)methyl)phenyl]-propanoic acid, and 2-(3-benzoyl-4-hydroxyphenyl)-propanoic acid. The latter was reported for the first time in biological systems. After 7 d of incubation, only small amounts of 2-[(3-hydroxy(phenyl)methyl)phenyl]-propanoic acid (0.08 mg) remained in the liquid medium in comparison with the initial ketoprofen dose (1.0 mg), suggesting possible mineralization of ketoprofen.


Ability of white-rot fungi to remove selected pharmaceuticals and identification of degradation products of ibuprofen by Trametes versicolor

E. Marco-Urrea*, M. Pérez-Trujillo, T. Vicent, G. Caminal

Chemosphere, 2009, 74, 765-772. DOI

A screening using four white-rot fungi (Trametes versicolor, Irpex lacteus, Ganoderma lucidum and Phanerochaete chrysosporium) was performed on the degradation of 10 mg L−1 of ibuprofen (IBU, anti-inflammatory), clofibric acid (CLOFI, lipid regulator) and carbamazepine (CARBA, antiepileptic/analgetic) after 7 d of incubation. Whereas IBU was extensively degraded by all the fungi tested, T. versicolor was the only strain able to degrade either CLOFI (∼91%) and CARBA (∼58%), although the latter was also degraded by G. lucidum (∼47%). In vitro experiments using manganese peroxidase and laccase-mediator system showed that extracellular fungal enzyme systems did not appear to play a role in the first step of degradation. During the very early stages of IBU degradation by T. versicolor, two hydroxylated metabolites were detected and identified by NMR spectroscopy: 1-hydroxy ibuprofen and 2-hydroxy ibuprofen. These byproducts were subsequently degraded by the fungus to 1,2-dihydroxy ibuprofen, that was not reported in biological systems to date. Furthermore, these results are of particular interest because CLOFI and CARBA are highly persistent in the aquatic environment and they pass unchanged or poorly transformed in wastewater treatment plants.


Dechlorination of 1, 2, 3-and 1, 2, 4-trichlorobenzene by the white-rot fungus Trametes versicolor

E. Marco-Urrea, M. Pérez-Trujillo, G. Caminal*, T. Vicent

Journal of hazardous materials, 2009, 166, 1141-1147. DOI

The degradation of 1,2,3-, 1,3,5- and 1,2,4-trichlorobenzene (TCB) by the white-rot fungus Trametes versicolor was studied. Time course experiments showed a degradation rate of 2.27 and 2.49 nmol d−1 mg−1 dry weight of biomass during the first 4 d of incubation in cultures spiked with 6 mg L−1 of 1,2,3- and 1,2,4-TCB, respectively. A high percent of degradation of 91.1% (1,2,3-TCB) and 79.6 (1,2,4-TCB) was obtained after 7 d. However, T. versicolor was not able to degrade 1,3,5-TCB under the conditions tested. For a range of concentrations of 1,2,4-TCB between 6.5 and 23 mg L−1, a complete dechlorination of the molecule was observed. Cytochrome P450 monooxygenase appears to be involve in the first step of 1,2,4-TCB degradation, as evidenced by marked inhibition of both dechlorination and degradation of 1,2,4-TCB in the presence of the known cyt P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide. Four intermediates formed from 1,2,4-TCB degradation were detected the second day of incubation, which did not appear the seventh day: 2,3,5-trichloromuconate, its corresponding carboxymethylenebutenolide, 2- or 5-chloro-4-oxo-2-hexendioic acid and 2- or 5-chloro-5-hydroxy-4-oxo-2-pentenoic acid. Based on these results, a degradation pathway of 1,2,4-TCB through cyt P450 monooxygenase and epoxide hydrolase was proposed.