Eurico J. Cabrita

The qualitative probing of hydrogen bond strength by diffusion-ordered NMR spectroscopy

Abstract The feasibility of using diffusion-ordered NMR spectroscopy (DOSY) as a useful probe to qualitatively understand the relative strength of hydrogen bonds between various components in a mixture and a ligand in non-aqueous solutions has been demonstrated. 1 H-detected DOSY and 31 P-detected DOSY were used.

Characterization of Reactive Intermediates by Diffusion‐Ordered NMR Spectroscopy: A Snapshot of the Reaction of 13CO2 with [Cp2Zr(Cl)H]

In cases in which the structural characterization of reagents generated in situ or of reactive intermediates is not feasible with conventional methods, DOSY (diffusion ordered spectroscopy) NMR spectroscopy provides a valuable method to obtain additional information.[1] Until now, pulsed field gradient (PFG) spin-echo NMR experiments were employed mainly in organic and pharmaceutical chemistry for the study of aggregation and binding processes.[1d, 2] More recently, however, with the introduction of DOSY NMR spectroscopy the traditional diffusion experiment was subject to a remarkable advance.[3] By presenting the results in a π2D spectrumTM–the chemical shift is displayed in one dimension, the diffusion in the other–the resolution and assignment of even complex mixtures are greatly facilitated. The surprising similarity of this concept as well as the manner of presentation led to the description of DOSY NMR spectroscopy as πNMR chromatographyTM. Although this technique is also extraordinarily well-suited for applications in organometallic and inorganic chemistry in solution, it has attracted little attention.[4] Herein we demonstrate the usefulness of DOSY NMR spectroscopy by presenting a new application. In the course of the present investigation, DOSY NMR spectroscopy has been successfully applied for the first time to determine the molecularity of an intermediate in the reaction of [Cp2Zr(Cl)H] (1) with CO2 (Scheme 1). Recently, we

Inhibition of LOX by flavonoids: a structure-activity relationship study.

The lipoxygenase (LOX) products have been identified as mediators of a series of inflammatory diseases, namely rheumatoid arthritis, inflammatory bowel disease, psoriasis, allergic rhinitis, atherosclerosis and certain types of cancer. Hence, LOX inhibitors are of interest for the modulation of these phenomena and resolution of the inflammatory processes. During LOX activity, peroxyl radical complexes are part of the reaction and may function as sources of free radicals. Thus antioxidants, such as flavonoids, capable of inhibiting lipid peroxidation and scavenging free radicals, may act as LOX inhibitors. The aim of this work was to assess the structure-activity relationship among a series of flavonoids concerning 5-LOX inhibition, through a systematic study of the inhibition of the formation of LTB4 in human neutrophils. The type of inhibition of the flavonoids was further studied using soybean LOX, type I, and Saturation Transfer Difference (1)H NMR (STD-(1)H NMR) was used to characterize the binding epitopes of the compounds to LOX-1. The obtained results reinforce flavonoids as effective inhibitors of LTB4 production in human neutrophils. It was also possible to establish a structure/activity relationship for the inhibitory activity and the type of inhibition.

Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

The organic acids present in beer provide important information on the products quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

Solvation of Carbon Dioxide in [C4mim][BF4] and [C4mim][PF6] Ionic Liquids Revealed by High-Pressure NMR Spectroscopy†

Where is CO2 ? The intermolecular interactions of [C4 mim]BF4 and [C4 mim]PF6 ionic liquids and CO2 have been determined by high-pressure NMR spectroscopy in combination with molecular dynamic simulations. The anion and the cation are both engaged in interactions with CO2 . A detailed picture of CO2 solvation in these ILs is provided. CO2 solubility is essentially determined by the microscopic structure of the IL.

Binding of Ibuprofen, Ketorolac, and Diclofenac to COX-1 and COX-2 Studied by Saturation Transfer Difference NMR

Saturation transfer difference NMR (STD-NMR) spectroscopy has emerged as a powerful screening tool and a straightforward way to study the binding epitopes of active compounds in early stage lead discovery in pharmaceutical research. Here we report the application of STD-NMR to characterize the binding of the anti-inflammatory drugs ibuprofen, diclofenac, and ketorolac to COX-1 and COX-2. Using well-studied COX inhibitors and by comparing STD signals with crystallographic structures, we show that there is a relation between the orientations of ibuprofen and diclofenac in the COX-2 active site and the relative STD responses detected in the NMR experiments. On the basis of this analysis, we propose that ketorolac should bind to the COX-2 active site in an orientation similar to that of diclofenac. We also show that the combination of STD-NMR with competition experiments constitutes a valuable tool to address the recently proposed behavior of COX-2 as functional heterodimers and complements enzyme activity studies in the effort to rationalize COX inhibition mechanisms.

Density functional study of proline-catalyzed intramolecular Baylis-Hillman reactions.

The mechanisms of proline-catalyzed and imidazole-co-catalyzed intramolecular Baylis-Hillman reactions have been studied by using density functional theory methods at the B3LYP/6-31G(d,p) level of theory. A polarizable continuum model (PCM B3LYP/6-31++G(d,p)//B3LYP/6-31G(d,p)) was used to describe solvent effects. Different reaction pathways were investigated, which indicated that water is an important catalyst in the imine/enamine conversion step in the absence of imidazole. When imidazole is used as a co-catalyst, water is still important in the imidazole addition step, but is not present in the Baylis-Hillman cyclization step. The computational data has allowed us to rationalize the experimental outcome of the intramolecular Baylis-Hillman reaction, validating some of the mechanistic steps proposed in the literature, as well as to propose new ones that considerably change and improve our understanding of the full reaction path.

A Rational Approach to CO2 Capture by Imidazolium Ionic Liquids: Tuning CO2 Solubility by Cation Alkyl Branching

Branching at the alkyl side chain of the imidazolium cation in ionic liquids (ILs) was evaluated towards its effect on carbon dioxide (CO2 ) solubilization at 10 and 80 bar (1 bar=1×10(5)  Pa). By combining high-pressure NMR spectroscopy measurements with molecular dynamics simulations, a full description of the molecular interactions that take place in the IL-CO2 mixtures can be obtained. The introduction of a methyl group has a significant effect on CO2 solubility in comparison with linear or fluorinated analogues. The differences in CO2 solubility arise from differences in liquid organization caused by structural changes in the cation. ILs with branched cations have similar short-range cation-anion orientations as those in ILs with linear side chains, but present differences in the long-range order. The introduction of CO2 does not cause perturbations in the former and benefits from the differences in the latter. Branching at the cation results in sponge-like ILs with enhanced capabilities for CO2 capture.

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules – an NMR, X‐ray crystallography and computational chemistry approach

The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi‐subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate‐binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X‐ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies.

Development of PMMA membranes functionalized with hydroxypropyl-β-cyclodextrins for controlled drug delivery using a supercritical CO2-assisted technology

Cyclodextrin-containing polymers have proved themselves to be useful for controlled release. Herein we describe the preparation of membranes of poly(methylmethacrylate) (PMMA) containing hydroxypropyl-beta-cyclodextrins (HP-beta-CDs) using a supercritical CO(2)-assisted phase inversion method, for potential application as drug delivery devices. Results are reported on the membrane preparation, physical properties, and drug elution profile of a model drug. The polymeric membranes were obtained with HP-beta-CD contents ranging from 0 to 33.4 wt%, by changing the composition of the casting solution, and were further impregnated with ibuprofen using supercritical carbon dioxide (scCO(2)) in batch mode. The influence of the membrane functionalization in the controlled release of ibuprofen was studied by performing in vitro experiments in buffer solution pH at 7.4. The release of the anti-inflammatory drug could be tuned by varying the cyclodextrin content on the membranes.