NMR Discrimination of d- and l-α-Amino Acids at Submicromolar Concentration via Parahydrogen-Induced Hyperpolarization.

Differentiation of enantiomers represents an important research area for pharmaceutical, chemical, and food industries. However, enantiomer separation is a laborious task that demands complex analytical techniques, specialized equipment, and expert personnel. In this respect, discrimination and quantification of d- and l-α-amino acids is no exception, generally requiring extensive sample manipulation, including isolation, functionalization, and chiral separation. This complex sample treatment results in high time costs and potential biases in the quantitative determination. Here, we present an approach based on the combination of non-hydrogenative parahydrogen-induced hyperpolarization and nuclear magnetic resonance that allows detection, discrimination, and quantification of d- and l-α-amino acids in complex mixtures such as biofluids and food extracts down to submicromolar concentrations. Importantly, this method can be directly applied to the system under investigation without any prior isolation, fractionation, or functionalization step.

Analysis of Complex Mixtures by Chemosensing NMR Using para-Hydrogen-Induced Hyperpolarization.

Nuclear magnetic resonance (NMR) is a powerful technique for chemical analysis. The use of NMR to investigate dilute analytes in complex systems is, however, hampered by its relatively low sensitivity. An additional obstacle is represented by the NMR signal overlap. Because solutes in a complex mixture are usually not isotopically labeled, NMR studies are often limited to 1H measurements, which, because of the modest dispersion of the 1H resonances (typically ∼10 ppm), can result in challenging signal crowding. The low NMR sensitivity issue can be alleviated by nuclear spin hyperpolarization (i.e., transiently increasing the differences in nuclear spin populations), which determines large NMR signal enhancements. This has been demonstrated for hyperpolarization methods such as dynamic nuclear polarization, spin-exchange optical pumping and para-hydrogen-induced polarization (PHIP). In particular, PHIP has grown into a fast, efficient, and versatile technique since the recent discovery of non-hydrogenative routes to achieve nuclear spin hyperpolarization.For instance, signal amplification by reversible exchange (SABRE) can generate proton as well as heteronuclear spin hyperpolarization in a few seconds in compounds that are able to transiently bind to an iridium catalyst in the presence of para-hydrogen in solution. The hyperpolarization transfer catalyst acts as a chemosensor in the sense that it is selective for analytes that can coordinate to the metal center, such as nitrogen-containing aromatic heterocycles, sulfur heteroaromatic compounds, nitriles, Schiff bases, diaziridines, carboxylic acids, and amines. We have demonstrated that the signal enhancement achieved by SABRE allows rapid NMR detection and quantification of a mixture of substrates down to low-micromolar concentration. Furthermore, in the transient complex, the spin configuration of p-H2 can be easily converted to spin hyperpolarization to produce up to 1000-fold enhanced NMR hydride signals. Because the hydrides' chemical shifts are highly sensitive to the structure of the analyte associating with the iridium complex, they can be employed as hyperpolarized "probes" to signal the presence of specific compounds in the mixture. This indirect detection of the analytes in solution provides important benefits in the case of complex systems, as hydrides resonate in a region of the 1H spectrum (at ca. -20 ppm) that is generally signal-free. The enhanced sensitivity provided by non-hydrogenative PHIP (nhPHIP), together with the absence of interference from the complex matrix (usually resonating between 0 and 10 ppm), set the detection limit for this NMR chemosensor down to sub-µM concentrations, approximately 3 orders of magnitude lower than for conventional NMR. This nhPHIP approach represents, therefore, a powerful tool for NMR analysis of dilute substrates in complex mixtures as it addresses at once the issues of signal crowding and NMR sensitivity. Importantly, being performed at high field inside the NMR spectrometer, the method allows for rapid acquisition of multiple scans, multidimensional hyperpolarized NMR spectra, in a fashion comparable to that of standard NMR measurements.In this Account, we focus on our chemosensing NMR technology, detailing its principles, advantages, and limitations and presenting a number of applications to real systems such as biofluids, beverages, and natural extracts.

Cationic Geminoid Peptide Amphiphiles Inhibit DENV2 Protease, Furin, and Viral Replication.

Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.

Parahydrogen Hyperpolarization Allows Direct NMR Detection of α-Amino Acids in Complex (Bio)mixtures.

The scope of non-hydrogenative parahydrogen hyperpolarization (nhPHIP) techniques has been expanding over the last years, with the continuous addition of important classes of substrates. For example, pyruvate can now be hyperpolarized using the Signal Amplification By Reversible Exchange (SABRE) technique, offering a fast, efficient and low-cost PHIP alternative to Dynamic Nuclear Polarization for metabolic imaging studies. Still, important biomolecules such as amino acids have so far resisted PHIP, unless properly functionalized. Here, we report on an approach to nhPHIP for unmodified α-amino acids that allows their detection and quantification in complex mixtures at sub-micromolar concentrations. This method was tested on human urine, in which natural α-amino acids could be measured after dilution with methanol without any additional sample treatment.

Monitoring Heterogeneously Catalyzed Hydrogenation Reactions at Elevated Pressures Using In-Line Flow NMR.

We present a novel setup that can be used for the in-line monitoring of solid-catalyzed gas-liquid reactions. The method combines the high sensitivity and resolution of a stripline NMR detector with a microfluidic network that can withstand elevated pressures. In our setup we dissolve hydrogen gas in the solvent, then flow it with the added substrate through a catalyst cartridge, and finally flow the reaction mixture directly through the stripline NMR detector. The method is quantitative and can be used to determine the solubility of hydrogen gas in liquids; it allows in-line monitoring of hydrogenation reactions and can be used to determine the reaction kinetics of these reactions. In this work, as proof of concept we demonstrate the optimization of the Pd-catalyzed hydrogenation reactions of styrene, phenylacetylene, cyclohexene, and hex-5-en-2-one in a microfluidic context.

Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus.

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

Trace analysis in water-alcohol mixtures by continuous p-H2 hyperpolarization at high magnetic field.

Nuclear magnetic resonance (NMR) studies of complex mixtures are often limited by the low sensitivity of the technique and by spectral overlap. We have recently reported on an NMR chemosensor on the basis of para-Hydrogen Induced Polarization that potentially addresses both these issues, albeit for specific classes of compounds. This approach makes use of Signal Amplification By Reversible Exchange (SABRE) catalysts in methanol and allows selective detection and quantification of dilute analytes in complex mixtures. Herein, we demonstrate that, despite a large decrease in attained hyperpolarization, this method can be extended to water-alcohol mixtures. Our approach was tested on whisky, where nitrogenous heterocyclic flavor components at low-micromolar concentration could be detected and quantified.

Direct Hyperpolarization of Nitrogen-15 in Aqueous Media with Parahydrogen in Reversible Exchange.

Signal amplification by reversible exchange (SABRE) is an inexpensive, fast, and even continuous hyperpolarization technique that uses para-hydrogen as hyperpolarization source. However, current SABRE faces a number of stumbling blocks for translation to biochemical and clinical settings. Difficulties include inefficient polarization in water, relatively short-lived 1H-polarization, and relatively limited substrate scope. Here we use a water-soluble polarization transfer catalyst to hyperpolarize nitrogen-15 in a variety of molecules with SABRE-SHEATH (SABRE in shield enables alignment transfer to heteronuclei). This strategy works in pure H2O or D2O solutions, on substrates that could not be hyperpolarized in traditional 1H-SABRE experiments, and we record 15N T1 relaxation times of up to 2 min.

Peptide-Appended Permethylated ß-Cyclodextrins with Hydrophilic and Hydrophobic Spacers.

A novel synthetic methodology, employing a combination of the strain-promoted azide-alkyne cycloaddition and maleimide-thiol reactions, for the preparation of permethylated ß-cyclodextrin-linker-peptidyl conjugates is reported. Two different bifunctional maleimide cross-linking probes, the polyethylene glycol containing hydrophilic linker bicyclo[6.1.0] nonyne-maleimide and the hydrophobic 5'-dibenzoazacyclooctyne-maleimide, were attached to azide-appended permethylated ß-cyclodextrin. The successfully introduced maleimide function was exploited to covalently graft a cysteine-containing peptide (Ac-Tyr-Arg-Cys-Amide) to produce the target conjugates. The final target compounds were isolated in high purity after purification by isocratic preparative reverse-phase high-performance liquid chromatography. This novel synthetic approach is expected to give access to many different cyclodextrin-linker peptides.

Design of Radioiodinated Pharmaceuticals: Structural Features Affecting Metabolic Stability towards in Vivo Deiodination.

Radioiodinated pharmaceuticals are convenient tracers for clinical and research investigations because of the relatively long half-lives of radioactive iodine isotopes (i.e., 123I, 124I, and 131I) and the ease of their chemical insertion. Their application in radionuclide imaging and therapy may, however, be hampered by poor in vivo stability of the C-I bond. After an overview of the use of iodine in biology and nuclear medicine, we present here a survey of the catabolic pathways for iodinated xenobiotics, including their biodistribution, accumulation, and biostability. We summarize successful rational improvements in the biostability and conclude with general guidelines for the design of stable radioiodinated pharmaceuticals. It appears to be necessary to consider the whole molecule, rather than the radioiodinated fragment alone. Iodine radionuclides are generally retained in vivo on sp2 carbon atoms in iodoarenes and iodovinyl moieties, but not in iodinated heterocycles or on sp3 carbon atoms. Iodoarene substituents also have an influence, with increased in vivo deiodination in the cases of iodophenols and iodoanilines, whereas methoxylation and difluorination improve biostability.

DOSY Analysis of Micromolar Analytes: Resolving Dilute Mixtures by SABRE Hyperpolarization.

DOSY is an NMR spectroscopy technique that resolves resonances according to the analytes' diffusion coefficients. It has found use in correlating NMR signals and estimating the number of components in mixtures. Applications of DOSY in dilute mixtures are, however, held back by excessively long measurement times. We demonstrate herein, how the enhanced NMR sensitivity provided by SABRE hyperpolarization allows DOSY analysis of low-micromolar mixtures, thus reducing the concentration requirements by at least 100-fold.

NMR-Based Chemosensing via p-H2 Hyperpolarization: Application to Natural Extracts.

When dealing with trace analysis of complex mixtures, NMR suffers from both low sensitivity and signal overlap. NMR chemosensing, in which the association between an analyte and a receptor is "signaled" by an NMR response, has been proposed as a valuable analytical tool for biofluids and natural extracts. Such chemosensors offer the possibility to simultaneously detect and distinguish different analytes in solution, which makes them particularly suitable for analytical applications on complex mixtures. In this study, we have combined NMR chemosensing with nuclear spin hyperpolarization. This was realized using an iridium complex as a receptor in the presence of parahydrogen: association of the target analytes to the metal center results in approximately 1000-fold enhancement of the NMR response. This amplification allows the detection, identification, and quantification of analytes at low-micromolar concentrations, provided they can weakly associate to the iridium chemosensor. Here, our NMR chemosensing approach was applied to the quantitative determination of several flavor components in methanol extracts of ground coffee.

A New Ir-NHC Catalyst for Signal Amplification by Reversible Exchange in D2 O.

NMR signal amplification by reversible exchange (SABRE) has been observed for pyridine, methyl nicotinate, N-methylnicotinamide, and nicotinamide in D2 O with the new catalyst [Ir(Cl)(IDEG)(COD)] (IDEG=1,3-bis(3,4,5-tris(diethyleneglycol)benzyl)imidazole-2-ylidene). During the activation and hyperpolarization steps, exclusively D2 O was used, resulting in the first fully biocompatible SABRE system. Hyperpolarized (1) H substrate signals were observed at 42.5 MHz upon pressurizing the solution with parahydrogen at close to the Earth's magnetic field, at concentrations yielding barely detectable thermal signals. Moreover, 42-, 26-, 22-, and 9-fold enhancements were observed for nicotinamide, pyridine, methyl nicotinate, and N-methylnicotinamide, respectively, in conventional 300 MHz studies. This research opens up new opportunities in a field in which SABRE has hitherto primarily been conducted in CD3 OD. This system uses simple hardware, leaves the substrate unaltered, and shows that SABRE is potentially suitable for clinical purposes.

Early stages of catalyst aging in the iridium mediated water oxidation reaction.

When exposed to a potential exceeding 1.5 V versus RHE for several minutes the molecular iridium bishydroxide complex bearing a pentamethylcyclopentadienyl and a N-dimethylimidazolin-2-ylidene ligand spontaneously adsorbs onto the surface of glassy carbon and gold electrodes. Simultaneously with the adsorption of the material on the electrode, the evolution of dioxygen is detected and modifications of the catalyst structure are observed. XPS and XAS studies reveal that the species present at the electrode interface is best described as a partly oxidized molecular species rather than the formation of large aggregates of iridium oxide. These findings are in line with the unique kinetic profile of the parent complex in the water oxidation reaction.

Computational (DFT) and Experimental (EXAFS) Study of the Interaction of [Ir(IMes)(H)2 (L)3 ] with Substrates and Co-substrates Relevant for SABRE in Dilute Systems.

Signal amplification by reversible exchange (SABRE) is an emerging hyperpolarization method in NMR spectroscopy, in which hyperpolarization is transferred through the scalar coupling network of para-hydrogen derived hydrides in a metal complex to a reversibly bound substrate. Substrates can even be hyperpolarized at concentrations below that of the metal complex by addition of a suitable co-substrate. Here we investigate the catalytic system used for trace detection in NMR spectroscopy with [Ir(IMes)(H)2 (L)3 ](+) (IMes=1,3-dimesitylimidazol-2-ylidene) as catalyst, pyridine as a substrate and 1-methyl-1,2,3-triazole as co-substrate in great detail. With density functional theory (DFT), validated by extended X-ray absorption fine structure (EXAFS) experiments, we provide explanations for the relative abundance of the observed metal complexes, as well as their contribution to SABRE. We have established that the interaction between iridium and ligands cis to IMes is weaker than that with the trans ligand, and that in mixed complexes with pyridine and triazole, the latter preferentially takes up the trans position.

2D†NMR Trace Analysis by Continuous Hyperpolarization at High Magnetic Field.

Nuclear magnetic resonance is often the technique of choice in chemical analysis because of its sensitivity to molecular structure, quantitative character, and straightforward sample preparation. However, determination of trace analytes in complex mixtures is generally limited by low sensitivity and extensive signal overlap. Here, we present an approach for continuous hyperpolarization at high magnetic field that is based on signal amplification by reversible exchange (SABRE) and can be straightforwardly incorporated in multidimensional NMR experiments. This method was implemented in a 2D correlation experiment that allows detection and quantification of analytes at nanomolar concentration in complex solutions.

Quantitative trace analysis of complex mixtures using SABRE hyperpolarization.

Signal amplification by reversible exchange (SABRE) is an emerging nuclear spin hyperpolarization technique that strongly enhances NMR signals of small molecules in solution. However, such signal enhancements have never been exploited for concentration determination, as the efficiency of SABRE can strongly vary between different substrates or even between nuclear spins in the same molecule. The first application of SABRE for the quantitative analysis of a complex mixture is now reported. Despite the inherent complexity of the system under investigation, which involves thousands of competing binding equilibria, analytes at concentrations in the low micromolar range could be quantified from single-scan SABRE spectra using a standard-addition approach.

Toward nanomolar detection by NMR through SABRE hyperpolarization.

SABRE is a nuclear spin hyperpolarization technique based on the reversible association of a substrate molecule and para-hydrogen (p-H2) to a metal complex. During the lifetime of such a complex, generally fractions of a second, the spin order of p-H2 is transferred to the nuclear spins of the substrate molecule via a transient scalar coupling network, resulting in strongly enhanced NMR signals. This technique is generally applied at relatively high concentrations (mM), in large excess of substrate with respect to metal complex. Dilution of substrate ligands below stoichiometry results in progressive decrease of signal enhancement, which precludes the direct application of SABRE to the NMR analysis of low concentration (µM) solutions. Here, we show that the efficiency of SABRE at low substrate concentrations can be restored by addition of a suitable coordinating ligand to the solution. The proposed method allowed NMR detection below 1 µM in a single scan.

The benefit of the European User Community from transnational access to national radiation facilities.

Transnational access (TNA) to national radiation sources is presently provided via programmes of the European Commission by BIOSTRUCT-X and CALIPSO with a major benefit for scientists from European countries. Entirely based on scientific merit, TNA allows all European scientists to realise synchrotron radiation experiments for addressing the Societal Challenges promoted in HORIZON2020. In addition, by TNA all European users directly take part in the development of the research infrastructure of facilities. The mutual interconnection of users and facilities is a strong prerequisite for future development of the research infrastructure of photon science. Taking into account the present programme structure of HORIZON2020, the European Synchrotron User Organization (ESUO) sees considerable dangers for the continuation of this successful collaboration in the future.

Solution scattering studies of the hierarchical assembly of porphyrin trimers based on benzene triscarboxamide.

The self-assembly of achiral and chiral porphyrin trimers based on benzene triscarboxamide in solution is studied with the help of NMR, FT-IR, UV-vis, and CD spectroscopy. These studies revealed that in apolar solvents the porphyrin trimers self-assembled in columnar stacks via a combination of hydrogen bonding and π-π stacking interactions. While the critical aggregation constant is about 0.2 mM in chloroform, aggregation already occurs at micromolar concentrations in n-hexane. Small angle neutron scattering (SANS) studies in chloroform, toluene, and n-hexane confirmed aggregation of the trimers into columnar stacks. In chloroform and n-hexane, but not in toluene, the trimers gelated the solvent. In chloroform the stacks of the achiral trimer were found to contain on average about 70 molecules, while in toluene the stacks were much smaller and contained on average 7-9 molecules. In n-hexane the SANS studies revealed that the chiral trimer formed a gel with an average mesh size of the transient network of chains of approximately 90 nm, with chains being built up from effective cylindrical aggregates with an average length of 20 nm.