Labile Hydrogen Atoms Research Articles

Hydrogen/deuterium exchange for the analysis of carbohydrates

Carbohydrates and glycans are integral to many biological processes, including cell-cell recognition and energy storage. However, carbohydrates are often difficult to analyze due to the high degree of isomerism present. One method being developed to distinguish these isomeric species is hydrogen/deuterium exchange-mass spectrometry (HDX-MS). In HDX-MS, carbohydrates are exposed to a deuterated reagent and the functional groups with labile hydrogen atoms, including hydroxyls and amides, exchange with the 1 amu heavier isotope, deuterium. These labels can then be detected by MS, which monitors the mass increase with the addition of D-labels. The observed rate of exchange is dependent on the exchanging functional group, the accessibility of the exchanging functional group, and the presence of hydrogen bonds. Herein, we discuss how HDX has been applied in the solution-phase, gas-phase, and during MS ionization to label carbohydrates and glycans. Additionally, we compare differences in the conformations that are labeled, the labeling timeframes, and applications of each of these methods. Finally, we comment on future opportunities for development and use of HDX-MS to analyze glycans and glycoconjugates.

Fragmentation pathways of deprotonated ortho-hydroxybenzyl alcohol.

The ortho, meta, and para isomers of hydroxybenzyl alcohol can be unequivocally distinguished by the collision-induced dissociation mass spectra of their anions. The presence of a prominent peak at m/z 121 for an elimination of a dihydrogen molecule renders the ortho-isomer spectrum markedly different from those of its meta and para congeners. Investigations carried out with deuterium-labeled isotopologues of the ortho isomer verified that the labile hydrogen atom on the hydroxyl group and one of the benzylic hydrogen atoms are specifically removed in the formation of the m/z 121 ion. The ortho-isomer spectrum also showed a prominent peak at m/z 93. Experimental data indicated that the m/z 93 product ion originates either from a two-step H2and CO elimination mechanism or from a direct loss of a HCHO molecule from the precursor anion. The intensity ratio of the m/z 93 and 94 peaks in the spectrum recorded from the m/z 124 ion generated from a sample of o-hydroxybenzyl alcohol dissolved in D2O supported the notion that the direct HCHO loss is the more dominant pathway for the generation of the phenolate ion under low activation conditions. In contrast, the two-step mechanism becomes the more dominant pathway under high collisional activation conditions. The spectrum also showed a weak peak at m/z 105 for a water loss. Based on computational data, the m/z 105 ion generated in this way appears to be a composite generated from a common ion-neutral complex intermediate in which a hydroxyl anion is positioned equidistantly between one of the benzylic hydrogens and a nearby hydrogen atom of the benzene ring. Upon activation, the complex dissociates to form either a phenide or a quinone methide anion. The reaction forming a carbon dioxide adduct under ion-mobility conditions was used to support the proposed water-loss mechanism.

Ionic conduction within non-stoichiometric N-Methylimidazole-Acetic Acid Pseudo-Protic ionic liquid mixtures

We recently suggested that proton conduction mechanisms, such as the Grotthuss mechanism, could occur in an equimolar mixture of N-methylimidazole (C1Im) and acetic acid (CH3COOH), which is a pseudo-protic ionic liquid (pPIL). Appropriate amounts of C1Im and CH3COOH were mixed in various molar ratios to investigate the concentration dependence of proton conduction. At a mole fraction of xC1Im = 0.2, the ionic conductivity was the highest, despite the highest viscosity. Proton dynamics and carriers are crucial in rapid proton conduction. To clarify the proton conduction mechanism and the identities of the chemical species within the non-equimolar pPILs [C1Im]x[CH3COOH](1 –x), pulsed-field gradient nuclear magnetic resonance and Raman spectroscopy were performed. C1HIm+ predominantly existed at xC1Im = 0.2 due to proton transfer from CH3COOH, and CH3COO–···CH3COOH hydrogen bonding occurred. With an increasing molar ratio of C1Im, the molecular complex C1Im-CH3COOH formed, with the species distribution function exhibiting a maximum at approximately xC1Im = 0.5. Although the self-diffusion coefficients were small and the viscosity increased with decreasing C1Im concentration, the ionic conductivity increased. In addition, the self-diffusion coefficients of the labile hydrogen atoms were the largest among all of the hydrogen atoms at xC1Im = 0.2, suggesting that a specific proton conduction mechanism occurred via hydrogen bonding. Furthermore, the proton conduction mechanism could be controlled by altering the composition of the pPIL.

Influence of Ultraviolet Radiation on Mechanical Properties of a Photoinitiator Compounded High Vinyl Styrene-Butadiene-Styrene Block Copolymer.

Ultraviolet curing of elastomers is a special curing technique that has gained importance over the conventional chemical crosslinking method, because the former process is faster, and thus, time-saving. Usually, a suitable photoinitiator is required to initiate the process. Ultraviolet radiation of required frequency and intensity excites the photoinitiator which abstracts labile hydrogen atoms from the polymer with the generation of free radicals. These radicals result in crosslinking of elastomers via radical–radical coupling. In the process, some photodegradation may also take place. In the present work, a high vinyl (~50%) styrene–butadiene–styrene (SBS) block copolymer which is a thermoplastic elastomer was used as the base polymer. An attempt was made to see the effect of ultraviolet radiation on the mechanical properties of the block copolymer. The process variables were time of exposure and photoinitiator concentration. Mechanical properties like tensile strength, elongation at break, modulus at different elongations and hardness of the irradiated samples were studied and compared with those of unirradiated ones. In this S-B-S block copolymer, a relatively low exposure time and low photoinitiator concentration were effective in obtaining optimized mechanical properties. Infrared spectroscopy, contact angle and scanning electron microscopy were used to characterize the results obtained from mechanical measurements.

Hydrogen-Mediated Noncovalent Interactions in Solids: What Can NMR Crystallography Tell About?

Hydrogen atoms play a crucial role in the aggregation of organic (bio)molecules through diverse number of noncovalent interactions that they mediate, such as electrostatic in proton transfer systems, hydrogen bonding, and CH–π interactions, to mention only the most prominent. To identify and adequately describe such low-energy interactions, increasingly sensitive methods have been developed over time, among which quantum chemical computations have witnessed impressive advances in recent years. For reaching the present state-of-the-art, computations had to rely on a pool of relevant experimental data, needed at least for validation, if not also for other purposes. In the case of molecular crystals, the best illustration for the synergy between computations and experiment is given by the so-called NMR crystallography approach. Originally designed to increase the confidence level in crystal structure determination of organic compounds from powders, NMR crystallography is able now to offer also a wealth of information regarding the noncovalent interactions that drive molecules to pack in a given crystalline pattern or another. This is particularly true for the noncovalent interactions which depend on the exact location of labile hydrogen atoms in the system: in such cases, NMR crystallography represents a valuable characterization tool, in some cases complementing even the standard single-crystal X-ray diffraction technique. A concise introduction in the field is made in this mini-review, which is aimed at providing a comprehensive picture with respect to the current accuracy level reached by NMR crystallography in the characterization of hydrogen-mediated noncovalent interactions in organic solids. Different types of practical applications are illustrated with the example of molecular crystals studied by our research group, but references to other representative developments reported in the literature are also made. By summarizing the major concepts and methodological progresses, the present work is also intended to be a guide to the practical potential of this relatively recent analytical tool for the scientists working in areas where crystal engineering represents the main approach for rational design of novel materials.

Hydrogen/Deuterium and 16O/18O-Exchange Mass Spectrometry Boosting the Reliability of Compound Identification.

Accurate and reliable identification of chemical compounds is the ultimate goal of mass spectrometry analyses. Currently, identification of compounds is usually based on the measurement of the accurate mass and fragmentation spectrum, chromatographic elution time, and collisional cross section. Unfortunately, despite the growth of databases of experimentally measured MS/MS spectra (such as MzCloud and Metlin) and developing software for predicting MS/MS fragments in silico from SMILES patterns (such as MetFrag, CFM-ID, and Ms-Finder), the problem of identification is still unsolved. The major issue is that the elution time and fragmentation spectra depend considerably on the equipment used and are not the same for different LC-MS systems. It means that any additional descriptors depending only on the structure of the chemical compound will be of big help for LC-MS/MS-based omics. Our approach is based on the characterization of compounds by the number of labile hydrogen and oxygen atoms in the molecule, which can be measured using hydrogen/deuterium and 16O/18O-exchange approaches. The number of labile atoms (those from -OH, -NH, ═O, and -COOH groups) can be predicted from SMILES patterns and serves as an additional structural descriptor when performing a database search. In addition, distribution of isotope labels among MS/MS fragments can be roughly predicted by software such as MetFrag or CFM-ID. Here, we present an approach utilizing the selection of structural candidates from a database on the basis of the number of functional groups and analysis of isotope labels distribution among fragments. It was found that our approach allows reduction of the search space by a factor of 10 and considerably increases the reliability of the compound identification.

Methods to Access 2-aminobenzimidazoles of Medicinal Importance

:Benzimidazole (BI) and derivatives are interesting because several of these compounds have been found to have a diversity of biological activities with clinical applications. In view of their importance, the synthesis of BI and its derivatives is still considered as a challenge for synthetic chemists. Examples of compounds used in medicinal chemistry containing BI, as important nucleus, are Astemizole (antihistaminic), Omeprazole (antiulcerative) and Rabendazole (fungicide), some of these compounds have the 2- aminobenzimidazole (2ABI) as base nucleus. The structure of 2ABI derivatives contains a cyclic guanidine moiety, which is interesting because of its free lone pairs, labile hydrogen atoms and planar delocalized structure. The delocalized 10-π electron system and the extension of the electron conjugation with the exocyclic amino group, in 2ABI, making these heterocycles to have amphoteric character. The 2ABI has been used as building blocks for the synthesis of several BI derivatives as medicinally important molecules. On these bases, herein, we present a bibliographic review concerning the recent methodologies used in the synthesis of 2ABIs, including the substituted ones.

Hydrogen-Deuterium Scrambling Based on Chemical Isotope Labeling Coupled with LC-MS: Application to Amine Metabolite Identification in Untargeted Metabolomics.

Identification of metabolites at the trace level in complex samples is still one of the major challenges in untargeted metabolomics. One formula in the metabolomic database is always corresponding to more than one candidate, which increases the difficulty and cost in the subsequent process of standard compound matching. In this study, we developed an effective method for amine metabolite identification by hydrogen-deuterium scrambling (HDS) based on chemical isotope labeling coupled with liquid chromatography-mass spectrometry (HDS-CIL-LC-MS). After d4-4-(N,N-dimethylamino)phenyl isothiocyanate (d4-DMAP) labeling, the labeled amine metabolites can produce HDS under collision-induced dissociation (CID). The HDS can effectively reflect the number of labile hydrogen atoms in amine metabolites and thus distinguish amine isomers with different functional groups. The developed HDS-CIL-LC-MS method was applied to the determination of amine metabolites in mice feces, in which the amine candidates obtained by the database based on chemical formula searching were reduced by 64% on average, which greatly reduces the cost of standard compound matching. Taken together, the developed HDS-CIL-LC-MS analysis was demonstrated to be a promising method for untargeted metabolomics and a novel strategy for deciphering tandem mass spectrometry (MS2) spectra.

Quinone methide dimers lacking labile hydrogen atoms are surprisingly excellent radical-trapping antioxidants.

Hydrogen atom transfer (HAT) is the mechanism by which the vast majority of radical-trapping antioxidants (RTAs), such as hindered phenols, inhibit autoxidation. As such, at least one weak O-H bond is the key structural feature which underlies the reactivity of phenolic RTAs. We recently observed that quinone methide dimers (QMDs) synthesized from hindered phenols are significantly more reactive RTAs than the phenols themselves despite lacking O-H bonds. Herein we describe our efforts to elucidate the mechanism by which they inhibit autoxidation. Four possible reaction paths were considered: (1) HAT from the C-H bonds on the carbon atoms which link the quinone methide moieties; (2) tautomerization or hydration of the quinone methide(s) in situ followed by HAT from the resultant phenolic O-H; (3) direct addition of peroxyl radicals to the quinone methide(s), and (4) homolysis of the weak central C-C bond in the QMD followed by combination of the resultant persistent phenoxyl radicals with peroxyl radicals. The insensitivity of the reactivity of the QMDs to substituent effects, solvent effects and a lack of kinetic isotope effects rule out the HAT reactions (mechanisms 1 and 2). Simple (monomeric) quinone methides, to which peroxyl radicals add, were found to be ca. 100-fold less reactive than the QMDs, ruling out mechanism 3. These facts, combined with the poor RTA activity we observe for a QMD with a stronger central C-C bond, support mechanism 4. The lack of solvent effects on the RTA activity of QMDs suggests that they may find application as additives to materials which contain H-bonding accepting moieties that can dramatically suppress the reactivity of conventional RTAs, such as phenols. This reactivity does not extend to biological membranes owing to the increased microviscosity of the phospholipid bilayer, which suppresses QMD dissociation in favour of recombination. Interestingly, the simple QMs were found to be very good RTAs in phospholipid bilayers - besting even the most potent form of vitamin E.

Water hydrogen uptake in biomolecules detected via nuclear magnetic phosphorescence

We introduce a new symmetry-based method for structural investigations of areas surrounding water-exchanging hydrogens in biomolecules by liquid-state nuclear magnetic resonance spectroscopy. Native structures of peptides and proteins can be solved by NMR with fair resolution, with the notable exception of labile hydrogen sites. The reason why biomolecular structures often remain elusive around exchangeable protons is that the dynamics of their exchange with the solvent hampers the observation of their signals. The new spectroscopic method we report allows to locate water-originating hydrogens in peptides and proteins via their effect on nuclear magnetic transitions similar to electronic phosphorescence, long-lived coherences. The sign of long-lived coherences excited in coupled protons can be switched by the experimenter. The different effect of water-exchanging hydrogens on long-lived coherences with opposed signs allows to pinpoint the position of these labile hydrogen atoms in the molecular framework of peptides and proteins.

Arginine Side-Chain Hydrogen Exchange: Quantifying Arginine Side-Chain Interactions in Solution.

The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side‐chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side‐chain 1Hϵ protons and thus present a method to gauge the strength of arginine side‐chain interactions. The method employs 13C‐detection and the one‐bond deuterium isotope shift observed for 15Nϵ to generate two exchanging species in 1H2O/2H2O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side‐chains in enzymes, in phase separation, and in protein interaction interfaces in general.

Charge-state Resolved Infrared Multiple Photon Dissociation (IRMPD) Spectroscopy of Ubiquitin Ions in the Gas Phase

In this study, we obtained for the first time the direct infrared multiple photon dissociation (IRMPD) spectra of ubiquitin ions in the range 2700–3750 cm−1. Ubiquitin ions with different charge states showed absorption in the two regions of 2940–3000 cm−1 and 3280–3400 cm−1. The increase of the charge state of ubiquitin ions broadened the absorption peak on the high-frequency side in the second region, indicating some hydrogen bonds were weakened due to Coulomb interaction. It is also found that the relative intensity of the absorption peak in the first region compared to the absorption peak in the second region increased with increasing charge state, making the IRMPD spectra charge-state resolved. Although it is usually reasonable to suggest the origin of the absorption in the range 2940–3000 cm−1 as the C-H bond stretching modes, the results show significantly reduced absorption after the deuteration of all labile hydrogen atoms. A possible explanation for this is that the coupling coefficients between the C-H vibrational mode and other selective modes decreased greatly after the deuteration, reducing the rate of energy redistribution and probability of consecutive IR absorption.

Synergistic hollow CoMo oxide dual catalysis for tandem oxygen transfer: Preferred aerobic epoxidation of cyclohexene to 1,2-epoxycyclohexane

The CoMo hollow bimetallic oxide has been exploited in the aerobic epoxidation of cyclohexene containing labile allylic hydrogen atoms using ethylbenzene as a solvent. Cooperativity between Co(II) and Mo(VI) sites consisting of CoOMo or Mo−Oδ−⋯Coδ+ unit on hollow oxide surfaces is investigated for controlling preferred epoxidation catalysis via tandem oxygen transfer. Various characterization techniques like XRD diffraction, N2 physisorption, TEM, and Raman, XPS, Infrared, and UV–vis spectroscopies are employed to reveal the relationship between structure of active sites and catalytic performance. Together with these comprehensive experimental and computational studies, in this unique tandem catalytic process, Co(II) sites effectively mediate the first step of the overall oxidation cycle yielding a phenylethylperoxy radical by oxygen activation [Co(II) to Co(III) O2− to phenylethyl radical and finally to a phenylethylperoxy radical]. On the other hand, Mo(VI) sites are shown to be the excellent catalytic species for the subsequent epoxidation step by the transfer of oxygen atom of the phenylethylperoxy radical to cyclohexene. Hence, a 59% epoxidation selectivity with 33% cyclohexene conversion is accomplished through the required combination of Co(II) and Mo(VI) (2:1), allowing to tune epoxidation efficiency toward higher driving forces (relative to oxygenation of allylic hydrogen atom). This remarkably different catalytic performances between Co(II) and Mo(VI) can be attributed to the stronger overall polarization ability of Mo(VI) toward σ* OO orbital of the OO unit derived from its d-type orbitals, which helps the donor-acceptor interactions with the double bond.

Characterization of stress degradation products of amodiaquine dihydrochloride by liquid chromatography with high-resolution mass spectrometry and prediction of their properties by using ADMET Predictor™.

The degradation behavior of amodiaquine dihydrochloride, an antimalarial drug, was investigated in solution as well as solid states. The drug was subjected to hydrolytic, photolytic, oxidative, and thermal stress conditions, according to International Conference on Harmonization guideline Q1A(R2). It showed extensive hydrolysis in acidic, alkaline, and neutral solutions both with and without light, while it proved to be stable to thermal and oxidative conditions. In total, six degradation products were formed, which were separated on a C8 column, employing a gradient reversed-phase high-performance liquid chromatography method in which acetonitrile and 10mM ammonium formate (pH 3.0) were used in the mobile phase. To characterize the degradation products, mass fragmentation behavior of the drug was established by direct infusion of solution to quadrupole time-of-flight and multiple-stage mass spectrometry systems. Liquid chromatography with high-resolution mass spectrometry studies were subsequently carried out on the stressed samples using the same gradient high-performance liquid chromatography method employed for the separation of the degradation products. Hydrogen/deuterium exchange studies were additionally conducted to determine the number of labile hydrogen atoms. The degradation pathway of the drug was delineated, justified by mechanistic explanation. Lastly, ADMET Predictor™ software was employed to predict relevant physicochemical and toxicity data for the degradation products.

UV-Induced Hydrogen-Atom-Transfer Processes in 3-Thio-1,2,4-triazole Isolated in Ar and H2 Low-Temperature Matrixes.

The UV-induced thione → thiol phototautomeric reaction has been studied for monomeric 3-thio-1,2,4-triazole (3-ST) isolated in low-temperature Ar and n-H2 (normal hydrogen) matrixes. Prior to any UV irradiation, monomers of 3-ST isolated in solid Ar or solid n-H2 adopted mainly the most stable thione tautomeric form, as revealed by the IR spectra. Upon UV (λ > 275 nm) irradiation of 3-ST isolated in Ar matrixes, the IR bands due to this thione form decreased, while a set of initially weak bands increased in intensity. Growing bands indicated generation of a photoproduct, which was identified as the thiol tautomer with labile hydrogen atoms attached to sulfur and N(2) atoms. The UV-induced spectral changes allowed also identification of another minor thiol tautomer of 3-ST, which was present in the matrix prior to any irradiation and did not change its population upon exposure to UV light. The identification of the observed isomeric forms was supported by comparison of their separated experimental IR spectra with the spectra theoretically predicted for the various structures of 3-ST. The thione → thiol phototautomerization that was the main UV-induced process observed for 3-ST in Ar matrixes did not occur for monomers of the compound trapped in solid n-H2.

Photo-oxidative degradation behavior of linseed oil based epoxy resin

The photo-oxidation behaviour of a biobased epoxy polymer has been studied under irradiation at wavelengths > 315 nm at 45°C and in the presence of oxygen. The resin is obtained after curing a blend of an epoxidized linseed oil (ELO) and epoxidized glycerol (EG) with methyltetrahydrophtalic anhydride (MTHPA). Molecular changes induced by the UV exposure were followed by infrared and UV-Vis spectroscopy. A mechanism involving the abstraction of the most labile hydrogen atoms of the structure and accounting for the formation of the photoproducts identified by FTIR spectroscopy has been proposed. Microhardness and Atomic Force Microscopy (AFM) equipped with a nanomechanical mapping mode were performed to determine the modification of the morphology and of the mechanical behaviour of the top surface. Microhardness and AFM results suggest that crosslinking reactions occur in competition with β scission reactions. The crosslinking reactions control the changes in mechanical properties of the top surface at short irradiation times while the β scission reactions become more competitive for long irradiation times and results in a decrease of stiffness of the surface.

Computational methods and challenges in hydrogen/deuterium exchange mass spectrometry.

Hydrogen/Deuterium exchange (HDX) has been applied, since the 1930s, as an analytical tool to study the structure and dynamics of (small) biomolecules. The popularity of using HDX to study proteins increased drastically in the last two decades due to the successful combination with mass spectrometry (MS). Together with this growth in popularity, several technological advances have been made, such as improved quenching and fragmentation. As a consequence of these experimental improvements and the increased use of protein-HDXMS, large amounts of complex data are generated, which require appropriate analysis. Computational analysis of HDXMS requires several steps. A typical workflow for proteins consists of identification of (non-)deuterated peptides or fragments of the protein under study (local analysis), or identification of the deuterated protein as a whole (global analysis); determination of the deuteration level; estimation of the protection extent or exchange rates of the labile backbone amide hydrogen atoms; and a statistically sound interpretation of the estimated protection extent or exchange rates. Several algorithms, specifically designed for HDX analysis, have been proposed. They range from procedures that focus on one specific step in the analysis of HDX data to complete HDX workflow analysis tools. In this review, we provide an overview of the computational methods and discuss outstanding challenges. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:649-667, 2017.

Stevens rearrangement of heterocyclic ammonium salts containing prop-2-yn-1-yl group

Stevens rearrangement was studied of ammonium salts where piperidinium or morpholinium groups are linked to prop-2-yn-1-yl moiety and labile hydrogen atom functions (methoxycarbonylmethyl, phenacyl, cyanomethyl). In the case of phenacyl analog furan derivatives were formed, in other cases dienamines were the reaction products.

Synthesis and Properties of Peroxy Oligomers Obtained by Telomerization Effects of the Presence of Bases

<p>New bifunctional oligomers bearing peroxy groups have been synthesized by telomerization in one step reaction. For obtaining oligoperoxides (PO) by telomerization method, epoxide compounds will react with substances containing labile hydrogen atom. Using the principle of stochiometric imbalance between diepoxy compounds and substances with mobile hydrogen atoms and employing a functional peroxide as telogen, the synthesis of PO was studied. 2,2–Di[4–(2,3–epoxy–1-propoxy)phenyl]–propane (diglycidyl ether of diphenylol propane - DGEDPP), 1,2–Di (2,3–epoxy–1-propoxy) ethane (diglycidyl ether of ethylene glycol - DGEEG) and 1,2–Epoxy–3–tert–butylperoxypropane (EP) have been synthesized by methods reported in the literature. Chemical structures have been confirmed by NMR and FTIR, number-average molecular weights M<sub>n</sub> of PO<sub>s</sub> by cryoscopy, active oxygen content [O]<sub>act</sub>. for PO<sub>s</sub> was determined by iodometry and epoxy number (<em>e.n</em>.) for PO<sub>s</sub> was measured via direct titration of PO samples. A 50% solution of potassium or sodium isopropylates in 2-propanol is used to catalyze the telomerisation. Different parameters such as ratio of components, temperature, reaction time have been optimized in vu to get well defined peroxy oligomers. The presence of peroxy groups in synthesized POs allows us to employ these compounds as curing agents for polymers containing unsaturated double bonds and may be used to improve performance of unsaturated polyester GFR systems.</p>

Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.