1H HR MAS NMR metabolomic and non-destructive 2D NMR relaxometry to assess internal quality in apples.

NMR can be considered a multi-scale multidimensional technology in the sense that it provides both spatial insight at macroscopic (MRI) or microscopic level (relaxometry), together with chemical characterization (HR-MAS). In this study 296 apples (from 4 cultivars) were MRI screened (20 slices per fruit) among which 7 fruits were used for metabolomic study by 1H HR MAS in order to assess various chemical shifts: malic acid, sucrose, glucose, fructose and ethanol. On the first season, tissue samples were taken from the sound and affected apples (near the core, centre and outer part of the mesocarp) belonging to sound and affected locations, while on the second season, tissue samples were focused on the comparison between sound and affected tissue. Beside, MRI and 2D non-destructive relaxometry (on whole fruits, and localized tissue) where performed on 72 and 12 apples respectively in order to compare features at macroscopic (tissue) and microscopic (subcellular) level. HR MAS shows higher content of ?-glucose, ?-glucose, malic acid and aromatic compounds in watercore affected tissues from both seasons, while sound tissue reflects higher sucrose. Microscopic (subcellular) degradation of tissue varies according to disorder development and is in good accordance with macroscopic characterization with MRI.

Application of NMR techniques for the evaluation of fruit internal quality and physical properties of food

Los alimentos son sistemas complejos, formados por diversas estructuras a diferentes escalas: macroscópica y microscópica. Muchas propiedades de los alimentos, que son importantes para su procesamiento, calidad y tratamiento postcosecha, están relacionados con su microestructura. La presente tesis doctoral propone una metodología completa para la determinación de la estructura de alimentos desde un punto de vista multi-escala, basándose en métodos de Resonancia Magnética Nuclear (NMR). Las técnicas de NMR son no invasivas y no destructivas y permiten el estudio tanto de macro- como de microestructura. Se han utilizado distintos procedimientos de NMR dependiendo del nivel que se desea estudiar. Para el nivel macroestructural, la Imagen de Resonancia Magnética (MRI) ha resultado ser muy útil para la caracterización de alimentos. Para el estudio microestructural, la MRI requiere altos tiempos de adquisición, lo que hace muy difícil la transferencia de esta técnica a aplicaciones en industria. Por tanto, la optimización de procedimientos de NMR basados en secuencias relaxometría 2D T1/T2 ha resultado ser una estrategia primordial en esta tesis. Estos protocolos de NMR se han implementado satisfactoriamente por primera vez en alto campo magnético. Se ha caracterizado la microestructura de productos alimentarios enteros por primera vez utilizando este tipo de protocolos. Como muestras, se han utilizado dos tipos de productos: modelos de alimentos y alimentos reales (manzanas). Además, como primer paso para su posterior implementación en la industria agroalimentaria, se ha mejorado una línea transportadora, especialmente diseñada para trabajar bajo condiciones de NMR en trabajos anteriores del grupo LPF-TAGRALIA. Se han estudiado y seleccionado las secuencias más rápidas y óptimas para la detección de dos tipos de desórdenes internos en manzanas: vitrescencia y roturas internas. La corrección de las imágenes en movimiento se realiza en tiempo real. Asimismo, se han utilizado protocolos de visión artificial para la clasificación automática de manzanas potencialmente afectadas por vitrescencia. El presente documento está dividido en diferentes capítulos: el Capítulo 2 explica los antecedentes de la presente tesis y el marco del proyecto en el que se ha desarrollado. El Capítulo 3 recoge el estado del arte. El Capítulo 4 establece los objetivos de esta tesis doctoral. Los resultados se dividen en cinco sub-secciones (dentro del Capítulo 5) que corresponden con los trabajos publicados bien en revistas revisadas por pares, bien en congresos internacionales o bien como capítulos de libros revisados por pares. La Sección 5.1. es un estudio del desarrollo de la vitrescencia en manzanas mediante MRI y lo relaciona con la posición de la fruta dentro de la copa del árbol. La Sección 5.2 presenta un trabajo sobre macro- y microestructura en modelos de alimentos. La Sección 5.3 es un artículo en revisión en una revista revisada por pares, en el que se hace un estudio microestrcutural no destructivo mediante relaxometría 2D T1/T2. la Sección 5.4, hace una comparación entre manzanas afectadas por vitrescencia mediante dos técnicas: tomografía de rayos X e MRI, en manzana. Por último, en la Sección 5.5 se muestra un trabajo en el que se hace un estudio de secuencias de MRI en línea para la evaluación de calidad interna en manzanas. Los siguientes capítulos ofrecen una discusión y conclusiones (Capítulo 6 y 7 respectivamente) de todos los capítulos de esta tesis doctoral. Finalmente, se han añadido tres apéndices: el primero con una introducción de los principios básicos de resonancia magnética nuclear (NMR) y en los otros dos, se presentan sendos estudios sobre el efecto de las fibras en la rehidratación de cereales de desayuno extrusionados, mediante diversas técnicas. Ambos trabajos se presentaron en un congreso internacional. Los resultados más relevantes de la presente tesis doctoral, se pueden dividir en tres grandes bloques: resultados sobre macroestructura, resultados sobre microestructura y resultados sobre MRI en línea. Resultados sobre macroestructura: - La imagen de resonancia magnética (MRI) se aplicó satisfactoriamente para la caracterización de macroestructura. En particular, la reconstrucción 3D de imágenes de resonancia magnética permitió identificar y caracterizar dos tipos distintos de vitrescencia en manzanas: central y radial, que se caracterizan por el porcentaje de daño y la conectividad (número de Euler). - La MRI proveía un mejor contraste para manzanas afectadas por vitrescencia que las imágenes de tomografía de rayos X (X-Ray CT), como se pudo verificar en muestras idénticas de manzana. Además, el tiempo de adquisición de la tomografía de rayos X fue alrededor de 12 veces mayor (25 minutos) que la adquisición de las imágenes de resonancia magnética (2 minutos 2 segundos). Resultados sobre microestructura: - Para el estudio de microestructura (nivel subcelular) se utilizaron con éxito secuencias de relaxometría 2D T1/T2. Estas secuencias se usaron por primera vez en alto campo y sobre piezas de alimento completo, convirtiéndose en una forma no destructiva de llevar a cabo estudios de microestructura. - El uso de MRI junto con relaxometría 2D T1/T2 permite realizar estudios multiescala en alimentos de forma no destructiva. Resultados sobre MRI en línea: - El uso de imagen de resonancia magnética en línea fue factible para la identificación de dos tipos de desórdenes internos en manzanas: vitrescencia y podredumbre interna. Las secuencias de imagen tipo FLASH resultaron adecuadas para la identificación en línea de vitrescencia en manzanas. Se realizó sin selección de corte, debido a que la vitrescencia puede desarrollarse en cualquier punto del volumen de la manzana. Se consiguió reducir el tiempo de adquisición, de modo que se llegaron a adquirir 1.3 frutos por segundos (758 ms por fruto). Las secuencias de imagen tipo UFLARE fueron adecuadas para la detección en línea de la podredumbre interna en manzanas. En este caso, se utilizó selección de corte, ya que se trata de un desorden que se suele localizar en la parte central del volumen de la manzana. Se consiguió reducir el tiempo de adquisicón hasta 0.67 frutos por segundo (1475 ms por fruto). En ambos casos (FLASH y UFLARE) fueron necesarios algoritmos para la corrección del movimiento de las imágenes en tiempo real. ABSTRACT Food is a complex system formed by several structures at different scales: macroscopic and microscopic. Many properties of foods that are relevant to process engineering or quality and postharvest treatments are related to their microstructure. This Ph.D Thesis proposes a complete methodology for food structure determination, in a multiscale way, based on the Nuclear Magnetic Resonance (NMR) phenomenon since NMR techniques are non-invasive and non-destructive, and allow both, macro- and micro-structure study. Different NMR procedures are used depending on the structure level under study. For the macrostructure level, Magnetic Resonance Imaging (MRI) revealed its usefulness for food characterization. For microstructure insight, MRI required high acquisition times, which is a hindrance for transference to industry applications. Therefore, optimization of NMR procedures based on T1/T2 relaxometry sequences was a key strategy in this Thesis. These NMR relaxometry protocols, are successfully implemented in high magnetic field. Microstructure of entire food products have been characterized for the first time using these protocols. Two different types of food products have been studied: food models and actual food (apples). Furthermore, as a first step for the food industry implementation, a grading line system, specially designed for working under NMR conditions in previous works of the LPF-TAGRALIA group, is improved. The study and selection of the most suitable rapid sequence to detect two different types of disorders in apples (watercore and internal breakdown) is performed and the real time image motion correction is applied. In addition, artificial vision protocols for the automatic classification of apples potentially affected by watercore are applied. This document is divided into seven different chapters: Chapter 2 explains the thesis background and the framework of the project in which it has been worked. Chapter 3 comprises the state of the art. Chapter 4 establishes de objectives of this Ph.D thesis. The results are divided into five different sections (in Chapter 5) that correspond to published peered reviewed works. Section 5.1 assesses the watercore development in apples with MRI and studies the effect of fruit location in the canopy. Section 5.2 is an MRI and 2D relaxometry study for macro- and microstructure assessment in food models. Section 5.3 is a non-destructive microstructural study using 2D T1/T2 relaxometry on watercore affected apples. Section 5.4 makes a comparison of X-ray CT and MRI on watercore disorder of different apple cultivars. Section 5.5, that is a study of online MRI sequences for the evaluation of apple internal quality. The subsequent chapters offer a general discussion and conclusions (Chapter 6 and Chapter 7 respectively) of all the works performed in the frame of this Ph.D thesis (two peer reviewed journals, one book chapter and one international congress).Finally, three appendices are included in which an introduction to NMR principles is offered and two published proceedings regarding the effect of fiber on the rehydration of extruded breakfast cereal are displayed. The most relevant results can be summarized into three sections: results on macrostructure, results on microstructure and results on on-line MRI. Results on macrostructure: - MRI was successfully used for macrostructure characterization. Indeed, 3D reconstruction of MRI in apples allows to identify two different types of watercore (radial and block), which are characterized by the percentage of damage and the connectivity (Euler number). - MRI provides better contrast for watercore than X-Ray CT as verified on identical samples. Furthermore, X-Ray CT images acquisition time was around 12 times higher (25 minutes) than MRI acquisition time (2 minutes 2 seconds). Results on microstructure: - 2D T1/T2 relaxometry were successfully applied for microstructure (subcellular level) characterization. 2D T1/T2 relaxometry sequences have been applied for the first time on high field for entire food pieces, being a non-destructive way to achieve microstructure study. - The use of MRI together with 2D T1/T2 relaxometry sequences allows a non-destructive multiscale study of food. Results on on-line MRI: - The use of on-line MRI was successful for the identification of two different internal disorders in apples: watercore and internal breakdown. FLASH imaging was a suitable technique for the on-line detection of watercore disorder in apples, with no slice selection, since watercore is a physiological disorder that may be developed anywhere in the apple volume. 1.3 fruits were imaged per second (768 ms per fruit). UFLARE imaging is a suitable sequence for the on-line detection of internal breakdown disorder in apples. Slice selection was used, as internal breakdown is usually located in the central slice of the apple volume. 0.67 fruits were imaged per second (1475 ms per fruit). In both cases (FLASH and UFLARE) motion correction was performed in real time, during the acquisition of the images.

Non-Destructive Global and Localized 2D T1/T2 NMR Relaxometry to Resolve Microstructure in Apples Affected by Watercore.

Apples can be considered as having a complex system formed by several structures at different organization levels: macroscale (mayor que100 ?m) and microscale (menor que100 ?m). This work implements 2D T1/T2 global and localized relaxometry sequences on whole apples to be able to perform an intensive non-destructive and non-invasive microstructure study. The 2D T1/T2 cross-correlation spectroscopy allows the extraction of quantitative information about the water compartmentation in different subcellular organelles. A clear difference is found as sound apples show neat peaks for water in different subcellular compartments, such as vacuolar, cytoplasmatic and extracellular water, while in watercore-affected tissues such compartments appear merged. Localized relaxometry allows for the predefinition of slices in order to understand the microstructure of a particular region of the fruit, providing information that cannot be derived from global 2D T1/T2 relaxometry.

Power absorption measurements during NMR experiments

The heating produced by the absorption of radiofrequency (RF) has been considered a secondary undesirable effect during MRI procedures. In this work, we have measured the power absorbed by distilled water, glycerol and egg-albumin during NMR and non-NMR experiments. The samples are dielectric and examples of different biological materials. The samples were irradiated using the same RF pulse sequence, whilst the magnetic field strength was the variable to be changed in the experiments. The measurements show a smooth increase of the thermal power as the magnetic field grows due to the magnetoresistive effect in the copper antenna, a coil around the probe, which is directly heating the sample. However, in the cases when the magnetic field was the adequate for the NMR to take place, some anomalies in the expected thermal powers were observed: the thermal power was higher in the cases of water and glycerol, and lower in the case of albumin. An ANOVA test demonstrated that the observed differences between the measured power and the expected power are significant.

Non-Destructive Global and Localized 2D T1/T2 NMR Relaxometry to Resolve Microstructure in Apples Affected by Watercore

Apples can be considered as having a complex system formed by several structures at different organization levels: macroscale (>100 μm) and microscale (<100 μm). This work implements 2D T1/T2 global and localized relaxometry sequences on whole apples to be able to perform an intensive non-destructive and non-invasive microstructure study. The 2D T1/T2 cross-correlation spectroscopy allows the extraction of quantitative information about the water compartmentation in different subcellular organelles. A clear difference is found as sound apples show neat peaks for water in different subcellular compartments, such as vacuolar, cytoplasmatic and extracellular water, while in watercore-affected tissues such compartments appear merged. Localized relaxometry allows for the predefinition of slices in order to understand the microstructure of a particular region of the fruit, providing information that cannot be derived from global 2D T1/T2 relaxometry.

NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains

Polymers of N-substituted glycines (“peptoids”) containing chiral centers at the α position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of ≈6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

Calculated 11B–13C NMR chemical shift relationship in hypercoordinate methonium and boronium ions

The boronium-carbonium continuum was extended to include hypercoordinated protonated methanes and their boron analogs. The 11B NMR chemical shifts of the hypercoordinated hydriodo boron compounds and the 13C NMR chemical shifts of the corresponding isoelectronic and isostructural carbocations were calculated by using the GIAO-MP2 method. The data show good linear correlation between 11B and 13C NMR chemical shifts, which indicates that the same factors that determine the chemical shifts of the boron nuclei also govern the chemical shifts of carbon nuclei of these hypercoordinated hydriodo compounds.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase

Nuclear magnetic resonance (NMR) of isolated lignins from an Arabidopsis mutant deficient in ferulate 5-hydroxylase (F5H) and transgenic plants derived from the mutant by overexpressing the F5H gene has provided detailed insight into the compositional and structural differences between these lignins. Wild-type Arabidopsis has a guaiacyl-rich, syringyl-guaiacyl lignin typical of other dicots, with prominent β-aryl ether (β–O–4), phenylcoumaran (β–5), resinol (β–β), biphenyl/dibenzodioxocin (5–5), and cinnamyl alcohol end-group structures. The lignin isolated from the F5H-deficient fah1–2 mutant contained only traces of syringyl units and consequently enhanced phenylcoumaran and dibenzodioxocin levels. In fah1–2 transgenics in which the F5H gene was overexpressed under the control of the cauliflower mosaic virus 35S promoter, a guaiacyl-rich, syringyl/guaiacyl lignin similar to the wild type was produced. In contrast, the isolated lignin from the fah1–2 transgenics in which F5H expression was driven by the cinnamate 4-hydroxylase promoter was almost entirely syringyl in nature. This simple lignin contained predominantly β-aryl ether units, mainly with erythro-stereochemistry, with some resinol structures. No phenylcoumaran or dibenzodioxocin structures (which require guaiacyl units) were detectable. The overexpression of syringyl units in this transgenic resulted in a lignin with a higher syringyl content than that in any other plant we have seen reported.

Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers

Complete resolution of the amide resonances in a three-dimensional solid-state NMR correlation spectrum of a uniformly 15N-labeled membrane protein in oriented phospholipid bilayers is demonstrated. The three orientationally dependent frequencies, 1H chemical shift, 1H–15N dipolar coupling, and 15N chemical shift, associated with each amide resonance are responsible for resolution among resonances and provide sufficient angular restrictions for protein structure determination. Because the protein is completely immobilized by the phospholipids on the relevant NMR time scales (10 kHz), the linewidths will not degrade in the spectra of larger proteins. Therefore, these results demonstrate that solid-state NMR experiments can overcome the correlation time problem and extend the range of proteins that can have their structures determined by NMR spectroscopy to include uniformly 15N-labeled membrane proteins in phospholipid bilayers.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Prion protein NMR structure and species barrier for prion diseases

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

NMR of laser-polarized xenon in human blood

By means of optical pumping with laser light it is possible to enhance the nuclear spin polarization of gaseous xenon by four to five orders of magnitude. The enhanced polarization has allowed advances in nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI), including polarization transfer to molecules and imaging of lungs and other void spaces. A critical issue for such applications is the delivery of xenon to the sample while maintaining the polarization. Described herein is an efficient method for the introduction of laser-polarized xenon into systems of biological and medical interest for the purpose of obtaining highly enhanced NMR/MRI signals. Using this method, we have made the first observation of the time-resolved process of xenon penetrating the red blood cells in fresh human blood—the xenon residence time constant in the red blood cells was measured to be 20.4 ± 2 ms. The potential of certain biologically compatible solvents for delivery of laser-polarized xenon to tissues for NMR/MRI is discussed in light of their respective relaxation and partitioning properties.

NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: Applicability of solution 19F NMR

We report high resolution solution 19F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF3-CH2-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6–10 mg), in dodecylmaltoside, were analyzed at 20°C by solution 19F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for 19F labels at positions 67 (−0.2 ppm) and 140 (−0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution 19F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.

NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

This paper describes the NMR observation of 15N—15N and 1H—15N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6–7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2–3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.