13C Nuclear Magnetic Resonance Signal Research Articles

NMR characterization of uniformly 13C- and/or 15N-labeled, unsulfated chondroitins with high molecular weights.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-β-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-β-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-β-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the β-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.

Molecular Characterization and Quantification of Saponins from Sugar Beet (Beta vulgaris ssp. vulgaris var. altissima)

Consumer demand for sustainably and ecologically produced food has grown in recent years. However, for numerous reasons, this demand has not always been met. New solutions are still being sought, particularly in the area of emulsifiers, an integral ingredient of many food products. The biggest challenge is the limited scope of application offered by current alternatives. While some progress has been made using foam active quillaja saponins, they are neither pleasant‐tasting nor sustainably produced. Only very little is known about other alternatives, and especially the behavior of saponins, particularly on a molecular level, is not very well understood. Moreover, it is often not known which parts of a plant contain the highest levels of saponins and are therefore most suitable for extraction.To expand the current level of knowledge about emulsifying and foam active extracts, saponin extracts were made from oat bran, beetroot as well as sugar beet and characterized in close cooperation with the Department of Food Physics and Meat Science at the University of Hohenheim. Measurements conducted on these extracts showed that foam activity is a good indicator of their emulsifying ability. The most promising one ‐ sugar beet extract ‐ was examined in more detail using taste dilution analysis, which revealed saponin fractions with a slight off‐taste and high foam activity. A series of eight saponins was obtained from these fractions and further characterized, together with three commercially available ones that were identified using non‐targeted screening. The unequivocal identification and structure elucidation was performed using a combination of liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and both one‐dimensional (1D) and two‐dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy. For most substances, this was the first time that a complete assignment of 1H and 13C NMR signals had been made. All of these saponins possess taste recognition values above 1000 μmol/L, which is higher than most values for the taste‐ active saponins reported in the literature. The foam activity of these substances was measured using a self‐developed small‐scale foam activity assay. It was measured for different concentrations of saponins in a buffer solution. The foam activity of numerous other saponins was also determined, resulting in the most comprehensive overview of the foam activities of individual saponins.In addition to these isolation and characterization activities, an LC‐MS/MS method of performing simultaneous quantitative analyses of all the obtained sugar beet saponins was developed and validated. These were quantified in several sugar beet varieties (root and leaves), different sugar beet compartments, and side streams originating during the sugar beet converting process. Great variations in mass fractions were found in these materials, ranging from 862 mg/kg to 2,452 mg/kg for the various sugar beet root varieties and from 907 mg/kg to 5,398 mg/kg for the sugar beet leave varieties. Sugar beet fiber was identified as the best saponin source, with a total saponin quantity of 12.7 g/kg, followed by dried sugar beet pulp, with a total quantity of 10.3 g/kg. As a by‐product stream, dried sugar beet pulp is highly suitable for the sustainable manufacture of saponins.The results obtained are of key significance to the utilization of sugar beet saponins as well as saponins extracted from other plant materials or by‐product streams for use as food additives.

Hyperpolarized 13C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization

AbstractHyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d‐DNP) provides a unique and general way to detect 13C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d‐DNP now encompasses the analysis of complex mixtures at natural 13C abundance. However, the application of d‐DNP in this area has been limited to metabolite extracts. Here, we report the first d‐DNP‐enhanced 13C NMR analysis of a biofluid ‐urine‐ at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Characterization of retinal chromophore and protonated Schiff base in Thermoplasmatales archaeon heliorhodopsin using solid-state NMR spectroscopy

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.

A Simplified Method of Synthesis to Obtain Zwitterionic Cellulose under Mild Conditions with Active Ionic Moieties.

A simplified procedure to synthesize zwitterionic cellulose by means of N-protected aspartic anhydride under mild conditions was developed. The preparation of modified cellulose samples was carried out under heterogeneous, aqueous conditions by reacting NH4OH-activated cellulose with aspartic anhydrides N-protected with trifluoroacetyl (TFAc) and carbobenzyloxy (Cbz). Modified cellulose samples Cel-Asp-N-TFAc and Cel-Asp-N-Cbz were characterized by Fourier Transform Infrared (FTIR) and 13C solid state Nuclear Magnetic Resonance (NMR) spectroscopy. The functionalization degree of each cellulose sample was determined by the 13C NMR signal integration values corresponding to the cellulose C1 vs. the Cα of the aspartate residue and corroborated by elemental analysis. In agreement, both analytical methods averaged a grafting degree of 20% for Cel-Asp-N-TFAc and 16% for Cel-Asp-N-Cbz. Conveniently, Cel-Asp-N-TFAc was concomitantly partially N-deprotected (65%) as determined by the ninhydrin method. The zwitterion character of this sample was confirmed by a potentiometric titration curve and the availability of these amino acid residues on the cellulose was inspected by adsorption kinetics method with a 100 mg L−1 cotton blue dye solution. In addition, the synthesis reported in the present work involves environmentally related advantages over previous methodologies developed in our group concerning to zwitterionic cellulose preparation.

The Amphotericin B-Ergosterol Complex Spans a Lipid Bilayer as a Single-Length Assembly.

Amphotericin B (AmB) is a polyene macrolide antibiotic clinically used as an antifungal drug. Its preferential complexation with ergosterol (Erg), the major sterol of fungal membranes, leads to the formation of a barrel-stave-like ion channel across a lipid bilayer. To gain a better understanding of the mechanism of action, the mode of lipid bilayer spanning provides essential information. However, because of the lack of methodologies to observe it directly, it has not been revealed for the Erg-containing channel assembly for many years. In this study, we disclosed that the AmB-Erg complex spans a lipid bilayer with a single-molecule length, using solid-state nuclear magnetic resonance (NMR) experiments. Paramagnetic relaxation enhancement by Mn2+ residing near the surface of lipid bilayers induced the depth-dependent decay of 13C NMR signals for individual carbon atoms of AmB. We found that both terminal segments, the 41-COOH group and C38-C40 methyl groups, come close to the lipid bilayer surfaces, suggesting that the AmB-Erg complex spans a palmitoyloleoylphosphatidylcholine (POPC) bilayer with a single-molecule length. Molecular dynamics simulation experiments further confirmed the stabilization of the AmB-Erg complex as a single-length spanning complex. These results provide experimental evidence of the single-length complex incorporated in the membrane by making thinner a POPC-Erg bilayer that mimics fungal membranes.

Multifunctional biocomposites based on collagen and keratin with properties for agriculture and industry applications

In this work, we optimize the performance of a previously proposed method for transferring parahydrogen induced polarization to “insensitive” spin-1/2 NMR (Nuclear Magnetic Resonance) nuclei, which have low gyromagnetic ratio and low natural abundance. By optimizing the reaction conditions and pressure of the parahydrogen gas and using adiabatically switched radiofrequency fields we achieve high polarization transfer efficiency and report carbon spin polarization of dimethyl acetylene dicarboxylate reaching 35%, which corresponds to 13C NMR signal enhancements of about 43,000 at 9.4 Tesla. Such polarization levels allow one to work with mM concentrations at natural carbon abundance and to detect 13C NMR signal in single scan. In combination with a pseudo phase cycle, the polarization transfer method used here also enables efficient suppression of unwanted background signals.

Polarization of low-γ nuclei by transferring spin order of parahydrogen at high magnetic fields.

In this work, we optimize the performance of a previously proposed method for transferring parahydrogen induced polarization to "insensitive" spin-1/2 NMR (Nuclear Magnetic Resonance) nuclei, which have low gyromagnetic ratio and low natural abundance. By optimizing the reaction conditions and pressure of the parahydrogen gas and using adiabatically switched radiofrequency fields we achieve high polarization transfer efficiency and report carbon spin polarization of dimethyl acetylene dicarboxylate reaching 35%, which corresponds to 13C NMR signal enhancements of about 43,000 at 9.4 Tesla. Such polarization levels allow one to work with mM concentrations at natural carbon abundance and to detect 13C NMR signal in single scan. In combination with a pseudo phase cycle, the polarization transfer method used here also enables efficient suppression of unwanted background signals.

Lactonization Method To Assign the Anomeric Configuration of the 3,4-Unsaturated Congeners of N-Acetylneuraminic Acid.

Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.

A Complete and Unambiguous 1H and 13C NMR Signals Assignment of para- Naphthoquinones, ortho- and para-Furanonaphthoquinones

A complete and unambiguous assignment of 1H and 13C nuclear magnetic resonance (NMR) signals of 29 naphthoquinones is reported on the basis of one- and two-dimensional NMR techniques (1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear multiple-bond correlation (HMBC)). This is the first report distinguishing data between para-naphthoquinones, ortho- and para-furanonaphthoquinones isomers.

Glyoxalase activity in human erythrocytes and mouse lymphoma, liver and brain probed with hyperpolarized 13C-methylglyoxal

Methylglyoxal is a faulty metabolite. It is a ubiquitous by-product of glucose and amino acid metabolism that spontaneously reacts with proximal amino groups in proteins and nucleic acids, leading to impairment of their function. The glyoxalase pathway evolved early in phylogeny to bring about rapid catabolism of methylglyoxal, and an understanding of the role of methylglyoxal and the glyoxalases in many diseases is beginning to emerge. Metabolic processing of methylglyoxal is very rapid in vivo and thus notoriously difficult to detect and quantify. Here we show that 13C nuclei in labeled methylglyoxal can be hyperpolarized using dynamic nuclear polarization, providing 13C nuclear magnetic resonance signal enhancements in the solution state close to 5,000-fold. We demonstrate the applications of this probe of metabolism for kinetic characterization of the glyoxalase system in isolated cells as well as mouse brain, liver and lymphoma in vivo.

Multinuclear Applications on 0.5 T Magnetic Resonance Scanner

The article describes experiments on the detection of nuclear magnetic resonance (NMR) signals from nuclei other than protons on low field (0.5 T) clinical MR scanner. The possibility of obtaining NMR spectra and MR imaging (MRI) from nuclei with a high natural abundance (19F, 31P, 11B, 23Na), as well as isotope-enriched samples (13C and 2H), is presented. The possibilities of recording NMR spectra for low-sensitivity nuclei (13C, 29Si, 2H, 17O, 14N) are shown. Special attention is paid to the detection of NMR signal from perfluorocarbons (PFC). The examples of 19F MRI of PFC obtained in the in vivo study of both animals and human are presented. The 19F MRI of fluorinated gas, which can be used as a contrast agent for lung imaging, was also obtained. The opportunity of increasing 13C NMR signal from PFC by polarization transfer from fluorine nuclei is demonstrated. It was shown that 23Na can be detected in NMR spectroscopy and MRI of animal and human. The prospects of 17O NMR for technology applications are noticed.

Solid-State NMR Characterization of the Structure of Self-Assembled Ile–Phe–OH

Solid-state nuclear magnetic resonance (NMR) spectroscopy provides significant structural information regarding the conformation and dynamics of a variety of solid samples. In this study, we recorded the 13C and 15N solid-state NMR spectra of a self-assembled isoleucine-phenylalanine (Ile–Phe–OH) dipeptide. Immediately after the addition of hexane to a solution of concentrated peptide in ethyl acetate, the peptide visually aggregated into a nanofiber. Then, we obtained well-resolved 13C and 15N NMR signals of the natural, isotopic-abundant Ile–Phe–OH peptide in the nanofiber. Furthermore, we calculated the chemical shift values of the reported crystal structure of the Ile–Phe dipeptide via the density functional theory (DFT) calculation and compared these results with the experimental values. Notably, the two sets of values were in good agreement with each other, which indicated that the self-assembled structure closely reflected the crystal structure. Therefore, herein, we demonstrated that solid-state NMR characterization combined with DFT calculations is a powerful method for the investigation of molecular structures in self-assembled short peptides.

13C Dynamic Nuclear Polarization Using a Trimeric Gd3+ Complex as an Additive.

Dissolution dynamic nuclear polarization (DNP) is one of the most successful techniques that resolves the insensitivity problem in liquid-state nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) by amplifying the signal by several thousand-fold. One way to further improve the DNP signal is the inclusion of trace amounts of lanthanides in DNP samples doped with trityl OX063 free radical as the polarizing agent. In practice, stable monomeric gadolinium complexes such as Gd-DOTA or Gd-HP-DO3A are used as beneficial additives in DNP samples, further boosting the DNP-enhanced solid-state 13C polarization by a factor of 2 or 3. Herein, we report on the use of a trimeric gadolinium complex as a dopant in 13C DNP samples to improve the 13C DNP signals in the solid-state at 3.35 T and 1.2 K and consequently, in the liquid-state at 9.4 T and 298 K after dissolution. Our results have shown that doping the 13C DNP sample with a complex which holds three Gd3+ ions led to an improvement of DNP-enhanced 13C polarization by a factor of 3.4 in the solid-state, on par with those achieved using monomeric Gd3+ complexes but only requires about one-fifth of the concentration. Upon dissolution, liquid-state 13C NMR signal enhancements close to 20 000-fold, approximately 3-fold the enhancement of the control samples, were recorded in the nearby 9.4 T high resolution NMR magnet at room temperature. Comparable reduction of 13C spin-lattice T1 relaxation time was observed in the liquid-state after dissolution for both the monomeric and trimeric Gd3+ complexes. Moreover, W-band electron paramagnetic resonance (EPR) data have revealed that 3-Gd doping significantly reduces the electron T1 of the trityl OX063 free radical, but produces negligible changes in the EPR spectrum, reminiscent of the results with monomeric Gd3+-complex doping. Our data suggest that the trimeric Gd3+ complex is a highly beneficial additive in 13C DNP samples and that its effect on DNP efficiency can be described in the context of the thermal mixing mechanism.

Characterization of brominated natural rubber by solution-state 2D NMR spectroscopy

Bromination of natural rubber was analyzed through nuclear magnetic resonance (NMR) spectroscopy, in which small 1H and 13C NMR signals, appearing after bromination of natural rubber, were positively assigned by 1D and 2D NMR measurements. The brominated natural rubber (BrDPNR) was prepared by bromination of deproteinized natural rubber (DPNR) with N-bromosuccinimide (NBS) in dichloromethane at 303K for 3h under a nitrogen atmosphere. The 13C NMR signals at 20.5, 22, 29, 29.8, 35.8, 40 and 112.4ppm were assigned to the primary and the secondary carbons of the brominated trans-1,4-isoprene units, whereas the signals at 24.5 and 32.6ppm were assigned to the primary and the secondary carbons of the brominated cis-1,4-isoprene units. During the bromination of DPNR with NBS, the isoprene units underwent cis-trans isomerization.

Low-temperature dynamic nuclear polarization with helium-cooled samples and nitrogen-driven magic-angle spinning

We describe novel instrumentation for low-temperature solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS), focusing on aspects of this instrumentation that have not been described in detail in previous publications. We characterize the performance of an extended interaction oscillator (EIO) microwave source, operating near 264GHz with 1.5W output power, which we use in conjunction with a quasi-optical microwave polarizing system and a MAS NMR probe that employs liquid helium for sample cooling and nitrogen gas for sample spinning. Enhancement factors for cross-polarized 13C NMR signals in the 100–200 range are demonstrated with DNP at 25K. The dependences of signal amplitudes on sample temperature, as well as microwave power, polarization, and frequency, are presented. We show that sample temperatures below 30K can be achieved with helium consumption rates below 1.3l/h. To illustrate potential applications of this instrumentation in structural studies of biochemical systems, we compare results from low-temperature DNP experiments on a calmodulin-binding peptide in its free and bound states.

Synthesis and evaluation of nitroxide-based oligoradicals for low-temperature dynamic nuclear polarization in solid state NMR

We describe the synthesis of new nitroxide-based biradical, triradical, and tetraradical compounds and the evaluation of their performance as paramagnetic dopants in dynamic nuclear polarization (DNP) experiments in solid state nuclear magnetic resonance (NMR) spectroscopy with magic-angle spinning (MAS). Under our experimental conditions, which include temperatures in the 25–30K range, a 9.4T magnetic field, MAS frequencies of 6.2–6.8kHz, and microwave irradiation at 264.0GHz from a 800mW extended interaction oscillator source, the most effective compounds are triradicals that are related to the previously-described compound DOTOPA-TEMPO (see Thurber et al., 2010), but have improved solubility in glycerol/water solvent near neutral pH. Using these compounds at 30mM total nitroxide concentration, we observe DNP enhancement factors of 92–128 for cross-polarized 13C NMR signals from 15N,13C-labeled melittin in partially protonated glycerol/water, and build-up times of 2.6–3.8s for 1H spin polarizations. Net sensitivity enhancements with biradical and tetraradical dopants, taking into account absolute 13C NMR signal amplitudes and build-up times, are approximately 2–4 times lower than with the best triradicals.

Loss of optical and molecular indicators of terrigenous dissolved organic matter during long-term photobleaching

Two water samples from the Great Dismal Swamp National Wildlife Refuge with high dissolved organic matter (DOM) concentrations (51 and 121 mg C L−1) were subjected to ultraviolet (UV) light for up to 110 days. During the course of the irradiations, 74–88 % of the original dissolved organic carbon was lost along with 95–99 % of the absorption at 300 nm. Based on changes observed during light exposure, three pools of DOM were identified: photo-labile, photo-refractory, and photo-produced compounds. Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to determine structural moieties characteristic to each of these pools. These analyses showed aromatic carbons were preferentially removed while carbohydrate-like and amide/peptide-like carbons were preserved during UV exposure. An increase in carbon normalized 13C NMR signal in the 0–50 ppm region suggests that alkyl moieties were produced, while FTIR signal at 1,745 cm−1 and two-dimensional 1H–13C NMR results confirmed the photochemical production of acetate. Several properties typically used to trace terrigenous DOM in ocean margin and marine environments were significantly altered. Optical properties, including absorption spectral slopes and fluorescence indices, as well as carbon-normalized lignin yields shifted from terrestrial values towards those more typical of coastal or open ocean samples. The loss of terrestrial signatures during irradiation highlights the difficulty faced when quantifying the contribution of terrigenous DOM to aquatic carbon pools.

Electron Transfer Mechanism of the Rieske Protein from Thermus thermophilus from Solution Nuclear Magnetic Resonance Investigations

We report nuclear magnetic resonance (NMR) data indicating that the Rieske protein from the cytochrome bc complex of Thermus thermophilus (TtRp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. To test models concerning the mechanism by which TtRp transfers between different sites on the complex, we monitored (1)H, (15)N, and (13)C NMR signals as a function of the redox state and molar ratio of added ligand. Our studies of full-length TtRp were conducted in the presence of dodecyl phosphocholine micelles to solvate the membrane anchor of the protein and the hydrophobic tail of the ligand (hydroubiquinone). NMR data indicated that hydroubiquinone binds to TtRp and stabilizes an altered protein conformation. We utilized a truncated form of the Rieske protein lacking the membrane anchor (trunc-TtRp) to investigate redox-state-dependent conformational changes. Local chemical shift perturbations suggested possible conformational changes at prolyl residues. Detailed investigations showed that all observable prolyl residues of oxidized trunc-TtRp have trans peptide bond configurations but that two of these peptide bonds (Cys151-Pro152 and Gly169-Pro170 located near the iron-sulfur cluster) become cis in the reduced protein. Changes in the chemical shifts of backbone signals provided evidence of redox-state- and ligand-dependent conformational changes localized near the iron-sulfur cluster. These structural changes may alter interactions between the Rieske protein and the cytochrome b and c sites and provide part of the driving force for movement of the Rieske protein between these two sites.

Solid state nuclear magnetic resonance with magic-angle spinning and dynamic nuclear polarization below 25 K

We describe an apparatus for solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS) at 20–25K and 9.4Tesla. The MAS NMR probe uses helium to cool the sample space and nitrogen gas for MAS drive and bearings, as described earlier [1], but also includes a corrugated waveguide for transmission of microwaves from below the probe to the sample. With a 30mW circularly polarized microwave source at 264GHz, MAS at 6.8kHz, and 21K sample temperature, greater than 25-fold enhancements of cross-polarized 13C NMR signals are observed in spectra of frozen glycerol/water solutions containing the triradical dopant DOTOPA-TEMPO when microwaves are applied. As demonstrations, we present DNP-enhanced one-dimensional and two-dimensional 13C MAS NMR spectra of frozen solutions of uniformly 13C-labeled l-alanine and melittin, a 26-residue helical peptide that we have synthesized with four uniformly 13C-labeled amino acids.